WorldWideScience

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.

  2. PLASMINOGEN ACTIVATOR OF YERSINIA PESTIS

    V. V. Evseeva

    2015-04-01

    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 fibrin clots preventing bacteria dissemination after bites of infected fleas or subcutaneous challenge is believed to be the main Y. pestis factor responsible for generalization of infectious process. Pla-mediated ability of Y. pestis for selective binding with extracellular matrix and basal membranes may promote further hydrolysis of these structures by the host’s plasmin and overcoming tissue barriers by the pathogen. Y. pestis plasminogen activator also hydrolyses C3 complement component, human antimicrobial peptide — cathelicidin LL-37 and such cytokines as tumor necrosis factor α, interferon γ, interleukin 8 and protein 1 of monocyte chemotaxis. The main endogenic TFPI tissue factor pathway inhibitor also highly susceptible to proteolytic action of Pla, and efficiency of TFPI inactivation is much higher than efficacy of plasminogen activation. The review also debates the possibility of using Pla as a molecular target for prophylaxis and treatment of plague. 

  3. Coevolutionary patterns in plasminogen activation

    Gladysheva, Inna P.; Turner, Ryan B.; Sazonova, Irina Y; Liu, Lin; Reed, Guy L

    2003-01-01

    The generation of plasmin by plasminogen (Pg) activators (PAs) is a physiologic process in animals that dissolves blood clots and promotes wound healing, blood vessel growth, and the migration of normal and cancerous cells. Pathogenic bacteria have evolved PAs [e.g., streptokinase (SK) and staphylokinase] that exploit the Pg system to infect animals. Animal PAs have a conserved ability to cleave a wide spectrum of animal Pgs, but the ability of bacterial PAs to cleave ...

  4. 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 the A1D12 F(ab region to Glu-plasminogen increases the catalytic efficiency of plasminogen activation by plasminogen activators. Therefore, it may be useful to apply clinically A1D12 for the therapy of thromboembolic events such as myocardial infarction by humanizing the F(ab fragment of the A1D12 antibody. Inhibition pattern of antibody MC2B8 obey the mixed type of enzyme inhibition by binding the antibody probably at, or near, the cleavage site of Glu-plasminogen.

  5. Plasminogen activation and cancer

    Danø, Keld; Behrendt, N.; Hoyer-Hansen, G.; Johnsen, Morten; Lund, L. R.; Ploug, M.; Nielsen, John Rømer

    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...

  6. 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

  7. Plasminogen and Plasminogen Activators Protect against Renal Injury in Crescentic Glomerulonephritis

    Kitching, A. Richard; Holdsworth, Stephen R.; Ploplis, Victoria A; Plow, Edward F; Collen, Désiré; Carmeliet, Peter; Tipping, Peter G.

    1997-01-01

    The plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of plasminogen and plasminogen activators in the development of inflammatory glomerular injury, GN was i...

  8. The Glycosylation of Plasminogen Activator Inhibitor-1

    Skottrup, Peter; Pedersen, Katrine Egelund; Christensen, Anni; Thøgersen, Ida; Andreasen, Peter; Enghild, Jan Johannes

    Plasminogen activator inhibitor type-1 (PAI-1) has three potential sites for N-linked glycosylation, including Asn209Tyr210Thr211, Asn265Met266Thr267, and Asn329Glu330Ser331. Using a HEK293 expression system, we have made mutants with Asp or Gln substitutions of the Asn residue in each of these...

  9. Flow Cytometric Assays for Quantifying Activated Ovine Platelets

    Johnson, Carl A.; Snyder, Trevor A.; Woolley, Joshua R.; Wagner, William R.

    2007-01-01

    Ovines are a common animal model for preclinical evaluation of cardiovascular devices including heart valves, endovascular grafts, and ventricular assist devices. Biocompatibility is essential to the success of these devices; however, tools to assess biocompatibility in ovines are limited. To address this need, antibodies that bind to activated human and bovine platelets and annexin V protein were evaluated for potential cross-reactivity to activated ovine platelets. These candidate markers w...

  10. Indium-111 labeled tissue plasminogen activator

    Although radiolabeled fibrinogen has been used successfully for detection of thromboemboli by imaging, plasminogen activators offer the advantage of clot detection after active fibrin deposition has ceased. The authors have labeled recombinant tissue plasminogen activator (rTPA) with indium-111 (In) and studied its properties in vivo. Using the cyclic anhydride and standard methodology, an average of about 1 DTPA group per molecule was attached and the protein was labeled with In to a specific activity of 2-3 mCi/mg. Size exclusion HPLC showed each preparation to be of high radiochemical purity and that the In was bound only to the DTPA groups. In vivo clot localization was investigated using a canine clot model in which thrombogenic catheters are placed in a femoral artery and/or in the jugular vein prior to the administration of labeled rTPA. After administration of 0.5-1.0 mg of In-rTPA, blood and serum activity levels were found to decrease with a half time of 8 +- 1 min. in good agreement with the literature value of 7 min. for unlabeled rTPA. Clot:blood and clot:serum ratios were found to increase rapidly, in one study reaching 6.3 after 31 min. Because of the short residence time of rTPA in blood, activity in cardiac and pulmonary blood pools decreased rapidly such that clots were visualized within minutes of injection. The authors conclude that rTPA may be labeled with In without destroying the molecule's ability to localize in clot and that, with this agent, useful images of forming clot may be obtained within one hour of administration. This protein may therefore be ideal for imaging pulmonary and cardiac thromboemboli by gamma scintigraphy

  11. 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. The activation of the plasminogen activator system may be one mechanism by which all-trans retinoic acid exerts beneficial effects in cutaneous wound healing. PMID:11348470

  12. Human monocytes can produce tissue-type plasminogen activator

    1989-01-01

    Evidence has previously been presented that monocytes and macrophages produce urokinase-type plasminogen activator. We have shown for the first time that human monocytes, when stimulated appropriately in vitro, can produce tissue type-plasminogen activator (t-PA) of 70 kD. Detection of t-PA mRNA was consistent with the biochemical and immunological characterization of t-PA produced by human monocytes.

  13. 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.

  14. Evaluation of Prognostic Values of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 in Crimean-Congo Hemorrhagic Fever Patients

    Gurbuz; Ozturk,, S; Tutuncu; Sencan; Cicek Senturk; Altay

    2015-01-01

    Background Crimean-Congo hemorrhagic fever (CCHF) is a widespread disease in Turkey, and was responsible for many deaths in endemic regions during the last decade. The pathogenesis of the disease is not fully understood yet. Objectives In this study we aimed to determine the levels of tissue plasminogen activator (tPA) and Plasminogen activator inhibitor-1 (PAI-1) as predictors of prognosis in CCHF. ...

  15. Photonic Activation of Plasminogen induced by low dose UVB

    Correia, Manuel Guiherme L.P. Marins; Snabe, Torben; Thiagarajan, Viruthachalam; Petersen, Steffen; Sara R. R. Campos, Sara R. R.; Baptista, António M; Neves Petersen, Teresa

    2015-01-01

    Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity w...

  16. [Role of plasminogen activator inhibitor type 1 in tumor angiogenesis].

    Bajou, K; Devy, L; Masson, V; Albert, V; Frankenne, F; Noël, A; Foidart, J M

    2001-01-01

    The plasminogen/plasmin system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumour cell migration. High levels of components of the plasminogen activation system, and paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI-1), have been correlated with a poor prognosis for patients with cancers of different types. Recent findings clearly suggest that PAI-1 is essential for capillary sprouting during tumour angiogenesis. Moreover, there is accumulating evidence that both the urokinase receptor and PAI-1 are multifunctional proteins involved not only in extracellular matrix proteolysis but also in cellular adhesion and migration through their binding site for vitronectin. The understanding of whether PAI-1 plays a regulatory role in angiogenesis by tightly controlling proteolytic activity or by influencing cell migration could allow a new anti-angiogenic approach for tumour therapy. PMID:11806282

  17. A Novel Interaction between Complement Inhibitor C4b-binding Protein and Plasminogen That Enhances Plasminogen Activation.

    Agarwal, Vaibhav; Talens, Simone; Grandits, Alexander M; Blom, Anna M

    2015-07-24

    The complement, coagulation, and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross-talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to Streptococcus pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the β-chain and protein S binds plasminogen much stronger than the main isoform containing the β-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the β-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, whereas the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation. PMID:26067271

  18. Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

    Jeon Hyejin; Kim Jong-Heon; Kim Jae-Hong; Lee Won-Ha; Lee Myung-Shik; Suk Kyoungho

    2012-01-01

    Abstract Background Plasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of urokinase type plasminogen activators (uPA) and tissue type plasminogen activators (tPA), which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system. Methods In this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatog...

  19. 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; Chang, T. S.; 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...

  20. 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....

  1. Treatment of subretinal hemorrhages with tissue plasminogen activator.

    Borrillo, J L; Regillo, C D

    2001-06-01

    Submacular hemorrhages pose a danger to visual acuity. Intervention may help prevent or mitigate severe visual loss. Pneumatic displacement and vitrectomy with direct evacuation are two methods of treating submacular hemorrhages. Tissue plasminogen activator may be an important adjunct to these techniques. PMID:11389348

  2. 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; Svenningsen, Per; Nielsen, Flemming; Hinrichs, Gitte Rye; Bistrup, Claus; Jensen, Boye L

    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...... (uPA) in vitro. It was hypothesized that uPA is abnormally filtered to pre-urine and is inhibited in urine by amiloride in nephrotic syndrome. This was tested by determination of Na+-balance, uPA protein and activity and amiloride concentration in urine from rats with puromycin aminonucleoside (PAN......) induced nephrotic syndrome. Urine samples from 6 adult and 18 pediatric patients with nephrotic syndrome were analyzed for uPA activity and protein. PAN-treatment induced significant proteinuria in rats which coincided with increased urine uPA protein and activity, increased urine protease activity and...

  3. Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

    Barazzone, C; Belin, D.; Piguet, P F; Vassalli, J D; Sappino, A. P.

    1996-01-01

    Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whe...

  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. 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

    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...

  6. Immunohistochemical localisation of tissue plasminogen activator in human brain tumours.

    Franks, A J; Ellis, E.

    1989-01-01

    The distribution of tissue plasminogen activator (t-PA) has been studied in a series of 38 human brain tumours and two specimens of cerebral cortex, using the monoclonal antibody ESP6. t-PA was localised in vascular endothelium in the majority of tumours and both the cortical specimens, confirmed by double staining with Ulex europaeus lectin (Uel) and Factor 8-related antigen. Nineteen out of 22 high grade astrocytomas showed strong endothelial staining whereas staining was weak or absent in ...

  7. Evaluation of Prognostic Values of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 in Crimean-Congo Hemorrhagic Fever Patients

    Gurbuz

    2015-10-01

    Full Text Available Background Crimean-Congo hemorrhagic fever (CCHF is a widespread disease in Turkey, and was responsible for many deaths in endemic regions during the last decade. The pathogenesis of the disease is not fully understood yet. Objectives In this study we aimed to determine the levels of tissue plasminogen activator (tPA and Plasminogen activator inhibitor-1 (PAI-1 as predictors of prognosis in CCHF. Patients and Methods Patients who were diagnosed by the polymerase chain reaction (PCR and IgM positivity in the reference laboratory were included in this study. Tissue Plasminogen activator and PAI-1 levels were measured by the enzyme linked immunosorbent assay (ELISA using a commercial kit (human t-PA ELISA and human PAL-1 ELISA; BioVendor research and diagnostic products, BioVendor-Laboratorni medicina a.s., Brno, Czech Republic. Results A total of 46 patients participated in this study. The significant differences between recovering patients and the patients who died, regarding Aspartate aminotransferase (AST, Creatine Phosphokinase (CPK, Lactate Dehydrogenase (LDH, Prothrombin Time (PT, activated Partial Thromboplastin time (aPTT, and thrombocyte and fibrinogen levels, were consistent with many clinical studies in the literature. The fatal cases were found to have higher tPA and PAI-1 levels in contrast to the patients who completely recovered. Conclusions We think that these findings may help the progress of understanding of CCHF pathogenesis.

  8. 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 ultraviolet B induces urokinase-type plasminogen activator receptor. The epidermal induction of components of the proteolytic urokinase system by ultraviolet B may help explain the photoinducibility of pemphigus lesions. PMID:10417621

  9. Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity

    Stillfried, Gillian E; Saunders, Darren N; Ranson, Marie

    2007-01-01

    Introduction The regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used t...

  10. Plasminogen activator in the rodent brain

    The cellular origin(s), the biochemical properties and the developmental pattern of the protease activator (PA) were investigated in the rodent brain. PA activity was localized in frozen brain sections by a novel autoradiographic technique. PA levels and electrophoretic mobility were determined in homogenates prepared from major regions of the developing and the mature brain, and both the localization and the specific activity of the enzyme were examined in X-irradiated brain regions. PA activity was shown to be correlated with cell bodies in neuronal-enriched regions and also with endothelial, meningeal and ependymal layers. PA levels increased in a transient manner and at different rates and time periods in the various brain regions that were analyzed. PA in neuronal, but not in epithelial cell layers was affected by X-irradiation and one of the brain PA species had a similar molecular weight to that of neuroblastoma cells. The authors' findings suggest that in the brain PA is produced by neurons and by epithelial cells, and that it may have additional functions to that of thrombolysis both in the developing and the mature brain. (Auth.)

  11. 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

  12. 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...... methodology. In the present study we investigated levels of plasminogen activator inhibitor type 1 (PAI-I) and urokinase plasminogen activator receptor (uPAR), as quantitated by ELISA in tumour extracts from 64 NSCLC patients (38 squamous cell carcinomas, 26 adenocarcinomas), and compared them to staining...

  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. 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.

  15. 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) ...

  16. 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...

  17. Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase

    Figueiredo, Barbara C.; Da'dara, Akram A.; Oliveira, Sergio C.; Skelly, Patrick J.

    2015-01-01

    Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females) can all promote significant plasminogen (PLMG) activation in the presence of tissue plasminogen activator (tPA). This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno) is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA) and phosphoenolpyruvate (PEP). The enzyme has maximal activity at pH 7.5, requires Mg2+ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasites ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation. PMID:26658895

  18. Tissue-type plasminogen activator in somatostatin cells of rat pancreas and hypothalamus

    Kristensen, P; Larsson, L I; Danø, K; Nielsen, Jens Høiriis

    1987-01-01

    Plasminogen activators (PAs) proteolytically convert plasminogen to plasmin, which, in turn, can degrade most proteins. This system has been implicated in a variety of biological processes. Using immunocytochemical methods, we here describe the localization of tissue-type PA (t-PA) in rat somatos...... hypothalamus. No t-PA immunoreactivity could be detected in somatostatin cells of the gastric and intestinal mucosa....

  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. Role of plasminogen activator-plasmin system in tumor angiogenesis.

    Rakic, J M; Maillard, C; Jost, M; Bajou, K; Masson, V; Devy, L; Lambert, V; Foidart, J M; Noël, A

    2003-03-01

    New blood formation or angiogenesis has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization. Angiogenesis is associated with important extracellular remodeling involving different proteolytic systems among which the plasminogen system plays an essential role. It belongs to the large serine proteinase family and can act directly or indirectly by activating matrix metalloproteinases or by liberating growth factors and cytokines sequestered within the extracellular matrix. Migration of endothelial cells is associated with significant upregulation of proteolysis and, conversely, immunoneutralization or chemical inhibition of the system reduces angiogenesis in vitro. On the other hand, genetically altered mice developed normally without overt vascular anomalies indicating the possibility of compensation by other proteases in vivo. Nevertheless, they have in some experimental settings revealed unanticipated roles for previously characterized proteinases or their inhibitors. In this review, the complex mechanisms of action of the serine proteases in pathological angiogenesis are summarized alongside possible therapeutic applications. PMID:12737307

  1. 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.

  2. Tissue plasminogen activator is a potent activator of PDGF-CC

    Fredriksson, Linda; Li, Hong; Fieber, Christina; Li, Xuri; Eriksson, Ulf

    2004-01-01

    Tissue plasminogen activator (tPA) is a serine protease involved in the degradation of blood clots through the activation of plasminogen to plasmin. Here we report on the identification of tPA as a specific protease able to activate platelet-derived growth factor C (PDGF-C). The newly identified PDGF-C is secreted as a latent dimeric factor (PDGF-CC) that upon proteolytic removal of the N-terminal CUB domains becomes a PDGF receptor α agonist. The CUB domains in PDGF-CC directly interact with...

