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Sample records for ovine plasminogen activator

  1. Insulin and insulin-like growth factor I exert different effects on plasminogen activator production or cell growth in the ovine thyroid cell line OVNIS.

    Degryse, B; Maisonobe, F; Hovsépian, S; Fayet, G

    1991-11-01

    Insulin and Insulin-like Growth Factor I (IGF-I) are evaluated for their capacity to affect cell proliferation and plasminogen activator (PA) activity production in an ovine thyroid cell line OVNIS. Insulin at physiological and supraphysiological doses induces cell proliferation and increases PA activity. IGF-I, which is also clearly mitogenic for these cells, surprisingly does not modulate PA activity. The results indicate that the growth promoting effect is mediated through the insulin and IGF-I receptors whereas PA activity is solely regulated via the insulin receptors. PMID:1802921

  2. Plasminogen activation in cancer

    Reijerkerk, Arie

    2004-01-01

    The subject of this thesis focusses on the role of the plasminogen activation system in angiogenesis and cancer. The plasminogen activation system regulates fibrinolysis and controls cell migration and invasion by plasmin-mediated matrix proteolysis. Plasmin is formed upon cleavage of the zymogen pl

  3. Characterization of plasmin, plasminogen and plasminogen activator in goat milk

    F. Polidori; G. Lebbroni; Polidori, P; Fantuz, F

    2011-01-01

    Plasmin (PL), a serine-proteinase, appears to be the predominant native proteinase in milk and it is mainly associated to casein micelles which represent its substrate (Bastian and Brown, 1996). Plasmin occurs in milk together with its inactive zymogene, plasminogen (PG) (Bastian et al. 1991). The cascade of reactions leading to plasminogen activation is regulated by a complex network of molecular interactions between plasminogen activators (PA) (tissue-type and urokinase-type) and at least t...

  4. PLASMINOGEN ACTIVATOR OF YERSINIA PESTIS

    V. V. Evseeva

    2015-01-01

    Full Text Available Plague has been the cause of three pandemics and has led to the death of millions of people. Plague is a typical zoonosis caused by Yersinia pestis that circulates in populations of wild rodents inhabiting natural plague foci on all continents except for Australia. Transmission of plague is provided by flea bites. Circulation of Y. pestis in natural plague foci is supported by a numerous of pathogenicity factors. This review explores one of them, plasminogen activator Pla. This protein is one of representatives of omptins, a family of enterobacterial outer membrane proteases that are responsible for colonization of specific organs or even infection generalization as a result of successful overcoming of the host innate immunity. The review reflects the history of its discovery and studying of its genetic control, biosynthesis, isolation and purification, physicochemical properties. Highly purified preparations of plasminogen activator are deficient in enzymatic activities but renaturation in the presence of Y. pestis lipooligosaccharide restores enzymatic properties of Pla. This pathogenicity factor is absent in representatives of the most ancient phylogenetic group of the plague pathogen, bv. caucasica, while the ancestor of other groups of Y. pestis subsp. microtus obtained in result of horizontal transfer Pla isoform with characteristics similar to properties of omptins from the less virulent enterobacteria. After that in the course of microevolution the “classic” isoform of Pla with increased protease activity was selected that is typical of all highly virulent for humans strains of Y. pestis subsp. pestis. The “classic” isoform of Pla Y. pestis is functionally similar to mammalian plasminogen activators transforming plasminogen into plasmin with the help of limited proteolysis. Pla protease activating plasminogen and also degrading the main plasmin inhibitor — α2-antiplasmin and, respectively, determining Y. pestis ability to lyse

  5. Characterization of plasmin, plasminogen and plasminogen activator in goat milk

    F. Polidori

    2011-03-01

    Full Text Available Plasmin (PL, a serine-proteinase, appears to be the predominant native proteinase in milk and it is mainly associated to casein micelles which represent its substrate (Bastian and Brown, 1996. Plasmin occurs in milk together with its inactive zymogene, plasminogen (PG (Bastian et al. 1991. The cascade of reactions leading to plasminogen activation is regulated by a complex network of molecular interactions between plasminogen activators (PA (tissue-type and urokinase-type and at least three types of specific PA inhibitors (Politis, 1996. Stage of lactation affects PL and PA activities: late lactation is associated with higher activity of PL and PA (Baldi et al., 1996. Plasmin in milk is responsible for the hydrolysis of α- and β-caseins (Aslam and Hurley, 1997; Trujillo et al., 1997...

  6. The effect of anti-human plasminogen monoclonal antibodies on Glu-plasminogen activation by plasminogen activators

    M. Akrami

    2006-07-01

    Full Text Available Background: Human plasminogen is a plasma glycoprotein synthesized mainly in the liver. Conversion of plasminogen to plasmin by plasminogen activators is a key event in the fibrinolytic system. In this study, we investigated the effects of two anti-human plasminogen monoclonal antibodies, A1D12 and MC2B8 on Glu-plasminogen activation in presence of u-PA, t-PA and streptokinase. Methods: Producing of Hybridoma antibodies was performed by fusion of spleen cells from BALB/C mice immunized with Glu-plasminogen and NS1 myeloma cells. Antibody binding to Human Glu-plasminogen was assessed using an ELISA assay. Activation of plasminogen was determined by measuring plasmin generation using the chromogenic substrate S-2251 and the effect of monoclonal antibodies, A1D12 and MC2B8 on plasminogen activation in solution was then evaluated. Initial rates and kinetic parameters of plasminogen activation in the presence of monoclonal antibodies were calculated. The effect of the monoclonal antibody MC2B8 on the rate of plasmin hydrolysis was measured. The effect of F(ab'2 fragment of A1D12 on u-PA catalyzed-plasminogen activation also compared with the effect of the whole antibody in this reaction. Results: ELISA assay showed that the antibodies reacted well with antigens. A1D12 increased the maximum velocity (Vmax of plasminogen activation by each of the three plasminogen activators and MC2B8 decreased it. In all activation reactions, the KM value of plasminogen activation did not significantly change in the presence of antibody A1D12 whereas antibody MC2B8 increased the KM value of plasminogen activation by u-PA, fibrin monomer dependent t-PA and streptokinase. Monoclonal antibody MC2B8 had no significant effect on plasmin hydrolysis rate of synthetic substrate S-2251. Activation rate of plasminogen by u-PA in the lower concentration of F (ab2 fragment of A1D12 was identical to activation in the presence of the whole antibody. Conclusion: The binding of

  7. Tissue plasminogen activator and urokinase plasminogen activator in human epileptogenic pathologies

    A.M. Iyer; E. Zurolo; K. Boer; J.C. Baayen; F. Giangaspero; A. Arcella; G.C. Di Gennaro; V. Esposito; W.G.M. Spliet; P.C. van Rijen; D. Troost; J.A. Gorter; E. Aronica

    2010-01-01

    A growing body of evidence demonstrates the involvement of plasminogen activators (PAs) in a number of physiologic and pathologic events in the CNS. Induction of both tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) has been observed in different experimental models of ep

  8. Plasminogen activation and cancer

    Danø, Keld; Behrendt, N.; Hoyer-Hansen, G.;

    2005-01-01

    Breakdown of the extracellular matrix is crucial for cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease plasmin and a number of matrix metalloproteases.The activity of each of these proteases is regulated by an array of...... regulation of extracellular proteolysis in cancer involves a complex interplay between cancer cells and non-malignant stromal cells in the expression of the molecular components involved. For some types of cancer, this cellular interplay mimics that observed in the tissue of ori- gin during non......-neoplastic tissue remodelling processes.We propose that cancer invasion can be considered as uncontrolled tissue remodelling. Inhibition of extracellular proteases is an attractive approach to cancer therapy. Because proteases have many different functions in the normal organism, efficient inhibition will have...

  9. Liposomal nanocarriers for plasminogen activators.

    Koudelka, Stepan; Mikulik, Robert; Mašek, Josef; Raška, Milan; Turánek Knotigová, Pavlína; Miller, Andrew D; Turánek, Jaroslav

    2016-04-10

    Several plasminogen activators (PAs) have been found effective in treating different thromboembolic diseases. However, administration of conventional thrombolytic therapy is limited by a low efficacy of present formulations of PAs. Conventional treatments using these therapeutic proteins are associated with several limitations including rapid inactivation and clearance, short half-life, bleeding complications or non-specific tissue targeting. Liposome-based formulations of PAs such as streptokinase, tissue-plasminogen activator and urokinase have been developed to improve the therapeutic efficacy of these proteins. Resulting liposomal formulations were found to preserve the original activity of PAs, promote their selective delivery and improve thrombus targeting. Therapeutic potential of these liposome-based PAs has been demonstrated successfully in various pre-clinical models in vivo. Reductions in unwanted side effects (e.g., hemorrhage or immunogenicity) as well as enhancements of efficacy and safety were achieved in comparison to currently existing treatment options based on conventional formulations of PAs. This review summarizes present achievements in: (i) preparation of liposome-based formulations of various PAs, (ii) development of PEGylated and targeted liposomal PAs, (iii) physico-chemical characterization of these developed systems, and (iv) testing of their thrombolytic efficacy. We also look to the future and the imminent arrival of theranostic liposomal formulations to move this field forward. PMID:26876783

  10. Innovative Approaches to Plasminogen Activator Therapy

    Haber, Edgar; Quertermous, Thomas; Matsueda, Gary R.; Runge, Marschall S.

    1989-01-01

    Plasminogen activator therapy for acute myocardial infarction has become standard medical practice. Bleeding complications, however, limit the utility of the currently available agents. This article reviews how the tools of molecular biology and protein engineering are being used to develop safer and more effective plasminogen activators.

  11. Molecular assembly of plasminogen and tissue-type plasminogen activator on an evolving fibrin surface

    Fleury, V.; Loyau, S.; Lijnen, H.R.; Nieuwenhuizen, W.; Anglés-Cano, E.

    1993-01-01

    A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue-type plasminogen activator (t-PA), plasminogen and fi

  12. Plasminogen activator and its assay of the activity

    Plasminogen activators (PA) are specific proteolytic enzymes. Which convert the inactive proenzyme to plasmin. The plasmin formed is a potent and nonspecific protease which cleaves blood fibrin clots into soluble polypeptide. The author describes some biochemical characteristic of the different components of the plasminogen activator system, current methods for assay of the activity of the PA. The potential application of PA as diagnostic tools in diseases of the thrombi

  13. Design of a novel chimeric tissue plasminogen activator with favorable Vampire bat plasminogen activator properties.

    Kazemali, MohammadReza; Majidzadeh-A, Keivan; Sardari, Soroush; Saadatirad, Amir Hossein; Khalaj, Vahid; Zarei, Najmeh; Barkhordari, Farzaneh; Adeli, Ahmad; Mahboudi, Fereidoun

    2014-12-01

    Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator. PMID:25442953

  14. Plasminogen activator inhibitor type 1 gene polymorphism and sepsis.

    Hermans, P.W.M.; Hazelzet, J.A.

    2005-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a 50-kilodalton glycoprotein of the serine protease inhibitor family. The primary role of PAI-1 in vivo is the inhibition of both tissue- and urokinase-type plasminogen activators. In addition to this function, PAI-1 acts as an acute-phase protein du

  15. Aberrant glomerular filtration of urokinase-plasminogen activator in nephrotic syndrome leads to amiloride-sensitive plasminogen activation in urine

    Stæhr, Mette; Buhl, Kristian Bergholt; Andersen, René F;

    2015-01-01

    In nephrotic syndrome, aberrant glomerular filtration of plasminogen and conversion to active plasmin in pre-urine is thought to activate proteolytically ENaC and contribute to sodium retention and edema. The ENaC blocker amiloride is an off-target inhibitor of urokinase-type plasminogen activator...

  16. Retinoic acid upregulates the plasminogen activator system in human epidermal keratinocytes.

    Braungart, E; Magdolen, V; Degitz, K

    2001-05-01

    The activation of the proteolytic plasminogen activator system is important for the re-epithelialization of skin wounds. Keratinocytes synthesize and secrete the urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. Receptor-bound urokinase-type plasminogen activator efficiently activates cell surface bound plasminogen. This results in pericellular proteolysis, which facilitates keratinocyte migration. Urokinase-type plasminogen activator activity is specifically controlled by plasminogen activator inhibitor-1 and -2. As retinoids have been reported to accelerate epithelialization of skin wounds in animal studies and clinical settings, we investigated the effects of all-trans retinoic acid on the plasminogen activator system in human epidermal keratinocytes. As tested in a chromogenic plasminogen activation assay, incubation with 10 microM all-trans retinoic acid caused a marked induction of cell-associated plasminogen activity after 24 h, and this induction was blocked by neutralizing anti-urokinase-type plasminogen activator antibodies, but not anti-tissue-type plasminogen activator antibodies. All-trans retinoic acid lead to a strong increase in urokinase-type plasminogen activator (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) after 24 h. At this time-point, tissue-type plasminogen activator and plasminogen activator inhibitor-1 and -2 proteins were not or only slightly increased. Northern blot analyses revealed that all-trans retinoic acid caused an early and short-lived increase of plasminogen activator inhibitor-1, but a prolonged induction of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA levels. Collectively, these data suggest that all-trans retinoic acid activates the plasminogen activator system in human epidermal keratinocytes by differentially regulating activating and inhibiting components

  17. Increased alveolar plasminogen activator in early asbestosis

    Cantin, A.; Allard, C.; Begin, R.

    1989-03-01

    Alveolar macrophage-derived plasminogen activator (PA) activity is decreased in some chronic interstitial lung diseases such as idiopathic pulmonary fibrosis and sarcoidosis but increased in experimental models of acute alveolitis. Although asbestos fibers can stimulate alveolar macrophages (AM) to release PA in vitro, the effect of chronic asbestos exposure of the lower respiratory tract on lung PA activity remains unknown. The present study was designed to evaluate PA activity of alveolar macrophages and bronchoalveolar lavage (BAL) fluid in asbestos-exposed sheep and asbestos workers. Forty-three sheep were exposed to either 100 mg UICC chrysotile B asbestos in 100 ml phosphate-buffered saline (PBS) or to 100 ml PBS by tracheal infusion every 2 wk for 18 months. At Month 18, chest roentgenograms were analyzed and alveolar macrophage and extracellular fluid PA activity were measured in samples obtained by BAL. Alveolar macrophage PA activity was increased in the asbestos-exposed sheep compared to control sheep (87.2 +/- 17.3 versus 41.1 +/- 7.2 U/10(5) AM-24 h, p less than 0.05) as was the BAL fluid PA activity (674.9 +/- 168.4 versus 81.3 +/- 19.7 U/mg alb-24 h, p less than 0.01). Among the asbestos-exposed sheep, 10 had normal chest roentgenograms (Group SA) and 15 had irregular interstitial opacities (Group SB). Strikingly, whereas Group SA did not differ from the control group in BAL cellularity or PA activity, Group SB had marked increases in alveolar macrophages (p less than 0.005), AM PA activity (p less than 0.02), and BAL PA activity (p less than 0.001) compared to the control group.

  18. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    Roldan, A L; Cubellis, M V; Masucci, M T;

    1990-01-01

    The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, an...

  19. Urokinase and type I plasminogen activator inhibitor production by normal human hepatocytes: modulation by inflammatory agents.

    Busso, N; Nicodeme, E; Chesne, C; Guillouzo, A; Belin, D; Hyafil, F

    1994-07-01

    We examined the effects of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-beta) on the plasminogen activator system (urokinase, tissue-type plasminogen activator, type 1 plasminogen activator inhibitor) in primary cultures of human hepatocytes. We show that interleukin-1 beta and tumor necrosis factor-alpha increase urokinase-type plasminogen activator production, reinforcing the concept that increased urokinase production is associated with inflammatory processes. By contrast, the same agents (i.e., interleukin-1 beta and tumor necrosis factor-alpha) do not stimulate plasminogen activator inhibitor type 1 production. This latter observation rules out hepatocytes as a major cellular source of plasmatic plasminogen activator inhibitor type 1 during acute-phase-related responses. Among the inflammatory agents used, transforming growth factor-beta was found to be the most effective modulator of both urokinase-type plasminogen activator and plasminogen activator inhibitor type 1, inducing severalfold increases of activity of urokinase-type plasminogen activator, antigen and the corresponding mRNA and increasing plasminogen activator inhibitor type 1 antigen and mRNA levels. Urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 modulation by transforming growth factor-beta may play a critical role in hepatic pathophysiology. PMID:8020888

  20. The topology of plasminogen binding and activation on the surface of human breast cancer cells

    Andronicos, N M; Ranson, M.

    2001-01-01

    The urokinase-dependent activation of plasminogen by breast cancer cells plays an important role in metastasis. We have previously shown that the metastatic breast cancer cell line MDA-MB-231 over-expresses urokinase and binds and efficiently activates plasminogen at the cell surface compared to non-metastatic cells. The aim of this study was to further characterise plasminogen binding and determine the topology of cell surface-bound plasminogen in terms of its potential for activation. The l...

  1. Aspirin inhibits vascular plasminogen activator activity in vivo. Studies utilizing a new assay to quantify plasminogen activator activity.

    Levin, R I; Harpel, P C; Weil, D; T. S. Chang; Rifkin, D. B.

    1984-01-01

    Vascular or tissue-type plasminogen activator (TPA) is a key enzyme in physiologic fibrinolysis. To study the role of prostaglandins in modulating the synthesis and release of TPA in vivo, we prospectively studied the effect of aspirin (650 mg/d X 2) on TPA activity in 13 human subjects before and after 10 min of forearm venous occlusion. TPA activity was quantified by a newly developed enzyme-linked immunosorbent assay that both measures and differentiates between TPA and urokinase (UK)-like...

  2. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter; Christensen, Anni; Naessens, Dominik; Deinum, Johanna; Enghild, Jan Johannes; Declerck, Paul; Andreasen, Peter

    2003-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous......-inactivating compounds of potential clinical importance....

  3. Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin alpha v beta 3 induces signal transduction and enhances plasminogen activation

    Tarui, Takehiko; Akakura, Nobuaki; Majumdar, Mousumi; Andronicos, Nicholas; Takagi, Junichi; Mazar, Andrew P.; Bdeir, Khalil; Kuo, Alice; Yarovoi, Serge V.; Cines, Douglas B.; Takada, Yoshikazu

    2006-01-01

    It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity recep...

  4. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive 125I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface

  5. Pericellular proteolytic cascade by plasmin/plasminogen activator system

    2001-01-01

    Plasmin/plasminogen activators (PA) are the serine enzyme which digests fibrin and/or fibrinogen. Plasmin is produced by the cleavage of its precursor, plasminogen by PAs (urokinase-type PA and tissue-type PA). These events are expected in the thrmbolytic therapy for thromboembolic deseases. Apart from the blood fibrinolysis mentioned above, new role of plasmin/plasminogen activators has been extensively investigated in the field of cellular biology. On the cell surface, the receptor for urokinase-type PA (u-PAR) was found (that for t-PA has not cloned yet). Then, plasmin as well as u-PA itself activates pro-form of matrix metalloproteinases (MMPs) around the pericellular space. These proteolytic activities by u-PA, plasmin and MMPs induce the degradation of extracellular matrix (ECM), affording the cells certain enviroment for their biological function. Further, the coupling of u-PA/u-PAR system and integrins can generate intracellular signal transductions which take part in the regulation of cell proliferation, attachment or migration followed by various physiological and pathophysiological functions. These serial mechanisms are the principle of pericellular proteolytic cascade.

  6. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  7. Photonic Activation of Plasminogen induced by low dose UVB

    Correia, Manuel Guiherme L.P. Marins; Snabe, Torben; Thiagarajan, Viruthachalam;

    2015-01-01

    products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show...... observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical.......3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads...

  8. Urokinase plasminogen activator and plasminogen activator inhibitor type-1 in nonsmall-cell lung cancer: relation to prognosis and angiogenesis

    Offersen, Birgitte Vrou; Pfeiffer, Per; Andreasen, Peter;

    2007-01-01

    BACKGROUND: Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have previously been suggested as prognostic markers in nonsmall-cell lung carcinomas (NSCLC). We investigate whether uPA and PAI-1 are prognostic markers in NSCLC and whether they are related to...... sandwich ELISA method. RESULTS: Both uPA and PAI-1 were independent of classical histopathological parameters as well as of microvessel density and vascular pattern. Using death within the first 5 years as endpoint, neither of the factors were prognostic markers in univariate analysis, however......, significantly higher levels of uPA and PAI-1 were seen in tumours with an angiogenic vascular pattern. In multivariate analysis, high disease stage (P<0.0001), adenocarcinoma (P=0.007), old age (P=0.02), and presence of an angiogenic pattern (P=0.05) were identified as independent markers of death within 5...

  9. UVB increases urokinase-type plasminogen activator receptor (uPAR) expression.

    Marschall, C; Lengyel, E; Nobutoh, T; Braungart, E; Douwes, K; Simon, A; Magdolen, V; Reuning, U; Degitz, K

    1999-07-01

    Keratinocytes synthesize and secrete urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. When bound to urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator proteolytically converts surface bound plasminogen to plasmin, which in turn cleaves many extracellular components leading to pericellular proteolysis. The activation of the urokinase system has been observed during re-epithelialization of skin wounds and in lesions of the autoimmune blistering skin disease pemphigus. As pemphigus is photoinducible, we investigated the effect of ultraviolet B on urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor expression in the epidermal keratinocyte cell line A431. Ultraviolet B increased cellular and secreted urokinase-type plasminogen activator protein (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) 24 h postirradiation. Northern blot analysis indicated that ultraviolet B increased urokinase-type plasminogen activator receptor mRNA. Compared with a more rapid mRNA induction by epidermal growth factor (maximal after 4 h) the ultraviolet B response was maximal after 24 h and prolonged up to 36 h. The mRNA induction was not dependent on protein synthesis as judged by cycloheximide incubation. Ultraviolet B did not influence urokinase-type plasminogen activator receptor mRNA stability (actinomycin D incubation). A transiently transfected chloramphenicol acetyltransferase-reporter construct containing a -398/+51 urokinase-type plasminogen activator receptor promoter fragment was activated when cells were exposed to ultraviolet B. This induction was almost completely abolished by mutating a -182/-176 AP-1 binding sequence. Ultraviolet B increased the binding capacity at this AP-1 motif in electrophoretic mobility shift assays. These data identify a distinct transcriptional mechanism by which

  10. Recombinant tissue plasminogen activator following paediatric cataract surgery

    Mehta, J; ADAMS, G

    2000-01-01

    BACKGROUND—The use of recombinant tissue plasminogen activator (r-TPA) has been advocated in the treatment of postsurgical fibrinous membrane formation following cataract surgery in adults. Its use in paediatric cases is not well documented.
METHOD—A retrospective review of paediatric cataract extractions performed at Moorfields Eye Hospital between 1 January 1997 and 4 April 1999 was carried out.
RESULTS—Cataract extractions were performed in 37 patients, 22 in males 15 in females. Four (9.2...

  11. Comparative molecular analysis of ovine and bovine Streptococcus uberis isolates.

    Gilchrist, T L; Smith, D G E; Fitzpatrick, J L; Zadoks, R N; Fontaine, M C

    2013-02-01

    Streptococcus uberis causes clinical and subclinical mastitis in cattle and sheep, but it is unknown whether the composition of Strep. uberis populations differs between host species. To address this, we characterized a collection of bovine and ovine Strep. uberis isolates with shared geographical and temporal origins by means of an expanded multilocus sequence typing scheme. Among 14 ovine and 35 bovine isolates, 35 allelic profiles were detected. Each allelic profile was associated with a single host species and all but one were new to the multilocus sequence typing database. The median number of new alleles per isolate was higher for ovine isolates than for bovine isolates. None of the ovine isolates belonged to the global clonal complexes 5 or 143, which are commonly associated with bovine mastitis and which have a wide geographical distribution. Ovine isolates also differed from bovine isolates in carriage of plasminogen activator genes, with significantly higher prevalence of pauB in ovine isolates. Isolates that were negative for yqiL, one of the targets of multilocus sequence typing, were found among ovine and bovine isolates and were not associated with a specific sequence type or global clonal complex. One bovine isolate carried a gapC allele that was probably acquired through lateral gene transfer, most likely from Streptococcus salivarius. We conclude that ovine isolates are distinct from bovine isolates of Strep. uberis, and that recombination between isolates from different host species or bacterial species could contribute to changes in virulence gene profiles with relevance for vaccine development. PMID:23200465

  12. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay

    A two-site immunoradiometric assay for human extrinsic (tissue-type) plasminogen activator was developed by using rabbit antibodies raised against plasminogen activator purified from human melanoma cell culture fluid. Samples of 100 μl containing 1 to 100 ng/ml plasminogen activator were incubated in the wells of polyvinyl chloride microtiter plates coated with antibody. The amount of bound extrinsic plasminogen activator was quantitated by the subsequent binding of 125I-labeled affinospecific antibody. The mean level of plasma samples taken at rest was 6.6 +/- 2.9 ng/ml (n = 54). This level increased approximately threefold by exhaustive physical exercise, venous occlusion, or infusion of DDAVP. Extrinsic plasminogen activator in plasma is composed of a fibrin-adsorbable and active component (1.9 +/- 1.1 ng/ml, n = 54, in resting conditions) and an inactive component that does not bind to a fibrin clot (probably extrinsic plasminogen activator-proteinase inhibitor complexes). The fibrin-adsorbable fraction increased approximately fivefold to eightfold after physical exercise, venous occlusion, or DDAVP injections. Potential applications of the immunoradiometric assay are illustrated by the measurement of extrinsic plasminogen activator in different tissue extracts, body fluids, and cell culture fluids and in oocyte translation products after injection with mRNA for plasminogen activator

  13. Plasma soluble urokinase plasminogen activator receptor in children with urinary tract infection

    Wittenhagen, Per; Andersen, Jesper Brandt; Hansen, Anita; Lindholm, Lone; Rønne, Frederik Malmborg; Theil, Jørn; Tvede, Michael; Eugen-Olsen, Jesper

    2011-01-01

    In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection.......In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection....

  14. Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinase-type plasminogen activator in human U-937 lymphoma cells

    1987-01-01

    The tumor-promoting phorbol ester PMA induces changes in the histiocytic human lymphoma cell line U-937 akin to cellular differentiation (Ralph, P., N. Williams, M. A. S. Moore, and P. B. Litcofsky, 1982, Cell. Immunol., 71:215-223) and concomitantly stimulates the biosynthesis of plasminogen activator inhibitor 2 (PAI 2) and of urokinase-type plasminogen activator (u-PA). PAI 2 is found in a nonglycosylated intracellular and a glycosylated secreted form. The former appears to be identical to...

  15. Urokinase plasminogen activator (uPA and plasminogen activator inhibitor type-1 (PAI-1 in breast cancer - correlation with traditional prognostic factors

    Lampelj Maja

    2015-12-01

    Full Text Available Background. Urokinase plasminogen activator (uPA and plasminogen activator inhibitor type-1 (PAI-1 play a key role in tumour invasion and metastasis. High levels of both proteolytic enzymes are associated with poor prognosis in breast cancer patients. The purpose of this study was to evaluate the correlation between traditional prognostic factors and uPA and PAI-1 expression in primary tumour of breast cancer patients.

  16. Effect of Plasminogen Activator Inhibitor-1 and Tissue Plasminogen Activator Polymorphisms on Susceptibility to Type 2 Diabetes in Malaysian Subjects

    Zaid Al-Hamodi

    2012-01-01

    Full Text Available Elevated activity of plasminogen activator inhibitor-1 (PAI-1 and decreased tissue plasminogen activator (tPA activity are considered to be important risk factors for type 2 diabetes mellitus (T2DM and metabolic syndrome (MetS. The aim of this study was to investigate the association of the PAI-1 4G/5G and tPA Alu-repeat I/D polymorphisms with T2DM in Malaysian subjects. Serum insulin, coronary risk panel, plasma glucose, and PAI-1 4G/5G and tPA Alu-repeat I/D polymorphisms were studied in 303 T2DM subjects (227 with MetS and 76 without MetS and 131 normal subjects without diabetes and MetS. Statistical analysis showed that the dominant and additive models of PAI-1 4G/5G polymorphism showed a weak association with T2DM without MetS (OR=2.35, P=0.045; OR=1.67, P=0.058. On the other hand, the recessive model of the tPA Alu-repeat I/D polymorphism showed an association with T2DM with MetS (OR=3.32, P=0.013 whereas the dominant and additive models of the tPA Alu-repeat I/D polymorphism were not associated with T2DM either with or without MetS.

  17. Expression of urokinase plasminogen activator, its receptor and type-1 inhibitor in malignant and benign prostate tissue

    Usher, Pernille Autzen; Thomsen, Ole Frøkjær; Iversen, Peter; Johnsen, Morten; Brünner, Nils; Høyer-Hansen, Gunilla; Andreasen, Peter; Danø, Keld; Nielsen, Boye Schnack

    2005-01-01

    The plasminogen activation (PA) cascade participates in degradation of extracellular matrix during cancer invasion. We have studied the expression of urokinase-type plasminogen activator (uPA) mRNA, uPA receptor (uPAR) mRNA and immunoreactivity, and type-1 plasminogen activator inhibitor (PAI-1) ...

  18. Metastasis of transgenic breast cancer in plasminogen activator inhibitor-1 gene-deficient mice

    Almholt, Kasper; Nielsen, Boye Schnack; Frandsen, Thomas Leth; Brunner, Nils; Danø, Keld; Johnsen, M.

    2003-01-01

    The plasminogen activator inhibitor-1 (PAI-1) blocks the activation of plasmin(ogen), an extracellular protease vital to cancer invasion. PAI-1 is like the corresponding plasminogen activator uPA (urokinase-type plasminogen activator) consistently expressed in human breast cancer. Paradoxically......, high levels of PAI-1 as well as uPA are equally associated with poor prognosis in cancer patients. PAI-1 is thought to play a vital role for the controlled extracellular proteolysis during tumor neovascularization. We have studied the effect of PAI-1 deficiency in a transgenic mouse model of...... metastasizing breast cancer. In these tumors, the expression pattern of uPA and PAI-1 resembles that of human ductal breast cancer and plasminogen is required for efficient metastasis. In a cohort of 63 transgenic mice that were either PAI-1-deficient or wild-type sibling controls, primary tumor growth and...

  19. Keeping the blood flowing—plasminogen activator genes and feeding behavior in vampire bats

    Tellgren-Roth, Åsa; Dittmar, Katharina; Massey, Steven E.; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A.; Liberles, David A.

    2009-01-01

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  20. Structural and functional peculiarities of plasminogen activator inhibitor PAI-1

    Kondratuk A. S.

    2010-07-01

    Full Text Available PAI-1, an important component of the hemostasis system, is a specific inhibitor of both urokinase type and tissue type plasminogen activators. PAI-1 belongs to the serpin family. The interaction between somatomedin-like domain of vitronectin and PAI-1 leads to stabilization of the latter. PAI-1 latency transition is related to the conformational changes in the reactive central loop. The inhibitory mechanism of PAI-1 is in accordance with the classic scheme of serpin action. PAI-1 blocks the adhesion mediated by UPA and integrins, so this inhibitor plays an important role in adhesion process and angiogenesis. An altered PAI-1level is associated with the development of cardiovascular diseases, kidney fibrosis, diabetis, cancerogenesis.

  1. Bicyclic Peptide Inhibitor of Urokinase-Type Plasminogen Activator

    Roodbeen, Renée; Paaske, Berit; Jiang, Longguang;

    2013-01-01

    The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptidebased inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established...... monocyclic peptide, upain-2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across-the-ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic...... burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain-2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain-2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also...

  2. Plasminogen activator activity and plasma-coagulum lysis measured by use of optimized fibrin gel structure preformed in microtiter plates

    Sidelmann, Johannes Jakobsen; Jespersen, J; Gram, J

    1995-01-01

    We introduce a new fibrin plate assay performed in microtiter plates. By means of spectroscopic studies we optimized the structure of the fibrin gel and then used the optimized fibrin gel to determine plasminogen activator activity. Plasminogen activator solutions were applied on top of the fibrin...

  3. Mean transit times and the sites of synthesis and catabolism of tissue plasminogen activator and plasminogen activator inhibitor type 1 in young subjects

    Jørgensen, M; Petersen, K R; Vinberg, N; Jespersen, J; Gram, J; Tønnesen, K H

    2001-01-01

    .8 min. No net extraction of PAI-1 antigen took place in the splanchnic circulation. In conclusion, we demonstrated that active t-PA and t-PA antigen are catabolized and active PAI-1 produced in the splanchnic circulation in young healthy subjects during steady state. Furthermore, our data show that......Using an invasive technique, we studied the mean transit time, the net quantitative turnover rate, and the sites of synthesis and catabolism of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in healthy young volunteers in the fasting, steady state. Blood was...

  4. Rapid neural regulation of muscle urokinase-like plasminogen activator as defined by nerve crush.

    Hantaï, D; Rao, J. S.; Festoff, B W

    1990-01-01

    Muscle plasminogen activators (PAs), such as urokinase-like PA and, to a lesser extent, tissue PA, increase dramatically after denervation induced by axotomy. The PA/plasmin system has also been implicated in degradation of specific components of the muscle fiber basement membrane after local activation of plasminogen. These results suggest that neural regulation of muscle extracellular matrix metabolism accompanies or precedes regeneration after injury and is mediated by activation of PAs. I...

  5. Tissue plasminogen activator in the treatment of superior vena caval thrombosis associated with parenteral nutrition.

    Barclay, G R; Allen, K.; Pennington, C R

    1990-01-01

    Two patients, one of whom was pregnant, developed superior vena caval thrombosis while receiving central parenteral nutrition. They were successfully treated with recombinant tissue plasminogen activator (t-PA).

  6. Plasminogen activator inhibitor-1 in sputum and nasal fluids increases in asthmatics during common colds

    Cho, Seong H.; Hong, Seung J.; Chen, Haimei; Habib, Ali; Cho, David; Lee, Sun H.; Kang, Joseph; Ward, Theresa; Boushey, Homer A.; Schleimer, Robert P.; Avila, Pedro C.

    2014-01-01

    Capsule Summary This study showed that sputum and nasal lavage levels of plasminogen activator inhibitor-1 (PAI-1) rise during a common cold in asthmatic patients. This rise may contribute to the progression of airway remodeling. PMID:24373352

  7. Unimpeded skin carcinogenesis in K14-HPV16 transgenic mice deficient for plasminogen activator inhibitor

    Masset, Anne; Maillard, Catherine; Sounni, Nor Eddine; Jacobs, Nathalie; Bruyére, Françoise; Delvenne, Philippe; Tacke, Marlene; Reinheckel, Thomas; Foidart, Jean-Michel; Coussens, Lisa M.; Noël, Agnès

    2011-01-01

    Angiogenesis, extracellular matrix remodeling and cell migration are associated with cancer progression and involve at least, the plasminogen activating system and its main physiological inhibitor, the plasminogen activator inhibitor-1 (PAI-1). Considering the recognized importance of PAI-1 in the regulation of tumor angiogenesis and invasion in murine models of skin tumor transplantation, we explored the functional significance of PAI-1 during early stages of neoplastic progression in the tr...

  8. Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

    The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis

  9. Tissue distribution and regulation of plasminogen activator inhibitor-1 in obese mice.

    Samad, F; Loskutoff, D J

    1996-01-01

    BACKGROUND: Although elevated plasminogen activator inhibitor-1 (PAI-1) is associated with obesity and may be a risk factor for cardiovascular disease, the mechanism(s) that lead to this elevation, and the tissue/cellular origins of this increase, remain to be defined. In this report, we have addressed these questions using genetically obese mice (ob/ob) and their lean counterparts (+/?). MATERIALS AND METHODS: PAI-1 activity and antigen levels were determined using a tissue-type plasminogen ...

  10. β2-Glycoprotein I Is a Cofactor for t-PA–Mediated Plasminogen Activation

    Bu, Chunya; Gao, Lei; Xie, Weidong; Zhang, Jainwei; He, Yuhong; Cai, Guoping; McCrae, Keith R

    2010-01-01

    Regulation of the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. Here, we report that the abundant plasma glycoprotein, β2-glycoprotein I (β2GPI), stimulates t-PA–dependent plasminogen activation in the fluid phase and within a fibrin gel. The region within β2GPI responsible for stimulating t-PA activity is at least partially contained within β2GPI domain V. β2GPI bound t-PA with high affinity (Kd ~ 20 nM), stimulated t-PA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (kcat/Km) of t-PA–mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of β2GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following β2GPI repletion. Finally, stimulation of t-PA–mediated plasminogen activity by β2GPI was inhibited by monoclonal anti-β2GPI antibodies, as well as by anti-β2GPI antibodies from patients with antiphospholipid syndrome (APS). These findings suggest that β2GPI may be an endogenous regulator of fibrinolysis. Impairment of β2GPI-stimulated fibrinolysis by anti-β2GPI antibodies may contribute to the development of thrombosis in patients with APS. PMID:19180513

  11. Tissue plasminogen activator attenuates ventilatorinduced lung injury in rats

    Liang-ti HUANG; Hsiu-chu CHOU; Leng-fang WANG; Chung-ming CHEN

    2012-01-01

    Aim:To test the hypothesis that the tissue plasminogen activator (tPA) may counteract the inhibitory effect ot plasminogen activator inhibitors (PAI) and attenuate lung injury in a rat model of ventilator-induced lung injury (VILI).Methods:Adult male Sprague-Dawley rats were ventilated with a HVZP (high-volume zero PEEP) protocol for 2 h at a tidal volume of 30 ml/kg,a respiratory rate of 25 breaths/min,and an inspired oxygen fraction of 21%.The rats were divided into 3 groups (n=7 for each):HVZP+tPA group receiving tPA (1.25 mg/kg,iv) 15 min before ventilation,HVZP group receiving HVZP+vehicle injection,and a control group receiving no ventilation.After 2 h of ventilation,the rats were killed; blood and lungs were collected for biochemical and histological analyses.Results:HVZP ventilation significantly increased total protein content and the concentration of macrophage inflammatory protein-2 (MIP-2) in the bronchoalveolar lavage fluid (BALF) as well as the lung injury score.Rats that received HVZP ventilation had significantly higher lung PAI-1 mRNA expression,plasma PAI-1and plasma D-dimer levels than the control animals,tPA treatment significantly reduced the BALF total protein and the lung injury score as compared to the HVZP group,tPA treatment also significantly decreased the plasma D-dimer levels and the HVZP ventilation-induced lung vascular fibrin thrombi,tPA treatment showed no effect on MIP-2 level in BALF.Conclusion:These results demonstrate that VILI increases lung PAI-1 mRNA expression,plasma levels of PAI-1 and D-limers,lung injury score and vascular fibrin deposition,tPA can attenuate VILI by decreasing capillary-alveolar protein leakage as well as local and systemic coagulation as shown by decreased lung vascular fibrin deposition and plasma D-dimers.

  12. Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin alpha v beta 3 induces signal transduction and enhances plasminogen activation.

    Tarui, Takehiko; Akakura, Nobuaki; Majumdar, Mousumi; Andronicos, Nicholas; Takagi, Junichi; Mazar, Andrew P; Bdeir, Khalil; Kuo, Alice; Yarovoi, Serge V; Cines, Douglas B; Takada, Yoshikazu

    2006-03-01

    It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity receptors for the uPA kringle. We found that uPA binds specifically to integrin alpha v beta 3 on CHO cells depleted of uPAR. The binding of uPA to alpha v beta 3 required the uPA kringle domain. The isolated uPA kringle domain binds specifically to purified, recombinant soluble, and cell surface alpha v beta 3, and other integrins (alpha 4 beta 1 and alpha 9 beta 1), and induced migration of CHO cells in an alpha v beta 3-dependent manner. The binding of the uPA kringle to alpha v beta 3 and uPA kringle-induced alpha v beta 3-dependent cell migration were blocked by homologous plasminogen kringles 1-3 or 1-4 (angiostatin), a known integrin antagonist. We studied whether the binding of uPA to integrin alpha v beta 3 through the kringle domain plays a role in plasminogen activation. On CHO cell depleted of uPAR, uPA enhanced plasminogen activation in a kringle and alpha v beta 3-dependent manner. Endothelial cells bound to and migrated on uPA and uPA kringle in an alpha v beta 3-dependent manner. These results suggest that uPA binding to integrins through the kringle domain plays an important role in both plasminogen activation and uPA-induced intracellular signaling. The uPA kringle-integrin interaction may represent a novel therapeutic target for cancer, inflammation, and vascular remodeling. PMID:16525582

  13. Human breast cancer cell-mediated bone collagen degradation requires plasminogen activation and matrix metalloproteinase activity

    Hill Peter A

    2005-02-01

    Full Text Available Abstract Background Breast cancer cells frequently metastasize to the skeleton and induce extensive bone destruction. Cancer cells produce proteinases, including matrix metalloproteinases (MMPs and the plasminogen activator system (PAS which promote invasion of extracellular matrices, but whether these proteinases degrade bone matrix is unclear. To characterize the role that breast cancer cell proteinases play in bone degradation we compared the effects of three human breast cancer cell lines, MDA-MB-231, ZR-75-1 and MCF-7 with those of a normal breast epithelial cell line, HME. The cell lines were cultured atop radiolabelled matrices of either mineralized or non-mineralized bone or type I collagen, the principal organic constituent of bone. Results The 3 breast cancer cell lines all produced significant degradation of the 3 collagenous extracellular matrices (ECMs whilst the normal breast cell line was without effect. Breast cancer cells displayed an absolute requirement for serum to dissolve collagen. Degradation of collagen was abolished in plasminogen-depleted serum and could be restored by the addition of exogenous plasminogen. Localization of plasmin activity to the cell surface was critical for the degradation process as aprotinin, but not α2 antiplasmin, prevented collagen dissolution. During ECM degradation breast cancer cell lines expressed urokinase-type plasminogen activator (u-PA and uPA receptor, and MMPs-1, -3, -9,-13, and -14. The normal breast epithelial cell line expressed low levels of MMPs-1, and -3, uPA and uPA receptor. Inhibitors of both the PAS (aprotinin and PA inhibitor-1 and MMPs (CT1166 and tisue inhibitor of metalloproteinase blocked collagen degradation, demonstrating the requirement of both plasminogen activation and MMP activity for degradation. The activation of MMP-13 in human breast cancer cells was prevented by plasminogen activator inhibitor-1 but not by tissue inhibitor of metalloproteinase-1, suggesting

  14. Phenotypic overlap between MMP-13 and the plasminogen activation system during wound healing in mice.

    Juncker-Jensen, Anna; Lund, Leif R.

    2011-01-01

    Background Proteolytic degradation of extracellular matrix is a crucial step in the healing of incisional skin wounds. Thus, healing of skin wounds is delayed by either plasminogen-deficiency or by treatment with the broad-spectrum metalloproteinase (MP) inhibitor Galardin alone, while the two perturbations combined completely prevent wound healing. Both urokinase-type plasminogen activator and several matrix metallo proteinases (MMPs), such as MMP-3, -9 and -13, are expressed in the leading-...

  15. Pneumatic displacement without tissue plasminogen activator in premacular subhyaloid hemorrhage

    Rumita S. Kadarisman

    2007-06-01

    Full Text Available To assess the efficacy and safety of intravitreal injection of Sulfur Hexafluoride (SF6 gas without the use of tissue Plasminogen Activator (tPA in premacular Subhyaloid Hemorrhage (SHH, 5 eyes of 5 patients with premacular SHH were enrolled. After performing paracentesis of the anterior chamber, 0.3 ml pure SF6 gas was injected through pars plana with a 30 gauge needle. Facedown position was maintained for 5 days. Subhyaloid Hemorrhage was displaced in 4/5 (80% eyes with a duration of SHH less than 2 weeks. The pre-injection visual acuity of all 5 eyes was finger counting and improved in 4/5 ( 80% eyes within 3 days to 7 days post-injection to 6/20 - 6/6. The underlying disease was hypercoagulation in 1 patient, diabetes mellitus in 2 patients, hypertension in 1 patient and unknown in 1 patient. No complications were encountered. In conclusion, SF6 gas injected into the vitreous without the use of tPA, can displace SHH if performed within 14 days of duration, and results in rapid visual recovery. This procedure is proven to be safe. (Med J Indones 2007; 16:104-7 Keywords: subhyaloid hemorrhage, pneumatic displacement, sulfur hexafluoride gas

  16. Adipokines (adiponectin and plasminogen activator inhhibitor-1 in metabolic syndrome

    M K Garg

    2012-01-01

    Full Text Available Background: The clustering of cardiovascular risk factors is termed the metabolic syndrome (MS, which strongly predicts the risk of diabetes and cardiovascular disease (CVD. Adipokines may contribute to the development of obesity and insulin resistance and may be a causal link between MS, diabetes and CVD. Hence, we studied the adipokines - adiponectin and plasminogen activator inhibitor-1 (PAI-1 - in subjects with MS. Materials and Methods: We studied 50 subjects with MS diagnosed by International Diabetes Federation (IDF criteria and 24 healthy age- and sex-matched controls. Clinical evaluation included anthropometry, body fat analysis by bioimpedance, highly sensitive C-reactive protein, insulin, adiponectin, and PAI-1 measurement. Results: Subjects with MS had lower adiponectin (4.01 ± 2.24 vs. 8.7 ± 1.77 μg/ml; P < 0.0001 and higher PAI-1 (53.85 ± 16.45 vs. 17.35 ± 4.45 ng/ml; P < 0.0001 levels than controls. Both were related with the number of metabolic abnormalities. Adiponectin was negatively and PAI-1 was positively associated with body mass index, waist hip ratio (WHR, body fat mass, percent body fat, and all the parameters of MS, except HDL where the pattern reversed. WHR and triglycerides were independent predictors of adipokines in multiple regression analysis. Receiver operating characteristic curve analysis showed that adiponectin (6.7 μg/ml and PAI-1 (25.0 ng/ml levels predicted the MS with high sensitivity, specificity and accuracy in Indian population. Conclusions: Subjects with MS have lower adiponectin and higher PAI-1 levels compared to healthy controls. Lifestyle measures have been shown to improve the various components of MS, and hence there is an urgent need for public health measures to prevent the ongoing epidemic of diabetes and CVD.

  17. The pro-urokinase plasminogen-activation system in the presence of serpin-type inhibitors and the urokinase receptor

    Behrendt, Niels; List, Karin; Andreasen, Peter A; Danø, Keld

    The reciprocal pro-enzyme activation system of plasmin, urokinase-type plasminogen activator (uPA) and their respective zymogens is a potent mechanism in the generation of extracellular proteolytic activity. Plasminogen activator inhibitor type 1 (PAI-1) acts as a negative regulator. This system ...

  18. Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer

    Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (uPA) are involved in colorectal cancer invasion and metastasis. There is still debate whether the activity of MMP-2 and MMP-9 differs between tumors located in the colon and rectum. We designed this study to determine any differences in the expression of MMP-2, MMP-9 and uPA system between colon and rectal cancer tissues. Cancer tissue samples were obtained from colon carcinoma (n = 12) and rectal carcinomas (n = 10). MMP-2 and MMP-9 levels were examined using gelatin zymography and Western blotting; their endogenous inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed by Western blotting. uPA, uPAR and PAI-1 were examined using enzyme-linked immunosorbent assay (ELISA). The activity of uPA was assessed by casein-plasminogen zymography. In both colon and rectal tumors, MMP-2, MMP-9 and TIMP-1 protein levels were higher than in corresponding paired normal mucosa, while TIMP-2 level in tumors was significantly lower than in normal mucosa. The enzyme activities or protein levels of MMP-2, MMP-9 and their endogenous inhibitors did not reach a statistically significant difference between colon and rectal cancer compared with their normal mucosa. In rectal tumors, there was an increased activity of uPA compared with the activity in colon tumors (P = 0.0266), however urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) showed no significant difference between colon and rectal cancer tissues. These findings suggest that uPA may be expressed differentially in colon and rectal cancers, however, the activities or protein levels of MMP-2, MMP-9, TIMP-1, TIMP-2, PAI-1 and uPAR are not affected by tumor location in the colon or the rectum

  19. Plasminogen activator inhibitor-1 (PAI-1 and urokinase plasminogen activator (uPA in sputum of allergic asthma patients.

    Sebastian Zukowski

    2008-06-01

    Full Text Available Urokinase plasminogen activator (uPA and its inhibitor (PAI-1 have been associated with asthma. The aim of this study was to evaluate concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs. The study was performed on 19 HDM-AAs and 8 healthy nonatopic controls (HCs. Concentration of uPA and PAI-1 was evaluated in induced sputum supernatants using ELISA method. In HDM-AAs the median sputum concentration of uPA (128 pg/ml; 95% CI 99 to 183 pg/ml and PAI-1 (4063 pg/ml; 95%CI 3319 to 4784 pg/ml were significantly greater than in HCs (17 pg/ml; 95%CI 12 to 32 pg/ml; p<0.001 and 626 pg/ml; 95%CI 357 to 961 pg/ml; p<0.001 for uPA and PAI-1 respectively. The sputum concentration of uPA correlated with sputum total cell count (r=0.781; p=0.0001 and with logarithmically transformed exhaled nitric oxide concentration (eNO (r=0.486; p=0.035 but not with FEV1 or bronchial reactivity to histamine. On the contrary, the sputum PAI-1 concentration correlated with FEV1 (r=-0,718; p=0.0005 and bronchial reactivity to histamine expressed as log(PC20 (r=-0.824; p<0.0001 but did not correlate with sputum total cell count or eNO. The results of this study support previous observations linking PAI-1 with airway remodeling and uPA with cellular inflammation. Moreover, the observed effect of uPA seems to be independent of its fibrynolytic activity.

  20. Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor

    Ellis, V; Behrendt, N; Danø, K

    1991-01-01

    The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by uPA spe...

  1. Overexpression of hepatic plasminogen activator inhibitor type 1 mRNA in rabbits with fatty liver

    Jian-Gao Fan; Liang-Hua Chen; Zheng-Jie Xu; Min-De Zeng

    2001-01-01

    @@ INTRODUCTION Plasminogen activator inhibitor type 1 ( PAI-I ), an approximately Mr 50000 glycoprotein, is the major physiological inhibitor of plasminogen activators. It is not only the priming factor for atherosclerosis and coronary thrombosis[1-3] , but also participates in the genesis of chronic hepatitis and liver fibrosis[4-11] . However, there has been no available report yet about the research of hepatic PAl-1 gene expression in hyperlipidemia and fatty liver. The present study aimed to explore the change of hepatic PAl-1 mRNA and its plasma activity by means of animal model.

  2. Tranexamic acid, an inhibitor of plasminogen activation, reduces urinary collagen cross-link excretion in both experimental and rheumatoid arthritis

    Ronday, H.K.; TeKoppele, J.M.; Greenwald, R.A.; Moak, S.A.; Roos, J.A.D.M. de; Dijkmans, B.A.C.; Breedveld, F.C.; Verheijen, J.H.

    1998-01-01

    The plasminogen activation system is one of the enzyme systems held responsible for bone and cartilage degradation in rheumatoid arthritis (RA). In this study, we evaluated the effect of tranexamic acid (TEA), an inhibitor of plasminogen activation, on urinary collagen cross-link excretion and radio

  3. Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53*

    Shetty, Sreerama; Shetty, Praveenkumar; Idell, Steven; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P.; Shetty, Rashmi S.

    2008-01-01

    H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53-/- cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53-/- cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-...

  4. Tissue plasminogen activator-independent roles of neuroserpin in the central nervous system

    Jiao Ma; Yu Tong; Dan Yu; Meng Mao

    2012-01-01

    A number of studies have confirmed the existence of tissue-type plasminogen activator-independent roles of neuroserpin, a member of the serine protease inhibitor superfamily. In this review article, we aim to clarify this role. These unique roles of neuroserpin are involved in its neuroprotective effect during ischemic brain injury, its regulation of tumorigenesis, and the mediation of emotion and cognition through the inhibition of urokinase-type plasminogen activator and fibrinolysin, modification of Th cells, reducing plaque formation, promoting process growth and intracellular adhesion, and altering the expression of cadherin and nuclear factor kappa B.

  5. Levels of plasminogen activator inhibitor type 1 and urokinase plasminogen activator receptor in non-small cell lung cancer as measured by quantitative ELISA and semiquantitative immunohistochemistry

    Pappot, Helle; Skov, Birgit Guldhammer; Pyke, Charles; Grøndahl-Hansen, Jan

    The components of the plasminogen activation system have been reported to have prognostic impact in several cancer types, e.g. breast-, colon-, gastric- and lung cancer. Most of these studies have used quantification by enzyme-linked immunosorbent assay (ELISA) on tumour tissue extracts. However......, results in non-small cell lung cancer (NSCLC) studies obtained by quantitative ELISA and semiquantitative immunohistochemistry differ. If the prognostic value of the components of the plasminogen activation system is to be exploited clinically in the future, it is important to choose an easy and valid...... aim of predicting prognosis. In conclusion, a larger comparative study is needed to clarify the relationship between ELISA and immunohistochemical results, before a methodology for clinical use can be chosen in non-small cell lung cancer....

  6. Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells

    The effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells were examined. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner. The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP)-abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 560C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of 125I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa

  7. Interconversion of Active and Inactive Conformations of Urokinase-Type Plasminogen Activator

    Liu, Zhuo; Kromann-Hansen, Tobias; Lund, Ida K;

    2012-01-01

    The catalytic activity of serine proteases depends on a salt-bridge between the amino group of residue 16 and the side chain of Asp194. The salt-bridge stabilizes the oxyanion hole and the S1 specificity pocket of the protease. Some serine proteases exist in only partially active forms, in which...... the amino group of residue 16 is exposed to the solvent. Such a partially active state is assumed by a truncated form of the murine urokinase-type plasminogen activator (muPA), consisting of residues 16-243. Here we investigated the allosteric interconversion between partially active states and the...

  8. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    Barnathan, E S; Kuo, A; Karikó, K;

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  9. Doxycycline inhibits MMPs via modulation of plasminogen activators in focal cerebral ischemia.

    Burggraf, Dorothe; Trinkl, Andreas; Dichgans, Martin; Hamann, Gerhard F

    2007-03-01

    Tetracyclines inhibit matrix metalloproteinases (MMPs) and reduce infarction volume following cerebral ischemia. In this thesis an involvement of urokinase could be proven. Cerebral ischemia in rats was induced for 3 h followed by 24 h reperfusion (suture model). Each 6 animals received orally either doxycycline or water. Doxycycline treatment began 10 days before ischemia. MMP-2 and MMP-9 were substantially decreased. The possibility of involvement of the endogenous MMP inhibitors in the MMP inhibiting mechanisms was excluded. The plasminogen activator uPA was significantly decreased by doxycycline indicating an MMP inhibiting mechanism including the plasminogen/plasmin system. In the doxycycline group, this resulted in a decreased damage to the cerebral microvessels and less loss of the basal lamina antigen collagen type IV. Hemoglobin extravasation was also significantly reduced. Our results suggest that doxycycline may have a potential use as an anti-ischemic compound since it provides microvascular protection by inhibiting the plasminogen system. PMID:17166729

  10. Production of Plasminogen Activator in Cultures of Superior Cervical Ganglia and Isolated Schwann Cells

    Alvarez-Buylla, Arturo; Valinsky, Jay E.

    1985-05-01

    Plasminogen activator has been implicated in tissue remodeling and cell migration during embryogenesis. In the developing nervous system, these processes are evident in the migration of neurons, axonal extension, Schwann cell migration, and the ensheathment and myelination of nerves. We have studied the production of plasminogen activator in cultures of superior cervical ganglia under conditions in which both neurons and glia are present. We have found that a principal source of the enzyme in these cultures is the glial cells and that the enzyme could not be detected at the growing tips of neurites. Plasminogen activator is also produced by Schwann cells isolated from neonatal rat sciatic nerve. The production of the enzyme by these cells is stimulated 6- to 10-fold by cholera toxin. Isolated Schwann cells and glial cells in the ganglion explant cultures produce the tissue form of plasminogen activator, a form of the enzyme not often found in nonmalignant cells. Preliminary experiments suggest that neuronal-glial interactions may regulate enzyme production by Schwann cells.

  11. Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration

    Ohki, Makiko; Ohki, Yuichi; Ishihara, Makoto;

    2010-01-01

    Ischemia of the heart, brain, and limbs is a leading cause of morbidity and mortality worldwide. Treatment with tissue type plasminogen activator (tPA) can dissolve blood clots and can ameliorate the clinical outcome in ischemic diseases. But the underlying mechanism by which tPA improves ischemi...

  12. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

    Behrendt, N; Rønne, E; Ploug, M;

    1990-01-01

    The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. ...

  13. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    Behrendt, N; Ploug, M; Patthy, L;

    1991-01-01

    The purified urokinase plasminogen activator receptor (u-PAR) was cleaved into two fragments by mild chymotrypsin treatment. The smaller fragment (apparent Mr 16,000) possessed the ligand-binding capability, as shown by chemical cross-linking analysis. This fragment constituted the NH2-terminal p...

  14. Relationship between plasminogen activator inhibitor type-1 (PAI-1 gene polymorphisms and osteoporosis in Turkish women

    Merih Ozgen

    2012-11-01

    Full Text Available OBJECTIVE: The development of osteoporosis is associated with several risk factors, such as genetic structures that affect bone turnover and bone mass. The impact of genetic structures on osteoporosis is not known. Plasminogen activator inhibitor type-1 regulates the bone matrix and bone balance. This study assessed the correlation between plasminogen activator inhibitor type-1 gene 4G/5G polymorphisms and osteoporosis in a population of Turkish women. METHODS: A total of 195 postmenopausal female patients who were diagnosed with osteoporosis (Group I based on bone mineral density measurements via dual-energy x-ray absorptiometry and 90 females with no osteoporosis (Group II were included in this study. Correlations between PAI-1 gene 4G/5G polymorphisms and osteoporosis were investigated through the identification of PAI-1 gene 4G/5G polymorphism genotypes using the polymerase chain reaction. RESULTS: No significant differences in the genotype and allele frequency of 4G/5G plasminogen activator inhibitor type-1 polymorphisms were observed between the two groups, and both groups exhibited the most frequently observed 4G5G genotype. CONCLUSION: No correlation between the development of osteoporosis in the female Turkish population and 4G/5G plasminogen activator inhibitor type-1 gene polymorphisms was observed.

  15. Amiloride lowers blood pressure and attenuates urine plasminogen activation in patients with treatment-resistant hypertension

    Stolzenburg Oxlund, Christina; Buhl, Kristian Bergholt; Jacobsen, Ib A;

    2014-01-01

    daytime BP was reduced by 6.3/3.0 mm Hg. Seven of 80 cases (9%) discontinued amiloride due to hyperkalemia >5.5 mol/L, the most frequent adverse event. Urinary plasmin(ogen) and albumin excretions were significantly reduced after amiloride treatment (P < .0001). Urokinase activity was detectable in...

  16. Femoral artery recanalisation with percutaneous angioplasty and segmentally enclosed plasminogen activator

    Jørgensen, B; Tønnesen, K H; Bülow, J; Nielsen, J D; Jørgensen, M; Holstein, P; Andersen, E

    1989-01-01

    tissue-type plasminogen activator (rt-PA) and 1000 IU heparin were then infused into the enclosed space for 30 minutes, followed by intravenous heparin for 24 hours. At 10 and 30 days all 6 patients had evidence of recanalisation and remission of symptoms. Mean ankle-arm pressure index improved by 72% at...

  17. Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands

    Pedersen, Katrine E; Einholm, Anja P; Christensen, Anni;

    2003-01-01

    compared with native PAI-1, the polymers exhibited an increased resistance to temperature-induced unfolding. Polymerization was associated with specific changes in patterns of digestion with non-target proteases. During incubation with urokinase-type plasminogen activator, the polymers were slowly...

  18. Tissue plasminogen activator modulates the cellular and behavioral response to cocaine

    Maiya, Rajani; Zhou, Yan; Norris, Erin H.; Kreek, Mary Jeanne; Strickland, Sidney

    2009-01-01

    Cocaine exposure induces long-lasting molecular and structural adaptations in the brain. In this study, we show that tissue plasminogen activator (tPA), an extracellular protease involved in neuronal plasticity, modulates the biochemical and behavioral response to cocaine. When injected in the acute binge paradigm, cocaine enhanced tPA activity in the amygdala, which required activation of corticotropin-releasing factor type-1 (CRF-R1) receptors. Compared with WT mice, tPA−/− mice injected wi...

  19. Gene expression of fibrinolytic factors urokinase plasminogen activator and plasminogen activator inhibitor-1 in rabbit temporo-mandibular joint cartilage with disc displacement

    ZHAN Jing; GU Zhi-yuan; WU Li-qun; ZHANG Yin-kai; HU Ji-an

    2005-01-01

    Background The urokinase plasminogen activator system is believed to play an important role in degradation of the extracellular matrix associated with cartilage and bone destruction; however its precise roles in temporomandibular disorders have not yet been clarified. The aims of this study were to investigate the gene expression of fibrinolytic factors urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the articular cartilage of rabbit temporomandibular joint (TMJ) with disc displacement (DD) and to probe the relationship between fibrinolytic activity and cartilage remodeling. Methods Disc displacement of right joints was performed in 36 of 78 rabbits under investigation. The animals were sacrificed at 4 days and 1, 2, 4, 8 and 12 weeks after surgery, respectively. The right joints of these animals were harvested and processed for the examination of mRNA expression of uPA and PAI-1 in articular cartilage using in situ hybridization techniques. Results The expression of uPA and PAI-1 was co-expressed weakly in the chondrocytes from transitive zone to hypertrophic zone and mineralized zone, while no hybridizing signals were shown in proliferative zone and superficial zone in control rabbits. The most striking was the up-regulation of uPA and PAI-1 mRNA in 4-day rabbits postoperatively at the onset of cartilage degeneration. The strongest hybridizing signals for uPA and PAI-1 were seen in 2-week rabbits postoperatively. After 2 weeks, the expression of uPA and PAI-1 began to decrease and reached nearly normal level at 12 weeks. Conclusions The expression of the uPA/PAI-1 system coincides with the pathological changes in condylar cartilage after DD. The uPA/PAI-1 system may be one of the essential mediators in articular cartilage remodeling.

  20. Regulation of programmed cell death by plasminogen activator inhibitor type 1 (PAI-1)

    Lademann, Ulrik Axel; Rømer, Maria Unni Koefoed

    2008-01-01

    numbers of reports suggest that PAI-1 also can regulate programmed cell death (PCD) in cancer cells and normal cells. A number of reports suggest that PAI-1 can inhibit PCD through its pro-adhesive/anti-proteolytic property whereas other reports suggest that PAI-1 induces PCD through its anti......-adhesive property.Furthermore,it has been suggested that PAI-1 can either induce or inhibit PCD though activation of cell signalling pathways.This review will focus on the regulation of programmed cell death by PAI-1 in both normal cells and cancer cells.......Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with poor prognosis in cancer. An explanation to the elevated levels of PAI-1 could be a protective response to the increased proteolytic activity, caused by elevated levels of urokinase- type plasminogen activator (u...

  1. Induction of macrophage plasminogen activator by asbestos is independent of PKC activation

    Lison, D.; Raguzzi, F.; Lauwerys, R. (Louvain Univ., Brussels (Belgium). Industrial Toxicology and Occupational Medicine Unit)

    1991-07-01

    This study was undertaken to assess whether plasminogen activator (PA) induction in macrophages exposed to chrysotile fibers is mediated by protein kinase C (PKC) activation. In PKC depleted J774 cells, PA induction could be elicited by chrysotile whereas, as expected, the response to phorbol myristate acetate (PMA) was abolished. The effect of PMA and chrysotile on the distribution of PKC activity in the J774 cell line was also compared by measuring the enzyme catalytic activity and phorbol dibutyrate (PDBu) binding sites. No redistribution of PKC was observed after simulation with PA inducing doses of chrysotile, whereas a clear translocation was observed with PMA. It is concluded that the mechanism of PA induction by chrysotile in this macrophage-like cell line is independent of PKC activation. (orig.).