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

    Stolzenburg Oxlund, Christina; Buhl, Kristian Bergholt; Jacobsen, Ib A; Hansen, Mie R; Gram, Jeppe; Henriksen, Jan Erik; Schousboe, Karoline; Tarnow, Lise; Jensen, Boye L

    2014-01-01

    inhibitor of the renin-angiotensin-aldosterone system) and increased to 10 mg if BP control was not achieved at 4 weeks. Complete dataset for urine analysis was available in 60 patients. Systolic and diastolic BP measured by ambulatory BP monitoring and office monitoring were significantly reduced. Average....../proteinuria to resistant hypertension. Amiloride, an ENaC inhibitor, inhibits urokinase-type plasminogen activator. We hypothesized that amiloride (1) reduces blood pressure (BP); (2) attenuates plasminogen-to-plasmin activation; and (3) inhibits urine urokinase-type plasminogen activator in patients with...... macroalbuminuric urine, with a tendency toward reduction in activity after amiloride treatment. Amiloride lowers BP, urine plasminogen excretion and activation, and albumin/creatinine ratio, and is a relevant add-on medication for the treatment of resistant hypertension in patients with T2DM and microalbuminuria....

  4. Plasma plasminogen activator inhibitor-1 predicts myocardial infarction in HIV-1-infected individuals

    Knudsen, Andreas; Katzenstein, Terese L; Benfield, Thomas; Jørgensen, Niklas R; Kronborg, Gitte; Gerstoft, Jan; Obel, Niels; Kjær, Andreas; Lebech, Anne-Mette

    2014-01-01

    antiretroviral therapy, sex, smoking and no known cardiovascular disease. Levels of high-sensitivity C-reactive protein, soluble endothelial selectin, soluble vascular cell adhesion molecule, soluble intercellular adhesion molecule, matrix metalloprotease 9, myeloperoxidase, and plasminogen activator inhibitor 1...

  5. 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

  6. Characterization of a plasminogen activator from human melanoma cells cultured in vitro

    This thesis describes the work that have been done on the isolation and characterization of a plasminogen activator, Mel-PA, that is released by human melanoma cells cultured in vitro. This enzyme was compared to the urinary plasminogen activator, urokinase. The human melanoma cell line released large amounts of Mel-PA into the surrounding medium when cultured under serum-free conditions. These cells released only one type of plasminogen activator. A technique was developed in which plasminogen activators were seperated electrophoretically and detected in polyacrylamide gel slabs. Mel-PA was concentrated and partially purified by affinity chromatography on benzamidine-sepharose. A study of the distribution of plasminogen activators in tissues and body fluids showed that all mammals examined had two immunochemically distinct plasminogen activators that corresponded, in their distribution, to the urokinase-like and Mel-PA like enzymes of man. A comparitive study of the kinetic behaviour of Mel-PA and urokinase showed numerous differences between the catalytic activities of these two enzymes

  7. Upregulation of Monocyte Urokinase Plasminogen Activator Receptor during Human Endotoxemia

    Dekkers, Pascale E. P.; ten Hove, Tessa; te Velde, Anje A.; van Deventer, Sander J. H.; van der Poll, Tom

    2000-01-01

    The receptor for urokinase-type plasminogen activator (uPAR) (CD87) plays an important role in leukocyte adhesion and migration. To assess the effect of endotoxin on cellular uPAR, uPAR expression was determined on leukocytes by fluorescence-activated cell sorter analysis in seven healthy subjects following intravenous injection of endotoxin (lot G; 4 ng/kg). Endotoxin induced a transient increase in uPAR expression on monocytes, reaching a 92% ± 46% increase over baseline expression after 6 h (P < 0.05). Endotoxin did not influence uPAR expression on granulocytes, while uPAR remained undetectable on lymphocytes. Endotoxin also increased soluble uPAR levels in plasma (P < 0.05). Stimulation of human whole blood with endotoxin or gram-positive stimuli in vitro also resulted in an upregulation of monocyte uPAR expression. Although tumor necrosis factor alpha (TNF) upregulated monocyte uPAR expression, anti-TNF did not influence the endotoxin-induced increase in monocyte uPAR expression. These data suggest that infectious stimuli may influence monocyte function in vivo by enhancing the expression of uPAR. PMID:10722614

  8. 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 that plasmin activates MMP-13 directly. Conclusions These data demonstrate that breast cancer cells dissolve type I collagen and that there is an absolute requirement for plasminogen activation and MMP activity in the degradation process.

  9. 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

  10. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert (UMASS, MED)

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  11. 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

  12. 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.

  13. Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by Rickettsia conorii- and Rickettsia rickettsii-infected cultured endothelial cells.

    Drancourt, M.; Alessi, M.C.; Levy, P. Y.; Juhan-Vague, I; Raoult, D

    1990-01-01

    Hemostasis abnormalities have been described in patients with Mediterranean spotted fever and Rocky Mountain spotted fever. Evidence of the activation of the fibrinolytic system has been obtained in both diseases. After experimental Rocky Mountain spotted fever, an elevated level of fibrinogen was found in parallel with the activation of the fibrinolytic system and transient elevation of the tissue-type plasminogen activator. Later protein is mainly synthesized by endothelial cells. The abili...

  14. Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro.

    Wojta, J.; Holzer, M.; Hufnagl, P.; Christ, G.; Hoover, R. L.; Binder, B R

    1991-01-01

    The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42 degrees C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by a four- to fivefold increase in PAI-1 specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and ant...

  15. 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...

  16. Staphylokinase Control of Staphylococcus aureus Biofilm Formation and Detachment Through Host Plasminogen Activation.

    Kwiecinski, Jakub; Peetermans, Marijke; Liesenborghs, Laurens; Na, Manli; Bjrnsdottir, Halla; Zhu, Xuefeng; Jacobsson, Gunnar; Johansson, Bengt R; Geoghegan, Joan A; Foster, Timothy J; Josefsson, Elisabet; Bylund, Johan; Verhamme, Peter; Jin, Tao

    2016-01-01

    Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components. PMID:26136471

  17. Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from Streptococcus uberis

    Ward, Philip N.; Leigh, James A.

    2002-01-01

    A bovine plasminogen activator of atypical molecular mass (∼45 kDa) from Streptococcus uberis strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072–1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of S. uberis strains. Analysis of the locus normally occupied by pauA revealed an absence of the pa...

  18. 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

  19. Endogenous Bradykinin Contributes to Increased Plasminogen Activator Inhibitor 1 Antigen following Hemodialysis

    Marney, Annis M.; Ma, Ji; Luther, James M.; Ikizler, T. Alp; Brown, Nancy J.

    2009-01-01

    Oxidative stress and inflammation predict cardiovascular events in chronic hemodialysis patients. Hemodialysis activates the kallikrein-kinin system, increasing bradykinin. Bradykinin promotes inflammation but also stimulates endothelial release of tissue-plasminogen activator and inhibits platelet aggregation. Understanding the detrimental and beneficial effects of endogenous bradykinin during hemodialysis has implications for the treatment of cardiovascular disease in the hemodialysis popul...

  20. Increased tissue-type plasminogen activator activity in orthotopic but not heterotopic liver transplantation: the role of the anhepatic period.

    Bakker, C M; Metselaar, H J; Groenland, T N; Gomes, M J; Knot, E A; Hesselink, E J; Schalm, S W; Stibbe, J; Terpstra, O T

    1992-08-01

    The major cause of the increased tissue-type plasminogen activator activity during orthotopic liver transplantation is still unclear. Both the lack of hepatic clearance of tissue-type plasminogen activator in the anhepatic period and increased endothelial release from the graft on reperfusion have been proposed as the major causes. Heterotopic liver transplantation avoids the resection of the host liver and is a useful model to help differentiate between these two possibilities. In this study the fibrinolytic system was evaluated in 10 orthotopic liver transplantations, 18 heterotopic liver transplantations and a control group of 10 partial hepatic resections. A marked increment in tissue-type plasminogen activator activity, from 0.2 to 5.2 IU/ml (p less than 0.02), was observed during the anhepatic period of orthotopic liver transplantation, which rapidly normalized after reperfusion. In contrast, tissue-type plasminogen activator activity levels remained normal in heterotopic liver transplantation and partial hepatic resections. In orthotopic liver transplantation and in heterotopic liver transplantation no increase occurred in tissue-type plasminogen activator activity after reperfusion. The first venous hepatic outflow after reperfusion did not contain elevated tissue-type plasminogen activator activity levels. Plasma degradation products of fibrin and fibrinogen increased during the anhepatic period of orthotopic liver transplantation (from 2.60 to 8.80 micrograms/ml [p less than 0.008] and from 0.40 to 1.60 micrograms/ml [p less than 0.04], respectively) and remained elevated thereafter. In heterotopic liver transplantation and partial hepatic resections these levels remained low. In conclusion, the lack of hepatic clearance during the anhepatic period is probably the most important factor in the evolution of increased tissue-type plasminogen activator activity during orthotopic liver transplantation. PMID:1639350

  1. The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent.

    Devy, Laetitia; Blacher, Silvia; Grignet-Debrus, Christine; Bajou, Khalid; Masson, Veronique; Gerard, Robert D; Gils, Ann; Carmeliet, Geert; Carmeliet, Peter; Declerck, Paul J; Nöel, Agnès; Foidart, Jean-Michel

    2002-02-01

    Plasminogen activator inhibitor 1 (PAI-1) is believed to control proteolytic activity and cell migration during angiogenesis. We previously demonstrated in vivo that this inhibitor is necessary for optimal tumor invasion and vascularization. We also showed that PAI-1 angiogenic activity is associated with its control of plasminogen activation but not with the regulation of cell-matrix interaction. To dissect the role of the various components of the plasminogen activation system during angiogenesis, we have adapted the aortic ring assay to use vessels from gene-inactivated mice. The single deficiency of tPA, uPA, or uPAR, as well as combined deficiencies of uPA and tPA, did not dramatically affect microvessel formation. Deficiency of plasminogen delayed microvessel outgrowth. Lack of PAI-1 completely abolished angiogenesis, demonstrating its importance in the control of plasmin-mediated proteolysis. Microvessel outgrowth from PAI-1-/- aortic rings could be restored by adding exogenous PAI-1 (wild-type serum or purified recombinant PAI-1). Addition of recombinant PAI-1 led to a bell-shaped angiogenic response clearly showing that PAI-1 is proangiogenic at physiological concentrations and antiangiogenic at higher levels. Using specific PAI-1 mutants, we could demonstrate that PAI-1 promotes angiogenesis at physiological (nanomolar) concentrations through its antiproteolytic activity rather than by interacting with vitronectin. PMID:11818362

  2. Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

    Jeon Hyejin

    2012-06-01

    Full Text Available Abstract Background Plasminogen activator inhibitor type 1 (PAI-1 is the primary inhibitor of urokinase type plasminogen activators (uPA and tissue type plasminogen activators (tPA, which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system. Methods In this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatography and tandem mass spectrometry. Secretion of PAI-1 from glial cultures was detected by ELISA and western blotting analysis. Cell migration was evaluated by in vitro scratch-wound healing assay or Boyden chamber assay and an in vivo stab wound injury model. Phagocytic activity was measured by uptake of zymosan particles. Results The levels of PAI-1 mRNA and protein expression were increased by lipopolysaccharide and interferon-? stimulation in both microglia and astrocytes. PAI-1 promoted the migration of microglial cells in culture via the low-density lipoprotein receptor-related protein (LRP 1/Janus kinase (JAK/signal transducer and activator of transcription (STAT1 axis. PAI-1 also increased microglial migration in vivo when injected into mouse brain. PAI-1-mediated microglial migration was independent of protease inhibition, because an R346A mutant of PAI-1 with impaired PA inhibitory activity also promoted microglial migration. Moreover, PAI-1 was able to modulate microglial phagocytic activity. PAI-1 inhibited microglial engulfment of zymosan particles in a vitronectin- and Toll-like receptor 2/6-dependent manner. Conclusion Our results indicate that glia-derived PAI-1 may regulate microglial migration and phagocytosis in an autocrine or paracrine manner. This may have important implications in the regulation of brain microglial activities in health and disease.

  3. Soluble Urokinase Plasminogen Activator Receptor for Risk Prediction in Patients Admitted with Acute Chest Pain

    Lyngbæk, Stig; Andersson, Charlotte; Marott, Jacob L; Møller, Daniél V; Christiansen, Michael; Iversen, Kasper K; Clemmensen, Peter; Eugen-Olsen, Jesper; Hansen, Peter R; Jeppesen, Jørgen L

    2013-01-01

    Plasma concentrations of soluble urokinase plasminogen activator receptor (suPAR) predict mortality in several clinical settings, but the long-term prognostic importance of suPAR in chest pain patients admitted on suspicion of non-ST-segment elevation acute coronary syndrome (NSTEACS) is uncertain....

  4. 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.

  5. Soluble Urokinase-Type Plasminogen Activator Receptor Levels in Patients With Schizophrenia

    Nielsen, Jimmi; Røge, Rasmus; Pristed, Sofie Gry; Viuff, Anne Grethe; Ullum, Henrik; Thørner, Lise Wegner; Werge, Thomas; Vang, Torkel

    2015-01-01

    BACKGROUND: The etiology of schizophrenia remains largely unknown but alterations in the immune system may be involved. In addition to the psychiatric symptoms, schizophrenia is also associated with up to 20 years reduction in life span. Soluble urokinase-type plasminogen activator receptor (suPA...

  6. Effects of inhaled plasminogen activator on the balance between coagulation and fibrinolysis in traumatized pigs

    Münster, A-M B; Rasmussen, L; Sidelmann, Johannes Jakobsen; Ingemann Jensen, J; Bech, B; Gram, J

    2002-01-01

    effect of severe trauma on the alveolar fibrinolytic/coagulation balance, and the effect here-upon of inhalation of single-chain urokinase plasminogen activator (scu-PA) in pigs. The study shows an increased concentration of scu-PA in the bronchoalveolar lavage fluid of the treated animals in association...

  7. Presence of urokinase-type plasminogen activator (u-PA) in tissue extracts of antrochoanal polyp.

    Yamashiro, Y; Nakamura, M; Huang, G W; Kosugi, T

    1992-09-01

    Using a biochemical technique, the authors characterized and identified a plasminogen activator (PA) derived from tissue extracts of antrochoanal polyp (AP) and paranasal mucous membrane (PMM) with chronic sinusitis. The results of fibrin zymography indicated that the tissue extracts of AP revealed two lytic zones and that those of PMM revealed a single lytic zone on fibrin-agarose plates. One of the AP zones exhibited the same relative mobility as the PMM zone (molecular weight: 65 kd), while the other AP zone had a smaller molecular weight (about 54 kd). Goat immunoglobulin G (IgG) fraction of antihuman uterine tissue-type plasminogen activator (t-PA) inhibited the 65-kd lytic zones of AP and PMM. Antihuman low-molecular-weight urokinase inhibited only the 54-kd lytic zone of AP, and nonspecific goat IgG failed to inhibit any of the lytic zones. On the other hand, 10(-2) mol trans 4-(aminomethyl)cyclohexane-carboxylic acid (t-AMCHA) inhibited all of the lytic zones. No lytic zones could be observed on plasminogen-free fibrin-agarose plates. These findings confirmed that the tissue extracts of PMM contained t-PA, and that those of AP contained both t-PA and urokinase-type plasminogen activator (u-PA). In addition, it appeared that u-PA in inflammatory tissue was related to proliferative changes of the mucous membrane. PMID:1518351

  8. NMR secondary structure of the plasminogen activator protein staphylokinase

    Staphylokinase (Sak) is a 15.5 kDa protein secreted by several strains of Staphylococcusaureus. Due to its ability to convert plasminogen, the inactive proenzyme of the fibrinolytic system, into plasmin, Sak is presently undergoing clinical trials for blood clot lysis in the treatment of thrombovascular disorders. With a view to developing a better understanding of the mode of action of Sak, we have initiated a structural investigation of Sak via multidimensional heteronuclear NMR spectroscopy employing uniformly 15N- and 15N, 13C-labelled Sak. Sequence-specific resonance assignments have been made employing 15N-edited TOCSY and NOE experiments and from HNCACB, CBCA(CO)NH, HBHA(CBCACO)NH and CC(CO)NH sets of experiments. From an analysis of the chemical shifts, 3JHNHα scalar coupling constants, NOEs and HN exchange data, the secondary structural elements of Sak have been characterized

  9. Urokinase binds to a plasminogen activator inhibitor type-2-like molecule in placental microvillous membranes

    Jensen, Poul Henning; Nykjaer, A; Andreasen, P A; Lund, Lars; Astedt, B; Lecander, I; Gliemann, J

    1989-01-01

    activity, did not bind. Catalytically active tissue-type plasminogen activator did compete with 125I-labelled urokinase for binding although less efficiently than urokinase. Binding activity remained in the 100,000 x g pellet after treatment of the membranes with 3 M KCl, alkaline stripping at pH 12 or......Placental microvillous membranes exhibited saturable binding of urokinase-type plasminogen activator with plateau achieved by 30 min at 4 degrees C and 10 min at 37 degrees C. The binding was essentially irreversible. The capacity was about 8 pmol urokinase per mg membrane protein. Half......-maximal displacement of 125I-labelled urokinase was achieved with about 1.0 nM unlabelled urokinase when using 75 micrograms membrane protein/ml. 125I-labelled urokinase did not bind when treated with diisopropylfluorophosphate to block the catalytic activity. Single-chain urokinase (prourokinase), devoid of catalytic...