  2. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration.

    Hayashi, Moyuru; Tamura, Yosuke; Dohmae, Naoshi; Kojima, Soichi; Shimonaka, Motoyuki

    2008-05-01

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides. PMID:18294956

  3. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides

  4. Technetium-99m-labeled recombinant tissue plasminogen activator for the imaging of emboli in vivo

    Takahashi, Akihiro; Itoh, Kazuo; Tsukamoto, Eriko; Furudate, Masayori; Kamiyama, Hiroyasu; Abe, Hiroshi (Hokkaido Univ., Sapporo (Japan). School of Medicine)

    1993-07-01

    Tissue-type plasminogen activator (t-PA) effectively lyses activate thrombus by direct action. Recombinant t-PA (rt-PA) was labeled with technetium-99m ([sup 99m]Tc) to investigate the in vivo binding to fibrin clots in a feline cerebral embolism model created by insertion of an artificial fibrin clot within the carotid artery. [sup 99m]Tc-rt-PA administered intravenously provided clearer imaging of clots after priming with cold rt-PA, with uptake peaking 5-10 minutes after the injection. [sup 99m]Tc-labeled human serum albumin was not retained at clot sites. Systemically administered [sup 99m]Tc-rt-PA binds to fibrin clots within carotid arteries in our feline model. Our results suggest that the interaction of intrinsic plasminogen activator inhibitors with extrinsically administered rt-PA may regulate the demonstration of a clot, although the precise mechanism is unclear. (author).

  5. Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinase-type plasminogen activator in human U-937 lymphoma cells

    Genton, C.; Kruithof, E.K.; Schleuning, W.D.

    1987-03-01

    The tumor-promoting phorbol ester PMA induces changes in the histiocytic human lymphoma cell line U-937 akin to cellular differentiation and concomitantly stimulates the biosynthesis of plasminogen activator inhibitor 2 (PAI 2) and of urokinase-type plasminogen activator (u-PA). PAI 2 is found in a nonglycosylated intracellular and a glycosylated secreted form. The former appears to be identical to PAI 2 previously purified from placental extracts and large-scale U-937 cell cultures. The sixfold increase of PAI 2 antigen measured 24 h after PMA treatment in cell extracts and conditioned media is accompanied by an equal increase of active PAI 2 mRNA, whereas the 6 to 13-fold increase of u-PA antigen in the same samples is associated with only a 1.5-fold mRNA increase. The increase of PAI 2, but not of u-PA, biosynthesis requires transcription. A 50-fold molar excess of PAI 2 over u-PA is found in both extracts and conditioned media of PMA-treated cells. PAI 2 represents at least 0.3% of total de novo synthesized protein 24 h after induction with PMA. Thus, PAI 2, but not u-PA, is an abundant product of this precursor analogue of the mononuclear phagocyte lineage, and might represent a new marker for monocyte/macrophage differentiation.

  6. Urokinase-type plasminogen activator receptor as a predictor of poor outcome in patients with systemic inflammatory response syndrome

    Wu, Xiao-Ling; Long, Ding; Yu, Li; Yang, Jun-hui; Zhang, Yuan-chao; Geng, Feng

    2013-01-01

    BACKGROUND: Urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR) are known as important factors, which mediate a variety of functions in terms of vascular homeostasis, inflammation and tissue repair. However, their role in systemic inflammatory response syndrome (SIRS) has been less well studied. This study aimed to test the hypothesis that the abnormalities of fibrinolysis and degradation of extracellular matrix mediated by uPA and uPAR are dire...

  7. Triglyceride concentration and waist circumference influence alcohol-related plasminogen activator inhibitor-1 activity increase in black South Africans

    Pieters, Marlien; de Lange, Zelda; Hoekstra, Tiny; Ellis, Suria M.; Kruger, Annamarie

    2010-01-01

    We investigated the association between alcohol consumption and plasminogen activator inhibitor-1 activity (PAI-1(act)) and fibrinogen concentration in a black South African population presenting with lower PAI-1(act) and higher fibrinogen than what is typically observed in white populations. We, fu

  8. Association of Geographical Factors With Administration of Tissue Plasminogen Activator for Acute Ischemic Stroke

    Kunisawa, Susumu; Morishima, Toshitaka; Ukawa, Naoto; Ikai, Hiroshi; Otsubo, Tetsuya; Ishikawa, Koichi B.; Yokota, Chiaki; Minematsu, Kazuo; Fushimi, Kiyohide; Imanaka, Yuichi

    2013-01-01

    Background Intravenous tissue plasminogen activator (tPA) is an effective treatment for acute ischemic stroke if administered within a few hours of stroke onset. Because of this time restriction, tPA administration remains infrequent. Ambulance use is an effective strategy for increasing tPA administration but may be influenced by geographical factors. The objectives of this study are to investigate the relationship between tPA administration and ambulance use and to examine how patient trave...

  9. C-reactive protein increases plasminogen activator inhibitor–1 expression in human endothelial cells

    Chen, Changyi; Nan, Bicheng; Lin, Peter; Yao, Qizhi

    2007-01-01

    C-reactive protein (CRP) is an inflammatory marker which predicts cardiovascular disease. However, it is not fully understood whether CRP has direct effects on endothelial functions and gene expression. The purpose of current study was to determine the effects and molecular mechanisms of CRP on the expression of plasminogen activator inhibitor-1 (PAI-1) in human endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated with CRP at clinically relevant concentrations for d...

  10. Dictyostelium discoideum: a model for testing novel inhibitors of urokinase-type plasminogen activator

    Thompson, Elinor

    2013-01-01

    The social amoeba Dictyostelium discoideum is a useful non-animal eukaryote for testing novel compounds and dissecting cell regulatory molecular networks. We used this model organism to investigate the effect of a series of arylboronic acids and pinacol esters on development, chemotaxis and viability. These compounds were studied in parallel by collaborators for serine protease and urokinase-type plasminogen activator (uPA) inhibition, both in vitro and in vivo. In those biochemical assays, t...

  11. A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization

    Wilczynska, Malgorzata; Lobov, Sergei; Ohlsson, Per-Ingvar; Ny, Tor

    2003-01-01

    Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize....

  12. A Novel Serine Protease Secreted by Medicinal Maggots Enhances Plasminogen Activator-Induced Fibrinolysis

    van der Plas, Mariena J. A.; Andersen, Anders S.; Nazir, Sheresma; van Tilburg, Nico H.; Oestergaard, Peter R.; Krogfelt, Karen A.; van Dissel, Jaap T.; Hensbergen, Paul J.; Rogier M Bertina; Nibbering, Peter H.

    2014-01-01

    Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As haemostatic processes play an important role in wound healing, this study focused on the effects of maggot secretions on coagulation and fibrinolysis. The results showed that maggot secretions enhance plasminogen activator-induced formation of plasmin and fibrinolysis in a dose- and time-dependent manner. By contrast, coagulation was not affected by secretions. Biochemical studies indicated that a novel s...

  13. Effect of Amiloride to Retinal Toxicity Induced by Tissue Plasminogen Activator

    Kim, Ungsoo Samuel; Oh, Hyun-Sub; Kwon, Oh Woong; Chung, In; Lee, Sung-Ho; Lee, Joon Haeng

    2012-01-01

    Purpose The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated. Methods Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentr...

  14. Regulation of Plasminogen Activator Inhibitor-1 Expression in Endothelial Cells with Exposure to Metal Nanoparticles

    Yu, Min; Mo, Yiqun; Wan, Rong; Chien, Sufan; Zhang, Xing; Zhang, Qunwei

    2010-01-01

    Recent studies demonstrated that exposure to nanoparticles could enhance the adhesion of endothelial cells and modify the membrane structure of vascular endothelium. The endothelium plays an important role in the regulation of fibrinolysis, and imbalance of the fibrinolysis system potential contributes to the development of thrombosis. Plasminogen activator inhibitor-1 (PAI-1) is the most potent endogenous inhibitor of fibrinolysis and is involved in the pathogenesis of several cardiovascular...

  15. Interaction of Plasminogen Activator Inhibitor Type-1 (PAI-1) with Vitronectin

    Schröck, Florian Rudolf

    2005-01-01

    The serpin plasminogen activator inhibitor type-1 (PAI-1) is of importance in physiological processes such as fibrinolysis and thrombolysis as well as in pathophysiological processes like thrombosis, tumor cell adhesion or invasion, and metastasis. The interaction of PAI-1 with the extracellular matrix protein vitronectin (Vn) was implicated to play an important role in several of these processes and is therefore a possible target for therapeutic strategies. Understanding the PAI-1/Vn interac...

  16. Tryptophan Properties in Fluorescence and Functional Stability of Plasminogen Activator Inhibitor 1

    Verheyden, Stefan; Sillen, Alain; Gils, Ann; Paul J Declerck; Engelborghs, Yves

    2003-01-01

    Plasminogen activator inhibitor 1 harbors four tryptophan residues at positions 86, 139, 175, and 262. To investigate the contribution of each tryptophan residue to the total fluorescence and to reveal the mutual interactions of the tryptophan residues and interactions with the other amino acids, 15 mutants in which tryptophan residues have been replaced by phenylalanines were constructed, purified, and characterized. Conformational distribution analysis revealed that the tryptophan mutants h...

  17. Penta-L-lysine Potentiates Fibrin-Independent Activity of Human Tissue Plasminogen Activator.

    Rehan, Mohammad; Sagar, Amin; Sharma, Vandna; Mishra, Sanskruti; Ashish; Sahni, Girish

    2015-10-22

    The therapeutic action of tissue plasminogen activator (t-PA) is a two-step process: (1) binding to lysine-rich fibrin (Km event) and (2) converting local plasminogen into plasmin (Kcat event). Overcoming limitations of other structural biophysics methods, we wanted to employ small-angle X-ray scattering (SAXS) to visualize what shape changes occur/accompany t-PA activation, but the prime hurdle was the polydisperse nature of the fibrin, which occluded scattering information from t-PA. Earlier, larger polylysine peptides have been used to potentiate activation of t-PA, so while screening short polylysine peptides as alternatives to fibrin or larger peptides, we found that penta-polylysine (P5) specifically activates t-PA in a dose-dependent manner, averaging to almost 3-fold more than in the absence of any peptide. SAXS data analysis confirmed that P5 does not induce association of t-PA molecules, and a narrower peak profile of the Kratky plot indicated that P5 binding quenches inherent motion in t-PA. Shape reconstruction of t-PA ∓ P5 revealed that P5 closes the "gap" between the two gross domains of t-PA, viz. fused F/E, K1 and K2 domains, and the P domain. Docking experiments suggested that, while other polylysine peptides preferentially interacted with the surfaces of kringle domains, P5 "slipped into" the gap/groove between K2 and P domains, thereby mediating a substantial increase in the number of long-range interactions between the K2 domain and exosites in the P domain. We report here dissection of shape events involved in between Km/Kcat steps of t-PA activation, which can pave the way toward the search for small molecule function regulator(s) of t-PA. PMID:26447340

  18. Host Plasminogen Activator Inhibitor-1 Promotes Human Skin Carcinoma Progression in a Stage-Dependent Manner

    Catherine Maillard

    2005-01-01

    Full Text Available Angiogenesis and tumor expansion are associated with extracellular matrix remodeling and involve various proteases such as the plasminogen (Pig/plasminogen activator (PA system. Recently, several experimental data have implicated the plasminogen activator inhibitor-1 (PAI-1 in tumor angiogenesis in murine systems. However, little is known about PAI-1 functions in human skin carcinoma progression. By generating immunodeficient mice (in Rag-1-/- or nude background deleted for PAI-1 gene (PAI-1-/- , we have evaluated the impact of host PAI-1 deficiency on the tumorigenicity of two malignant human skin keratinocyte cell lines HaCaT II-4 and HaCaT A5-RT3 forming low-grade and high-grade carcinomas, respectively. When using the surface transplantation model, angiogenesis and tumor invasion of these two cell lines are strongly reduced in PAI-1-deficient mice as compared to the wild-type control animals. After subcutaneous injection in PAI-1-/- mice, the tumor incidence is reduced for HaCaT II-4 cells, but not for those formed by HaCaT A5-RT3 cells. These data indicate that PAI-1 produced by host cells is an important contributor to earlier stages of human skin carcinoma progression. It exerts its tumor-promoting effect in a tumor stage-dependent manner, but PAI-1 deficiency is not sufficient to prevent neoplastic growth of aggressive tumors of the human skin.

  19. Prognostic significance of urokinase plasminogen activator and plasminogen activator inhibitor-1 mRNA expression in lymph node- and hormone receptor-positive breast cancer

    One of the most thoroughly studied systems in relation to its prognostic relevance in patients with breast cancer, is the plasminogen activation system that comprises of, among others, the urokinase Plasminogen Activator (uPA) and its main inhibitor, the Plasminogen Activator Inhibitor-1 (PAI-1). In this study, we investigated the prognostic value of uPA and PAI-1 at the mRNA level in lymph node- and hormone receptor-positive breast cancer. The study included a retrospective series of 87 patients with hormone-receptor positive and axillary lymph node-positive breast cancer. All patients received radiotherapy, adjuvant anthracycline-based chemotherapy and five years of tamoxifen treatment. The median patient age was 54 and the median follow-up time was 79 months. Distant relapse occurred in 30 patients and 22 patients died from breast cancer during follow-up. We investigated the prognostic value of uPA and PAI-1 at the mRNA level as measured by real-time quantitative RT-PCR. uPA and PAI-1 gene expression was not found to be correlated with any of the established clinical and pathological factors. Metastasis-free Survival (MFS) and Breast Cancer specific Survival (BCS) were significantly shorter in patients expressing high levels of PAI-1 mRNA (p < 0.0001; p < 0.0001; respectively). In Cox multivariate analysis, the level of PAI-1 mRNA appeared to be the strongest prognostic factor for MFS (Hazard Ratio (HR) = 10.12; p = 0.0002) and for BCS (HR = 13.17; p = 0.0003). Furthermore, uPA gene expression was not significantly associated neither with MFS (p = 0.41) nor with BCS (p = 0.19). In a Cox-multivariate regression analysis, uPA expression did not demonstrate significant independent prognostic value. These findings indicate that high PAI-1 mRNA expression represents a strong and independent unfavorable prognostic factor for the development of metastases and for breast cancer specific survival in a population of hormone receptor- and lymph node-positive breast cancer

  20. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C;

    2011-01-01

    Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types...... to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies...

  1. Construction and expression of a recombinant antibody-targeted plasminogen activator

    Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin β chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent that t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, the authors have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the γ2b constant region and the catalytic β chain of t-PA. This construct was transfected into heavy chain loss variant cells derived form the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots and radioimmunoassay. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. IN a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, they have produced a hybrid protein capable of high affinity fibrin binding and plasminogen activation

  2. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    Barnathan, E S; Kuo, A; Karikó, K; Rosenfeld, L; Murray, S C; Behrendt, N; Rønne, E; Weiner, D; Henkin, J; Cines, D B

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...... endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence of a...

  3. Does low protein concentration of tissue-type plasminogen activator predict a low risk of spontaneous deep vein thrombosis?

    Gram, J; Sidelmann, Johannes Jakobsen; Jespersen, J

    1995-01-01

    Many reports have demonstrated an abnormal fibrinolysis in a subset of patients with deep vein thrombosis. We have studied systemic global fibrinolytic activity and protein concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma of 25...... activity (p < 0.02), and significantly higher protein concentrations of t-PA (p < 0.0001) and PAI-1 (p < 0.0006). We used probit scale plots to identify the consequence of different cut-off points to separate patients from controls. Reasonable separation could be obtained for t-PA with a cut-off point of 5...

  4. Urokinase-type plasminogen activator receptor (uPAR) as a promising new imaging target

    Persson, Morten; Kjaer, Andreas

    2013-01-01

    Urokinase-type plasminogen activator receptor (uPAR) has been shown to be of special importance during cancer invasion and metastasis. However, currently, tissue samples are needed for measurement of uPAR expression limiting the potential as a clinical routine. Therefore, non-invasive methods are...... the cell surface. Due to the importance of uPAR in cancer invasion and metastasis, a number of high-affinity ligands have been identified during the last decades. These ligands have recently been used as starting point for the development of a number of ligands for imaging of uPAR using various...

  5. Plasminogen Activator Inhibitor-1 and Susceptibility to Lung Cancer: A Population Genetics Perspective

    Bayramoglu, Aysegul; Gunes, Hasan Veysi; Metintas, Muzaffer; Degirmenci, Irfan; Guler, Halil Ibrahim; Ustuner, Cengiz; Musmul, Ahmet

    2014-01-01

    Aim: The aim of this study was to investigate the polymorphism frequency of plasminogen activator inhibitor-1 (PAI-1) (rs1799889) 4G/5G in patients with lung cancer. Methods: In this study, 286 genomic DNAs (154 lung cancer patients+132 subjects without lung cancer) were analyzed. Polymorphisms were determined by using the polymerase chain reaction (PCR) method, with 4G and 5G allele-specific primers. PCR products were assessed by a charge-coupled device camera and exposed to 2% agarose gel e...

  6. Soluble Urokinase Plasminogen Activator Receptor Levels in Tuberculosis Patients at High Risk for Multidrug Resistance

    Tri Yudani Mardining Raras; Triwahju Astuti; Iin Noor Chozin

    2012-01-01

    The soluble urokinase plasminogen activator receptor (suPAR) has been shown to be a strong prognostic biomarker for tuberculosis (TB). In the present study, the profiles of plasma suPAR levels in pulmonary TB patients at high risk for multidrug resistance were analyzed and compared with those in multidrug resistant (MDR)-TB patients. Forty patients were prospectively included, consisting of 10 MDR-TB patients and 30 TB patients at high risk for MDR, underwent clinical assesment. Plasma suPAR ...

  7. Does intravenous administration of recombinant tissue plasminogen activator for ischemic stroke can cause inferior myocardial infarction?

    Mostafa Almasi

    2016-06-01

    Full Text Available Recombinant tissue plasminogen activator (rTPA is one of the main portions of acute ischemic stroke management, but unfortunately has some complications. Myocardial infarction (MI is a hazardous complication of administration of intravenous rTPA that has been reported recently. A 78-year-old lady was admitted for elective coronary artery bypass graft surgery. On the second day of admission, she developed acute left hemiparesis and intravenous rTPA was administered within 120 minutes. Three hours later, she has had chest pain. Rescue percutaneous coronary intervention was performed on right coronary artery due to diagnosis of inferior MI, and the symptoms were resolved.

  8. Angiostatin generating capacity and anti-tumour effects of D-penicillamine and plasminogen activators

    Upregulation of endogenous angiostatin levels may constitute a novel anti-angiogenic, and therefore anti-tumor therapy. In vitro, angiostatin generation is a two-step process, starting with the conversion of plasminogen to plasmin by plasminogen activators (PAs). Next, plasmin excises angiostatin from other plasmin molecules, a process requiring a donor of a free sulfhydryl group. In previous studies, it has been demonstrated that administration of PA in combination with the free sulfhydryl donor (FSD) agents captopril or N-acetyl cysteine, resulted in angiostatin generation, and anti-angiogenic and anti-tumour activity in murine models. In this study we have investigated the angiostatin generating capacities of several FSDs. D-penicillamine proved to be most efficient in supporting the conversion of plasminogen to angiostatin in vitro. Next, from the optimal concentrations of tPA and D-penicillamine in vitro, equivalent dosages were administered to healthy Balb/c mice to explore upregulation of circulating angiostatin levels. Finally, anti-tumor effects of treatment with tPA and D-penicillamine were determined in a human melanoma xenograft model. Surprisingly, we found that despite the superior angiostatin generating capacity of D-penicillamine in vitro, both in vivo angiostatin generation and anti-tumour effects of tPA/D-penicillamine treatment were impaired compared to our previous studies with tPA and captopril. Our results indicate that selecting the most appropriate free sulfhydryl donor for anti-angiogenic therapy in a (pre)clinical setting should be performed by in vivo rather than by in vitro studies. We conclude that D-penicillamine is not suitable for this type of therapy

  9. Angiostatin generating capacity and anti-tumour effects of D-penicillamine and plasminogen activators

    Maass Cathy N

    2006-06-01

    Full Text Available Abstract Background Upregulation of endogenous angiostatin levels may constitute a novel anti-angiogenic, and therefore anti-tumor therapy. In vitro, angiostatin generation is a two-step process, starting with the conversion of plasminogen to plasmin by plasminogen activators (PAs. Next, plasmin excises angiostatin from other plasmin molecules, a process requiring a donor of a free sulfhydryl group. In previous studies, it has been demonstrated that administration of PA in combination with the free sulfhydryl donor (FSD agents captopril or N-acetyl cysteine, resulted in angiostatin generation, and anti-angiogenic and anti-tumour activity in murine models. Methods In this study we have investigated the angiostatin generating capacities of several FSDs. D-penicillamine proved to be most efficient in supporting the conversion of plasminogen to angiostatin in vitro. Next, from the optimal concentrations of tPA and D-penicillamine in vitro, equivalent dosages were administered to healthy Balb/c mice to explore upregulation of circulating angiostatin levels. Finally, anti-tumor effects of treatment with tPA and D-penicillamine were determined in a human melanoma xenograft model. Results Surprisingly, we found that despite the superior angiostatin generating capacity of D-penicillamine in vitro, both in vivo angiostatin generation and anti-tumour effects of tPA/D-penicillamine treatment were impaired compared to our previous studies with tPA and captopril. Conclusion Our results indicate that selecting the most appropriate free sulfhydryl donor for anti-angiogenic therapy in a (preclinical setting should be performed by in vivo rather than by in vitro studies. We conclude that D-penicillamine is not suitable for this type of therapy.

  10. The nature of interactions between tissue-type plasminogen activator and platelets

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves

  11. Modulation of NR2B-regulated contextual fear in the hippocampus by the tissue plasminogen activator system

    Norris, Erin H.; Strickland, Sidney

    2007-01-01

    Contextual fear conditioning is regulated by the hippocampus, and NR2B, a subunit of the NMDA receptor (NR), is involved in this process. We show that acute stress modulates tissue plasminogen activator (tPA) activity in the hippocampus by inducing expression of its inhibitor, plasminogen activator inhibitor-1. Acute stress increases NR2B expression and ERK1/2 phosphorylation, a classical marker of postsynaptic plasticity, in the hippocampus. tPA forms a complex with NR2B and is necessary for...

  12. Roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy

    Shu-Ling; Wu; Dong-Mei; Zhan; Shu-Hong; Xi; Xiang-Lian; He

    2014-01-01

    AIM:To investigate the role of tissue plasminogen activator(t-PA) and plasminogen activator inhibitor(PAI)in proliferative diabetic retinopathy(PDR) and to discuss the correlations among t-PA, PAI and vascular endothelial growth factor(VEGF) expressions.METHODS:A total of 36 vitreous samples were collected from 36 patients with PDR(PDR group), and 17 vitreous samples from 17 patients with idiopathic macular hole were used as control. The concentrations of t-PA, PAI and VEGF in samples were determined by ELISA method. The correlations among t-PA, PAI and VEGF expressions were discussed.RESULTS:The concentrations of t-PA, PAI and VEGF in the PDR group were significantly higher than those in the control group(P <0.001). The t-PA and PAI expressions were highly correlated with the VEGF expression(P <0.001).CONCLUSION:In addition to VEGF, a variety of bioactive substances, such as t-PA and PAI, are involved in the pathogenesis involved in the angiogenesis of PDR.VEGF can activate t-PA expression, resulting in collagen tissue degradation and angiogenesis. VEGF may also activate the mechanism for endogenous anti-neovascularization.

  13. Angiostatic activity of human plasminogen fragments is highly dependent on glycosylation

    To assess the importance of carbohydrate moieties to the anti-angiogenic activity of plasminogen fragments, we cloned the fragment corresponding to amino acids Val79 to Thr346 (Kint3-4) that presents the three glycosylation sites. The activity of glycosylated and unglycosylated Kint3-4 was tested in murine sponge implant model. We observed a significant decrease in the neovascularization on the sponge after treatment with Kint3-4 by histological examination and determination of the hemoglobin levels. The effects were more intense with the glycosylated than the unglycosylated protein. 99mTechnecium-labeled red blood cells confirmed the inhibition of cell infiltration in the implanted sponge. Studies using melanoma B16F1 implanted in a mouse demonstrated that treatment with glycosylated Kint3-4 (0.15 nmol/48 h) during 14 days suppresses tumor growth by 80%. The vascular endothelial growth factor mRNA levels on the tumor were reduced after treatment. Kint3-4 is a potent plasminogen fragment that has been found to inhibit tumor growth. (author)

  14. Therapeutic potential of an orally effective small molecule inhibitor of plasminogen activator inhibitor for asthma.

    Liu, Rui-Ming; Eldridge, Stephanie; Watanabe, Nobuo; Deshane, Jessy; Kuo, Hui-Chien; Jiang, Chunsun; Wang, Yong; Liu, Gang; Schwiebert, Lisa; Miyata, Toshio; Thannickal, Victor J

    2016-02-15

    Asthma is one of the most common respiratory diseases. Although progress has been made in our understanding of airway pathology and many drugs are available to relieve asthma symptoms, there is no cure for chronic asthma. Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators, has pleiotropic functions besides suppression of fibrinolysis. In this study, we show that administration of TM5275, an orally effective small-molecule PAI-1 inhibitor, 25 days after ovalbumin (OVA) sensitization-challenge, significantly ameliorated airway hyperresponsiveness in an OVA-induced chronic asthma model. Furthermore, we show that TM5275 administration significantly attenuated OVA-induced infiltration of inflammatory cells (neutrophils, eosinophils, and monocytes), the increase in the levels of OVA-specific IgE and Th2 cytokines (IL-4 and IL-5), the production of mucin in the airways, and airway subepithelial fibrosis. Together, the results suggest that the PAI-1 inhibitor TM5275 may have therapeutic potential for asthma through suppressing eosinophilic allergic response and ameliorating airway remodeling. PMID:26702150

  15. Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

    You, Weon-Kyoo; So, Seung-Ho; Sohn, Young-Doug; Lee, Hyosil; Park, Doo-Hong; Chung, Soo-Il; Chung, Kwang-Hoe

    2004-07-01

    Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis. PMID:15177278

  16. Engineering of plasminogen activators for targeting to thrombus and heightening thrombolytic efficacy.

    Absar, S; Gupta, N; Nahar, K; Ahsan, F

    2015-09-01

    Thrombotic occlusion of the coronary artery, which triggers acute myocardial infarction, is one of the major causes of death in the USA. Currently, arterial occlusions are treated with intravenous plasminogen activators (PAs), which dissolve the clot by activating plasminogen. However, PAs indiscriminately generate plasmin, which depletes critical clotting factors (fibrinogen, factor V, and factor VIII), precipitates a lytic state in the blood, and produces bleeding complications in a large patient population. PAs have been extensively investigated to achieve thrombus specificity, to attenuate the bleeding risk, and to widen their clinical applications. In this review, we discuss various strategies that have been pursued since the beginning of thrombolytic therapy. We review the biotechnological approaches that have been used to develop mutant and chimeric PAs for thrombus selectivity, including the use of specific antibodies for targeting thrombi. We discuss particulate carrier-based systems and triggered-release concepts. We propose new hypotheses and strategies to spur future studies in this research arena. Overall, we describe the approaches and accomplishments in the development of patient-friendly and workable delivery systems for thrombolytic drugs. PMID:26074048

  17. Plasminogen Activator System and Breast Cancer: Potential Role in Therapy Decision Making and Precision Medicine.

    Gouri, Adel; Dekaken, Aoulia; El Bairi, Khalid; Aissaoui, Arifa; Laabed, Nihad; Chefrour, Mohamed; Ciccolini, Joseph; Milano, Gérard; Benharkat, Sadek

    2016-01-01

    Shifting from the historical TNM paradigm to the determination of molecular and genetic subtypes of tumors has been a major improvement to better picture cancerous diseases. The sharper the picture is, the better will be the possibility to develop subsequent strategies, thus achieving higher efficacy and prolonged survival eventually. Recent studies suggest that urokinase-type plasminogen activator (uPA), uPA Receptor (uPAR), and plasmino-gen activator inhibitor-1 (PAI-1) may play a critical role in cancer invasion and metastasis. Consistent with their role in cancer dissemination, high levels of uPA, PAI-1, and uPAR in multiple cancer types correlate with dismal prognosis. In this respect, upfront determination of uPA and PAI-1 as invasion markers has further opened up the possibilities for individualized therapy of breast cancer. Indeed, uPA and PAI-1 could help to classify patients on their risk for metastatic spreading and subsequent relapse, thus helping clinicians in their decision-making process to propose, or not propose, adjuvant therapy. This review covers the implications for cancer diagnosis, prognosis, and therapy of uPA and PAI-1, and therefore how they could be major actors in the development of a precision medicine in breast cancer. PMID:27578963

  18. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene

    Soravia, E; Grebe, A; De Luca, P;

    1995-01-01

    The urokinase-type plasminogen activator receptor (uPAR) focuses at the cell surface the activation of pro-uPA and, hence, the formation of plasmin, thus enhancing directional extracellular proteolysis. To characterize the transcriptional regulatory mechanisms that control receptor expression, we...

  19. Stability of Recombinant Tissue Plasminogen Activator at −30 °C Over One Year

    Alkatheri, Abdulmalik

    2012-01-01

    Recombinant tissue plasminogen activator (rt-PA) is used to restore patency and avoid inadvertent removal of peripheral and central venous catheters. rt-PA was reconstituted (1 mg/mL) then cryopreserved at −30 °C for 1, 2, 3, 6, 8, and 12 months and, then its stability was determined. After cryopreservation for one and two months, rt-PA kept more than 95% of its activity compared to standard samples, while cryopreservation for three months caused 8% loss of activity. However, after cryopreservation for six months or more, rt-PA retained only 87.5% or less activity compared to standard samples. Therefore, it is recommended that reconstituted rt-PA be cryopreserved at −30 °C for a maximum period of three months. PMID:24275785

  20. Clinical significance of plasminogen activator inhibitor activity in patients with exercise-induced ischemia

    To assess the fibrinolytic system in patients with exercise-induced ischemia and its relation to ischemia and severity of coronary artery disease (CAD), 47 patients with CAD confirmed by results of coronary angiography underwent symptom-limited multistage exercise thallium-201 emission computed tomography. All patients with CAD had exercise-induced ischemia as assessed from thallium-201 images. Pre- and peak exercise blood samples from each patient and preexercise blood samples from control subjects were assayed for several fibrinolytic components and were also assayed for plasma adrenaline. The extent of ischemia was defined as delta visual uptake score (total visual uptake score in delayed images minus total visual uptake score in initial images) and the severity of CAD as the number of diseased vessels. In the basal condition, plasminogen activator inhibitor (PAI) activity was significantly higher in patients with exercise-induced ischemia as compared to control subjects (p less than 0.01), although there were no significant differences in other fibrinolytic variables between the two groups. Moreover, PAI activity in the basal condition displayed a significantly positive correlation with the extent of ischemia (r = 0.47, p less than 0.01). Patients with exercise-induced ischemia were divided into two groups (24 with single-vessel disease and 23 with multivessel disease). There were no significant differences in coronary risk factors, hemodynamics, or plasma adrenaline levels during exercise between single-vessel and multivessel disease except that delta visual uptake score was significantly higher in multivessel disease (p less than 0.01)

  1. Interaction of Plasminogen Activator Inhibitor-2 and Proteasome Subunit, Beta Type 1

    Jing FAN; Yu-Qing ZHANG; Ping LI; Min HOU; Li TAN; Xia WANG; Yun-Song ZHU

    2004-01-01

    The apoptosis protection by plasminogen activator inhibitor-2(PAI-2) is dependent on a 33 amino acid fragment between helix C and D of PAI-2 which is probably due to the interaction of PAI-2 with unknown intracellular proteins. In this study, we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cell cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone encoding 241 amino acids of proteasome (prosome, macropain) subunit, beta type 1(PSMβ1) which plays important roles in NF-κB activation. GST-pulldown experiments confirmed the interaction between PAI-2 and PSMβ1 in vitro. These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interation with PSMβ1.

  2. Probiotic in rennet paste can affect lipase activity of rennet and lipolysis in ovine cheese

    Marzia Albenzio

    2010-01-01

    Full Text Available Lambs were subjected to three different feeding regimes (mother suckling MS, artificial rearing AR, and artificial rearing with 7log10 cfu/ml Lactobacillus acidophilus supplementation to the milk substitute ARLb and slaughtered at 20d and 40d of age for each feeding treatment. Lambs abomasa were processed to rennet paste and lipases activity was evaluated. Rennet paste was used for Pecorino cheese production. Free fatty acids (FFAs and conjugated linoleic acids (CLAs were detected in cheese at 60d of ripening. Lipase activity was found higher in ARLb than in MS and AR rennet from lambs slaughtered at an older age. A reduction of all FFAs was observed in all cheeses when passing from 20 d to 40d of slaughtering. CLAs were more abundant in ARLb cheeses at both 20 and 40d. Milk substitute with Lb. acidophilus improves enzymatic features of rennet, and health and nutritional characteristics of ovine cheese.

  3. The liberated domain I of urokinase plasminogen activator receptor--a new tumour marker in small cell lung cancer

    Almasi, Charlotte E; Drivsholm, Lars; Pappot, Helle;

    2013-01-01

    The prognosis of small cell lung cancer (SCLC) remains poor with a 5-year survival rate of 4-6%. In non-small cell lung cancer (NSCLC), high levels of intact and cleaved forms of the receptor for urokinase plasminogen activator (uPAR) are significantly associated with short overall survival. Our...

  4. Procollagen-III in serum, plasminogen activation and fibronectin in bronchoalveolar lavage fluid during and following irradiation of human lung

    In the search for predictors of late radiation-induced lung injury we studied procollagen type III peptide concentration (P-III-P) in serum as well as fibronectin and plasminogen activation in bronchoalveolar lavage (BAL) fluid during and following irradiation of human lung. The patients received either high-dose hemithorax irradiation for pleural mesothelioma (11 patients) or high-dose irradiation with individually shaped fields for non-small cell lung cancer (12 patients). The severity of radiation fibrosis was assessed clinically from CT scans 6 months and 12 months after treatment. Four scores were used: severe, moderate, mild, or normal. Radiological lung injury varied from 'severe' (9 patients) to near absence of injury-'normal' (6 patients). Serum levels of P-III-P, when measured weekly during the 5-week period of radiotherapy or at several time-points after treatment, did not show consistent changes, nor did the levels correlate with the score for radiation fibrosis as assessed by CT scanning. Changes in fibronectin levels or in markers of plasminogen activation in BAL fluid did not correlate with the development of late lung injury. The levels of BAL fluid plasmin and plasminogen activator as assessed zymographically, but not the free net enzyme values, showed a tendency to be elevated in patients with severe radiation-induced lung injury, suggesting a possible role for inhibitors of the plasminogen activation cascade in the process of radiation-induced lung injury

  5. Intravenous tissue plasminogen activator in patients with stroke increases the bioavailability of insulin-like growth factor-1

    Wilczak, Nadine; Elting, Jan Willem; Chesik, Daniel; Kema, Ido P.; De Keyser, Jacques

    2006-01-01

    Background and Purpose-Insulin-like growth factor (IGF)-1 has potent neuroprotective properties. We investigated the effects of intravenous administration of tissue plasminogen activator (tPA) on serum levels of IGF-1 and IGF-binding protein (IGFBP)-3 in patients with acute ischemic stroke. Methods-

  6. Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol

    Ploug, M; Rønne, E; Behrendt, N;

    1991-01-01

    The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid a...

  7. Studies on functional and structural role of urokinase receptor and other components of the plasminogen activation system in malignancy

    Weidle, U H; Wöllisch, E; Rønne, E;

    1994-01-01

    Using immunohistochemistry and in-situ hybridization, we studied the expression of the components of the plasminogen activation system during progression to malignant melanoma with fresh melanocytic lesions. Expression of these components is confined to late stages of melanoma. t-PA expression is...

  8. Elevated plasma levels of vascular endothelial growth factor and plasminogen activator inhibitor-1 decrease during improvement of psoriasis

    Nielsen, Hans Jørgen; Christensen, Ib Jarle; Svendsen, M N;

    2002-01-01

    months. METHODS: Vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) were determined by ELISA methods in plasma collected from the patients before treatment and after 1, 3 and 6 months. Vessel counts were performed in biopsies from affected skin areas taken before...

  9. Tissue plasminogen activator and neuroserpin are widely expressed in the human central nervous system.

    Teesalu, Tambet; Kulla, Andres; Simisker, Aadu; Sirén, Vappu; Lawrence, Daniel A; Asser, Toomas; Vaheri, Antti

    2004-08-01

    Tissue plasminogen activator (tPA) is increasingly recognized to play important roles in various physiological and pathological processes in the central nervous system (CNS). Much of the data on the involvement of plasminogen activators in neurophysiology and -pathology have been derived from studies on experimental animals. We have now performed a systematic characterization of the expression of tPA and its inhibitor, neuroserpin, in normal human CNS. Brain and spinal cord samples from 30-36 anatomic locations covering all major brain regions were collected at 9 autopsies of donors with no neurological disease. Tissues were embedded in paraffin and tissue arrays were constructed. In two cases parallel samples were snap-frozen for biochemical analysis. Expression and activity profiling of tPA and neuroserpin were performed by immunohistochemistry, in situ hybridization, immunocapture and zymography assays. In the adult CNS, tPA was expressed at the mRNA and protein levels in many types of neurons, in particular in thalamus, cortex of cerebellum, pontine nuclei, neocortex, limbic system, and medulla oblongata. Interestingly, tPA was often co-expressed with its CNS inhibitor, neuroserpin. Despite overlapping expression of tPA and neuroserpin, zymography and immunocapture assays demonstrated that human neural tissue is a rich source of active tPA. Our analysis documents a detailed map of expression of tPA and its inhibitor in the human CNS and is compatible with the view that tPA is a key player in CNS physiology and pathology. PMID:15269833

  10. Plasminogen activation system in oral cancer: Relevance in prognosis and therapy (Review).

    Wyganowska-Świątkowska, Marzena; Jankun, Jerzy

    2015-07-01

    Research on carcinogenesis and progress in cancer treatment have reduced mortality of cancer patients. Mortality rates decreased by 1.5% per year from 2001 through 2010 for most types of cancer in men and women. However, oral cancer is still a significant global health problem since incidence and mortality rates are increasing. Oral cavity cancer is ranked the 8th in men and the 14th in women based on data collected between 2006 and 2010 by the National Institute of Health. Furthermore, an increasing incidence of head and neck neoplasms, particularly the tongue cancer among young adults has been reported recently. It is most likely due to increasing human papillomavirus (HPV) infection or the early start of tobacco and alcohol consumption. Treatment of oral cancer patients is mainly surgical and often leads to esthetic and functional deformities, with severe impact on the quality of life. Thus, novel form of treatments and selection of patients with high and low risk of mortality is of high priority for clinical studies. The expression of proteolytic enzymes in tumor and stromal tissues has been shown to have prognostic significance in many human cancers and inhibiting proteolysis can reduce tumor growth in many in vivo and in vitro models. Plasmin, with its activators and inhibitors are of great importance in many human malignances and collectively are called plasminogen activation system (PAS). In this comprehensive review we examine expression, possible prognostic markers and importance for therapy of the PAS members in oral cancer. Literature review suggests that overexpression of urokinase and its receptor are markers of poor outcome, thus, their inhibition can be explored in oral cancer therapy. Role of plasminogen activator inhibitor (PAI-1) is complex and depends on its concentration. Overexpression of PAI-1 favors angiogenesis, metastasis and poor prognosis, although when applied in very high concentrations it inhibits angiogenesis and tumor growth, the

  11. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L

    2016-04-01

    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis. PMID:26820595

  12. Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains*

    Bager, René; Kristensen, Thomas K.; Jensen, Jan K.; Szczur, Agnieszka; Christensen, Anni; Andersen, Lisbeth M.; Johansen, Jesper S.; Larsen, Niels; Baatrup, Erik; Huang, Mingdong; Ploug, Michael; Andreasen, Peter A.

    2012-01-01

    Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation. PMID:22733817

  13. Myocardial infarction following recombinant tissue plasminogen activator treatment for acute ischemic stroke: a dangerous complication

    ZHOU Zhi-gang; WANG Rui-lan; YU Kang-long

    2012-01-01

    Thrombolysis with intravenous tissue plasminogen activator (t-PA) is currently an approved therapy for patients with acute ischemic stroke.Acute myocardial infarction (AMI) immediately following t-PA treatment for stroke is a rare but serious complication.A case of acute myocardial infarction (MI) following IV t-PA infusion for acute stroke was observed.This is a 52-year-old male with a known history of hypertension and chest pain,who subsequently developed MI four hours after IV t-PA was administered for acute ischemic stroke.The disruption of intra-cardiac thrombus and subsequent embolization to the coronary arteries may be an important mechanism.In addition.spontaneous recanalization of infarct-related arteries may be associated with 9reater myocardial salvage and better prognosis.

  14. Interaction between Plasminogen Activator Inhibitor Type-2 and Pre-mRNA Processing Factor 8

    Jing FAN; Yu-Qing ZHANG; Ping LI; Chang TONG; Li TAN; Yun-Song ZHU

    2004-01-01

    The plasminogen activator inhibitor type-2 (PAI-2) dependent apoptosis protection is due to the 33 amino acids fragment located between helix C and D of PAI-2, this fragment may interact with some unknown intracellular proteins. In this study we used the fragment between helix C and D of PAI-2 as a bait to perform a yeast two-hybrid screen using a cDNA library constructed with HeLa cells during apoptosis,and retrieved a clone encoding 94 amino acid residues of C-terminus of pre-mRNA processing factor 8(PRPF8). Co-immunoprecipitation experiments confirmed that PAI-2 could interact with PRPF8 in vivo.PAI-2 could bind PRPF8 C-terminal in both the inside and outside of nuclear. These results suggested that the interaction between these two proteins might not be involved in the apoptosis process.

  15. Plasminogen activator inhibitor-1: physiologic role, regulation, and the influence of common pharmacologic agents.

    Tsikouris, James P; Suarez, Jose A; Meyerrose, Gary E

    2002-11-01

    Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of endogenous thrombolysis, thereby promoting thrombosis. PAI-1 is also a primary contributor to the development and recurrence of acute myocardial infarction. The renin angiotensin system, hypertriglyceridemia, hyperglycemia and hyperinsulinemia, and estrogen all influence the fibrinolytic system and PAI-1 in particular. Available data strongly suggest that angiotensin-converting enzyme (ACE) inhibitors and hormone replacement therapy with estrogen beneficially reduce PAI-1 production. Metformin, an agent commonly used for non-insulin-dependent diabetes mellitus (NIDDM), appears to favorably decrease PAI-1 production in NIDDM patients but not nondiabetic patients. Among the cholesterol-lowering statins, clinical literature evaluating pravastatin provides the most compelling data to support this agent's favorable effect on PAI-1. Other available statins either have not displayed an effect on PAI-1 or do not have clear data to conclusively define their effects on the fibrinolytic system. PMID:12412817

  16. STUDY OF HEARING OUTCOMES IN SUDDEN SENSORINEURAL HEARING LOSS TREATED WITH TISSUE PLASMINOGEN ACTIVATOR (TPA

    Rama Krishna

    2015-09-01

    Full Text Available Sudden Sensorineural Hearing Loss (SSHNL is a clinical condition that requires immediate management. There are many treatment options, which may not always revert the hearing to normal. Not only recording the degree of hearing loss, but also establishing the concurrent dysfunction of saccule by VEMP has facilitated a new approach to treatment strategy. Recombinant tissue Plasminogen Activator ((rtPA proved its efficacy in stroke and subsequently considered an option in the management of ISSNHL. The curren t study, conducted at different centres, on 15 patients utilized rtPA. The results showed a promising trend when saccular pathology is also evident by VEMP in association with Hearing loss. We recommend use of rtPA as primary modality in cases of ISSNHL wi th Saccular involvement.

  17. Tissue Plasminogen Activator Expression Is Restricted to Subsets of Excitatory Pyramidal Glutamatergic Neurons.

    Louessard, Morgane; Lacroix, Alexandre; Martineau, Magalie; Mondielli, Gregoire; Montagne, Axel; Lesept, Flavie; Lambolez, Bertrand; Cauli, Bruno; Mothet, Jean-Pierre; Vivien, Denis; Maubert, Eric

    2016-09-01

    Although the extracellular serine protease tissue plasminogen activator (tPA) is involved in pathophysiological processes such as learning and memory, anxiety, epilepsy, stroke, and Alzheimer's disease, information about its regional, cellular, and subcellular distribution in vivo is lacking. In the present study, we observed, in healthy mice and rats, the presence of tPA in endothelial cells, oligodendrocytes, mastocytes, and ependymocytes, but not in pericytes, microglial cells, and astrocytes. Moreover, blockage of the axo-dendritic transport unmasked tPA expression in neurons of cortical and hippocampal areas. Interestingly, combined electrophysiological recordings, single-cell reverse transcription polymerase chain reaction (RT-PCR), and immunohistological analyses revealed that the presence of tPA is restricted to subsets of excitatory pyramidal glutamatergic neurons. We further evidenced that tPA is stored in synaptobrevin-2-positive glutamatergic synaptic vesicles. Based on all these data, we propose the existence of tPA-ergic neurons in the mature brain. PMID:26377106

  18. Concentrations of plasminogen activator inhibitor-1 (PAI-1 and urokinase plasminogen activator (uPA in induced sputum of asthma patients after allergen challenge

    Krzysztof Kowal,

    2010-04-01

    Full Text Available Urokinase plasminogen activator (uPA and its inhibitor (PAI-1 are involved in tiisue remodeling and repairprocesses associated with acute and chronic inflammation. The aim of the study was to evaluate the effect of allergen challengeon concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs. ThirtyHDM-AAs and ten healthy persons (HCswere recruited for the study. In 24 HDM-AAs bronchial challenge with Dermatophagoidespteronyssinus (Dp and in 6 HDM-AAs sham challenege with saline were performed. In HDM-AAs sputumwas induced 24 hours before (T0 and 24 hours (T24 after the challenge. Concentration of uPA and PAI-1 in induced sputumwere determined using immunoenzymatic assays. At T0 in HDM-AAs mean sputum uPA (151±96 pg/ml and PAI-1(4341±1262 pg/ml concentrations were higher than in HC (18.8±6.7 pg/ml; p=0.0002 and 596±180 pg/ml; p<0.0001; foruPA and PAI-1 respectively. After allergen challenge further increase in sputum uPA (187±144 pg/ml; p=0.03 and PAI-1(6252±2323 pg/ml; p<0.0001 concentrations were observed. Moreover, in Dp challenged, but not in saline challengedHDM-AAs the mean uPA/PAI-1 ratio decreased significantly at T24. No significant increase in the studied parameters werefound in sham challenged patients. In HDM-AAs allergen exposure leads to activation of the plasmin system in the airways.Greater increase of the PAI-1 concentration than uPA concentration after allergen challenge may promote airway remodelingand play an important role in the development of bronchial hyperreactivity.

  19. Concentrations of plasminogen activator inhibitor-1 (PAI-1 and urokinase plasminogen activator (uPA in induced sputum of asthma patients after allergen challenge.

    Marcin Moniuszko

    2011-04-01

    Full Text Available Urokinase plasminogen activator (uPA and its inhibitor (PAI-1 are involved in tiisue remodeling and repair processes associated with acute and chronic inflammation. The aim of the study was to evaluate the effect of allergen challenge on concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs. Thirty HDM-AAs and ten healthy persons (HCswere recruited for the study. In 24 HDM-AAs bronchial challenge with Dermatophagoides pteronyssinus (Dp and in 6 HDM-AAs sham challenege with saline were performed. In HDM-AAs sputum was induced 24 hours before (T0 and 24 hours (T24 after the challenge. Concentration of uPA and PAI-1 in induced sputum were determined using immunoenzymatic assays. At T0 in HDM-AAs mean sputum uPA (151 Âą 96 pg/ml and PAI-1 (4341 Âą 1262 pg/ml concentrations were higher than in HC (18.8 Âą 6.7 pg/ml; p=0.0002 and 596 Âą 180 pg/ml; p<0.0001; for uPA and PAI-1 respectively. After allergen challenge further increase in sputum uPA (187 Âą 144 pg/ml; p=0.03 and PAI-1 (6252 Âą 2323 pg/ml; p<0.0001 concentrations were observed. Moreover, in Dp challenged, but not in saline challenged HDM-AAs the mean uPA/PAI-1 ratio decreased significantly at T24. No significant increase in the studied parameters were found in sham challenged patients. In HDM-AAs allergen exposure leads to activation of the plasmin system in the airways. Greater increase of the PAI-1 concentration than uPA concentration after allergen challenge may promote airway remodeling and play an important role in the development of bronchial hyperreactivity.

  20. The Prognostic Significance of Soluble Urokinase Plasminogen Activator Receptor in Acute Myeloid Leukemia

    Nergiz Erkut

    2016-05-01

    Full Text Available Objective: The soluble urokinase plasminogen activator receptor (suPAR is a soluble form of the urokinase plasminogen activator receptor expressed in various immune and cancer cells. The levels of suPAR have been demonstrated to correlate with prognosis in various cancers. This study was intended to investigate serum suPAR levels and their effect on prognosis in patients with acute myeloid leukemia (AML. Materials and Methods: Thirty newly diagnosed patients with AML and 29 healthy individuals were enrolled. Serum suPAR levels were analyzed by enzyme-linked immunosorbent assay. Results: Serum suPAR levels were significantly higher in patients with AML than in healthy individuals (9±5.9 ng/mL and 2.4±1.4 ng/mL, respectively; p<0.001. Positive correlation was determined between suPAR levels and white blood cell counts (p<0.01. Serum suPAR levels were lower in patients who achieved complete response than in patients not achieving complete response (5.5±2.2 ng/mL and 12±6.6 ng/mL, respectively; p<0.001. The median overall survival was longer in patients with serum suPAR levels below 6.71 ng/mL than in those with serum suPAR levels above 6.71 ng/mL (12.6±13.2 months and 1.71±0.6 months, respectively; p=0.02. Multivariate Cox regression analysis showed that suPAR had independent prognostic value (95% confidence interval: 1.029-6.259; p<0.05 in AML. Conclusion: Serum suPAR levels can be used as a prognostic marker in AML.

  1. Early pregnancy factor: large scale isolation of rosette inhibition test-active polypeptides from ovine placental extracts.

    Clarke, F M; Wilson, S; McCarthy, R; Perkins, T; Orozco, C

    1987-02-01

    Protocols are described for the isolation of substantial (mg) amounts of a rosette inhibition test (RIT)-active polypeptide fraction from ovine placental extracts. The main component of the preparation is a 12K polypeptide which contains a highly reactive thiol group. Oxidation may occur during isolation with the result that the final preparation is a mixture of the 12K polypeptide and a 25K disulphide linked dimer. The highly reactive thiol group was found to be directly involved in activity expression since gentle reduction followed by iodoacetylation resulted in a complete loss of activity. Antisera were prepared and the antibodies removed all the RIT activity from fresh ovine placental extracts, indicating that molecules related to those in the isolated preparation were responsible for all the activity in crude extracts. The antibodies also removed all the RIT activity from ovine and murine pregnancy sera, obtained both before and after implantation. Since EPF is defined as an RIT activity detected in pregnancy serum, these results establish that EPF activity is due to molecules similar to those isolated from the placental extracts. The availability of the preparative protocol and antibodies should hasten the biochemical definition of the EPF phenomenon. PMID:3598977

  2. Structure, function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor, uPAR

    Plesner, T; Behrendt, N; Ploug, M

    1997-01-01

    Several important functions have been assigned to the receptor for urokinase-type plasminogen activator, uPAR. As implied by the name, uPAR was first identified as a high affinity cellular receptor for urokinase plasminogen activator (uPA). It mediates the binding of the zymogen, pro-uPA, to the...... shown that urokinase-type plasminogen activator (uPAR) is expressed as a differentiation antigen on cells of the myelomonocytic lineage and as an activation antigen on monocytes and T lymphocytes. Neutrophils contain intracellular reservoirs of uPAR that are translocated to the plasma membrane upon...... activation, and neutrophils from patients with the rare blood disease paroxysmal nocturnal hemoglobinuria (PNH) that fail to express glycosyl-phosphatidylinositol-anchored proteins including uPAR, show a very significantly reduced transmigration over an endothelial barrier. Cell-associated plasminogen...

  3. In vivo gene delivery of urokinase-type plasminogen activator with regulatable lentivirus induces behavioural changes in chronic cocaine administration

    Bahi, Amine; Boyer, Frederic; Gumy, Christèle; Kafri, Tal; Dreyer, Jean-Luc

    2005-01-01

    Serine proteases play a key function in extracellular processes affecting central nervous system plasticity. Recently, the role of extracellular proteolytic processes in regulating synaptic structure and function has been described. However, to date direct evidence linking extracellular serine protease activity with drug-related behavioural changes has not been documented. Importantly, in a screening for genes induced after drug treatment we found that urokinase plasminogen-type activator (uP...

  4. Improvement of Psychotic Symptoms and the Role of Tissue Plasminogen Activator.

    Hoirisch-Clapauch, Silvia; Nardi, Antonio E

    2015-01-01

    Tissue plasminogen activator (tPA) mediates a number of processes that are pivotal for synaptogenesis and remodeling of synapses, including proteolysis of the brain extracellular matrix, degradation of adhesion molecules, activation of neurotrophins, and activation of the N-methyl-d-aspartate receptor. Abnormalities in these processes have been consistently described in psychotic disorders. In this paper, we review the physiological roles of tPA, focusing on conditions characterized by low tPA activity, which are prevalent in schizophrenia. We then describe how tPA activity is influenced by lifestyle interventions and nutritional supplements that may ameliorate psychotic symptoms. Next, we analyze the role of tPA in the mechanism of action of hormones and medications effective in mitigating psychotic symptoms, such as pregnenolone, estrogen, oxytocin, dopamine D3 receptor antagonists, retinoic acid, valproic acid, cannabidiol, sodium nitroprusside, N-acetyl cysteine, and warfarin. We also review evidence that tPA participates in the mechanism by which electroconvulsive therapy and cigarette smoking may reduce psychotic symptoms. PMID:26593907

  5. Improvement of Psychotic Symptoms and the Role of Tissue Plasminogen Activator

    Silvia Hoirisch-Clapauch

    2015-11-01

    Full Text Available Tissue plasminogen activator (tPA mediates a number of processes that are pivotal for synaptogenesis and remodeling of synapses, including proteolysis of the brain extracellular matrix, degradation of adhesion molecules, activation of neurotrophins, and activation of the N-methyl-d-aspartate receptor. Abnormalities in these processes have been consistently described in psychotic disorders. In this paper, we review the physiological roles of tPA, focusing on conditions characterized by low tPA activity, which are prevalent in schizophrenia. We then describe how tPA activity is influenced by lifestyle interventions and nutritional supplements that may ameliorate psychotic symptoms. Next, we analyze the role of tPA in the mechanism of action of hormones and medications effective in mitigating psychotic symptoms, such as pregnenolone, estrogen, oxytocin, dopamine D3 receptor antagonists, retinoic acid, valproic acid, cannabidiol, sodium nitroprusside, N-acetyl cysteine, and warfarin. We also review evidence that tPA participates in the mechanism by which electroconvulsive therapy and cigarette smoking may reduce psychotic symptoms.

  6. Presence of urokinase plasminogen activator, its inhibitor and receptor in small cell lung cancer and non-small cell lung cancer

    Pappot, H.; Pfeiffer, P.; Grøndahl Hansen, J.;

    1997-01-01

    Spreading of cancer cells is dependent on the combined action of several proteolytic enzymes, such as serine proteases, comprising the urokinase pathway of plasminogen activation. Previous studies of lung cancer indicate that expression, localization and prognostic impact of the components of the...... plasminogen activation system differ in the different non-small cell lung cancer (NSCLC) types, whereas the expression of the components in small cell lung cancer (SCLC) has only sparingly been investigated. In the present study we investigate the presence of the components of the plasminogen activation...... the clinical parameters. This is the first report of a study using a quantitative method to compare levels of the components of the plasminogen activation system in tissue extracts from the two major lung cancer groups. The study shows that uPA, PAI-1 and uPAR are present in SCLC-tissue, suggesting...

  7. Validation of an ELISA for the quantitation of lanoteplase, a novel plasminogen activator.

    Stouffer, B; Habte, S; Vachharajani, N; Tay, L

    1999-11-01

    An ELISA was developed and validated for the quantitation of lanoteplase in human citrated plasma. The ELISA employed a monoclonal anti-lanoteplase antibody absorbed onto 96-well microtiter plates to capture lanoteplase in citrated human plasma samples containing PPACK, a protease inhibitor. The captured lanoteplase was detected using a biotinylated rabbit anti-lanoteplase polyclonal antibody. The standard curve range in human plasma for the ELISA was 7-100 ng/ml. Assessment of individual standard curve variability indicated reproducible responses with r2 values of > or = 0.985. The accuracy (% DEV) and precision (%RSD) estimates for the ELISA based on the predicted values from quality control (QC) samples were within 7.3% and 11%, respectively. Cross-reactivity with t-PA was determined to be less than 11% by ELISA. The stability of lanoteplase was established in human citrated PPACK plasma for 24 hours at 4 degrees C, for 2 months at -20 degrees C, for 22 months at -70 degrees C, three weeks at room temperature, and through four freeze/thaw cycles. To quantify lanoteplase plasminogen activator (PA) activity, a commercially available chromogenic activity assay was also validated. This method and its application is described briefly here. The lanoteplase ELISA as well as the commercial activity method were successfully employed to evaluate the pharmacokinetic parameters of lanoteplase in support of clinical Phase II/III studies. PMID:10595857

  8. Characterization of indium-111 labeled recombinant tissue plasminogen activator for the imaging of thrombi

    The in vitro functional properties of recombinant tissue plasminogen activator (rt-PA), its biodistribution in mice, and its pharmacokinetics and clot localization properties in dogs have been investigated after labeling rt-PA with 111In. The rt-PA was coupled with the cyclic anhydride of DTPA using standard methodology. Amidolytic and fibrinolytic assays showed retention of protein activity when rt-PA was conjugated with an average of one DTPA group or less per molecule. Size exclusion HPLC showed each preparation to be radiochemically pure with 111In bound exlusively to the attached DTPA groups. Biodistribution in mice showed major accumulation of activity in the liver and kidneys. After administration of 0.5-1.0 mg of the labeled protein to dogs, blood activity decreased with a half time of approximately 5 min in agreement with previous reports of rapid blood clearance. Largely because of decreased blood levels, clot:blood ratios of labeled protein increased rapidly, in one study reaching 6.3 after 31 min, and satisfactory images of fibrin thrombi were obtained. The rt-PA may be labeled with 111In without destroying the ability of the protein to loacalize n clot and images of forming clot can be obtained with this agent within 1 h after administration. (orig.)

  9. Abrogation of plasminogen activator inhibitor-1-vitronectin interaction ameliorates acute kidney injury in murine endotoxemia.

    Kamlesh K Gupta

    Full Text Available Sepsis-induced acute kidney injury (AKI contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1 and vitronectin (Vn are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/- or expressing a PAI-1-mutant (PAI-1R101A/Q123K in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

  10. Abrogation of Plasminogen Activator Inhibitor-1-Vitronectin Interaction Ameliorates Acute Kidney Injury in Murine Endotoxemia

    Gupta, Kamlesh K.; Donahue, Deborah L.; Sandoval-Cooper, Mayra J.; Castellino, Francis J.; Ploplis, Victoria A.

    2015-01-01

    Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1−/−) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1−/− and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1−/− mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity. PMID:25799354

  11. Plasminogen activator inhibitor type 1 derived peptide, EEIIMD, diminishes cortical infarct but fails to improve neurological function in aged rats following middle cerebral artery occlusion

    Tan, Zhenjun; Li, Xinlan; Kelly, Kimberly A.; Rosen, Charles L.; Huber, Jason D.

    2009-01-01

    Age is a primary risk factor in stroke that is often overlooked in animal studies. We contend that using aged animals yields insight into aspects of stroke injury and recovery that are masked, or not elicited, in younger animals. In this study, we examined effects of co-administration of a plasminogen activator inhibitor type 1 derived peptide, EEIIMD, with tissue plasminogen activator (tPA) on infarct volume and functional outcome in aged rats following a transient middle cerebral artery occ...

  12. Effects of urokinase-type plasminogen activator in the acquisition, expression and reinstatement of cocaine-induced conditioned-place preference

    Bahi, Amine; Kusnecov, Alexander W; Dreyer, Jean-Luc

    2008-01-01

    Cocaine and many other psychostimulants strongly induce urokinase-type plasminogen activator (uPA) expression in the mesolimbic dopaminergic pathway, which plays a major role in drug-mediated behavioral plasticity [Bahi A, Boyer F, Gumy C, Kafri T, Dreyer JL. In vivo gene delivery of urokinase-type plasminogen activator with regulatable lentivirus induces behavioral changes in chronic cocaine administration. Eur J Neurosci 2004;20:3473–88; Bahi A, Boyer F, Kafri T, Dreyer JL. Silencing urokin...

  13. Increased expression of urokinase plasminogen activator and its cognate receptor in human seminomas

    The urokinase plasminogen activating system (uPAS) is implicated in neoplastic progression and high tissue levels of uPAS components correlate with a poor prognosis in different human cancers. Despite that, relative few studies are available on the expression and function of the uPAS components in human seminomas. In the present study we characterized the expression of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and the uPA inhibitors PAI-1 and PAI-2 in normal human testis and seminomas. The expression of the above genes was evaluated by means of quantitative RT-PCR, western blot, zymographic analysis and immunohistochemistry. Quantitative RT-PCR analysis of 14 seminomas demonstrated that uPA and uPAR mRNAs were, with respect to control tissues, increased in tumor tissues by 3.80 ± 0.74 (p < 0.01) and 6.25 ± 1.18 (p < 0.01) fold, respectively. On the other hand, PAI-1 mRNA level was unchanged (1.02 ± 0.24 fold), while that of PAI-2 was significantly reduced to 0.34 ± 0.18 (p < 0.01) fold. Western blot experiments performed with protein extracts of three seminomas and normal tissues from the same patients showed that uPA protein levels were low or undetectable in normal tissues and induced in tumor tissues. On the same samples, zymographic analysis demonstrated increased uPA activity in tumor tissue extracts. Western blot experiments showed that also the uPAR protein was increased in tumor tissues by 1.83 ± 0.15 fold (p < 0.01). The increased expression of uPA and uPAR was further confirmed by immunohistochemical staining performed in 10 seminomas and autologous uninvolved peritumoral tissues. Finally, variation in the mRNA level of PAI-1 significantly correlated with tumor size. We demonstrated the increased expression of uPA and uPAR in human seminomas with respect to normal testis tissues, which may be relevant in testicular cancer progression

  14. Elevated levels of plasminogen activators in the pathogenesis of delayed radiation damage in rat cervical spinal cord in vivo

    The pathophysiology of the cellular basis of radiation-induced demyelination and white-matter necrosis of the central nervous system (CNS) is poorly understood. Preliminary data suggest that tissue damage is partly mediated through changes in the proteolytic enzymes. In this study, we irradiated rat cervical spinal cords with single doses of 24 Gy of 18 MV photons or 20 MeV electrons and measured the levels of plasminogen activators at days 2, 7, 30, 60, 90, 120, 130 and 145 after irradiation, using appropriate controls at each time. Fibrin zymography revealed fibrinolytic bands representing molecular weights of 68,000 and 48,000 in controls and irradiated samples; these bands increased significantly at days 120, 130 and 145 after irradiation. Inhibition of these enzymatic bands with specific antibodies against tissue-type plasminogen activator (tPA) and amiloride, an inhibitor for urokinase plasminogen activator (uPA), confirmed that these bands were tPA and uPA. Enzymatic levels quantified by densitometry showed a twofold elevation in the levels of tPA and more than a tenfold increase in uPA after 120 days' irradiation. Activity of uPA was increased threefold by day 2 and increased steadily with time compared to nonirradiated control samples. Enzyme-linked immunosorbent assay (ELISA) also showed a threefold increase in the tPA content in the extracts of irradiated rat cervical spinal cords at days 120, 130 and 145. This study adds additional information to the proposed role of plasminogen activators in the pathogenic pathways of radiation damage in the CNS. 38 refs., 6 figs

  15. Sustained thrombolysis with DNA-recombinant tissue type plasminogen activator in rabbits

    Tissue type plasminogen activator (t-PA) is an effective thrombolytic agent in experimental animals. The duration of the thrombolytic effect of infused t-PA is unknown. The authors compared the duration of the thrombolytic effect of t-PA with streptokinase by measuring the lysis of 125I-fibrin-labeled thrombi in rabbit jugular veins at different times after a bolus injection of the fibrinolytic agents. The pharmacodynamics of both thrombolytic agents were determined in rabbits using a sensitive ex vivo fibrinolytic assay. Streptokinase and t-PA were given as a bolus dose of 15,000 U/kg. There was no detectable circulating fibrinolytic activity 30 minutes after the bolus dose of t-PA and 120 minutes after the bolus dose of streptokinase. The t-PA injection produced 34% thrombolysis at 30 minutes, 90% thrombolysis at 120 minutes, and 96% thrombolysis at 240 minutes. The streptokinase injection produced 17% thrombolysis at 30 minutes, 34% at 120 minutes, and 34% at 240 minutes. These observations indicate that the thrombolytic effect of t-PA is sustained beyond its time of clearance from the circulation whereas the thrombolytic effect of streptokinase closely parallels its activity in the circulation

  16. PGE2 reduces MMP-14 and increases plasminogen activator inhibitor-1 in cardiac fibroblasts.

    Kassem, Kamal M; Clevenger, Margarette H; Szandzik, David L; Peterson, Edward; Harding, Pamela

    2014-10-01

    Prostaglandin E2 (PGE2) is elevated during cardiac injury and we have previously shown that mice lacking the PGE2 EP4 receptor display dilated cardiomyopathy (DCM) with increased expression of the membrane type matrix metalloproteinase, MMP-14. We thus hypothesized that PGE2 regulates expression of MMP-14 and also affects fibroblast migration. Primary cultures of neonatal rat ventricular fibroblasts (NVFs) were used to test the effects of PGE2. Gene and protein expression was assessed by real time RT-PCR and Western blot, MMP activity was determined by zymography and migration of NVF was assessed by motility in a transwell system. PGE2 reduced expression of MMP-14 and these effects were antagonized by an EP4 antagonist. An EP4 agonist mimicked the effect of PGE2. PGE2 also increased mRNA and protein levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of MMP activation. However, PGE2-stimulation of PAI-1 was mediated by the EP1/EP3 receptor and not EP4. Migration of NVF was assessed by motility in a transwell system. Treatment of NVFs with PGE2 reduced the number of cells migrating toward 10% FCS. Treatment with the EP2 agonist also reduced migration but did not affect MMP-14 expression or PAI-1. Our results suggest that PGE2 utilizes different receptors and mechanisms to ultimately decrease MMP expression and NVF migration. PMID:25263346

  17. Quantitative PET of human urokinase-type plasminogen activator receptor with 64Cu-DOTA-AE105

    Persson, Morten; Madsen, Jacob; Østergaard, Søren;

    2012-01-01

    Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expressi...... levels of uPAR across different human cancer xenograft models in mice and to illustrate the clinical potential of uPAR PET in future settings for individualized therapy.......Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expression...

  18. Add-on rosiglitazone therapy improves plasminogen activity and high-density lipoprotein cholesterol in type 2 diabetes mellitus.

    Mustaffa, Nazri; Ibrahim, Suhairi; Abdullah, Wan Zaidah; Yusof, Zurkurnai

    2011-09-01

    Rosiglitazone is an oral hypoglycaemic agent of the thiazolidinedione group. This study aimed to assess changes in the diabetic prothrombotic state via plasminogen activity and changes in surrogate markers of atherosclerotic burden via ankle-brachial pressure index (ABPI) measurements after rosiglitazone was added to a pre-existing type 2 diabetes mellitus treatment regime. A nonblinded interventional study was designed. Fifty-nine patients were enrolled. Rosiglitazone-naïve patients were prescribed oral rosiglitazone 4 mg daily for 10 weeks. ABPI, plasminogen activity, glycosylated haemoglobin (HbA1c) and fasting lipid profile were measured pretreatment and post-treatment. Forty-eight patients completed the study. At the end of this study, mean plasminogen activity improvement was nearly 16% (PABPI improvement was 0.01 (P=0.439), mean HbA1c reduction was 0.51% (P<0.05), mean total cholesterol (TC) increase was 0.36 mmol/l (P<0.05), mean high-density lipoprotein cholesterol (HDL-C) increase was 0.15 mmol/l (P<0.05) and mean low-density lipoprotein cholesterol increased by 0.19 mmol/l (P=0.098). Rosiglitazone significantly improved plasminogen activity. There was also significant HbA1c reduction, and rise in both TC and HDL-C. Thus, rosiglitazone potentially improves the atherosclerotic burden and prothrombotic state. In future, more studies are needed to confirm the relationship between rosiglitazone, fibrinolytic system and atheromatous reduction in type 2 diabetes mellitus. PMID:21537159

  19. Plasminogen activator inhibitor-1 4G/5G polymorphism and retinopathy risk in type 2 diabetes: a meta-analysis

    Zhang Tengyue; Pang Chong; Li Ningdong; Zhou Elaine; Zhao Kanxing

    2013-01-01

    Abstract Background Mounting evidence has suggested that plasminogen activator inhibitor-1 (PAI-1) is a candidate for increased risk of diabetic retinopathy. Studies have reported that insertion/deletion polymorphism in the PAI-1 gene may influence the risk of this disease. To comprehensively address this issue, we performed a meta-analysis to evaluate the association of PAI-1 4G/5G polymorphism with diabetic retinopathy in type 2 diabetes. Methods Data were retrieved in a systematic manner a...

  20. Temporal changes in circulating P-selectin, plasminogen activator inhibitor-1, magnesium, and creatine kinase after percutaneous coronary intervention*

    Ying, Shu-qin; Xiang, Mei-xiang; Fang, Lu; Wang, Jian-an

    2010-01-01

    Objective: This study aims to determine the mechanisms underlying restenosis and ischemia-reperfusion injury of the myocardium after percutaneous coronary intervention (PCI). Methods: The present study examined serial changes (5 min, 30 min, 2 h, 6 h, and 24 h after PCI) in circulating P-selectin, plasminogen activator inhibitor-1 (PAI-1), magnesium (Mg), and creatine kinase-myocardial band fraction (CK-MB) levels, which may be associated with restenosis and myocardial injury in patients unde...

  1. The plasminogen activator system: involvement in central nervous system inflammation and a potential site for therapeutic intervention

    Gur-Wahnon, Devorah; Mizrachi, Tehila; Maaravi-Pinto, Florence-Yehudith; Lourbopoulos, Athanasis; Grigoriadis, Nikolaos; Higazi, Abd -Al Roof; Brenner, Talma

    2013-01-01

    Background Extracellular proteases such as plasminogen activators (PAs) and matrix metalloproteinases modulate cell-cell and cell-matrix interactions. Components of the PA/plasmin system have been shown to be increased in areas of inflammation, and have been suggested to play a role in inflammatory neurologic disorders such as epilepsy, stroke, brain trauma, Alzheimer's' disease and multiple sclerosis (MS). In the present study, we evaluated the involvement of the PA system in the animal mode...

  2. Intravenous Recombinant Tissue Plasminogen Activator Thrombolysis in a Patient with Acute Ischemic Stroke Secondary to Aortic Dissection

    Hong, Keun-Sik; Park, So-Young; Whang, Seon-Il; Seo, So-Young; Lee, Dong-Ha; Kim, Han-Joon; Cho, Joong-Yang; Cho, Yong-Jin; Jang, Woo-Ik; Kim, Chang Young

    2008-01-01

    Background Acute ischemic stroke secondary to aortic dissection (AoD) is challenging in the era of thrombolysis owing to the diagnostic difficulty within a narrow time window and the high risk of complications. Case Report A 64-year-old woman with middle cerebral artery occlusion syndrome admitted to the emergency room within intravenous recombinant tissue plasminogen activator (rt-PA) time window. Her neurological symptoms improved during thrombolysis, but chest and abdominal pain developed....

  3. An In Silico Approach towards the Prediction of Druglikeness Properties of Inhibitors of Plasminogen Activator Inhibitor1

    Umadevi Subramanian; Ashok Sivapunniyam; Ayyasamy Pudukadu Munusamy; Rajakumar Sundaram

    2014-01-01

    Diabetic retinopathy is the leading cause of blindness worldwide. It is caused by the abnormal growth of the retinal blood vessels. Plasminogen activator inhibitor1 (PAI1) is the key growth factor and the inhibition of PAI1 can reduce the angiogenesis. In this study, currently available inhibitors are taken and tested for the toxicity, binding affinity, and bioactivities of the compounds by in silico approach. Five toxic free inhibitors were identified, among which N-acetyl-D-glucosamine show...

  4. Localization of urokinase-type plasminogen activator in stromal cells in adenocarcinomas of the colon in humans.

    Grøndahl-Hansen, J.; Ralfkiaer, E; Kirkeby, L. T.; P. Kristensen; Lund, L. R.; Danø, K

    1991-01-01

    Human colon adenocarcinomas and adjacent normal colon tissues were stained immunohistochemically with three different monoclonal antibodies and one preparation of polyclonal antibodies against each of the two plasminogen activators, uPA (urokinase type) and tPA (tissue type). The staining patterns seen with the respective sets of antibodies were identical. In all of 10 cases, staining for uPA in the normal colon tissue was confined to scattered fibroblastlike cells in the lamina propria. Othe...

  5. Intraocular Lens Opacification following Intracameral Injection of Recombinant Tissue Plasminogen Activator to Treat Inflammatory Membranes after Cataract Surgery

    2015-01-01

    Purpose. To report 7 cases of intraocular lens (IOL) opacification following treatment of postoperative anterior chamber fibrin with recombinant tissue plasminogen activator (rtPA) after cataract surgery. Methods. Retrospective case series of 7 eyes in 7 patients who developed IOL opacification after receiving rtPA for anterior chamber inflammatory membrane formation resulting from phacoemulsification cataract surgery. Three explanted IOLs were investigated with light microscopy, histochemica...

  6. Tissue plasminogen activator as a key effector in neurobiology and neuropathology.

    Teesalu, T; Kulla, A; Asser, T; Koskiniemi, M; Vaheri, A

    2002-04-01

    Tissue plasminogen activator (tPA) is expressed by many types of neurons in the developing and adult rodent brain. We have now mapped tPA transcripts and protein in the human central nervous system using tissue arrays and find widespread expression, in particular in neocortical mantle, thalamus, amygdala, and hippocampal pyramidal neurons. The abundant presence of tPA protein in cellular vesicles implies that its acute release, e.g. upon ischaemic stroke or trauma, could play a role in neuronal damage. We also found in patients with multiple sclerosis (MS), and to a lesser extent patients with leukaemia and encephalitis, prominently elevated tPA activity in the cerebrospinal fluid and in MS in neurons in the proximity of areas of demyelination elevated tPA mRNA and antigen levels. In addition, we observed up-regulation of tPA expression in a mouse model of MS, experimental autoimmune encephalomyelitis. Accumulating evidence implies roles for tPA in normal neural function, as well as in neurodestructive processes in humans, such as occur in MS and brain tumours and warrant further studies on expression of tPA and its regulatory molecules in neurodegenerative diseases. PMID:12023848

  7. Leukemia inhibitory factor promote trophoblast invasion via urokinase-type plasminogen activator receptor in preeclampsia.

    Zheng, Qin; Dai, Kuixing; Cui, Xinyuan; Yu, Ming; Yang, Xuesong; Yan, Bin; Liu, Shuai; Yan, Qiu

    2016-05-01

    Preeclampsia is a pregnancy-related syndrome which can cause perinatal mortality and morbidity. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta, even result in preeclampsia. Understanding the molecular mechanisms underlying placentation facilitates the better intervention of preeclampsia. Urokinase-type plasminogen activator receptor (uPAR) is involved in the physiological and pathological processes. Leukemia inhibitory factor (LIF) is an important regulator in the establishment of pregnancy. However, the expression of uPAR in preeclamptic patients and its relationship with LIF remains unclear. In the current study, we found that the level of uPAR was relatively lower in the placentas from preeclamptic patients as compared with normal pregnant women. LIF promoted trophoblast cell outgrowth by upregulating uPAR in an explants culture, and LIF also enhanced migration and invasion potential through uPAR in trophoblast JAR and JEG-3 cell lines, and with increased gelatinolytic activities of matrix metalloproteinase 2 (MMP-2). The effect of LIF and uPAR on trophoblast migration and invasion was mediated by PI3K/AKT signaling pathway. Our data indicates the roles of LIF in promoting trophoblast migration and invasion through uPAR and suggest that abnormal expression of uPAR might be associated with the etiology of preeclampsia. PMID:27133045

  8. Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies

    Rasch, M.G.; Pass, J.; Illemann, M.;

    2008-01-01

    The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological...... murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this...... mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in...

  9. Biological and binding activities of ovine and porcine prolactins in porcine mammary tissue

    The concentration of prolactin receptors may play a critical role in regulating growth and development of the mammary gland during gestation and tumor development; however, the discrepancy between specific binding of ovine prolactin (oPRL) and porcine prolactin (pPRL) in porcine mammary tissue was disturbing. It was possible that 125I-oPRL may be an unsuitable ligand for the procine prolactin receptor. The validate the use of oPRL in binding assays, the biological and binding activities of oPRL and pPRL were compared. A lactogenic bioassay of pPRL was developed using porcine mammary explants cultured in Medium 199 containing insulin, cortisol, and pPRL. The potencies of oPRL and pPRL were compared using this bioassay. Oxidation of glucose and incorporation of glucose into lipids were similarly enhanced by physiological concentrations of both oPRL and pPRL. However, specific binding of 125I-oPRL was 20%, while less than 1% of 125I-pPRL was bound. 125I-oPRL bound to high affinity sites

  10. Induction of plasminogen activator by UV light in normal and xeroderma pigmentosum fibroblasts

    Normal and DNA repair-deficient human fibroblasts have been used to study induction of plasminogen activator (PA) by DNA damage. UV light induced the synthesis of PA in skin fibroblasts of all types of xeroderma pigmentosum (XP) in XP heterozygotes and in human amniotic cells. Enzyme induction was, however, not observed in fibroblasts of normal adults. In classical XP, which are deficient in excision repair, PA synthesis occurred in a narrow range of low-UV fluences. In such strains, the level of enzyme produced was correlated with the extent of repair deficiency. UV fluences required for PA induction in XP variants and XP heteozygotes were at least 10 times those inducing enzyme synthesis in excision-deficient XP. Maximum enzyme induction occurred 48 hr after irradiation, and the highest levels of enzyme produced were 15-20 times those of PA baseline levels. Electrophoretic analysis showed that UV irradiation enhances the synthesis of the M/sub r/ 60,000 human urokinase-type PA, which is present in low amounts in untreated cells. Our results suggest that PA induction in human cells is caused by unrepaired DNA damage and represents a eukaryotic SOS-like function. In addition, PA induction may provide a sensitive assay for detection of cellular DNA repair deficiencies and identification of XP heterozygotes

  11. Role of plasminogen activator inhibitor type-1 in radiation-induced normal tissues injury

    Radiotherapy is an essential tool for cancer treatment, but there is a balance between benefits and risks related to the use of ionizing radiation: the objective is to deliver a maximum dose to the tumour to destroy or to sterilize it while protecting surrounding normal tissues. Radio-induced damages to normal tissues are therefore a limiting factor when increasing the dose delivered to the tumour. One of the objectives of this research thesis is to bring to the fore a relationship between the initiation of lesions and the development of late damages, more particularly in the intestine, and to identify the involved molecular actors and their inter-connectivity. After a first part presenting ionizing radiation, describing biological effects of ionizing radiation and their use in radiotherapy, presenting the intestine and the endothelium and discussing the intestine radio-sensitivity, discussing the radio-induced intestine damages and radiotherapy-induced complications, and presenting the plasminogen activator inhibitor (PAI-1) and its behaviour in presence of ionizing radiation, two articles are reproduced. The first one addresses the effect of a pharmacological inhibition and of genetic deficiency in PAI-1 on the evolution of radio-induced intestine lesions. The second one discusses the fact that radio-induced PAI-1-related death of endothelial cells determines the severity of early radio-induced intestine lesions

  12. Early intracardiac thrombosis in preterm infants and thrombolysis with recombinant tissue type plasminogen activator

    Ferrari, F; Vagnarelli, F; Gargano, G; Roversi, M; Biagioni, O; Ranzi, A; Cavazzuti, G

    2001-01-01

    OBJECTIVES—To determine the incidence of catheter related thrombosis and to test the efficacy of recombinant tissue type plasminogen activator (rt-PA) in preterm infants.
STUDY DESIGN—From January 1995 to December 1998, echocardiography was performed in the first few days of life in 76 very low birthweight (⩽ 1500 g) infants out of a total of 147 having an umbilical catheter placed. When intracardiac thrombosis was diagnosed, rt-PA infusion was performed.
RESULTS—Four infants (5%) developed an intracardiac thrombosis during the first few days of life. In three of them, rt-PA at a dose of 0.4-0.5 mg/kg in a 20-30 minute bolus led to dissolution of the clot. One patient received a three hour infusion after the bolus, at a dose of 0.1 mg/kg/h, with resolution of the thrombus. No systemic effects were observed after rt-PA infusion.
CONCLUSIONS—Early thrombosis may occur as a complication of umbilical catheterisation in preterm infants; early echocardiographic detection of this disorder allows complete, safe, and rapid lysis with rt-PA.

 PMID:11420328

  13. Plasminogen Activator Inhibitor-1 in depression: Results from Animal and Clinical Studies.

    Jiang, Haitang; Li, Xiaoli; Chen, Suzhen; Lu, Na; Yue, Yingying; Liang, Jinfeng; Zhang, Zhijun; Yuan, Yonggui

    2016-01-01

    Evidence suggests that plasminogen activator inhibitor-1 (PAI-1) is a stress-related factor, and serum PAI-1 levels are increased in patients with major depressive disorders (MDD). Herein, we analysed PAI-1 protein levels in the brain, cerebrospinal fluid (CSF) and serum of rodents exposed to chronic unpredictable mild stress or treated with escitalopram. In addition, we examined PAI-1 concentrations in serum obtained from 17 drug-free depressed patients before and after escitalopram treatment. We found that PAI-1 expression was increased in area 1 of the cingulate cortex and prelimbic cortex of the medial prefrontal cortex as well as in the hippocampal cornu ammonis 3 and dentate gyrus in stressed rats. A downregulation of PAI-1 following chronic escitalopram treatment was also found. PAI-1 levels were higher in the CSF and serum in stressed rats than in controls, although the difference did not reach statistical significance in the serum. Escitalopram treatment significantly decreased PAI-1 levels in the serum, but not in the CSF. MDD patients had significantly greater serum PAI-1 concentrations than controls. Our results suggest that PAI-1 is implicated in the pathophysiology of depression. PMID:27456456

  14. Factors predicting intracerebral hemorrhage of patients treated with intravenous recombinant tissue plasminogen activator

    The use of recombinant tissue plasminogen activator (rt-PA) was approved in Japan in October 2005, and has had a marked effect on the treatment of patients presenting with acute ischemic stroke. Since the administration of rt-PA might cause intracerebral hemorrhage (ICH) and a poor prognosis, it is necessary to identify predictors of ICH after treatment with rt-PA. In this article, we examined 58 consecutive patients with acute ischemic stroke treated with intravenous rt-PA within 3 hours of symptom onset for 45 months, March 2006 to November 2009. In principle, we evaluated patients before and one day after rt-PA with MRI. We made a retrospective comparison of 21 patients with hemorrhagic change on CT and MRI T2* within 36 hours and 37 patients without hemorrhagic change. The rate of ICH with or without symptoms was increased with a higher National Institutes of Health Stroke Scale (NIHSS) and infarction range, defined by diffusion weighted imaging (DWI) Alberta Stroke Programme Early CT Score (ASPECTS). Major artery occlusion and reperfusion, including partial recanalization in MR angiography (MRA), were taken as factors in the hemorrhage group. In conclusion, DWI ASPECTS and NIHSS were useful predictors of ICH after rt-PA administration. (author)

  15. Dynamic Enhancer Methylation--A Previously Unrecognized Switch for Tissue-Type Plasminogen Activator Expression.

    Mia Magnusson

    Full Text Available Tissue-type plasminogen activator (t-PA, which is synthesized in the endothelial cells lining the blood vessel walls, is a key player in the fibrinolytic system protecting the circulation against occluding thrombus formation. Although classical gene regulation has been quite extensively studied in order to understand the mechanisms behind t-PA regulation, epigenetics, including DNA methylation, still is a largely unexplored field. The aim of this study was to establish the methylation pattern in the t-PA promoter and enhancer in non-cultured compared to cultured human umbilical vein endothelial cells (HUVECs, and to simultaneously examine the level of t-PA gene expression. Bisulphite sequencing was used to evaluate the methylation status, and real-time RT-PCR to determine the gene expression level. While the t-PA promoter was stably unmethylated, we surprisingly observed a rapid reduction in the amount of methylation in the enhancer during cell culturing. This demethylation was in strong negative correlation with a pronounced (by a factor of approximately 25 increase in t-PA gene expression levels. In this study, we show that the methylation level in the t-PA enhancer appears to act as a previously unrecognized switch controlling t-PA expression. Our findings, which suggest that DNA methylation is quite dynamic, have implications also for the interpretation of cell culture experiments in general, as well as in a wider biological context.

  16. Binding of tissue plasminogen activator to human umbilical vein endothelial cells

    The binding of purified, recombinant tissue plasminogen activator (tPA) to human umbilical vein endothelial cells (HUVEC) was studied in vitro using immunofluorescence as well as radiolabeled tPA. Immunofluorescence was performed on HUVEC grown on round glass coverslips using rabbit anti-human tPA and fluorescein-conjugated anti-rabbit immunoglobulin. Positive fluorescence was observed only after incubation of HUVEC with tPA. HUVEC were grown to confluence in 24-well tissue culture plates, washed, and incubated with a constant amount of 125I-tPA and various concentrations of unlabeled tPA. The binding of tPA to HUVEC was found to be specific, saturable, and reversible. Scatchard analysis yielded as equilibrium constant (K/sub eq/) of 4.2 x 106 M-1 and 1.2 x 107 binding sites per cell. Binding was inhibited by positively charged amino acids and by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone but not by carbohydrates including mannose, galactose, N-acetyl glucosamine and N-acetyl galactosamine. Neat human plasma abrogates but does not totally inhibit binding of tPA to HUVEC. Binding was neither enhanced nor inhibited by fibronectin. Although the affinity of binding of tPA to HUVEC is low, the endothelial cell may be involved in regulating plasma levels of tPA in vivo which may have therapeutic significance

  17. Timing of tissue plasminogen activator for acute ischemic stroke: outcomes-based recommendations for practice.

    Hanselman, Carol J

    2014-12-01

    Acute ischemic stroke (AIS) is a major cause of death and disability in the United States. Tissue plasminogen activator (t-PA) is an intravenously administered therapy that can prevent death and disability for patients presenting within early onset of AIS. There has been a debate around the exact time parameters for administration, because very few patients present to the hospital within the initial 0- to 3-hour window of time. Not all of the current national guidelines for timing of AIS in the United States are in agreement with regards to this issue. To the nurse caring for patients with neurologic illnesses, this topic is of utmost importance. Nurse are not only involved in determining the time of stroke symptom onset, but nurses also hold responsibility for a working knowledge of the latest eligibility and exclusion criteria for t-PA administration. This article examines the central body of research related to the timing of t-PA and makes recommendations for eligible candidates based on this literature. PMID:25365047

  18. Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum

    Smirnov Vladimir N

    2002-10-01

    Full Text Available Abstract Background Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown. Results We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation. Conclusions The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

  19. EXPRESSION AND SIGNIFICANCE OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR IN BREAST CANCER

    XIAO Jiping; ZHANG Guangde; XIA Wenhua; CHENG Deji

    1999-01-01

    Objective: To study the expression and clinical significance of urokinase-type plasminogen activator (uPA) in breast cancer. Methods: Applying streptavidin-biotin complex (SABC) immunohistochemical technique, expression of uPA was studied in 100 patients with primary breast cancer. Results: There were 55 patients with high uPA expression, and 45 with lower expression. There was significant correlation between uPA expression and TNM stage, lymph node status, and the tumor size. Neither age, menopausal status, nor ER status was significantly related with level of uPA expression. The patients with high expression of uPA had significantly shorter disease-free survival (DFS)and overall survival (OS) than did those with low expression of uPA. Univariate analysis showed that uPA as a prognostic factor was of similar magnitude to lymph node status and TNM stage, but stronger than that of ER status and tumor size. UPA was an independent prognostic factor affecting disease-free survival and overall survival. Conclusion: uPA appears to be a strong and independent biologic marker for predicting prognosis of breast cancer.

  20. Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking.

    Anna E Daniel

    Full Text Available Plasminogen activator inhibitor-1 (PAI-1, a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact.We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus.Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.

  1. Pneumatic Displacement of a Dense Submacular Hemorrhage with or without Tissue Plasminogen Activator

    Po-Min Yang

    2005-12-01

    Full Text Available Background: To evaluate the efficacy of treating a dense submacular hemorrhage withpneumatic displacement with or without tissue plasminogen activator (tPA.Methods: Twenty-four patients with a dense submacular hemorrhage were treated withintravitreal expansile gas, with or without an intravitreal injection of tPA, inorder to displace the submacular blood. The main outcome measurementsinclude preoperative and postoperative visual acuity, postoperative fluoresceinangiography (FAG results and additional postoperative treatments.Results: Total or subtotal subfoveal blood displacement was achieved in all 24 eyes.After a mean follow-up of 15.5 months (range 6-50 months, final visualacuity had improved two or more lines in 11 (45.8% of the 24 eyes, andmeasured 20/100 or better in 10 (4l.7% of the 11 eyes. Based on the FAGresults for 14 cases, nine eyes (64.3% received additional postoperativelaser treatment. Final visual acuity of 20/100 or better was achieved in four(40% of the 10 eyes, with a choroidal neovascular membrane (CNVMdetected on FAG, and dye leakage not detected in three (75% of the foureyes.Conclusions: Pneumatic displacement, with or without intravitreal injection of tPA, seemsuseful in displacing dense submacular hemorrhage and facilitating visualimprovement, although the visual result is often limited by the progression ofthe underlying macular disease. In patients with age-related macular degeneration,more treatable CNVM may be detected on postoperative FAG.

  2. Increased concentration of plasminogen activator inhibitor-1 and fibrinogen in individuals with metabolic syndrome.

    Palomo, Iván G; Gutiérrez, César L; Alarcón, Marcelo L; Jaramillo, Julio C; Segovia, Fabián M; Leiva, Elba M; Mujica, Verónica E; Icaza, Gloria N; Díaz, Nora S; Moore-Carrasco, Rodrigo

    2009-01-01

    Metabolic syndrome (MS) is closely linked to a generalized metabolic disorder referred to as insulin resistance. Disturbances in the hemostasis and fibrinolytic systems are a feature of MS. The aim of this study was to determine the concentration levels of fibrinogen and plasminogen activator inhibitor-1 (PAI-1) in a group of patients with MS with respect to a non-MS group, and to evaluate their possible relation with other risk factors in MS. The study was carried out in a total of 186 male and female non-smoking individuals aged 45-64 years, 93 with MS (ATP III criteria) and 93 without MS. Plasmatic levels of PAI-1 were measured by ELISA, and those of fibrinogen by the Claus method. The plasmatic levels of PAI-1 (men 49.2±19.8 vs. 35.0±12.2 ng/ml and women 42.0±19.7 vs. 31.6±14.6 ng/ml; p=0.0026) and fibrinogen (274.0±82.1 vs. 232.7±66.6 ng/ml; p=0.0002) were significantly higher in the MS group than in the non-MS group. PAI-1 was significantly associated with diastolic blood pressure, triglycerides and waist circumference. Fibrinogen was negatively associated with HDL-c. High plasmatic levels of PAI-1 and fibrinogen contribute to the cardiovascular risk that characterizes individuals with MS. PMID:21475821

  3. Genetic association of urokinase-type plasminogen activator gene rs2227564 site polymorphism with sporadic Alzheimer's disease in the Han Chinese population

    Xuelian Ji; Longfei Jia; Jianping Jia; Li Qi

    2012-01-01

    A missense C/T polymorphism in exon 6 (the NCBI rsID is rs2227564) of the urokinase-type plasminogen activator gene has been identified as a possible hot spot for Alzheimer's disease risk.The present study analyzed urokinase-type plasminogen gene polymorphisms of rs2227564 with sporadic Alzheimer's disease by PCR-restriction fragment length polymorphism.Results showed that CC,CT and TT genotype distribution frequencies had significant differences between sporadic Alzheimer's disease patients and healthy controls.In-depth analysis of the association between urokinase-type plasminogen gene rs2227564 polymorphisms and sporadic Alzheimer's disease indicated that people with the C-positive genotype CC + CT were at a higher risk for developing sporadic Alzheimer's disease.These results support the contribution of the polymorphisms of rs2227564 in the urokinase-type plasminogen gene to the pathogenesis of sporadic Alzheimer's disease in the Han Chinese population.

  4. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells.

    Zhang, J C; Sakthivel, R; Kniss, D; Graham, C H; Strickland, D K; McCrae, K R

    1998-11-27

    The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/alpha2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/alpha2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/alpha2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA.PAI complexes) in an LRP/alpha2MR-dependent manner, which was inhibited by the LRP/alpha2MR receptor-associated protein. Receptor-associated protein also caused a approximately 50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/alpha2MR antibodies. These results demonstrate that LRP/alpha2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA.PAI complexes and regeneration of unoccupied cell surface uPAR. PMID:9822706

  5. Enhancing the function of CD34(+ cells by targeting plasminogen activator inhibitor-1.

    Sugata Hazra

    Full Text Available Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34(+ cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1, a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34(+ cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34(+ cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO reduced PAI-1 mRNA in diabetic (p<0.01 and non-diabetic (p=0.05 CD34(+ cells. To reduce PAI-1 in human CD34(+ cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34(+ cell proliferation and migration in vitro, likely through increased PI3(K activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+ cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.

  6. A plasminogen activator inhibitor-1 inhibitor reduces airway remodeling in a murine model of chronic asthma.

    Lee, Sun H; Eren, Mesut; Vaughan, Douglas E; Schleimer, Robert P; Cho, Seong H

    2012-06-01

    We previously reported that plasminogen activator inhibitor (PAI)-1 deficiency prevents collagen deposition in the airways of ovalbumin (OVA)-challenged mice. In this study, we explored the therapeutic utility of blocking PAI-1 in preventing airway remodeling, using a specific PAI-1 inhibitor, tiplaxtinin. C57BL/6J mice were immunized with intraperitoneal injections of OVA on Days 0, 3, and 6. Starting on Day 11, mice were challenged with phosphate-buffered saline or OVA by nebulization three times per week for 4 weeks. Tiplaxtinin was mixed with chow and administered orally from 1 day before the phosphate-buffered saline or OVA challenge. Lung tissues were harvested after challenge and characterized histologically for infiltrating inflammatory cells, mucus-secreting goblet cells, and collagen deposition. Airway hyperresponsiveness was measured using whole-body plethysmography. Tiplaxtinin treatment significantly decreased levels of PAI-1 activity in bronchoalveolar lavage fluids, which indicates successful blockage of PAI-1 activity in the airways. The number of infiltrated inflammatory cells was reduced by tiplaxtinin treatment in the lungs of the OVA-challenged mice. Furthermore, oral administration of tiplaxtinin significantly attenuated the degree of goblet cell hyperplasia and collagen deposition in the airways of the OVA-challenged mice, and methacholine-induced airway hyperresponsiveness was effectively reduced by tiplaxtinin in these animals. This study supports our previous findings that PAI-1 promotes airway remodeling in a murine model of chronic asthma, and suggests that PAI-1 may be a novel target of treatment of airway remodeling in asthma. PMID:22323366

  7. Plasminogen Activator Inhibitor-1 Mitigates Brain Injury in a Rat Model of Infection-Sensitized Neonatal Hypoxia–Ischemia

    Yang, Dianer; Sun, Yu-Yo; Nemkul, Niza; Baumann, Jessica M.; Shereen, Ahmed; Dunn, R. Scott; Wills-Karp, Marsha; Lawrence, Daniel A.; Lindquist, Diana M.; Kuan, Chia-Yi

    2012-01-01

    Intrauterine infection exacerbates neonatal hypoxic–ischemic (HI) brain injury and impairs the development of cerebral cortex. Here we used low-dose lipopolysaccharide (LPS) pre-exposure followed by unilateral cerebral HI insult in 7-day-old rats to study the pathogenic mechanisms. We found that LPS pre-exposure blocked the HI-induced proteolytic activity of tissue-type plasminogen activator (tPA), but significantly enhanced NF-κB signaling, microglia activation, and the production of pro-inf...

  8. Cytokines in cerebrospinal fluid of neurosyphilis patients: Identification of Urokinase plasminogen activator using antibody microarrays.

    Lu, Ping; Zheng, Dao-Cheng; Fang, Chang; Huang, Jin-Mei; Ke, Wu-Jian; Wang, Liu-Yuan; Zeng, Wei-Ying; Zheng, He-Ping; Yang, Bin

    2016-04-15

    Little is known regarding protein responses to syphilis infection in cerebrospinal fluid (CSF) of patients presenting with neurosyphilis. Protein and antibody arrays offer a new opportunity to gain insights into global protein expression profiles in these patients. Here we obtained CSF samples from 46 syphilis patients, 25 of which diagnosed as having central nervous system involvement based on clinical and laboratory findings. The CSF samples were then analyzed using a RayBioH L-Series 507 Antibody Array system designed to simultaneously analyze 507 specific cytokines. The results indicated that 41 molecules showed higher levels in patients with neurosyphilis in comparison with patients without neural involvement. For validation by single target ELISA, we selected five of them (MIP-1a, I-TAC/CXCL11, Urokinase plasminogen activator [uPA], and Oncostatin M) because they have previously been found to be involved in central nervous system (CNS) disorders. The ELISA tests confirmed that uPA levels were significantly higher in the CSF of neurosyphilis patients (109.1±7.88pg/ml) versus patients without CNS involvement (63.86±4.53pg/ml, p<0.0001). There was also a clear correlation between CSF uPA levels and CSF protein levels (p=0.0128) as well as CSF-VDRL titers (p=0.0074) used to diagnose neurosyphilis. No significant difference between the two groups of patients, however, was found in uPA levels in the serum, suggesting specific activation of the inflammatory system in the CNS but not the periphery in neurosyphilis patients. We conclude that measurements of uPA levels in CSF may be an additional parameter for diagnosing neurosyphilis. PMID:27049560

  9. Intravenous Tissue Plasminogen Activator Can Be Safely Given without Complete Blood Count Results Back.

    Yi Dong

    Full Text Available It is well known that the efficacy of intravenous (i.v. tissue plasminogen activator (tPA is time-dependent when used to treat patients with acute ischemic strokes.Our study examines the safety issue of giving IV tPA without complete blood count (CBC resulted.This is a retrospective observational study by examining the database from Huashan Hospital in China and OSF/INI Comprehensive Stroke Center in United States. Patient data collected included demographics, occurrence of symptomatic intracranial hemorrhage, door to needle intervals, National Institute of Health Stroke Scale scores on admission, CBC results on admission and follow-up modified Rankin Scale scores. Linear regression and multivariable logistic regression analysis were used to identify factors that would have an impact on door-to-needle intervals.Our study included 120 patients from Huashan Hospital and 123 patients from INI. Among them, 36 in Huashan Hospital and 51 in INI received i.v. tPA prior to their CBC resulted. Normal platelet count was found in 98.8% patients after tPA was given. One patient had thrombocytopenia but no hemorrhagic event. A significantly shorter door to needle interval (DTN was found in the group without CBC resulted. There was also a difference in treatment interval between the two hospitals. Door to needle intervals had a strong correlation to onset to treatment intervals and NIHSS scores on admission.In patients presented with acute ischemic stroke, the risk of developing hemorrhagic event is low if i.v. tPA is given before CBC has resulted. The door to needle intervals can be significantly reduced.

  10. Recombinant human erythropoietin reduces plasminogen activator inhibitor and ameliorates pro-inflammatory responses following trauma

    M Mojtahedzadeh

    2011-05-01

    Full Text Available "n  "n Background and the purpose of the study: Besides its hematopoietic effects, erythropoietin (EPO by mobilization of iron and modulation of some inflammatory cytokines has antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate these effects of erythropoietin and its impact on organ function in traumatized patients. "n Methods: Twenty-six ICU-admitted traumatized patients within 24 hrs after trauma were randomly assigned to the EPO (received EPO, 300 units/Kg/day and Control (not received EPO groups. The inflammatory biomarkers including Tumor Necrosis Factor alpha (TNF-α, Interleukin 1 (IL-1, Plasminogen Activator Inhibitor 1 (PAI-1 and Nitrotyrosine were recorded at the admission, 3, 6 and 9 days thereafter. Acute Physiology and Chronic Health Evaluation (APACHE II and Sequential Organ Failure Assessment (SOFA scores were also recorded. "n Results: Among 12 patients (EPO group TNF-α level at the day of 9 (P=0.046, and within EPO group at the days of 3 (P=0.026 ameliorate, 6 (P=0.016, and 9 (P=0.052 were significantly lowered. Level of IL-1 and PAI-1 decreased significantly at days of 3, 6 and 9 post intervention. Also there were significant differences between two groups in the SOFA score during three measured time intervals (the first, third and seventh days. "n Conclusion: From the results of this study it seems that injection of erythrocyte stimulating agent is well tolerated and inhibits the inflammatory response and oxidative stress following trauma.

  11. Tissue Plasminogen Activator Binding to Superparamagnetic Iron Oxide Nanoparticle-Covalent Versus Adsorptive Approach.

    Friedrich, Ralf P; Zaloga, Jan; Schreiber, Eveline; Tóth, Ildikó Y; Tombácz, Etelka; Lyer, Stefan; Alexiou, Christoph

    2016-12-01

    Functionalized superparamagnetic iron oxide nanoparticles are frequently used to develop vehicles for drug delivery, hyperthermia, and photodynamic therapy and as tools used for magnetic separation and purification of proteins or for biomolecular imaging. Depending on the application, there are various possible covalent and non-covalent approaches for the functionalization of particles, each of them shows different advantages and disadvantages for drug release and activity at the desired location.Particularly important for the production of adsorptive and covalent bound drugs to nanoparticles is the pureness of the involved formulation. Especially the covalent binding strategy demands defined chemistry of the drug, which is stabilized by excess free amino acids which could reduce reaction efficiency. In this study, we therefore used tangential flow filtration (TFF) method to purify the drugs before the reaction and used the frequently applied and clinically available recombinant tissue plasminogen activator (tPA; Actilyse(®)) as a proof of concept. We then coupled the tPA preparation to polyacrylic acid-co-maleic acid (PAM)-coated superparamagnetic iron oxide nanoparticles (SPIONs) using an amino-reactive activated ester reaction and compared these particles to PAM-coated SPIONs with electrostatically adsorbed tPA.Using dynamic light scattering (DLS) and pH-dependent electrokinetic mobility measurements, we showed that surface properties of the SPIONs were significantly greater affected after activation of the particles compared to the adsorption controls. Different in vitro assays were used to investigate the activity of tPA after coupling to the particles and purification of the ferrofluid. Covalent linkage significantly improves the reactivity and long-term stability of the conjugated SPION-tPA system compared to simple adsorption. In conclusion, we have shown an effective way to produce SPIONs with covalent and non-covalent ultra-filtrated drugs. We showed

  12. Plasminogen activator inhibitor with very long half-life (VLHL PAI-1) can reduce bleeding in PAI-1-deficient patients.

    Jankun, Jerzy; Skrzypczak-Jankun, Ewa

    2013-08-01

    This review summarizes our current knowledge of plasminogen activator inhibitor (PAI-1) deficiency and proposes some novel treatments for this condition. PAI-1 is a fast acting inhibitor of tissue and urokinase plasminogen activators (tPA and uPA). PAI-1 controls/slows clot lysis triggered by tPA activated plasminogen. PAI-1 deficiency was once considered to be an extremely rare disorder characterized by frequent and prolonged bleeding episodes. PAI-1 deficiency is now thought to be more frequent than initially reported and is known to be caused by mutations in the PAI-1 gene that produce a dysfunctional PAI-1 protein or slow the secretion of PAI-1 into the circulation. PAI-1 deficiency is characterized by hyperfibrinolysis that results in frequent bleeding episodes. Patients with this condition form normal blood clots that are quickly lysed by unopposed tPA-activated plasmin. Spontaneous bleeding is rare in PAI-1 deficient patients, but moderate hemorrhaging of the knees, elbows, nose, and gums can be triggered by mild trauma. Additionally, prolonged bleeding after surgery is common and menstrual bleeding may be severe. Moderate PAI-1 deficiency is associated with a lifelong bleeding tendency, but severe deficiencies can be life-threatening. The diagnosis of this disorder remains challenging due to the lack of a clear definition of PAI-1 deficiency as well as a lack of standardized tests. Patients with mild PAI-1 deficiency may be treated with antifibrinolytic agents (ε-aminocaproic acid or tranexamic acid); however, not all patients respond well to these treatments. These patients may be treated with wild-type PAI-1; however, this molecule quickly converts into its inactive form. We propose to use PAI-1 with an extended half-life to treat these patients. PMID:23988002

  13. Ethanol-withdrawal seizures are controlled by tissue plasminogen activator via modulation of NR2B-containing NMDA receptors

    Pawlak, Robert; Melchor, Jerry P.; Matys, Tomasz; Skrzypiec, Anna E.; Strickland, Sidney

    2005-01-01

    Chronic ethanol abuse causes up-regulation of NMDA receptors, which underlies seizures and brain damage upon ethanol withdrawal (EW). Here we show that tissue-plasminogen activator (tPA), a protease implicated in neuronal plasticity and seizures, is induced in the limbic system by chronic ethanol consumption, temporally coinciding with up-regulation of NMDA receptors. tPA interacts with NR2B-containing NMDA receptors and is required for up-regulation of the NR2B subunit in response to ethanol...

  14. Plasminogen Acquisition and Activation at the Surface of Leptospira Species Lead to Fibronectin Degradation ▿

    Vieira, Monica L.; Vasconcellos, Silvio A.; Gonçales, Amane P.; de Morais, Zenaide M.; Nascimento, Ana L. T. O.

    2009-01-01

    Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-ami...

  15. Nucleotide sequence of the plasminogen activator gene of Yersinia pestis: relationship to ompT of Escherichia coli and gene E of Salmonella typhimurium.

    Sodeinde, O A; Goguen, J.D.

    1989-01-01

    We have determined the nucleotide sequence of the 1.4-kilobase DNA fragment containing the plasminogen activator gene (pla) of Yersinia pestis, which determines both plasminogen activator and coagulase activities of the species. The sequence revealed the presence of a 936-base-pair open reading frame that constitutes the pla gene. This reading frame encodes a 312-amino-acid protein of 34.6 kilodaltons and containing a putative 20-amino-acid signal sequence. The presence of a single large open...

  16. Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells

    Kiran Kumar Velpula

    2013-02-01

    Full Text Available Our previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA expression in the progression of meningioma. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Initially, radiation treatment was found to induce hypomethylation in meningioma cells with a decrease in DNA (cytosine-5-methyltransferase 1 (DNMT1 and methyl-CpG binding domain protein (MBD expression. However, oxidative damage by H2O2 or pretreatment of irradiated cells with N-acetyl cysteine (NAC did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression, whereas pre-treatment with sodium butyrate inhibited radiation-induced uPA expression, which complemented our prior results. Methylation-specific polymerase chain reaction on bisulfite-treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells. Transfection with small hairpin RNA (shRNA-expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decrease in invasion and proliferation of meningioma cells. Further, radiation treatment was found to recruit SP1 transcription factor, which was abrogated by shRNA treatment. Analysis on signaling events demonstrated the activation of MAP kinase kinase (MEK-extracellular signal-regulated kinase (ERK in radiation-treated cells, while U0126 (MEK/ERK inhibitor blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors treated with radiation compared to untreated tumors. In conclusion, our data suggest that radiation-mediated hypomethylation

  17. Inhibitory effect of interferon-gamma activated ovine umbilical vein endothelial cells on the intracellular replication of Toxoplasma gondii.

    Dimier, I H; Bout, D T

    1996-01-01

    Toxoplasma gondii is an obligate intracellular parasite that is a major cause of abortion and neonatal mortality in sheep. In congenital toxoplasmosis, T gondii first invades the umbilical vein endothelial cells and are then disseminated throughout the fetus. Treatment of ovine umbilical vein endothelial cells with bovine recombinant gamma-interferon (IFN-gamma) blocked the growth of T gondii. Growth of the parasite was measured by 3H-uracil incorporation 18 h after the onset of the infection and by microscopic enumeration of parallel cultures. This assay revealed that when the cells were pretreated with IFN-gamma in concentrations ranging from 0.15-1,250 U/mL, a high degree of inhibition of T gondii replication was observed with the effect being dose-dependent. Maximum activation was achieved by incubating with 625 U/mL IFN-gamma and no activity was present at 0.15 U/mL. This technique could be of relevance as a first line of defense against congenital ovine Toxoplasma infection. Inhibition of T gondii replication is due to a different mechanism from that existing in mouse macrophages and human fibroblasts. L-Arginine-dependent production of reactive nitrogen and oxygen intermediates was not responsible for the inhibition of T gondii replication. Supplements of five amino acids were able to overcome the inhibition partially but significantly. The mechanism of the inhibition remains to be elucidated. PMID:8822620

  18. Interferons Induce STAT1-Dependent Expression of Tissue Plasminogen Activator, a Pathogenicity Factor in Puumala Hantavirus Disease.

    Strandin, Tomas; Hepojoki, Jussi; Laine, Outi; Mäkelä, Satu; Klingström, Jonas; Lundkvist, Åke; Julkunen, Ilkka; Mustonen, Jukka; Vaheri, Antti

    2016-05-15

    Hantaviruses are zoonotic viruses that show various degrees of vasculopathy in humans. In this study, we analyzed the regulation of 2 fibrinolytic parameters, tissue plasminogen activator (tPA) and its physiological inhibitor, plasminogen activator inhibitor 1 (PAI-1), in Puumala hantavirus (PUUV)-infected patients and in human microvascular endothelial cells. We detected strong upregulation of tPA in the acute phase of illness and in PUUV-infected macaques and found the tPA level to positively correlate with disease severity. The median levels of PAI-1 during the acute stage did not differ from those during the recovery phase. In concordance, hantaviruses induced tPA but not PAI-1 in microvascular endothelial cells, and the induction was demonstrated to be dependent on type I interferon. Importantly, type I and II interferons directly upregulated tPA through signal transducer and activator of transcription 1 (STAT1), which regulated tPA gene expression via a STAT1-responsive enhancer element. These results suggest that tPA may be a general factor in the immunological response to viruses. PMID:26704613

  19. Plasminogen activator inhibitor-1 removal using dextran sulphate columns. Evidence of PAI-1 homeostasis.

    Maher, Vincent M G

    2009-08-01

    Patients with high plasma plasminogen activator inhibitor-1 (PAI-1) antigen levels are prone to develop thrombosis. Lowering PAI-1 levels may offer a therapeutic option and help to better understand PAI-1 metabolism. We examined the effect on plasma PAI-1 levels of LDL-apheresis using dextran sulphate (DS) columns in 12 patients (9 male, 3 female, 49 +\\/- 10 years) with heterozygous familial hypercholesterolaemia and coronary artery disease. One plasma volume equivalent (2.3-4.0 l) was treated during each procedure (at flow rates of 23 +\\/- 2 ml\\/min). Lipids and PAI-1 antigen levels were measured in plasma before and immediately after 19 aphereses (once in 7 patients, twice in 3 patients and three times in 2 patients) and also at 3 and 7 days post apheresis in five of these patients and in the column eluates from 8 of these patients. DS-apheresis reduced plasma cholesterol (50 +\\/- 8%), triglyceride (45 +\\/- 27%), apolipoprotein B (59 +\\/- 10%) and PAI-1 antigen levels from 10.2 +\\/- 5.2 to 6.0 +\\/- 3.1 ng\\/ml (P = 0.005). The PAI-I changes were independent of circadian variation. PAI-I bound to the DS-columns (3.51 +\\/- 1.03 ng\\/ml filtered plasma) and the percent of filtered PAI-1 that was bound correlated inversely (r = -0.81, P < 0.02) with basal PAI-1 levels indicating a high affinity saturable binding process. In four patients, plasma PAI-1 levels post-apheresis were higher than expected based on the amount of PAI-removed by the DS columns. The difference between the expected and actual PAI-1 level post apheresis, reflecting PAI-1 secretion or extracellular redistribution, correlated inversely with basal PAI-1 levels (r = -0.83, P = 0.01). PAI-1 levels returned to baseline pre-apheresis values 7 days post apheresis. PAI-1 antigen may be removed from plasma without adverse effect, resulting temporarily in its extracellular redistribution and restoration to baseline levels over one week. PAI-1 redistribution particularly when baseline pre

  20. Thrombolysis by intravenous tissue plasminogen activator (t-PA). Current status and future direction

    In Japan, the intravenous tissue plasminogen activator (t-PA) Alteplase (0.6 mg/kg) administration of the within 3 h of the onset of acute ischemic stroke was approved for therapeutic use in the year 2006. t-PA induces thrombolysis in patients with acute ischemic stroke, and this method has gradually gained recognition among physicians and the general population. However, the number of patients who were treated using Alteplase is low (4,000-5,000 patients/year), and this figure accounts for only 2-3% of the annual number of cases of ischemic stroke. There is little doubt that Alteplase treatment is a potentially effective modality for some patients with acute ischemic stroke. The post-marketing surveillance of 4,749 Japanese patients treated using Alteplase showed that 33% of the patients had modified Rankin scale (mRS) scores of 0-1, 17% of patients died and 4.5% presented with symptomatic intracerebral hemorrhage (ICH); these results were comparable to those from other countries. The expansion of the therapeutic time window has been a matter of concern. The investigators of the European Cooperative Acute Stroke Study (ECASS) have reported that there was significant improvement in the clinical outcomes of patients with acute ischemie stroke when Alteplase was administered 3-4.5 h after the onset of the symptoms. Mismatches in perfusion- and diffusion-weighted (DW) magnetic resonance imaging (MRI) images have been used for selecting patients 3 h after the onset of symptoms, and the findings from MRI, dwimages (DWI) and MR angiography are practical predictors of t-PA therapy within 3 h of onset. The Middle Cerebral Artery Embolism Local Fibrinolytic Intervention Trial (MELT) Japan study showed that local intra-arterial fibrinolysis is effective in patients with embolic MCA occlusion within 6 h of the onset of symptoms. Combining the initiation of intravenous t-PA administration with further intra-arterial fibrinolysis or mechanical thrombolectomy may improve the

  1. Soluble urokinase-type plasminogen activator receptor is a novel biomarker predicting acute exacerbation in COPD

    Gumus A

    2015-02-01

    Full Text Available Aziz Gumus,1 Nejat Altintas,2 Halit Cinarka,1 Aynur Kirbas,3 Muge Haziroglu,1 Mevlut Karatas,1 Unal Sahin1 1Department of Pulmonary Medicine, School of Medicine, Recep Tayyip Erdogan University, Rize, Turkey; 2Department of Pulmonary Medicine, School of Medicine, Namik Kemal University, Tekirdag, Turkey; 3Department of Clinical Biochemistry, School of Medicine, Recep Tayyip Erdogan University, Rize, Turkey Background: Chronic obstructive pulmonary disease (COPD is a chronic inflammatory condition, and progresses with acute exacerbations. (AE. During AE, levels of acute phase reactants such as C-reactive protein (CRP and inflammatory cells in the circulation increase. Soluble urokinase-type plasminogen activator receptor (suPAR levels increase in acute viral and bacterial infections and in diseases involving chronic inflammation. The purpose of this study was to investigate the effectiveness of suPAR in predicting diagnosis of AE of COPD (AE-COPD and response to treatment. Methods: The study population consisted of 43 patients diagnosed with AE-COPD and 30 healthy controls. suPAR, CRP, and fibrinogen levels were measured on the first day of hospitalization and on the seventh day of treatment. Results: We found that fibrinogen (P<0.001, CRP (P<0.001, and suPAR (P<0.001 were significantly higher in patients with AE-COPD than in healthy controls. Fibrinogen (P<0.001, CRP (P=0.001, and suPAR (P<0.001 were significantly decreased by the seventh day of treatment. However, the area under receiver operator characteristic curve showed that suPAR is superior to CRP and fibrinogen in distinguishing AE-COPD. There was a correlation between fibrinogen, CRP, and suPAR. However, only fibrinogen was a powerful predictor of suPAR in multiple linear regression. In multiple logistic regression, only suPAR and fibrinogen were strong predictors of AE-COPD (P=0.002 and P=0.014, respectively. Serum suPAR was negatively correlated with forced expiratory volume in 1

  2. [Variations in hemostasis and fibrinolysis during the treatment of acute myocardial infarct (AMI) with tissue-type plasminogen activator (TTPA). A study of 17 cases].

    Izaguirre Avila, R; Ruiz de Chávez Cervantes, A; Villavicencio, R; Gómez Trigos, A; Mar Chavira, R; Spíndola, M del C; Casanova, J M

    1993-01-01

    The aim of this trial was to estimate changes in the coagulation and fibrinolysis systems during the thrombolytic treatment with recombinant human tissue-type plasminogen activator (rt-PA) in patients with acute myocardial infarction and correlate with hemorrhagic complications. We studied 17 patients with a 3 hours-continuous systemic infusion of 100 mg of rt-PA. Prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen splits products, plasminogen, alfa-2-antiplasmin (a-2AP) and antithrombin III (AT-III) were performed before, during and after infusion. Most patients showed lengthening coagulation times. Fibrinogen and plasminogen were decreased and PDF was increased. No variations in alpha-2AP or AT-III were observed. The recuperation of fibrinogen levels occurred in 3 hours and there was hyperfibrinogenemia after day 3. No hemorrhagic complication was observed in patients with abnormalities in these coagulation or fibrinolytic tests. PMID:8347053

  3. Fibrin membrane pupillary-block glaucoma after uneventful cataract surgery treated with intracameral tissue plasminogen activator: a case report

    Yoshino Hideaki

    2012-03-01

    Full Text Available Abstract Background Fibrin pupillary-block glaucoma is a rare complication after cataract surgery. The treatment for this condition is still controversial, since Nd:YAG laser fibrin membranotomy tends to reocclude and laser peripheral iridotomy entails the risk of damaging the corneal endothelium in the presence of corneal edema associated with elevated intraocular pressure. Case presentation A 62-year-old man with diabetes mellitus developed acute elevation of intraocular pressure with a shallow anterior chamber five days after uneventful cataract surgery. Initially, slit lamp examination provided only limited information due to severe corneal edema. After resolution of corneal edema with systemic glaucoma therapy, a complete fibrin membrane was observed across the pupil by slit lamp examination. Anterior segment optic coherence tomography clearly revealed a thin fibrin membrane covering the entire pupillary space, a shallow anterior chamber, and a deep posterior chamber. The intraocular lens was not observed by anterior segment optic coherence tomography. In contrast, ultrasound biomicroscopy, which has superior penetration depth, was able to visualize the intraocular lens deep in the posterior chamber. Injection of tissue plasminogen activator into the anterior chamber resulted in complete fibrinolysis and released the pupillary block. Conclusion This case suggests that ocular anterior segment imaging modalities, especially ultrasound biomicroscopy, serve as powerful diagnostic tools to identify mechanisms of acute angle closure glaucoma, which is often accompanied by poor intraocular visibility. This is the first reported case of fibrin pupillary-block glaucoma after cataract surgery successfully treated with intracameral tissue plasminogen activator.

  4. Soluble urokinase plasminogen activator receptor as a prognostic marker of all-cause and cardiovascular mortality in a black population

    Botha, Shani; Fourie, Carla M T; Schutte, Rudolph; Eugen-Olsen, Jesper; Pretorius, Ronel; Schutte, Aletta E

    2015-01-01

    BACKGROUND: Elevated inflammatory markers such as C-reactive protein (CRP) and interleukin-6 (IL-6) are well-known risk factors for cardiovascular mortality. The less familiar marker, soluble urokinase plasminogen activator receptor (suPAR), is known to predict cancer, infections and all-cause mo......BACKGROUND: Elevated inflammatory markers such as C-reactive protein (CRP) and interleukin-6 (IL-6) are well-known risk factors for cardiovascular mortality. The less familiar marker, soluble urokinase plasminogen activator receptor (suPAR), is known to predict cancer, infections and all.......39, 95% CI 1.17-1.65) predicted all-cause mortality, while only suPAR (HR 1.40, 95% CI 1.04-1.87) and IL-6 (HR 1.61,95% CI 1.10-2.35) predicted cardiovascular mortality. The prognostic value of suPAR was independent of IL-6 and CRP (P≤0.015). CONCLUSION: SuPAR predicted both all-cause and cardiovascular...

  5. Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases

    Illemann, Martin; Bird, Nigel; Majeed, Ali;

    2009-01-01

    Metastatic growth and invasion by colon cancer cells in the liver requires the ability of the cancer cells to interact with the new tissue environment. Plasmin(ogen) is activated on cell surfaces by urokinase-type PA (uPA), and is regulated by uPAR and plasminogen activator inhibitor-1 (PAI-1......). To compare the expression patterns of uPA, uPAR and PAI-1 in colon cancer with that in their liver metastases, we analysed matched samples from 14 patients. In all 14 primary colon cancers, we found upregulation of uPAR, uPA mRNA and PAI-1 in primarily stromal cells at the invasive front. In 5 of the 14......, whereas 8 of the remaining 9 showed direct contact between the cancer cells and the liver parenchyma. We conclude that there are 2 distinct patterns of expression of uPAR, uPA and PAI-1 in colon cancer liver metastases and that these correlate closely with 2 morphological growth patterns. These findings...

  6. Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

    Trelle, Morten Beck; Hirschberg, Daniel; Jansson, Anna; Ploug, Michael; Roepstorff, Peter; Andreasen, Peter; Jørgensen, Thomas

    2012-01-01

    The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and...

  7. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis

    Bazzi, Zainab A.; Lanoue, Danielle; El-Youssef, Mouhanned; Romagnuolo, Rocco; Tubman, Janice; Cavallo-Medved, Dora; Porter, Lisa A.; Boffa, Michael B.

    2016-01-01

    Background Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues ...

  8. Preclinical evaluation of a urokinase plasminogen activator receptor-targeted nanoprobe in rhesus monkeys

    Chen Y

    2015-10-01

    Full Text Available Yushu Chen,1 Li Gong,2 Ning Gao,3 Jichun Liao,1 Jiayu Sun,1 Yuqing Wang,1 Lei Wang,1 Pengjin Zhu,1 Qing Fan,1 Yongqiang Andrew Wang,4 Wen Zeng,2 Hui Mao,3 Lily Yang,5 Fabao Gao11Molecular Imaging Center, Department of Radiology, West China Hospital, Sichuan University, Chengdu, 2Sichuan Primed Bio-Tech Group Co, Ltd, Chengdu, People’s Republic of China; 3Department of Radiology and Imaging Sciences, Emory University School of Medicine, Atlanta, GA, 4Ocean NanoTech, LLC, San Diego, CA, 5Department of Surgery, Emory University School of Medicine, Atlanta, GA, USAPurpose: To translate a recombinant peptide containing the amino-terminal fragment (ATF of urokinase plasminogen activator receptor-targeted magnetic iron oxide (IO nanoparticles (uPAR-targeted human ATF-IONPs into clinical applications, we conducted a pilot study to evaluate the toxicity and pharmacokinetics of this nanoparticle in normal rhesus monkeys.Methods: We assessed the changes in the following: magnetic resonance imaging (MRI signals from pretreatment stage to 14 days posttreatment, serum iron concentrations from 5 minutes posttreatment to 12 weeks posttreatment, routine blood examination and serum chemistry analysis results from pretreatment stage to 12 weeks after administration, and results of staining of the liver with Perls’ Prussian Blue and hematoxylin–eosin at 24 hours and 3 months posttreatment in two rhesus monkeys following an intravenous administration of the targeted nanoparticles either with a polyethylene glycol (ATF-PEG-IONP or without a PEG (ATF-IONP coating.Results: The levels of alkaline phosphatase, alanine transaminase, and direct bilirubin in the two monkeys increased immediately after the administration of the IONPs but returned to normal within 20 days and stayed within the normal reference range 3 months after the injection. The creatinine levels of the two monkeys stayed within the normal range during the study. In addition, red blood cells

  9. Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability.

    Bucci, Joel C; Trelle, Morten Beck; McClintock, Carlee S; Qureshi, Tihami; Jørgensen, Thomas J D; Peterson, Cynthia B

    2016-08-01

    Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function. PMID:27416303

  10. Prognostic value of plasma soluble urokinase plasminogen activator receptor (suPAR) in Danish patients with recurrent epithelial ovarian cancer (REOC)

    Begum, Farah Diba; Høgdall, Estrid V S; Riisbo, Rikke;

    2006-01-01

    The level of the soluble urokinase plasminogen activator receptor (suPAR) is elevated in tumour tissue from several types of cancer. This is the first study aiming to predict the prognosis for survival by the use of a pre-chemotherapeutic plasma suPAR value in 71 patients with recurrent epithelial...

  11. Maternal soluble fms-like tyrosine kinase-1, placental growth factor, plasminogen activator inhibitor-2, and folate concentrations and early fetal size : the Generation R study

    Bouwland-Both, Marieke I.; Steegers, Eric A. P.; Lindemans, Jan; Russcher, Henk; Hofman, Albert; Geurts-Moespot, Anneke J.; Sweep, Fred C. G. J.; Jaddoe, Vincent W. V.; Steegers-Theunissen, Regine P. M.