  10. 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...

  11. An enzyme-immunobinding assay for fast screening of expression of tissue plasminogen activator cDNA in E. coli

    Tissue plasminogen activator (TPA) has been isolated from normal human tissues and certain human cell lines in culture. The enzyme is a serine protease which converts an inactive zymogen, plasminogen to plasmin, and causes lysis of fibrin clots. The high affinity of TPA for fibrin indicates that it is a potential thrombolytic agent and is superior to urokinase-like plasminogen activators. Recently, TPA has been cloned and expressed in E. coli. Using TPA as a model protein, the authors report here the development of a direct, sensitive enzyme-immunoassay for the screening of a cDNA expression library using specific antibodies and peroxidase-labeled second antibody

  12. 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

    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....../min, respectively. The extraction fraction and the mean transit time in the splanchnic circulation were, respectively, 0.63 and 5.6 min for active t-PA and 0.17 and 21 min for t-PA antigen. Active PAI-1 was synthesized in the splanchnic circulation at a rate of 890 IU/min and had a mean transit time of about 9.......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...

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

    Lademann, Ulrik Axel; Rømer, Maria Unni Koefoed

    2008-01-01

    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...... 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....

  14. Candesartan reduces the hemorrhage associated with delayed tissue plasminogen activator treatment in rat embolic stroke

    Ishrat, Tauheed; Pillai, Bindu; Ergul, Adviye; Hafez, Sherif; Susan C. Fagan

    2013-01-01

    We have previously reported that angiotensin receptor blockade reduces reperfusion hemorrhage in a suture occlusion model of stroke, despite increasing matrix metalloproteinase (MMP-9) activity. We hypothesized that candesartan will also decrease hemorrhage associated with delayed (6h) tissue plasminogen activator (tPA) administration after embolic stroke, widening the therapeutic time window of tPA. Adult male Wistar rats were subjected to embolic middle cerebral artery occ...

  15. Terminalia catappa attenuates urokinase-type plasminogen activator expression through Erk pathways in Hepatocellular carcinoma

    Yeh, Chao-Bin; Yu, Yung-luen; Lin, Chiao-Wen; Chiou, Hui-Ling; Hsieh, Ming-Ju; Yang, Shun-Fa

    2014-01-01

    Background The survival rate of malignant tumors, and especially hepatocellular carcinoma (HCC), has not improved primarily because of uncontrolled metastasis. In our previous studies, we have reported that Terminalia catappa leaf extract (TCE) exerts antimetastasis effects on HCC cells. However, the molecular mechanisms of urokinase-type plasminogen activator (u-PA) in HCC metastasis have not been thoroughly investigated, and remain poorly understood. Methods The activities and protein level...

  16. Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator

    Kaczmarek, Jakub; Skottrup, Peter Durand

    2015-01-01

    Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like...... the nanobodies were determined by competitive ELISA to 80 nM and 522 nM, respectively. The best binder did not inhibit uPA (nAb-C3), however the lowest affinity binder (nAb-C8) was able to inhibit the uPA-mediated cleavage of the substrate S-2444. The results validate the naïve library as a resource...... for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure–function relationships....

  17. 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.).

  18. Simultaneous Combined Intravenous Recombinant Tissue Plasminogen Activator and Endovascular Therapy for Hyperacute Middle Cerebral Artery M1 occlusion

    Toyota, S.; Sugiura, S.; Iwaisako, K.

    2011-01-01

    We investigated the efficacy and safety of combined intravenous (IV) recombinant tissue plasminogen activator (rtPA) and simultaneous endovascular therapy (ET) for hyperacute middle cerebral artery (MCA) M1 occlusion.

  19. Purification and activation of caprine and canine plasminogens: Comparison with human plasminogen / Purificacin y activacin de los plasmingenos caprino y canino: comparacin con el plasmingeno humano

    Omaira, Caas Bermdez; Alfonso, Quijano Parra; Luis Fernando, Arbelez Ramrez.

    2011-06-01

    Full Text Available Objetivo: unificar la purificacin y activacin de los plasmingenos de tres especies diferentes, a saber: humana, caprina y canina. Materiales y mtodos: se usaron Lysina-Sefarosa 4B y Sefacel DEAE para las cromatografas de afinidad y de intercambio inico, respectivamente. Se determin la secuenc [...] ia terminal-N tanto de los plasmingenos intactos como de los degradados. Resultados: en las tres especies se identificaron bandas de 92 kDa correspondientes a los plasmingenos nativos. Se hall que sus secuencias terminales-N eran EPLDDY, DPLDDY y XXLDDY para los plasmingenos humano, caprino y canino, respectivamente. Adems, se purificaron los plasmingenos degradados circulantes, cuyas secuencias terminales-N fueron, en el mismo orden, KVYLSE, RITLL Y RIYSL. Conclusin: la activacin de los tres plasmingenos confirm la formacin de las bandas electroforticas tpicas de la plasmina humana correspondientes a las cadenas pesada A y liviana B, que tambin se identificaron en las plasminas caprina y canina. Este nuevo mtodo de purificacin facilita la comparacin y el esclarecimiento de los sistemas fibrinolticos de los mamferos. Abstract in english Objective: To unify the purification and activation of plasminogens from three different species, namely: human, caprine and canine. Materials and methods: Lysine-Sepharose 4B and sephacel DEAE were used, for affinity and ion-exchange chromatography, respectively. The N-terminal sequence was determi [...] ned for both the intact and degraded plasminogens. Results: Bands of 92 kDa corresponding to native plasminogens were identified in the three species. Their N-terminal sequences were found to be EPLDDY, DPLDDY and XXLDDY for human, caprine and canine plasminogen, respectively. Furthermore, the degraded in vivo circulating plasminogens from the three species were purified and their N-terminal sequences were KVYLSE, RITLL and RIYLS for the human, caprine and canine, in that order. Conclusion: Activation of the three plasminogens confirmed the formation of the typical electrophoretic bands for human plasmin corresponding to the heavy A and the light B chains which were also identified in the caprine and canine plasmins. This new purification methodology facilitates the comparison and further elucidation of the fibrinolytic systems in mammals.

  20. Urokinase plasminogen activator receptor on invasive cancer cells: A prognostic factor in distal gastric adenocarcinoma

    Alpizar, Warner Enrique Alpizar; Christensen, Ib Jarle; Santoni-Rugiu, Eric; Skarstein, Arne; Ovrebo, Kjell; Laerum, Ole Didrik; Illemann, Martin; Lærum, Ole D

    2012-01-01

    Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by...... mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. u...... association between the expression of uPAR on tumor cells in the peripheral invasion zone and overall survival of gastric cancer patients (HR = 2.16; 95% CI: 1.13-4.14; p = 0.02). Multivariate analysis showed that uPAR immunoreactivity in cancer cells at the invasive front is an independent prognostic factor...

  1. Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

    Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between 125I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase

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

    Kamlesh K. Gupta; 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-1R...

  3. Increased Expression of Plasminogen Activator Inhibitor-1 in Cardiomyocytes Contributes to Cardiac Fibrosis after Myocardial Infarction

    Takeshita, Kyosuke; HAYASHI, MUTSUHARU; Iino, Shigeo; Kondo, Takahisa; Inden, Yasuya; Iwase, Mitsunori; KOJIMA, TETSUHITO; HIRAI, Makoto; Ito, Masafumi; Loskutoff, David J.; Saito, Hidehiko; Murohara, Toyoaki; Yamamoto, Koji

    2004-01-01

    Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in tissue fibrosis by inactivating matrix metalloproteinases, which might effect on the progression of left ventricular dysfunction. However, little has been known about the expression of PAI-1 during cardiac remodeling. We used a mouse model of myocardial infarction (MI) by coronary ligation, in which the progression of left ventricular remodeling was confirmed by echocardiography. Histological examination showed that interstiti...

  4. 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...

  5. Stereotactic fibrinolysis of spontaneous intracerebral hematoma using infusion of recombinant tissue plasminogen activator

    Nasser Jos Augusto; Falavigna Asdrubal; Bezerra Mrcio; Martinez Victor; Freitas Gabriel; Alaminos Armando; Bonatelli Antnio; Ferraz Fernando

    2002-01-01

    PURPOSE: The authors present a prospective study on 10 patients with stereotactic infusion of tissue plasminogen activator (rtPA) intraparenchimal hemorrhage. METHODS: Between 1999 and 2000, 10 patients with deep seated hematomas in the basal ganglia were selected for stereotactic infusion of rtPA and spontaneous clot drainage. RESULTS: All cases had about 80% reduction of the hematoma volume in the CT scan at the third day. The intracranial pressure was normalized by the third day too. There...

  6. Medicolegal Considerations with Intravenous Tissue Plasminogen Activator in Stroke: A Systematic Review

    Archit Bhatt; Adnan Safdar; Dhara Chaudhari; Diane Clark; Amber Pollak; Arshad Majid; Mounzer Kassab

    2013-01-01

    Background. Intravenous tPA (tissue plasminogen activator) therapy remains underutilized in patients with Acute Ischemic Stroke (AIS). Anecdotal data indicates that physicians are increasingly liable for administering and for failure to administer tPA. Methods. An extensive search of Medline, Embase, Westlaw, LexisNexis Legal, and Google Scholar databases was performed. Case studies that involved malpractice litigation in ischemic stroke and thrombolytic therapy were analyzed systematically. ...

  7. 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...

  8. Hormonal regulation of plasminogen activator production in porcine kidney cells (LLC-PK1)

    An attempt is made to study the molecular events regulating and accompanying the hormonally modulated plasminogen activator (PA) gene expression. The model system of interest is a porcine kidney cell line, LLC-PK1, responding to calcitonin in the PA production. The plasminogen activator secreted by calcitonin treated pig kidney cells has been purified, characterized, and compared with human urinary urokinase. The purified enzyme resembles the 53 k MW components of human urokinase. PA induction in LLC-PK1 cells is sensitive to inhibition by actinomycin D, suggesting the enhanced transcription of PA-mRNA. This hypothesis was tested by measuring PA-mRNA sequences in Xenopus oocyte system which showed a 15-20 fold enhanced PA synthesis when supplied with poly (A)+ RNA from induced cells, above that obtained from uninduced cell RNA. A pleiotropic response to calcitonin in LLC-PK1 cells has been examined using two-dimensional gel electrophoresis of 35S methionine labeled polypeptides. A set of twelve intracellular polypeptides, ten induced and two repressed, has been identified in calcitonin stimulated cells. One of the induced polypeptides has been identified as plasminogen activator by two dimensional tryptic peptide mapping. Other calcitonin induced polypeptides have been identified as cytokeratins by their solubility properties and cross-reactivity with antiserum against human keratin. The results indicate that the experimental system presented here is a useful and valid one for the study of molecular events regulating and accompanying the hormonally modulated PA gene expression

  9. O-linked L-fucose is present in Desmodus rotundus salivary plasminogen activator.

    Gohlke, M; Baude, G; Nuck, R; Grunow, D; Kannicht, C; Bringmann, P; Donner, P; Reutter, W

    1996-03-29

    DSPAalpha1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPAalpha1 and its recombinant form isolated from Chinese hamster ovary cells. In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPAalpha1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins. Anew strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPAalpha1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator. PMID:8631761

  10. 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

  11. Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism

    Stubblefield, William B.; Alves, Nathan J.; Rondina, Matthew T.; Kline, Jeffrey A.

    2016-01-01

    Background We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE). Methods Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, ?2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405). Results Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT. Conclusion The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT. PMID:26866684

  12. 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 patients

  13. Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells

    Jensen, Poul Henning; Christensen, Erik Ilsø; Ebbesen, P.; Gliemann, Jørgen; Andreasen, Peter A.

    1990-01-01

    We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u-PA-inhibitor......We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u...

  14. Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding.

    Jiao, J; Yu, M; Ru, B

    2001-04-01

    A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent. PMID:11295444

  15. Immunohistochemical localization of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and ?2-antiplasmin in human corneal perforation: a case report

    Sugioka Koji

    2012-11-01

    Full Text Available Abstract Background Corneal ulceration leading to perforation is associated with infectious and non-infectious destructive conditions in the cornea. The fibrinolytic (plasminogen/plasmin system is considered to contribute to tissue remodeling in the wound healing process and it is believed to play an important role in proteolysis and fibrosis. To determine the localization of urokinase-type plasminogen activator (u-PA, u-PA receptor (u-PAR and ?2-antiplasmin (?2AP in the tissue of a corneal perforation, we investigated immunohistochemical expressions of u-PA, u-PAR, ?2AP, CD68, and ?-smooth muscle actin (?-SMA in a patient with corneal perforation that developed from an ulcer of no clear cause. Case presentation The patient was a 77-year-old woman who presented with a perforated corneal ulcer in her right eye. The cause of her corneal ulcer was unknown. Double immunohistochemistry was performed for the combinations of u-PA with u-PAR, CD68 or ?-SMA and ?2AP with CD68 or ?-SMA to detect the localization of u-PA and ?2AP. u-PA and u-PAR co-localization was seen in the corneal ulceration area. u-PA was mainly observed in CD68-positive cells and in some ?-SMA positive cells. On the other hand, ?2AP was not expressed in CD68-positive cells, but was expressed in ?-SMA positive cells. Conclusion We identified expression of the u-PA/u-PAR complex and ?2AP in a patient with a corneal ulcer. These two molecules are believed to play a crucial role in inflammatory cell recruitment, ECM synthesis and degradation during corneal wound healing.

  16. Distal hinge of plasminogen activator inhibitor-1 involves its latency transition and specificities toward serine proteases

    Shaltiel Shmuel

    2003-07-01

    Full Text Available Abstract Background The plasminogen activator inhibitor-1 (PAI-1 spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347, which interacts with serine residue of tissue-type plasminogen activator (tPA with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases. Results To understand more about the role of distal hinge for PAI-1 specificities towards serine proteases and for its conformational transition, wild type PAI-1 and its mutants were expressed in baculovirus system. WtPAI-1 was found to be about 12 fold more active than the fibrosarcoma PAI-1. Single site mutants within the Asp355-Arg356-Pro357 segment of PAI-1 yield guanidine activatable inhibitors (a that can still form SDS stable complexes with tPA and urokinase plasminogen activator (uPA, and (b that have inhibition rate constants towards plasminogen activators which resemble those of the fibrosarcoma inhibitor. More importantly, latency conversion rate of these mutants was found to be ~3–4 fold faster than that of wtPAI-1. We also tested if Glu351 is important for serine protease specificity. The functional stability of wtPAI-1, Glu351Ala, Glu351Arg was about 18 ± 5, 90 ± 8 and 14 ± 3 minutes, respectively, which correlated well with both their corresponding specific activities (84 ± 15 U/ug, 112 ± 18 U/ug and 68 ± 9 U/ug, respectively and amount of SDS-stable complex formed with tPA after denatured by Guanidine-HCl and dialyzed against 50 mM sodium acetate at 4°C. The second-order rate constants of inhibition for uPA, plasmin and thrombin by Glu351Ala and Glu351Arg were increased about 2–10 folds compared to wtPAI-1, but there was no change for tPA. Conclusion The Asp355-Pro357 segment and Glu351 in distal hinge are involved in maintaining the inhibitory conformation of PAI-1. Glu351 is a specificity determinant of PAI-1 toward uPA, plasmin and thrombin, but not for tPA.

  17. 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...

  18. Clinical evaluation of tissue plasminogen activator (t-PA) levels in patients with liver diseases.

    Okabe, K; Kato, I; Sato, S; Kashiwabara, T; Furuta, S; Sato, H; Tanikawa, K; Kumashiro, R; Kanayama, M

    1992-02-01

    Tissue plasminogen activator (t-PA) levels in plasma or serum were studied in 416 patients with liver diseases: acute hepatitis (AH, n = 30); fulminant hepatitis (FH, n = 36); chronic inactive hepatitis (CIH, n = 57); chronic active hepatitis (CAH, n = 39); compensated liver cirrhosis (cLC, n = 78); decompensated liver cirrhosis (dLC, n = 84); hepatocellular carcinoma (HCC, n = 64); advanced hepatocellular carcinoma (aHCC, n = 28); and compared with that of a control group (n = 106) of healthy subjects. The t-PA levels showed significant increase in patients with AH, FH, CAH, cLC, dLC and HCC, compared with normal controls. The abnormal rates in t-PA levels (higher than 8.3 ng/ml) for each type of liver diseases were 86.1% in FH, 46.2% in CAH, 50% in cLC, 85.7% in dLC, 67.2% in HCC, and 89.3% in aHCC. t-PA levels tended to be higher in more advanced liver diseases. t-PA levels significantly correlated positively with plasminogen activator inhibitor (PAI-1) in AH, cLC, dLC, HCC and aHCC, and negatively with plasmin alpha 1-plasmin inhibitor complex (PIC), plasminogen (Plg), FDP, AT III and alpha 2-plasmin inhibitor (alpha 2-PI) in dLC, prothrombin time (PT) and fibrinogen (Fbg) in HCC. t-PA levels in patients with FH, CAH and dLC were significantly higher than those in patients with AH, CIH and cLC, respectively. Moreover, the changes of t-PA levels in the clinical courses of various liver diseases revealed that t-PA levels increased sensitively with progression of liver diseases or in advanced liver diseases.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1313384

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

    Pedersen, Katrine E; Einholm, Anja P; Christensen, Anni; Schack, Lotte; Wind, Troels; Kenney, John M; Andreasen, Peter A

    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...... converted to reactive centre-cleaved monomers, indicating substrate behaviour of the terminal PAI-1 molecules in the polymers. A quadruple mutant of PAI-1 with a retarded rate of latency transition also had a retarded rate of polymerization. Studying a number of serpins by native gel electrophoresis, ligand...