    2013-01-01

    OBJECTIVE: Fetal growth is dependent on adequate development of the placenta. Impaired angiogenesis and vasculogenesis in early pregnancy compromises placental and embryonic development. The proteins soluble fms-like tyrosine kinase (sFlt)-1, placental growth factor (PlGF), and plasminogen activator

  12. A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification

    Nielsen, L S; Kellerman, G M; Behrendt, N;

    1988-01-01

    The iodinated Mr approximately equal to 15,000 amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (u-PA) molecule bound specifically to the cell surface of all of seven cultured human tumor cell lines studied. Cross-linking of iodinated ATF to the cell surface using a bifun...

  13. Effects of a high-fat diet on spontaneous metastasis of Lewis lung carcinoma in plasminogen activator inhibitor-1 deficient and wild-type mice

    We investigated the effects of plasminogen activator inhibitor-1 (PAI-1) deficiency on spontaneous metastasis of Lewis lung carcinoma (LLC) in PAI-1 deficient (PAI-1-/-) and wildtype mice (C57BL/6J background) fed the AIN93G diet or that diet modified with 45% calories from fat. The high-fat diet i...

  14. Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L-cells.

    M.J. Brown (Morris); A.W.R. Tyrrell; C.G. Chapman; J.E. Carey; D.M. Glover; F.G. Grosveld (Frank); I. Dodd; J.H. Robinson

    1985-01-01

    textabstractWe have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libra

  15. Presence of urokinase plasminogen activator, its inhibitor and receptor in small cell lung cancer and non-small cell lung cancer

    Pappot, H.; Pfeiffer, P.; Grøndahl Hansen, J.; Skov, B.G.

    1997-01-01

    Spreading of cancer cells is dependent on the combined action of several proteolytic enzymes, such as serine proteases, comprising the urokinase pathway of plasminogen activation. Previous studies of lung cancer indicate that expression, localization and prognostic impact of the components of the...

  16. Risk Factors Associated with Serum Levels of the Inflammatory Biomarker Soluble Urokinase Plasminogen Activator Receptor in a General Population

    Haupt, Thomas H; Kallemose, Thomas; Ladelund, Steen;

    2014-01-01

    .001). An unhealthy diet and alcohol abstinence in men were also associated with higher suPAR levels. Physical activity in leisure time had a modest impact on suPAR levels in univariate analysis, but not in the final adjusted model. In conclusion, smoking and morbid obesity were strongly associated with higher serum...... suPAR levels in this general population. Diet and alcohol consumption also seemed to impact suPAR levels. Lifestyle changes are likely to affect suPAR since ex-smokers had suPAR levels comparable to those of never-smokers.......The soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of mortality risk in various patient populations. However, little is known about the implications of lifestyle for suPAR levels in the general population. Lifestyle, demographic, and cardiovascular disease (CVD) risk factor...

  17. Hypoxia-ischemia or excitotoxin-induced tissue plasminogen activator- dependent gelatinase activation in mice neonate brain microvessels.

    Priscilla L Omouendze

    Full Text Available Hypoxia-ischemia (HI and excitotoxicity are validated causes of neonatal brain injuries and tissue plasminogen activator (t-PA participates in the processes through proteolytic and receptor-mediated pathways. Brain microvascular endothelial cells from neonates in culture, contain and release more t-PA and gelatinases upon glutamate challenge than adult cells. We have studied t-PA to gelatinase (MMP-2 and MMP-9 activity links in HI and excitotoxicity lesion models in 5 day-old pups in wild type and in t-PA or its inhibitor (PAI-1 genes inactivated mice. Gelatinolytic activities were detected in SDS-PAGE zymograms and by in situ fluorescent DQ-gelatin microscopic zymographies. HI was achieved by unilateral carotid ligature followed by a 40 min hypoxia (8%O₂. Excitotoxic lesions were produced by intra parenchymal cortical (i.c. injections of 10 µg ibotenate (Ibo. Gel zymograms in WT cortex revealed progressive extinction of MMP-2 and MMP-9 activities near day 15 or day 8 respectively. MMP-2 expression was the same in all strains while MMP-9 activity was barely detectable in t-PA⁻/⁻ and enhanced in PAI-1⁻/⁻ mice. HI or Ibo produced activation of MMP-2 activities 6 hours post-insult, in cortices of WT mice but not in t-PA⁻/⁻ mice. In PAI-1⁻/⁻ mice, HI or vehicle i.c. injection increased MMP-2 and MMP-9 activities. In situ zymograms using DQ-gelatin revealed vessel associated gelatinolytic activity in lesioned areas in PAI-1⁻/⁻ and in WT mice. In WT brain slices incubated ex vivo, glutamate (200 µM induced DQ-gelatin activation in vessels. The effect was not detected in t-PA⁻/⁻ mice, but was restored by concomitant exposure to recombinant t-PA (20 µg/mL. In summary, neonatal brain lesion paradigms and ex vivo excitotoxic glutamate evoked t-PA-dependent gelatinases activation in vessels. Both MMP-2 and MMP-9 activities appeared t-PA-dependent. The data suggest that vascular directed protease inhibition may have

  18. Activation of the zymogen to urokinase-type plasminogen activator is associated with increased interdomain flexibility

    Behrens, Manja A; Bøtkjær, Kenneth Alrø; Goswami, Sumit;

    2011-01-01

    A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catal...

  19. Clinical significance of the plasminogen activator system in relation to grade of tumor and treatment response in colorectal carcinoma patients.

    Halamkova, J; Kiss, I; Pavlovsky, Z; Tomasek, J; Jarkovsky, J; Cech, Z; Tucek, S; Hanakova, L; Moulis, M; Zavrelova, J; Man, M; Benda, P; Robek, O; Kala, Z; Penka, M

    2011-01-01

    Urokinase (uPA) plays an essential role in the activation of plasminogen to plasmin, and together with its receptor (uPAR), tissue activator (tPA) and urokinase inhibitors (PAI 1, PAI 2, PAI 3 and protease nexin) forms the plasminogen activator system (PAS), a component of metastatic cascade importantly contributing to the invasive growth and angiogenesis of malignant tumours. In our project we examined the expression of uPA, uPAR, PAI 1 and PAI 2 in tumor tissue and we also studied the plasma levels of PAI 1 before and after the initiation of therapy in patients with colorectal carcinoma in relationship to grade of tumor and the treatment response. In our prospective evaluation we included 80 patients treated for adenocarcinoma of the colon and rectum. Analysis of collected data revealed statistically significant evidence of a relationship between the level of PAI 1 in plasma before treatment and grade of the tumor, which increases with tumor grade (p=0.025). We demonstrated that there exists a statistically significant relationship between the expression of PAI 2 (p<0.001) and uPAR (p=0.031) and grade of tumor. We also confirmed a statistically significant relationship between soluble levels of PAI 1 before treatment and therapeutic response (p=0.021). In our group of patients the expression of uPA, uPAR, PAI 1 and 2 in tumor tissue in relation to response to treatment was also assessed. Our results suggest that the greater expression of these parameters in tumor tissue is linked to a worse response to therapy. In conclusion, PAS factors help as a prognostic indicators and could also act as a predictive factor in colorectal carcinoma. PMID:21744990

  20. Lack of association between level of Plasminogen Activator Inhibitor-1 and estimates of tumor angiogenesis in early breast cancer

    Offersen, Birgitte Vrou; Riisbro, Rikke; Knoop, Ann;

    2007-01-01

    Plasminogen Activator Inhibitor type-1 (PAI-1) is involved in tumor invasion and progression. High levels of PAI-1 are associated with poor prognosis in breast cancer, and PAI-1 has been shown to play a role in angiogenic processes. Since estimates of tumor angiogenesis may predict poor prognosis...... we studied the relationship between PAI-1 and estimates of angiogenesis in breast cancer. Tumor tissue specimens from 438 breast cancer patients were included. Median follow-up was 10.3 years. Protein levels of PAI-1 were measured using an ELISA. Angiogenesis scores were performed using a Chalkley.......009) were independent markers of death from breast cancer. This study confirms high PAI-1 or high Chalkley counts as markers of poor prognosis in breast cancer patients, and suggests that the prognostic impact of PAI-1 is independent of its supposed involvement in tumor angiogenesis. Udgivelsesdato: 2007...

  1. Efficacy of recombinant tissue-type plasminogen activator thrombolysis and primary coronary stenting after acute myocardial infarction

    陈步星; 王伟民; 赵红; 胡大一; 徐成斌; 赵明中; 卢明瑜; 刘健; 吴淳

    2003-01-01

    Objective To compare the efficacy of low dose recombinant tissue-type plasminogen activator (rt-PA) thrombolysis with primary coronary stenting after acute myocardial infarction.Methods Of 261 patients with first acute myocardial infarction, 131 were given low dose rt-PA intravenous thrombolysis, and 130 primary coronary stenting.Results The age, time from onset of chest pain to hospital presentation and infarct location between these two groups were comparable. The patency rate of the infarct-related artery (IRA) in patients in the thrombolysis group was significantly lower than that of patients in the primary stenting group (P0.05).Conclusion Comparing with low dose rt-PA thrombolytic therapy after acute myocardial infarction, primary coronary stenting has a higher patency rate of the IRA, better cardiac function and shorter hospitalization time.

  2. Elevated plasma levels of vascular endothelial growth factor and plasminogen activator inhibitor-1 decrease during improvement of psoriasis

    Nielsen, Hans Jørgen; Christensen, Ib Jarle; Svendsen, M N; Hansen, Ulla; Werther, K; Brünner, N; Petersen, L J; Kristensen, J K

    2002-01-01

    OBJECTIVE AND DESIGN: An evaluation of angiogenesis related molecules during open treatment of psoriasis. MATERIALS AND SUBJECTS: Plasma samples and skin biopsies from 16 patients with psoriasis and plasma samples from 13 healthy controls. TREATMENT: Ranitidine 300 mg orally twice daily for 6...... months. METHODS: Vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) were determined by ELISA methods in plasma collected from the patients before treatment and after 1, 3 and 6 months. Vessel counts were performed in biopsies from affected skin areas taken before...... treatment and after 3 and 6 months. The results were compared to simultaneous PASI scores. RESULTS: Pre-treatment plasma levels of VEGF and PAI-1 were significantly elevated in patients compared with levels in healthy persons (p = 0.02 and p = 0.04, respectively). The plasma levels decreased significantly...

  3. Soluble urokinase plasminogen activator receptor levels are elevated and associated with complications in patients with type 1 diabetes

    Theilade, S; Lyngbaek, S; Hansen, T W;

    2015-01-01

    OBJECTIVES: Soluble urokinase plasminogen activator receptor (suPAR) is a marker of inflammation and endothelial dysfunction. We investigated the associations between suPAR and diabetes, including diabetes duration and complications, in patients with type 1 diabetes. DESIGN, SETTING AND SUBJECTS...... diabetes and is associated with diabetes duration and complications independent of other risk factors. suPAR is a potential novel risk marker for the management of diabetes.......: From 2009 to 2011, 667 patients with type 1 diabetes and 51 nondiabetic control subjects were included in a cross-sectional study at Steno Diabetes Center, Gentofte, Denmark. suPAR levels were measured with an enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: The investigated diabetic...

  4. An In Silico Approach towards the Prediction of Druglikeness Properties of Inhibitors of Plasminogen Activator Inhibitor1

    Umadevi Subramanian

    2014-01-01

    Full Text Available Diabetic retinopathy is the leading cause of blindness worldwide. It is caused by the abnormal growth of the retinal blood vessels. Plasminogen activator inhibitor1 (PAI1 is the key growth factor and the inhibition of PAI1 can reduce the angiogenesis. In this study, currently available inhibitors are taken and tested for the toxicity, binding affinity, and bioactivities of the compounds by in silico approach. Five toxic free inhibitors were identified, among which N-acetyl-D-glucosamine shows the significant binding affinity and two of the molecules are having the better bioactivity properties. The molecular optimization of 2-(acetylamino-2-deoxy-A-D-glucopyranose and alpha-L-fucose can be used for the treatment of diabetic retinopathy.

  5. Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis

    Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the

  6. Relationship between plasminogen activator inhibitor-1 gene polymorphism and early local hemostatic activation in patients with percutaneous coronary intervention procedure

    Objective: To investigate the relationship between plasminogen activatorinhibitor-1 (PAI-1) 4 G/5 G gene polymorphism and local homeostatic activation of PAI-1, D-dimers (DD), activated factor VII (F VII Ia) and P-Selectin (CD62P), on patients under percutaneous coronary intervention (PCI)procedures, and to evaluate its prognostic value on acute stent thrombosis by gene polymorphism analysis. Methods: 20 stable angina patients with a 70% diameter stenosis by visual estimation during angiography and a clinical indication for revascularization were selected. Lesions were treated with the use of standard interventional techniques, both stents implantation underwent with adjunctive balloon angioplasty. Simultaneous blood samples were drawn in sequence from the ostium of the coronary artery before balloon angioplasty through guiding catheter, from the distal coronary artery just beyond the dilated segment after balloon angioplasty and after stent implantation, through aspiration catheter. Markers of PAI-1 and CD62P were measured by ELISA. Markers of F VII and DD were measured by technique chronometrique and ELISA VIDAS respectively. Prevalence of the 4 G/5 G polymorphism was investigated using DNA analysis. Results: The distribution of PAI-1 genotypes in French people was as follows: 4 G/4 G in 30.0%, 4 G/5 G in 60.0% and 5 G/5 G in 10.0%. Among the patients, the frequency of the 4 G and 5 G allele were 0.60 and 0.40 respectively. In patients with the 4 G/5 G polymorphism of PAI-1 gene, the activities of the PAI-1, DD and F VIIa in the coronary circulation were significantly increased after balloon angioplasty in comparing with those before balloon angioplasty (P=0.01, respectively). However, there were no significant differences between the levels of hemostatic activation at ostium before balloon angioplasty and distal to lesion after stent implantation in patients with the 4 G/5 G genotype. Conclusions: Balloon angioplasty more easily induces vessel shrinkage and

  7. Tumour microenvironments induce expression of urokinase plasminogen activator receptor (uPAR and concomitant activation of gelatinolytic enzymes.

    Synnøve Magnussen

    Full Text Available The urokinase plasminogen activator receptor (uPAR is associated with poor prognosis in oral squamous cell carcinoma (OSCC, and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells' expression level of uPAR affected the activity of gelatinolytic enzymes.The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography.We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes.Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational

  8. 1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

    The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli. The spectrum of t-Pa kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identified side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka ∼ 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degree C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. Overall, the data indicate a highly structured conformation for the recombinant t-PA kringle 2 that is closely related to that of the previously investigated PGN kringles 1, 4, and 5

  9. Targeting tumor cell invasion and dissemination in vivo by an aptamer that inhibits urokinase-type plasminogen activator through a novel multifunctional mechanism

    Botkjaer, Kenneth A; Deryugina, Elena I; Dupont, Daniel Miotto;

    2012-01-01

    Data accumulated over the latest two decades have established that the serine protease urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. When designing inhibitors of the proteolytic activity of serine proteases, obtaining sufficient specificity is problematic......, because the topology of the proteases' active sites are highly similar. In an effort to generate highly specific uPA inhibitors with new inhibitory modalities, we isolated uPA-binding RNA aptamers by screening a library of 35 nucleotides long 2'-fluoro-pyrimidine RNA molecules using a version of human pro......-uPA lacking the epidermal growth factor-like and kringle domains as bait. One pro-uPA-binding aptamer sequence, referred to as upanap-126, proved to be highly specific for human uPA. Upanap-126 delayed the proteolytic conversion of human pro-uPA to active uPA, but did not inhibit plasminogen activation...

  10. Mean transit times and the sites of synthesis and catabolism of tissue plasminogen activator and plasminogen activator inhibitor type 1 in young subjects

    Jørgensen, M; Petersen, K.R.; Vinberg, N; Jespersen, J; Gram, Jørgen Brodersen; Tønnesen, K H

    2001-01-01

    sampled simultaneously from a large hepatic vein, an artery and the inferior caval vein, while measuring the splanchnic plasma flow rate and the plasma volume. We found that the catabolism of active t-PA and t-PA antigen took place in the splanchnic circulation with net rates of 7.2 and 6.3 pmol...

  11. EXPRESSION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR IN SMOOTH MUSCLE CELLS OF INJURED ILIAC ARTERIES IN RABBITS

    马晓莉; 黄文英; 佘铭鹏; 李晓惠; 笪冀平

    1996-01-01

    In this experiment, expression of tissue-type plasminogen activator (t-PA) in smooth muscle cells(SMCs) was measured at different iutervals after the arterial injury. In the normal lilac arteries, only low levels of t-PA activity were estimated, t-PA activity in extracts of the iliac arteries increased significantly at the 4th day after the injury, equivalent to the process that SMCs migrated from the media to the intima,and the t-PA activity was then decreased approximately to the normal level at the 7th day. Coexistent to the above data, results from in situ hybridization showed that the expression of t-PA mRNA in the intimaas well as media increased also significantly nr the 4th day after the arterial injury, and at the 7th day, t-PA mRNA was detected only in those SMCs locating closely adjacent to the internal elastic lamina. These results suggest that t-PA might play an important role in SMC migration following endothelial injury, and antagcaaism of t-PA expression and/or activity within the vessel wall might be helpful in intervening the devnlopment of restenosis following angioplasty.

  12. CCl4 induces tissue-type plasminogen activator in rat brain; protective effects of oregano, rosemary or vitamin E.

    Lavrentiadou, Sophia N; Tsantarliotou, Maria P; Zervos, Ioannis A; Nikolaidis, Efstathios; Georgiadis, Marios P; Taitzoglou, Ioannis A

    2013-11-01

    The high metabolic rate and relatively low antioxidant defenses of the lipid-rich brain tissue render it highly susceptible to reactive oxygen species (ROS) and oxidative stress, whereas the implication of ROS in the pathogenesis of several diseases in the central nervous system is well-established. The plasminogen activator (PA) system is a key modulator of extracellular proteolysis, extracellular matrix remodeling and neuronal cell signaling and has been implicated in the pathogenesis of these diseases. This study evaluates the role of tissue-type PA (t-PA) in oxidative stress and the protective role of dietary antioxidants in the rat brain. We used the CCl4 experimental model of ROS-induced lipid peroxidation and evaluated the antioxidant effect of oregano, rosemary or vitamin E. CCl4-treated Wistar rats exhibited elevated brain t-PA activity, which was decreased upon long-term administration of oregano, rosemary or vitamin E. PA inhibitor-1 (PAI-1) activity was also slightly elevated by CCl4, but this increase was not affected by the antioxidants. We hypothesize that the CCl4-induced t-PA activity indicates extracellular proteolytic activity that may be linked to neuronal cell death and brain damage. Vitamin E or antioxidants present in oregano or rosemary are effective in inhibiting t-PA elevation and can be considered as a potential protection against neuronal damage. PMID:23831191

  13. The Single Substitution I259T, Conserved in the Plasminogen Activator Pla of Pandemic Yersinia pestis Branches, Enhances Fibrinolytic Activity

    Haiko, Johanna; Kukkonen, Maini; Ravantti, Janne J.; Westerlund-Wikström, Benita; Korhonen, Timo K.

    2009-01-01

    The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla β-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the β-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259...

  14. High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro.

    Song, Shaozheng; Ge, Xin; Cheng, Yaobin; Lu, Rui; Zhang, Ting; Yu, Baoli; Ji, Xueqiao; Qi, Zhengqiang; Rong, Yao; Yuan, Yuguo; Cheng, Yong

    2016-08-01

    The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits. PMID:27230577

  15. IMD-4690, a Novel Specific Inhibitor for Plasminogen Activator Inhibitor-1, Reduces Allergic Airway Remodeling in a Mouse Model of Chronic Asthma via Regulating Angiogenesis and Remodeling-Related Mediators

    Tezuka, Toshifumi; Ogawa, Hirohisa; AZUMA, MASAHIKO; GOTO, HISATSUGU; Uehara, Hisanori; Aono, Yoshinori; Hanibuchi, Masaki; Yamaguchi, Yoichi; Fujikawa, Tomoyuki; Itai, Akiko; Nishioka, Yasuhiko

    2015-01-01

    Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen...

  16. Different radiolabelling methods alter the pharmacokinetic and biodistribution properties of Plasminogen Activator Inhibitor Type 2 (PAI-2) forms

    Introduction: Tumour-associated urokinase plasminogen activator (uPA) is a critical marker of invasion and metastasis, and it is recognised as having strong prognostic relevance as well as being a therapeutic target. The specific uPA inhibitor plasminogen activator inhibitor type-2 (PAI-2, SerpinB2) specifically targets cell bound uPA and is internalised. Furthermore, preclinical studies have established the “proof-of-principle” of uPA-targeting by PAI-2-cytotoxin conjugates in human carcinoma models. However, these studies also suggest that PAI-2 is rapidly cleared via the renal system with low total dose reaching the tumour. In this study, a comparative single photon emission computed tomography (SPECT) and biodistribution (BD) analysis of different forms of PAI-2 labelled with the radioisotopes iodine-123 (123I) and technetium-99m (99mTc) was undertaken. Methods: The pharmacokinetic (PK) properties and BD of wild-type, ΔCD-loop and PEGylated ΔCD-loop PAI-2 labelled with the commonly used diagnostic SPECT radioisotopes 99mTc or 123I were compared in mouse models of human prostate carcinoma. Whole body SPECT imaging was also performed. Results: Both wild-type and the shorter but active ΔCD-loop form of PAI-2 123I-labelled indirectly via conjugation to free amine groups (termed 123I-Bn-PAI-2) exhibited low tumour uptake, rapid excretion and similar PK profiles. Preliminary studies with a short branched-chain PEGylated 123I-Bn-PAI-2 ΔCD-loop indicated an increase in blood retention time and tumour uptake. All 123I-Bn-labelled radiotracers were largely excreted through the kidneys. By comparison, both wild-type 123I-PAI-2 (labelled directly via tyrosine residues) and 99mTc-PAI-2 displayed different PK/BD patterns compared to 123I-Bn-PAI-2, suggesting greater liver based catabolism and thus slower elimination. SPECT imaging mimicked the BD results of all radiotracers. Conclusion: The different labelling methods gave distinct PAI-2 BD and tumour uptake profiles

  17. Urokinase-type plasminogen activator-like proteases in teleosts lack genuine receptor-binding epidermal growth factor-like domains

    Bager, René; Kristensen, Thomas K.; Jensen, Jan; Szczur, Agnieszka; Christensen, Anni; Andersen, Lisbeth; Johansen, Jesper Sanderhoff; Larsen, Niels; Baatrup, Erik; Huang, Mingdong; Ploug, Michael; Andreasen, Peter A.

    2012-01-01

    zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian...... uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding...

  18. Induction of oxidative stress and inhibition of plasminogen activator inhibitor-1 production in endothelial cells following exposure to organic extracts of diesel exhaust particles and urban fine particles

    Furuyama, Akiko; Koike, Eiko [National Institute for Environmental Studies, Inhalation toxicology Team, Tsukuba (Japan); Hirano, Seishiro [National Institute for Environmental Studies, Environmental Health Sciences Division, Tsukuba (Japan); Kobayashi, Takahiro [National Institute for Environmental Studies, Inhalation toxicology Team, Tsukuba (Japan); National Institute for Environmental Studies, Environmental Health Sciences Division, Tsukuba (Japan)

    2006-03-15

    Endothelial cells play important roles in anticoagulant and fibrinolytic systems. Recent studies suggest that increases in ambient particulate matter (PM) levels have been associated with an increase in mortality rate from cardiovascular diseases. We examined the production of heme oxygenase-1 (HO-1) and factors related to the fibrinolytic function by rat heart microvessel endothelial cells exposed to organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) to investigate the direct effects of these soluble organic fractions in these PM on the fibrinolytic function of endothelial cells. The cell monolayer exposed to 10 {mu}g/ml OE-DEP produced a larger amount of HO-1 than cells exposed to 10 {mu}g/ml OE-UFP. OE-DEP and OE-UFP exposure reduced plasminogen activator inhibitor-1 (PAI-1) production by the cells but did not affect the production of thrombomodulin, tissue-type plasminogen activator, or urokinase-type plasminogen activator. Increased PAI-1 synthesis in response to treatment with 1.0 ng/ml tumor necrosis factor-{alpha} or 0.5 ng/ml transforming growth factor-{beta}1 was reduced by OE-DEP exposure. Suppression of PAI-1 production by OE-DEP exposure was mediated through oxidative stress and was independent of HO-1 activity. These results suggest that exposure to the soluble organic fraction of PM and DEP induced oxidative stress and reduced the PAI-1 production of endothelial cells. (orig.)

  19. Nucleotide sequence of the plasminogen activator gene of Yersinia pestis: relationship to ompT of Escherichia coli and gene E of Salmonella typhimurium.

    Sodeinde, O A; Goguen, J D

    1989-05-01

    We have determined the nucleotide sequence of the 1.4-kilobase DNA fragment containing the plasminogen activator gene (pla) of Yersinia pestis, which determines both plasminogen activator and coagulase activities of the species. The sequence revealed the presence of a 936-base-pair open reading frame that constitutes the pla gene. This reading frame encodes a 312-amino-acid protein of 34.6 kilodaltons and containing a putative 20-amino-acid signal sequence. The presence of a single large open reading frame is consistent with our previous conclusion that the two Pla proteins which appear in the outer membrane of pla+ Y. pestis are derived from a common precursor. The deduced amino acid sequence of Pla revealed that it possesses a high degree of homology to the products of gene E of Salmonella typhimurium and ompT of Escherichia coli but does not possess significant homology to other plasminogen activators of known sequence. We also identified a transcription unit that resides on the complimentary strand and overlaps the pla gene. PMID:2651310

  20. Subretinal recombinant tissue plasminogen activator and pneumatic displacement for the management of subretinal hemorrhage occurring after anti-VEGF injections for wet AMD

    Tognetto, D

    2011-01-01

    Daniele Tognetto, Eirini Skiadaresi, Paolo Cecchini, Giuseppe RavalicoEye Clinic, University of Trieste, Trieste, ItalyAbstract: We describe three cases of submacular hemorrhage that occurred two to four days after anti-VEGF intravitreal injection for occult choroidal neovascularisation in age-related macular degeneration and their management with 25 gauge pars plana vitrectomy with injection of subretinal recombinant tissue plasminogen activator (rTPA) followed by fluid-air exchange and post...

  1. Transconjunctival sutureless vitrectomy with tissue plasminogen activator, gas and intravitreal bevacizumab in the management of predominantly hemorrhagic age-related macular degeneration

    Arias, Luis

    2010-01-01

    Luis Arias1,2, Jordi Monés11Institut de la Màcula i de la Retina, Centro Médico Teknon, Barcelona; 2Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, BarcelonaPurpose: To determine the efficacy and safety of treating predominantly hemorrhagic age-related macular degeneration (AMD) with transconjunctival sutureless vitrectomy (TSV), tissue plasminogen activator (tPA), sulphur hexafluoride (SF6), and intravitreal bevacizumab.Methods: Retro...

  2. [Massive endocardial thrombosis in a patient with a peritoneo-jugular shunt for refractory ascites: the therapeutic success of tissue plasminogen activator and defibrotide].

    Sacchetti, C; Pederzoli, S; Tamborrino, E; Grandi, M

    1994-01-01

    The authors report a case of a patient with a refractory ascites due to extrahepatic portal thrombosis in course of idiopathic thrombocythemia. A peritoneovenous shunt was applied and as a late complication a massive thrombosis of the intracardiac portion of the duct developed. Thrombolysis was obtained with tissue plasminogen activator at doses usually administered for acute myocardial infarction. Prophylaxis of recurrence was pursued with pictomide and defibrotide. PMID:8079040

  3. The −675 4G/5G Polymorphism in Plasminogen Activator Inhibitor-1 Gene Is Associated with Risk of Asthma: A Meta-Analysis

    Wei Nie; Bing Li; Qing-Yu Xiu

    2012-01-01

    BACKGROUND: A number of studies assessed the association of -675 4G/5G polymorphism in the promoter region of plasminogen activator inhibitor (PAI)-1 gene with asthma in different populations. However, most studies reported inconclusive results. A meta-analysis was conducted to investigate the association between polymorphism in the PAI-1 gene and asthma susceptibility. METHODS: Databases including Pubmed, EMBASE, HuGE Literature Finder, Wanfang Database, China National Knowledge Infrastructu...

  4. Association of the 4 g/5 g polymorphism of plasminogen activator inhibitor-1 gene with sudden sensorineural hearing loss. A case control study

    Cho Seong; Chen Haimei; Kim Il; Yokose Chio; Kang Joseph; Cho David; Cai Chun; Palma Silvia; Busi Micol; Martini Alessandro; Yoo Tae J

    2012-01-01

    Abstract Background The 5 G/5 G genotype of PAI-1 polymorphism is linked to decreased plasminogen activator inhibitor-1 (PAI-1) levels and it has been suggested that lower PAI-1 levels may provide protective effects on inflammation, local microcirculatory disturbance, and fibrotic changes, which are likely associated with development of sudden sensorineural hearing loss (SSNHL). Methods The association of the 4 G/5 G PAI-1 polymorphism with the development and clinical outcome of SSNHL is eva...

  5. The therapeutic effect and prognosis of acute cerebral infarction patients with atrial fibrillation treated by intravenous thrombolysis with recombinant tissue plasminogen activator

    尤寿江

    2013-01-01

    Objective To investigate the efficacy and safety of intravenous thrombolysis with recombinant tissue plasminogen activator (rt-PA) in acute cerebral infarct patients with atrial fibrillation (AF) and the predicting factors of poor prognosis.Methods Totally 162 patients with acute cerebral infarct were treated with rt-PA within 4.5hours from the onset.According to past history and the electrocardiogram,the patients was classified into AF

  6. Interleukin-1ß-induced expression of the urokinase-type plasminogen activator receptor and its co-localization with MMPs in human articular chondrocytes

    Schwab, W.; Schulze-Tanzil, G.; Mobasheri, A; Dressler, J.; Kotzsch, M.; Shakibaei, M.

    2004-01-01

    The urokinase-type plasminogen activator receptor (uPAR) plays a critical role in cartilage degradation during osteoarthritis as it regulates pericellular proteolysis mediated by serine proteinases. Another important family of proteinases responsible for ECM destruction in arthritis are the matrix metalloproteinases (MMPs). MMPs are regulated by IL- 1ß, a cytokine that plays a pivotal role in pathogenesis of osteoarthritis. This study was undertaken to address ...

  7. Soluble urokinase-type plasminogen activator receptor levels in patients with burn injuries and inhalation trauma requiring mechanical ventilation: an observational cohort study

    Backes, Yara; van der Sluijs, Koenraad F; Tuip de Boer, Anita M; Hofstra, Jorrit Jan; Vlaar, Alexander PJ; Determann, Rogier M; Knape, Paul; David P Mackie; Schultz, Marcus J.

    2011-01-01

    Introduction Soluble urokinase-type plasminogen activator receptor (suPAR) has been proposed as a biologic marker of fibrinolysis and inflammation. The aim of this study was to investigate the diagnostic and prognostic value of systemic and pulmonary levels of suPAR in burn patients with inhalation trauma who need mechanical ventilation. Methods suPAR was measured in plasma and nondirected lung-lavage fluid of mechanically ventilated burn patients with inhalation trauma. The samples were obta...

  8. Evaluation of 12-Lipoxygenase (12-LOX and Plasminogen Activator Inhibitor 1 (PAI-1 as Prognostic Markers in Prostate Cancer

    Tomasz Gondek

    2014-01-01

    Full Text Available In carcinoma of prostate, a causative role of platelet 12-lipoxygenase (12-LOX and plasminogen activator inhibitor 1 (PAI-1 for tumor progression has been firmly established in tumor and/or adjacent tissue. Our goal was to investigate if 12-LOX and/or PAI-1 in patient’s plasma could be used to predict outcome of the disease. The study comprised 149 patients (age 70±9 divided into two groups: a study group with carcinoma confirmed by positive biopsy of prostate (n=116 and a reference group (n=33 with benign prostatic hyperplasia (BPH. The following parameters were determined by the laboratory test in plasma or platelet-rich plasma: protein level of 12-LOX, PAI-1, thromboglobulin (TGB, prostate specific antigen (PSA, C-reactive protein (CRP, hemoglobin (HGB, and hematocrit (HCT, as well as red (RBC and white blood cells (WBC, number of platelets (PLT, international normalized ratio of blood clotting (INR, and activated partial thromboplastin time (APTT. The only difference of significance was noticed in the concentration of 12-LOX in platelet rich plasma, which was lower in cancer than in BPH group. Standardization to TGB and platelet count increases the sensitivity of the test that might be used as a biomarker to assess risk for prostate cancer in periodically monitored patients.

  9. The human urokinase-plasminogen activator gene (PLAU) is located on chromosome 10q24 centromeric to the HOX11 gene

    Stein, P.M.; Stass, S.A.; Kagan, J. (Univ. of Texas, Houston (United States))

    1993-04-01

    Urokinase-plasminogen activator is one of two soluble serine proteases that are produced by humans and that convert plasminogen, an inactive proenzyme present in plasma and other extracellular fluids, to plasmin, a protease with broad substrate specificities. Its activity is involved in processes requiring localized extracellular proteolysis such as fibrinolysis, tissue remodeling, and cell migration. Increased production of urokinase has been associated with cancer metastases. The gene for urokinase-plasminogen activator, PLAU, was mapped to chromosome 10q24-qter. By employing somatic cell genetics, polymerase chain reaction (PCR), and Southern blot analysis, the authors assign PLAU to chromosome 10q24. Human chromosome segment 10q23-q25 contains the genes for terminal deoxynucleotidyltransferase, cytochrome P450IIC, glutamic-oxaloacetic transaminase, and plasma retinol binding protein, which form a syntenic group on murine chromosome 19. It is therfore of interest that PLAU and glutamate dehydrogenase, which are on murine chromosome 14, also map in or close to this region of human chromosome 10.

  10. Apolipoprotein(a) inhibits in vitro tube formation in endothelial cells: identification of roles for Kringle V and the plasminogen activation system.

    Liu, Lei; Boffa, Michael B; Koschinsky, Marlys L

    2013-01-01

    Elevated plasma concentrations of lipoprotein(a) are associated with increased risk for atherothrombotic diseases. Apolipoprotein(a), the unique glycoprotein component of lipoprotein(a), is characterized by the presence of multiple kringle domains, and shares a high degree of sequence homology with the serine protease zymogen plasminogen. It has been shown that angiostatin, a proteolytic fragment of plasminogen containing kringles 1-4, can effectively inhibit angiogenesis. Moreover, proteolytic fragments of plasminogen containing kringle 5 are even more potent inhibitors of angiogenesis than angiostatin. Despite its strong similarity with plasminogen, the role of apolipoprotein(a) in angiogenesis remains controversial, with both pro- and anti-angiogenic effects reported. In the current study, we evaluated the ability of apolipoprotein(a) to inhibit VEGF- and angiopoietin-induced tube formation in human umbilical cord endothelial cells. A 17 kringle-containing form of recombinant apo(a) (17K), corresponding to a well-characterized, physiologically-relevant form of the molecule, effectively inhibited tube formation induced by either VEGF or angiopoietin-1. Using additional recombinant apolipoprotein(a) (r-apo(a)) variants, we demonstrated that this effect was dependent on the presence of an intact lysine-binding site in kringle V domain of apo(a), but not on the presence of the functional lysine-binding site in apo(a) kringle IV type 10; sequences within in the amino-terminal half of the molecule were also not required for the inhibitory effects of apo(a). We also showed that the apo(a)-mediated inhibition tube formation could be reversed, in part by the addition of plasmin or urokinase plasminogen activator, or by removal of plasminogen from the system. Further, we demonstrated that apo(a) treated with glycosidases to remove sialic acid was significantly less effective in inhibiting tube formation. This is the first report of a functional role for the glycosylation

  11. Apolipoprotein(a inhibits in vitro tube formation in endothelial cells: identification of roles for Kringle V and the plasminogen activation system.

    Lei Liu

    Full Text Available Elevated plasma concentrations of lipoprotein(a are associated with increased risk for atherothrombotic diseases. Apolipoprotein(a, the unique glycoprotein component of lipoprotein(a, is characterized by the presence of multiple kringle domains, and shares a high degree of sequence homology with the serine protease zymogen plasminogen. It has been shown that angiostatin, a proteolytic fragment of plasminogen containing kringles 1-4, can effectively inhibit angiogenesis. Moreover, proteolytic fragments of plasminogen containing kringle 5 are even more potent inhibitors of angiogenesis than angiostatin. Despite its strong similarity with plasminogen, the role of apolipoprotein(a in angiogenesis remains controversial, with both pro- and anti-angiogenic effects reported. In the current study, we evaluated the ability of apolipoprotein(a to inhibit VEGF- and angiopoietin-induced tube formation in human umbilical cord endothelial cells. A 17 kringle-containing form of recombinant apo(a (17K, corresponding to a well-characterized, physiologically-relevant form of the molecule, effectively inhibited tube formation induced by either VEGF or angiopoietin-1. Using additional recombinant apolipoprotein(a (r-apo(a variants, we demonstrated that this effect was dependent on the presence of an intact lysine-binding site in kringle V domain of apo(a, but not on the presence of the functional lysine-binding site in apo(a kringle IV type 10; sequences within in the amino-terminal half of the molecule were also not required for the inhibitory effects of apo(a. We also showed that the apo(a-mediated inhibition tube formation could be reversed, in part by the addition of plasmin or urokinase plasminogen activator, or by removal of plasminogen from the system. Further, we demonstrated that apo(a treated with glycosidases to remove sialic acid was significantly less effective in inhibiting tube formation. This is the first report of a functional role for the

  12. Plasminogen activator inhibitor-1 4G/5G gene polymorphism in patients with myocardial or cerebrovascular infarction in Tianjin, China

    战梅; 周玉玲; 韩忠朝

    2003-01-01

    Objective To investigate the association between the plasminogen activator inhibitor-1 (PAI-1) 4G/5G gene polymorphism and the occurrence of myocardial and cerebrovascular infarctions in individuals from Tianjin, China.Methods The PAI-1 genotype was determined using allele-specific polymerase chain reaction (AS-PCR) in 56 myocardial infarction (MI) patients, 54 cerebrovascular infarction(CI) patients and 83 unrelated healthy controls. All subjects ' clinical features and plasma PAI-1 activity levels were determined.Results The PAI-1 genotype distribution frequency of the single guanine deletion/insertion 4G/5G polymorphism (located -675 bp upstream from the start of transcription) significantly differed between the patients and healthy controls. In the MI group, the 4G/4G-genotype frequency was increased, but the 4G/5G-genotype is decreased when compared to the control group. In the CI group, both the 4G/4G- and 4G/5G -genotypes occurred at a lower frequency than those in the control group (P<0.001). The plasma PAI-1 activity level in the MI group was lowered as the presence of the 4G allele decreases. In the CI group, the frequency of 5G/5G was much higher than that of the control group (P<0.001). The plasma PAI-1 activity level in the CI group was elevated as the presence of the 5G allele increased. Furthermore, positive correlation between triglyceride, glucose levels and PAI-1 activity were found in all three groups (P<0.001).Conclusions The PAI-1 4G/5G gene polymorphism is associated with a higher risk of MI and CI in individuals in Tianjin, China. The deletion/insertion polymorphism is probably an important hereditary risk factor for heart diseases. Moreover, triglyceride and glucose levels of plasma have functional importance in regulating PAI-1 activity.

  13. Distribution patterns of Gd-DTPA-enhanced magnetic resonance imaging after intravenous tissue plasminogen activator therapy for acute myocardial infarction

    Fukuzawa, Shigeru; Watanabe, Hiroyuki; Shimada, Kazuhiro; Katagiri, Nakoto; Ozawa, Shun (Funabashi Municipal Medical Center, Chiba (Japan))

    1994-03-01

    In patients who received thrombolytic therapy for acute myocardial infarction (AMI), we observed 3 distinct patterns in gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced magnetic resonance (MR) imaging. To clarify the significance of these distribution patterns of Gd-DTPA, 20 consecutive patients underwent Gd-DTPA-enhanced MR imaging 7-10 days after AMI. All of the patients received intravenous recombinant tissue plasminogen activator (IVTPA) within 6 h of onset. Echocardiograms were obtained prior to and serially over 10 days, and interpreted for regional wall motion. Coronary angiograms were obtained the day before discharge. None of the 6 patients with a closed infarct-related artery, and 9 of the 14 patients with an open artery, demonstrated subendocardial enhancement (p<0.05). All of these latter 9 patients demonstrated a significant improvement in wall motion between days 1 and 10 after AMI. In contrast, only 1 of the 7 patients with transmural enhancement and none of the 4 patients with non-homogeneous enhancement demonstrated improvement of wall motion on day 10 (p<0.05). We concluded that subendocardial enhancement was a fair prognostic sign for restoration of regional cardiac function in patients who received IVTPA during AMI. (author).

  14. Independent prognostic value of angiogenesis and the level of plasminogen activator inhibitor type 1 in breast cancer patients

    Hansen, S; Overgaard, J; Rose, C;

    2003-01-01

    Tumour angiogenesis and the levels of plasminogen activator inhibitor type 1 (PAI-1) are both informative prognostic markers in breast cancer. In cell cultures and in animal model systems, PAI-1 has a proangiogenic effect. To evaluate the interrelationship of angiogenesis and the PAI-1 level in b...... Chalkley count are independent prognostic markers for recurrence-free survival in patients with primary breast cancer, suggesting that the prognostic impact of PAI-1 is not only based on its involvement in angiogenesis....... breast cancer, we have evaluated the prognostic value of those factors in a total of 228 patients with primary, unilateral, invasive breast cancer, evaluated at a median follow-up time of 12 years. Microvessels were immunohistochemically stained by antibodies against CD34 and quantitated by the Chalkley...... overall survival. In the multivariate analysis, the uPA level did not show independent prognostic impact for any of the analysed end points. In contrast, the risk of recurrence was independently and significantly predicted by both the PAI-1 level and the Chalkley count, with a hazard ratio (95% CI) of 1...

  15. Cigarette Smoke and the Induction of Urokinase Plasminogen Activator Receptor In Vivo: Selective Contribution of Isoforms to Bronchial Epithelial Phenotype.

    Portelli, Michael A; Stewart, Ceri E; Hall, Ian P; Brightling, Christopher E; Sayers, Ian

    2015-08-01

    The urokinase plasminogen activator receptor (uPAR) gene (PLAUR) has been identified as an asthma susceptibility gene, with polymorphisms within that gene being associated with baseline lung function, lung function decline, and lung function in a smoking population. Soluble cleaved uPAR (scuPAR), a molecule identified as a marker of increased morbidity and mortality in a number of diseases, has been shown to be elevated in the airways of patients with asthma and in patients with chronic obstructive pulmonary disease. However, the functionality of soluble receptor isoforms and their relationship with an important initiator for obstructive lung disease, cigarette smoke, remains undefined. In this study, we set out to determine the effect of cigarette smoke on soluble uPAR isoforms, its regulatory pathway and the resultant effect on bronchial epithelial cell function. We identified a positive association between cigarette pack-years and uPAR expression in the airway bronchial epithelium of biopsies from patients with asthma (n = 27; P = 0.0485). In vitro, cigarette smoke promoted cleavage of uPAR from the surface of bronchial epithelial cells (1.5× induction; P bronchial epithelial cells. This suggests that cigarette smoke elevates soluble receptor isoforms in bronchial epithelial cells through direct (cleavage) and indirect (messenger RNA expression) means. These findings provide further insight into how cigarette smoke may influence changes in the airways of importance to airway remodeling and obstructive lung disease progression. PMID:25490122

  16. Prognostic significance of circulating intact and cleaved forms of urokinase plasminogen activator receptor in inoperable chemotherapy treated cholangiocarcinoma patients

    Grunnet, M; Christensen, I J; Lassen, U;

    2014-01-01

    determine if pre-treatment serum levels of uPAR forms and a decrease in levels during chemotherapy are predictive of survival in patients with inoperable cholangiocarcinoma. DESIGN AND METHODS: Patients with inoperable cholangiocarcinoma were consecutively included in the training set (n=108). A test set......PAR(I-III)+uPAR(II-III) after 2cycles of chemotherapy was associated with poor survival (HR=1.79, 95% CI:1.08-2.97, p=0.023, n=57). This predictor, however, was not significant in the test set (p=0.21, 26 events in 27 patients). CONCLUSION: The baseline level of uPAR(I-III)+uPAR(II-III) is a predictor of survival in inoperable......BACKGROUND: High levels of intact and cleaved forms of the urokinase-type plasminogen activator receptor (uPAR) in both tissue and blood are associated with poor survival in several cancer diseases. The prognostic significance of uPAR in cholangiocarcinoma is unknown. The aims of this study were to...

  17. Dissolution of emboli in rats with experimental cerebral thromboembolism by recombinant human tissue plasminogen activator (TD-2061)

    Tissue plasminogen activator (t-PA) is frequently administered clinically as thrombolytic therapy. We injected recombinant t-PA into rats with cerebral 125I-labeled blood clot emboli to evaluate the dissolutive effect of recombinant human single-chain t-PA (rt-PA; TD-2061) on such emboli and to examine the possibility of improving neurological damage in patients with cerebral thrombosis. When rt-PA was given intravenously at a dose of 350,000 IU/kg 2 minutes before embolization, radioactivity in the affected cerebral hemisphere decreased to 20% of that in the vehicle control 2 hours after embolization. A significant decrease in radioactivity in the cerebral hemisphere was also found on the administration of 700,000 IU/kg of rt-PA 30 or 60 minutes after embolization, but not when rt-PA was administered 2 minutes after embolization. Marked inhibition of abnormal behavior such as hemiplegia was seen on treatment with rt-PA 2 minutes before embolization, but not at all when rt-PA treatment was given 30 or 60 minutes after embolization. The findings suggest that rt-PA can dissolve blood clot emboli in cerebral vessels and that prompt thrombolytic therapy is important to minimize neurological dysfunction in cases of cerebral thromboembolism

  18. Serum levels of soluble urokinase plasminogen activator receptor is associated with parasitemia in children with acute Plasmodium falciparum malaria infection

    Perch, M; Kofoed, Pe; Fischer, Torge; Có, F; Rombo, L; Aaby, P; Eugen-Olsen, J

    2004-01-01

    Serum levels of soluble urokinase plasminogen activator receptor (suPAR) are significantly elevated and of prognostic value in patients suffering from serious infectious diseases such as HIV and tuberculosis. Our objective was to investigate suPAR levels during symptomatic malaria infection and 7...... days after treatment. Children younger than 6 years who presented with fever or other symptoms compatible with malaria were enrolled. Blood films and samples were collected on day 0 and day 7. Twenty-five children were allocated to each of three groups according to the amount of Plasmodium falciparum...... group 1 after 7 days of treatment. All became malaria negative in their blood slides and all decreased in suPAR level to median 3.48 ng/mL (IQR: 3.08-3.91) (P <0.0001). Group 2 consisted of 25 children with 1-20 parasites in their blood slide. The suPAR level was median 2.91 ng/mL (IQR: 2.27-4.40) and...

  19. Hemothorax under thrombolytic therapy with recombinant tissue: plasminogen activator (rt-PA) in a 16-year-old girl.

    Varnholt, V; Ringe, H; Nietsch, L; Gaedicke, G

    1999-12-01

    We present the case of a 16-year-old girl with an extended thrombosis of the femoral and iliac vein and the inferior vena cava during pleuropneumonia; predisposing risk factors for thrombophilia were: use of contraceptives, nicotine abuse and congenital deficiency of antithrombin III (not previously diagnosed). Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA; initial dose: 0.08 mg/kg/h) was started. 2 days later--after diagnosis of an extended hemothorax: 1500 ml blood were obtained after thoracocentesis, transfusion of packed red blood cells was necessary--rt-PA was stopped, with only heparin (400 U/kg/d) being administered. 36 h later--the thrombosis had not yet changed--the thrombolytic therapy with rt-PA was continued in a markedly reduced dose (0.015 mg/kg/d) with no further bleeding complications. 8 days later--after successful thrombolysis--t-PA was stopped, heparin was given for another 10 days, then cumarin was administered orally. PMID:10650854

  20. Plasminogen activator and serine protease inhibitor-E2 (protease nexin-1) expression by bovine granulosa cells in vitro.

    Cao, Mingju; Sahmi, Malha; Lussier, Jacques G; Price, Christopher A

    2004-09-01

    Remodeling of the extracellular matrix (ECM) occurs during antral follicle growth, and the plasminogen activators (PA) have been implicated in this process in rodents. In the present study, we measured the expression and secretion of PA and the PA inhibitor protease nexin-1 (SerpinE2) in antral and basal bovine granulosa cells from small (8 mm) during 6 days of culture in serum-free medium. Casein zymography revealed that the cells secreted predominantly tissue-type PA (tPA) with urokinase (uPA) being associated mainly with cell lysates, and Western blot demonstrated that the cells secreted SerpinE2. Overall, secreted tPA activity was higher in cultures of cells from small follicles compared with large follicles, and secreted SerpinE2 levels were higher in cultures of cells from large follicles. In cultures of cells from small follicles, secreted tPA levels increased with time of culture for antral but not basal cells, and SerpinE2 levels increased with time for basal but not antral cells. In cultures of granulosa cells from large follicles, tPA activity increased significantly with time of culture, whereas SerpinE2 levels decreased. Cell-associated uPA activity decreased with time in cells from medium and large follicles. Reverse-transcription polymerase chain reaction and Northern blot analysis showed that SerpinE2 secretion was regulated largely at the transcriptional level, whereas tPA secretion was not. The data suggest stage-dependent regulation of granulosa cell PA and SerpinE2 production, consistent with a role in ECM remodeling during follicle growth. PMID:15128599

  1. Dengue Virus Nonstructural Protein 1-Induced Antibodies Cross-React with Human Plasminogen and Enhance Its Activation.

    Chuang, Yung-Chun; Lin, Jessica; Lin, Yee-Shin; Wang, Shuying; Yeh, Trai-Ming

    2016-02-01

    Dengue virus (DENV) infection is the most common mosquito-borne viral disease, and it can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks of DHF/DSS. However, the mechanism underlying hemorrhage in DHF/DSS remains elusive. In previous studies, plasminogen (Plg) cross-reactive Abs, which can recognize DENV nonstructural protein (NS) 1, have been found in dengue patients. However, it is unclear whether these Abs are indeed induced by DENV NS1. Thus, we immunized mice with recombinant NS1 from both bacteria and drosophila to determine whether NS1 can induce Plg cross-reactive Abs. The results from the NS1-immunized mouse sera indicated that NS1 immunization induced Abs that could cross-react with Plg. To study the effects of these NS1-induced Plg cross-reactive Abs on fibrinolysis, we isolated several Plg cross-reactive anti-NS1 mAbs from these mice and found that some of them could enhance Plg activation. In addition, epitope mapping with a phage-displayed random peptide library revealed that one of these mAbs (2A5) could recognize NS1 C-terminal residues 305-311, which share sequence homology with Plg residues 590-597. A synthetic peptide of NS1 residues 305-311 could inhibit the binding of both 2A5 and its Fab to Plg and its enhanced activation. Thus, our results suggest that DENV NS1 can induce Plg cross-reactive Abs through molecular mimicry, which can enhance Plg activation and may contribute to the pathogenesis of DHF/DSS. PMID:26712948

  2. Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system.

    Killeen, S D

    2009-05-19

    Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.

  3. Tumor marker utility and prognostic relevance of cathepsin B, cathepsin L, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, CEA and CA 19-9 in colorectal cancer

    Cathepsin B and L (CATB, CATL), urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 play an important role in colorectal cancer invasion. The tumor marker utility and prognostic relevance of these proteases have not been evaluated in the same experimental setting and compared with that of CEA and CA-19-9. Protease, CEA and CA 19-9 serum or plasma levels were determined in 56 patients with colorectal cancer, 25 patients with ulcerative colitis, 26 patients with colorectal adenomas and 35 tumor-free control patients. Protease, CEA, CA 19-9 levels have been determined by ELISA and electrochemiluminescence immunoassay, respectively; their sensitivity, specificity, diagnostic accuracy have been calculated and correlated with clinicopathological staging. The protease antigen levels were significantly higher in colorectal cancer compared with other groups. Sensitivity of PAI-1 (94%), CATB (82%), uPA (69%), CATL (41%) were higher than those of CEA or CA 19-9 (30% and 18%, respectively). PAI-1, CATB and uPA demonstrated a better accuracy than CEA or CA 19-9. A combination of PAI-1 with CATB or uPA exhibited the highest sensitivity value (98%). High CATB, PAI-1, CEA and CA 19-9 levels correlated with advanced Dukes stages. CATB (P = 0.0004), CATL (P = 0.02), PAI-1 (P = 0.01) and CA 19-9 (P = 0.004) had a significant prognostic impact. PAI-1 (P = 0.001), CATB (P = 0.04) and CA 19-9 (P = 0.02) proved as independent prognostic variables. At the time of clinical detection proteases are more sensitive indicators for colorectal cancer than the commonly used tumor markers. Determinations of CATB, CATL and PAI-1 have a major prognostic impact in patients with colorectal cancer

  4. Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes

    Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1α (HIF-1α), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia-induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H2O2)-induced dysregulation of adiponectin and PAI-1 production. H2O2 treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein (C/EBPα), but had no effect on HIF-1α, whereas hypoxia stabilized HIF-1α and decreased expression of C/EBPα, but not PPARγ. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases

  5. CFTR suppresses tumor progression through miR-193b targeting urokinase plasminogen activator (uPA) in prostate cancer.

    Xie, C; Jiang, X H; Zhang, J T; Sun, T T; Dong, J D; Sanders, A J; Diao, R Y; Wang, Y; Fok, K L; Tsang, L L; Yu, M K; Zhang, X H; Chung, Y W; Ye, L; Zhao, M Y; Guo, J H; Xiao, Z J; Lan, H Y; Ng, C F; Lau, K M; Cai, Z M; Jiang, W G; Chan, H C

    2013-05-01

    Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is expressed in the epithelial cells of a wide range of organs/tissues from which most cancers are derived. Although accumulating reports have indicated the association of cancer incidence with genetic variations in CFTR gene, the exact role of CFTR in cancer development and the possible underlying mechanism have not been elucidated. Here, we report that CFTR expression is significantly decreased in both prostate cancer cell lines and human prostate cancer tissue samples. Overexpression of CFTR in prostate cancer cell lines suppresses tumor progression (cell growth, adhesion and migration), whereas knockdown of CFTR leads to enhanced malignancies both in vitro and in vivo. In addition, we demonstrate that CFTR knockdown-enhanced cell proliferation, cell invasion and migration are significantly reversed by antibodies against either urokinase plasminogen activator (uPA) or uPA receptor (uPAR), which are known to be involved in various malignant traits of cancer development. More interestingly, overexpression of CFTR suppresses uPA by upregulating the recently described tumor suppressor microRNA-193b (miR-193b), and overexpression of pre-miR-193b significantly reverses CFTR knockdown-enhanced malignant phenotype and abrogates elevated uPA activity in prostate cancer cell line. Finally, we show that CFTR gene transfer results in significant tumor repression in prostate cancer xenografts in vivo. Taken together, the present study has demonstrated a previously undefined tumor-suppressing role of CFTR and its involvement in regulation of miR-193b in prostate cancer development. PMID:22797075

  6. Southern copperhead venom enhances tissue-type plasminogen activator induced fibrinolysis but does not directly lyse human plasma thrombi.

    Nielsen, Vance G

    2016-07-01

    In addition to degrading fibrinogen as a source of consumptive coagulopathy, purified fractions of southern copperhead (Agkistrodon contortrix contortrix; A. c. contortrix) venom has been demonstrated to enhance fibrinolysis. The goal of this investigation was to characterize the kinetic fibrinolytic profile of A. c. contortrix venom in the absence and presence of tissue-type plasminogen activator (tPA) to determine if intact venom had tPA independent fibrinolytic properties. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to A. c. contortrix venom (0-6 μg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, plasma was exposed to 0-6 μg/ml of venom without tPA added and coagulation observed for 3 h. Venom significantly prolonged the onset of coagulation, decreased the velocity of thrombus growth but did not significantly decrease clot strength. In the presence of tPA, venom significantly decreased clot strength, shortened the time of onset of fibrinolysis, decreased clot lysis time but did not significantly affect the maximum rate of lysis. Lastly, while venom exposure in the absence of tPA significantly prolonged the onset of coagulation and decreased the velocity of clot growth, venom exposure did not result in detectable fibrinolysis over the 3 h observation period. A. c. contortrix venom enhances tPA mediated fibrinolysis by degrading plasma coagulation kinetics. Intact A. c. contortrix venom does not possess sufficient fibrinolytic activity to cause fibrinolysis in human plasma at the concentration tested. PMID:26407681

  7. Cytosolic Proteins Contribute to Surface Plasminogen Recruitment of Neisseria meningitidis

    Knaust, Andreas; Weber, Martin V. R.; Hammerschmidt, Sven; Bergmann, Simone; Frosch, Matthias; Kurzai, Oliver

    2007-01-01

    Plasminogen recruitment is a common strategy of pathogenic bacteria and results in a broad-spectrum surface-associated protease activity. Neisseria meningitidis has previously been shown to bind plasminogen. In this study, we show by several complementary approaches that endolase, DnaK, and peroxiredoxin, which are usually intracellular proteins, can also be located in the outer membrane and act as plasminogen receptors. Internal binding motifs, rather than C-terminal lysine residues, are res...

  8. Preliminary experience with air transfer of patients for rescue endovascular therapy after failure of intravenous tissue plasminogen activator.

    Tsujimoto, Masanori; Yoshimura, Shinichi; Enomoto, Yukiko; Yamada, Noriaki; Matsumaru, Naoki; Kumada, Keisuke; Toyoda, Izumi; Ogura, Shinji; Iwama, Toru

    2015-01-01

    The present report describes our experience with air transfer of patients with acute ischemic stroke in whom intravenous tissue plasminogen activator (IV t-PA) failed for rescue endovascular therapy (EVT). Twenty-three consecutive patients in whom IV t-PA failed were transferred to our hospital for rescue EVT between February 2011 and April 2013. The amount of time required for transfer, distance, clinical outcomes, and complications were compared between patients transferred by ground (TG group; n = 17) and by air (TA group; n = 6). Computed tomography imaging on arrival revealed hemorrhagic transformation in 1 (5.9%) patient in the TG group, whereas none of the patients in the TA group developed any type of complication. The remaining 22 patients received rescue EVT. The elapsed time from the request call to arrival at our hospital did not significantly differ between the TG and TA groups (45.8 ± 4.9 min vs. 41.6 ± 2.3 min). However, the distance from the primary hospital to our institution was significantly longer for the TA group than for the TG group (38.8 ± 10.4 km vs. 13.5 ± 1.2 km, p = 0.001). The frequency of favorable outcomes (modified Rankin Scale 0-1 at 90 days after onset) in the TG and TA groups were 25.0% and 50.0%, respectively (p = 0.267). Air transfer for patients after IV t-PA failure allowed for more rapid delivery of patients over longer distances than ground transfer. PMID:25739430

  9. Prognostic value analysis of urokinase-type plasminogen activator receptor in oral squamous cell carcinoma: an immunohistochemical study

    Oral squamous cell carcinoma (OSCC) represents the most common oral malignancy. Despite recent advances in therapy, up to 50% of the cases have relapse and/or metastasis. There is therefore a strong need for the identification of new biological markers able to predict the clinical behaviour of these lesions in order to improve quality of life and overall survival. Among tumour progression biomarkers, already known for their involvement in other neoplasia, a crucial role is ascribed to the urokinase-type plasminogen activator receptor (uPAR), which plays a multiple role in extracellular proteolysis, cell migration and tissue remodelling not only as a receptor for the zymogen pro-uPA but also as a component for cell adhesion and as a chemoattractant. The purpose of this study was to gain information on the expression of uPAR in OSCC and to verify whether this molecule can have a role as a prognostic/predictive marker for this neoplasia. In a retrospective study, a cohort of 189 OSCC patients was investigated for uPAR expression and its cellular localization by immunohistochemistry. As standard controls, 8 normal oral mucosal tissues free of malignancy, obtained from patients with no evidence or history of oral cavity tumours, were similarly investigated. After grouping for uPAR expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G), tumour size, recurrence, TNM staging and overall survival rate. In our immunohistochemical study, 74 cases (39.1%) of OSCC showed a mostly cytoplasmic positivity for uPAR, whereas 115 were negative. uPAR expression correlated with tumour differentiation grade and prognosis: percentage of positive cases was the greatest in G3 (70.4%) and patients positives for uPAR expression had an expectation of life lower than those for uPAR negatives. The results obtained in this study suggest a role of uPAR as a potential biomarker useful to identify higher risk subgroups of OSCC patients

  10. Hyperacute thrombolysis with recombinant tissue plasminogen activator of acute ischemic stroke: Feasibility and effectivity from an Indian perspective

    Sharma S

    2008-01-01

    Full Text Available Given the constraints of resources, thrombolysis for acute ischemic stroke (AIS is under evaluation in developing countries like India, especially in areas such as western Utter Pradesh, where it is overly crowded and there is poor affordability. Aim: This study was done to evaluate recombinant tissue plasminogen activator r-tpa in acute ischemic stroke in hyper acute phase, in selected patients of western Utter Pradesh, in terms of feasibility and effectivity. Design: Open, non randomized study. Materials and Methods: Thirty two patients were classified using Trial of ORG 10172 in Acute Stroke treatment (TOAST criteria (large artery atherosclerotic = 8; cardio embolic = 6; small vessel occlusion = 14; other determined etiology = 2; undetermined etiology = 2. The mean time to reach the hospital was 2 h (1.15-3.0, the mean door to CT scan 20 min (10-40 and door to r-tpa injection was 30 min (24-68. The National Institute of Health Stroke Scale (NIHSS scores ranged from 11-22 (mean 15.5 +2.7. The dose of r-tpa administered was 0.9 mg/kg. Results: Twenty one patients (65.6% showed significant improvement on the NIHSS score, at 48 h (4 points. (Mean change = 10; range = 4-17. At one month, 25 (78% recorded improvement on the Barthel index (mean change = 45%. One developed frontal lobe hemorrhage and another developed recurrent stroke; one died of aspiration; and four showed no improvement. Modified Rankin score (m RS was administered at the end of three months to 28 patients (90%; however, the rest could not be directly observed. The average modified Rankin Score was 1.2 (0-2. Conclusions: Hyperacute thrombolysis was found feasible and effective in selected patients with AIS from western Utter Pradesh and who had poor affordability.

  11. Relationship of plasminogen activator inhibitor 1 gene 4G/5G polymorphisms to hypertension in Korean women

    Kyu-nam Kim; Kwang-min Kim; Bom-taeck Kim; Nam-seok Joo; Doo-yeoun Cho; Duck-joo Lee

    2012-01-01

    Background Hypertension (HTN) is a major determinant of various cardiovascular events.Plasma levels of plasminogen activator inhibitor 1 (PAl-1) modulate this risk.A deletion/insertion polymorphism within the PAl-1 loci (4G/4G,4G/5G,5G/5G) affects the expression of this gene.The present study investigated the association between PAl-1 loci polymorphisms and HTN in Korean women.@@Methods Korean women (n=1312) were enrolled in this study to evaluate the association between PAl-1 4G/5G gene polymorphisms and HTN as well as other metabolic risk factors.PAl-1 loci polymorphisms were investigated using polymerase chain reaction amplification and single-strand conformation polymorphism analysis.@@Results The three genotype groups differed with respect to systolic blood pressure (P=0.043),and diastolic blood pressure (P=0.009) but not with respect to age,body mass index,total cholesterol,low or high density lipoprotein cholesterol,triglycerides,or fasting blood glucose.Carriers of the PAl-1 4G allele had more hypertension significantly (PAl-1 4G/5G vs.PAl-1 5G/5G,P=0.032; PAl-1 4G/4G vs.PAl-1 5G/5G,P=0.034).When stratified according to PAl-1 4G/5G polymorphism,there was no significant difference in all metabolic parameters among PAl-1 genotype groups in patients with HTN as well as subjects with normal blood pressure.The estimated odds ratio of the 4G/4G genotype and 4G/5G for HTN was 1.7 (P=0.005),and 1.6 (P=0.015),respectively.@@Conclusion These findings might indicate that PAl-1 loci polymorphisms independently contribute to HTN and that gene-environmental interaction may be not associated in Korean women.