  20. 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

  1. 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.

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

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E. (Washington Univ., St. Louis, MO (USA))

    1990-07-15

    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.

  3. 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

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

    Shu-Ling Wu

    2014-10-01

    Full Text Available 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.

  5. Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

    Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

    2006-05-01

    Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis. PMID:16676078

  6. Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

    Jensen, Jan Kristian; Malmendal, Anders; Schitt, Birgit; Skeldal, Sune; Pedersen, Katrine E; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A; Wind, Troels

    2006-01-01

    The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family...

  7. A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

    Runge, M. S.; QUERTERMOUS, T.; Zavodny, P J; Love, T W; Bode, C.; Freitag, M.; Shaw, S. Y.; Huang, P. L.; Chou, C C; Mullins, D.

    1991-01-01

    A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)] and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the ...

  8. Role of urokinase plasminogen activator and plasminogen activator inhibitor mRNA expression as prognostic factors in molecular subtypes of breast cancer

    Witzel I

    2014-11-01

    Full Text Available Isabell Witzel,1 Karin Milde-Langosch,1 Marcus Schmidt,2 Thomas Karn,3 Sven Becker,3 Ralph Wirtz,4 Achim Rody,5 Elena Laakmann,1 Dina Schtze,1 Fritz Jnicke,1 Volkmar Mller1 1Department of Gynecology, University Medical Center, Hamburg, 2Department of Obstetrics and Gynecology, University Hospital, Mainz, 3Department of Obstetrics and Gynecology, University Hospital, Frankfurt, 4STRATIFYER Molecular Pathology GmbH, Cologne, 5Department of Obstetrics and Gynecology, University Medical Center Schleswig-Holstein, Luebeck, Germany Background: Protein levels of urokinase plasminogen activator (uPA and its inhibitor (PAI-1 determined by enzyme-linked immunosorbent assay from fresh-frozen tumor tissue have been evaluated as prognostic factors in prospectively randomized trials in breast cancer. However, the role of uPA and PAI-1 in the context of breast cancer subtypes and for mRNA expression of these factors is less clear. Methods: We evaluated uPA and PAI-1 mRNA expression using the Affymetrix HG-U 133A array within molecular subgroups of breast cancer in cohorts of patients with systemic treatment (cohort A, n=362 and without systemic treatment (cohort B, n=200. We validated mRNA expression in a cohort of HER2-positive breast cancer patients (cohort C, n=290. Luminal, triple-negative, and HER2-positive subcohorts were defined by ESR1 and ERBB2 mRNA expression using predefined cutoffs. Results: In the entire cohort A, elevated PAI-1 but not uPA mRNA expression was associated with shorter disease-free survival (P=0.007 for PAI and 0.069 for uPA. Regarding different molecular subgroups, 67% (n=244 of tumors were luminal, 14% (n=49 were HER2-positive, and 19% (n=69 were triple-negative. Elevated PAI-1 mRNA expression was associated with shorter disease-free survival only in the HER2-positive subgroup (P=0.031. The same disease-free survival results were found for uPA in HER2-positive patients (P=0.011. In contrast, no association between either marker and survival was observed in the luminal or triple-negative subgroups. In the HER2-positive validation cohort C, elevated uPA and PAI-1 mRNA expression also showed strong associations with shorter disease-free survival (P=0.014 for PAI-1, P<0.001 for uPA. Conclusion: In this study, the prognostic impact of uPA and PAI-1 expression was mainly observed in patients with HER2-positive tumors. Keywords: urokinase plasminogen activator, urokinase plasminogen activator inhibitor-1, HER2, breast cancer, prognosis

  9. Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation.

    Kwiecinski, Jakub; Na, Manli; Jarneborn, Anders; Jacobsson, Gunnar; Peetermans, Marijke; Verhamme, Peter; Jin, Tao

    2015-01-01

    Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices. PMID:26519394

  10. Rapid localization of indium-111-labeled inhibited recombinant tissue plasminogen activator in a rabbit thrombosis model.

    Butler, S P; Kader, K L; Owen, J; Wang, T S; Fawwaz, R A; Alderson, P O

    1991-03-01

    The thrombus localizing properties of indium-111-recombinant tissue plasminogen activator (111In-rt-PA) have been investigated in an effort to achieve prompt and accurate detection of thrombi. Unlike previous studies with rt-PA, the active plasminogen catalytic site was permanently inhibited with peptides of chloromethyl ketone so that the radiotracer binds to fibrin without causing fibrinolysis. Thrombi were created in the external jugular vein of 14 male New Zealand white rabbits followed by injection of 111In-rt-PA. The agent cleared rapidly in vivo with a half-time of 4.6 min. The thrombus: blood ratio in nonheparinized rabbits (n = 7) was 6.39 +/- 0.86. The ratio in heparinized rabbits (n = 4) was 3.11 +/- 0.23. Thrombi were clearly visible in the planar images of both groups 1 hr postinjection. The combination of rapid thrombus localization and positive images, especially in the presence of anticoagulation, suggests that further work is warranted with rt-PA thrombus imaging. PMID:1900886

  11. Rapid localization of indium-111-labeled inhibited recombinant tissue plasminogen activator in a rabbit thrombosis model

    The thrombus localizing properties of indium-111-recombinant tissue plasminogen activator (111In-rt-PA) have been investigated in an effort to achieve prompt and accurate detection of thrombi. Unlike previous studies with rt-PA, the active plasminogen catalytic site was permanently inhibited with peptides of chloromethyl ketone so that the radiotracer binds to fibrin without causing fibrinolysis. Thrombi were created in the external jugular vein of 14 male New Zealand white rabbits followed by injection of 111In-rt-PA. The agent cleared rapidly in vivo with a half-time of 4.6 min. The thrombus: blood ratio in nonheparinized rabbits (n = 7) was 6.39 +/- 0.86. The ratio in heparinized rabbits (n = 4) was 3.11 +/- 0.23. Thrombi were clearly visible in the planar images of both groups 1 hr postinjection. The combination of rapid thrombus localization and positive images, especially in the presence of anticoagulation, suggests that further work is warranted with rt-PA thrombus imaging

  12. Rapid localization of indium-111-labeled inhibited recombinant tissue plasminogen activator in a rabbit thrombosis model

    Butler, S.P.; Kader, K.L.; Owen, J.; Wang, T.S.; Fawwaz, R.A.; Alderson, P.O. (Columbia-Presbyterian Medical Center, New York, NY (USA))

    1991-03-01

    The thrombus localizing properties of indium-111-recombinant tissue plasminogen activator ({sup 111}In-rt-PA) have been investigated in an effort to achieve prompt and accurate detection of thrombi. Unlike previous studies with rt-PA, the active plasminogen catalytic site was permanently inhibited with peptides of chloromethyl ketone so that the radiotracer binds to fibrin without causing fibrinolysis. Thrombi were created in the external jugular vein of 14 male New Zealand white rabbits followed by injection of {sup 111}In-rt-PA. The agent cleared rapidly in vivo with a half-time of 4.6 min. The thrombus: blood ratio in nonheparinized rabbits (n = 7) was 6.39 +/- 0.86. The ratio in heparinized rabbits (n = 4) was 3.11 +/- 0.23. Thrombi were clearly visible in the planar images of both groups 1 hr postinjection. The combination of rapid thrombus localization and positive images, especially in the presence of anticoagulation, suggests that further work is warranted with rt-PA thrombus imaging.

  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. Detection of deep venous thrombosis with indium 111-labelled monoclonal antibody against tissue plasminogen activator

    The administration of a radiolabelled monoclonal antibody against tissue plasminogen activator allows detection of areas with increased fibrinolytic activity, i.e. those with an active thrombotic lesion. Eight patients with phlebographically verified deep venous thrombosis were examined. At the time of immunoscintigraphy study they were examined receiving anticoagulant therapy. Some 75-85 MBq 111In-labelled antibody were injected, and scintigrams were obtained after 30 min and after 24 h. The precise site of the thrombus could not be visualized after 30 min due to high background activity, whereas after 24 h it was detectable in all patients. The thrombus/background ratios achieved are twice as high as those observed in a human antifibrin antibody study. These preliminary data suggest a high sensitivity of our PA-specific antibody for the detection of active deep venous thrombosis in man, and our antibody seems to offer theoretical advantages over both platelet and fibrin-specific antibodies. (orig.)

  15. Effect of single-chain ovine gonadotropins with dual activity on ovarian function in sheep.

    Rutigliano, Heloisa M; Adams, Betty M; Jablonka-Shariff, Albina; Boime, Irving; Adams, Thomas E

    2014-08-01

    We examined the half-life and biological activity of two single-chain proteins that combined portions of ovine FSH and LH. We proposed the hypothesis that these chimeric proteins would display LH and FSH activities and would promote follicle maturation in ewes. Estrus activity was synchronized using progestogen-impregnated vaginal pessaries. To negate the impact of endogenous LH and FSH, animals received serum-containing antibodies against GNRH 1 day before pessary removal (PR). At PR sheep (five animals per group) received a single injection (10 IU/kg, i.v.) of either the ovine-based (oFcLcα) gonadotropin analog, an ovine-based analog containing oLHβ truncated at the carboxyl terminus (oFcL(ΔT)cα), or a human-based gonadotropin analog (hFcLcα). Control animals received a comparable amount of gonadotropin-free protein. Ovulation was induced 3 days after PR using human chorionic gonadotropin (1000 IU, i.v.). Ovaries were collected 11 days after PR. Neither estradiol (E2) or progesterone (P4) production, development of preovulatory follicles or corpora lutea (CL) were noted in control animals receiving gonadotropin-free protein. Significant increase in the synthesis of E2 and P4 was noted in sheep receiving the dually active gonadotropin analogs. The number of CLs present 11 days after PR was significantly increased in sheep receiving the chimeric glycoproteins compared with control animals. The magnitude of the secretory and ovarian responses did not differ between hFcLcα and oFcLcα or between oFcLcα and oFcL(ΔT)cα. Immunoactivity of LH and FSH was low in control animals, but was significantly elevated in sheep receiving the gonadotropin analogs. In conclusion, ovine-based gonadotropin analogs are functionally active in sheep and a single injection is adequate to induce the development of multiple ovulatory follicles. PMID:24811780

  16. Proteolytic cleavage of urokinase-type plasminogen activator by stromelysin-1 (MMP-3).

    Ugwu, F; Van Hoef, B; Bini, A; Collen, D; Lijnen, H R

    1998-05-19

    Matrix metalloproteinase-3 (MMP-3, or stromelysin-1) specifically hydrolyzes the Glu143-Leu144 peptide bond in 45-kDa single-chain urokinase-type plasminogen activator (scu-PA) and in its two-chain (tcu-PA) derivative, yielding a 17-kDa NH2-terminal domain comprising the u-PA receptor (u-PAR) binding site and a 32-kDa COOH-terminal moiety containing the serine proteinase domain of u-PA. The conversion is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. Biospecific interaction analysis indicates that binding of MMP-3 occurs through the 32-kDa fragment. The 32-kDa fragment derived from scu-PA (scu-PA-32k) has a specific activity of 32k) with a specific activity of 79 000 IU/mg. tcu-PA and tcu-PA-32k moieties derived from scu-PA-32k by plasmin or from tcu-PA by MMP-3 have comparable amidolytic activities toward the chromogenic substrate S-2444 (kcat/Km of 110 and 160 mM-1 s-1, respectively) and similar plasminogen activating activities in a coupled chromogenic substrate assay. Specific binding of the 17-kDa NH2-terminal domain to THP-1 monocytoid cells is completely abolished by competition with scu-PA but is not affected by scu-PA-32k (residual binding of 88 +/- 9% (mean +/- SEM; n = 3) with 25-fold molar excess). Thus, MMP-3 removes a functional NH2-terminal u-PAR-binding domain from u-PA without affecting its enzymatic properties. PMID:9585535

  17. 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...

  18. 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

  19. Successful thrombolysis using recombinant tissue plasminogen activator in cases of severe pulmonary embolism with mobile thrombi in the right atrium

    Şatiroğlu, Ömer; Durakoğlugil, Murtaza Emre; Uğurlu, Yavuz; Şahin, İsmail; Doğan, Sitki; Ergül, Elif; KARADAĞ, Zakir; Bostan, Mehmet

    2014-01-01

    Hereby, we report two cases of acute pulmonary embolism with concomitant right-sided thrombus, which were successfully treated using recombinant tissue plasminogen activator (rtPA). These patients had life-threatening acute right ventricular failure, which dramatically improved within hours following thrombolysis. These cases emphasize the clinical utility of rtPA for the treatment of life-threatening pulmonary embolism.

  20. A flexible multidomain structure drives the function of the urokinase-type plasminogen activator receptor (uPAR)

    Mertens, Haydyn D.T.; Kjærgaard, Magnus; Mysling, Simon; Gårdsvoll, Henrik; Jørgensen, Thomas J. D.; Svergun, Dimitri I.; Ploug, Michael

    2012-01-01

    The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin mediated adhesion to vitronectin. These processes are however tightly linked as the high-affinity binding of urokinase regulates the binding of u...

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

    Persson, Morten; Madsen, Jacob; Østergaard, Søren; Jensen, Mette Munk; Jørgensen, Jesper Tranekjær; Juhl, Karina; Lehmann, Charlotte; Ploug, Michael; Kjaer, Andreas

    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...

  2. Effect of retinoic acid on plasminogen activator expression in human melanoma cells.

    Alexander, C L; Edward, M; MacKie, R M

    1999-08-01

    The plasminogen activation system comprises various proteases that contribute to the invasive potential and metastatic spread of the tumour cell. Two such proteases are tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. Both these enzymes convert plasminogen into the active zymogen plasmin, which has a broad substrate specificity and is capable of degrading a wide range of extracellular matrix molecules. In this study, we examined the effect of retinoic acid (RA) on uPA and tPA secretion in the highly metastatic C8161 and the poorly metastatic Hs294T human melanoma cell lines using a specific enzyme-linked immunosorbent assay (ELISA) detection system, and correlated this production with RA receptor (RAR) expression. Over a range of dilutions, we were able to show that the highly metastatic C8161 cells secreted 0.95 ng of uPA/cell compared with 4.41 fg/cell for the Hs294T cells, whereas the Hs294T cells secreted 24.5 fg of tPA/cell compared with 4.35 fg/cell for the C8161 cells. On exposure of the cells to RA (10(-10)-10(-5) M) for 4 days, uPA secretion was increased 3.4-fold in the C8161 cell line and 1.6-fold in the Hs294T cell line using 10(-8) M RA. In addition, tPA expression was increased in both cell lines by 3.7-fold in the C8161 cells and 3.8-fold in the Hs294T cells with 10(-6) M RA treatment. Increases in PA expression by RA have been reported to involve RAR alpha and RAR beta expression. We were able to detect RAR beta and gamma expression in both cell lines, with and without RA treatment, but were unable to detect expression of RAR alpha. This suggests that another mechanism must exist to regulate the RA modulation of tPA and uPA secretion in these cell lines that does not require RAR alpha expression. PMID:10504054

  3. Plasminogen activator inhibitor-1 suppresses endogenous fibrinolysis in a canine model of pulmonary embolism

    Plasminogen activator inhibitor-1 (PAI-1), the specific, fast-acting inhibitor of tissue-type plasminogen activator (t-PA), binds to fibrin and has been found in high concentrations within arterial thrombi. These findings suggest that the localization of PAI-1 to a thrombus protects that same thrombus from fibrinolysis. In this study, clot-bound PAI-1 was assessed for its ability to suppress clot lysis in vivo. Autologous, canine whole blood clots were formed in the presence of increasing amounts of activated PAI-1 (0-30 micrograms/ml). Approximately 6-8% of the PAI-1 bound to the clots under the experimental conditions. Control and PAI-1-enriched clots containing iodine-125-labeled fibrin (ogen) were homogenized, washed to remove nonbound elements, and delivered to the lungs of anesthetized dogs where the homogenates subsequently underwent lysis by the endogeneous fibrinolytic system. 125I-labeled fibrin degradation products appeared in the blood of control animals within 10 minutes and were maximal by 90 minutes. PAI-1 reduced fibrin degradation product release in a dose-responsive manner at all times between 30 minutes and 5 hours (greater than or equal to 76% inhibition at 30 minutes, PAI-1 greater than or equal to 6 micrograms/ml). PAI-1 also suppressed D-dimer release from clots containing small amounts of human fibrin (ogen). t-PA administration attenuated the effects of PAI-1, whereas latent PAI-1 (20 micrograms/ml) had no effect on clot lysis. Blood levels of PA and PAI activity remained unaltered during these experiments. The results indicate that PAI-1 markedly inhibits endogenous fibrinolysis in vivo and, moreover, suggest that the localization of PAI-1 to a forming thrombus is an important physiological mechanism for subsequent thrombus stabilization

  4. 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

  5. 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

  6. 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

  7. 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.