  12. Para-aminobenzamidine linked regenerated cellulose membranes for plasminogen activator purification: Effect of spacer arm length and ligand density

    Fasoli, Ezio; Reyes, Yiaslin Ruiz; Guzman, Osiris Martinez; Rosado, Alexandra; Cruz, Vivian Rodriguez; Borges, Amaris; Martinez, Edmarie; Bansal, Vibha

    2013-01-01

    Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; ii) the affinity ligand (pABA) density on membrane surface (5–25 nmoles per cm2). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi- component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14- atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmole of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in monocomponent system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11±1.0 nmoles per cm2) in mono- and multi- component systems for 7- as well as 14- atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use. PMID:23703544

  13. Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis.

    Nicolas Pelisch

    Full Text Available Multiple sclerosis (MS is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS. Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1 have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB. The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.

  14. Association of plasminogen activator inhibitor-1 and angiotensin converting enzyme polymorphisms with recurrent pregnancy loss in Iranian women

    Fatemeh Shakarami

    2015-10-01

    Full Text Available Background: Recurrent pregnancy loss (RPL defined by two or more failed pregnancies before 20 weeks of gestation. Several factors play a role in RPL including thrombophilic conditions which can be influenced by gene polymorphisms. Plasminogen activator inhibitor-1 (PAI-1 and angiotensin converting enzyme (ACE genes are closely related to fibrinolytic process, embryonic development and pregnancy success. Objective: The aim of this study was to investigate the relationship between RPL and common polymorphisms in ACE and PAI-1 genes. Materials and Methods: In this case control study, 100 women with recurrent abortions (at least two were selected as cases and 100 healthy women with two or more normal term deliveries without a history of abortion as controls. Total genomic DNA was isolated from blood leukocytes. The status of the PAI-1 4G/5G and ACE (D/I polymorphism was determined by PCR-RFLP. Results: Homozygosity for PAI-1 4G polymorphism was seen in 17 cases (17%, and 5 controls (5% (p=0.006 so patients with homozygote 4G mutation were significantly more prone to RPL in contrast to control group (OR: 4.63, % 95 CI: 1.55-13.84. In addition, 7 patients (7 %, and no one from the control group, were homozygote (I/I for ACE polymorphism (p=0.034, suggesting no significant associations between ACE D allele or DD genotype and RPL. Conclusion: Considering these results, because 4G/4G polymorphism for PAI-1 gene could be a thrombophilic variant leading to abortion, analysis of this mutation and other susceptibility factors are recommended in patients with RPL.

  15. Transcatheter Thrombolysis with High-Dose Bolus Tissue Plasminogen Activator in Iatrogenic Arterial Occlusion after Femoral Arterial Catheterization

    Purpose: To assess the efficacy of percutaneous local thrombolysis with high-dose bolus recombinant tissue plasminogen activator (rt-PA) in patients with acute limb ischemia due to arterial thrombosis after cardiac catheterization.Methods: We treated eight patients (7 men; mean age 56 years) with thrombotic occlusion of both the common femoral artery (CFA) and external iliac artery (EIA) in six patients and of the CFA only in two patients. Two 5 mg boluses of rt-PA were injected into the proximal clot through a 5 Fr end-hole catheter and subsequently two additional boluses of 5 mg rt-PA were given through a catheter with multiple side-holes. In case of a significant amount of residual thrombus, a continuous infusion of 2.5 mg/hr of rt-PA was started.Results: Successful lysis was achieved in all patients. The mean duration of lysis was 2 hr 41 min. The mean total amount of rt-PA delivered was 23.16 mg. In four patients unmasked flow-limited dissections confined to the CFA were managed by prolonged balloon dilatation, while in the remaining four patients with extension of the dissection to the external iliac artery one or two Easy Wallstents were implanted. There was prompt relief of lower limb ischemic symptoms and signs in all patients. Two groin hematomas were conservatively treated.Clinical and color Doppler flow imaging follow-up with a mean duration of 15 months, showed no reappearance of ischemic symptoms or development of restenosis in any of the patients. One patient died 6 months after thrombolysis.Conclusions: Transcatheter thrombolysis with high-dose bolus rt-PA is a safe and effective treatment inpatients with iatrogenic arterial occlusion after femoral catheterization. Underlying dissections should be treated by prolonged balloon dilatation but stent implantation is often required

  16. Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis.

    Pelisch, Nicolas; Dan, Takashi; Ichimura, Atsuhiko; Sekiguchi, Hiroki; Vaughan, Douglas E; van Ypersele de Strihou, Charles; Miyata, Toshio

    2015-01-01

    Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS. PMID:25915660

  17. The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.

    Adrian O'Hara

    Full Text Available The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2 and regenerating protein 1 (Reg1. Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.

  18. Dissecting the effect of RNA aptamer binding on the dynamics of plasminogen activator inhibitor 1 using hydrogen/deuterium exchange mass spectrometry

    Trelle, Morten B; Dupont, Daniel Miotto; Madsen, Jeppe Buur;

    2014-01-01

    , about their effects on protein conformation and dynamics. We have employed hydrogen/deuterium exchange (HDX) mass spectrometry to study the effect of RNA aptamers on the structural flexibility of the serpin plasminogen activator inhibitor-1 (PAI-1). The aptamers have characteristic effects...... on the biochemical properties of PAI-1. In particular, they are potent inhibitors of the structural transition of PAI-1 from the active state to the inactive, so-called latent state. This transition is one of the largest conformational changes of a folded protein domain without covalent modification. Binding...

  19. Activity of Ginkgo biloba Extract and Quercetin on Thrombomodulin Expression and Tissue-type Plasminogen Activator Secretion by Human Umbilical Vein Endothelial Cells

    WEN-JUN LAN; XIAO-XIANG ZHENG

    2006-01-01

    In order to investigate the pharmacological properties of Ginkgo biloba extract (GBE) on improving blood circulation, the regulating action of GBE and quercetin (a main flavonoid ingredient in GBE) on thrombomodulin (TM)expression and tissue-type plasminogen activator (t-PA) secretion was studied. Methods Using flow cytometer and gel image system respectively, we evaluated the TM expression and the t-PA secretion by human umbilical vein endothelial cells (HUVECs) in vitro. Results The increase of TM expression on HUVECs surface was induced by GBE rather than quercetin in a dose- and time-dependent manner. Both GBE and quercetin increased the t-PA release significantly.Conclusion The effect of GBE on improving blood circulation may be partly attributed to its promoting TM expression and t-PA secretion by endothelial cells, and quercetin participated in the effect of GBE on t-PA secretion. However, the action of GBE on increasing TM expression needs further study.

  20. Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells.

    Stewart, M D; Stewart, D M; Johnson, G A; Vyhlidal, C A; Burghardt, R C; Safe, S H; Yu-Lee, L Y; Bazer, F W; Spencer, T E

    2001-01-01

    Interferon-tau (IFNtau), a type I IFN produced by sheep conceptus trophectoderm, is the signal for maternal recognition of pregnancy. Although it is clear that IFNtau suppresses transcription of the estrogen receptor alpha and oxytocin receptor genes and induces expression of various IFN-stimulated genes within the endometrial epithelium, little is known of the signal transduction pathway activated by the hormone. This study determined the effects of IFNtau on signal transducer and activator of transcription (STAT) activation, expression, DNA binding, and transcriptional activation using an ovine endometrial epithelial cell line. IFNtau induced persistent tyrosine phosphorylation and nuclear translocation of STAT1 and -2 (10 min to 48 h), but transient phosphorylation and nuclear translocation of STAT3, -5a/b, and -6 (10 to gene factor-3 and STAT1 homodimers formed and bound an IFN-stimulated response element (ISRE) and gamma-activated sequence (GAS) element, respectively. IFNtau increased transcription of GAS-driven promoters at 3 h, but suppressed their activity at 24 h. In contrast, the activity of an ISRE-driven promoter was increased at 3 and 24 h. These results indicate that IFNtau activates multiple STATs and has differential effects on ISRE- and GAS-driven gene transcription. PMID:11145571

  1. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography

    Baoyu Zhao; Sonu Gandhi; Cai Yuan; Zhipu Luo; Rui Li; Henrik Gårdsvoll; Valentina de Lorenzi; Nicolai Sidenius; Mingdong Huang; Michael Ploug

    2015-01-01

    The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two ...

  2. Caseinolytic activity of fruit extract from Opuntia ficus-indica on bovine, caprine, and ovine Sodium Caseinates

    Pintado, Ana I.; Macedo, Angela C.; Teixeira, Grimaneza; Pais, Salomé; Clemente, Alda; Malcata, F. Xavier

    2001-01-01

    The rates and extents of hydrolysis of RS- and â-caseins from bovine, caprine, and ovine sodium caseinates produced by an enzymatic extract of the fruit of Opuntia ficusindica, (L.) Miller were evaluated and compared with those produced by a commercial animal rennet. A mechanistic model based on a pseudo-first-order enzymatic reaction, in the presence of first-order deactivation of the enzyme, was postulated and successfully fitted to the experimental data. The animal rennet ex...

  3. THE INCREASE IN PLASMINOGEN ACTIV ATOR INHIBITOR TYPE 1 EXPRESSION BY STIMULATION OF ACTIVATORS FOR PEROXISOME PROLIFERATOR ACTIVA TED RECEPTORS IN HUMAN ENDOTHELIAL CELLS

    叶平; 胡晓晖; 赵亚力

    2002-01-01

    Objective.To investigate the effect of peroxisome proliferator activated receptors (PPARs) activators on plasminogen activator inhibitor type 1 (PAI 1) expression in human umbilical vein endothelial cells and the possible mechanism.Methods.Human umbilical vein endothelial cells (HUVECs) were obtained from normal fetus,and cultured conventionally.Then the HUVECs were exposed to test agents (linolenic acid,linoleic acid,oleic acid,stearic acid and prostaglandin J2 respectively) in varying concentrations with fresh media.RT- PCR and ELISA were applied to determine the expression of PPARs and PAI 1 in HUVECs.Results.PPAR α,PPAR δ and PPAR γ mRNA were detected by using RT PCR in HUVECs.Treatment of HUVECs with PPARα and PPAR γ activators- - linolenic acid,linoleic acid,oleic acid and prostaglandin J2 respectively,but not with stearic acid could augment PAI I mRNA expression and protein secretion in a concentration dependent manner.However,the mRNA expressions of 3 subclasses of PPAR with their activators in HUVECs were not changed compared with controls.Conclusion.HUVECs express PPARs.PPARs activators may increase PAI 1 expression in ECs,but the underlying mechanism remains unclear.Although PPARs expression was not enhanced after stimulated by their activators in ECs,the role of functionally active PPARs in regulating PAI 1 expression in ECs needs to be further investigated by using transient gene transfection assay.

  4. E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene

    Koziczak, M; Müller, H; Helin, K;

    2001-01-01

    -sensitive retinoblastoma protein (pRB), a shift to a permissive temperature induced PAI-1 mRNA expression. In U2OS cells stably expressing an E2F1-estrogen receptor chimeric protein that could be activated by tamoxifen, PAI-1 gene transcription was markedly reduced by tamoxifen even in the presence of cycloheximide. These...

  5. Influence of bone marrow fat embolism on coagulation activation in an ovine model of vertebroplasty

    Krebs, J; Ferguson, S. J.; Hoerstrup, S P; Goss, B G; Haeberli, A; Aebli, N

    2008-01-01

    BACKGROUND: Intraoperative cardiovascular deterioration as a result of pulmonary embolization of bone marrow fat is a potentially serious complication during vertebroplasty. The release of fatty material and thromboplastin from the bone marrow cavity during vertebroplasty may activate the coagulation cascade resulting in thrombogenesis, and pharmacological prophylaxis may therefore prevent cardiovascular complications. Thus, the effects of bone marrow fat embolism on coagulation activation du...

  6. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R;

    2007-01-01

    reorganization in mature osteoclasts. INTRODUCTION: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express uPAR and...... to mechanical tests. UPAR KO calvaria osteoblasts were characterized by proliferation assays, RT-PCR for important proteins secreted during differentiation, and immunoblot for activator protein 1 (AP-1) family members. In vitro osteoclast formation was tested with uPAR KO bone marrow monocytes in the...... osteoblasts showed a proliferative advantage with no difference in apoptosis, higher matrix mineralization, and earlier appearance of alkaline phosphatase (ALP). Surface RANKL expression at different stages of differentiation was not altered. AP-1 components, such as JunB and Fra-1, were upregulated in u...

  7. Tissue Plasminogen Activator Binding to Superparamagnetic Iron Oxide Nanoparticle—Covalent Versus Adsorptive Approach

    Friedrich, Ralf P.; Zaloga, Jan; Schreiber, Eveline; Tóth, Ildikó Y.; Tombácz, Etelka; Lyer, Stefan; Alexiou, Christoph

    2016-06-01

    Functionalized superparamagnetic iron oxide nanoparticles are frequently used to develop vehicles for drug delivery, hyperthermia, and photodynamic therapy and as tools used for magnetic separation and purification of proteins or for biomolecular imaging. Depending on the application, there are various possible covalent and non-covalent approaches for the functionalization of particles, each of them shows different advantages and disadvantages for drug release and activity at the desired location.

  8. Plasma levels of soluble urokinase-type plasminogen activator receptor associate with the clinical severity of acute Puumala hantavirus infection.

    Tuula K Outinen

    Full Text Available OBJECTIVES: Urokinase-type plasminogen activator receptor is a multifunctional glycoprotein, the expression of which is increased during inflammation. It is known to bind to β3-integrins, which are elementary for the cellular entry of hantaviruses. Plasma soluble form of the receptor (suPAR levels were evaluated as a predictor of severe Puumala hantavirus (PUUV infection and as a possible factor involved in the pathogenesis of the disease. DESIGN: A single-centre prospective cohort study. SUBJECTS AND METHODS: Plasma suPAR levels were measured twice during the acute phase and once during the convalescence in 97 patients with serologically confirmed acute PUUV infection using a commercial enzyme-linked immunosorbent assay (ELISA. RESULTS: The plasma suPAR levels were significantly higher during the acute phase compared to the control values after the hospitalization (median 8.7 ng/ml, range 4.0-18.2 ng/ml vs. median 4.7 ng/ml, range 2.4-12.2 ng/ml, P<0.001. The maximum suPAR levels correlated with several variables reflecting the severity of the disease. There was a positive correlation with maximum leukocyte count (r = 0.475, p<0.001, maximum plasma creatinine concentration (r = 0.378, p<0.001, change in weight during the hospitalization (r = 0.406, p<0.001 and the length of hospitalization (r = 0.325, p = 0.001, and an inverse correlation with minimum platelet count (r = -0.325, p = 0.001 and minimum hematocrit (r = -0.369, p<0.001. CONCLUSION: Plasma suPAR values are markedly increased during acute PUUV infection and associate with the severity of the disease. The overexpression of suPAR possibly activates β3-integrin in PUUV infection, and thus might be involved in the pathogenesis of the disease.

  9. Plasma Levels of Soluble Urokinase-Type Plasminogen Activator Receptor Associate with the Clinical Severity of Acute Puumala Hantavirus Infection

    Outinen, Tuula K.; Tervo, Laura; Mäkelä, Satu; Huttunen, Reetta; Mäenpää, Niina; Huhtala, Heini; Vaheri, Antti; Mustonen, Jukka; Aittoniemi, Janne

    2013-01-01

    Objectives Urokinase-type plasminogen activator receptor is a multifunctional glycoprotein, the expression of which is increased during inflammation. It is known to bind to β3-integrins, which are elementary for the cellular entry of hantaviruses. Plasma soluble form of the receptor (suPAR) levels were evaluated as a predictor of severe Puumala hantavirus (PUUV) infection and as a possible factor involved in the pathogenesis of the disease. Design A single-centre prospective cohort study. Subjects and Methods Plasma suPAR levels were measured twice during the acute phase and once during the convalescence in 97 patients with serologically confirmed acute PUUV infection using a commercial enzyme-linked immunosorbent assay (ELISA). Results The plasma suPAR levels were significantly higher during the acute phase compared to the control values after the hospitalization (median 8.7 ng/ml, range 4.0–18.2 ng/ml vs. median 4.7 ng/ml, range 2.4–12.2 ng/ml, P<0.001). The maximum suPAR levels correlated with several variables reflecting the severity of the disease. There was a positive correlation with maximum leukocyte count (r = 0.475, p<0.001), maximum plasma creatinine concentration (r = 0.378, p<0.001), change in weight during the hospitalization (r = 0.406, p<0.001) and the length of hospitalization (r = 0.325, p = 0.001), and an inverse correlation with minimum platelet count (r = −0.325, p = 0.001) and minimum hematocrit (r = −0.369, p<0.001). Conclusion Plasma suPAR values are markedly increased during acute PUUV infection and associate with the severity of the disease. The overexpression of suPAR possibly activates β3-integrin in PUUV infection, and thus might be involved in the pathogenesis of the disease. PMID:23990945

  10. Plasminogen is a critical regulator of cutaneous wound healing.

    Sulniute, Rima; Shen, Yue; Guo, Yong-Zhi; Fallah, Mahsa; Ahlskog, Nina; Ny, Lina; Rakhimova, Olena; Broden, Jessica; Boija, Hege; Moghaddam, Aliyeh; Li, Jinan; Wilczynska, Malgorzata; Ny, Tor

    2016-05-01

    Wound healing is a complicated biological process that consist of partially overlapping inflammatory, proliferation and tissue remodelling phases. A successful wound healing depends on a proper activation and subsequent termination of the inflammatory phase. The failure to terminate the inflammation halts the completion of wound healing and is a known reason for formation of chronic wounds. Previous studies have shown that wound closure is delayed in plasminogen-deficient mice, and a role for plasminogen in dissection of extracellular matrix was suggested. However, our finding that plasminogen is transported to the wound by inflammatory cells early during the healing process, where it potentiates inflammation, indicates that plasminogen may also have other roles in the wound healing process. Here we report that plasminogen-deficient mice have extensive fibrin and neutrophil depositions in the wounded area long after re-epithelialisation, indicating inefficient debridement and chronic inflammation. Delayed formation of granulation tissue suggests that fibroblast function is impaired in the absence of plasminogen. Therefore, in addition to its role in the activation of inflammation, plasminogen is also crucial for subsequent steps, including resolution of inflammation and activation of the proliferation phase. Importantly, supplementation of plasminogen-deficient mice with human plasminogen leads to a restored healing process that is comparable to that in wild-type mice. Besides of being an activator of the inflammatory phase during wound healing, plasminogen is also required for the subsequent termination of inflammation. Based on these results, we propose that plasminogen may be an important future therapeutic agent for wound treatment. PMID:26791370

  11. Trappin ovine molecule (TOM), the ovine ortholog of elafin, is an acute phase reactant in the lung.

    Brown, Thomas I; Mistry, Rohit; Collie, D David; Tate, Steven; Sallenave, Jean-Michel

    2004-09-16

    As large animal models continue to play an important role in translating lung-directed therapeutic strategies from laboratory animals to humans, there is an increasing interest in the analysis of endogenous regulators of inflammation at both a genomic and a therapeutic level. To this end, we have sought to characterize the ovine ortholog of elafin, an important regulator of inflammation in humans. We have isolated both the elafin cDNA and gene, which have a similar structure to other species' orthologs. Interestingly, we have isolated two alleles for ovine elafin, which contain a very high number of transglutamination repeats, thought to be important in binding elafin to the interstitium. The mainly mucosal mRNA distribution for ovine elafin suggests that ovine elafin may, like its human ortholog, have functions in innate immunity. This is supported by analysis of elafin and the related protein secretory leukocyte protease inhibitor (SLPI) in ovine bronchoalveolar fluid in response to locally administered lipopolysaccharide and confirmation of them acting as "alarm" antiproteases. We have also cloned the ovine elafin cDNA into an adenoviral vector and have demonstrated correct processing of the secreted protein as well as biological activity. Overexpression of ovine elafin in a lung-derived epithelial cell line has a protective effect against the enzymes human neutrophil and porcine pancreatic elastase. The identification of the ovine elafin gene and its translated protein are important in developing practical strategies aimed at regulating inflammation in the large mammalian lung. PMID:15292488

  12. Plasminogen activator inhibitor 1: Mechanisms of its synergistic regulation by growth factors

    Song, Xiaoling [Iowa State Univ., Ames, IA (United States)

    2010-01-01

    My research is on the synergistic regulation of PAI-1 by EGF and TGF-β. The mechanism of synergistic regulation of PAI-1 by EGF and TGF-β are addressed. Methods are described for effective identification of RNA accessible sites for antisense oligodexoxynucleotides (ODNs) and siRNA. In this study effective AS-ODN sequences for both Lcn2 and Bcl2 were identified by in vitro tiled microarray studies. Our results suggest that hybridization of ODN arrays to a target mRNA under physiological conditions might be used as a rapid and reliable in vitro method to accurately identify targets on mRNA molecules for effective antisense and potential siRNA activity in vivo.

  13. Intravenous thrombolysis with recombinant tissue plasminogen activator for ischemic stroke patients over 80 years old: the Fukuoka Stroke Registry.

    Ryu Matsuo

    Full Text Available The benefit of intravenous recombinant tissue plasminogen activator (rt-PA therapy for very old patients with acute ischemic stroke remains unclear. The aim of this study was to elucidate the efficacy and safety of intravenous rt-PA therapy for patients over 80 years old.Of 13,521 stroke patients registered in the Fukuoka Stroke Registry in Japan from June 1999 to February 2013, 953 ischemic stroke patients who were over 80 years old, hospitalized within 3 h of onset, and not treated with endovascular therapy were included in this study. Among them, 153 patients were treated with intravenous rt-PA (0.6 mg/kg. For propensity score (PS-matched case-control analysis, 148 patients treated with rt-PA and 148 PS-matched patients without rt-PA therapy were selected by 1:1 matching with propensity for using rt-PA. Clinical outcomes were neurological improvement, good functional outcome at discharge, in-hospital mortality, and hemorrhagic complications (any intracranial hemorrhage [ICH], symptomatic ICH, and gastrointestinal bleeding.In the full cohort of 953 patients, rt-PA use was associated positively with neurological improvement and good functional outcome, and negatively with in-hospital mortality after adjustment for multiple confounding factors. In PS-matched case-control analysis, patients treated with rt-PA were still at lower risk for unfavorable clinical outcomes than non-treated patients (neurological improvement, odds ratio 2.67, 95% confidence interval 1.61-4.40; good functional outcome, odds ratio 2.23, 95% confidence interval 1.16-4.29; in-hospital mortality, odds ratio 0.30, 95% confidence interval 0.13-0.65. There was no significant association between rt-PA use and risk of hemorrhagic complications in the full and PS-matched cohorts.Intravenous rt-PA therapy was associated with improved clinical outcomes without significant increase in risk of hemorrhagic complications in very old patients (aged>80 years with acute ischemic stroke.

  14. Plasminogen activator inhibitor-1 4G/5G polymorphism and retinopathy risk in type 2 diabetes: a meta-analysis

    Zhang Tengyue

    2013-01-01

    Full Text Available Abstract Background Mounting evidence has suggested that plasminogen activator inhibitor-1 (PAI-1 is a candidate for increased risk of diabetic retinopathy. Studies have reported that insertion/deletion polymorphism in the PAI-1 gene may influence the risk of this disease. To comprehensively address this issue, we performed a meta-analysis to evaluate the association of PAI-1 4G/5G polymorphism with diabetic retinopathy in type 2 diabetes. Methods Data were retrieved in a systematic manner and analyzed using Review Manager and STATA Statistical Software. Crude odds ratios (ORs with 95% confidence intervals (CIs were used to assess the strength of associations. Results Nine studies with 1, 217 cases and 1, 459 controls were included. Allelic and genotypic comparisons between cases and controls were evaluated. Overall analysis suggests a marginal association of the 4G/5G polymorphism with diabetic retinopathy (for 4G versus 5G: OR 1.13, 95%CI 1.01 to 1.26; for 4G/4G versus 5G/5G: OR 1.30, 95%CI 1.04 to 1.64; for 4G/4G versus 5G/5G + 4G/5G: OR 1.26, 95%CI 1.05 to 1.52. In subgroup analysis by ethnicity, we found an association among the Caucasian population (for 4G versus 5G: OR 1.14, 95% CI 1.00 to 1.30; for 4G/4G versus 5G/5G: OR 1.33, 95%CI 1.02 to 1.74; for 4G/4G versus 5G/5G + 4G/5G: OR 1.41, 95%CI 1.13 to 1.77. When stratified by the average duration of diabetes, patients with diabetes histories longer than 10 years have an elevated susceptibility to diabetic retinopathy than those with shorter histories (for 4G/4G versus 5G/5G: OR 1.47, 95%CI 1.08 to 2.00. We also detected a higher risk in hospital-based studies (for 4G/4G versus 5G/5G+4G/5G: OR 1.27, 95%CI 1.02 to 1.57. Conclusions The present meta-analysis suggested that 4G/5G polymorphism in the PAI-1 gene potentially increased the risk of diabetic retinopathy in type 2 diabetes and showed a discrepancy in different ethnicities. A higher susceptibility in patients with longer duration of

  15. Role of Urokinase-type Plasminogen Activator in the Precontact Sperm-egg Communication and Fertility of Mice in vitro

    2005-01-01

    Objective To explore the role of urokinase-type plasminogen activator(uPA) in precontact sperm-egg communication and fertility of mice in vitro.Methods Firstly, sperm chemotaxis (SC) induced by uPA was assayed by measuring the sperm densities in capillaries with a descending gradient or no gradient of uPA respectively. Secondly, the role of uPAR that exists in sperm plasma membrane in SC was studied by examining the change of sperm density in capillary after incubating spermatozoa with anti-uPAR antibody. Thirdly, SC induced by eggs, which had been treated with uPA, PAI-1 and anti-uPAR beforehand respectively, was assayed to study the role of uPA in PSEC. Lastly, the fertilization capability of spermatozoa treated with uPA was examined by counting the number of fertilized eggs.Results 1)The density of spermatozoa that migrated down the gradient of uPA into the capillary was significantly lower than that into the capillary containing no-gradient uPA. 2) When uPAR of spermatozoa was inhibited by anti-uPAR antibody, the density of spermatozoa that migrated into the capillary with ascending gradient of uPA decreased correspondingly. 3) The density of spermatozoa attracted by eggs, which were treated with uPA beforehand, increased significantly than that of attracted by non-treated eggs. On the contrary, the sperm density decreased correspondingly when the egg was treated with PAI-1. 4) The number of fertilized eggs increased significantly after the spermatozoa used here was treated with uPA beforehand.Conclusion uPA could induce SC of mice sperm in vitro through the uPAR on its membrane, enhance the capability of egg inducing SC, and promote spermatozoa to fertilize eggs. Thus, uPA may act as an attractant in PSEC, increase the chance encounter of spermatozoa and eggs, therefore, enhance the fertility success correspondingly.This study, in some degree, provides an evidence that uPA may be used as a new medicine and diagnostic reagent for male infertility.

  16. Collagenase and tissue plasminogen activator production in developing rat calvariae: normal progression despite fetal exposure to microgravity

    Davis, B. A.; Sipe, B.; Gershan, L. A.; Fiacco, G. J.; Lorenz, T. C.; Jeffrey, J. J.; Partridge, N. C.

    1998-01-01

    Exposure to zero gravity has been shown to cause a decrease in bone formation. This implicates osteoblasts as the gravity-sensing cell in bone. Osteoblasts also are known to produce neutral proteinases, including collagenase and tissue plasminogen activator (tPA), which are thought to be important in bone development and remodeling. The present study investigated the effects of zero gravity on development of calvariae and their expression of collagenase and tPA. After in utero exposure to zero gravity for 9 days on the NASA STS-70 space shuttle mission, the calvariae of rat pups were examined by immunohistochemistry for the presence and location of these two proteinases. The ages of the pups were from gestational day 20 (G20) to postnatal (PN) day 35. Both collagenase and tPA were found to be present at all ages examined, with the greatest amount of both proteinases present in the PN14 rats. At later ages, high amounts were maintained for tPA but collagenase decreased substantially between ages PN21 to PN35. The location of collagenase was found to be associated with bone-lining cells, osteoblasts, osteocytes, and in the matrix along cement lines. In contrast, tPA was associated with endothelial cells lining the blood vessels entering bone. The presence and developmental expression of these two proteinases appeared to be unaffected by the exposure to zero gravity. The calvarial thickness of the pups was also examined; again the exposure to zero gravity showed little to no effect on the growth of the calvariae. Notably, from G20 to PN14, calvarial thickness increased dramatically, reaching a plateau after this age. It was apparent that elevated collagenase expression correlated with rapid bone growth in the period from G20 to PN14. To conclude, collagenase and tPA are present during the development of rat calvariae. Despite being produced by the same cell in vitro, i.e., the osteoblast, they are located in distinctly different places in bone in vivo. Their presence

  17. Adrenergic Activation of Melatonin Secretion in Ovine Pineal Explants in Short-Term Superfusion Culture Occurs via Protein Synthesis Independent and Dependent Phenomena

    Bogdan Lewczuk

    2014-01-01

    Full Text Available The ovine pineal is generally considered as an interesting model for the study on adrenergic regulation of melatonin secretion due to some functional similarities with this gland in the human. The present investigations, performed in the superfusion culture of pineal explants, demonstrated that the norepinephrine-induced elevation of melatonin secretion in ovine pinealocytes comprised of two subsequent periods: a rapid increase phase and a slow increase phase. The first one included the quick rise in release of N-acetylserotonin and melatonin, occurring parallel to elevation of NE concentration in the medium surrounding explants. This rapid increase phase was not affected by inhibition of translation. The second, slow increase phase began after NE level had reached the maximum concentration in the culture medium and lasted about two hours. It was completely abolished by the treatment with translation inhibitors. The obtained results showed for the first time that the regulation of N-acetylserotonin synthesis in pinealocytes of some species like the sheep involves the on/off mechanism, which is completely independent of protein synthesis and works very fast. They provided strong evidence pointing to the need of revision of the current opinion that arylalkylamines N-acetyltransferase activity in pinealocytes is controlled exclusively by changes in enzyme abundance.

  18. In vivo kinetics of [sup 99m]Tc labeled recombinant tissue plasminogen activator in rabbits

    Itoh, Kazuo; Ieko, Masahiro; Hiraguchi, Etsuro; Kitayama, Hide; Tsukamoto, Eriko (Hokkaido Univ., Sapporo (Japan). School of Medicine)

    1994-08-01

    Our previous studies demonstrated that [sup 99m]Tc labeled recombinant tissue plasminiogen activator (rt-PA) retained high affinity with fibrin in vitro but showed unexpectedly low uptake in fresh thrombi in vivo. The present study was performed to determine the in vivo kinetics of radiolabeled t-PA in the rabbit. Sequential images and blood samples after the intravenous administration of [sup 99m]Tc labeled rt-PA in thrombus-bearing rabbits were taken. The radioactivity and immunological level of t-PA and PAI-1 in the solution eluted to each fraction by gel permeation chromatography were measured by means of a well scintillation counter and enzyme-linked immunosorbent assay (ELISA). Most of the radioactivity was eluted in the fraction (Fr.7) of larger molecular weight than that (Fr.9) of intact t-PA. The level of intact rt-PA was increased with a regimen involving the preadministration of cold rt-PA which was followed by the administration of hot rt-PA. The level of PAI-1 in plasma showed an increased rebound 15 minutes after the intravenous injection. These results suggest two possible reasons why rt-PA retains high affinity with fibrin in vitro, once radiolabeled, but was ineffective in delineating fresh thrombi with a gamma camera: (1) some plasma components such as PAI-1 combine with circulating radiolabeled rt-PA and form a larger molecule immediately and/or (2) radiolabeled rt-PA is modulated as a consequence of the radiolabeling and forms a larger molecule than intact rt-PA. (author).

  19. Cholinergic signal activated renin angiotensin system associated with cardiovascular changes in the ovine fetus

    Geng, Chunsong; Mao, Caiping; Wu, Lei; Cheng, Yu; Liu, Rulu; Chen, Bingxin; Chen, Ling; Zhang, Lubo; Xu, Zhice

    2010-01-01

    Aim: Cholinergic regulation is important in the control of cardiovascular and endocrine responses. The mechanisms behind cardiovascular responses induced by cholinergic activation are explored by studying hormonal systems, including renin-angiotensin and vasopressin (VP). Results: In chronically prepared fetal sheep, intravenous infusion of the cholinergic agonist carbachol increased fetal systolic, diastolic, and mean arterial pressure accompanied with bradycardia at near-term. Although int...

  20. Time profile of oxidative stress and neutrophil activation in ovine acute lung injury and sepsis

    Lange, Matthias; Szabo, Csaba; Traber, Daniel L.; Horvath, Eszter; Hamahata, Atsumori; Nakano, Yoshimitsu; Traber, Lillian D.; Cox, Robert A.; Schmalstieg, Frank C.; Herndon, David N.; Enkhbaatar, Perenlei

    2012-01-01

    The formation of oxidative stress in the lung and activation of neutrophils are major determinants in the development of respiratory failure following acute lung injury (ALI) and sepsis. However, the time changes of these pathogenic factors have not been sufficiently described. Twenty-four chronically instrumented sheep were subjected to cotton smoke inhalation injury and instillation of live Pseudomonas aeruginosa into both lungs. The sheep and were euthanized at 4, 8, 12, 18, and 24 hours p...

  1. Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2

    Berberine, a compound isolated from medicinal herbs, has been reported with many pharmacological effects related to anti-cancer and anti-inflammation capabilities. In this study, we observed that berberine exerted a dose- and time-dependent inhibitory effect on the motility and invasion ability of a highly metastatic A549 cells under non-cytotoxic concentrations. In cancer cell migration and invasion process, matrix-degrading proteinases are required. A549 cell treated with berberine at various concentrations showed reduced ECM proteinases including matrix metalloproteinase-2 (MMP2) and urokinase-plasminogen activator (u-PA) by gelatin and casein zymography analysis. The inhibitory effect is likely to be at the transcriptional level, since the reduction in the transcripts levels was corresponding to the proteins. Moreover, berberine also exerted its action via regulating tissue inhibitor of metalloproteinase-2 (TIMP-2) and urokinase-plasminogen activator inhibitor (PAI). The upstream mediators of the effect involved c-jun, c-fos and NF-κB, as evidenced by reduced phosphorylation of the proteins. These findings suggest that berberine possesses an anti-metastatic effect in non-small lung cancer cell and may, therefore, be helpful in clinical treatment

  2. Plasminogen activator inhibitor type 1 gene is located at region q21. 3-q22 of chromosome 7 and genetically linked with cystic fibrosis

    Klinger, K.W.; Winqvist, R.; Riccio, A.; Andreasen, P.A.; Sartorio, R.; Nielsen, L.S.; Stuart, N.; Stanislovitis, P.; Watkins, P.; Douglas, R.

    1987-12-01

    The regional chromosomal location of the human gene for plasminogen activator inhibitor type 1 (PAI1) was determined by three independent methods of gene mapping. PAI1 was localized first to 7cen-q32 and then to 7q21.3-q22 by Southern blot hybridization analysis of a panel of human and mouse somatic cell hybrids with a PAI1 cDNA probe and in situ hybridization, respectively. The authors frequent HindIII restriction fragment length polymorphism (RFLP) of the PAI1 gene with an information content of 0.369. In family studies using this polymorphism, genetic linkage was found between PAI1 and the loci for erythropoietin (EPO), paraoxonase (PON), the met protooncogene (MET), and cystic fibrosis (CF), all previously assigned to the middle part of the long arm of chromosome 7. The linkage with EPO was closest with an estimated genetic distance of 3 centimorgans, whereas that to CF was 20 centimorgans. A three-point genetic linkage analysis and data from previous studies showed that the most likely order of these loci is EPO, PAI1, PON, (MET, CF), with PAI1 being located centromeric to CF. The PAI1 RFLP may prove to be valuable in ordering genetic markers in the CF-linkage group and may also be valuable in genetic analysis of plasminogen activation-related diseases, such as certain thromboembolic disorders and cancer.

  3. Angiotensin II induces secretion of plasminogen activator inhibitor 1 and a tissue metalloprotease inhibitor-related protein from rat brain astrocytes

    The present study investigates angiotensin (Ang) II effects on secretory protein synthesis in brain astrocytes cultured from neonatal and 21-day-old rats. Ang II-induced changes in the de novo synthesis of [35S]methionine-labeled secretory proteins were visualized using two-dimensional NaDodSO4/PAGE. Astrocytes from 21-day-old rat brain possess specific high-affinity receptors for Ang II. These cells express two Ang II-induced secretory proteins with Mr 55,000 (AISP-55K) and Mr 30,000 (AISP-30K), which were time- and dose-dependent (EC50, 1 nM). [Sar1, Ile8]Ang II (where Sar is sarcosine) inhibited Ang II-induced secretion of AISP-55K but not AISP-30K. N-terminal amino acid sequencing indicates that AISP-55K is identical to rat plasminogen activator inhibitor 1, whereas AISP-30K exhibits 72-81% identity to three closely related proteins: human tissue inhibitor of metalloproteases, a rat phorbol ester-induced protein, and the murine growth-responsive protein 16C8. Immunofluorescent staining with rat plasminogen activator inhibitor 1 antibody was induced in the majority of cells in culture after Ang II treatment of astrocytes from 21-day-old rat brains. Absence of this response to Ang II in astrocytes from neonatal rat brain provides evidence that this action of Ang II on astrocytes is developmentally regulated

  4. Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor by Hypoxia-Inducible Factor 1 Is Crucial for Invasion of Pancreatic and Liver Cancer

    Peter Büchler

    2009-02-01

    Full Text Available Angioinvasion is critical for metastasis with urokinase-type plasminogen activator receptor (uPAR and tumor hypoxia-activated hypoxia-inducible factor 1 (HIF-1 as key players. Transcriptional control of uPAR expression by HIF has never been reported. The aim of the present study, therefore, was to test whether tumor hypoxia-induced HIF expression may be linked to transcriptional activation of uPAR and dependent angioinvasion. We used human pancreatic cancer cells and a model of parental and derived HIF-1β-deficient mouse liver cancer cell lines and performed Northern blot analysis, nuclear runoff assays, electrophoretic mobility shift assay, polymerase chain reaction-generated deletion mutants, luciferase assays, Matrigel invasion assays, and in vivo angioinvasion assays in the chorioallantoic membrane of fertilized chicken eggs. Urokinase-type plasminogen activator receptor promoter analysis resulted in four putative HIF binding sites. Hypoxia strongly induced de novo transcription of uPAR mRNA. With sequential deletion mutants of the uPAR promoter, it was possible to identify one HIF binding site causing a nearly 200-fold increase in luciferase activity. Hypoxia enhanced the number of invading tumor cells in vitro and in vivo. In contrast, HIF-1β-deficient cells failed to upregulate uPAR expression, to activate luciferase activity, and to invade on hypoxia. Taken together, we show for the first time that uPAR is under transcriptional control of HIF and that this is important for hypoxia-induced metastasis.

  5. Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina

    Highlights: ► Staurosporine mediates stimulation of RGC differentiation in vitro cultured retinal neuroblasts. ► Staurosporine mediates uPA activation during RGC differentiation in vitro. ► Inhibition of uPA blocks the staurosporine mediated RGC differentiation both in vitro and in ovo. ► Thus, uPA may play a role in the staurosporine-mediated stimulation of RGC differentiation. -- Abstract: Here, we investigated whether staurosporine-mediated urokinase-type plasminogen activator (uPA) activation is involved in retinal ganglion cell (RGC) differentiation. Retinal cells were isolated from developing chick retinas at embryonic day 6 (E6). Relatively few control cells grown in serum-free medium started to form processes by 12 h. In contrast, staurosporine-treated cells had processes within 3 h, and processes were evident at 8 h. Immunofluorescence staining showed that Tuj-1-positive cells with shorter neurites could be detected in control cultures at 18 h, whereas numerous Tuj-1 positive ganglion cells with longer neuritic extensions were seen in staurosporine-treated cultures. BrdU-positive proliferating cells were more numerous in control cultures than in staurosporine-treated cultures, and the BrdU staining was not detected in post-mitotic Tuj-1 positive ganglion cells. Western blotting of cell lysates showed that staurosporine induced high levels of the active form of uPA. The staurosporine-induced uPA signal was localized predominantly in the soma, neurites and axons of Tuj-1-positive ganglion cells. Amiloride, an inhibitor of uPA, markedly reduced staurosporine-induced Tuj-1 staining, neurite length, neurite number, and uPA staining versus controls. In developing retinas in ovo, amiloride administration remarkably reduced the staurosporine-induced uPA staining and RGC differentiation. Taken together, our in vitro and in vivo data collectively indicate that uPA plays a role in the staurosporine-mediated stimulation of RGC differentiation.

  6. A suppressive effect of prostaglandin E2 on the expression of SERPINE1/plasminogen activator inhibitor-1 in human articular chondrocytes: An in vitro pilot study

    Kayo Masuko

    2009-04-01

    Full Text Available Kayo Masuko1, Minako Murata2, Naoya Suematsu1, Kazuki Okamoto1, Kazuo Yudoh2, Hiroyuki Shimizu3, Moroe Beppu3, Hiroshi Nakamura4, Tomohiro Kato11Department of Biochemistry; 2Department of Frontier Medicine, Institute of Medical Science; 3Department of Orthopedic Surgery, St. Marianna University School of Medicine, Kawasaki-shi, Kanagawa, Japan; 4Department of Joint Disease and Rheumatism, Nippon Medical School, Bunkyo-ku, Tokyo, JapanAbstract: Prostaglandin E2 (PGE2 is expressed in articular joints with inflammatory arthropathy and may exert catabolic effects leading to cartilage degradation. As we observed in a preliminary experiment that PGE2 suppressed the expression of SERPINE1/plasminogen activator inhibitor (PAI-1 mRNA in chondrocytes, we focused on the effect of PGE2 on PAI-1 in a panel of cultured chondrocytes obtained from osteoarthritic patients. Specifically, articular cartilage specimens were obtained from patients with osteoarthritis who underwent joint surgery. Isolated chondrocytes were cultured in vitro as a monolayer and stimulated with PGE2. Stimulated cells and culture supernatants were analyzed using Western blotting and enzyme-linked immunosorbent assay. The results confirmed that the in vitro PGE2 stimulation suppressed the expression of PAI-1 in the tested chondrocyte samples. The inhibitory effect was partly abrogated by an antagonist of EP4 receptor of PGE2, but not by an EP2 antagonist. Although PGE2 induced activations of mitogen-activated protein kinases (MAPK, blocking of the MAPK did not abrogate the suppressive effect of PGE2, implying a distinct signaling pathway. In summary, prostaglandin is suggested to modulate the plasminogen system in chondrocytes. Further elucidation of the interaction might open a new avenue to understand the degradative process of cartilage.Keywords: chondrocyte, prostaglandin, PGE2, PAI-1

  7. Soluble Urokinase Plasminogen Activator Receptor Level Is an Independent Predictor of the Presence and Severity of Coronary Artery Disease and of Future Adverse Events

    Eapen, Danny J; Manocha, Pankaj; Ghasemzedah, Nima;

    2014-01-01

    INTRODUCTION: Soluble urokinase plasminogen activator receptor (suPAR) is an emerging inflammatory and immune biomarker. Whether suPAR level predicts the presence and the severity of coronary artery disease (CAD), and of incident death and myocardial infarction (MI) in subjects with suspected CAD......, is unknown. METHODS AND RESULTS: We measured plasma suPAR levels in 3367 subjects (67% with CAD) recruited in the Emory Cardiovascular Biobank and followed them for adverse cardiovascular (CV) outcomes of death and MI over a mean 2.1±1.1 years. Presence of angiographic CAD (≥50% stenosis in ≥1...... coronary artery) and its severity were quantitated using the Gensini score. Cox's proportional hazard survival and discrimination analyses were performed with models adjusted for established CV risk factors and C-reactive protein levels. Elevated suPAR levels were independently associated with the presence...

  8. Soluble urokinase plasminogen activator receptor (suPAR) is a novel, independent predictive marker of myocardial infarction in HIV-1-infected patients

    Rasmussen, Line Jee Hartmann; Knudsen, A; Katzenstein, T L;

    2016-01-01

    OBJECTIVES: Patients infected with HIV are at increased risk of myocardial infarction (MI). Increased plasma levels of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) have been associated with increased risk of cardiovascular diseases (CVD), including MI in the......, gender, duration of antiretroviral therapy (ART), smoking and no known CVD. suPAR was measured in the four plasma samples available for each patient at different time-points; 1, Before initiation of ART; 2, 3 months after initiation of ART; 3, 1 year before the case's MI; and 4, The last sample available...... before the case's MI. RESULTS: In unadjusted conditional regression analysis, higher levels of suPAR were associated with a significant increase in risk of MI at all time-points. Patients in the third and fourth suPAR quartiles had a three- to 10-fold higher risk of MI compared to patients in the lowest...

  9. Plasma soluble urokinase-type plasminogen activator receptor level is independently associated with coronary microvascular function in patients with non-obstructive coronary artery disease

    Mekonnen, Girum; Corban, Michel T; Hung, Olivia Y;

    2015-01-01

    BACKGROUND: Soluble urokinase-type plasminogen activator receptor (suPAR) is a novel biomarker released from leukocytes and endothelial cells that has been associated with atherosclerotic cardiovascular disease. We hypothesized that plasma suPAR level is an independent predictor of coronary...... microvascular function. METHODS: Coronary blood flow velocity and plasma suPAR levels were evaluated in patients with non-obstructive coronary artery disease. Coronary flow reserve (CFR) was calculated as the ratio of hyperemic to basal average peak blood flow velocity and coronary microvascular dysfunction was...... defined as CFR ≤ 2.0 in the setting of a fractional flow reserve value of ≥0.75. Plasma suPAR levels were measured using ELISA technique. The association between suPAR and CFR was investigated using univariate and multivariate regression analyses. RESULTS: In 66 patients, 47% were men, 26% had diabetes...

  10. Distinctive binding modes and inhibitory mechanisms of two peptidic inhibitors of urokinase-type plasminogen activator with isomeric P1 residues

    Jiang, Longguang; Zhao, Baoyu; Xu, Peng;

    2015-01-01

    Two isomeric piperidine derivatives (meta and para isomers) were used as arginine mimics in the P1 position of a cyclic peptidic inhibitor (CPAYSRYLDC) of urokinase-type plasminogen activator. The two resulting cyclic peptides showed vastly different affinities (~70 fold) to the target enzyme. X......-ray crystal structure analysis showed that the two P1 residues were inserted into the S1 specificity pocket in indistinguishable manners. However, the rest of the peptides bound in entirely different ways on the surface of the enzyme, and the two peptides have different conformations, despite the highly...... similar sequence. These results demonstrate how the subtle difference in P1 residue can dictate the exosite interactions and the potencies of peptidic inhibitors, and highlight the importance of P1 residue for protease inhibition. This study provides important information for the development of peptidic...

  11. Tissue-type plasminogen activator and C-reactive protein in acute coronary heart disease. A nested case-control study

    Gram, J; Bladbjerg, E-M; Møller, L; Sjøl, A; Jespersen, J

    2000-01-01

    258 controls. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Subjects with ischaemic heart disease identified in 1991 by the Danish National Hospital Register. Protein concentration of C-reactive protein (CRP) and tissue-type plasminogen activator (t-PA) were measured with ELISA methods in stored serum......OBJECTIVES: To study the importance of inflammation and fibrinolysis for evolution of ischaemic heart disease in a cohort of initially healthy subjects. DESIGN: Nested case-control study. Follow-up periods 7-15 years. SUBJECTS: Included in the study were 133 cases with coronary heart disease and...... independent predictor of coronary heart disease, whilst such a significance disappeared for CRP. With the use of ROC curves we determined that AUC for t-PA was 0.62, and for CRP 0.59, indicating that none of these two analytes has a high prognostic power in predicting future coronary events in an initially...

  12. TNFalpha-mediated plasminogen activation on neutrophils is involved in the high plasmin activity in mammary secretion of drying-off cows.

    Chou, Wen K; Yu, Ting C; Chen, Shuen E; Peh, Ho C; Liu, Wen B; Chen, Ming T; Nagahata, Hajime; Chang, Chai J

    2009-11-01

    Interactions between inflammatory cytokines and plasminogen (Pg) activation system on immune cells are yet to be established. In previous studies we reported a somatic cell-associated elevation of proteolytic activity in mammary secretion of drying-off goats and cows. The purposes of the present study were to examine the role of TNF-alpha in polymorphonuclear neutrophil (PMN)-associated Pg activation, and the significance of this activation pathway for overall plasmin (Pm) activity in mammary secretion of drying-off cows. Results of experiments in vitro showed that the spontaneous Pg activation observed on fresh preparations of bovine blood PMN was completely blocked by anti bovine TNF-alpha antibody, and was further up-regulated by exogenous bovine TNF-alpha. Monitoring the parameters of mammary secretion of drying-off cows revealed that both somatic cell counts and differential PMN ratio was significantly elevated at weeks 1, 2 and 3 of milk stasis. Nevertheless, specific activity of soluble Pm in mammary secretion increased and the level of 17-kDa TNF-alpha decreased immediately following milk stasis. Iimmunoblotting revealed that although both 26-kDa pro-TNF-alpha and 17-kDa TNF-alpha were consistently present in somatic cells of mammary secretion collected at weeks 0, 1, 2 and 3 of milk stasis, only 26-kDa pro-TNF-alpha was present in somatic cells of milk during lactation. In-vitro assay indicated that cell-free mammary secretion of drying-off cows exerted no Pg activation bioactivity towards bovine blood PMN. Altogether, the current study suggests the existence of an active TNF-alpha-Pg-Pm autocrine/paracrine loop on the massively infiltrated PMN inside udders of drying-off cows, which involves extensive binding and internalization of 17-kDa TNF-alpha on PMN and consequently activation of Pg, resulting in high Pm activity and low 17-kDa TNF-alpha level in mammary secretion. These coordinated mechanisms may play a role in the defence of drying-off mammary

  13. Transconjunctival sutureless vitrectomy with tissue plasminogen activator, gas and intravitreal bevacizumab in the management of predominantly hemorrhagic age-related macular degeneration

    Luis Arias

    2010-02-01

    Full Text Available Luis Arias1,2, Jordi Monés11Institut de la Màcula i de la Retina, Centro Médico Teknon, Barcelona; 2Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, BarcelonaPurpose: To determine the efficacy and safety of treating predominantly hemorrhagic age-related macular degeneration (AMD with transconjunctival sutureless vitrectomy (TSV, tissue plasminogen activator (tPA, sulphur hexafluoride (SF6, and intravitreal bevacizumab.Methods: Retrospective study, consecutive case series. Patients with acute hemorrhagic AMD treated with 25- or 23-gauge TSV, subretinal or intravitreal tPA, fluid-air-SF6 exchange and intravitreal injection of bevacizumab. All operations were performed within the first 5 days after the start of symptoms, which consisted of visual acuity (VA loss and central scotoma.Results: Fifteen eyes from 15 patients were included. The patients’ mean age was 79.6 years, and the mean follow-up was 11.8 months. Five patients (33% were receiving oral anticoagulant treatment. At baseline, the mean VA (logMAR values was 1.5 (20/640 Snellen equivalent. At the last follow-up visit, the mean VA was 1.1 (20/250 (P < 0.0001; paired t-test. The submacular hemorrhage was successfully displaced in all the cases. Complications consisted of three cases of vitreous hemorrhage and a tear or the retinal pigment epithelium. Twelve cases (80% did not require further treatment during the follow-up period.Conclusion: A surgical approach with 25- or 23-gauge TSV, tPA, SF6 and intravitreal bevacizumab is an efficacious and safe procedure in patients with hemorrhagic AMD. Early treatment is advisable for obtaining the optimal outcome.Keywords: Hemorrhagic age-related macular degeneration, tissue plasminogen activator, intravitreal bevacizumab; transconjunctival sutureless vitrectomy

  14. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR by surface plasmon resonance and X-ray crystallography

    Baoyu Zhao

    2015-12-01

    Full Text Available The urokinase-type plasminogen activator receptor (uPAR or CD87 is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]. uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU.This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA and the provisional matrix protein vitronectin (Vn (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2–4]. The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5] and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1 recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2 developing monoclonal antibodies with unique specificities using this protein as antigen; (3 mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]; and finally (4 solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively].

  15. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography.

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; Gårdsvoll, Henrik; de Lorenzi, Valentina; Sidenius, Nicolai; Huang, Mingdong; Ploug, Michael

    2015-12-01

    The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2-4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]); and finally (4) solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively]. PMID:26504891

  16. Recombinant Tissue Plasminogen Activator Induces Neurological Side Effects Independent on Thrombolysis in Mechanical Animal Models of Focal Cerebral Infarction: A Systematic Review and Meta-Analysis.

    Mei-Xue Dong

    Full Text Available Recombinant tissue plasminogen activator (rtPA is the only effective drug approved by US FDA to treat ischemic stroke, and it contains pleiotropic effects besides thrombolysis. We performed a meta-analysis to clarify effect of tissue plasminogen activator (tPA on cerebral infarction besides its thrombolysis property in mechanical animal stroke.Relevant studies were identified by two reviewers after searching online databases, including Pubmed, Embase, and ScienceDirect, from 1979 to 2016. We identified 6, 65, 17, 12, 16, 12 and 13 comparisons reporting effect of endogenous tPA on infarction volume and effects of rtPA on infarction volume, blood-brain barrier, brain edema, intracerebral hemorrhage, neurological function and mortality rate in all 47 included studies. Standardized mean differences for continuous measures and risk ratio for dichotomous measures were calculated to assess the effects of endogenous tPA and rtPA on cerebral infarction in animals. The quality of included studies was assessed using the Stroke Therapy Academic Industry Roundtable score. Subgroup analysis, meta-regression and sensitivity analysis were performed to explore sources of heterogeneity. Funnel plot, Trim and Fill method and Egger's test were obtained to detect publication bias.We found that both endogenous tPA and rtPA had not enlarged infarction volume, or deteriorated neurological function. However, rtPA would disrupt blood-brain barrier, aggravate brain edema, induce intracerebral hemorrhage and increase mortality rate.This meta-analysis reveals rtPA can lead to neurological side effects besides thrombolysis in mechanical animal stroke, which may account for clinical exacerbation for stroke patients that do not achieve vascular recanalization with rtPA.

  17. Influences on the activities of tissue- type plasminogen activator of mouse brain in asphyxia%窒息对鼠脑分泌组织型纤溶酶原激活物的影响

    徐剑文; 王玮; 康仲涵; 赵小贞; 张更

    2001-01-01

    Objective To observe the changes of the activity of tissue-type plasminogen activator(TPA) after asphyxia. Methods Asphyxia was induced in mouse pups by performing a‘ delayed cesarean section' . The experiment was designed for a control group and 4 asphyctic groups to detect the activity of TPA. Results TPA activity of brain increased after asphyxia (P<0.01). Conclusion TPA increased after asphyxia might be able to attack the basement membrane of microvessels, then opened the blood-brain barrier and induced neuronal damage.

  18. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  19. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H2O2) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H2O2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine

  20. The increase of plasminogen activator inhibitor activity is associated with graft occlusion in patients undergoing aorto-coronary bypass surgery

    Rifon, J. (José); Paramo, J. A.; Panizo, C. (Carlos); R. Montes; Rocha, E

    1997-01-01

    Early graft occlusion is a common complication in patients undergoing aorto-coronary bypass surgery. Both mechanical and haemostatic factors play a role in the pathogenesis of thrombotic occlusion. Several studies have demonstrated a relationship between fibrinolytic activity and venous or arterial thrombosis. We undertook this study to evaluate the possible contribution of the fibrinolytic system to postoperative occlusion in patients undergoing aorto-coronary bypass graft (CABG). A venous o...

  1. Urokinase plasminogen activator inhibits HIV virion release from macrophage-differentiated chronically infected cells via activation of RhoA and PKCε.

    Francesca Graziano

    Full Text Available BACKGROUND: HIV replication in mononuclear phagocytes is a multi-step process regulated by viral and cellular proteins with the peculiar feature of virion budding and accumulation in intra-cytoplasmic vesicles. Interaction of urokinase-type plasminogen activator (uPA with its cell surface receptor (uPAR has been shown to favor virion accumulation in such sub-cellular compartment in primary monocyte-derived macrophages and chronically infected promonocytic U1 cells differentiated into macrophage-like cells by stimulation with phorbol myristate acetate (PMA. By adopting this latter model system, we have here investigated which intracellular signaling pathways were triggered by uPA/uPAR interaction leading the redirection of virion accumulation in intra-cytoplasmic vesicles. RESULTS: uPA induced activation of RhoA, PKCδ and PKCε in PMA-differentiated U1 cells. In the same conditions, RhoA, PKCδ and PKCε modulated uPA-induced cell adhesion and polarization, whereas only RhoA and PKCε were also responsible for the redirection of virions in intracellular vesicles. Distribution of G and F actin revealed that uPA reorganized the cytoskeleton in both adherent and polarized cells. The role of G and F actin isoforms was unveiled by the use of cytochalasin D, a cell-permeable fungal toxin that prevents F actin polymerization. Receptor-independent cytoskeleton remodeling by Cytochalasin D resulted in cell adhesion, polarization and intracellular accumulation of HIV virions similar to the effects gained with uPA. CONCLUSIONS: These findings illustrate the potential contribution of the uPA/uPAR system in the generation and/or maintenance of intra-cytoplasmic vesicles that actively accumulate virions, thus sustaining the presence of HIV reservoirs of macrophage origin. In addition, our observations also provide evidences that pathways controlling cytoskeleton remodeling and activation of PKCε bear relevance for the design of new antiviral strategies aimed

  2. Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina

    Kim, Yeoun-Hee [Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Chang, Yongmin [Department of Molecular Medicine, Kyungpook National University College of Medicine, Kyungpook National University, 200 Dongduk-Ro Jung-Gu, Daegu 700-714 (Korea, Republic of); Jung, Jae-Chang, E-mail: jcjung@knu.ac.kr [Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701 (Korea, Republic of)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Staurosporine mediates stimulation of RGC differentiation in vitro cultured retinal neuroblasts. Black-Right-Pointing-Pointer Staurosporine mediates uPA activation during RGC differentiation in vitro. Black-Right-Pointing-Pointer Inhibition of uPA blocks the staurosporine mediated RGC differentiation both in vitro and in ovo. Black-Right-Pointing-Pointer Thus, uPA may play a role in the staurosporine-mediated stimulation of RGC differentiation. -- Abstract: Here, we investigated whether staurosporine-mediated urokinase-type plasminogen activator (uPA) activation is involved in retinal ganglion cell (RGC) differentiation. Retinal cells were isolated from developing chick retinas at embryonic day 6 (E6). Relatively few control cells grown in serum-free medium started to form processes by 12 h. In contrast, staurosporine-treated cells had processes within 3 h, and processes were evident at 8 h. Immunofluorescence staining showed that Tuj-1-positive cells with shorter neurites could be detected in control cultures at 18 h, whereas numerous Tuj-1 positive ganglion cells with longer neuritic extensions were seen in staurosporine-treated cultures. BrdU-positive proliferating cells were more numerous in control cultures than in staurosporine-treated cultures, and the BrdU staining was not detected in post-mitotic Tuj-1 positive ganglion cells. Western blotting of cell lysates showed that staurosporine induced high levels of the active form of uPA. The staurosporine-induced uPA signal was localized predominantly in the soma, neurites and axons of Tuj-1-positive ganglion cells. Amiloride, an inhibitor of uPA, markedly reduced staurosporine-induced Tuj-1 staining, neurite length, neurite number, and uPA staining versus controls. In developing retinas in ovo, amiloride administration remarkably reduced the staurosporine-induced uPA staining and RGC differentiation. Taken together, our in vitro and in vivo data collectively indicate that

  3. Characterization of the ovine ortholog of secretory leukoprotease inhibitor.

    Brown, Thomas I; Mistry, Rohit; Gray, Robert; Imrie, Margaret; Collie, David D; Sallenave, Jean-Michel

    2005-08-01

    There is great interest in the use of the sheep as a model for the investigation of inflammation in the lung. The serine antiproteases secretory leukoprotease inhibitor (SLPI) and elafin are important "alarm antiproteases" in the lung and have potentially important roles in the innate immune response. SLPI was first characterized in man and subsequently in murine, porcine, and rat tissues. Here we present the first data concerning the gene and cDNA sequence encoding for the ovine ortholog of SLPI, a protein of 132 amino acids with 66% sequence identity at the amino acid level with human SLPI. A 24-amino-acid signal sequence signifies that, like the other mammalian orthologs, ovine SLPI is a secreted protein. Tissue distribution of expression is demonstrated by reverse transcription polymerase chain reaction (RT-PCR) and shows features similar to SLPI expression in other mammals, specifically at mucosal surfaces such as the upper respiratory and intestinal tracts, and also the skin, liver, and kidney. This distribution lends credence to SLPI having important roles in innate immunity. We have also cloned the ovine SLPI cDNA into an expression vector and expressed the ovine SLPI protein in vitro. This has enabled us to demonstrate that ovine SLPI is correctly processed (Western blot analysis and SELDI-TOF mass spectrometry analysis) and has biological antihuman neutrophil elastase activity. In summary, the ovine ortholog of SLPI shows similarities to other members of the SLPI family and has all the features of a modulator of innate immunity. PMID:16180144

  4. Amelioration of glomerulosclerosis with all-trans retinoic acid is linked to decreased plasminogen activator inhibitor-1 and α-smooth muscle actin

    Xia LIU; Lei L(U); Bei-bei TAO; Ai-ling ZHOU; Yi-chun ZHU

    2011-01-01

    Aim:To examine the effects of all-trans retinoic acid (atRA) on renal morphology and function as well as on renal plasminogen activator inhibitor-1 (PAI-1) expression and plasmin activity in rats with 5/6 nephrectomy.Methods:Adult male Sprague Dawley rats were given 5/6 nephrectomy or sham operation. Renal function was measured 2 weeks later. The nephrectomized rats were assigned to groups matched for proteinuria and treated with vehicle or atRA (5 or 10 mg/kg by gastric gavage once daily) for the next 12 weeks. Rats with sham operation were treated with vehicle. At the end of the treatments,kidneys were collected for histological examination, Western blot analysis, and enzymatic activity measurements.Results:The 5/6 nephrectomy promoted hypertension, renal dysfunction, and glomerulosclerosis. These changes were significantly reduced in the atRA-treated group. The expressions of PAI-1 and α-smooth muscle actin (α-SMA) were significantly increased in the vehicle-treated nephrectomized rats. Treatment with atRA significantly reduced the expressions of PAI-1 and α-SMA. However, piasmin activity remained unchanged following atRA treatment.Conclusion:Treatment with atRA ameliorates glomerulosclerosis and improves renal function in rats with 5/6 nephrectomy. This is associated with a decrease in PAI-1 and α-SMA, but not with a change in plasmin activity.

  5. Apolipoprotein(a) Inhibits In Vitro Tube Formation in Endothelial Cells: Identification of Roles for Kringle V and the Plasminogen Activation System

    Lei Liu; Boffa, Michael B.; Koschinsky, Marlys L.

    2013-01-01

    Elevated plasma concentrations of lipoprotein(a) are associated with increased risk for atherothrombotic diseases. Apolipoprotein(a), the unique glycoprotein component of lipoprotein(a), is characterized by the presence of multiple kringle domains, and shares a high degree of sequence homology with the serine protease zymogen plasminogen. It has been shown that angiostatin, a proteolytic fragment of plasminogen containing kringles 1-4, can effectively inhibit angiogenesis. Moreover, proteolyt...

  6. Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

    The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 μg/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 μg/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1β (83%) or TNF-α (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity

  7. Distribution of trichloroacetic acid precipitated radioactivity in rabbits after intravenous injection of 125I-labelled single-chain human recombinant urokinase-type plasminogen activator

    Objective: To study tissue distribution profiles of trichloroacetic acid (TCA) precipitated radioactivity in rabbits following intravenous injection of 125I-labelled single-chain human recombinant urokinase-type plasminogen activator (125I-rh-sc-uPA). Methods: 125I-rh-su-uPA was prepared by Iodogen method and was purified by Saphacryl HR S-200. The radio-chemical purity was measured by reverse phase high performance liquid chromatography (RP-HPLC). Biological activity of labelled protein was measured by thrombolytic activity on fibrin plate. Results: The identification of 125I-rh-sc-uPA revealed that it was a 94.4% radio-chemically purified thrombolytic active protein. The AUC of radioactivity gradients in tissues and other specimens were as follows: kidney>urine>plasma>liver>spleen>adrenal gland>bone marrow>heart>urinary bladder>lungs>gonad>small intestinal wall>fat>lymph node>gall bladder>thymus gland>intestinal content>muscle>brain>feces in colon. Conclusion: The highest TCA precipitated radioactivity was found in plasma following I.V. of 125I-rh-u-PA, which might be favorable for the drug action. The main excretion route was urinary system

  8. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

    Gately, Stephen; Twardowski, Przemyslaw; Stack, M. Sharon; Cundiff, Deborah L.; Grella, Davida; Castellino, Francis J.; Enghild, Jan; Kwaan, Hau C.; Lee, Francis; Kramer, Robert A.; Volpert, Olga; Bouck, Noel; Soff, Gerald A.

    1997-01-01

    Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [u...

  9. Inhibition of urokinase plasminogen activator “uPA” activity alters ethanol consumption and conditioned place preference in mice

    Al Maamari E

    2014-09-01

    Full Text Available Elyazia Al Maamari,* Mouza Al Ameri, Shamma Al Mansouri, Amine Bahi*Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates*These authors contributed equally to this workAbstract: Urokinase plasminogen activator, uPA, is a serine protease implicated in addiction to drugs of abuse. Using its specific inhibitor, B428, we and others have characterized the role of uPA in the rewarding properties of psychostimulants, including cocaine and amphetamine, but none have examined the role of uPA in ethanol use disorders. Therefore, in the current study, we extended our observations to the role of uPA in ethanol consumption and ethanol-induced conditioned place preference. The general aim of the present series of experiments was to investigate the effects of the administration of the B428 on voluntary alcohol intake and ethanol conditioned reward. A two-bottle choice, unlimited-access paradigm was used to compare ethanol intake between vehicle- and 3, 10, and 30 mg/kg B428-administered mice. For this purpose, the mice were presented with an ethanol solution (2.5%–20% and water, at each concentration for 4 days, and their consumption was measured daily. Consumption of saccharin and quinine solutions was also measured. Systemic administration of B428 dose-dependently decreased ethanol intake and preference. Additionally, B428 mice did not differ from vehicle mice in their intake of graded solutions of tastants, suggesting that the uPA inhibition did not alter taste function. Also, ethanol metabolism was not affected following B428 injection. More importantly, 1.5 g/kg ethanol-induced conditioned place preference acquisition was blocked following B428 administration. Taken together, our results are the first to implicate uPA inhibition in the regulation of ethanol consumption and preference, and suggest that uPA may be considered as a possible therapeutic drug target for alcoholism and

  10. Evaluation of Serum Fibrinogen, Plasminogen, α2-Anti-Plasmin, and Plasminogen Activator Inhibitor Levels (PAI and Their Correlation with Presence of Retinopathy in Patients with Type 1 DM

    Sefika Burcak Polat

    2014-01-01

    Full Text Available Background. Diabetic retinopathy (DR is the leading cause of blindness in the world. Retinopathy can still progress despite optimal metabolic control. The aim of the study was to determine whether different degrees of DR (proliferative or nonproliferative were associated with abnormally modulated hemostatic parameters in patients with T1DM. Method. 52 T1DM patients and 40 healthy controls were enrolled in the study. Patients were subdivided into three categories. Group I was defined as those without retinopathy, group II with NPRP, and group III with PRP. We compared these subgroups with each other and the control group (Group IV according to the serum fibrinogen, plasminogen, alpha2-anti-plasmin (α2-anti-plasmin, and PAI. Results. We detected that PAI-1, serum fibrinogen, and plasminogen levels were similar between the diabetic and control groups (P=0.209, P=0.224, and P=0.244, resp., whereas α2-anti-plasmin was higher in Groups I, II, and III compared to the control group (P<0.01, P<0.05, and P<0.001, resp.. There was a positive correlation between serum α2-anti-plasmin and HbA1c levels (r=0,268, P=0.031. Conclusion. To our knowledge there is scarce data in the literature about α2-anti-plasmin levels in type 1 diabetes. A positive correlation between α2-anti-plasmin with HbA1c suggests that fibrinolytic markers may improve with disease regulation and better glycemic control.

  11. Cross-talk between human mast cells and bronchial epithelial cells in plasminogen activator inhibitor-1 production via transforming growth factor-β1.

    Cho, Seong H; Lee, Sun H; Kato, Atsushi; Takabayashi, Tetsuji; Kulka, Marianna; Shin, Soon C; Schleimer, Robert P

    2015-01-01

    Previous reports suggest that plasminogen activator inhibitor-1 (PAI-1) promotes airway remodeling and that human and mouse mast cells (MCs) are an important source of PAI-1. In the present study we investigated MC-epithelial cell (EC) interactions in the production of PAI-1. We stimulated the human MC line LAD2 with IgE-receptor cross-linking and collected the supernatants. We incubated the human bronchial EC line BEAS-2B with the LAD2 supernatants and measured the level of PAI-1. When the supernatants from IgE-stimulated LAD2 were added to BEAS-2B, there was a significant enhancement of PAI-1 production by BEAS-2B. When we treated the MC supernatants with a transforming growth factor (TGF)-β1 neutralizing antibody, the MC-derived induction of PAI-1 from BEAS-2B was completely abrogated. Although TGF-β1 mRNA was constitutively expressed in resting LAD2, it was not highly induced by IgE-mediated stimulation. Nonetheless, active TGF-β1 protein was significantly increased in LAD2 after IgE-mediated stimulation. Active TGF-β1 produced by primary cultured human MCs was significantly reduced in the presence of a chymase inhibitor, suggesting a role of MC chymase as an activator of latent TGF-β1. This study indicates that stimulation of human MCs by IgE receptor cross-linking triggers activation of TGF-β1, at least in part via chymase, which in turn induces the production of PAI-1 by bronchial ECs. Our data suggest that human MCs may play an important role in airway remodeling in asthma as a direct source of PAI-1 and by activating bronchial ECs to produce further PAI-1 via a TGF-β1-mediated activation pathway. PMID:24987792

  12. The diagnostic value of soluble urokinase plasminogen activator receptor compared with C-reactive protein and procalcitonin in children with febrile neutropenia.

    Sirinoglu, Melis; Soysal, Ahmet; Karaaslan, Ayşe; Kepenekli Kadayifci, Eda; Cinel, Ismail; Koç, Ahmet; Tokuç, Gülnur; Yaman, Ali; Haklar, Goncagül; Şirikçi, Önder; Turan, Serap; Altınkanat Gelmez, Gülşen; Söyletir, Güner; Bakır, Mustafa

    2016-04-01

    The aim of the present study was to determine the diagnostic value of soluble urokinase plasminogen activator receptor (suPAR) in pediatric patients with febrile neutropenia. A prospective case-control study was performed. Patients included 29 children with febrile neutropenia (FN) and 27 control subjects without any infection or immunosuppressive condition. Blood samples were obtained on the day of admission and on the 4th to 7th days of the hospital stay. The median (minimum-maximum) serum levels of suPAR obtained on the first day of the admission were 2.08 (0.93-9.42) and 2.22 (1.08-5.13) ng/mL for the FN group and the control group, respectively. The median serum levels of suPAR in the FN and control groups were not significantly different (P = .053). The mean serum suPAR level was significantly higher in nonsurvivors than in survivors in the FN group (P < .05). In the FN group, the area under the receiver operating characteristics curve (AUCROC) for suPAR was 0.546, but no optimum cutoff value, sensitivity, specificity, negative predictive value (NPV), or positive predictive value (PPV) was obtained. We conclude that suPAR is not useful as a diagnostic biomarker in children with febrile neutropenia; however, persistent high serum suPAR level may predict mortality in FN in children. PMID:27057782

  13. Camptothecin induces urokinase-type plasminogen activator gene-expression in human RC-K8 malignant lymphoma and H69 small cell lung cancer cells.

    Shibakura M

    2002-10-01

    Full Text Available We previously reported that anthracyclines, which could generate reactive oxygen species (ROS, could induce the urokinase-type plasminogen activator (uPA gene expression in human RC-K8 malignant lymphoma cells and in H69 small cell lung cancer (SCLC cells. In screening other uPA-inducible anti-cancer agents, we found that camptothecin (CPT and its derivative, SN38, could induce uPA in RC-K8 and H69 cells. CPT and SN38, which are also used for the treatment of lymphoma and SCLC, significantly increased the uPA accumulation in the conditioned media of both cells in a dose-dependent manner. The maximum induction of uPA mRNA levels was observed 24 h after stimulation. Pretreatment with pyrrolidine dithiocarbamate (PDTC, an anti-oxidant, inhibited the CPT-induced uPA mRNA expression. Thus, CPT induces uPA through gene expression, and, therefore, CPT may influence the tumor-cell biology by up-regulating the uPA/plasmin system.

  14. Interrelations between blood-brain barrier permeability and matrix metalloproteinases are differently affected by tissue plasminogen activator and hyperoxia in a rat model of embolic stroke

    Michalski Dominik

    2012-01-01

    Full Text Available Abstract Background In ischemic stroke, blood-brain barrier (BBB regulations, typically involving matrix metalloproteinases (MMPs and inhibitors (TIMPs as mediators, became interesting since tissue plasminogen activator (tPA-related BBB breakdown with risk of secondary hemorrhage was considered to involve these mediators too. Despite high clinical relevance, detailed interactions are purely understood. After a pilot study addressing hyperoxia as potential neuroprotective co-treatment to tPA, we analyzed interrelations between BBB permeability (BBB-P, MMPs and TIMPs. Findings Rats underwent embolic middle cerebral artery occlusion (eMCAO and treatment with normobaric (NBO or hyperbaric oxygen (HBO, tPA, tPA+HBO, or no treatment. BBB-P was assessed by intravenously applied FITC-albumin at 4 or 24 hours. MMP-2/-9 and TIMP-1/-2 serum levels were determined at 5 or 25 hours. Time point-corrected partial correlations were used to explore interrelations of BBB-P in ischemic regions (extra-/intravasal FITC-albumin ratio and related serum markers. BBB-P correlated positively with MMP-2 and MMP-9 in controls, whereas hyperoxia led to an inverse association, most pronounced for HBO/MMP-9 (r = -0.606; P Conclusions HBO was found to reverse the positively directed interrelation of BBB-P and MMPs after eMCAO, but this effect failed to sustain in the expected amount when HBO and tPA were given simultaneously.

  15. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation

  16. Extracellular Matrix Biomarker, Fibulin-1, Is Closely Related to NT-proBNP and Soluble Urokinase Plasminogen Activator Receptor in Patients with Aortic Valve Stenosis (The SEAS Study)

    Kruger, Ruan; Rasmussen, Lars M; Argraves, William S;

    2014-01-01

    BACKGROUND: Fibulin-1, a circulating extracellular matrix glycoprotein, has been associated with arterial disease and elevated N-terminal prohormone B-type natriuretic peptide (NT-proBNP) in diabetes. Soluble urokinase plasminogen activator receptor (suPAR), a marker of inflammation, has been...... associated with subclinical atherosclerosis. Therefore, we aimed to explore the interplay between these biomarkers and mild to moderate aortic valve stenosis (AS). METHODS: In 374 patients with mild to moderate AS, we investigated the relationship of fibulin-1 with NT-proBNP, levels of suPAR and the degree...... of AS at baseline and after one and four years of treatment with Simvastatin 40 mg and Ezetimibe 10 mg or placebo. RESULTS: During treatment, fibulin-1 became more closely associated with NT-proBNP (βyear0 = 0.10, p = 0.08, βyear1 = 0.16, p = 0.005, βyear4 = 0.22, p

  17. TISSUE INHIBITOR OF METALLOPROTEINASE 1, MATRIX METALLOPROTEINASE 9, ALPHA-1 ANTITRYPSIN, METALLOTHIONEIN AND UROKINASE TYPE PLASMINOGEN ACTIVATOR RECEPTOR IN SKIN BIOPSIES FROM PATIENTS AFFECTED BY AUTOIMMUNE BLISTERING DISEASES

    Ana Maria Abreu Velez

    2013-07-01

    Full Text Available Introduction: Proteinases and proteinase inhibitors have been described to play a role in autoimmune skin blistering diseases. We studied skin lesional biopsies from patients affected by several autoimmune skin blistering diseases for proteinases and proteinase inhibitors. Methods: We utilized immunohistochemistry to evaluate biopsies for alpha-1-antitrypsin, human matrix metalloproteinase 9 (MMP9, human tissue inhibitor of metalloproteinases 1 (TIMP-1, metallothionein and urokinase type plasminogen activator receptor (uPAR. We tested 30 patients affected by endemic pemphigus, 30 controls from the endemic area, and 15 normal controls. We also tested 30 biopsies from patients with bullous pemphigoid (BP, 20 with pemphigus vulgaris (PV, 8 with pemphigus foliaceus, and 14 with dermatitis herpetiformis (DH. Results: Contrary to findings in the current literature, most autoimmune skin blistering disease biopsies were negative for uPAR and MMP9. Only some chronic patients with El Bagre-EPF were positive to MMP9 in the dermis, in proximity to telocytes. TIMP-1 and metallothionein were positive in half of the biopsies from BP patients at the basement membrane of the skin, within several skin appendices, in areas of dermal blood vessel inflammation and within dermal mesenchymal-epithelial cell junctions.

  18. Neuroproteome changes after ischemia/reperfusion injury and tissue plasminogen activator administration in rats: a quantitative iTRAQ proteomics study.

    Zamir Merali

    Full Text Available The thrombolytic, recombinant tissue plasminogen activator (rt-PA is the only approved therapy for acute ischemic stroke (AIS. When administered after AIS, rt-PA has many adverse pleiotropic actions, which are currently poorly understood. The identification of proteins showing differential expression after rt-PA administration may provide insight into these pleiotropic actions. In this study we used a 2D-LC MS/MS iTRAQ proteomic analysis, western blotting, and pathway analysis to analyze changes in protein expression 24-hours after rt-PA administration in the cortical brain tissue of 36 rats that underwent a sham or transient middle cerebral artery occlusion surgery. After rt-PA administration we reported alterations in the expressions of 18 proteins, many of which were involved in excitatory neurotransmitter function or cytoskeletal structure. The expression changes of GAD2 and EAAT1 were validated with western blot. The interactions between the identified proteins were analyzed with the IPA pathway analysis tool and three proteins: DPYSL2, RTN4, and the NF-kB complex, were found to have characteristics of being key proteins in the network. The differential protein expressions we observed may reflect pleiotropic actions of rt-PA after experimental stroke, and shine light on the mechanisms of rt-PA's adverse effects. This may have important implications in clinical settings where thrombolytic therapy is used to treat AIS.

  19. The receptor for urokinase-plasminogen activator (uPAR) controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style.

    Margheri, Francesca; Luciani, Cristina; Taddei, Maria Letizia; Giannoni, Elisa; Laurenzana, Anna; Biagioni, Alessio; Chillà, Anastasia; Chiarugi, Paola; Fibbi, Gabriella; Del Rosso, Mario

    2014-03-30

    The receptor for the urokinase plasminogen activator (uPAR) is up-regulated in malignant tumors. Historically the function of uPAR in cancer cell invasion is strictly related to its property to promote uPA-dependent proteolysis of extracellular matrix and to open a path to malignant cells. These features are typical of mesenchymal motility. Here we show that the full-length form of uPAR is required when prostate and melanoma cancer cells convert their migration style from the "path generating" mesenchymal to the "path finding" amoeboid one, thus conferring a plasticity to tumor cell invasiveness across three-dimensional matrices. Indeed, in response to a protease inhibitors-rich milieu, prostate and melanoma cells activated an amoeboid invasion program connoted by retraction of cell protrusions, RhoA-mediated rounding of the cell body, formation of a cortical ring of actin and a reduction of Rac-1 activation. While the mesenchymal movement was reduced upon silencing of uPAR expression, the amoeboid one was almost completely abolished, in parallel with a deregulation of small Rho-GTPases activity. In melanoma and prostate cancer cells we have shown uPAR colocalization with β1/β3 integrins and actin cytoskeleton, as well integrins-actin co-localization under both mesenchymal and amoeboid conditions. Such co-localizations were lost upon treatment of cells with a peptide that inhibits uPAR-integrin interactions. Similarly to uPAR silencing, the peptide reduced mesenchymal invasion and almost abolished the amoeboid one. These results indicate that full-length uPAR bridges the mesenchymal and amoeboid style of movement by an inward-oriented activity based on its property to promote integrin-actin interactions and the following cytoskeleton assembly. PMID:24681666

  20. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T.; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A.; Valdez-Cruz, Norma A.

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system. PMID:26991106

  1. Irradiation-Induced Regulation of Plasminogen Activator Inhibitor Type-1 and Vascular Endothelial Growth Factor in Six Human Squamous Cell Carcinoma Lines of the Head and Neck

    Purpose: It has been shown that plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are involved in neo-angiogenesis. The aim of this study was to investigate the irradiation-induced regulation of PAI-1 and VEGF in squamous cell carcinomas of the head and neck (SCCHN) cell lines of varying radiation sensitivity. Methods and Materials: Six cell lines derived from SCCHN were investigated in vitro. The colorimetric AlamarBlue assay was used to detect metabolic activity of cell lines during irradiation as a surrogate marker for radiation sensitivity. PAI-1 and VEGF secretion levels were measured by enzyme-linked immunosorbent assay 24, 48, and 72 h after irradiation with 0, 2, 6, and 10 Gy. The direct radioprotective effect of exogenous PAI-1 was measured using the clonogenic assay. For regulation studies, transforming growth factor-β1 (TGF-β1), hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), or both HIF-1α and HIF-2α were downregulated using siRNA. Results: Although baseline levels varied greatly, irradiation led to a comparable dose-dependent increase in PAI-1 and VEGF secretion in all six cell lines. Addition of exogenous stable PAI-1 to the low PAI-1-expressing cell lines, XF354 and FaDu, did not lead to a radioprotective effect. Downregulation of TGF-β1 significantly decreased VEGF secretion in radiation-sensitive XF354 cells, and downregulation of HIF-1α and HIF-2α reduced PAI-1 and VEGF secretion in radiation-resistant SAS cells. Conclusions: Irradiation dose-dependently increased PAI-1 and VEGF secretion in all SCCHN cell lines tested regardless of their basal levels and radiation sensitivity. In addition, TGF-β1 and HIF-1α could be partly responsible for VEGF and PAI-1 upregulation after irradiation.

  2. Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice

    List, K; Jensen, O N; Bugge, T H;

    2000-01-01

    alpha(1)-antitrypsin. We suggest that mouse glandular kallikrein mGK-6 is an activator of pro-uPA in the mouse urinary tract in vivo. Since this kallikrein is expressed in a number of tissues and also occurs in plasma, it can also be considered a candidate for a physiological pro-uPA activator in other...

  3. Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway

    Freeman Robert S

    2011-02-01

    Full Text Available Abstract Background Thrombolytic therapy with tissue plasminogen activator (tPA benefits patients with acute ischemic stroke. However, tPA increases the risk for intracerebral bleeding and enhances post-ischemic neuronal injury if administered 3-4 hours after stroke. Therefore, combination therapies with tPA and neuroprotective agents have been considered to increase tPA's therapeutic window and reduce toxicity. The anticoagulant factor protein S (PS protects neurons from hypoxic/ischemic injury. PS also inhibits N-methyl-D-aspartate (NMDA excitotoxicity by phosphorylating Bad and Mdm2 which blocks the downstream steps in the intrinsic apoptotic cascade. To test whether PS can protect neurons from tPA toxicity we studied its effects on tPA/NMDA combined injury which in contrast to NMDA alone kills neurons by activating the extrinsic apoptotic pathway. Neither Bad nor Mdm2 which are PS's targets and control the intrinsic apoptotic pathway can influence the extrinsic cascade. Thus, based on published data one cannot predict whether PS can protect neurons from tPA/NMDA injury by blocking the extrinsic pathway. Neurons express all three TAM (Tyro3, Axl, Mer receptors that can potentially interact with PS. Therefore, we studied whether PS can activate TAM receptors during a tPA/NMDA insult. Results We show that PS protects neurons from tPA/NMDA-induced apoptosis by suppressing Fas-ligand (FasL production and FasL-dependent caspase-8 activation within the extrinsic apoptotic pathway. By transducing neurons with adenoviral vectors expressing the kinase-deficient Akt mutant AktK179A and a triple FKHRL1 Akt phosphorylation site mutant (FKHRL1-TM, we show that Akt activation and Akt-mediated phosphorylation of FKHRL1, a member of the Forkhead family of transcription factors, are critical for FasL down-regulation and caspase-8 inhibition. Using cultured neurons from Tyro3, Axl and Mer mutants, we show that Tyro3, but not Axl and Mer, mediates

  4. Role of connective growth factor in plasminogen activator inhibitor-1 and fibronectin expression induced by transforming growth factor β1 in renal tubular cells

    张春; 孟宪芳; 朱忠华; 杨晓; 邓安国

    2004-01-01

    Background Connective tissue growth factor (CTGF) contributes greatly to renal tubulointerstitial fibrosis, which is the final event leading to end-stage renal failure. This study was designed to investigate the effects of CTGF antisense oligodeoxynucleotides (ODNs) on the expressions of plasminogen activator inhibitor-1 (PAI-1) and fibronectin in renal tubular cells induced by transforming growth factor β1 (TGF-β1) in addition to the role of CTGF in the accumulation and degradation of renal extracellular matrix (ECM).Methods A human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODNs were transfected into HKC cells. After HKC cells were stimulated with TGF-β1 (5 μg/L), the mRNA levels of PAI-1 and fibronectin were measured by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 and fibronectin in the medium were determined by Western blot and ELISA, respectively.Results TGF-β1 was found to induce tubular CTGF, PAI-1, and fibronectin mRNA expression. PAI-1 and fibronectin mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODNs. CTGF antisense ODNs also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 and fibronectin protein secreted into the medium.Conclusions CTGF may play a crucial role in the accumulation and degradation of excessive ECM during tubulointerstitial fibrosis, and transfecting CTGF antisense ODNs may be an effective way to prevent renal fibrosis.

  5. Fructose Rich Diet-Induced High Plasminogen Activator Inhibitor-1 (PAI-1 Production in the Adult Female Rat: Protective Effect of Progesterone

    Eduardo Spinedi

    2012-08-01

    Full Text Available The effect of progesterone (P4 on fructose rich diet (FRD intake-induced metabolic, endocrine and parametrial adipose tissue (PMAT dysfunctions was studied in the adult female rat. Sixty day-old rats were i.m. treated with oil alone (control, CT or containing P4 (12 mg/kg. Rats ate Purina chow-diet ad libitum throughout the entire experiment and, between 100 and 120 days of age drank ad libitum tap water alone (normal diet; CT-ND and P4-ND or containing fructose (10% w/v; CT-FRD and P4-FRD. At age 120 days, animals were subjected to a glucose tolerance test or decapitated. Plasma concentrations of various biomarkers and PMAT gene abundance were monitored. P4-ND (vs. CT-ND rats showed elevated circulating levels of lipids. CT-FRD rats displayed high (vs. CT-ND plasma concentrations of lipids, leptin, adiponectin and plasminogen activator inhibitor-1 (PAI-1. Lipidemia and adiponectinemia were high (vs. P4-ND in P4-FRD rats. Although P4 failed to prevent FRD-induced hyperleptinemia, it was fully protective on FRD-enhanced plasma PAI-1 levels. PMAT leptin and adiponectin mRNAs were high in CT-FRD and P4-FRD rats. While FRD enhanced PMAT PAI-1 mRNA abundance in CT rats, this effect was absent in P4 rats. Our study supports that a preceding P4-enriched milieu prevented the enhanced prothrombotic risk induced by FRD-elicited high PAI-1 production.

  6. The -675 4G/5G polymorphism in plasminogen activator inhibitor-1 gene is associated with risk of asthma: a meta-analysis.

    Wei Nie

    Full Text Available BACKGROUND: A number of studies assessed the association of -675 4G/5G polymorphism in the promoter region of plasminogen activator inhibitor (PAI-1 gene with asthma in different populations. However, most studies reported inconclusive results. A meta-analysis was conducted to investigate the association between polymorphism in the PAI-1 gene and asthma susceptibility. METHODS: Databases including Pubmed, EMBASE, HuGE Literature Finder, Wanfang Database, China National Knowledge Infrastructure (CNKI and Weipu Database were searched to find relevant studies. Odds ratios (ORs with 95% confidence intervals (CIs were used to assess the strength of association in the dominant model, recessive model, codominant model, and additive model. RESULTS: Eight studies involving 1817 cases and 2327 controls were included. Overall, significant association between 4G/5G polymorphism and asthma susceptibility was observed for 4G4G+4G5G vs. 5G5G (OR = 1.56, 95% CI 1.12-2.18, P = 0.008, 4G/4G vs. 4G/5G+5G/5G (OR = 1.38, 95% CI 1.06-1.80, P = 0.02, 4G/4G vs. 5G/5G (OR = 1.80, 95% CI 1.17-2.76, P = 0.007, 4G/5G vs. 5G/5G (OR = 1.40, 95% CI 1.07-1.84, P = 0.02, and 4G vs. 5G (OR = 1.35, 95% CI 1.08-1.68, P = 0.008. CONCLUSIONS: This meta-analysis suggested that the -675 4G/5G polymorphism of PAI-1 gene was a risk factor of asthma.

  7. Elevated urinary levels of urokinase-type plasminogen activator receptor (uPAR) in pancreatic ductal adenocarcinoma identify a clinically high-risk group

    The urokinase plasminogen activator receptor is highly expressed and its gene is amplified in about 50% of pancreatic ductal adenocarcinomas; this last feature is associated with worse prognosis. It is unknown whether the level of its soluble form (suPAR) in urine may be a diagnostic-prognostic marker in these patients. The urinary level of suPAR was measured in 146 patients, 94 pancreatic ductal adenocarcinoma and 52 chronic pancreatitis. Urine from 104 healthy subjects with similar age and gender distribution served as controls. suPAR levels were normalized with creatinine levels (suPAR/creatinine, ng/mg) to remove urine dilution effect. Urinary suPAR/creatinine values of pancreatic ductal adenocarcinoma patients were significantly higher (median 9.8; 25th-75th percentiles 5.3-20.7) than those of either healthy donors (median 0; 0-0.5) or chronic pancreatitis patients (median 2.7; 0.9-4.7). The distribution of values among cancer patients was widespread and asymmetric, 53% subjects having values beyond the 95th percentile of healthy donors. The values of suPAR/creatinine did not correlate with tumour stage, Ca19-9 or CEA levels. Higher values correlated with poor prognosis among non-resected patients at univariate analysis; multivariate Cox regression identified high urinary suPAR/creatinine as an independent predictor of poor survival among all cancer patients (odds ratio 2.10, p = 0.0023), together with tumour stage (stage III odds ratio 2.65, p = 0.0017; stage IV odds ratio 4.61, p < 0.0001) and female gender (odds ratio 1.85, p = 0.01). A high urinary suPAR/creatinine ratio represents a useful marker for the identification of a subset of patients with poorer outcome

  8. Elevated urinary levels of urokinase-type plasminogen activator receptor (uPAR in pancreatic ductal adenocarcinoma identify a clinically high-risk group

    Brocco Giorgio

    2011-10-01

    Full Text Available Abstract Background The urokinase plasminogen activator receptor is highly expressed and its gene is amplified in about 50% of pancreatic ductal adenocarcinomas; this last feature is associated with worse prognosis. It is unknown whether the level of its soluble form (suPAR in urine may be a diagnostic-prognostic marker in these patients. Methods The urinary level of suPAR was measured in 146 patients, 94 pancreatic ductal adenocarcinoma and 52 chronic pancreatitis. Urine from 104 healthy subjects with similar age and gender distribution served as controls. suPAR levels were normalized with creatinine levels (suPAR/creatinine, ng/mg to remove urine dilution effect. Results Urinary suPAR/creatinine values of pancreatic ductal adenocarcinoma patients were significantly higher (median 9.8; 25th-75th percentiles 5.3-20.7 than those of either healthy donors (median 0; 0-0.5 or chronic pancreatitis patients (median 2.7; 0.9-4.7. The distribution of values among cancer patients was widespread and asymmetric, 53% subjects having values beyond the 95th percentile of healthy donors. The values of suPAR/creatinine did not correlate with tumour stage, Ca19-9 or CEA levels. Higher values correlated with poor prognosis among non-resected patients at univariate analysis; multivariate Cox regression identified high urinary suPAR/creatinine as an independent predictor of poor survival among all cancer patients (odds ratio 2.10, p = 0.0023, together with tumour stage (stage III odds ratio 2.65, p = 0.0017; stage IV odds ratio 4.61, p Conclusions A high urinary suPAR/creatinine ratio represents a useful marker for the identification of a subset of patients with poorer outcome.

  9. Hesperetin and its sulfate and glucuronide metabolites inhibit TNF-α induced human aortic endothelial cell migration and decrease plasminogen activator inhibitor-1 (PAI-1) levels.

    Giménez-Bastida, Juan Antonio; González-Sarrías, Antonio; Vallejo, Fernando; Espín, Juan Carlos; Tomás-Barberán, Francisco A

    2016-01-01

    Epidemiological, clinical and preclinical studies have reported the protection offered by citrus consumption, mainly orange, against cardiovascular diseases, which is primarily mediated by the antiatherogenic and vasculoprotective effects of the flavanone hesperetin-7-O-rutinoside (hesperidin). However, flavanone aglycones or glycosides are not present in the bloodstream but their derived phase-II metabolites could be the actual bioactive molecules. To date, only a few studies have explored the effects of circulating hesperetin-derived metabolites (glucuronides and sulfates) on endothelial cells. Herein, we describe for the first time the effects of hesperetin 3'-O-glucuronide, hesperetin 7-O-glucuronide, hesperetin 3'-O-sulfate, hesperetin 7-O-sulfate and hesperetin on human aortic endothelial cell (HAEC) migration upon pro-inflammatory stimuli as an essential step to angiogenesis. Hesperetin and its derived metabolites, at physiologically relevant concentrations (1-10 μM), significantly attenuated cell migration in the presence of the pro-inflammatory cytokine TNF-α (50 ng mL(-1)), which was accompanied and perhaps mediated by a significant decrease in the levels of the thrombogenic plasminogen activator inhibitor-1 (PAI-1). However, hesperetin metabolites did not counteract the TNF-α-induced production of pro-inflammatory interleukin-6 (IL-6) and IL-8. We also study here for the first time, the metabolism of hesperetin and its derived metabolites by HAEC with and without a pro-inflammatory stimulus. All these results reinforce the concept according to which circulating phase-II hesperetin metabolites are critical molecules contributing to the cardioprotective effects upon consumption of citrus fruits such as orange. PMID:26456097

  10. Impact of statin therapy on plasma levels of plasminogen activator inhibitor-1. A systematic review and meta-analysis of randomised controlled trials.

    Sahebkar, Amirhossein; Catena, Cristiana; Ray, Kausik K; Vallejo-Vaz, Antonio J; Reiner, Željko; Sechi, Leonardo A; Colussi, GianLuca

    2016-07-01

    Elevated plasma levels of the pro-thrombotic and pro-inflammatory factor plasminogen activator inhibitor-1 (PAI-1) may contribute to the pathogenesis of atherosclerotic cardiovascular disease. Beyond their lipid-lowering effect, statins have been shown to modulate plasma PAI-1 levels but evidence from individual randomised controlled trials (RCTs) is controversial. Therefore, we aimed to assess the potential effects of statin therapy on plasma PAI-1 concentration through a meta-analysis of RCTs. We searched Medline and SCOPUS databases (up to October 3, 2014) to identify RCTs investigating the effect of statin therapy on plasma PAI-1 concentrations. We performed random-effects meta-analysis and assessed heterogeneity (I² test, subgroup and sensitivity analyses) and publication bias (funnel plot, Egger and "trim and fill" tests). Sixteen RCTs (comprising 19 treatment arms) were included and pooled analyses showed a significant effect of statins in reducing plasma PAI-1 concentrations (weighted mean difference WMD: -15.72 ng/ml, 95 % confidence interval [CI]: -25.01, -6.43,). In subgroup analysis, this effect remained significant in with lipophilic statins (atorvastatin and simvastatin) (WMD: -21.32 ng/ml, 95 % CI: -32.73, -9.91, I²=99 %) and particularly atorvastatin (WMD: -20.88 ng/mL, 95 % CI: -28.79, -12.97, I2=97 %). In the meta-regression analysis, the impact of statins on PAI-1 did not correlate with the administered dose, duration of treatment and changes in plasma LDL-cholesterol concentrations. Finally, evidence of publication bias was observed. In conclusion, taking into account the limit of heterogeneity between studies, the present meta-analysis suggests that statin therapy (mainly atorvastatin) significantly lowers plasma PAI-1 concentrations. PMID:27009446

  11. Analgesic drug delivery via recombinant tissue plasminogen activator and microRNA-183-triggered opening of the blood-nerve barrier.

    Yang, Shaobing; Krug, Susanne M; Heitmann, Johanna; Hu, Liu; Reinhold, Ann Kristin; Sauer, Solange; Bosten, Judith; Sommer, Claudia; Fromm, Michael; Brack, Alexander; Rittner, Heike L

    2016-03-01

    The peripheral nerve contains three barriers which include the blood-nerve barrier consisting of endoneurial vessels and the perineurium as well as autotypic junctions in Schwann cells. The perineurium prevents diffusion of perineurally injected drugs that can be used for selective regional pain control. It is composed of a basal membrane and layers of perineurial cells sealed by tight junction proteins like claudin-1. Claudin-1 expression and barrier function are regulated via low-density lipoprotein receptor-related protein (LRP-1). Perisciatic application of recombinant tissue plasminogen activator (rtPA) or the catalytically inactive rtPAi - both agonists of LRP-1 - reduced claudin-1 mRNA and protein expression in the rat nerve. This facilitated an increase of nociceptive thresholds after local application of hydrophilic opioids or the voltage gated sodium channel blocker (NaV1.7) ProToxin-II without apparent nerve toxicity. RtPA-induced barrier opening was mediated by LRP-1 and intracellularly by Erk phosphorylation. In silico, microRNA (miR)-rno-29b-2-5p and rno-miR-183-5p were identified as potential regulators of claudin-1 transcription in the rat. RtPA application increased miR-183-5p in the sciatic nerve. MiR-183-5p mimics functionally opened the perineurium and downregulated claudin-1 expression in vivo. In vitro, hsa-miR-183-3p mimics reduced claudin-1 expression in human HT-29/B6 cells. Overall, rtPA regulates perineurial barrier tightness via LRP-1, Erk phosphorylation and miR-183-5p/3p. This mechanism might serve as a new principle to facilitate drug delivery to peripheral nerves in humans. PMID:26735170

  12. Reduced uptake of plasminogen activators during formation of whole blood thrombi by a bovine polypeptide of uterine origin: in vitro study using Chandler's loop method

    The authors have previously isolated and purified by high performance liquid chromatography low molecular weight polypeptide of uterine origin (LMW-UDF) which promotes metastasis in tumor-bearing mice. In order to determine what effects this polypeptide could have on the coagulation systems they generated whole blood thrombi in vitro in the presence (1 μg/ml - 100 μg/ml) or absence of test protein. Using trace amount of radiolabeled plasminogen activators, 125I-Urokinase and 125I-pro-Urokinase (single chain urokinase) inhibition of uptake to the thrombi was observed vs control groups. Also, specific radioactivity of the thrombi pre-incubated with LMW-UDF was statistically significantly lower than control thrombi (p 125I-fibrinogen revealed no net increase or decrease of the uptake into the thrombi when LMW-UDF was used. Gross morphological differences were noted in the group with 1 μg/ml LMW-UDF, where weight was significantly greater (243.56 mg +/- SD 50.44) and size was significantly larger (3.5 cm +/- SD 0.6) when compared to the control groups (61.45 mg +/- SD 10.26 and 0.8 cm +/- SD 0.3), respectively. It appears that LMW-UDF has an influence on the physical formation of whole blood thrombi and suggests that this effect is mediated possibly by the uptake of Urokinase and pro-Urokinase. The actual mechanism of the action is unknown but may be caused by altered fibrin cross-linking or platelet aggregation

  13. 9 CFR 113.301 - Ovine Ecthyma Vaccine.

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Ovine Ecthyma Vaccine. 113.301 Section... Virus Vaccines § 113.301 Ovine Ecthyma Vaccine. Ovine Ecthyma Vaccine shall be prepared from tissue... inoculation with virulent ovine ecthyma virus. Ovine Ecthyma Vaccine is exempt from the...

  14. Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration

    Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis. Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p

  15. The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface

    Behrendt, N; Rønne, E; Danø, K

    1995-01-01

    PA receptor, uPAR, is a cell-surface protein which plays an important role in the localization and regulation of these processes. In the present article a number of established conclusions concerning the structure and function of uPAR are presented, and in addition various models are discussed which might...... domain is directly involved in the molecular contact with uPA. The receptor binds uPA as well as its proenzyme, pro-uPA, in such a manner that the activation cascade can occur directly on the cell surface. Furthermore, the activation rates are enhanced relative to the situation in solution, probably due...

  16. Stability of the octameric structure affects plasminogen-binding capacity of streptococcal enolase.

    Amanda J Cork

    Full Text Available Group A Streptococcus (GAS is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen. Streptococcal surface enolase (SEN is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen to its binding sites, leading to more efficient plasmin(ogen binding and activation.

  17. Stability of the Octameric Structure Affects Plasminogen-Binding Capacity of Streptococcal Enolase

    Law, Ruby H. P.; Casey, Lachlan W.; Valkov, Eugene; Bertozzi, Carlo; Stamp, Anna; Jovcevski, Blagojce; Aquilina, J. Andrew; Whisstock, James C.; Walker, Mark J.; Kobe, Bostjan

    2015-01-01

    Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation. PMID:25807546

  18. IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

    Tezuka, Toshifumi; Ogawa, Hirohisa; Azuma, Masahiko; Goto, Hisatsugu; Uehara, Hisanori; Aono, Yoshinori; Hanibuchi, Masaki; Yamaguchi, Yoichi; Fujikawa, Tomoyuki; Itai, Akiko; Nishioka, Yasuhiko

    2015-01-01

    Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling. PMID:25785861

  19. IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

    Toshifumi Tezuka

    Full Text Available Plasminogen activator inhibitor (PAI-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp. IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

  20. A combination of desmopressin and docetaxel inhibit cell proliferation and invasion mediated by urokinase-type plasminogen activator (uPA) in human prostate cancer cells

    Sasaki, Hiroshi; Klotz, Laurence H. [Division of Urology, Sunnybrook Health Sciences Center, Toronto, ON (Canada); Sugar, Linda M. [Department of Pathology, Sunnybrook Health Sciences Center, Toronto, ON (Canada); Kiss, Alexander [Department of Research Design and Biostatistics, Institute for Clinical Evaluative Sciences, Sunnybrook Health Sciences Center, Toronto, ON (Canada); Venkateswaran, Vasundara, E-mail: vasundara.venkateswaran@sunnybrook.ca [Division of Urology, Sunnybrook Health Sciences Center, Toronto, ON (Canada)

    2015-08-28

    Background: This study was designed to assess the effectiveness of a combination treatment using both desmopressin and docetaxel in prostate cancer treatment. Desmopressin is a well-known synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in in vivo models. Docetaxel is widely used for the treatment of castration resistant prostate cancer (CRPC) patients. However, durable responses have been uncommon to date. In this study, we investigated the anti-tumor effect of desmopressin in combination with docetaxel in vitro and in vivo. Methods: Two prostate cancer cells (PC3, LNCaP) were treated with different concentrations of desmopressin alone, docetaxel alone, and a combination of desmopressin and docetaxel. Cell proliferation was determined by MTS assay. The anti-invasive and anti-migration potential of desmopressin and in combination with docetaxel were examined by wound healing assay, migration chamber assay, and matrigel invasion assay. Results: The combination of desmopressin and docetaxel resulted in a significant inhibition of PC3 and LNCaP cell proliferation (p < 0.01). Additionally, cell migration and invasion were also inhibited by the combination when compared to that of either treatment alone in PC3 cells (p < 0.01). The anti-tumor effect of this combination treatment was associated with down-regulation of both urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP-2 and MMP-9) in PC3 cells. Conclusions: We are the first to elucidate the anti-tumor and anti-metastatic potential of desmopressin in combination with docetaxel in a prostate cancer model via the uPA-MMP pathway. Our finding could potentially contribute to the therapeutic profile of desmopressin and enhance the efficacy of docetaxel based treatment for CRPC. - Highlights: • Desmopressin inhibits cell proliferation in prostate cancer cells. • The expression of cyclin A and CDK2

  1. Association of the 4 g/5 g polymorphism of plasminogen activator inhibitor-1 gene with sudden sensorineural hearing loss. A case control study

    Cho Seong

    2012-06-01

    Full Text Available Abstract Background The 5 G/5 G genotype of PAI-1 polymorphism is linked to decreased plasminogen activator inhibitor-1 (PAI-1 levels and it has been suggested that lower PAI-1 levels may provide protective effects on inflammation, local microcirculatory disturbance, and fibrotic changes, which are likely associated with development of sudden sensorineural hearing loss (SSNHL. Methods The association of the 4 G/5 G PAI-1 polymorphism with the development and clinical outcome of SSNHL is evaluated via a case control study. 103 patients with SSNHL and 113 age and sex-matched controls were enrolled at University of Ferrara, Italy and hearing loss outcome was measured at least 3 months after the onset of hearing loss. DNA was isolated from peripheral blood using the QIAamp kit and the 4 G/5 G polymorphism in the −675 promoter region was genotyped with an allele-specific PCR. Genotype distribution was tested in patients and compared to controls by chi-square and odd-ratio analysis. The codominant and recessive models were used for the multiple logistic regression analyses of the PAI-1 gene allele. Results In this population, 5 G/5 G genotype had a two-time lower frequency in SSNHL patients compared to healthy controls (15.5% vs 30.1% and was associated with decreased odds compared to 4 G/5 G genotype (OR 0.37, 95% CI 0.19-0.75, p = 0.005. In addition, the patients with 5 G/5 G genotype showed a trend of more than 2 times higher ratio of hearing recovery (> 20 dB after systemic corticosteroid treatment compared to 4 G/5 G genotype (OR 2.3, 95% CI 0.32 - 16.83, p = 0.39, suggesting a better clinical outcome. Conclusions The 5 G/5 G genotype of PAI-1 may be associated with a reduced risk of SSNHL in the Italian population.

  2. Small Interfering RNA-mediated Caveolin-1 Knockout on Plasminogen Activator Inhibitor-1 Expression in Insulin-stimulated Human Vascular Endothelial Cells

    Huiling YANG; Gebo WEN; Weixin HU; Shuya HE; Zhihua QUAN; Weixia PENG; Bin YAN; Jianghua LIU; Fang WEN; Renxian CAO; Yangyan XU

    2007-01-01

    Using human vascular endothelial cells (ECV304) as the target,we studied the effect of caveolin(CAV)-1 in the course of insulin-stimulated expression of plasminogen activator inhibitor(PAI)-1.The appropriate single-stranded oligonucleotides representing the RNAi CAV-1 gene were analyzed by Ambion software.After annealing to generate double-stranded oligonucleotides (ds oligo),it was cloned into the pENTR/U6 entry vector containing RNA polymerase Ⅲ expression element by T4 DNA ligase.The short hairpin (shRNA) sequences transferred from the pENTR/U6 entry were cloned into the pLenti6/BLOCK-iTDEST vector with an LR recombination reaction.After identification by sequencing,we successfully constructed the CAV-1 RNAi lentiviral expression system using Gateway technology.Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction,immunofluorescence staining and Western blotting.ECV304 cells were cultured in the medium containing different concentrations of insulin(1×10-9 to 1×10-7M)with the CAV-1 gene silenced or not.The expression level and subcellular localization of PAI-1 and CAV-1 were compared using reverse transcription-polymerase chain reaction,immunofluorescence staining and Western blot assay.The results showed that the potent inhibition of CAV-1 expression could reach 85%,and it was specific to the CAV-1-derived shRNA,not the S100A13-derived shRNA.There was no dramatic difference in PAI-1 expression between the RNAi+ and RNAi-ECV304 cells incubated with physiological insulin,but PAI-1 protein did accumulate under the cell membrane.As the concentration of insulin increased,the expression of PAI-1 was up-regulated,whereas the expression of CAV-1 attenuated.Furthermore,PAl-1 clearly augmented after CAV-1 knockdown.These results indicated that hyperinsulinism could promote PAI-1 expression by inhibiting CAV-1,and stabilizing or up-regulating CAV-1 expression in endothelial cells might reduce complications of the great vessels

  3. A combination of desmopressin and docetaxel inhibit cell proliferation and invasion mediated by urokinase-type plasminogen activator (uPA) in human prostate cancer cells

    Background: This study was designed to assess the effectiveness of a combination treatment using both desmopressin and docetaxel in prostate cancer treatment. Desmopressin is a well-known synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in in vivo models. Docetaxel is widely used for the treatment of castration resistant prostate cancer (CRPC) patients. However, durable responses have been uncommon to date. In this study, we investigated the anti-tumor effect of desmopressin in combination with docetaxel in vitro and in vivo. Methods: Two prostate cancer cells (PC3, LNCaP) were treated with different concentrations of desmopressin alone, docetaxel alone, and a combination of desmopressin and docetaxel. Cell proliferation was determined by MTS assay. The anti-invasive and anti-migration potential of desmopressin and in combination with docetaxel were examined by wound healing assay, migration chamber assay, and matrigel invasion assay. Results: The combination of desmopressin and docetaxel resulted in a significant inhibition of PC3 and LNCaP cell proliferation (p < 0.01). Additionally, cell migration and invasion were also inhibited by the combination when compared to that of either treatment alone in PC3 cells (p < 0.01). The anti-tumor effect of this combination treatment was associated with down-regulation of both urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP-2 and MMP-9) in PC3 cells. Conclusions: We are the first to elucidate the anti-tumor and anti-metastatic potential of desmopressin in combination with docetaxel in a prostate cancer model via the uPA-MMP pathway. Our finding could potentially contribute to the therapeutic profile of desmopressin and enhance the efficacy of docetaxel based treatment for CRPC. - Highlights: • Desmopressin inhibits cell proliferation in prostate cancer cells. • The expression of cyclin A and CDK2

  4. Ultrasound-targeted transfection of tissue-type plasminogen activator gene carried by albumin nanoparticles to dog myocardium to prevent thrombosis after heart mechanical valve replacement

    Ji J

    2012-06-01

    Full Text Available Ji Jun, Ji Shang-Yi, Yang Jian-An, He Xia, Yang Xiao-Han, Ling Wen-Ping, Chen Xiao-LingDepartment of Pathology and Cardiovascular Surgery, Shenzhen Sun Yat-Sen Cardiovascular Hospital, Shenzhen, Guangdong, People's Republic of ChinaBackground: There are more than 300,000 prosthetic heart valve replacements each year worldwide. These patients are faced with a higher risk of thromboembolic events after heart valve surgery and long-term or even life-long anticoagulative and antiplatelet therapies are necessary. Some severe complications such as hemorrhaging or rebound thrombosis can occur when the therapy ceases. Tissue-type plasminogen activator (t-PA is a thrombolytic agent. One of the best strategies is gene therapy, which offers a local high expression of t-PA over a prolonged time period to avoid both systemic hemorrhaging and local rebound thrombosis. There are some issues with t-PA that need to be addressed: currently, there is no up-to-date report on how the t-PA gene targets the heart in vivo and the gene vector for t-PA needs to be determined.Aims: To fabricate an albumin nano-t-PA gene ultrasound-targeted agent and investigate its targeting effect on prevention of thrombosis after heart mechanic valve replacement under therapeutic ultrasound.Methods: A dog model of mechanical tricuspid valve replacement was constructed. A highly expressive t-PA gene plasmid was constructed and packaged by nanoparticles prepared with bovine serum albumin. This nanopackaged t-PA gene plasmid was further cross-linked to ultrasonic microbubbles prepared with sucrose and bovine serum albumin to form the ultrasonic-targeted agent for t-PA gene transfection. The agent was given intravenously followed by a therapeutic ultrasound treatment (1 MHz, 1.5 w/cm2, 10 minutes of the heart soon after valve replacement had been performed. The expression of t-PA in myocardium was detected with multiclonal antibodies to t-PA by the indirect immunohistochemical method

  5. EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression

    An elevated level of EMMPRIN in cancer tissues have been correlated with tumor invasion in numerous cancers including oral cavity and larynx. Although EMMPRIN's effect has been generally attributed to its MMP inducing activity, we have previously demonstrated in breast cancer model that EMMPRIN can also enhance invasion by upregulating uPA. In this study, the role of EMMPRIN in regulating uPA and invasion was investigated in oral squamous cell carcinoma (OSCC) progression. Precancerous and invasive oral tumoral tissues were used as well as the corresponding cell lines, DOK and SCC-9 respectively. The paracrine regulation of uPA by EMMPRIN was investigated by treating culture cells with EMMPRIN-enriched membrane vesicles. UPA expression was analyzed by qPCR and immunostaining and the consequence on the invasion capacity was studied using modified Boyden chamber assay, in the presence or absence of EMMPRIN blocking antibody, the uPA inhibitor amiloride or the MMP inhibitor marimastat. OSCC tumors were shown to express more EMMPRIN and uPA compared to dysplastic lesions. The corresponding cell models, SCC-9 and DOK cells, displayed similar expression pattern. In both cell types EMMPRIN upregulated the expression of uPA as well as that of MMP-2 and MMP-9. EMMPRIN treatment led to a significant increase in cell invasion both in the invasive SCC-9 and in the less invasive dysplastic DOK cells, in an MMP and uPA dependent manner. Our results suggest that the upregulation of uPA contributes to EMMPRIN's effect in promoting oral tumor invasion

  6. Data in support of a central role of plasminogen activator inhibitor-2 polymorphism in recurrent cardiovascular disease risk in the setting of high HDL cholesterol and C-reactive protein using Bayesian network modeling.

    Corsetti, James P; Salzman, Peter; Ryan, Dan; Moss, Arthur J; Zareba, Wojciech; Sparks, Charles E

    2016-09-01

    Data is presented that was utilized as the basis for Bayesian network modeling of influence pathways focusing on the central role of a polymorphism of plasminogen activator inhibitor-2 (PAI-2) on recurrent cardiovascular disease risk in patients with high levels of HDL cholesterol and C-reactive protein (CRP) as a marker of inflammation, "Influences on Plasminogen Activator Inhibitor-2 Polymorphism-Associated Recurrent Cardiovascular Disease Risk in Patients with High HDL Cholesterol and Inflammation" (Corsetti et al., 2016; [1]). The data consist of occurrence of recurrent coronary events in 166 post myocardial infarction patients along with 1. clinical data on gender, race, age, and body mass index; 2. blood level data on 17 biomarkers; and 3. genotype data on 53 presumptive CVD-related single nucleotide polymorphisms. Additionally, a flow diagram of the Bayesian modeling procedure is presented along with Bayesian network subgraphs (root nodes to outcome events) utilized as the data from which PAI-2 associated influence pathways were derived (Corsetti et al., 2016; [1]). PMID:27284570

  7. Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors Type 1 and 2 in human and rhesus monkey fetal membranes.

    Liu, Y X; Hu, Z Y; Liu, K; Byrne, S; Zou, R J; Ny, T; d'Lacey, C; Ockleford, C D

    1998-01-01

    Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-1 and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA was also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was

  8. The X-ray Crystal Structure of Full-Length Human Plasminogen

    Ruby H.P. Law

    2012-03-01

    Full Text Available Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp domain, five kringle domains (KR1-5, and a serine protease (SP domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.

  9. Flavonol-enriched fraction from Vaccinium macrocarpon fruit inhibits matrix metalloproteinase-2, matrix metalloproteinase-9 and urokinase-type plasminogen activator expression in human prostate cancer cells in vitro

    James MacPhee

    2014-11-01

    Full Text Available Background: Prostate cancer, amongst other cancer types has a genetic and environmental component, which can contribute to prostate cancer development and progression. Vaccinum macrocarpon (American cranberry is a botanical that contains several phytochemicals which have been suggested to play a role in preventing cardiovascular disease, cancer, and urinary tract infections as well as in the maintenance of oral health. Context and purpose of this study: This investigation evaluated the effects of a flavonolenriched fraction (FL from the American cranberry (Vaccinium macrocarpon containing quercetin and myricetin glycosides on matrix metalloproteinase (MMP and urokinase-type plasminogen activator (uPA activities and their associated regulatory proteins in DU145 human prostate cancer cells in vitro. Results: A flavonol-enriched fraction (FL was prepared from Vaccinium macrocarpon berries and the effect of this fraction on prostate cancer cell behaviour was assessed using biochemical and molecular approaches including cytotoxicity assays and Western blot analysis to determine protein expression. Cranberry FL decreased cellular viability of DU145 cells at a concentration of 25 ug/ml by 20% after 6 hours of treatment. Further investigations determined that associated with this cytotoxicity, cranberry FL decreases matrix metalloproteinase (MMP ( specifically MMP-2 and MMP-9 activity and urokinase plasminogen activator (uPA activity through effects on specific temporal MMP regulators and uPA regulators and by affecting either the phosphorylation status and/or expression of specific MAP kinase, PI-3 kinase, NF-kB and AP-1 pathway associated proteins. Conclusion: This study demonstrates, for the first time, the ability of Vaccinium macrocarpon flavonols to modulate cellular pathways associated with migration, invasion, and proliferation, suggesting that cranberry (Vaccinium macrocarpon is a viable candidate for further research as a natural product that

  10. Role of Plasminogen in Propagation of Scrapie

    Salmona, Mario; Capobianco, Raffaella; Colombo, Laura; De Luigi, Ada; Rossi, Giacomina; Mangieri, Michela; Giaccone, Giorgio; Quaglio, Elena; Chiesa, Roberto; Donati, Maria Benedetta; Tagliavini, Fabrizio; Forloni, Gianluigi

    2005-01-01

    To investigate whether plasminogen may feature in scrapie infection, we inoculated plasminogen-deficient (Plg−/−), heterozygous plasminogen-deficient (Plg+/−), and wild-type (Plg+/+) mice by the intracerebral or intraperitoneal (i.p.) route with the RML scrapie strain and monitored the onset of neurological signs of disease, survival time, brain, and accumulation of scrapie disease-associated forms of the prion protein (PrPSc). Only after i.p. inoculation, a slight, although significant, diff...

  11. Plasminogen Improves Mouse IVF by Interactions with Inner Acrosomal Membrane-Bound MMP2 and SAMP14.

    Ferrer, Marvin J S; Xu, Wei; Shetty, Jagathpala; Herr, John; Oko, Richard

    2016-04-01

    Spermatozoa must penetrate the outer investments of the oocyte, the cumulus oophorus and the zona pellucida (ZP), in order for fertilization to occur. This may require exposure of enzymes on the sperm's inner acrosomal membrane (IAM), one of which is matrix metalloproteinase (MMP) 2, to factors in oviductal fluid. Plasminogen is present in oviductal fluid and activates MMP2 in somatic tissues. The objectives of this study were: 1) to examine possible interactions between plasminogen and IAM-bound plasminogen activator receptor (SAMP14) and -MMP2, 2) to demonstrate plasminogen's presence in the extracellular environment at the site of fertilization, and 3) to provide evidence that plasminogen plays a role in fertilization. Zymographs of sonicated bull and rat sperm extracts incubated with plasmin and/or plasminogen (plasmin/ogen) showed acceleration of initiation of MMP2 activity in concentrations as low as 1 μg/ml. Immunohistochemical and immunofluorescence analysis of plasmin/ogen revealed its presence in the cytoplasm of mouse ovarian and oviductal oocytes, oviductal epithelium, around the ZP, and amongst the cumulus cells. We modified the standard in vitro fertilization (IVF) approach to more closely mimic natural fertilization by reducing sperm concentration during insemination by ∼100× and also comparing cumulus-intact and denuded oocytes. In mice, addition of plasminogen in IVF medium significantly improved fertilization, while MMP2 antibody significantly inhibited sperm penetration in these conditions. IVF improvement by plasminogen was blocked by SAMP14 antibody. Furthermore, MMP2 antibody inhibition was coincident with a failure by spermatozoa to disperse the cumulus oophorus. We provide evidence that plasminogen on its own and through an MMP2-related mechanism improves the ability of oocytes to be fertilized, and demonstrate its effect in sperm penetration of oocyte investments. PMID:26935599

  12. Novel in vitro systems for prediction of veterinary drug residues in ovine milk and dairy products.

    González-Lobato, L; Real, R; Herrero, D; de la Fuente, A; Prieto, J G; Marqués, M M; Alvarez, A I; Merino, G

    2014-01-01

    A new in vitro tool was developed for the identification of veterinary substrates of the main drug transporter in the mammary gland. These drugs have a much higher chance of being concentrated into ovine milk and thus should be detectable in dairy products. Complementarily, a cell model for the identification of compounds that can inhibit the secretion of drugs into ovine milk, and thus reduce milk residues, was also generated. The ATP-binding cassette transporter G2 (ABCG2) is responsible for the concentration of its substrates into milk. The need to predict potential drug residues in ruminant milk has prompted the development of in vitro cell models over-expressing ABCG2 for these species to detect veterinary drugs that interact with this transporter. Using these models, several substrates for bovine and caprine ABCG2 have been found, and differences in activity between species have been reported. However, despite being of great toxicological relevance, no suitable in vitro model to predict substrates of ovine ABCG2 was available. New MDCKII and MEF3.8 cell models over-expressing ovine ABCG2 were generated for the identification of substrates and inhibitors of ovine ABCG2. Five widely used veterinary antibiotics (marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin and difloxacin) were discovered as new substrates of ovine ABCG2. These results were confirmed for the bovine transporter and its Y581S variant using previously generated cell models. In addition, the avermectin doramectin was described as a new inhibitor of ruminant ABCG2. This new rapid assay to identify veterinary drugs that can be concentrated into ovine milk will potentially improve detection and monitoring of veterinary drug residues in ovine milk and dairy products. PMID:24679113

  13. The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2

    Background: Different types of pulmonary hypertension (PH) share the same process of pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play an important role in this process. Objectives: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration. Methods: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured human PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated. Results: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level. In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly, and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased PASMCs proliferation and migration, inhibited the apoptosis. Conclusions: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2. - Highlights: • sGC activator BAY41-2272 up regulated PAI-2 in PASMCs, on the mRNA and protein level. • PAI-2 overexpression inhibits the proliferation and migration of human PASMCs. • PAI-2 overexpression promotes the apoptosis of human PASMCs. • sGC activator might alleviate the vascular remodeling in pulmonary hypertension

  14. The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2

    Zhang, Shuai [Beijing Institute of Respiratory Medicine, Beijing Chao-yang Hospital, Capital Medical University, 8 Gongti South Rd, Beijing (China); Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, 8 Gongti South Rd, Beijing (China); Zou, Lihui [Institute of Geriatrics, Beijing Hospital, 1 Dahua Rd, Beijing (China); National Clinical Research Center for Respiratory Diseases, 1 Dahua Rd, Beijing (China); Yang, Ting; Yang, Yuanhua; Zhai, Zhenguo [Beijing Institute of Respiratory Medicine, Beijing Chao-yang Hospital, Capital Medical University, 8 Gongti South Rd, Beijing (China); Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, 8 Gongti South Rd, Beijing (China); Xiao, Fei [Institute of Geriatrics, Beijing Hospital, 1 Dahua Rd, Beijing (China); National Clinical Research Center for Respiratory Diseases, 1 Dahua Rd, Beijing (China); Wang, Chen, E-mail: chenwangcjfh@163.com [Beijing Institute of Respiratory Medicine, Beijing Chao-yang Hospital, Capital Medical University, 8 Gongti South Rd, Beijing (China); Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, 8 Gongti South Rd, Beijing (China); National Clinical Research Center for Respiratory Diseases, 1 Dahua Rd, Beijing (China)

    2015-03-15

    Background: Different types of pulmonary hypertension (PH) share the same process of pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play an important role in this process. Objectives: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration. Methods: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured human PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated. Results: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level. In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly, and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased PASMCs proliferation and migration, inhibited the apoptosis. Conclusions: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2. - Highlights: • sGC activator BAY41-2272 up regulated PAI-2 in PASMCs, on the mRNA and protein level. • PAI-2 overexpression inhibits the proliferation and migration of human PASMCs. • PAI-2 overexpression promotes the apoptosis of human PASMCs. • sGC activator might alleviate the vascular remodeling in pulmonary hypertension.

  15. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. α2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [125I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

  16. Interventional effect of flunarizine on the expression of cyclooxygenase-2 and plasminogen activator inhibitor type-1 during experimental Cerebral ischemia/reperfusion in gerbils

    Wensheng Zhou; Zhiping Hu; Yan Hong

    2006-01-01

    BACKGROUND:Some researches suggest that induced cyclooxygenase-2 (COX-2) can cause brain injury through a series of ways at the phase of cerebral ischemia/hypoxia.Plasminogen activator inhibitor type-1(PAI-1)is a kind of inhibitor of serine stretch protein enzyme and is able to protect cell surface and microvascular basement membrane from degradation of protease and also protect contact surface among cells so as to maintain integrality of tissue structure.However,correlation of protective effect of flunarizine on brain with COX-2 and PAI-1 should be studied further.OBJECTIVE:To observe the effect of flunadzine on expressions of COX-2 and PAI-1 protein in forebrain and degree of brain injury among gerbils after cerebral ischemia.DESIGN:A randomized controlled animal study.SEITING:Department of Neurology,the Second Xiangya Hospital of Central South University;Department of Neurology,Mawangdui Hospital of Hunan Province.MATERIALS:A total of 40 healthy gerbils,of both genders,aged 9 months,weighing(90±10)g,were selected in this study.Anti-COX-2 multi-antibody,anti-PAI-1 multi-antibody,SABC immunohistochemical kit and DAB kit were provided by Wuhan Boster Biological Engineering Co.,Ltd.;and flunarizine capsule was provided by Xi'an Yangsen Pharmaceutical Company(batch number:041018726,dosage:5 mg/pill).METHODS:The experiment was Carried out in Laboratory of Mental Disease,Hunan Provincial Gedatdcs Institute affiliated by Hunan Provincial Mawangdui Hospital from January 2004 to March 2005.① All gerbils were randomly divided into cerebral ischemia group,flunarizine intervention group,sham operation group and normal control group with 10 in each group.Gerbils in normal control group were only cut off their heads.Gerbils in sham operation group were only dissected their bilateral common carotid arteries and sacdficad 1 day later.Gerbils in cerebral ischemia group and flunanzine intervention group were anesthetized,centrally cut open skin of neck,bluntly dissected

  17. Ginkgo biloba Extract (EGb 761®) Inhibits Glutamate-induced Up-regulation of Tissue Plasminogen Activator Through Inhibition of c-Fos Translocation in Rat Primary Cortical Neurons.