  8. Effect of recombinant tissue-type plasminogen activator on acute myocardial infarction

    Radionuclide studies were performed in 18 patients with acute myocardial infarction receiving i.v. injection of recombinant tissue-type plasminogen activator (rt-PA) within 12 hr after an attack. Thallium-201 single photon emission computed tomography revealed that infarct size decreased by 42% in the rt-PA treated group, as compared with 25% in the control group. Left ventricular ejection fraction, as found on first-pass radionuclide angiography with Tc-99m PYP, was significantly higher in the rt-PA treated group than the control group (49% vs 38%). Radionuclide imagings were helpful in confirming myocardial salvage after rt-PA intravenous therapy. It was also considered necessary to perform rt-PA therapy as early as possible after an acute myocardial attack. (N.K.)

  9. 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.

  10. 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.

  11. Endogenous Nitric Oxide Contributes to Bradykinin-Stimulated Glucose Uptake but Attenuates Vascular Tissue-Type Plasminogen Activator Release

    Pretorius, Mias; Brown, Nancy J.

    2010-01-01

    Bradykinin causes vasodilation, stimulates tissue-type plasminogen activator (t-PA) release and, in rodents, increases muscle glucose uptake. Although bradykinin causes vasodilation partly by activating nitric-oxide synthase (NOS), the role of nitric oxide in regulating bradykinin-stimulated t-PA release is uncertain. This study examined the effect of high-dose NOS inhibition on bradykinin-stimulated t-PA release and glucose uptake in humans. We studied 24 healthy (12 women and 12 men), overw...

  12. A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimmentation Velocity Analysis*

    Minor, Kenneth H.; Schar, Christine R.; Blouse, Grant E; Shore, Joseph D.; Lawrence, Daniel A.; Schuck, Peter; Peterson, Cynthia B.

    2005-01-01

    Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and peri-cellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complex...

  13. 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...

  14. 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.

  15. Plasminogen activator inhibitor 1 is an intracellular inhibitor of furin proprotein convertase.

    Bernot, Denis; Stalin, Jimmy; Stocker, Pierre; Bonardo, Bernadette; Scroyen, Ilse; Alessi, Marie-Christine; Peiretti, Franck

    2011-04-15

    Proprotein convertases (PCs) are a family of serine proteases that are involved in the post-translational processing and activation of a wide range of regulatory proteins. The upstream role of PCs in the control of many physiological and pathological processes generates a growing interest in understanding their regulation. Here, we demonstrate that the serine protease inhibitor plasminogen activator inhibitor 1 (PAI-1) forms an SDS-stable complex with the PC furin, which leads to the inhibition of the intra-Golgi activity of furin. It is known that elevated PAI-1 plasma levels are correlated with the occurrence of the metabolic syndrome and type 2 diabetes, and we show that PAI-1 reduces the furin-dependent maturation and activity of the insulin receptor and ADAM17: two proteins involved in the onset of these metabolic disorders. In addition to demonstrating that PAI-1 is an intracellular inhibitor of furin, this study also provides arguments in favor of an active role for PAI-1 in the development of metabolic disorders. PMID:21406565

  16. 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

  17. Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis

    Berry, C. N.; Lunven, C.; Lechaire, I; Girardot, C.; O'Connor, S E

    1998-01-01

    Elevated plasminogen activator inhibitor 1 (PAI-1) is a risk factor for thrombosis, and inhibitors of the interaction between PAI-1 and tissue plasminogen activator (t-PA) have antithrombotic and pro-thrombolytic activity in animals. We describe the antithrombotic effects in the rat of a monoclonal antibody (MA33H1) which converts PAI-1 to a non-inhibitory substrate.The activity of MA33H1 against rat PAI-1 was confirmed using two-chain t-PA and a chromogenic substrate. MA33H1 was evaluated in...

  18. Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

    Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275 → Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-rho-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275 → Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with →2-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has a increased fibrin binding compared to the two-chain form

  19. 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...... 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...

  20. Cathepsin-D, urokinase plasminogen activator and type-1 plasminogen activator inhibitor in early breast cancer: an immunohistochemical study of prognostic value and relations to tenascin-C and other factors

    Jahkola, T.; Toivonen, T.; Smitten, K von; Virtanen, I.; Wasenius, V-M; Blomqvist, C.

    1999-01-01

    Cytosolic determinations of cathepsin-D (cath-D), urokinase plasminogen activator (uPA) and its specific inhibitor PAI-1 have shown an association with adverse prognosis in breast cancer. Our aim was to study the distribution of these markers in small axillary node-negative breast carcinomas using immunohistochemistry and relate the semiquantitative results to known prognostic factors, the expression of tenascin-C (Tn-C) in invasion border of the tumour and prognosis. All the 158 women (159 t...

  1. Targeted delivery of tissue plasminogen activator by binding to silica-coated magnetic nanoparticle

    Chen JP

    2012-09-01

    Full Text Available Jyh-Ping Chen,1 Pei-Ching Yang,1 Yunn-Hwa Ma,2 Su-Ju Tu,3 Yu-Jen Lu1,41Department of Chemical and Materials Engineering, 2Department of Physiology and Pharmacology, 3Department of Medical Imaging and Radiological Sciences, Chang Gung University, Kwei-San, Taoyuan, Taiwan, Republic of China; 4Department of Neurosurgery, Chang Gung Memorial Hospital, Kwei-San, Taoyuan, Taiwan, Republic of ChinaBackground and methods: Silica-coated magnetic nanoparticle (SiO2-MNP prepared by the sol-gel method was studied as a nanocarrier for targeted delivery of tissue plasminogen activator (tPA. The nanocarrier consists of a superparamagnetic iron oxide core and an SiO2 shell and is characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, superconducting quantum interference device, and thermogravimetric analysis. An amine-terminated surface silanizing agent (3-aminopropyltrimethoxysilane was used to functionalize the SiO2 surface, which provides abundant —NH2 functional groups for conjugating with tPA.Results: The optimum drug loading is reached when 0.5 mg/mL tPA is conjugated with 5 mg SiO2-MNP where 94% tPA is attached to the carrier with 86% retention of amidolytic activity and full retention of fibrinolytic activity. In vitro biocompatibility determined by lactate dehydrogenase release and cell proliferation indicated that SiO2-MNP does not elicit cytotoxicity. Hematological analysis of blood samples withdrawn from mice after venous administration indicates that tPA-conjugated SiO2-MNP (SiO2-MNP-tPA did not alter blood component concentrations. After conjugating to SiO2-MNP, tPA showed enhanced storage stability in buffer and operation stability in whole blood up to 9.5 and 2.8-fold, respectively. Effective thrombolysis with SiO2-MNP-tPA under magnetic guidance is demonstrated in an ex vivo thrombolysis model where 34% and 40% reductions in blood clot lysis time were observed compared with runs without magnetic targeting and with free tPA, respectively, using the same drug dosage. Enhanced penetration of SiO2-MNP-tPA into blood clots under magnetic guidance was confirmed from microcomputed tomography analysis.Conclusion: Biocompatible SiO2-MNP developed in this study will be useful as a magnetic targeting drug carrier to improve clinical thrombolytic therapy.Keywords: magnetic nanoparticles, drug delivery, thrombolysis, tissue plasminogen activator, silica

  2. 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

  3. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing. PMID:25991438

  4. The effect of 40 kHz ultrasound on tissue plasminogen activator-induced clot lysis in three in vitro models

    Pieters, Marlien; Hekkenberg, Rob T.; Barrett-Bergshoeff, Marrie; Rijken, Dingeman C.

    2005-04-01

    In previous work from the same laboratory, high-frequency ultrasound (US) (3 MHz) was shown to promote in vitro fibrinolysis through enhanced supply of plasminogen to the clot surface. The application of high-frequency US is limited in vivo due to tissue heating. Low-frequency US, however, has less tissue heating and improved penetration. Internal plasma clot lysis and external lysis with compacted and non-compacted plasma clots were used to determine the magnitude of the effect of low-frequency US (40 kHz; 0.5 W/cm2) on tissue plasminogen activator-induced lysis and to elucidate the mechanisms behind the effect. Ultrasound enhanced lysis in all three models, with the largest effects (4-fold) in the external lysis model with compacted plasminogen-poor clots. The acceleration effect of ultrasound in this model decreased with increasing t-PA-and decreasing plasminogen concentrations. Ultrasound had a much smaller effect in this model when compacted plasminogen-rich clots were used. In the external lysis, non-compacted clot model, ultrasound resulted in consistently higher lysis rates. The acceleration effect of lysis, increased slightly (1.3 to 1.8-fold) with increasing t-PA-and decreasing plasminogen concentrations. Plasminogen supply to the clot surface was again shown to be an important contributor to ultrasound-enhanced lysis. [M. Pieters, R. T. Hekkenberg, M. Barrett-Bergshoeff, and D. C. Rijken. Ultrasound in Med & Biol 30, 1545-1552 (2004).

  5. 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...

  6. Simvastatin suppresses dexamethasone-induced secretion of plasminogen activator inhibitor-1 in human bone marrow adipocytes

    Baba Hideo

    2011-04-01

    Full Text Available Abstract Background Osteonecrosis of the femoral head is a common complication of high-dose glucocorticoid treatment. Intravascular thrombosis is thought to be associated with the ischemic state of the femoral head. Plasminogen activator inhibitor-1 (PAI-1 is an adipokine, which are physiologically active substances secreted from visceral and subcutaneous adipocytes. PAI-1 suppresses fibrinolysis by binding tissue-type plasminogen activator. Several reports have described the relationship between PAI-1 and steroid-induced osteonecrosis of the femoral head, and the preventive effects of lipid-lowering agents (statins against steroid-induced osteonecrosis of the femoral head. We previously reported that adipokines and dexamethasone induced PAI-1 secretion from bone marrow adipocytes. The purpose of the present study is to examine the effects of simvastatin on PAI-1 secretion from human bone marrow adipocytes in vitro. Methods Primary bone marrow adipocytes were extracted from collagenase-treated bone marrow fluid obtained from the femoral necks of 40 patients (6 men, 34 women; age range, 52-81 years undergoing hip joint replacement surgery. After suspended culture with or without dexamethasone or simvastatin, PAI-1 mRNA expression was assessed by real-time RT-PCR. Total PAI-1 protein secretion in culture medium was assessed by enzyme-linked immunosorbent assay. Results PAI-1 mRNA expression was up-regulated by 388% (P = 0.002 with dexamethasone, and down-regulated by 45% (P = 0.002 with simvastatin, as compared to control levels. Dexamethasone increased total PAI-1 secretion by 166% (P = 0.001 and simvastatin decreased total PAI-1 secretion by 64% (P = 0.002. No significant changes were observed in adiponectin mRNA expression and secretion by dexamethasone and simvastatin, while pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion by 89%, as compared to control levels. Conclusion The present study confirmed the suppressive effects of simvastatin on PAI-1 expression and secretion from bone marrow adipocytes. Furthermore, pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion. Simvastatin may thus exhibit preventive effects against steroid-induced osteonecrosis of the femoral head by suppressing PAI-1 secretion.

  7. 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

  8. 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

  9. Urokinase plasminogen activator receptor is expressed in invasive cells in gastric carcinomas from high- and low-risk countries

    Alpízar-Alpízar, Warner; Nielsen, Boye Schnack; Sierra, Rafaela; Illemann, Martin; Ramírez, Jose A; Arias, Adriana; Durán, Sundry; Skarstein, Arne; Ovrebo, Kjell; Lund, Leif R; Laerum, Ole D

    2010-01-01

    Gastric cancer is the second cancer causing death worldwide. Both incidence and mortality rates vary according to geographical regions. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation......, and its presence on gastric cancer cells is linked to micro-metastasis and poor prognosis. Immunohistochemical analyses of a set of 44 gastric cancer lesions from Costa Rica showed expression of uPAR in cancer cells in both intestinal subtype (14 of 27) and diffuse subtype (10 of 17). We compared the...... expression pattern of uPAR in gastric cancers from a high-risk country (Costa Rica) with a low-risk country (Norway). We found uPAR on gastric cancer cells in 24 of 44 cases (54%) from Costa Rica and in 13 of 23 cases (56%) from Norway. uPAR was seen in macrophages and neutrophils in all cases. We also...

  10. Tissue Plasminogen Activator Alters Intracellular Sequestration of Zinc through Interaction with the Transporter ZIP4

    Emmetsberger, Jaime; Mirrione, Martine M.; Zhou, Chun; Fernandez-Monreal, Monica; Siddiq, Mustafa M.; Ji, Kyungmin; Tsirka, Stella E. (SBU)

    2010-09-17

    Glutamatergic neurons contain free zinc packaged into neurotransmitter-loaded synaptic vesicles. Upon neuronal activation, the vesicular contents are released into the synaptic space, whereby the zinc modulates activity of postsynaptic neurons though interactions with receptors, transporters and exchangers. However, high extracellular concentrations of zinc trigger seizures and are neurotoxic if substantial amounts of zinc reenter the cells via ion channels and accumulate in the cytoplasm. Tissue plasminogen activator (tPA), a secreted serine protease, is also proepileptic and excitotoxic. However, tPA counters zinc toxicity by promoting zinc import back into the neurons in a sequestered form that is nontoxic. Here, we identify the zinc influx transporter, ZIP4, as the pathway through which tPA mediates the zinc uptake. We show that ZIP4 is upregulated after excitotoxin stimulation of the mouse, male and female, hippocampus. ZIP4 physically interacts with tPA, correlating with an increased intracellular zinc influx and lysosomal sequestration. Changes in prosurvival signals support the idea that this sequestration results in neuroprotection. These experiments identify a mechanism via which neurons use tPA to efficiently neutralize the toxic effects of excessive concentrations of free zinc.

  11. 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

  12. Cloning and Expression of a Human Tissue Plasminogen Activator Variant:K2S in Escherichia coli

    Hamid Mir Mohammad Sadeghi

    2007-01-01

    Full Text Available The DNA sequence of Kringle-2 and serine protease domains of the human tissue plasminogen activator (reteplase, K2S was PCR amplified. This product was then cloned into the expression vector pET15b plasmid. The presence of the insert was confirmed by restriction digestion, PCR and determination of the nucleotide sequence. By using isopropyl ?-D thiogalactopyranoside (IPTG, reteplase was induced in E. coli BL21 cells and analyzed using polyacrylamide gel electrophoresis (PAGE.

  13. 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

  14. 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

  15. 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

  16. Differential effects of low-dose tissue plasminogen activator and streptokinase on platelet aggregation.

    Berridge, D C; Burgess-Wilson, M E; Westby, J C; Hopkinson, B R; Makin, G S

    1989-10-01

    Despite increasing success with low-dose intra-arterial thrombolysis, early rethrombosis still occurs. Platelet aggregation is thought to play a major part in this process. We have therefore investigated the effects of recombinant tissue plasminogen activator (rt-PA) and streptokinase on platelet function at doses currently used for peripheral arterial thrombolysis. Platelet-rich plasma was stirred at 37 degrees C, with either streptokinase (100, 300 or 1000 units ml-1) or rt-PA (10 (T10), 30 (T30) and 100 (T100) mg l-1), with immediate addition of an agonist for platelet aggregation (thrombin, collagen, adenosine diphosphate (ADP) or adrenaline) at a predetermined threshold dose. Significant inhibition of collagen-induced and adrenaline-induced platelet aggregation was produced with rt-PA at all doses used (P less than 0.05). With adrenaline as the agonist, T100 produced disaggregation to a mean (s.d.) level of 26 per cent. Thrombin-stimulated platelet aggregation was significantly reduced by T100 (P less than 0.001) and T30 (P less than 0.01) only, disaggregation being dose-dependent and complete with T100. Using ADP as the agonist, T100 produced a significant reduction in maximum platelet aggregation (P less than 0.01), and disaggregation was achieved to a mean (s.d.) level of 48(13) per cent. Streptokinase did not produce any significant changes in any parameter of aggregation. PMID:2513082

  17. Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells.