    Cho, Kyu Suk; Lee, Ian Myungwon; Sim, Seobo; Lee, Eun Joo; Gonzales, Edson Luck; Ryu, Jong Hoon; Cheong, Jae Hoon; Shin, Chan Young; Kwon, Kyoung Ja; Han, Seol-Heui

    2016-01-01

    EGb 761(®) , a standardized extract of Ginkgo biloba leaves, has antioxidant and antiinflammatory properties in experimental models of neurodegenerative disorders such as stroke and Alzheimer's disease. Tissue plasminogen activator (tPA) acts a neuromodulator and plays a crucial role in the manifestation of neurotoxicity leading to exaggerated neuronal cell death in neurological insult conditions. In this study, we investigated the effects of EGb 761 on the basal and glutamate-induced activity and expression of tPA in rat primary cortical neurons. Under basal condition, EGb 761 inhibited both secreted and cellular tPA activities, without altering tPA mRNA level, as modulated by the activation of p38. Compared with basal condition, EGb 761 inhibited the glutamate-induced up-regulation of tPA mRNA resulting in the normalization of overt tPA activity and expression. c-Fos is a component of AP-1, which plays a critical role in the modulation of tPA expression. Interestingly, EGb 761 inhibited c-Fos nuclear translocation without affecting c-Fos expression in glutamate-induced rat primary cortical neurons. These results demonstrated that EGb 761 can modulate tPA activity under basal and glutamate-stimulated conditions by both translational and transcriptional mechanisms. Thus, EGb 761 could be a potential and effective therapeutic strategy in tPA-excessive neurotoxic conditions. PMID:26478151

  18. NKp46 defines ovine cells that have characteristics corresponding to NK cells

    Connelley Timothy

    2011-02-01

    Full Text Available Abstract Natural killer (NK cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and γδ+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, but not all, express CD16 - characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+ populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+ populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+ cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species.

  19. A regulatory hydrophobic area in the flexible joint region of plasminogen activator inhibitor-1, defined with fluorescent activity-neutralizing ligands. Ligand-induced serpin polymerization

    Egelund, R; Einholm, A P; Pedersen, K E; Nielsen, R W; Christensen, A; Deinum, J; Andreasen, Peter A

    2001-01-01

    all tested nonfluorescent neutralizers, indicating that all neutralizers bind to a common hydrophobic area preferentially accessible in active PAI-1. Activity neutralization proceeded through two consecutive steps as follows: first step is conversion to forms displaying substrate behavior toward u...... having overlapping, but not identical, binding sites in the region around alpha-helices D and E and beta-strand 1A, known to act as a flexible joint when beta-sheet A opens and the reactive center loop inserts as beta-strand 4A during reaction with target proteinases. The defined binding area may be a...

  20. Type 1 plasminogen activator inhibitor (PAI-1 in clear cell renal cell carcinoma (CCRCC and its impact on angiogenesis, progression and patient survival after radical nephrectomy

    Seidal Tomas

    2010-12-01

    Full Text Available Abstract Background To examine the expression of type 1 plasminogen inhibitor (PAI-1 in clear cell renal cell carcinoma (CCRCC, and its possible association with microvessel density (MVD, the expression of thrombospondin-1 (TSP-1, nuclear grade, tumour stage, continuously coded tumour size (CCTS and to assess the value of PAI as a prognostic marker in 162 patients with CCRCC treated with radical nephrectomy. Methods A total of 172 consecutive patients with CCRCC treated with radical nephrectomy were enrolled in the study. The expression of PAI-1, TSP-1 and factor VIII were analysed on formalin-fixed, paraffin-embedded tissues without knowledge of the clinical outcome. Ten cases, where PAI-1 immunohistochemistry was not possible due to technical problems and lack of material, were excluded. Sixty-nine patients (43% died of RCC, while 47 patients (29% died of other diseases. Median follow-up was 13.8 years for the surviving 46 patients (28%. Results Nine percent of the tumours showed PAI-1 positivity. High expression of PAI-1 was significantly inversely correlated with TSP-1 (p = 0.046 and directly with advanced stage (p = 0.008, high NG (3+4 (p = 0.002, tumour size (p = 0.011, microvessel density (p = 0.049 and disease progression (p = 0.002. In univariate analysis PAI-1 was a significant prognosticator of cancer-specific survival (CSS (p Conclusions PAI-1 was found to be an independently significant prognosticator of CSS and a promoter of tumour angiogenesis, aggressiveness and progression in CCRCC.

  1. Role of plasminogen in propagation of scrapie.

    Salmona, Mario; Capobianco, Raffaella; Colombo, Laura; De Luigi, Ada; Rossi, Giacomina; Mangieri, Michela; Giaccone, Giorgio; Quaglio, Elena; Chiesa, Roberto; Donati, Maria Benedetta; Tagliavini, Fabrizio; Forloni, Gianluigi

    2005-09-01

    To investigate whether plasminogen may feature in scrapie infection, we inoculated plasminogen-deficient (Plg(-/-)), heterozygous plasminogen-deficient (Plg(+/-)), and wild-type (Plg(+/+)) mice by the intracerebral or intraperitoneal (i.p.) route with the RML scrapie strain and monitored the onset of neurological signs of disease, survival time, brain, and accumulation of scrapie disease-associated forms of the prion protein (PrP(Sc)). Only after i.p. inoculation, a slight, although significant, difference in survival (P follicular dendritic or lymphoid cells in Plg(-/-) mice. Once the disease progressed and mice began to die of infection, differences were no longer apparent in either brains or spleens. In conclusion, our data indicate that plasminogen has no major effect on the survival of scrapie agent-infected mice. PMID:16103174

  2. Effects of urokinase type plasminogen activator and plasminogen activator inhibitor-1 expressions on the formation of aneurysm of perimembranous ventricular septal defect%尿激酶型纤溶酶原激活物及其抑制物表达在膜周型室间隔缺损自发闭合中的作用

    钱娟; 李本尚; 殷敏智; 沈萍; 孙锟

    2015-01-01

    0.05).结论 uPA及抑制物系统在VSA形成过程中起重要作用,参与瘤体的形成和纤维增殖过程.%Objective The exact mechanisms of defect closure in patients with perimembranous ventricular septal defect (PMVSD) remain unknown.We hypothesized that the expression of urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) may mediate extracellular matrix (ECM) remodeling in aneurysms.Method Seven normal heart tricuspid septal leaflet and 33 aneurysms were collected in Shanghai Renji Hospital and Shanghai Children's Medical Center from January 2008 to June 2010.Immunohistochemical expression of uPA and PAI-1 in 4 normal heart valvular tissues and 15 aneurysms was detected with immunohistochemical methods.The expression of uPA and PAI-1 mRNA in 3 normal heart valvular tissues and 7 aneurysms was studied by real time fluorescent PCR;the protein expression of uPA and PAI-1 in 4 normal heart valvular tissues and 11 aneurysms was tested with Western blotting.Result The surface of the aneurysms were completely covered by endothelial cells.Two types of granulation tissue,myxoid and fibrous,were associated with the aneurismal formation.uPA were recognized predominantly in valvar interstitial cells (VICs) which located mainly in regions adjacent to the endothelium and smooth muscle cells of blood vessels.PAI-1 was found in both VICs which located mainly in granulation tissue and endothelial cells.Nine aneurysms expressed a higher uPA activity than 4 normal valvular tissues ((74.6 ± 11.8) % vs.(49.5 ± 7.4) %;t =3.87,P =0.003) and six aneurysms expressed a low uPA activity ((10.3±3.1)% vs.(49.5±7.4)%;t=11.78,P=0.000) andahighPAI-1 activity ((55.2±1.7) % vs.(50.8 ± 3.8) %;t =2.55,P =0.034) using immunohistochemical methods.uPA / PAI-1 ratio of protein expression tested by Western blot was 0.88 ± 0.22 in four normal heart vavular tissues;five aneurysms expressed high uPA activity and low PAI-1 activity and u

  3. Therapeutic benefits of combined treatment with tissue plasminogen activator and 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside in an animal model of ischemic stroke.

    Yu, Shu; Liu, Xin; Shen, Yuntian; Xu, Hui; Yang, Yumin; Ding, Fei

    2016-07-01

    Tissue plasminogen activator (tPA) is the only approved therapy for acute ischemic stroke, but tPA therapy is limited by a short therapeutic window and some adverse side effects. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside, a salidroside analog (code-named SalA-4g), has shown potent neuroprotective effects. In this study, a rat model of embolic middle cerebral artery occlusion (MCAO) was used to mimic ischemic stroke. The embolic MCAO rats were intravenously (iv) injected with tPA alone, SalA-4g alone, or a combination of tPA and SalA-4g. Compared to treatment with tPA alone at 4h post MCAO, combined treatment with tPA at 4h post MCAO and SalA-4g starting at 4h post MCAO and continuing for 3days at an interval of 24h significantly reduced neurological deficits and infarct volume, and significantly inhibited the intracerebral bleeding, edema formation, neuronal loss, and cellular apoptosis in the ischemic brain. Our results suggested that additive neuroprotective actions of SalA-4g contributed to widening the therapeutic window of tPA therapy and ameliorating its side effects in treating MCAO rats. The therapeutic benefits of combined treatment with tPA and SalA-4g for ischemic stroke might be associated with its effects on cerebral glucose metabolism. PMID:27060484

  4. Characterization of Plasminogen Binding to NB4 Promyelocytic Cells Using Monoclonal Antibodies against Receptor-Induced Binding Sites in Cell-Bound Plasminogen

    Mercè Jardí

    2012-01-01

    Full Text Available The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17 that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20–26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment.

  5. Mannheimia Species Associated with Ovine Mastitis▿

    Omaleki, Lida; Barber, Stuart R.; Allen, Joanne L.; Browning, Glenn F.

    2010-01-01

    Mannheimia glucosida, M. haemolytica, and M. ruminalis were isolated from cases of acute mastitis in ewes. M. glucosida was found to be a common cause of clinical mastitis in sheep. Selected phenotypic tests in addition to genotyping were needed to definitively identify Mannheimia species causing ovine mastitis.

  6. Functional heterodimerization of prolactin and growth hormone receptors by ovine placental lactogen.

    Herman, A; Bignon, C; Daniel, N; Grosclaude, J; Gertler, A; Djiane, J

    2000-03-01

    Although homo- or heterodimerization are common mechanisms for activation of cytokine receptors, cross-talk between two distinct receptors in this superfamily has been never shown. Here we show a physiologically relevant example indicating that such an interaction does occurs, thus raising the hypothesis that heterodimerization between distinct cytokine receptors may be a novel mechanism contributing to the diversity of cytokine signaling. These findings were documented using both surface plasmon resonance and gel filtration experiments and show that ovine placental lactogen (PL) heterodimerizes the extracellular domains (ECDs) of ruminant growth hormone receptor (GHR) and prolactin receptor (PRLR). We also show that PL or PL analogues that exhibit little or no activity in cells transfected with PRLRs and no activity in cells transfected with ovine GHRs exhibit largely enhanced activity in cells cotransfected with both PRLRs and GHRs. Furthermore, chimeric receptors consisting of cytosolic and transmembrane part of ovine GHR or ovine PRLR and ECDs of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha or beta were constructed. Upon transfection into Chinese hamster ovary cells along with reporter luciferase gene and stimulation by GM-CSF, a significant increase in luciferase activity occurred when GM-CSFR-alpha-PRLR and GM-CSFR-beta-GHR or GM-CSFR-alpha-GHR and GM-CSRR-beta-PRLR were cotransfected. In conclusion, we show that ovine PL is capable of functional heterodimerization of GHR and PRLR and that when their cytosolic parts, coupled to the ECD of GM-CSF receptors, are heterodimerized by GM-CSF, they are capable of transducing biological signal. PMID:10692427

  7. Plasminogen activator inhibitor-I-related regulation of procollagen I (α1 and α2) by antitransforming growth factor-β1 treatment during radiation-impaired wound healing

    Purpose: Plasminogen activator inhibitor (PAI)-1 mediates transforming growth factor-β1 (TGF-β1)-related signaling by stimulating collagen Type I synthesis in radiation-impaired wound healing. The regulation of α(I)-procollagen is contradictory in fibroblasts of different fibrotic lesions. It is not known whether anti-TGF-β1 treatment specifically inhibits α(I)-procollagen synthesis. We used an experimental wound healing study to address anti-TGF-β1-associated influence on α(I)-procollagen synthesis. Methods and Materials: A free flap was transplanted into the preirradiated (40 Gy) or nonirradiated neck region of Wistar rats: Group 1 (n = 8) surgery alone; Group 2 (n = 14) irradiation and surgery; Group 3 (n = 8) irradiation and surgery and anti-TGF-β1 treatment. On the 14th postoperative day, skin samples were processed for fibroblast culture, in situ hybridization for TGF-β1, immunohistochemistry, and immunoblotting for PAI-1, α1/α2(I)-procollagen. Results: Anti-TGF-β1 significantly reduced TGF-β1 mRNA (p 1 treatment in vivo significantly reduced α1(I)-procollagen protein (p 2(I)-procollagen expression. Conclusion: These results emphasize anti-TGF-β1 treatment to reduce radiation-induced fibrosis by decreasing α1(I)-procollagen synthesis in vivo. α1(I)-procollagen and α2(I)-procollagen might be differentially regulated by anti-TGF-β1 treatment. Increased TGF-β signaling in irradiated skin fibroblasts seemed to be reversible, as shown by a reduction in PAI-1 expression after anti-TGF-β1 treatment

  8. Antithrombin III (AT and recombinant tissue plasminogen activator (R-TPA used singly and in combination versus supportive care for treatment of endotoxin-induced disseminated intravascular coagulation (DIC in the neonatal pig

    Gardner Renee'

    2006-05-01

    Full Text Available Abstract Background Disseminated intravascular coagulation (DIC is a pathological disturbance of the complex balance between coagulation and anticoagulation that is precipitated by vascular injury, acidosis, endotoxin release and/or sepsis and characterized by severe bleeding and excessive clotting. The innately low levels of coagulation factors found in newborn infants place them at extremely high risk for DIC. Anecdotal reports suggest that either anticoagulant or fibrinolytic therapy may alleviate some of the manifestations of DIC. To test the hypothesis that replacement of both anticoagulants and fibrinolytics may improve survival and outcome better than either single agent or supportive care alone, we utilized a neonatal piglet model of endotoxin-induced DIC. Methods DIC was induced in twenty-seven neonatal pigs (7 to 14 days of age by intravenous administration of E. coli endotoxin (800 μg/kg over 30 min. The piglets were divided into 4 groups on the basis of treatment protocol [A: supportive care alone; B: Antithrombin III (AT, 50 μg/kg bolus, 25 μg/kg per hr continuous infusion and supportive care; C: Recombinant Tissue Plasminogen Activator (R-TPA, 25 μg/kg per hr continuous infusion and supportive care; D: AT, R-TPA and supportive care] and monitored for 3 primary outcome parameters (survival time, macroscopic and microscopic organ involvement and 4 secondary outcome parameters (hematocrit; platelet count; fibrinogen level; and antithrombin III level. Results Compared with supportive care alone, combination therapy with AT and R-TPA resulted in a significant improvement of survival time, hematocrit, AT level, macroscopic and microscopic organ involvement, p Conclusion The findings suggest that combining AT, R-TPA and supportive care may prove more advantageous in treating the clinical manifestations of DIC in this neonatal pig model than either single modality or supportive care alone.

  9. Protease inhibitors prevent plasminogen-mediated, but not pemphigus vulgaris-induced, acantholysis in human epidermis.

    Schuh, Theda; Besch, Robert; Braungart, Evelyn; Flaig, Michael J; Douwes, Kathrin; Sander, Christian A; Magdolen, Viktor; Probst, Christopher; Wosikowski, Katja; Degitz, Klaus

    2003-02-01

    Pemphigus is an autoimmune blistering disease of the skin and mucous membranes. It is caused by autoantibodies directed against desmosomes, which are the principal adhesion structures between epidermal keratinocytes. Binding of autoantibodies leads to the destruction of desmosomes resulting in the loss of cell-cell adhesion (acantholysis) and epidermal blisters. The plasminogen activator system has been implicated as a proteolytic effector in pemphigus. We have tested inhibitors of the plasminogen activator system with regard to their potential to prevent pemphigus-induced cutaneous pathology. In a human split skin culture system, IgG preparations of sera from pemphigus vulgaris patients caused histopathologic changes (acantholysis) similar to those observed in the original pemphigus disease. All inhibitors that were tested (active site inhibitors directed against uPA, tPA, and/or plasmin; antibodies neutralizing the enzymatic activity of uPA or tPA; substances interfering with the binding of uPA to its specific cell surface receptor uPAR) failed to prevent pemphigus vulgaris IgG-mediated acantholysis. Plasminogen-mediated acantholysis, however, was effectively antagonized by the synthetic active site serine protease inhibitor WX-UK1 or by p-aminomethylbenzoic acid. Our data argue against applying anti-plasminogen activator/anti-plasmin strategies in the management of pemphigus. PMID:12675525

  10. The Influence of Fibrin on the Reaction of Plasminogen Activation by Mut ant of Pro-Urokinase%纤维蛋白对尿激酶原激活纤溶酶 原反应动力学的影响

    党昕; 杨晶鑫; 茹强; 袁洪生; 茹炳根

    2001-01-01

    Because the influence of fibrin on the reaction of plasm inogen activation by various plasminogen activators is different, the kinetic co nstant of the reaction of plasminogen activation catalyzed by InB with and witho ut fibrin were detected. The result is: Kfibrinm=4.2 μmol*L -1,greater than the normal Km=0.379 μmol*L-1; kfib rincat=0.107 s-1,greater than the normal kcat=0.0165 s-1. The results suggest that existence of fibrin in the reaction system of plasminogen activation depress the affinity between InB and plasminog en, but accelerates the hydrolysis of plasminogen by InB. The count up effect is inhibition.%反应体系中存在的纤维蛋白(fibrin)对尿激酶(UK)、scu-PA以及组织 型纤溶酶原激活剂(t-PA)激活纤溶酶原(plasminogen)的反应有不同的作用:UK、t-PA激活plasminogen的反应可被反应体系中存在的fibrin所加强;fibrin对scu-PA激活 plasminogen反应的动力学常数无明显影响;但对小分子质量scu-PA与单链抗体的嵌合分子 激活plasminogen的反应起明显的抑制作用.为确定反应体系中存在的fibrin对scu-PA的K区 插 入突变体-InB激活plasminogen反应的影响,测定了在反应体系中存在fibrin的情况下的In B激活plasminogen反应的Kfibrinm以及kfibrincat.Kfibrinm=4.2 μmol*L-1,远远大于无fibrin时的Km=0.379 μmol*L-1,说明有fibrin存在时突变体InB与天然底物plasminogen的亲和 性降低了.kfibrincat=0.107 s-1,也远远大于无fibrin时kcat=0.0165 s-1,说明有fibrin存在时突变体InB对plasminogen的反 应活性增强了.原因可能是:与fibrin结合的plasminogen的构象发生了有利于被纤溶酶原激 活剂水解的变化.

  11. Evaluation of ovine milk clotting aptitude

    A. Caroli; Martini, M.

    2010-01-01

    A comparative study of the lactodynamographic parameters was carried out on ovine milk. Besides evaluating the repeatability and reproducibility of the analytical method, the influence of some variables such as the genetic type (three breeds), the kind of milk (whole or skimmed), and its concentration after reconstitution (12g or 20g /100 ml) was evaluated. The working plan involved 6 laboratories for the final statistic analyses, by the use of freeze-dried milk samples (adequately reconstitu...

  12. Problems concerning ovine milk clotting aptitude

    Martini, M.; Fortina, R.; Di Stasio, L; Chiofalo, L; A. Caroli; BOLLA, P.; E. Duranti; V. Piccolo; A. Zullo

    2003-01-01

    A comparative study of the lactodynamographic parameters was carried out on ovine milk. Besides evaluating the repeatability and reproducibility of the analytical method, the influence of some variables such as the genetic type (three breeds), the kind of milk (whole or skimmed), and its concentration after reconstitution (12g or 20g /100 ml) was evaluated. The working plan involved 6 laboratories for the final statistic analyses, by the use of freeze-dried milk samples (adequately reconstitu...

  13. Elevated Plasma Tissue-type Plasminogen Activator (t-PA) and Soluble Throm-bomodulin in Patients Suffering From Severe Acute Respiratory Syndrome (SARS) as a Possible Index for Prognosis and Treatment Strategy

    2005-01-01

    Objective To detect the presence of endothelial injury in patients with severe acute respiratory syndrome (SARS) via enhanced levels of tissue-type plasminogen activator (t-PA) and soluble thrombomodulin (sTM). Methods Case patients were from Xuanwu Hospital (Capital University of Medical Sciences, Beijing, China), and all of them met clinical criteria for SARS. Healthy controls were some of the hospital employees. Endothelial injury bio-markers tPA and sTM were detected by commercial ELISA-methods. Results Classic plasma markers of endothelial injury, tPA and sTM significantly elevated in SARS patients in comparison to controls [t-PA: 1.48±0.16 nmol/L versus 0.25±0.03 nmol/L (P<0.0001), and sTM: 0.26±0.06 nmol/L versus 0.14±0.02 nmol/L (P<0.05)]. The only patient who died had extremely high levels of these endothelial injury markers (t-PA: 2.77 nmol/L and sTM: 1.01 nmol/L). The likelihood ratio analysis indicated the excellent discriminating power for SARS at the optimal cut-point of 0.49 nmol/L for tPA and 0.20 nmol/L for sTM, respectively. Significant numerical correlations were found among these endothelial injury markers in SARS patients. The numerical coefficient of correlation Pearson r between t-PA and sTM was 0.5867 (P<0.05). Conclusion Increased plasma concentrations of tPA and sTM in patients with SARS suggest the possibility of endothelial injury. SARS patients might need anticoagulant therapy or fibrinolytic therapy in order to reverse intraalveolar coagulation, microthrombi formation, alveolar and interstitial fibrin deposition. It may not only provide a useful treatment and prognostic index but also allow a further understanding of the pathological condition of the disease.

  14. Combined mRNA expression levels of members of the urokinase plasminogen activator (uPA) system correlate with disease-associated survival of soft-tissue sarcoma patients

    Members of the urokinase-type plasminogen activator (uPA) system are up-regulated in various solid malignant tumors. High antigen levels of uPA, its inhibitor PAI-1 and its receptor uPAR have recently been shown to be associated with poor prognosis in soft-tissue sarcoma (STS) patients. However, the mRNA expression of uPA system components has not yet been comprehensively investigated in STS patients. The mRNA expression level of uPA, PAI-1, uPAR and an uPAR splice variant, uPAR-del4/5, was analyzed in tumor tissue from 78 STS patients by quantitative PCR. Elevated mRNA expression levels of PAI-1 and uPAR-del4/5 were significantly associated with clinical parameters such as histological subtype (P = 0.037 and P < 0.001, respectively) and higher tumor grade (P = 0.017 and P = 0.003, respectively). In addition, high uPAR-del4/5 mRNA values were significantly related to higher tumor stage of STS patients (P = 0.031). On the other hand, mRNA expression of uPA system components was not significantly associated with patients' survival. However, in STS patients with complete tumor resection (R0), high PAI-1 and uPAR-del4/5 mRNA levels were associated with a distinctly increased risk of tumor-related death (RR = 6.55, P = 0.054 and RR = 6.00, P = 0.088, respectively). Strikingly, R0 patients with both high PAI-1 and uPAR-del4/5 mRNA expression levels showed a significant, 19-fold increased risk of tumor-related death (P = 0.044) compared to the low expression group. Our results suggest that PAI-1 and uPAR-del4/5 mRNA levels may add prognostic information in STS patients with R0 status and distinguish a subgroup of R0 patients with low PAI-1 and/or low uPAR-del4/5 values who have a better outcome compared to patients with high marker levels

  15. Identification of a peroxisome proliferator responsive element (PPRE)-like cis-element in mouse plasminogen activator inhibitor-1 gene promoter

    PAI-1 is expressed and secreted by adipose tissue which may mediate the pathogenesis of obesity-associated cardiovascular complications. Evidence is presented in this report that PAI-1 is not expressed by preadipocyte, but significantly induced during 3T3-L1 adipocyte differentiation and the PAI-1 expression correlates with the induction of peroxisome proliferator-activated receptor γ (PPARγ). A peroxisome proliferator responsive element (PPRE)-like cis-element (-206TCCCCCATGCCCT-194) is identified in the mouse PAI-1 gene promoter by electrophoretic mobility shift assay (EMSA) combined with transient transfection experiments; the PPRE-like cis-element forms a specific DNA-protein complex only with adipocyte nuclear extracts, not with preadipocyte nuclear extracts; the DNA-protein complex can be totally competed away by non-labeled consensus PPRE, and can be supershifted with PPARγ antibody. Mutation of this PPRE-like cis-element can abolish the transactivation of mouse PAI-1 promoter mediated by PPARγ. Specific PPARγ ligand Pioglitazone can significantly induce the PAI-1 expression, and stimulate the secretion of PAI-1 into medium

  16. Parthenogenetic activation of zona-free ovine oocytes%绵羊去透明带卵母细胞的孤雌激活

    杜晨光; 张小宇; 郭志凯

    2012-01-01

    In this research the effects of two activation methods and different doses of activators on zona-intact and zona-free oocytes parthenogenetic development were compared to establish a simple nuclear transfer procedure. Zona-free oocytes were treated with 2.5μmol/L ionomycin and 2mmol/L 6-DMAP for the parthenogenetic activation,respectively,the rate of cleavage was 78.9%,and the development rate to blastocysts was 13.7%.Zona-intact oocytes were treated with 5μmol/L ionomycin and 2mmol/L 6-DMAP,respectively,and the rate of cleavage was 88.5% and the development rate to blastocysts was 14.7%.Zona-intact and zona-free oocytes cultured in WOWs were treated with 5μmol/L ionomycin and 6-DMAP for parthenogenetic activation,and the rates of cleavage were 70.4% and 69.2%,respectively.Zona-intact and zona-free oocytes were treated with 7% ethanol and 6-DMAP for parthenogenetic activation,and the rates of cleavage were 33.3% and 23.0%,respectively.The cleavage rates of oocytes treated with ionomycin were significant high than that with 7% ethanol.The results indicated that cleavage rate was not affected by zona-intact or zona-free of oocytes,but the cleavage oocytes cultured in WOWs did not develop to blastocyst.%为探索一种简化的绵羊核移植操作程序,比较了不同浓度离子霉素、两种不同激活方法对保留和去除透明带的绵羊卵母细胞孤雌发育的影响。结果,2.5μmol/L离子霉素+2mmol/L 6-DMAP激活去除透明带卵母细胞时,卵裂率为78.9%,囊胚发育率为13.7%;采用5μmol/L离子霉素+2mmol/L 6-DMAP方法激活保留透明带的卵母细胞时,卵裂率为88.5%,囊胚发育率为14.7%。在WOWs培养中,对去除和保留透明带的卵母细胞,用5μmol/L离子霉素+2mmol/L 6-DMAP激活时,卵裂率分别为70.4%和69.2%,用70mL/L无水乙醇+2mmol/L 6-DMAP激活时,卵裂率分别为33.3%和23.0%,离子霉素处理组的卵裂率显著高于无水乙醇处理组。结果表明,透明带的去除与否对

  17. Minimum intravenous infectious dose of ovine progressive pneumonia virus (OPPV)

    The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6 month-old lambs. Twelve groups of two 6 month-old lambs were inoculated intravenously with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titer...

  18. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection

    Dowall, Stuart David; Callan, Jo; Zeltina, Antra; Al-Abdulla, Ibrahim; Strecker, Thomas; Sarah K Fehling; Krähling, Verena; Bosworth, Andrew; Rayner, Emma; Taylor, Irene; Charlton, Sue; Landon, John; Cameron, Ian; Hewson, Roger; Nasidi, Abdulsalami

    2015-01-01

    The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOT...

  19. Plasminogen deficiency causes reduced corticospinal axonal plasticity and functional recovery after stroke in mice.

    Zhongwu Liu

    Full Text Available Tissue plasminogen activator (tPA has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg into plasmin. In this study, using plasminogen knockout (Plg-/- mice and their Plg-native littermates (Plg+/+, we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg+/+ and Plg-/- mice (n = 10/group were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo. A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA was injected into the left motor cortex to anterogradely label the corticospinal tract (CST. Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg+/+ and Plg-/- embryos. In Plg+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg-/- mice was significantly impaired compared to Plg+/+ mice (p0.82, p<0.01. Plg-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.

  20. Two types of abnormal genes for plasminogen in families with a predisposition for thrombosis

    The gene coding for plasminogen has been compared with several abnormal genes from Japanese patients by the polymerase chain reaction and DNA sequence analysis. Two types of abnormal genes coding for plasminogen were identified in these patients. In the type I mutation, a guanosine in GCT coding for Ala-601 near the active-site histidine was replaced by an adenosine resulting in ACT coding for threonine. This mutation was also shown by the loss of a cleavage site for Fnu4HI endonuclease, a restriction enzyme that recognizes GCTGC but not ACTGC. In the type II mutation, a guanosine in GTC coding for Val-355 was replaced by a thymidine resulting in TTC coding for phenylalanine. This change was readily shown by digestion with Ava II endonuclease, a restriction enzyme that recognized GGTCC and not GTTCC. The type I mutation has been found to be identical to a plasminogen variant identified in Japanese patients by amino acid sequence analysis and also detected by isoelectric focusing, whereas the type II mutation is a unique amino acid substitution in the connecting region between the third and fourth kringles in plasminogen. DNA sequence analysis also revealed that the abnormal genes carry several silent nucleotide substitutions located primarily within introns and 5' and 3' flanking regions

  1. The Glycosylation of Plasminogen Activator Inhibitor-1

    Skottrup, Peter; Pedersen, Katrine Egelund; Christensen, Anni; Thøgersen, Ida; Andreasen, Peter; Enghild, Jan Johannes

    sequences. Analyses of these mutants for the content of N-acetyl glucosamine showed that Asn209 and Asn265, but not Asn329, are glycosylated, in agreement with previous suggestions made on the basis of X-ray crystal structure analysis of PAI-1 expressed in CHO cells (Xue et al. (1998) Structure 6, 627......-636). In contrast, PAI-1, containing a total of 26 Ser and 26 Thr residues, which are potential targets for O-linked glycosylation, was found to be devoid of N-acetyl-galactosamine, demonstrating the absence of O-linked glycosylation. Analysis of PAI-1 variants with mutational inactivation of each of the...

  2. The Glycosylation of Plasminogen Activator Inhibitor-1

    Skottrup, Peter; Pedersen, Katrine Egelund; Christensen, Anni; Thøgersen, Ida; Andreasen, Peter; Enghild, Jan J.

    2002-01-01

    sequences. Analyses of these mutants for the content of N-acetyl glucosamine showed that Asn209 and Asn265, but not Asn329, are glycosylated, in agreement with previous suggestions made on the basis of X-ray crystal structure analysis of PAI-1 expressed in CHO cells (Xue et al. (1998) Structure 6, 627......-636). In contrast, PAI-1, containing a total of 26 Ser and 26 Thr residues, which are potential targets for O-linked glycosylation, was found to be devoid of N-acetyl-galactosamine, demonstrating the absence of O-linked glycosylation. Analysis of PAI-1 variants with mutational inactivation of each of the...

  3. The Glycosylation of Plasminogen Activator Inhibitor-1

    Skottrup, Peter Durand; Pedersen, Katrine Egelund; Christensen, Anni; Thøgersen, Ida; Andreasen, Peter; Enghild, Jan Johannes

    sequences. Analyses of these mutants for the content of N-acetyl glucosamine showed that Asn209 and Asn265, but not Asn329, are glycosylated, in agreement with previous suggestions made on the basis of X-ray crystal structure analysis of PAI-1 expressed in CHO cells (Xue et al. (1998) Structure 6, 627...

  4. The Glycosylation of Plasminogen Activator Inhibitor-1

    Skottrup, Peter; Pedersen, Katrine Egelund; Christensen, Anni; Thøgersen, Ida; Andreasen, Peter; Enghild, Jan Johannes

    sequences. Analyses of these mutants for the content of N-acetyl glucosamine showed that Asn209 and Asn265, but not Asn329, are glycosylated, in agreement with previous suggestions made on the basis of X-ray crystal structure analysis of PAI-1 expressed in CHO cells (Xue et al. (1998) Structure 6, 627...... sequences utilized for N-linked glycosylation by Fluorophore Assisted Carbohydrate Electrophoresis (FACE), showed a different N-linked glycosylation profile of the glycans at each of the 2 sites. The exact structure of the carbohydrate chains at each of these 2 sequences are being determined using MALDI...

  5. Does plasminogen activator inhibitor-1 drive lymphangiogenesis?

    Bruyère, Françoise; Melen-Lamalle, Laurence; Blacher, Silvia; Detry, Benoît; Masset, Anne; Lecomte, Julie; Lambert, Vincent; Maillard, Catherine; Høyer-Hansen, Gunilla; Lund, Leif R; Foidart, Jean-Michel; Noël, Agnès

    2010-01-01

    by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To...... induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by...

  6. Glucose metabolism and effect of acetate in ovine adipocytes.

    Yang, Y T; White, L S; Muir, L A

    1982-08-01

    Isolated ovine adipocytes were incubated in vitro with specifically labeled 14C-glucose in the presence or absence of acetate. The flux patterns of glucose carbon through major metabolic pathways were estimated. When glucose was added as the sole substrate, approximately equal portions of glucose carbon (10%) were oxidized to CO2 in the pentose phosphate pathway, in the pyruvate dehydrogenase reaction and in the citrate cycle. Fifteen percent of the glucose carbon was incorporated into fatty acids and 43% was released as lactate and pyruvate. Addition of acetate to the medium increased glucose carbon uptake by 1.5-fold. Most of this increase was accounted for by a sevenfold increase in the activity of the pentose phosphate pathway. Acetate increased glucose carbon fluxes via pentose phosphate pathway to triose phosphates, from triose phosphate to pyruvate, into glyceride glycerol, into lactate and pyruvate and into pyruvate dehydrogenase and citrate cycle CO2. Glucose carbon incorporated into fatty acids was decreased 50% by acetate while, carbon fluxes through the phosphofructokinase-aldolase reactions were not significantly increased. Results of this study suggest that, when glucose is the sole substrate, the conversion of glucose to fatty acids in ovine adipocytes may not be limited by the maximum capacity of hexokinase, the pentose phosphate pathway or enzymes involved in the conversion of triose phosphates to pyruvate and of pyruvate to fatty acid. Acetate increased glucose utilization apparently by increasing activity of the pentose phosphate pathway as a result of enhanced NADPH utilization for fatty acid synthesis. PMID:7142048

  7. Determination of Some Hydrodynamic Parameters of Ovine Serum Albumin Solutions Using Viscometric Measurements

    Monkos, Karol

    2005-01-01

    The influence of protein concentration and temperature on the viscosity of ovine serum albumin (OSA) solutions was studied. The Mooney equation and a modified Arrhenius formula were used to described the viscosity-concentration and viscosity-temperature dependence of the solutions, respectively. The effective specific volume, the activation energy and entropy of viscous flow for hydrated OSA were calculated. The axial ratio and the dimensions of the main semi-axes of hydrated OSA were establi...

  8. Production of bacteriocin-like substances by lactic acid bacteria isolated from regional ovine cheese

    Cássia Regina Nespolo; Adriano Brandelli

    2010-01-01

    Lactic acid bacteria (LAB) were isolated from ovine milk and cheeses manufactured in the South Region of Brazil. Among 112 bacterial isolates investigated, 59 were chosen through a screening for LAB. Among these 59 strains of LAB, 21% showed antimicrobial, proteolytic and lipolytic activities. Based on this screening, Lactobacillus plantarum LCN 17 and Lactobacillus rhamnosus LCN 43 were selected and tested for the production of bacteriocin-like substances (BLS). The BLS produced by both isol...

  9. Expression of ovine gamma interferon in Escherichia coli and Corynebacterium glutamicum.

    Billman-Jacobe, H; Hodgson, A L; Lightowlers, M; Wood, P. R.; Radford, A J

    1994-01-01

    Bacteria of two species, Escherichia coli and Corynebacterium glutamicum, were used as hosts to express recombinant ovine gamma interferon as a fusion protein with glutathione S-transferase. The recombinant gamma interferon produced by both bacteria was biologically active in vitro and was recognized by anti-gamma interferon monoclonal antibodies. E. coli produced large amounts of soluble recombinant protein which could be purified by a simple affinity chromatography method. Only a small frac...

  10. Molecular pathogenesis of plasminogen Hakodate: the second Japanese family case of severe type I plasminogen deficiency manifested late-onset multi-organic chronic pseudomembranous mucositis.

    Osaki, Tsukasa; Souri, Masayoshi; Song, Young-Seok; Izumi, Naohiro; Law, Ruby; Ichinose, Akitada

    2016-08-01

    A 64-year-old man first developed ligneous conjunctivitis at the age of 58 years after right pulmonary resection because of suspected cancer; otherwise, he had been healthy. Since then, he began to suffer from various forms of chronic pseudomembranous mucositis. Laboratory tests demonstrated that he had 7.8 % of plasminogen activity and 5.9 % of the normal antigen level. Thus, he was diagnosed as having severe type I plasminogen deficiency, making him the third case in Japan. DNA sequencing and PCR-restriction fragment length polymorphism analyses revealed that this patient was a compound heterozygote of a G-to-A missense mutation (G266E) in exon VIII and a g-to-a mutation at the obligatory splicing acceptor site in intron 12 (IVS12-1g>a). These two mutations were confirmed to be novel. Molecular modeling and splice site strength calculation predicted conformational disorder(s) for the Glu266 mutant and a drastic decrease in splicing efficiency for intron 12, respectively. Western blot analysis demonstrated that the patient contained a small amount of the normal-sized plasminogen protein. Mass spectrometric analysis of the patient's plasminogen revealed a peptide containing the wild-type Gly266 residue and no peptides with mutations at Glu266. However, he had never suffered from thrombosis. Low levels of fibrinogen/fibrin degradation products (FDP), D-dimer, and plasmin-α2-plasmin inhibitor complex clearly indicated a hypo-fibrinolytic condition. However, his plasma concentration of elastase-digested crosslinked FDPs was 4.8 U/mL, suggesting the presence of an on-going plasmin(ogen)-independent "alternative" fibrinolytic system, which may protect the patient from thrombosis. The patient has been free from recurrence of ligneous conjunctivitis for approximately 2.5 years. PMID:27193180

  11. A new homologous radioimmunoassay for ovine follicle stimulating hormone: development and characterization

    The development and characterization of an homologous radioimmunoassay for ovine FSH is described in detail. Two antisera obtained by immunization of rabbits either with 'native' FSH or by periodate oxidized FSH have been compared. These two antisera were chosen for their low cross-reactions with plasma proteins in the presence of highly purified labelled FSH. After saturation with a purified ovine LH preparation, these antisera measured similar FSH activities in the plasma. Cross-reactions with pituitary proteins were low (1-2% TSH, 0.02% LH and lower for other pituitary hormones tested: GH, Prol. and ACTH) and probably result from FSH contamination of these preparations. With each antiserum, inhibition curves with ovine plasma, crude pituitary extracts or purified preparations of FSH were parallel. The total recovery of exogenous FSH added to plasma demonstrated that plasma proteins do not interfere with the assay. Activities of purified FSH preparations by bioassay or by radioimmunoassay were similar for some and different for others. This observation is discussed. Lastly, a study of the effect of incubation volumes on the precision and the sensitivity facilitated a complete automatisation of the pipetting phase of the assay. Under these conditions, the intra and inter-assay precision were 5.5 to 8% and 17.5 to 19.7% within the range of 25 to 70% B/Bo and 46 to 60% B/Bo respectively and the sensitivity was 2.4 ng NIH-FSH-S3/ml plasma. These results suggest that the present assay, using either antiserum, can be used for specific quantitation of ovine FSH

  12. Effect of vacuum-assisted closure on the expression of urokinase-type plaminogen activator and urokinase-type plasminogen activator receptor in acute and chronic wounds healing%封闭负压引流技术对创面愈合过程纤溶酶原激活剂级联表达的影响

    李跃军; 曹大勇; 陈绍宗

    2006-01-01

    目的研究封闭负压引流技术对急慢性创面纤溶酶原激活剂(plasminogen activator,PA)级联中尿激酶型纤溶酶激活剂(urokinase-type plaminogen activator,uPA)和尿激酶型纤溶酶激活剂受体(urokinase-type plasminogen activator receptor,uPA)表达的影响.方法雄性小家猪5头,背部两侧形成急性创面,分为实验组和对照组,仅实验组动物接受封闭负压引流治疗.分别在治疗前和治疗后第1、3、6、9、12、18、25天于创缘切取标本,用兔抗人uPA和uPAR单克隆抗体按ABC程序进行免疫组织化学染色,并计算uPAA和uPAR的标记指数.人慢性创面6例,清创后进行持续封闭负压引流治疗,在治疗后第1、3、5、7天采集创面渗出液,并用ELISA法检测渗出液中uPA和uPAR的含量.结果猪急性创面在封闭负压引流治疗后uPA和uPAR均增加,在第3天达到高峰,然后急速下降,但实验组的基因表达量和染色强度均显著高于对照组.6例人慢性创面进行治疗前uPA和uPAR表达较多,随着治疗时间的延长而逐渐减少.结论封闭负压引流治疗既能上调急性创面伤口周围的表皮角质形成细胞的uPA和uPAR表达,使之迅速增殖迁移;又能通过抑制慢性创面uPA和uPAR表达,从而减少细胞外基质的降解.

  13. Protease inhibitors prevent plasminogen-mediated, but not pemphigus vulgaris-induced, acantholysis in human epidermis

    Schuh, T.; Besch, R; Braungart, E.; Flaig, M. J.; Douwes, K.; Sander, C. A.; Magdolen, V; Probst, C.; Wosikowski, K.; Degitz, K.

    2003-01-01

    Pemphigus is an autoimmune blistering disease of the skin and mucous membranes. It is caused by autoantibodies directed against desmosomes, which are the principal adhesion structures between epidermal keratinocytes. Binding of autoantibodies leads to the destruction of desmosomes resulting in the loss of cell-cell adhesion (acantholysis) and epidermal blisters. The plasminogen activator system has been implicated as a proteolytic effector in pemphigus. We have tested inhibitors of the plasmi...

  14. Proteomics-driven analysis of ovine whey colostrum.

    Domenica Scumaci

    Full Text Available The aim of this study was to shed light in to the complexity of the ovine colostrum proteome, with a specific focus on the low abundance proteins. The ovine colostrum is characterized by a few dominating proteins, as the immunoglobulins, but it also contains less represented protein species, equally important for the correct development of neonates. Ovine colostrum, collected immediately after lambing, was separated by 1D SDS-PAGE. Proteins bands were digested with trypsin and the resulting peptides were analyzed by LC-MS/MS. On the basis of the Swiss-Prot database, a total of 343 unique proteins were identified. To our knowledge, this study represents the most comprehensive analysis of ovine colostrum proteome.

  15. Histomorphometric comparison of the human, swine, and ovine collecting systems.

    Simões, Michele; de Souza, Diogo B; Gallo, Carla B M; Pereira-Sampaio, Marco A; Costa, Waldemar S; Sampaio, Francisco J

    2016-07-01

    The ovine kidney has been recently determined to be a better model than the swine kidney for the study of collecting system healing after partial nephrectomy. However, there is no histological study comparing the collecting systems of these species. To compare human, swine, and ovine collecting systems using histomorphometry. The collecting systems of 10 kidneys from each species (human, swine, and ovine) were processed for histomorphometry. The thickness of the three layers (mucosal connective tissue, submucosal muscular, and adventitial connective tissue) were measured. The densities of smooth muscle fibers, elastic system fibers, and cells were also measured. Additionally, blood vessel density in the adventitial connective tissue was measured. Analysis of the collecting systems from the three species presented several differences. The adventitial connective tissue from the swine samples was thicker, with more blood vessels and smooth muscle fibers, compared with that from the human and ovine samples. Swine also had higher density of elastic fibers on the submucosal muscular layer. Ovine and human collecting systems shared several similar features, such as blood vessel and elastic fiber density in all layers and the density of cellular and muscular fibers in the submucosal muscular and adventitial connective tissue layers. The collecting system of the ovine kidney is more similar to that of the human kidney compared with that of the swine kidney. This may explain the differences between the healing mechanisms in swine and those in humans and sheep after partial nephrectomy. Anat Rec, 299:967-972, 2016. © 2016 Wiley Periodicals, Inc. PMID:27111677

  16. Transmissibility of caprine scrapie in ovine transgenic mice

    O’Rourke Katherine I

    2012-04-01

    Full Text Available Abstract Background The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338 expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats. Results First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports. Conclusions These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.

  17. Construction and identification of urokinase-type plasminogen activator biosensor plasmid%尿激酶型纤溶酶原激活物生物传感器的构建及鉴定

    崔钢华; 饶烽; 王琰; 曹薇薇; 刘伟; 王维山; 史晨辉

    2015-01-01

    目的:构建含有增强型青色荧光蛋白-尿激酶型纤溶酶原激活物(uPA)作用底物(substrate)-黄色荧光蛋白变体(YPet)融合蛋白的真核表达载体(ECFP-uPA substrate-linker-YPet),即 uPA 的生物传感器。方法:以 Src-biosensor 为模板,Primer Premier 5.0软件设计 YPet 引物,设计时5′端引入 uPA 底物序列及Linker,两端连接酶切位点及保护碱基。以 pMDTM-18T 为中间载体,通过基因工程方法构建含有 ECFP-uPA substrate-linker-YPet 的真核表达载体。然后转染293T 细胞,24 h 后观察转染效率和融合蛋白表达情况,在荧光显微镜下,应用 MetaFlour FRET 4.6软件观察并测量 uPA 生物传感器荧光共振能量转移(FRET)。结果:经过 PCR和双酶切鉴定,克隆片段和酶切片段均与 uPA substrate 分子大小相符。细胞转染后转染效率达40%。免疫荧光检测,uPA 生物传感器在293T 细胞膜表达,用重组人 uPA (rhuPA)刺激转染细胞可以检测到FRET 现象。结论:成功构建 uPA 生物传感器,该生物传感器能够作为活细胞分子探针用于研究 uPA 的时空变化。%Objective To construct the eukaryotic expression vector urokinase-type plasminogen activator (uPA) biosensor which was the composition of the fusion protein enhanced cyan fluorescent protein-uPA (substrate)-yellow fluorescent protein variant (ECFP-uPA substrate-linker-YPet).Methods By the template Src-biosensor, the YPet primers were designed by Primer Premier 5.0 software,and the restriction enzyme sites,uPA substrate gene sequence and linker were added in its 5′ end. With the intermediate vector pDMTM-18T, an eukaryotic expression vector which contained a fusion protein of ECFP-uPA substrate-linker-YPet was constructed by genetic engineering.Then the uPA biosensor was transfected into 293T cells.The transfection efficiency and expression of fusion proteins were observed after 24 h

  18. Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro.

    Morioka, Hideo; Morii, Takeshi; Vogel, Tikva; Hornicek, Francis J; Weissbach, Lawrence

    2003-07-01

    Plasminogen-related protein B (PRP-B) closely resembles the N-terminal plasminogen activation peptide, which is released from plasminogen during conversion to plasmin. We have previously demonstrated that the steady-state level of mRNA encoding PRP-B is increased within tumor tissues, and that recombinant PRP-B antagonizes neoplastic growth when administered systemically to mice harboring tumors, but no insights into the cell targets of PRP-B have been presented. Employing serum-free medium optimized for culturing human endothelial or smooth muscle cells, we show that recombinant PRP-B inhibits basic fibroblast growth factor-dependent cell migration for both cell types, as well as tube formation of endothelial cells. Comparison with the angiogenesis inhibitors angiostatin and endostatin revealed similar results. Recombinant PRP-B is effective in promoting cell attachment of endothelial and smooth muscle cells, and antibody interference experiments reveal that the interaction of recombinant PRP-B with endothelial cells is mediated at least in part by alpha(v)-containing integrins. Inhibition of angiogenesis in vivo by PRP-B was demonstrated in the chicken chorioallantoic membrane assay. PRP-B and other antiangiogenic molecules may elicit metabolic perturbations in endothelial cells as well as perivascular mesenchymal cells such as smooth muscle cells and pericytes. PMID:12799192

  19. The Biochemistry and Regulation of S100A10: A Multifunctional Plasminogen Receptor Involved in Oncogenesis

    Patricia A. Madureira

    2012-01-01

    Full Text Available The plasminogen receptors mediate the production and localization to the cell surface of the broad spectrum proteinase, plasmin. S100A10 is a key regulator of cellular plasmin production and may account for as much as 50% of cellular plasmin generation. In parallel to plasminogen, the plasminogen-binding site on S100A10 is highly conserved from mammals to fish. S100A10 is constitutively expressed in many cells and is also induced by many diverse factors and physiological stimuli including dexamethasone, epidermal growth factor, transforming growth factor-α, interferon-γ, nerve growth factor, keratinocyte growth factor, retinoic acid, and thrombin. Therefore, S100A10 is utilized by cells to regulate plasmin proteolytic activity in response to a wide diversity of physiological stimuli. The expression of the oncogenes, PML-RARα and KRas, also stimulates the levels of S100A10, suggesting a role for S100A10 in pathophysiological processes such as in the oncogenic-mediated increases in plasmin production. The S100A10-null mouse model system has established the critical role that S100A10 plays as a regulator of fibrinolysis and oncogenesis. S100A10 plays two major roles in oncogenesis, first as a regulator of cancer cell invasion and metastasis and secondly as a regulator of the recruitment of tumor-associated cells, such as macrophages, to the tumor site.

  20. Genetic architecture of gene expression in ovine skeletal muscle

    Kogelman Lisette JA

    2011-12-01

    Full Text Available Abstract Background In livestock populations the genetic contribution to muscling is intensively monitored in the progeny of industry sires and used as a tool in selective breeding programs. The genes and pathways conferring this genetic merit are largely undefined. Genetic variation within a population has potential, amongst other mechanisms, to alter gene expression via cis- or trans-acting mechanisms in a manner that impacts the functional activities of specific pathways that contribute to muscling traits. By integrating sire-based genetic merit information for a muscling trait with progeny-based gene expression data we directly tested the hypothesis that there is genetic structure in the gene expression program in ovine skeletal muscle. Results The genetic performance of six sires for a well defined muscling trait, longissimus lumborum muscle depth, was measured using extensive progeny testing and expressed as an Estimated Breeding Value by comparison with contemporary sires. Microarray gene expression data were obtained for longissimus lumborum samples taken from forty progeny of the six sires (4-8 progeny/sire. Initial unsupervised hierarchical clustering analysis revealed strong genetic architecture to the gene expression data, which also discriminated the sire-based Estimated Breeding Value for the trait. An integrated systems biology approach was then used to identify the major functional pathways contributing to the genetics of enhanced muscling by using both Estimated Breeding Value weighted gene co-expression network analysis and a differential gene co-expression network analysis. The modules of genes revealed by these analyses were enriched for a number of functional terms summarised as muscle sarcomere organisation and development, protein catabolism (proteosome, RNA processing, mitochondrial function and transcriptional regulation. Conclusions This study has revealed strong genetic structure in the gene expression program within

  1. Storage and lyophilization effects of extracts of Cynara cardunculus on the degradation of ovine and caprine caseins

    Tavaria, Freni K.; Sousa, M. José; Malcata, F. Xavier

    2001-01-01

    Clotting and proteolytic activities are important parameters when evaluating rennets for cheesemaking. Both these activities were determined for extracts of the plant Cynara cardunculus in fresh form and after lyophilization followed by reconstitution, either in water or in citrate buffer (pH 5.4) and stored for up to 4 weeks at 4°C. The patterns of degradation of ovine and caprine caseins were followed by urea polyacrylamide gel electrophoresis in attempts to qualitatively differentiate the ...

  2. Expression and characterization of novel ovine orthologs of bovine placental prolactin-related proteins

    Ohkoshi Katsuhiro

    2007-10-01

    Full Text Available Abstract Background The prolactin-related proteins (PRPs are non-classical placental-specific members of the prolactin/growth hormone family. Among ruminants, they are expressed in the cotyledonary villi of cattle and goat. We investigated placental PRP in sheep in order to gain a comprehensive understanding of the function and evolution of these molecules. We also examined the sequence properties, expression and lactogenic activation of the cloned genes. Results We cloned two novel ovine PRPs, named oPRP1 and oPRP2. oPRP2 had a typical PRP sequence similar to bovine PRP1 (bPRP1. oPRP1 had a short sequence identical with bovine or caprine type PRP but the reading frame was shifted. Both oPRPs were expressed in trophoblast giant binucleate cells (BNC as in cattle and goat. oPRP1 expression declined from the early to the middle stage of gestation. In contrast, oPRP2 expression remained constant throughout the gestation period. oPRP2 was translated to form a mature protein in a mammalian cell expression system. Western blotting showed a molecular mass of 35 kDa for the FLAG-tag fusion oPRP2 protein. This recombinant protein and bPRP1 were bioassayed using Nb2 lymphoma cells; it was confirmed that neither ruminant PRP had lactogenic activity because the Nb2 lymphoma cells did not proliferate. Conclusion We have identified two novel PRPs, oPRP1 and oPRP2, in ovine placenta. Both these ovine PRPs were localized and quantitatively expressed in BNC. Absence of lactogenic activity was confirmed for the oPRP2 molecule. It is anticipated that novel and known ruminant PRPs have common functions, except for lactogenic activity.

  3. 77Se NMR studies on ovine erythrocyte glutathione peroxidase

    To facilitate 77Se NMR observation of the endogenous active site selenium in ovine erythrocyte glutathione peroxidase (GSHPx), lambs have been maintained on an artificial diet deficient in selenium and supplemented with 0.2 ppm 92atom% 77Se , as selenite. After 5 months, preparations of GSHPx showed that incorporation of selenium from the artificial diet represented 88% of the GSHPx selenium. Each monthly bleeding of two sheep routinely yielded 20mg of pure 77Se-enriched GSHPx. Limitations on the solubility of the enzyme have so far prevented observation of 77Se resonances from the intact enzyme. Upon denaturation, a broad resonance is observed at 277 ppm, indicating that the selenium is involved in mixed selenide sulfide bonds both inter and intramolecularly. Reduction of the SeS bonds with dithiothreitol resulted in an upfield shift of the 77Se resonance to -212 ppm at pH 8 and -55ppm at pH4.2, consistent with formation of Se- and SeH respectively. It is concluded that the selenium is most probably in the SeS or Se- form in the intact enzyme. Relaxation time measurements were made at field strengths of 4.7 and 9.4T, which demonstrated the dominance of chemical shift anisotropy (CSA) relaxation for the selenium in GSHPx. A value of ≤ 262 ppm was determined for the CSA of the iodoacetamide derivative of GSHPx

  4. Staphylococcus aureus manganese transport protein C (MntC is an extracellular matrix- and plasminogen-binding protein.

    Natália Salazar

    Full Text Available Infections caused by Staphylococcus aureus--particularly nosocomial infections--represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM. Manganese transport protein C (MntC, a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA. The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

  5. Aberrant DNA methylation in cloned ovine embryos

    LIU Lei; HOU Jian; LEI TingHua; BAI JiaHua; GUAN Hong; AN XiaoRong

    2008-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of cloned ovine embryos. The em-bryos derived from in vitro fertilization were also examined for reference purpose. The results showed that: (1) during the pre-implantation development, cloned embryos displayed a similar demethylation profile to the fertilized embryos; that is, the methylation level decreased to the lowest at 8-cell stage, and then increased again at morulae stage. However, methylation level was obviously higher in cloned embryos than in stage-matched fertilized embryos, especially at 8-cell stage and afterwards; (2) at blastocyst stage, the methylation pattern in cloned embryos was different from that in fertilized em-bryos. In cloned blastocyst, inner cell mass (ICM) exhibited a comparable level to trophectoderm cells (TE), while in in-vitro fertilized blastocyst the methylation level of ICM was lower than that of TE, which is not consistent with that reported by other authors. These results indicate that DNA methylation is abnormally reprogrammed in cloned embryos, implying that aberrant DNA methylation reprogramming may be one of the factors causing cloned embryos developmental failure.

  6. Evaluation of ovine milk clotting aptitude

    A. Caroli

    2010-01-01

    Full Text Available A comparative study of the lactodynamographic parameters was carried out on ovine milk. Besides evaluating the repeatability and reproducibility of the analytical method, the influence of some variables such as the genetic type (three breeds, the kind of milk (whole or skimmed, and its concentration after reconstitution (12g or 20g /100 ml was evaluated. The working plan involved 6 laboratories for the final statistic analyses, by the use of freeze-dried milk samples (adequately reconstituted on the basis of established methods from Sardinia, Comisana, and Massese ewes. All the considered variability factors showed a highly significant effect (P<0.001 on the lactodynamographic parameters considered. In particular, Massese ewe milk showed the shortest curd speed (k20 and the best coagulum strength (a30 and a45, although clotting time (CT was the highest one. The same trend was registered for skimmed milk and for the most concentrated one (20g. Repeatability values within laboratories were 96% and 97% for CT and k20, lowering for a30 e a45, (respectively 87% and 85%. Much lower coefficients were found for the among laboratories reproducibility, ranging from a maximum of 58% for CT to a minimum of 18% for k20. The wide variability observed indicates that lactodynamographic parameters are comparable only within the same lab. Further investigation is needed to compare different labs in order to obtain more homogeneous results.

  7. Problems concerning ovine milk clotting aptitude

    M. Martini

    2003-01-01

    Full Text Available A comparative study of the lactodynamographic parameters was carried out on ovine milk. Besides evaluating the repeatability and reproducibility of the analytical method, the influence of some variables such as the genetic type (three breeds, the kind of milk (whole or skimmed, and its concentration after reconstitution (12g or 20g /100 ml was evaluated. The working plan involved 6 laboratories for the final statistic analyses, by the use of freeze-dried milk samples (adequately reconstituted on the basis of established methods from Sardinia, Comisana, and Massese ewes. All the considered variability factors showed a highly significant effect (P<0.001 on the lactodynamographic parameters considered. In particular, Massese ewe milk showed the shortest curd speed (k20 and the best coagulum strength (a30 and a45, although clotting time (CT was the highest one. The same trend was registered for skimmed milk and for the most concentrated one (20g. Repeatability values within laboratories were 96% and 97% for CT and k20, lowering for a30 e a45, (respectively 87% and 85%. Much lower coefficients were found for the among laboratories reproducibility, ranging from a maximum of 58% for CT to a minimum of 18% for k20. The wide variability observed indicates that lactodynamographic parameters are comparable only within the same lab. Further investigation is needed to compare different labs in order to obtain more homogeneous results.

  8. Chemical modification studies of the streptokinase-plasminogen interaction

    The streptokinase (SK) interaction with human plasminogen (Pg) was investigated by differential chemical modification. In separate experiments, available lysine residues in both free streptokinase and streptokinase in complex with Pg were trace labeled by reaction with high specific activity [3H]-acetic anhydride at a reagent-to-lysine molar ratio of 0.5%. The [3H]-acetyl-SK from the complex was reisolated. Both the free and complex forms of 3H-acetyl-SK were then each mixed with uniformly and quantitatively modified [14C]-acetyl-SK in a 3H/14C isotopic ratio of ten-to-one. Each of the SK forms was fragmented by reaction with CNBr. These CNBr fragments, which were purified by Sephadex G-75 chromatography, were further cleaved by proteases to produce peptides containing a minimum number of lysines. After the isolation of peptides by reversed-phase HPLC, the 3H/14C ratios of lysines were individually determined. By comparison of the 3H/14C ratio's in the free-SK and complex-SK it was found that the majority of lysines did not change its reactivities in free or complex forms. However, several lysines were relatively unreactive in the SK complexed with Pg as compared to that in free SK. This suggests that these residues are either in the complex binding interface or they were less reactive as a result of conformational change induced by complex formation

  9. Leptin Matures Aspects of Lung Structure and Function in the Ovine Fetus.

    De Blasio, Miles J; Boije, Maria; Kempster, Sarah L; Smith, Gordon C S; Charnock-Jones, D Stephen; Denyer, Alice; Hughes, Alexandra; Wooding, F B Peter; Blache, Dominique; Fowden, Abigail L; Forhead, Alison J

    2016-01-01

    In human and ovine fetuses, glucocorticoids stimulate leptin secretion, although the extent to which leptin mediates the maturational effects of glucocorticoids on pulmonary development is unclear. This study investigated the effects of leptin administration on indices of lung structure and function before birth. Chronically catheterized singleton sheep fetuses were infused iv for 5 days with either saline or recombinant ovine leptin (0.5 mg/kg · d leptin (LEP), 0.5 LEP or 1.0 mg/kg · d, 1.0 LEP) from 125 days of gestation (term ∼145 d). Over the infusion, leptin administration increased plasma leptin, but not cortisol, concentrations. On the fifth day of infusion, 0.5 LEP reduced alveolar wall thickness and increased the volume at closing pressure of the pressure-volume deflation curve, interalveolar septal elastin content, secondary septal crest density, and the mRNA abundance of the leptin receptor (Ob-R) and surfactant protein (SP) B. Neither treatment influenced static lung compliance, maximal lung volume at 40 cmH2O, lung compartment volumes, alveolar surface area, pulmonary glycogen, protein content of the long form signaling Ob-Rb or phosphorylated signal transducers and activators of transcription-3, or mRNA levels of SP-A, C, or D, elastin, vascular endothelial growth factor-A, the vascular endothelial growth factor receptor 2, angiotensin-converting enzyme, peroxisome proliferator-activated receptor γ, or parathyroid hormone-related peptide. Leptin administration in the ovine fetus during late gestation promotes aspects of lung maturation, including up-regulation of SP-B. PMID:26479186

  10. Late results of catheter-directed recombinant tissue plasminogen activator fibrinolytic therapy of iliofemoral deep venous thrombosis Resultados de longo prazo do tratamento fibrinolítico da trombose venosa iliacofemoral por infusão seletiva do ativador de plasminogênio tissular recombinante em baixas doses

    Ivan Benaduce Casella

    2007-02-01

    Full Text Available PURPOSE: To evaluate the efficacy of catheter-directed low-dose recombinant tissue-type plasminogen activator infusion in the treatment of iliofemoral deep venous thrombosis and prevention of post-thrombotic syndrome. METHOD: Eighteen patients (out of 260 evaluated with acute iliofemoral deep venous thrombosis and no previous evidence of venous insufficiency were prospectively selected for thrombolytic therapy. Catheter-directed low-dose recombinant tissue-type plasminogen activator (1 mg/h was infused into the thrombotic segments. RESULTS: Effective fibrinolysis was achieved in 14 of 18 cases, with correlation between effective fibrinolysis and major/complete resolution of acute signs and symptoms (P OBJETIVOS: Avaliar a eficácia da infusão seletiva por cateter do ativador de plasminogênio tecidual recombinante em baixas doses no tratamento da trombose venosa iliacofemoral e na prevenção da síndrome pós-trombótica. MÉTODO: Dezoito pacientes (de 260 avaliados portadores de trombose venosa profunda iliacofemoral sem evidência prévia de insuficiência venosa foram selecionados para terapia fibrinolítica e submetidos a infusão seletiva por cateter do ativador de plasminogênio tecidual recombinante na dose de 1mg/dl nos segmentos venosos trombóticos. RESULTADOS: Quatorze pacientes apresentaram fibrinólise efetiva; observamos correlação entre o grau de melhora clínica observado e a redução percentual do volume trombótico (P<.01. Não houve episódios de complicações graves. Quatro pacientes apresentaram retrombose precoce (1 a 8 semanas. Os pacientes foram seguidos por um período de até 131 semanas (média 85.2. A incidência de sinais e sintomas clínicos de insuficiência venosa e os achados ecográficos de refluxo valvular foram significativamente menores nos pacientes em que a terapia fibrinolítica foi efetiva e a perviedade mantida ao longo do período de seguimento, na comparação com os casos de falha aguda ou de

  11. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection

    Dowall, Stuart David; Callan, Jo; Zeltina, Antra; Al-Abdulla, Ibrahim; Strecker, Thomas; Fehling, Sarah K.; Krähling, Verena; Bosworth, Andrew; Rayner, Emma; Taylor, Irene; Charlton, Sue; Landon, John; Cameron, Ian; Hewson, Roger; Nasidi, Abdulsalami; Bowden, Thomas A.; Carroll, Miles W.

    2016-01-01

    The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease. PMID:26715676

  12. Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue.

    Cavalcante, A Y P; Gouveia, B B; Barberino, R S; Lins, T L B G; Santos, L P; Gonçalves, R J S; Celestino, J J H; Matos, M H T

    2016-08-01

    This study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days of in vitro culture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) after in vitro culture of ovine ovarian tissue. PMID:26503557

  13. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection.

    Dowall, Stuart David; Callan, Jo; Zeltina, Antra; Al-Abdulla, Ibrahim; Strecker, Thomas; Fehling, Sarah K; Krähling, Verena; Bosworth, Andrew; Rayner, Emma; Taylor, Irene; Charlton, Sue; Landon, John; Cameron, Ian; Hewson, Roger; Nasidi, Abdulsalami; Bowden, Thomas A; Carroll, Miles W

    2016-04-01

    The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease. PMID:26715676

  14. 78 FR 68327 - Importation of Ovine Meat From Uruguay

    2013-11-14

    ... effective and published in the Federal Register on May 29, 2003 (68 FR 31940-31949, Docket No. 02-109-3... FR 10266-10269, Docket No. APHIS-2008-0085), we proposed to also allow the importation of fresh ovine... establishment, with no evidence found of vesicular disease. (f) The meat consists only of bovine parts or...

  15. Experimental infection of cattle with ovine Dichelobacter nodosus isolates

    Knappe-Poindecker, Maren; Jørgensen, Hannah Joan; Jensen, Tim Kåre;

    2015-01-01

    Dichelobacter nodosus is the main causative agent of ovine footrot, and there are strong indications that the bacterium can be transferred to cattle grazing on the same pasture as sheep. The aim of this study was to investigate if benign and virulent D. nodosus strains isolated from sheep can be ...

  16. 76 FR 10266 - Importation of Ovine Meat From Uruguay

    2011-02-24

    ... (68 FR 31940-31949, Docket No. 02-109-3), we amended the regulations to authorize the importation of... premises. (c) The meat comes from bovines and sheep that originate from premises where foot-and-mouth... beef and ovine meat to the United States. (d) The meat comes from bovines and sheep that were...

  17. Isolation and characterization of bifidobacteria from ovine cheese

    Bunešová, V.; Killer, Jiří; Vlková, E.; Musilová, S.; Tomáška, M.; Rada, V.; Kmeť, V.

    2014-01-01

    Roč. 188, č. 1 (2014), s. 26-30. ISSN 0168-1605 R&D Projects: GA ČR GA13-08803S Institutional support: RVO:67985904 Keywords : Bifidobacterium sp. * ovine cheese * cultivation Subject RIV: EE - Microbiology, Virology Impact factor: 3.082, year: 2014

  18. PbsP, a cell wall-anchored protein that binds plasminogen to promote hematogenous dissemination of group B Streptococcus.

    Buscetta, Marco; Firon, Arnaud; Pietrocola, Giampiero; Biondo, Carmelo; Mancuso, Giuseppe; Midiri, Angelina; Romeo, Letizia; Galbo, Roberta; Venza, Mario; Venza, Isabella; Kaminski, Pierre-Alexandre; Gominet, Myriam; Teti, Giuseppe; Speziale, Pietro; Trieu-Cuot, Patrick; Beninati, Concetta

    2016-07-01

    Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine-rich region, and a LPXTG cell wall-anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface-bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non-CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium. PMID:26888569

  19. A genetically engineered human Kunitz protease inhibitor with increased kallikrein inhibition in an ovine model of cardiopulmonary bypass.

    Ohri, S K; Parratt, R; White, T; Becket, J; Brannan, J J; Hunt, B J; Taylor, K M

    2001-05-01

    A recombinant human serine protease inhibitor known as Kunitz protease inhibitor (KPI) wild type has functional similarities to the bovine Kunitz inhibitor, aprotinin, and had shown a potential to reduce bleeding in an ovine model of cardiopulmonary bypass (CPB). The aim of this study was to assess KPI-185, a modification of KPI-wild type that differs from KPI-wild type in two amino acid residues and which enhances anti-kallikrein activity in a further double-blind, randomized study in an ovine model of CPB, and to compare with our previous study of KPI-wild type and aprotinin in the same ovine model. Post-operative drain losses and subjective assessment of wound 'dryness' showed no significant differences between KPI-185 and KPI-wild type, despite the significant enhancement of kallikrein inhibition using KPI-185 seen in serial kallikrein inhibition assays. These preliminary findings support the hypothesis that kallikrein inhibition is not the major mechanism by which Kunitz inhibitors such as aprotinin reduce perioperative bleeding. PMID:11419655

  20. Ingestive Behavior of Ovine Fed with Marandu Grass Silage Added with Naturally Dehydrated Brewery Residue.

    de Jesus Ferreira, Daniele; de Moura Zanine, Anderson; de Paula Lana, Rogério; Lima de Souza, Alexandre; Divino Ribeiro, Marinaldo; Mattos Negrão, Fagton; Castro, Wanderson José Rodrigues; Nunes Parente, Henrique; Valério Geron, Luiz Juliano; de Azevedo Câmara, Larissa Rodrigues

    2016-01-01

    The objective was to evaluate the ingestive behavior of ovine fed Marandu grass silage with dehydrated brewery residue added. The experiment had a completely randomized design with five treatments and four repetitions, with the treatments levels of inclusion being of 0, 10, 20, 30, and 40% natural matter of naturally dehydrated brewery residue for 36 hours to the marandu grass silage. 20 ovines were used and the experimental period was 21 days, 15 being for adaptation to diets. The use of brewery byproduct promoted quadratic effect (P rumination efficiency of dry matter (g DM/hour) were significant (P rumination efficiency showed crescent linear behavior. The DM and NDF consumption expressed in kg/meal and in minutes/kg were also significant (P Rumination activity expressed in g DM and NDF/piece was influenced (P < 0.05) by the adding of brewery residue in marandu grass silage in quadratic way, with maximum value estimated of 1.57 g DM/bolus chewed in inclusion of 24.72% additive in grass silage. The conclusion is that intermediary levels adding of 20 to 25% dehydrated brewery residue affects certain parameters of ingestive behavior. PMID:27547811

  1. Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.

    Tomasz Brzoska

    Full Text Available The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP. The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.

  2. Detection of streptomycin residues in local meat of bovine and ovine

    O. A. Abdullah; A. M. Shareef; O. H. Sheet

    2012-01-01

    From meat retails in Mosul province, forty-five meat samples of local ovine and bovine (23 bovine samples and 22 ovine samples) were collected. The period of collection was during November 2010 to May 2011, by means of multistage random sampling for detection of streptomycin residues. Enzyme linked immunosorbent assay (ELISA) was used for detection of streptomycin residues. The results revealed that eleven ovine meat samples (50%) were positive to streptomycin residue, with a mean value 35.06...

  3. High level expression of kringle 5 fragment of plasminogen in Pichia pastoris.

    Zhou, Yufei; Zheng, Quan; Gao, Jin; Gu, Jun

    2005-02-01

    Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300 mgl(-1). Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5. PMID:15717125

  4. Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

    Gozzo A.J.

    2003-01-01

    Full Text Available Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates reduced (1.2 to 3.0 times the catalytic efficiency of kallikrein (in a nanomolar range on the hydrolysis of plasminogen (0.3 to 1.8 µM and increased (1.9 to 7.7 times the enzyme efficiency in factor XII (0.1 to 10 µM activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times kallikrein inhibition by antithrombin (1.4 µM, while chondroitin 4- and 6-sulfates reduced it (1.3 times. Heparin and heparan sulfate increased (1.4 times the enzyme inhibition by the C1-inhibitor (150 nM.

  5. A monoclonal antibody to pestviruses in bovine and ovine sera

    An enzyme-linked immunoabsorbent assay (ELISA) has been developed to defeat antibodies to pestviruses in bovine and ovine sera. Single sera from 211 cattle and 22 sheep from 7 different farms were tested using ELISA and Serum Neutralisation Test (SNT). 17 Monoclonal antibodies (Mabs) directed against P80, gp48 and gp53 were tested for ability to coat ELISA plates and capture the bovine viral diarrhea antigen. 5 mabs(WB 103, WB, 105, WB 112 against P80 kDa protein, WB 210 and WB 214 directed against gp48 and gp 53 kDa protein. Specific antibody to BVDV was detected by rabbit anti-bovine and anti-ovine IgG antisera. The quantitative correlation between two tests was good

  6. Novel plasminogen gene mutations in Turkish patients with type I plasminogen deficiency.

    Dönmez-Demir, Buket; Celkan, Tiraje; Sarper, Nazan; Deda, Gülhis; İnce, Elif; Çalişkan, Ümran; Öztürk, Gülyüz; Karagün, Barbaros; Küpesiz, Alphan; Tokgöz, Hüseyin; Akar, Nejat; Özdağ, Hilal

    2016-09-01

    The plasminogen (Plg) protein is the inactive proenzyme form of plasmin that dissolves fibrin thrombi by a process called fibrinolysis. It has been shown that homozygous or compound-heterozygous deficiency of this protein is a major cause of a rare inflammatory disease affecting mainly mucous membranes found in different body sites. In this study, five individual Turkish patients and nine Turkish families with type 1 Plg deficiency were investigated for PLG gene mutations. All of the coding regions of the PLG gene mutations were screened for mutations using denaturing high-pressure liquid chromatography (DHPLC). Samples showing a different DHPLC profile were subjected to DNA sequencing analysis. Here, we described five novel mutations namely, Cys49Ter, +1 IVS6 G>A, Gly218Val, Tyr283Cys, and Gly703Asp. Previously identified five nonsynonymous (Lys38Glu, Glu180Lys, Gly420Asp, Asp453Asn, Pro763Ser), five synonymous (330 C>T, 582 C>T, 771 T>C, 1083 A>G, 2286 T>G), and a 3' untranslated region (3' UTR) mutation (c.*45 A>G) were also reported in this present study. In this study, we have identified a total of eight mutations, five of which are novel. The mutations/polymorphisms identified in eight of the patients do not explain the disease phenotype. These cases probably carry other pathological mutations (homozygous or compound heterozygous) that cannot be detected by DHPLC. PMID:26340456

  7. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

    Mariola M Marcinkiewicz

    Full Text Available The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age. Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.

  8. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

    Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  9. Casein fraction of ovine milk from indigenous Greek breeds

    Moatsou, Golfo; Samolada, Maria; Katsabeki, Alexandra; Anifantakis, Emmanuel

    2004-01-01

    Whole casein fractions isoelectrically prepared from bulk milks from four different indigenous Greek ovine breeds were analysed by urea-PAGE and reversed-phase HPLC. Individual caseins prepared by fractionation on a cation-exchange column were used to locate the peaks on the chromatograms. Apart from heterogeneity that was depicted in the peak shape of caseins, there was also quantitative variability regarding the $\\alpha$s- and $\\beta$-casein contents. According to the RP-HPLC results, the r...

  10. An epidemiological investigation on serum levels of leptin, adiponectin, interleukin-6and plasminogen activator inhibitor-1 in middle aged and elderly people%中老年血清瘦素、脂联素、白介素-6及纤溶酶原激活物抑制物-1的流行病学调查

    鲍蓓; 李志海; 陈卫文; 王立; 靳玉凤; 王晓燕

    2012-01-01

    目的 对广州市中老年人血清瘦素( leptin)、脂联素(adiponectin)、白介素-6(interleukin-6,IL-6)及纤溶酶原激活物抑制物-1(plasminogen activator inhibitor-1,PAI-1)进行流行病学调查.方法 在广州市生物库第三期注册登记的10 027名中老年人中随机抽取1996名中老年人进行问卷调查和健康体检.以酶联免疫吸附双抗夹心( ELISA)法测定leptin、adiponectin、IL-6、PAI-1.结果 广州市中老年人leptin、adiponectin、IL-6、PAI-1分别为( 10.46±9.75) μg/L、(9115.5±9812.91) μg/L、(13.13±6.50) pg/mL和(163.71±110.04)μg/L.校正年龄因素后,男、女性别间leptin、adiponectin、IL-6和PAI-1差异均有显著性(P<0.05).结论 本次大样本的中老年leptin、adiponectin、IL-6、PAI -1的流行病学调查数据可靠,为后期的生活方式与慢性心、脑血管和代谢性疾病关系的研究和公共卫生干预提供了依据.%Objective To conduct an epidemiological investigation on leptin,adiponectin,interleukin-6 ( IL-6 ),and plasminogen activator inhibitor-1 ( PAI-1 ) in the middle aged and elderly people in Guangzhou.Methods 1996 of 10 027 middle aged and elderly people registered at Guangzhou Database were randomly selected to be surveyed by questionnaires and to have physical examination.Levels of leptin,adiponectin,IL-6,and PAI-1 were determined by enzyme-linked immunosorbent assay ( ELISA ).Results Leptin was ( 10.46 ± 9.75 ) μg/L, adiponectin was ( 9115.5 ± 9812.91 ) μg/L,IL-6 was ( 13.13 ± 6.50 ) pg/mL,and PAI-1 was ( 163.71 ± 110.04 ) μg/L in Guangzhou middle aged and elderly people.There were significant differences in levels of leptin,adiponectin,IL-6,and PAI-1 between male and female after age adjustment.Conclusions Reliable data of leptin,adiponectin,IL-6,and PAI-1 in this large-scale epidemiological investigation provides support for the future research on the relationship between lifestyle and chronic cardiovascular,cardiocerebrovascular diseases

  11. Plasmin in nephrotic urine activates the epithelial sodium channel

    Svenningsen, Per; Bistrup, Claus; Friis, Ulla G; Bertog, Marko; Haerteis, Silke; Krueger, Bettina; Stubbe, Jane; Jensen, Ole Nørregaard; Thiesson, Helle C; Uhrenholt, Torben R; Jespersen, Bente; Jensen, Boye L; Korbmacher, Christoph; Skøtt, Ole

    2008-01-01

    Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus...... plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen...

  12. Production of bacteriocin-like substances by lactic acid bacteria isolated from regional ovine cheese

    Cássia Regina Nespolo

    2010-12-01

    Full Text Available Lactic acid bacteria (LAB were isolated from ovine milk and cheeses manufactured in the South Region of Brazil. Among 112 bacterial isolates investigated, 59 were chosen through a screening for LAB. Among these 59 strains of LAB, 21% showed antimicrobial, proteolytic and lipolytic activities. Based on this screening, Lactobacillus plantarum LCN 17 and Lactobacillus rhamnosus LCN 43 were selected and tested for the production of bacteriocin-like substances (BLS. The BLS produced by both isolates showed antimicrobial activity against Listeria monocytogenes, whereas that produced by L. plantarum LCN 17 presented higher stability to different temperature, pH and enzyme treatments. These strains present potential for production of BLS, and for use as starter cultures.

  13. Trappin ovine molecule (TOM), the ovine ortholog of elafin, is an acute phase reactant in the lung

    Brown, Thomas I; Mistry, Rohit; Collie, D David; Tate, Steven; Sallenave, Jean-Michel

    2004-01-01

    As large animal models continue to play an important role in translating lung-directed therapeutic strategies from laboratory animals to humans, there is an increasing interest in the analysis of endogenous regulators of inflammation at both a genomic and a therapeutic level. To this end, we have sought to characterize the ovine ortholog of elafin, an important regulator of inflammation in humans. We have isolated both the elafin cDNA and gene, which have a similar structure to other species'...

  14. The bacteriological quality of goat and ovine milk

    Kateřina Bogdanovičová

    2015-05-01

    Full Text Available This study concentrates on information concerning the microbiological hazards that can be present in raw milk from animal species other than cows. A total of 54 (23 of ovine and 31 of goat bulk tank milk samples from 10 farms in the Czech Republic were collected in years 2013 - 2014. The sampling was done at regular time intervals during the whole year, with five to eight samples collected from each of the 10 dairy farms involved in the study. All milk samples were collected into sterile sampling bottles and transported in a cooler sampling case to the laboratory for immediate examination. Farms were randomly selected to cover the whole area of the Czech Republic. The prevalence and characteristic of Escherichia coli, Staphylococcus aureus, Salmonella spp., Campylobacter spp. and Listeria monocytogenes was studied. Raw cow's milk can be contaminated by E. coli intramammarily during clinical or subclinical mastitis and either directly through animal feces or indirectly during milk collection through farm employees or the milking equipment. E. coli was detected in 90.3% of the goat milk and 95.7% of the ovine milk samples. The genes encoding Shiga toxins 1 and 2- (stx1, stx2 were not detected and no STEC was identified. The Eae was the detected in 3 (4.6% isolates. S. aureus was detected in 9 (29.0% samples of goat milk and 8 (34.8% samples of ovine milk. A total 12 (57.1% enterotoxin positive S. aureus were obtained; 6 (28.6% were positive for the production of sec encoding enterotoxin SEC; in 4 (19.0% isolates the gene seh was detected; 2 (9.5% isolates were proven positive for seg (4.8% and combination seg and sei (4.8%. The presence of MRSA was not detected in the tested samples in our study. L. monocytogenes was detected in 1 (3.2% samples of goat milk and 1 (4.3% samples of ovine milk. The serotype (1/2a, 1/2b was detected in our study. Campylobacter spp. and Salmonella spp. were not isolated from any of the samples. These results form

  15. Hexamethylenebisacetamide (HMBA) is a growth factor for human, ovine and porcine thyroid cells.

    Fayet, G; Amphoux-Fazekas, T; Aouani, A; Hovsépian, S

    1996-03-01

    Hexamethylenebisacetamide (HMBA) provokes in murine erythroleukemia cells (MELC) a commitment to terminal differentiation leading to the activation of the expression of hemoglobin. HMBA has been tested also in other cells from colon cancer, melanoma or lung cancer. However it has not yet been tested in the thyroid. We demonstrate in this paper that HMBA in kinetics and concentration-response experiments increases the proliferation of human thyroid cells isolated from Graves'-Basedow patients. It also acts like a growth factor for ovine and porcine thyroid cells, respectively, from the OVNIS line and the ATHOS line. This molecule which is a differentiating factor in the MELC system and a growth factor in human thyroid cell cultures represents a potential to get human thyroid cell lines expressing specialized functions. PMID:8734479

  16. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    CLAUDIA TEREZIA SOCOL; A. VLAIC; VIORICA COSIER

    2013-01-01

    An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, cont...

  17. Genetic architecture of gene expression in ovine skeletal muscle

    Kogelman, Lisette Johanna Antonia; Byrne, Keren; Vuocolo, Tony;

    2011-01-01

    -based gene expression data we directly tested the hypothesis that there is genetic structure in the gene expression program in ovine skeletal muscle.Results: The genetic performance of six sires for a well defined muscling trait, longissimus lumborum muscle depth, was measured using extensive progeny testing...... architecture to the gene expression data, which also discriminated the sire-based Estimated Breeding Value for the trait. An integrated systems biology approach was then used to identify the major functional pathways contributing to the genetics of enhanced muscling by using both Estimated Breeding Value...

  18. Percutaneous osseointegrated prostheses for amputees: Limb compensation in a 12-month ovine model.

    Shelton, Trevor J; Beck, J Peter; Bloebaum, Roy D; Bachus, Kent N

    2011-10-13

    Percutaneous osseointegrated prostheses are being investigated as an alternative strategy to attach prosthetic limbs to patients. Although the use of these implants has shown to be promising in clinical trials, the ability to maintain a skin seal around an osseointegrated implant interface is a major challenge to prevent superficial and deep periprosthetic infections. The specific aim of this study was to establish a translational load-bearing ovine model to assess postoperative limb compensation and gait symmetry following a percutaneous osseointegrated implant. We tested the following hypotheses: (1) the animals would return to pre-amputation limb loads within 12-months; (2) the animals would return to a symmetrical gait pattern (stride length and time in stance) within 12-months. The results demonstrated that one month following surgery, the sheep loaded their amputated limb to a mean value of nearly 80% of their pre-amputation loading condition; by 12-months, this mean had dropped to approximately 74%. There was no statistical differences between the symmetry of the amputated forelimb and the contralateral forelimb at any time point for the animals stride length or the time spent in the stance phase of their gait cycle. Thus, the data showed that while the animals maintained symmetric gait patterns, they did not return to full weight-bearing after 12-months. The results of this study showed that a large animal load-bearing model had a symmetric gait and was weight bearing for up to 12 months. While the current investigation utilizes an ovine model, the data show that osseointegrated implant technology with postoperative follow-up can help our human patients return to symmetric gait and maintain an active lifestyle, leading to an improvement in their quality of life following amputation. PMID:21920525

  19. Tetranectin, a trimeric plasminogen-binding C-type lectin

    Holtet, T L; Graversen, Jonas Heilskov; Clemmensen, I;

    1997-01-01

    Tetranectin, a plasminogen-binding protein belonging to the family of C-type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin-as well as natural tetranectin from human plasma-was shown by chemical cross......-linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization....

  20. Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen-plasmin system.

    Abdullah, Zuraidah; Bayraktutan, Ulvi

    2016-07-01

    Ischaemic stroke, accompanied by neuroinflammation, impairs blood-brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood-brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen-plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane. PMID:27094771

  1. Production of monoclonal antibodies reactive with ovine eosinophils

    Meeusen Els NT

    2007-09-01

    Full Text Available Abstract Background There is strong evidence implicating eosinophils in host defence against parasites as well as allergic disease pathologies. However, a lack of reagents such as monoclonal antibodies (mAbs specific for eosinophils has made it difficult to confirm the functional role of eosinophils in such disease conditions. Using an established mammary model of allergic inflammation in sheep, large numbers of inflammatory cells enriched for eosinophils were collected from parasite-stimulated mammary glands and used for the generation of mAbs against ovine eosinophils. Results A panel of mAbs was raised against ovine eosinophils of which two were shown to be highly specific for eosinophils. The reactivity of mAbs 3.252 and 1.2 identified eosinophils from various cell and tissue preparations with no detectable reactivity on cells of myeloid or lymphoid lineage, tissue mast cells, dendritic cells, epithelial cells or other connective tissues. Two other mAbs generated in this study (mAbs 4.4 and 4.10 were found to have reactivity for both eosinophils and neutrophils. Conclusion This study describes the production of new reagents to identify eosinophils (as well as granulocytes in sheep that will be useful in studying the role of eosinophils in disease pathologies in parasite and allergy models.

  2. Polyamine profile in ovine and caprine colostrum and milk.

    Galitsopoulou, Augustina; Michaelidou, Alexandra-Maria; Menexes, George; Alichanidis, Efstathios

    2015-04-15

    The objective of this study was to monitor the post-partum variation of polyamine content, in ovine and caprine milk, from indigenous Greek breeds. Twenty samples of ewe and 20 samples of goat colostrum and milk were collected at the 1st, 2nd, 3rd, 4th, 5th and 15th day post-partum. Putrescine, spermidine and spermine were measured as dansylated derivatives by high-performance liquid chromatography. Putrescine was the least concentrated of these substances in both milk types. Spermidine was the prevailing polyamine in caprine samples, reaching levels up to 4.41 μmol/l on the 3rd day post-partum. In ovine milk, the profile of the mean concentrations showed greater levels of spermine than spermidine, except for the 5th day post-partum. These data suggest that goat colostrum and ewe milk (15th day) could be considered as good natural sources for these bioactive growth factors, and may become useful raw materials for designing tailored dairy products for specific population groups. PMID:25465997

  3. Molecular and Biomorphometrical Identification of Ovine Babesiosis in Iran

    F Noorollahi

    2010-12-01

    Full Text Available Background: Ovine babesiosis is the most important haemoparasitic tick-borne disease of small ruminants in Iran caused by Babesia ovis, B. motasi, and B. crassa. The aim of this study was to characterize the species of ovine Babesia species isolated from different geographical region of Iran.Methods: One hundred fifty four blood samples collected from animals, which demonstrated the pale mucous membranes or hyperthermia. The specimens were transferred to the laboratory and the blood smears stained with Geimsa, the morphological and biometrical data of parasite in any infected erythrocyte have been considered. Extracted DNA from each blood samples were used in PCR and semi nested- PCR in order to confirm the presence of the species.Results: The results of the PCR assays showed nine (5.85%, 81 (53% and 18 (11.7% were distinguished as Babesia, Theileria and mixed infection, respectively. Semi nested- PCR did not confirm the presence of B. motasi.Conclusion: The causative organism of many cases of haemoprotozoal diseases, which recorded in previous studies, could be B. ovis or Theileria lestoquardi. The result confirmed that B. ovis was only species which causes babesiosis in the study areas. It seems that the biometrical polymor­phisms could exist in B. ovis in Iran. This polymorphism could be a main problem in differen­tiation between B. ovis and B. motasi and it could be dissolved by specific PCR analysis.

  4. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter;

    2003-01-01

    glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

  5. Soluble urokinase plasminogen activator receptor during allogeneic stem cell transplantation

    Haastrup, E; Andersen, J; Ostrowski, S R; Høyer-Hansen, G; Heilmann, C; Ullum, H; Müller, K; Jacobsen, N

    2011-01-01

    course of allogeneic stem cell transplantation (SCT). Twenty SCT patients were included in the study. suPAR was measured by ELISA in daily taken plasma samples during the pretransplant conditioning with chemotherapy and weekly for 1 month after infusion of the graft. suPAR levels before the start of the...

  6. Detection of streptomycin residues in local meat of bovine and ovine

    O. A. Abdullah

    2012-01-01

    Full Text Available From meat retails in Mosul province, forty-five meat samples of local ovine and bovine (23 bovine samples and 22 ovine samples were collected. The period of collection was during November 2010 to May 2011, by means of multistage random sampling for detection of streptomycin residues. Enzyme linked immunosorbent assay (ELISA was used for detection of streptomycin residues. The results revealed that eleven ovine meat samples (50% were positive to streptomycin residue, with a mean value 35.06 µg kg-1, while 14 bovine meat samples (60.86% were positive to residual streptomycin with a mean value 59.56 µg kg-1. From the results, it is clear that all tested meat samples (ovine and bovine were safe enough for human consumption.

  7. Genome sequences of mannheimia haemolytica serotype A2: ovine and bovine isolates

    This report describes the genome sequences of Mannheimia haemolytica, serotype A2 isolated from pneumonic lungs of two different ruminant species, one from Ovis aries, designated as Ovine (O) and the other from Bos taurus, designated as Bovine (B)....

  8. The ovine respiratory syncytial virus F gene sequence and its diagnostic application.

    Eleraky, N Z; Kania, S A; Potgieter, L N

    2001-11-01

    Ruminant respiratory syncytial viruses (RSVs) are classified into 2 subgroups, ovine RSV and bovine RSV. Although ovine RSV infects cattle, its contribution to bovine respiratory tract disease has not been established, which is an important issue for vaccine development in cattle. Diagnosis by virus isolation or serology has low or variable sensitivity and/or specificity and polymerase chain reaction (PCR) has been recommended as a rapid and sensitive technique for RSV detection. A simple procedure has been developed to detect and identify bovine and ovine RSVs. First, the nucleotide sequence of the ovine RSV fusion (F) gene was determined and compared with representative strains of bovine RSV and human RSV subgroups A and B. The ovine RSV F gene has 85 and 72-73% nucleotide identity with those of bovine RSV and human RSV, respectively. The predicted amino acid sequence of the ovine RSV F gene has 94 and 83-84% amino acid identity with those of bovine RSV and human RSV, respectively. Then PCR primers targeting a specific F gene fragment of bovine and ovine RSV were designed. The primers represented bases 85-103 and the complementary sequence to bases 510-493 of the ovine RSV F gene. A similar PCR product (426 bp) was obtained on agarose gel electrophoresis from bovine RSV and from ovine RSV. The products, however, were unique to the parent virus and could be distinguished by EcoRI or MspI restriction endonuclease cleavage. EcoRI cleaved the ovine product into 2 bands (285 and 141 bp) but failed to affect the bovine RSV PCR product. However, MspI cleaved the bovine product into 2 bands (229 and 197 bp) but had no effect on the ovine product. Also, this assay did not amplify any PCR product with human RSV. The reverse transcription-polymerase chain reaction (RT-PCR) followed by restriction enzyme digestion is a useful and practical approach for detection and differentiation of ruminant respiratory syncytial viruses. PMID:11724134

  9. Induction of ovine trophoblast cell fusion by fematrin-1 in vitro.

    Koshi, Katsuo; Nakaya, Yuki; Kizaki, Keiichiro; Ishiguro-Oonuma, Toshina; Miyazawa, Takayuki; Spencer, Thomas E; Hashizume, Kazuyoshi

    2016-03-01

    Endogenous retroviruses present in the genomes take a specific role in placental formation in various vertebrates, including bovine and sheep. Fematrin-1, which is the envelope (Env) protein of bovine endogenous retrovirus found in bovine placenta, is involved in the formation of fetomaternal hybrid cells in cattle placenta. This study was conducted to clarify whether fematrin-1 possesses fusogenic activity in trophoblast cells. Another question is whether Env proteins only have species-specific activity or not. For this, fematrin-1 gene was transfected in ovine trophoblast cells, and we examined fusogenic activity with Cos-7 cells. Although fematrin-1 fusogenic activity was detected in both neutral and acidic pH conditions, acidic condition significantly enhanced it. These activities were rather weaker than those of vesicular stomatitis virus G protein as a positive control. However, the ratio of fematrin-1 and vesicular stomatitis virus G protein fusion index was confirmed similar to those in the previous reports. Some fusion cells showed multinucleate cells. These results imply that fematrin-1 is involved in the formation of trophoblast hybrid cells even in different species trophoblastic cells. PMID:26212859

  10. Discovery and genomic characterization of a novel ovine partetravirus and a new genotype of bovine partetravirus

    Tse, Herman; Tsoi, Hoi-wah; Teng, Jade L. L.; Chen, Xin-Chun; Liu, Haiying; Zhou, Boping; Zheng, Bo-Jian; Woo, Patrick C. Y.; Lau, Susanna K. P.; Yuen, Kwok-Yung

    2011-01-01

    Partetravirus is a recently described group of animal parvoviruses which include the human partetravirus, bovine partetravirus and porcine partetravirus (previously known as human parvovirus 4, bovine hokovirus and porcine hokovirus respectively). In this report, we describe the discovery and genomic characterization of partetraviruses in bovine and ovine samples from China. These partetraviruses were detected by PCR in 1.8% of bovine liver samples, 66.7% of ovine liver samples and 71.4% of o...

  11. In vitro culture medium (IVC) supplementation with sericin improves developmental competence of ovine zygotes.

    Aghaz, Faranak; Hajarian, Hadi; KaramiShabankareh, Hamed

    2016-03-01

    This study was carried out to investigate the effects of supplementation of potassium simplex optimized medium (KSOM-aa) with various sericin concentrations (0, 0.1, 0.5, 1 and 2.5%) on ovine zygotes. The results indicate that the supplementation of oocyte in vitro culture medium with optimal concentration of sericin (0.1 and 0.5%) may have beneficial effects on developmental competence of in vitro-derived ovine embryos. PMID:26952758

  12. Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins

    Jönsson, Klas; Guo, Betty P.; Monstein, Hans-Jürg; Mekalanos, John J.; Kronvall, Göran

    2004-01-01

    Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two H. pylori plasminogen-binding proteins have been described; however, no genes were identified. Here we report the use of a phage display library to clone two genes from the H. pylori CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with H. pylori 26695 HP0508. A s...

  13. Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

    Selwyn A. Headley

    2012-12-01

    Full Text Available Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2 in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2 tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

  14. Long-term physicochemical and immunological stability of a liquid formulated intact ovine immunoglobulin-based antivenom.

    Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

    2013-03-15

    An antivenom should be stable under the conditions that it will be both transferred and stored. Thus instability may lead to a loss of efficacy and an increased incidence and severity of adverse effects. Stability is a particular problem in countries where the temperatures and humidity are high. Here we investigate the stability of a liquid-formulated, intact ovine immunoglobulin-based antivenom, EchiTAbG™, which is used extensively in Nigeria to treat envenoming by the West African saw-scaled viper, Echis ocellatus. Ampoules of antivenom were assessed as to their specific antibody content by small scale affinity chromatography and their purity by size exclusion gel filtration and turbidity. Three different batches of the antivenom revealed no significant changes, using these assessment techniques, during 42 months storage at 4 °C or at ambient temperature, followed by one month at 37 °C. These real-time studies indicate that the antivenom remains stable for a minimum of 3.5 years and that it can be exposed to tropical temperatures without any loss in immunoglobulin binding activity. This further highlights the clinical utility of liquid formulated ovine IgG antivenoms by demonstrating their retention of potency in the event of a short term failing in the cold chain. PMID:23319075

  15. Ovine model for studying pulmonary immune responses

    Joel, D.D.; Chanana, A.D.

    1984-11-25

    Anatomical features of the sheep lung make it an excellent model for studying pulmonary immunity. Four specific lung segments were identified which drain exclusively to three separate lymph nodes. One of these segments, the dorsal basal segment of the right lung, is drained by the caudal mediastinal lymph node (CMLN). Cannulation of the efferent lymph duct of the CMLN along with highly localized intrabronchial instillation of antigen provides a functional unit with which to study factors involved in development of pulmonary immune responses. Following intrabronchial immunization there was an increased output of lymphoblasts and specific antibody-forming cells in efferent CMLN lymph. Continuous divergence of efferent lymph eliminated the serum antibody response but did not totally eliminate the appearance of specific antibody in fluid obtained by bronchoalveolar lavage. In these studies localized immunization of the right cranial lobe served as a control. Efferent lymphoblasts produced in response to intrabronchial antigen were labeled with /sup 125/I-iododeoxyuridine and their migrational patterns and tissue distribution compared to lymphoblasts obtained from the thoracic duct. The results indicated that pulmonary immunoblasts tend to relocate in lung tissue and reappear with a higher specific activity in pulmonary lymph than in thoracic duct lymph. The reverse was observed with labeled intestinal lymphoblasts. 35 references, 2 figures, 3 tables.

  16. Ovine model for studying pulmonary immune responses

    Anatomical features of the sheep lung make it an excellent model for studying pulmonary immunity. Four specific lung segments were identified which drain exclusively to three separate lymph nodes. One of these segments, the dorsal basal segment of the right lung, is drained by the caudal mediastinal lymph node (CMLN). Cannulation of the efferent lymph duct of the CMLN along with highly localized intrabronchial instillation of antigen provides a functional unit with which to study factors involved in development of pulmonary immune responses. Following intrabronchial immunization there was an increased output of lymphoblasts and specific antibody-forming cells in efferent CMLN lymph. Continuous divergence of efferent lymph eliminated the serum antibody response but did not totally eliminate the appearance of specific antibody in fluid obtained by bronchoalveolar lavage. In these studies localized immunization of the right cranial lobe served as a control. Efferent lymphoblasts produced in response to intrabronchial antigen were labeled with 125I-iododeoxyuridine and their migrational patterns and tissue distribution compared to lymphoblasts obtained from the thoracic duct. The results indicated that pulmonary immunoblasts tend to relocate in lung tissue and reappear with a higher specific activity in pulmonary lymph than in thoracic duct lymph. The reverse was observed with labeled intestinal lymphoblasts. 35 references, 2 figures, 3 tables

  17. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    CLAUDIA TEREZIA SOCOL

    2013-12-01

    Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

  18. Effects of angiotensin Ⅱ receptor antagonist on expression of plasminogen activator inhibitor-1 in the renal biopsy of patients with chronic kidney disease%血管紧张素Ⅱ受体拮抗剂对慢性肾脏病患者肾组织中纤溶酶原激活物抑制物-1表达的影响

    王文革; 李侠

    2010-01-01

    Objective To evaluate the effects of angiotensin Ⅱreceptor antagonist (ARB) on expression of plasminogen activator inhibitor(PAI)-1 in the human renal biopsy of patients with chronic kidney disease(CKD)and analyse its clinical significance.Methods PAI-1 expression of 63 specimens with CKD was detected by immunohistochemistry and quantitative analysis was achieved by image analysis system. meanwhile, the patients'clinical data were collected,and PAI-1 expression of 63 specimens with CKD was compared with that of normal renal tissue(19 cases).Results Compared with the normal renal tissue,the expression of PAI-1 in CKD 1,2 and 3 stage wag respectively drastically increased(P<0.05).The expression of PAI-1 in CKD 3 stage was higher than that in CKD 1 and 2 stage(P<0.05).The expression of PAI-1 in the patients treated by valsartan[renal corpuscle(4.73±1.18)%,renal tubule(37.16±6.81)%]was lower than that with no treatment[renal corpuscle(5.81±1.95)%,renal tubule(43.22 ±10.25)%](P<0.05).Conclusions It suggests that the abnormal expression of PAI-1 in CKD play an important role in the progression of glomerular and tubulointerstitiai sclerosis in patients with CKD.ARB may prevent the development of renal inflammation and sclerosis by inhibiting the production of PAI-1.ARB may contribute to the prevention and therapy of CKD.%目的 探讨血管紧张索Ⅱ受体拮抗剂(ARB)对慢性肾脏病(CKD)患者肾组织中纤溶酶原激活物抑制物(PAI)-1表达的影响及临床意义.方法 采用免疫组化法检测63例CKD患者肾穿刺标本中PAI-1的表达水平,用图像分析法对PAI-1进行定量测定,同时收集患者的相关临床资料,并与因肾肿瘤切除肾脏的远离肿瘤部分的正常肾组织19例比较.结果 CKD 1、2、3期患者PAI-1在肾组织中的表达均较正常肾组织显著增多(P<0.05);而CKD 3期患者PAI-1在肾组织中的表达明显高于CKDI期和2期患者(P<0.05).应用缬沙坦治疗的CKD 16例患者肾组织中PAI-1

  19. Impaired arterial neointima formation in mice with disruption of the plasminogen gene.

    Carmeliet, P; Moons, L; Ploplis, V; Plow, E; Collen, D

    1997-01-01

    To define the role of plasminogen (Plg) in the smooth muscle cell response after arterial wall injury, neointima formation was evaluated after electric injury of the femoral artery in plasminogen-deficient (Plg-/-) mice. The injury destroyed all medial smooth muscle cells, denuded the injured segment of intact endothelium, and induced transient platelet-rich mural thrombosis. In wild-type (Plg+/+) mice, vascular wound healing was characterized by lysis of the thrombus, transient infiltration ...

  20. Elimination of [14C]heptachlor from body stores of lactating ewes treated with ovine growth hormone

    Elimination of [14C]heptachlor from body burdens of sheep was measured using mature ewes nursing single offspring, and the influence of exogenous ovine growth hormone (oGH) on elimination was studied. Six ewes (62 +/- 2.5 kg BW) were dosed (i.p.) once with [14C]heptachlor (2.04 mg/kg Bw; .88 microCi/mg heptachlor) and three were treated additionally with oGH (oGH; 5 mg/d) for 21 d. Three additional ewes served as controls. Excreta were collected each day for 21 d. Milk and blood were collected every 3rd d until ewes were euthanized at d 21. 14C activity was measured in excreta, milk, blood and tissues. Total cumulative activity of [14C]heptachlor and(or) metabolites in excreta (21 d) did not differ (P greater than .20) in ewes given oGH (25 +/- 2%) vs none (23 +/- 2%). Milk yield and protein content were unaffected (P greater than .10) by oGH. Ewes given oGH eliminated 2.2 +/- 2% of total 14C dosage into milk during 21 d, whereas ewes untreated with oGH eliminated 1.3 +/- .2% (P less than .10); total 14C activity eliminated into milk plus excreta was similar for ewes given oGH or none (P greater than .10). For all six ewes, half-times (T1/2) for distribution and elimination of 14C activity (heptachlor and metabolites) were 1.5 d and 11.7 d, respectively. Blood concentrations of 14C activity during 21 d yielded elimination half-time as 23 d. Unlike bovines, which eliminate heptachlor slowly (T1/2 approximately 70 to 80 d) and mainly into milk fat, lactating ovines eliminated heptachlor and(or) metabolites mainly into excreta and about sixfold faster than bovines

  1. Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM.

    Youn, Mi-Ran; Park, Mee-Hee; Choi, Chang-Ki; Ahn, Byung-Cheol; Kim, Hak Yong; Kang, Sang Sun; Hong, Yong-Kil; Joe, Young Ae; Kim, Jong-Soo; You, Weon-Kyoo; Lee, Hyo-Sil; Chung, Soo-Il; Chang, Soo-Ik

    2006-05-12

    Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, Kd, are 0.89 and 0.18 microM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin. PMID:16564503

  2. A diffusion and T2 relaxation MRI study of the ovine lumbar intervertebral disc under compression in vitro

    Drew, Simon C.; Silva, Pujitha; Crozier, Stuart; Pearcy, Mark J.

    2004-08-01

    The ovine lumbar intervertebral disc is a useful model for the human lumbar disc. We present preliminary estimates of diffusion coefficients and T2 relaxation times in a pilot MRI study of the ovine lumbar intervertebral disc during uniaxial compression in vitro, and identify factors that hamper the ability to accurately monitor the temporal evolution of the effective diffusion tensor at high spatial resolution.

  3. Determining Human Clot Lysis Time (in vitro with Plasminogen/Plasmin from Four Species (Human, Bovine, Goat, and Swine

    Omaira Cañas Bermúdez

    2015-05-01

    Full Text Available Cardiovascular disease is the leading cause of death worldwide, including failures in the plasminogen/plasmin system which is an important factor in poor lysis of blood clots. This article studies the fibrinolytic system in four species of mammals, and it identifies human plasminogen with highest thrombolysis efficiency. It examines plasminogen from four species (human, bovine, goat, and swine and identifies the most efficient one in human clot lysis in vitro. All plasminogens were identically purified by affinity chromatography. Human fibrinogen was purified by fractionation with ethanol. The purification of both plasminogen and fibrinogen was characterized by one-dimensional SDS-PAGE (10%. Human clot formation in vitro and its dissolution by plasminogen/plasmin consisted of determining lysis time from clot formation to its dilution. Purification of proteins showed greater than 95% purity, human plasminogen showed greater ability to lyse clot than animal plasminogen. The article concludes that human plasminogen/plasmin has the greatest catalysis and efficiency, as it dissolves human clot up to three times faster than that of irrational species.

  4. Biosynthesis of lutropin in ovine pituitary slices: incorporation of [35S]sulfate in carbohydrate units

    Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H2(35)SO4. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the alpha and beta subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [35S]sulfate was also incorporated into several other proteins in addition to LH. The location of 35SO/sub 2-(4)/ in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[35S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[35S] sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of 35SO/sub 2-(4)/ by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH

  5. A preliminary proteomic characterisation of extracellular vesicles released by the ovine parasitic nematode, Teladorsagia circumcincta

    Tzelos, Thomas; Matthews, Jacqueline B.; Buck, Amy H.; Simbari, Fabio; Frew, David; Inglis, Neil F.; McLean, Kevin; Nisbet, Alasdair J.; Whitelaw, C. Bruce A.; Knox, David P.; McNeilly, Tom N.

    2016-01-01

    Teladorsagia circumcincta is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The development of high levels of anthelmintic resistance in this nematode species challenges its future control. Recent research indicates that many parasite species release extracellular vesicles into their environment, many of which have been classified as endocytic in origin, termed exosomes. These vesicles are considered to play important roles in the intercellular communication between parasites and their hosts, and thus represent potentially useful targets for novel control strategies. Here, we demonstrate that exosome-like extracellular vesicles can be isolated from excretory-secretory (ES) products released by T. circumcincta fourth stage larvae (Tci-L4ES). Furthermore, we perform a comparative proteomic analysis of vesicle-enriched and vesicle-free Tci-L4ES. Approximately 73% of the proteins identified in the vesicle-enriched fraction were unique to this fraction, whilst the remaining 27% were present in both vesicle-enriched and vesicle-free fraction. These unique proteins included structural proteins, nuclear proteins, metabolic proteins, proteolytic enzymes and activation-associated secreted proteins. Finally, we demonstrate that molecules present within the vesicles-enriched material are targets of the IgA and IgG response in T. circumcincta infected sheep, and could potentially represent useful targets for future vaccine intervention studies. PMID:27084478

  6. The effect of cholecystokinin octapeptide upon the migrating myoelectric complex in the ovine small bowel

    Romanski Krzysztof W.

    2007-01-01

    Full Text Available There is no evident and precise data regarding the effect of cholecystokinin octapeptide (CCK-OP on the migrating myoelectric complex (MMC in sheep. Thus, in five rams seven bipolar platinum electrodes were implanted in the abomasal antrum and entire small intestine. The strain gauge force transducer was also attached near the duodenal electrode in three of these animals. During the experiments the myoelectric and motor activity was continuously recorded in fasted and non-fasted sheep. During the recordings two normal consecutive phases 3 of the MMC were recorded. Then, slow injections of CCK-OP of 0.02; 0.2 and 2.0 _g/kg of body weight were given intravenously during phase 1, 2a or 2b of the MMC, identified in the duodenum, until next two consecutive phases 3 MMC were recorded. The moderate dose of CCK-OP administered during phase 2a in non-fasted animals shortened the MMC cycles significantly while the highest dose of the hormone increased the cycle duration and inhibited phase 3 MMC in the duodenum. No such effect was observed in the jejunum. However, duration of phase 3 in this region was decreased mostly by the highest dose of CCK-OP. It can be concluded that CCK inhibits the MMC in ovine small bowel and its effect in the duodenum is most pronounced.

  7. A preliminary proteomic characterisation of extracellular vesicles released by the ovine parasitic nematode, Teladorsagia circumcincta.

    Tzelos, Thomas; Matthews, Jacqueline B; Buck, Amy H; Simbari, Fabio; Frew, David; Inglis, Neil F; McLean, Kevin; Nisbet, Alasdair J; Whitelaw, C Bruce A; Knox, David P; McNeilly, Tom N

    2016-05-15

    Teladorsagia circumcincta is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The development of high levels of anthelmintic resistance in this nematode species challenges its future control. Recent research indicates that many parasite species release extracellular vesicles into their environment, many of which have been classified as endocytic in origin, termed exosomes. These vesicles are considered to play important roles in the intercellular communication between parasites and their hosts, and thus represent potentially useful targets for novel control strategies. Here, we demonstrate that exosome-like extracellular vesicles can be isolated from excretory-secretory (ES) products released by T. circumcincta fourth stage larvae (Tci-L4ES). Furthermore, we perform a comparative proteomic analysis of vesicle-enriched and vesicle-free Tci-L4ES. Approximately 73% of the proteins identified in the vesicle-enriched fraction were unique to this fraction, whilst the remaining 27% were present in both vesicle-enriched and vesicle-free fraction. These unique proteins included structural proteins, nuclear proteins, metabolic proteins, proteolytic enzymes and activation-associated secreted proteins. Finally, we demonstrate that molecules present within the vesicles-enriched material are targets of the IgA and IgG response in T. circumcincta infected sheep, and could potentially represent useful targets for future vaccine intervention studies. PMID:27084478

  8. Plasmin enzymatic activity in the presence of actin

    Yusova E. I.

    2015-10-01

    Full Text Available Aim. To study the changes in the plasmin activity towards substrates with high and low molecular mass in the presence of actin. Methods. The proteins used for this investigation were obtained by affinity chromatography and gel-filtration. The plasmin enzymatic activity was determined by a turbidimetric assay and a chromogenic substrate-based assay. The enzyme linked immunosorbent assay and biotin-avidin-phosphatase system were used to study the interaction of plasminogen and its fragments with actin. Results. It was shown that G-actin causes 1.5-fold decrease in the rate of polymeric fibrin hydrolysis by plasmin and Glu-plasminogen activated by the tissue plasminogen activator. However, actin did not impede plasmin autolysis and had no influence on its amidase activity. We have studied an interaction of biotinylated Glu-plasminogen and its fragments (kringle 1-3, kringle 4 and mini-plasminogen with immobilized G-actin. Glu-plasminogen and kringle 4 had a high affinity towards actin (C50 is 113 and 117 nM correspondingly. Mini-plasminogen and kringe 4 did not bind to actin. A similar affinity of Glu-plasminogen and kringle 1-3 towards actin proves the involvement of the kringle 1-3 lysine-binding sites of the native plasminogen form in the actin interaction. Conclusions. Actin can modulate plasmin specificity towards high molecular mass substrates through its interaction with lysine-binding sites of the enzyme kringle domains. Actin inhibition of the fibrinolytic activity of plasmin is due to its competition with fibrin for thelysine binding sites of plasminogen/plasmin.

  9. Molecular screening of ovine mastitis in different breeds.

    Guerreiro, O; Velez, Z; Alvarenga, N; Matos, C; Duarte, M

    2013-02-01

    Clinical and subclinical mastitis directly affect mammary gland function and have a great economic impact on the sheep and goat dairy industries. The present study explores molecular diagnosis of ovine subclinical mastitis as a faster and more precise screening method compared with microbiology and biochemical techniques to assess the molecular and chemical properties of raw milk samples from healthy animals from 3 breeds of sheep raised in Portugal. Based on 16S ribosomal RNA screening by PCR, milk samples from all sheep were categorized as contaminated (n=123) or noncontaminated (n=104). For contaminated milk, different specific primers were used for pathogen identification (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis). Streptococcus agalactiae was identified as the most frequent agent. We further assessed whether contaminated versus noncontaminated samples were chemically different in terms of fat, protein, lactose, pH, and solids-not-fat. This molecular screening method allowed rapid and efficient identification of contaminated raw sheep milk, including pathogen identification, before significant alterations in milk chemical properties could be detected. This methodology may lead to a specific and efficient animal treatment and consequently less expensive flock management. PMID:23245954

  10. Ewe welfare and ovine milk and cheese quality

    A. Sevi

    2010-04-01

    Full Text Available Causes of welfare reduction in dairy sheep flocks are presented and their impact on ovine milk and cheese quality is discussed. Attention is focused on climatic extremes, poor housing and milking hygiene, and nutritional imbalance: mechanisms are outlined through which stress-induced reduction of immune function can result in poor milk composition, deteriorated renneting ability of milk and altered proteolysis in cheese during ripening. In particular, the impact is brought out of exposure to high ambient temperature on the nutritional properties of ewe milk, in terms of increased short-chain and saturated fatty acids, and decreased unsaturated to saturated fatty acid ratio. As well, the relationship is highlighted between ewe welfare and udder health. Especially under poor hygiene conditions the risk of mastitis markedly increases due to reduction of the natural defense mechanisms of the teat and mammary gland and increased number and pathogenicity of the micro-organisms in contact with the entrance of the teat canal. Evidence is provided that rise in milk somatic cell count, in response to bacteria penetration into the udder, can lead to decreased milk yield and altered composition of milk and cheese, due to extensive epithelium secretory cell damage.

  11. Haplotypes and Sequence Variation in the Ovine Adiponectin Gene (ADIPOQ

    Qing-Ming An

    2015-11-01

    Full Text Available The adiponectin gene (ADIPOQ plays an important role in energy homeostasis. In this study five separate regions (regions 1 to 5 of ovine ADIPOQ were analysed using PCR-SSCP. Four different PCR-SSCP patterns (A1-D1, A2-D2 were detected in region-1 and region-2, respectively, with seven and six SNPs being revealed. In region-3, three different patterns (A3-C3 and three SNPs were observed. Two patterns (A4-B4, A5-B5 and two and one SNPs were observed in region-4 and region-5, respectively. In total, nineteen SNPs were detected, with five of them in the coding region and two (c.46T/C and c.515G/A putatively resulting in amino acid changes (p.Tyr16His and p.Lys172Arg. In region-1, -2 and -3 of 316 sheep from eight New Zealand breeds, variants A1, A2 and A3 were the most common, although variant frequencies differed in the eight breeds. Across region-1 and region-3, nine haplotypes were identified and haplotypes A1-A3, A1-C3, B1-A3 and B1-C3 were most common. These results indicate that the ADIPOQ gene is polymorphic and suggest that further analysis is required to see if the variation in the gene is associated with animal production traits.

  12. Proteolysis of ovine and caprine Caseins in solution by enzymatic extracts from flowers of Cynara cardunculuso

    Sousa, M. José; Malcata, F. Xavier

    1998-01-01

    Primary proteolysis of ovine and caprine Na-caseinate at 30°C in phosphate buffer at pH 6.5 or 5.5 in the absence of NaCl and at pH 5.2 with 5% (w/v) NaCl by cardosins in aqueous extracts of Cynara cardunculus flowers was investigated using urea-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Caprine caseinate underwent more extensive degradation than ovine caseinate under the same conditions (pH 6.5 and pH 5.5); proteolysis of b- and ...

  13. Recombinant kringle 5 from plasminogen antagonises hepatocyte growth factor-mediated signalling.

    Ansell, Peter J; Zhang, Haiying; Davidson, Don J; Harlan, John E; Xue, John; Brodjian, Sevan; Lesniewski, Rick; McKeegan, Evelyn

    2010-03-01

    The blood protein plasminogen is proteolytically cleaved to produce angiostatin and kringle 5 (K5), both of which are known angiogenesis inhibitors. A common structural element between K5, angiostatin and other endogenous angiogenesis inhibitors is the presence of the kringle protein-interacting domain. Another kringle domain-containing protein, hepatocyte growth factor (HGF), promotes angiogenesis by binding to and stimulating the tyrosine kinase receptor Met. HGF binding to Met is dependent on the kringle domains of HGF. Because both K5 and HGF contain kringle motifs and because these proteins have opposite effects on angiogenesis, we hypothesised that K5 can antagonise HGF-mediated signalling in a Met-dependent manner. We determined that K5 binding to H1299 cells is competed by HGF suggesting that these two proteins bind to the same protein. Purified K5 immunoprecipitates with Met and this interaction is abolished by increasing doses of HGF. Using proliferation, phosphorylation of Met and Akt as markers of HGF activity, we determined that K5 inhibits HGF-mediated signalling. Taken together, these data support a model by which K5 binds to Met and functions as a competitive antagonist of HGF signalling and presents a novel mechanism of action of K5. PMID:20061137

  14. 组织型纤溶酶原激活剂基因在牛卵丘—卵母复合体体外成熟进程中的表达%Expression of Tissue-type Plasminogen Activator in Bovine Cumulus-oocytes during in vitro Maturation

    关洪敏; 米焱; 张伶俐; 李海军; 曹贵方

    2012-01-01

    In the present study,four experiments were conducted to investigate the potential effects of tPA on bovine cumulus and oocytes during in vitro maturation (IVM) (0,8,16,24 h). The expressions of tPA were detected by relative Real-time RT-PCR experiments in bovine cumulus and oocytes both after 0,8,16,24 h of IVM and under different FSH concentrations after 16 h of IVM separately. Meanwhile,the degree of bovine cumulus cell expansion and the percentages of first polar body in oocyte were assessed respectively. TPA mRNA levels were higher in cumulus and oocytes at 16 h and 24 h than those at 0 h and 8 h of IVM significantly (P<0. 05). Among those groups that under different FSH concentrations (0,0. 01,0. 1 and 1 IU/mL) after 16 h of IVM.tPA mRNA levels of cumulus was increased following the elevated FSH concentrations,as well as the cumulus expansion,tPA mRNA of cumulus cells in 1 IU/mL FSH group was significantly higher than others. The results indicated that the expression of tPA in bovine cumulus was regulated positively by FSH.tPA could facilitate the cumulus expansion during IVM. However.it needed to be further investigated that tPA expression might be involved in first polar body excrusion of bovine oocytes.%为了初步了解组织型纤溶酶原激活剂( tissue-plasminogen activator,tPA)在牛卵丘—卵母复合体体外成熟进程中的潜在作用,试验运用实时定量RT-PCR技术分别检测体外成熟过程中不同时间段(0、8、16、24 h)以及添加不同浓度FSH成熟培养16 h后的牛卵丘细胞和卵母细胞中tPA基因相对表达变化,观察添加不同浓度FSH成熟培养16h后卵丘细胞膨胀程度差异,并统计卵母细胞第一极体排出情况.结果显示,卵丘—卵母复合体在体外成熟初期,tPA mRNA相对表达水平在体外成熟16和24 h的卵丘细胞和卵母细胞中显著高于0和8h组;在添加不同浓度FSH(0、0.01、0.1和1 IU/mL)体外成熟16h的各处理组,卵丘细胞中tPA mRNA相对表达

  15. Urokinase plasminogen receptor and the fibrinolytic complex play a role in nerve repair after nerve crush in mice, and in human neuropathies.

    Cristina Rivellini

    Full Text Available Remodeling of extracellular matrix (ECM is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.

  16. Antenatal maternal long-term hypoxia: acclimatization responses with altered gene expression in ovine fetal carotid arteries.

    Ravi Goyal

    Full Text Available In humans and other species, long-term hypoxia (LTH during pregnancy can lead to intrauterine growth restriction with reduced body/brain weight, dysregulation of cerebral blood flow (CBF, and other problems. To identify the signal transduction pathways and critical molecules, which may be involved in acclimatization to high altitude LTH, we conducted microarray with advanced bioinformatic analysis on carotid arteries (CA from the normoxic near-term ovine fetus at sea-level and those acclimatized to high altitude for 110+ days during gestation. In response to LTH acclimatization, in fetal CA we identified mRNA from 38 genes upregulated >2 fold (P2-fold (P<0.05. The major genes with upregulated mRNA were SLC1A3, Insulin-like growth factor (IGF binding protein 3, IGF type 2 receptor, transforming growth factor (TGF Beta-3, and genes involved in the AKT and BCL2 signal transduction networks. Most genes with upregulated mRNA have a common motif for Pbx/Knotted homeobox in the promoter region, and Sox family binding sites in the 3' un translated region (UTR. Genes with downregulated mRNA included those involved in the P53 pathway and 5-lipoxygenase activating proteins. The promoter region of all genes with downregulated mRNA, had a common 49 bp region with a binding site for DOT6 and TOD6, components of the RPD3 histone deacetylase complex RPD3C(L. We also identified miRNA complementary to a number of the altered genes. Thus, the present study identified molecules in the ovine fetus, which may play a role in the acclimatization response to high-altitude associated LTH.

  17. Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

    Craig, A M; Latham, C J; Blythe, L L; Schmotzer, W B; O'Connor, O A

    1992-01-01

    The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1444382

  18. Androgen receptor and histone lysine demethylases in ovine placenta.

    Cleys, Ellane R; Halleran, Jennifer L; Enriquez, Vanessa A; da Silveira, Juliano C; West, Rachel C; Winger, Quinton A; Anthony, Russell V; Bruemmer, Jason E; Clay, Colin M; Bouma, Gerrit J

    2015-01-01

    Sex steroid hormones regulate developmental programming in many tissues, including programming gene expression during prenatal development. While estradiol is known to regulate placentation, little is known about the role of testosterone and androgen signaling in placental development despite the fact that testosterone rises in maternal circulation during pregnancy and in placenta-induced pregnancy disorders. We investigated the role of testosterone in placental gene expression, and focused on androgen receptor (AR). Prenatal androgenization decreased global DNA methylation in gestational day 90 placentomes, and increased placental expression of AR as well as genes involved in epigenetic regulation, angiogenesis, and growth. As AR complexes with histone lysine demethylases (KDMs) to regulate AR target genes in human cancers, we also investigated if the same mechanism is present in the ovine placenta. AR co-immunoprecipitated with KDM1A and KDM4D in sheep placentomes, and AR-KDM1A complexes were recruited to a half-site for androgen response element (ARE) in the promoter region of VEGFA. Androgenized ewes also had increased cotyledonary VEGFA. Finally, in human first trimester placental samples KDM1A and KDM4D immunolocalized to the syncytiotrophoblast, with nuclear KDM1A and KDM4D immunostaining also present in the villous stroma. In conclusion, placental androgen signaling, possibly through AR-KDM complex recruitment to AREs, regulates placental VEGFA expression. AR and KDMs are also present in first trimester human placenta. Androgens appear to be an important regulator of trophoblast differentiation and placental development, and aberrant androgen signaling may contribute to the development of placental disorders. PMID:25675430

  19. Decellularized ovine arteries as small-diameter vascular grafts

    Atherosclerosis and its complications still represent the leading cause of death in the developed countries. While autologous blood vessels may be regarded as the best solution for peripheral and coronary bypass, they are unavailable in most patients. Even though tissue engineering techniques are often applied to the development of small-diameter vascular grafts, limiting factors of this approach are represented by the lack of essential extracellular matrix proteins and/or poor biomechanical properties of the scaffolds used. Along these lines, the aim of this study was to develop a decellularization protocol for ovine carotids to be used as suitable small-diameter vascular grafts. Samples were treated either with sodium dodecyl sulphate (SDS) or with Trypsin and Triton X-100; a final nuclease digestion was performed for both protocols. Morphological analyses demonstrate complete removal of nuclei and cellular components in treated vessels, also confirmed by significant reduction in wall thickness and DNA content. Essential extracellular matrix proteins such as collagen, elastin, and fibronectin are well preserved after decellularization. From a mechanical point of view, Trypsin and Triton X-100 treated arteries show elastic modules and compliance comparable to native carotids, whereas the use of SDS makes samples stiffer, with a significant decrease in the compliance mean value and an increase in longitudinal and circumferential Young’s modules. It is demonstrated that the treatment where Trypsin and Triton X-100 are combined guarantees complete decellularization of carotids, with no significant alteration of biomechanical and structural properties, thus preserving a suitable environment for adhesion, proliferation, and migration of cells. (paper)

  20. Reduction of canine plasminogen leads to an expanded molecule which precipitates.

    Jack A Kornblatt

    Full Text Available Canine plasminogen is made up of seven domains. In each domain there are several cysteines that are linked by disulfide bonds. Reduction of a limited number of the cystines destabilizes the protein such that it precipitates. The bond or bonds that are broken provide about 14 kcal of stabilization energy. Circular dichroism and dynamic light scattering indicate that there is probably an intermediate that is formed prior to precipitation and that the intermediate is somewhat larger than the compact form of plasminogen.