    Hansen, Line; Larsen, Esben Kjær Unmack; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr; Ploug, Michael; Kjems, Jørgen

    2013-09-01

    Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery. PMID:23835641

  18. Angiotensinogen and Plasminogen Activator Inhibitor-1 Gene Polymorphism in Relation to Renovascular Disease

    The present study was designed to evaluate angiotensinogen (AGT) M235T and plasminogen activator inhibitor-1 (PAI-1) (4G/5G) polymorphisims in relation to the occurrence of atherosclerotic renal artery stenosis (ARAS) and recurrent stenosis. In this study, 30 patients were enrolled after angiographic demonstration of ARAS; 100 healthy subjects for AGT polymorphism and 80 healthy subjects for PAI-1 polymorphism were considered the control group. The patients were followed for a mean 46.1 ± 9.2 months. The patients had significantly higher frequencies of the MT genotype and the T allele than control group (χ2 = 18.2, p 2 = 11.5 p 2= 2.45, p = 0.29 and χ2 = 0.019, p = 0.89). There were no significant differences in the genotype and allele findings for the patients with and without restenosis (p > 0.05). The C-reactive protein (CRP) level was higher in the patients with restenosis than in the patients without restenosis (7.694 ± 0.39 mg/L and 1.56 ± 1.08 mg/L) (p = 0.001). Our results suggest that the M235T MT genotype and T allele might be associated with increased risk of atherosclerotic renal artery stenosis. The CRP level might be an independent predictor for recurrent stenosis

  19. 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.

  20. TFRC and ACTB as the best reference genes to quantify Urokinase Plasminogen Activator in breast cancer

    Abdoli Nasrin

    2011-06-01

    Full Text Available Abstract Background Biomedical researchers have long looked for ways to diagnose and treat cancer patients at the early stages through biomarkers. Although conventional techniques are routinely applied in the detection of biomarkers, attitudes towards using Real-Time PCR techniques in detection of many biomarkers are increasing. Normalization of quantitative Real-Time PCR is necessary to validate non-biological alteration occurring during the steps of RNA quantification. Selection of variably expressed housekeeping genes (HKs will affect the validity of the data. The aim of the present study was to identify uniformly expressed housekeeping genes in order to use in the breast cancer gene expression studies. Urokinase Plasminogen Activator was used as a gene of interest. Findings The expression of six HKs (TFRC, GUSB, GAPDH, ACTB, HPRT1 and RPLP0 was investigated using geNorm and NormFinder softwares in forty breast tumor, four normal and eight adjacent tissues. RPLP0 and GAPDH revealed maximum M value, while TFRC demonstrated lowest M value. Conclusions In the present study the most and the least stable genes were TFRC and RPLP0 respectively. TFRC and ACTB were verified as the best combination of two genes for breast cancer quantification. The result of this study shows that in each gene expression analysis HKs selection should be done based on experiment conditions.

  1. 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

  2. DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light

    Human fetal fibroblasts have been tested for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus. Skin fibroblasts derived at 12 week gestation were more repair proficient than at 8 weeks. However, they exhibited a lower rate of repair than non-fetal cells. Enhancement of protease plasminogen activator (PA) was higher after u.v. irradiation in skin fibroblasts derived at 8 weeks than at 12 weeks gestation and was absent in non-fetal skin fibroblasts. Excision repair and PA inducibility depended on the tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s). (author)

  3. 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

  4. Annexin A2 in primary afferents contributes to neuropathic pain associated with tissue type plasminogen activator.

    Yamanaka, H; Kobayashi, K; Okubo, M; Noguchi, K

    2016-02-01

    Annexin A2 (ANX2) is a calcium (Ca(2+))-binding protein that binds to acidic phospholipids and is known to play a crucial role in many cellular regulatory processes. In particular, ANX2 has been described as a crucial receptor for thrombolysis by the tissue-type plasminogen activator (tPA) and plasmin system. In the nervous system, tPA is involved in processes of neuronal plasticity such as hippocampal long-term potentiation (LTP) and in the dorsal horn pain in several pain models. We investigated detailed changes in expression of ANX2 after nerve injury and evaluated the interaction with tPA using the rat spared nerve injury (SNI) model. SNI-induced the expression of ANX2 in L4/5 dorsal root ganglia (DRG) neurons. In the spinal cord, constitutive ANX2-immunoreactivity was expressed in laminae I-II. Peripheral nerve injury increased the ANX2 immunoreactive terminals mainly in laminae I-V of the dorsal horn on the side ipsilateral to the nerve injury. Double-labeling analysis revealed the co-localization of ANX2 with tPA in the axons of primary afferents in the dorsal horn. Experimental inhibition of ANX2 and tPA interaction by intrathecal administration of homocysteine significantly prevented and reversed SNI-induced mechanical allodynia. Thus, alterations of ANX2 may be involved in tPA-dependent plasticity after peripheral nerve injury and have an important role in neuropathic pain. PMID:26642807

  5. Evaluation of Cerebral Perfusion in Patients Undergoing Intravenous Recombinant Tissue Plasminogen Activator Thrombolysis

    HIRANO, Teruyuki

    2015-01-01

    Currently, the indication for thrombolytic therapy using intravenous recombinant tissue plasminogen activator (rt-PA) is restricted strictly to patients with acute ischemic stroke within 4.5 h of onset. The effect of rt-PA declines over time; therefore, we need to minimize the time delay while generating imaging information. The use of cerebral blood flow imaging is not recommended within this time window. Conversely, the balance of efficacy and the risk of bleeding complications differ among patients > 4.5 h after onset. Several ongoing studies are using mismatch concepts to extend the therapeutic time window for rt-PA. Long-awaited reliable software, such as RAPID and PMA, are now available to analyze computed tomography/magnetic resonance perfusion data. Patients with wake-up stroke (WUS) are another group that can be used to expand rt-PA candidates. Diffusion fluid- attenuated inversion recovery mismatch is a promising imaging surrogate to select good candidates with WUS. These trials will cause a therapeutic paradigm shift from time-based to tissue-based strategies in the near future. PMID:26369875

  6. 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.

  7. Dynamic structural and functional relationships in recombinant plasminogen activator inhibitor-1 (rPAI-1).

    Vaughan, D E; Declerck, P J; Reilly, T M; Park, K; Collen, D; Fasman, G D

    1993-10-01

    The conformational characteristics of active, latent, and denatured recombinant plasminogen activator inhibitor-1 (rPAI-1) were compared using UV spectroscopy, spectrofluorimetry and circular dichroism (CD) techniques. The UV absorbance wavelength maxima in all preparations approximated 280 nm, while the extinction coefficients of active and latent rPAI-1 differed by up to 60%. When incubated at 37 degrees C, the A280 of latent rPAI-1 was quite stable while the A280 of active rPAI-1 spontaneously increased, eventually approximating that of latent rPAI-1. Alkali difference spectroscopy yielded markedly divergent titration patterns for active and latent rPAI-1, suggesting that the tyrosine residues present in active and latent rPAI-1 differ in terms of solvent exposure. At an excitation wavelength of 280 nm, active rPAI-1 exhibited the greatest relative fluorescence quantum yield. The relative fluorescence of latent and denatured rPAI-1 were less than that of active PAI-1, and the emission maxima of both species were slightly red-shifted in comparison to that of active rPAI-1, suggesting that at least one of the four tryptophan residues present in rPAI-1 is less exposed to the aqueous environment in the active form of the molecule. In contrast, the derived secondary structures based on CD of active and latent rPAI-1 were nearly identical, with both moieties exhibiting approx. 40% alpha-helix and 15% beta-sheet. Taken together, these spectroscopic data provide evidence supporting the hypothesis that active and latent PAI-1 differ in terms of their tertiary conformation and aromatic residue exposure, while their secondary structures appear generally comparable. Furthermore, denaturant-induced reactivation of latent rPAI-1 produces a partially active rPAI-1 with spectroscopic properties similar to that of latent rPAI-1, suggesting that denatured rPAI-1 more closely resembles the latent rPAI-1 conformation after refolding. The spontaneous spectroscopic changes observed in rPAI-1 may reflect conformational transitions that are critical to the regulation of endogenous PAI-1 activity. PMID:8399383

  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. Prevention of obesity and insulin resistance in mice lacking plasminogen activator inhibitor 1.

    Ma, Li-Jun; Mao, Su-Li; Taylor, Kevin L; Kanjanabuch, Talerngsak; Guan, YouFei; Zhang, YaHua; Brown, Nancy J; Swift, Larry L; McGuinness, Owen P; Wasserman, David H; Vaughan, Douglas E; Fogo, Agnes B

    2004-02-01

    Increased plasminogen activator inhibitor 1 (PAI-1) has been linked to not only thrombosis and fibrosis but also to obesity and insulin resistance. Increased PAI-1 levels have been presumed to be consequent to obesity. We investigated the interrelationships of PAI-1, obesity, and insulin resistance in a high-fat/high-carbohydrate (HF) diet-induced obesity model in wild-type (WT) and PAI-1-deficient mice (PAI-1(-/-)). Obesity and insulin resistance developing in WT mice on an HF diet were completely prevented in mice lacking PAI-1. PAI-1(-/-) mice on an HF diet had increased resting metabolic rates and total energy expenditure compared with WT mice, along with a marked increase in uncoupling protein 3 mRNA expression in skeletal muscle, likely mechanisms contributing to the prevention of obesity. In addition, insulin sensitivity was enhanced significantly in PAI-1(-/-) mice on an HF diet, as shown by euglycemic-hyperinsulinemic clamp studies. Peroxisome proliferator-activated receptor (PPAR)-gamma and adiponectin mRNA, key control molecules in lipid metabolism and insulin sensitivity, were maintained in response to an HF diet in white adipose tissue in PAI-1(-/-) mice, contrasting with downregulation in WT mice. This maintenance of PPAR-gamma and adiponectin may also contribute to the observed maintenance of body weight and insulin sensitivity in PAI-1(-/-) mice. Treatment in WT mice on an HF diet with the angiotensin type 1 receptor antagonist to downregulate PAI-1 indeed inhibited PAI-1 increases and ameliorated diet-induced obesity, hyperglycemia, and hyperinsulinemia. PAI-1 deficiency also enhanced basal and insulin-stimulated glucose uptake in adipose cells in vitro. Our data suggest that PAI-1 may not merely increase in response to obesity and insulin resistance, but may have a direct causal role in obesity and insulin resistance. Inhibition of PAI-1 might provide a novel anti-obesity and anti-insulin resistance treatment. PMID:14747283

  10. Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator

    Deck Lorraine M

    2006-02-01

    Full Text Available Abstract Background Urokinase-type plasminogen activator (uPA plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents. Results 4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups. Conclusion This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors.

  11. Induction of plasminogen activator inhibitor type 1 in murine lupus-like glomerulonephritis.

    Moll, S; Menoud, P A; Fulpius, T; Pastore, Y; Takahashi, S; Fossati, L; Vassalli, J D; Sappino, A P; Schifferli, J A; Izui, S

    1995-11-01

    Three major components of the plasminogen activators (PA)/plasmin system are synthesized physiologically in glomeruli, and can be involved in glomerular proteolysis and extracellular matrix metabolism: tissue-type PA (tPA), urokinase (uPA) and PA inhibitor type 1 (PAI-1). To explore the possible role of a dysregulation of the plasmin protease system in the development and progression of lupus-like glomerulonephritis, we studied the expression of the renal plasmin protease components during the course of the disease, either acute, induced by IgG3 monoclonal cryoglobulins, or chronic, occurring spontaneously in three different lupus-prone mice: (NZBxNZW)F1, BXSB and MRL-lpr/lpr. RNase protection assays and in situ hybridizations revealed a marked glomerular induction of PAI-1 mRNA abundance without any significant changes in renal tPA and uPA mRNA levels in the two different types of lupus-like glomerulonephritis. The overexpression of PAI-1 mRNA occurred in parallel with a significant decrease in glomerular tPA-catalyzed enzymatic activity as determined by zymographic analysis. In addition, a concomitant increase in glomerular expression of transforming growth factor beta 1 (TGF-beta 1) mRNA was observed. The demonstration of a close correlation between the PAI-1 and TGF-beta 1 mRNA levels and the severity of lupus-like glomerular lesions suggests that a pertubation of the glomerular PA/PAI balance, resulting from a marked TGF-beta 1-mediated induction of PAI-1 gene expression, plays an important role in the progression of lupus-like glomerular lesions, leading to glomerulosclerosis. PMID:8544402

  12. Segregation analysis of plasminogen activator inhibitor-1 and fibrinogen levels in the NHLBI family heart study.

    Pankow, J S; Folsom, A R; Province, M A; Rao, D C; Williams, R R; Eckfeldt, J; Sellers, T A

    1998-10-01

    Elevated plasminogen activator inhibitor-1 (PAI-1) and fibrinogen concentrations are risk factors for coronary heart disease. We investigated environmental, familial, and genetic influences on PAI-1 antigen and fibrinogen concentrations in 2029 adults from 512 randomly ascertained families in 4 US communities. We used maximum-likelihood segregation analysis to fit several genetic and nongenetic modes of inheritance to the data to determine whether mendelian inheritance of a major gene could best explain the familial distributions of these 2 hemostatic factors. Age- and gender-adjusted familial correlations for PAI-1 antigen level averaged 0.16 in first-degree relatives (95% CI=0.11 to 0.21); the spouse correlation was positive but not statistically significant (r=0.10, 95% CI=-0.02 to 0.23). Complex segregation analysis indicated a major gene associated with higher PAI-1 concentrations in 65% of individuals from these families. Demographic, anthropometric, lifestyle, and metabolic characteristics together explained 37% to 47% of the variation in PAI-1 antigen levels, and the inferred major gene explained an additional 17% of the variance. Positive and statistically significant age- and gender-adjusted familial correlations in first-degree relatives indicated a possible heritable component influencing plasma fibrinogen concentration (r=0. 17, 95% CI=0.13 to 0.22); however, segregation analysis did not provide statistical evidence of a major gene controlling fibrinogen level. These family data suggest that there are modest familial and genetic effects on the concentration of PAI-1. PMID:9763527

  13. Interaction of mutants of tissue-type plasminogen activator with liver cells: effect of domain deletions.

    Kuiper, J; Van't Hof, A; Otter, M; Biessen, E A; Rijken, D C; van Berkel, T J

    1996-02-01

    The fibrin-specific thrombolyticum tissue-type plasminogen activator (t-PA) has proven to be a potent drug in several clinical trials, but its clinical application is complicated by the rapid clearance of t-PA from the circulation. The rapid plasma clearance of t-PA results from the uptake of t-PA in the liver. t-PA consists of several domains which may be involved in the interaction with the liver. Three domain-deletion mutants, which were produced by the use of a cassette gene system, were studied in vivo and in vitro for their capacity to bind to the various types of rat liver cells. The three mutants lacked, in comparison to control t-PA, the epidermal growth factor (G) domain, the finger (F) domain or the G domain plus the first kringle (K1). The plasma clearance of the three mutants was slower than that of control t-PA. The slower plasma clearance resulted from a decreased liver uptake: 50 and 80% for t-PA mutants and control t-PA respectively. It was found that the K1 domain was of major importance for the uptake of t-PA by liver endothelial cells in vivo and in vitro. The high-affinity binding of t-PA (and t-PA mutants) to parenchymal liver cells depended largely on the presence of the G domain. Other domain(s), like the F, K2 or protease domain, may be responsible for low-affinity, t-PA-specific binding to rat parenchymal liver cells. PMID:8611154

  14. Soluble urokinase plasminogen activator receptor and hypertension among black South Africans after 5 years.

    Botha, Shani; Fourie, Carla Mt; Schutte, Rudolph; Eugen-Olsen, Jesper; Schutte, Aletta E

    2015-06-01

    Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker that links inflammation with cardiovascular risk. However, studies linking suPAR and hypertension are scant. First, we determined whether baseline suPAR is elevated in normotensive black South Africans who developed hypertension over 5 years, compared with those who remained normotensive; and second, whether hypertension is associated with suPAR. This substudy is embedded in the South African leg of the Prospective Urban and Rural Epidemiology study, performed in the North West Province. We investigated 429 normotensive individuals, of which 191 developed hypertension and 238 remained normotensive over 5 years. We determined suPAR from plasma (ethylenediaminetetraacetic acid) samples with the suPARnostic ELISA Kit and blood pressure with an OMRON HEM-757 device. Despite similar mean baseline suPAR levels (P=0.43), suPAR increased more in the group that developed hypertension compared with those who remained normotensive (14.2% vs. 6.94%; P=0.007). Five-year percentage change in systolic blood pressure correlated positively (r=0.23; P=0.002) and associated independently with baseline suPAR (β=0.14; P=0.043), only in participants who developed hypertension. Participants were 1.41 times more likely (P=0.015) to develop hypertension with 1 s.d. increase in percentage change in suPAR levels over 5 years. Change in systolic blood pressure was associated with baseline suPAR in hypertensive participants and change in suPAR with hypertensive status. This study highlights the need for more research on the role of suPAR in hypertension and cardiovascular disease development in black South Africans. PMID:25740290

  15. 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.

  16. DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light

    We have tested human fetal fibroblasts for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus; this was evident by a delay in both the relaxation and the restoration of DNA supercoiling in nucleoids after irradiation. Skin fibroblasts derived at 12 week gestation were more repair proficient than those derived at 8 week gestation. However, they exhibited a somewhat lower rate of repair than non-fetal cells. The same fetal and non-fetal cells were also tested for induction of the protease plasminogen activator (PA) after u.v. irradiation. Enhancement of PA was higher in skin fibroblasts derived at 8 week than in those derived at 12 week gestation and was absent in non-fetal skin fibroblasts. These results are consistent with our previous findings that in human cells u.v. light-induced PA synthesis is correlated with reduced DNA repair capacity. Excision repair and PA inducibility were found to depend on tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. Lung compared to skin fibroblasts exhibited lower repair rates and produced higher levels of PA after irradiation. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s)

  17. The hepatic clearance of recombinant tissue-type plasminogen activator decreases after an inflammatory stimulus

    M.R. Nagaoka

    2000-01-01

    Full Text Available We have shown that tissue-type plasminogen activator (tPA and plasma kallikrein share a common pathway for liver clearance and that the hepatic clearance rate of plasma kallikrein increases during the acute-phase (AP response. We now report the clearance of tPA from the circulation and by the isolated, exsanguinated and in situ perfused rat liver during the AP response (48-h ex-turpentine treatment. For the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of 125I-tPA from the circulation of turpentine-treated rats (2.2 0.2 ml/min, N = 7 decreases significantly (P = 0.016 when compared to normal rats (3.2 0.3 ml/min, N = 6. The AP response does not modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80% cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of turpentine-treated rats (15.5 1.3 g/min, N = 4 was slower (P = 0.003 than the clearance rate by the liver of normal rats (22.5 0.7 g/min, N = 10. After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment, tPA was cleared by the perfused liver at 16.2 2.4 g/min (N = 5, suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kallikrein were not altered during the AP response. These results contribute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered.

  18. Ischaemiareperfusion injury impairs tissue plasminogen activator release in man

    Pedersen, Christian M.; Barnes, Gareth; Schmidt, Michael R.; Btker, Hans Erik; Kharbanda, Rajesh K.; Newby, David E.; Cruden, Nicholas L.

    2012-01-01

    Aims Ischaemiareperfusion (IR) injury causes endothelium-dependent vasomotor dysfunction that can be prevented by ischaemic preconditioning. The effects of IR injury and preconditioning on endothelium-dependent tissue plasminogen activator (t-PA) release, an important mediator of endogenous fibrinolysis, remain unknown. Methods and results Ischaemiareperfusion injury (limb occlusion at 200 mmHg for 20 min) was induced in 22 healthy subjects. In 12 subjects, IR injury was preceded by local or remote ischaemic preconditioning (three 5 min episodes of ipsilateral or contralateral limb occlusion, respectively) or sham in a randomized, cross-over trial. Forearm blood flow (FBF) and endothelial t-PA release were assessed using venous occlusion plethysmography and venous blood sampling during intra-arterial infusion of acetylcholine (520 g/min) or substance P (28 pmol/min). Acetylcholine and substance P caused dose-dependent increases in FBF (P<0.05 for all). Substance P caused a dose-dependent increase in t-PA release (P<0.05 for all). Acetylcholine and substanceP-mediated vasodilatation and substanceP-mediated t-PA release were impaired following IR injury (P<0.05 for all). Neither local nor remote ischaemic preconditioning protected against the impairment of substance P-mediated vasodilatation or t-PA release. Conclusion Ischaemiareperfusion injury induced substanceP-mediated, endothelium-dependent vasomotor and fibrinolytic dysfunction in man that could not be prevented by ischaemic preconditioning. Clinical Trial Registration Information: Reference number: NCT00789243, URL: http://clinicaltrials.gov/ct2/show/NCT00789243?term=NCT00789243&rank=1 PMID:21990263

  19. Tissue and urokinase plasminogen activators instigate the degeneration of retinal ganglion cells in a mouse model of glaucoma.

    Chintala, Shravan K

    2016-02-01

    Elevated intraocular pressure (IOP) promotes the degeneration of retinal ganglion cells (RGCs) during the progression of Primary Open-Angle Glaucoma (POAG). However, the molecular mechanisms underpinning IOP-mediated degeneration of RGCs remain unclear. Therefore, by employing a mouse model of POAG, this study examined whether elevated IOP promotes the degeneration of RGCs by up-regulating tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the retina. IOP was elevated in mouse eyes by injecting fluorescent-microbeads into the anterior chamber. Once a week, for eight weeks, IOP in mouse eyes was measured by using Tono-Pen XL. At various time periods after injecting microbeads, proteolytic activity of tPA and uPA in retinal protein extracts was determined by fibrinogen/plasminogen zymography assays. Localization of tPA and uPA, and their receptor LRP-1 (low-density receptor-related protein-1) in the retina was determined by immunohistochemistry. RGCs' degeneration was assessed by immunostaining with antibodies against Brn3a. Injection of microbeads into the anterior chamber led to a progressive elevation in IOP, increased the proteolytic activity of tPA and uPA in the retina, activated plasminogen into plasmin, and promoted a significant degeneration of RGCs. Elevated IOP up-regulated tPA and LRP-1 in RGCs, and uPA in astrocytes. At four weeks after injecting microbeads, RAP (receptor associated protein; 0.5 and 1.0 μM) or tPA-Stop (1.0 and 4.0 μM) was injected into the vitreous humor. Treatment of IOP-elevated eyes with RAP led to a significant decrease in proteolytic activity of both tPA and uPA, and a significant decrease in IOP-mediated degeneration of RGCs. Also, treatment of IOP-elevated eyes with tPA-Stop decreased the proteolytic activity of both tPA and uPA, and, in turn, significantly attenuated IOP-mediated degeneration of RGCs. Results presented in this study provide evidence that elevated IOP promotes the degeneration of RGCs by up-regulating the levels of proteolytically active tPA and uPA. PMID:26474495

  20. 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

  1. Intravenous Thrombolysis with Recombinant Tissue Plasminogen Activator for Ischemic Stroke Patients over 80 Years Old: The Fukuoka Stroke Registry

    Matsuo, Ryu; Kamouchi, Masahiro; Fukuda, Haruhisa; Hata, Jun; Wakisaka, Yoshinobu; Kuroda, Junya; Ago, Tetsuro; Kitazono, Takanari; .. .

    2014-01-01

    Objectives 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. Methods 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...

  2. Serum soluble urokinase-type plasminogen activator receptor levels in male patients with acute exacerbation of schizophrenia.

    Genc, Abdullah; Kalelioglu, Tevfik; Karamustafalioglu, Nesrin; Tasdemir, Akif; Genc, Esra Sena; Akkus, Mustafa; Emul, Murat

    2016-02-28

    Inflammatory abnormalities have been shown in the pathogenesis of schizophrenia. Soluble urokinase-type plasminogen activator receptor (suPAR) is a protein that is measurable in the circulating blood and reflects the inflammation in the body. We aimed to investigate serum suPAR levels in patients with schizophrenia who were in acute state and to compare with healthy controls. Forty five patients and 43 healthy controls were included in the study. We found no significant difference in suPAR levels between patients and controls, suggesting that suPAR as an inflammatory marker does not have a role in the inflammatory process of acute schizophrenia. PMID:26774189

  3. Synergistic and multidimensional regulation of plasminogen activator inhibitor type 1 expression by transforming growth factor type ? and epidermal growth factor

    Song, Xiaoling; Thalacker, F.W.; Nilsen-Hamilton, Marit

    2012-04-06

    The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type ? (TGF?) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGF? regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGF? also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGF? pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGF? are able to promote large and rapid changes in PAI-1 expression.

  4. 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 triggers uPA expression in meningioma cells.

  5. ROLE OF GENE POLYMORFISM OF PLASMINOGEN ACTIVATOR INGIBITOR TYPE I AS A RISK FACTOR FOR PREMATURE RUPTURE OF MEMBRANE AT TERM PREGNANCY

    M. G. Nikolayeva

    2016-01-01

    Full Text Available The retrospective study was designed to identify association of premature rupture of the fetal membranes (PROM with carrying polymorphisms in genes encoding folate metabolism and hemostasis in 717 women. More than one hundred potential predictors were analyzed including carriage of thrombogenic genes polymorphisms and genes encoding folate metabolism: FV[Arg506Gln], F II [20210 G/A], MTHFR [Ala222Val], (PAI-I[-675 5G/4G]. Study revealed that plasminogen activator ingibitor-1 gene polymorphism increases significantly the risk of premature rupture of the fetal membranes in term pregnancy (PROM: heterozygous plasminogen activator ingibitor-1 gene polymorphism is associated with 3.6-fold (95% CI 2.4–5.4; p < 0.001, homozygous plasminogen activator ingibitor-1 gene polymorphism – with 1.7-fold (95% CI 1.1–2.6; p = 0.01 risk rise of PROM.

  6. 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

  7. 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 second (r=-478, P=0.001. Conclusion: suPAR is a marker of acute inflammation. It is well correlated with such inflammation markers as CRP and fibrinogen. suPAR can be used as a predictor of AE-COPD and in monitoring response to treatment. Keywords: biomarker, chronic obstructive pulmonary disease, inflammation, suPAR

  8. 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-apheresis values were low may reflect a homeostatic mechanism to maintain sufficient PAI-1 levels. Procedures that could selectively remove PAI-1 from plasma may offer a treatment option for those with very high plasma PAI-1 levels and thrombosis.

  9. Plasminogen carbohydrate side chains in receptor binding and enzyme activation: A study of C6 glioma cells and primary cultures of rat hepatocytes

    The human [Glu1]-plasminogen carbohydrate isozymes, plasminogen type I (Pg 1) and plasminogen type II (Pg 2), were separated by chromatography and studied in cell binding experiments at 4 degrees C with primary cultures of rat hepatocytes and rat C6 glioma cells. In both cell systems, Pg 1 and Pg 2 bound to an equivalent number of receptors, apparently representing the same population of surface molecules. The affinity for Pg 2 was slightly higher. With hepatocytes, the KD for Pg 1 was 3.2 +/- 0.2 microM, and the KD for Pg 2 was 1.9 +/- 0.1 microM, as determined from Scatchard transformations of the binding isotherms. The Bmax was approximately the same for both isozymes. With C6 cells, the KD for Pg 1 was 2.2 +/- 0.1 microM vs. 1.5 +/- 0.2 microM for Pg 2. Again, the Bmax was similar with both isozymes. 125I-Pg 1 and 125I-Pg 2 were displaced from specific binding sites by either nonradiolabeled isozyme. The KI for Pg 2 was slightly lower than the KI for Pg 1 with hepatocytes (0.9 vs. 1.3 microM) and with C6 cells (0.6 vs. 1.1 microM). No displacement was detected with miniplasminogen at concentrations up to 5.0 microM. Activation of Pg 1 and Pg 2 by recombinant two-chain tissue-plasminogen activator (rt-PA) was enhanced by hepatocyte cultures. The enhancing effect was greater with Pg 2. Hepatocyte cultures did not affect the activation of miniplasminogen by rt-PA or the activation of plasminogen by streptokinase. Unlike the hepatocytes, C6 cells did not enhance the activation of plasminogen by rt-PA or streptokinase; however, plasmin generated in the presence of C6 cells reacted less readily with alpha 2-antiplasmin

  10. The Use of Low-Dose Recombinant Tissue Plasminogen Activator to Treat a Preterm Infant with an Intrauterine Spontaneous Arterial Thromboembolis

    Yaşar Demirelli

    2015-12-01

    Full Text Available Neonatal thromboembolic events are rare, and only a few cases of intrauterine spontaneous arterial thromboembolisms have been reported in the literature. Thrombolytic therapy with recombinant tissue plasminogen activator is usually the preferred treatment because it has a short half-life, fewer systemic side effects, and a strong, specific affinity for fibrin. Protocols vary from center to center, but there is still no consensus regarding the proper dosage or treatment duration. Herein, we present the case of an intrauterine spontaneous arterial thromboembolism in a preterm infant that completely resolved after being treated with low-dose recombinant tissue plasminogen activator (0.02 mg/kg/h.

  11. Assignment of the urokinase-type plasminogen activator receptor gene (PLAUR) to chromosome 19q13.1-q13.2.

    Børglum, A D; Byskov, A; Ragno, P; Roldan, A L; Tripputi, P; Cassani, G.; Danø, K.; F. Blasi; Bolund, L.; Kruse, T. A.

    1992-01-01

    The urokinase-type plasminogen activator receptor (uPAR) is a key molecule in the regulation of cell-surface plasminogen activation and, as such, plays an important role in many normal as well as pathological processes. We applied a cDNA probe from the corresponding gene (PLAUR) in a location analysis using a panel of human/rodent cell hybrids and in a multipoint linkage analysis of 40 CEPH families. These two independent studies both found PLAUR to be located on chromosome 19. The cell hybri...

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

    Illemann, Martin; Bird, Nigel; Majeed, Ali; Laerum, Ole D; Lund, Leif R; Danø, Keld; Nielsen, Boye Schnack

    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...... reaction, 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...

  13. Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

    Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins

  14. The Plasminogen Activator/Plasmin System Is Essential for Development of the Joint Inflammatory Phase of Collagen Type II-Induced Arthritis

    Li, Jinan; Ny, Annelii; Leonardsson, Gran; Nandakumar, Kutty Selva; Holmdahl, Rikard; Ny, Tor

    2005-01-01

    The plasminogen activator (PA) system has been proposed to have important roles in rheumatoid arthritis. Here we have used the autoimmune collagen type II (CII)-induced arthritis (CIA) model and mice deficient for urokinase-type PA (uPA) or plasminogen to investigate the role of the PA system for development of arthritis. Our data revealed that uPA-deficient mice have a lower severity and incidence of CIA than wild-type mice. Furthermore, although >80% of wild-type control mice developed CIA,...

  15. 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, white blood cells, hemoglobin level, and platelets remained normal during the 3 months of the study.Conclusion: All of the results suggest that a transient injury in terms of normal organ functions, but no microscopic necrotic lesions, was observed at a systemic delivery dose of 5 mg/kg of iron equivalent concentration in the acute phase, and that no chronic toxicity was found 3 months after the injection. Therefore, we conclude that uPAR-targeted IONPs have the potential to be used as receptor-targeted MRI contrasts as well as theranostic agents for the detection and treatment of human cancers in future studies. Keywords: uPAR-IONP, nonhuman primates, transient harm, self-healing

  16. 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; Patel, Riyaz S; Al Kassem, Hatem; Hammadah, Muhammad; Veledar, Emir; Le, Ngoc-Anh; Pielak, Tomasz; Thorball, Christian W; Velegraki, Aristea; Kremastinos, Dimitrios T; Lerakis, Stamatios; Sperling, Laurence; Quyyumi, Arshed A

    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...

  17. 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; Eshtehardi, Parham; Eapen, Danny J; Al-Kassem, Hatem; Rasoul-Arzrumly, Emad; Gogas, Bill D; McDaniel, Michael C; Pielak, Tomasz; Thorball, Christian W; Sperling, Laurence; Quyyumi, Arshed A; Samady, Habib

    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 micro...

  18. Activation of Urokinase Plasminogen Activator and Its Receptor Axis Is Essential for Macrophage Infiltration in a Prostate Cancer Mouse Model

    Jian Zhang

    2011-01-01

    Full Text Available Macrophages within the tumor microenvironment promote angiogenesis, extracellular matrix breakdown, and tumor cell migration, invasion, and metastasis. Activation of the urokinase plasminogen activator (uPA and its receptor (uPAR axis promotes prostate cancer tumorigenicity, invasion, metastasis, and survival within the tumor microenvironment. The link between macrophage infiltration and the uPA/uPAR axis in prostate cancer development has not been established, although it has been reported that uPA plays a critical role in monocyte and macrophage chemotaxis. In this study, murine prostate cancer RM-1 cells were subcutaneously inoculated into wild-type (WT, uPA?/?, and uPAR?/? mice. Tumor volume was significantly diminished in both uPA?/? and uPAR?/? mice compared with WT controls. Greater inhibition of tumor volume was also observed in uPA?/? mice compared with uPAR?/? mice, suggesting the important contribution of stromal-derived uPA to sustain the tumor growth. Immunohistochemical staining revealed that tumors in uPA?/? and uPAR?/? mice displayed significantly lower proliferative indices, higher apoptotic indices, and less neovascularity compared with the tumors in WT mice. Tumors in uPA?/? and uPAR?/? mice displayed significantly less macrophage infiltration as demonstrated by F4/80 staining and MAC3+ cell numbers by flow cytometry compared with the tumors from WT mice. These findings suggest that the uPA/uPAR axis acts in both autocrine and paracrine manners in the tumor microenvironment, and activation of uPA/uPAR axis is essential for macrophage infiltration into prostate tumors.

  19. 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

  20. Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis

    A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above

  1. 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; Brünner, Nils; Overgaard, Jens

    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...

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

    Hansen, S; Overgaard, J; Rose, C; Knoop, Ann; Laenkholm, A-V; Andersen, J; Sørensen, Flemming Brandt; Andreasen, P A

    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...... 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...... 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....

  3. Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor

    Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the λ P/sub L/ promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated M/sub r/ of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators

  4. The Research of Plasminogen Activator Inhibitor-1 Serumic Level to Response One Month Combined Endurance Training and Vitamin E Consumption

    S. Ahmadizad

    2013-10-01

    Full Text Available Introduction & Objective: The plasminogen activator inhibitor-1 (PAI-1 is an important regula-tor of fibrinolysis at sites of vascular injury and has been implicated in the pathogenesis of thrombosis at the arterial sites. The aim of this study was to examine the effect one month aerobic exercise and vitamin E consumption on plasminogen activator inhibitor-1. Materials & Methods: In this quasi experimental study thirty inactive healthy collegiate fe-males voluntarily participated in the study and were randomly divided into three groups: training with vitamin (endurance training accompanied by 400 IU/day vitamin E, vitamin (400 IU/day vitamin E and control (No vitamin and no exercise training. Endurance train-ing included 4 weeks of running on treadmill, 3 days a week. The duration and intensity of exercise in each session for the first week was 30 minutes at 65% of maximum heart rat and for the three remaining weeks were increased by 5 min and 5%, respectively. Two blood samples were taken before and 48 hours after training for measuring PAI-1 antigen. Results: Data analysis revealed that PAI-1 antigen increased in control group significantly (P?0.05, while PAI-1 decreases and increases in training with vitamin, and vitamin groups, respectively were not statistically significant. Changes in PAI-1 were significantly different among the three groups (P?0.05, but using analysis of covariance it was revealed that these differences were due to differences in resting levels of PAI-1. Conclusion: Based on the findings of present study, it could be concluded that short-term regular training combined with vitamin E consumption and chronic vitamin E consumption singly result in improvements in fibrinolytic system due to decrease or prevention of PAI-1 antigen. (Sci J Hamadan Univ Med Sci 2013; 20 (3:247-255

  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 plasminogen activator protease cascade warrants further investigation

  6. 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

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

    Liu, Zhuo; Kromann-Hansen, Tobias; Lund, Ida K; Hosseini, Masood; Jensen, Knud J; Høyer-Hansen, Gunilla; Andreasen, Peter A; Sørensen, Hans Peter

    2012-01-01

    fully active state. Both a monoclonal antibody (mU3) and a peptidic inhibitor (mupain-1--16) stabilize the active state. The epitope of mU3 is located in the 37- and 70-loops at a site homologous to exosite I of thrombin. The N-terminus((Ile16)) of muPA((16--243)) was less exposed upon binding of mU3 or...... mupain-1--16. In contrast, introduction of the mutations F40Y or E137A into muPA((16--243)) increased exposure of the N-terminus((Ile16)) and resulted in large changes in the thermodynamic parameters for mupain-1--16 binding. We conclude that the distorted state of muPA((16--243)) is conformationally...... ordered upon binding of ligands to the active site and upon binding of mU3 to the 37- and 70-loops. Our study establishes the 37- and 70-loops as a unique site for binding to compounds stabilizing the active state of serine proteases....

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

    Behrens, Manja A; Bøtkjær, Kenneth Alrø; Goswami, Sumit; Oliveira, Cristiano L P; Jensen, Jan K; Schar, Christine R; Declerck, Paul J; Peterson, Cynthia B; Andreasen, Peter A; Pedersen, Jan Skov

    2011-01-01

    study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived using available atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect...

  9. Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes

    Andreasen, P A; Sottrup-Jensen, L; Kjller, L; Nykjaer, A; Moestrup, S K; Petersen, C M; Gliemann, J

    1994-01-01

    uncomplexed t-PA bind to the multi-ligand receptors alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u-PA/PAI-1 complex bound to the glycosyl phosphatidyl inositol......-anchored urokinase receptor (u-PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI-1 complexes and uncomplexed t-PA from blood plasma. The alpha 2MR- and gp330-mediated endocytosis of a ligand (u-PA/PAI-1 complex) initially bound to another receptor (u...

  10. Decreased expression of the plasminogen activator inhibitor type1 is involved in degradation of extracellular matrix surrounding cervical cancer stem cells.

    Sato, Masakazu; Kawana, Kei; Adachi, Katsuyuki; Fujimoto, Asaha; Yoshida, Mitsuyo; Nakamura, Hiroe; Nishida, Haruka; Inoue, Tomoko; Taguchi, Ayumi; Takahashi, Juri; Kojima, Satoko; Yamashita, Aki; Tomio, Kensuke; Nagamatsu, Takeshi; Wada-Hiraike, Osamu; Oda, Katsutoshi; Osuga, Yutaka; Fujii, Tomoyuki

    2016-02-01

    The plasminogen activator (PA) system consists of plasminogen activator inhibitor type1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR). PAI-1 inhibits the activation of uPA (which converts plasminogen to plasmin), and is involved in cancer invasion and metastasis, by remodeling the extracellular matrix (ECM) through regulating plasmin. Cancer stem cells (CSCs) are a small subset of cells within tumors, and are thought to be involved in tumor recurrence and metastasis. Considering these facts, we investigated the relationship between PAI-1 and cervical CSCs. We used ALDH1 as a marker of cervical CSCs. First, we demonstrated that culturing ALDH1-high cells and ALDH-low cells on collagen IV-coted plates increased their expression of active PAI-1 (ELISA), and these increases were suggested to be at mRNA expression levels (RT-qPCR). Secondly, we demonstrated PAI-1 was indeed involved in the ECM maintenance. With gelatin zymography assays, we found that ALDH1-high cells and ALDH-low cells expressed pro-matrix metalloproteinase-2 (pro-MMP-2) irrespective of their coatings. With gelatinase/collagenase assay kit, we confirmed that collagenase activity was increased when ALDH1-low cells were exposed to TM5275, a small molecule inhibitor of PAI-1. Putting the data together, we hypothesized that cancer cells adhered to basal membrane secrete abundant PAI-1, on the other hand, cancer cells (especially CSCs rather than non-CSCs) distant from basal membrane secrete less PAI-1, which makes the ECM surrounding CSCs more susceptible to degradation. Our study could be an explanation of conflicting reports, where some researchers found negative impacts of PAI-1 expression on clinical outcomes and others not, by considering the concept of CSCs. PMID:26676222

  11. Anti-tissue plasminogen activator (tPA) as an effective therapy of neonatal hypoxia-ischemia with and without inflammation.

    Yang, Dianer; Kuan, Chia-Yi

    2015-04-01

    Hypoxic-ischemic brain injury is an important cause of neurodevelopmental deficits in neonates. Intrauterine infection and the ensuing fetal inflammatory responses augment hypoxic-ischemic brain injury and attenuate the efficacy of therapeutic hypothermia. Here, we review evidences from preclinical studies suggesting that the induction of brain parenchymal tissue-type plasminogen activator (tPA) plays an important pathogenic role in these conditions. Moreover, administration of a stable-mutant form of plasminogen activator inhibitor-1 called CPAI confers potent protection against hypoxic-ischemic injury with and without inflammation via different mechanisms. Besides intracerebroventricular injection, CPAI can also be administered into the brain using a noninvasive intranasal delivery strategy, adding to its applicability in clinical use. In sum, the therapeutic potential of CPAI in neonatal care merits further investigation with large-animal models of hypoxia-ischemia and cerebral palsy. PMID:25475942

  12. 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.)

  13. 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; Griffin John H; Fernndez Jos A; Zhong Zhihui; Barrett Theresa M; Guo Huang; Zlokovic Berislav V

    2011-01-01

    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 i...

  14. 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...

  15. Stereotactic fibrinolysis of spontaneous intracerebral hematoma using infusion of recombinant tissue plasminogen activator Fibrinlise com infuso de rtPA e drenagem estereotxica de hematoma intracerebral espontneo profundo

    Jos Augusto Nasser; Asdrubal Falavigna; Mrcio Bezerra; Victor Martinez; Gabriel Freitas; Armando Alaminos; Antnio Bonatelli; Fernando Ferraz

    2002-01-01

    PURPOSE: The authors present a prospective study on 10 patients with stereotactic infusion of tissue plasminogen activator (rtPA) intraparenchimal hemorrhage. METHODS: Between 1999 and 2000, 10 patients with deep seated hematomas in the basal ganglia were selected for stereotactic infusion of rtPA and spontaneous clot drainage. RESULTS: All cases had about 80% reduction of the hematoma volume in the CT scan at the third day. The intracranial pressure was normalized by the third day too. There...

  16. Radio-enzymatic-assay method of plasminogen

    Plasmin obtained from activating plasminogen is used to hydrolyze 125I-fibrin into soluble 125I-polypeptide, thus developing a radioenzymatic-assay method of plasminogen. The activity of plasmin can be measured and the content of 125I-fibrin can be determined by the method. This method is simple, quick, specific and sensitive for evaluating thrombophilia in clinical study of thrombin disease

  17. A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimmentation Velocity Analysis*

    Minor, Kenneth H.; Schar, Christine R.; Blouse, Grant E.; Shore, Joseph D.; Lawrence, Daniel A.; Schuck, Peter; Peterson, Cynthia B.

    2005-01-01

    Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and peri-cellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81—86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes. PMID:15905170

  18. A mechanism for assembly of complexes of vitronectin and plasminogen activator inhibitor-1 from sedimentation velocity analysis.

    Minor, Kenneth H; Schar, Christine R; Blouse, Grant E; Shore, Joseph D; Lawrence, Daniel A; Schuck, Peter; Peterson, Cynthia B

    2005-08-01

    Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and pericellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81-86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes. PMID:15905170

  19. Tissue-Plasminogen Activator Attenuates Alzheimer's Disease-Related Pathology Development in APPswe/PS1 Mice.

    ElAli, Ayman; Bordeleau, Maude; Thriault, Peter; Filali, Mohammed; Lampron, Antoine; Rivest, Serge

    2016-04-01

    Alzheimer's disease (AD) is the leading cause of dementia among elderly population. AD is characterized by the accumulation of beta-amyloid (A?) peptides, which aggregate over time to form amyloid plaques in the brain. Reducing soluble A? levels and consequently amyloid plaques constitute an attractive therapeutic avenue to, at least, stabilize AD pathogenesis. The brain possesses several mechanisms involved in controlling cerebral A? levels, among which are the tissue-plasminogen activator (t-PA)/plasmin system and microglia. However, these mechanisms are impaired and ineffective in AD. Here we show that the systemic chronic administration of recombinant t-PA (Activase rt-PA) attenuates AD-related pathology in APPswe/PS1 transgenic mice by reducing cerebral A? levels and improving the cognitive function of treated mice. Interestingly, these effects do not appear to be mediated by rt-PA-induced plasmin and matrix metalloproteinases 2/9 activation. We observed that rt-PA essentially mediated a slight transient increase in the frequency of patrolling monocytes in the circulation and stimulated microglia in the brain to adopt a neuroprotective phenotype, both of which contribute to A? elimination. Our study unraveled a new role of rt-PA in maintaining the phagocytic capacity of microglia without exacerbating the inflammatory response and therefore might constitute an interesting approach to stimulate the key populations of cells involved in A? clearance from the brain. PMID:26349911

  20. Treatment of hepatic venoocclusive disease with recombinant human tissue plasminogen activator or orthotopic liver transplantation after allogeneic bone marrow transplantation.

    Hgglund, H; Ringdn, O; Ericzon, B G; Duraj, F; Ljungman, P; Lnnqvist, B; Winiarski, J; Tydn, G

    1996-10-27

    Ten allogeneic bone marrow transplant (BMT) recipients with hepatic venoocclusive disease (VOD) were treated with recombinant human tissue plasminogen activator (rt-PA). Two of them subsequently underwent orthotopic liver transplantation (OLT). One additional patient with VOD underwent OLT without prior rt-PA treatment. Treatment with rt-PA was started a median of 14 (1--35) days after BMT. The dose of rt-PA given to adults was 10-50 mg i.v. and that given to children was 3-10 mg i.v. Treatment was given for 2-4 days. In three patients, the dose was administered over a longer period or it was repeated. Four patients responded to rt-PA therapy and six did not. Eight patients suffered from hemorrhages, one intracranial and three gastrointestinal. Four patients required blood transfusions. Four had minor subcutaneous hemorrhages and/or epistaxis. One patient died of intracranial hemorrhage and five from hepatic and/or multiorgan failure. Two patients treated with rt-PA, 10 mg/day for 4 days, are alive; one is alive and well 3 months after BMT, the other has relapsed after 7 months. The three patients undergoing OLT died of chronic hepatic failure, cerebral edema, and pneumonia. Our experience suggests that rt-PA should not be administered in high doses and that the treatment should not be given over a longer period, because of the risk of severe hemorrhages. PMID:8900305

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

    Hara, T.; Iwamoto, M.; Ogawa, H.; Yamamoto, A.; Tomikawa, M. (Research Institute, Daiichi Pharmaceutical Co., Ltd., Tokyo, (Japan))

    1990-08-15

    Tissue plasminogen activator (t-PA) is frequently administered clinically as thrombolytic therapy. We injected recombinant t-PA into rats with cerebral {sup 125}I-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.

  2. Negative correlation between D-dimer and plasminogen activator inhibitor-1 levels is absent in obese women.

    Ceo, Priscilla T; Petruceli, Eveline; Bosco, Adriana Ap; Andrade-Fernandes, Claudia M; Russi, Cristiane V C; Carvalho, Maria das G; Sandrim, Valeria C

    2012-07-01

    Obesity is characterized by alterations in haemostatic processes that lead to a prothrombotic state. D-dimer (D-Di) is the last product of the fibrinolysis and may reflect the haemostatic balance. As the plasminogen activator inhibitor (PAI)-1 is the main inhibitor of fibrinolysis and it is elevated in obese, we hypothesize that negative correlation exists between PAI-1 and D-Di. In addition, we evaluated if plasma levels of PAI-1 and D-Di may be correlated with clinical parameters of adiposity [waist circumference and waist-to-hip ratio (WHR)]. We measured plasma PAI-1 and D-Di concentrations using ELISA in 60 women: 21 lean women without comorbidities and 39 obese women. We found higher levels of D-Di and PAI-1 in obese groups compared to control group (P < 0.05). No differences were observed between obese and obese untreated hypertensives. PAI-1 levels, but not of D-Di, are positively correlated with BMI (control, r = 0.44) and WHR (all obese, r = 0.40). Negative correlation was found between PAI-1 and D-Di in control (r = -0.56), no association was observed in obese, signalizing to a particular attention regarding the clinical use of D-Di. Our results indicate the magnitude of central obesity as a risk factor for development of disorders related to prothrombotic states. PMID:22527291

  3. High-fat Diet Enhances and Plasminogen Activator Inhibitor-1 Deficiency Attenuates Bone Loss in Mice with Lewis Lung Carcinoma.

    Yan, Lin; Nielsen, Forrest H; Sundaram, Sneha; Cao, Jay

    2015-07-01

    This study determined the effects of a high-fat diet and plasminogen activator inhibitor-1 deficiency (Pai1(-/-)) on the bone structure in male C57BL/6 mice bearing Lewis lung carcinoma (LLC) in lungs. Significant reduction in bone volume fraction (BV/TV), trabecular number (Tb.N) and bone mineral density (BMD) in femurs and vertebrae were found in LLC-bearing mice compared to non-tumor-bearing mice. In LLC-bearing mice, the high-fat diet compared to the AIN93G control diet significantly reduced BV/TV, Tb.N and BMD in femurs and BV/TV in vertebrae. The high-fat diet significantly reduced BMD in vertebrae in wild-type mice but not in Pai1(-/-) mice. Compared to wild-type mice, PAI1 deficiency significantly increased BV/TV and Tb.N in femurs. The plasma concentration of osteocalcin was significantly lower and that of tartrate-resistant acid phosphatase 5b (TRAP5b) was significantly higher in LLC-bearing mice. The high-fat diet significantly reduced plasma osteocalcin and increased TRAP5b. Deficiency in PAI1 prevented the high-fat diet-induced increases in plasma TRAP5b. These findings demonstrate that a high-fat diet enhances, whereas PAI1 deficiency, attenuates metastasis-associated bone loss, indicating that a high-fat diet and PAI1 contribute to metastasis-associated bone deterioration. PMID:26124329

  4. 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; Eugen-Olsen, J; Fenger, M; Rossing, P; Jeppesen, J L

    2015-01-01

    % confidence interval) values per 1 ln unit increase in suPAR were as follows: 2.5 (1.1-5.7) for CVD: 2.7 (1.2-6.2) for autonomic dysfunction; 3.8 (1.3-10.9) for albuminuria and 2.5 (1.1-6.1) for a high degree of arterial stiffness (P ≤ 0.039). CONCLUSION: The suPAR level is higher in patients with type 1......: 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...... urokinase plasminogen activator receptor levels were lower in control subjects versus all patients, in control subjects versus normoalbuminuric patients (UAER <30 mg/24 h), in normoalbuminuric patients with short (<10 years) versus long diabetes duration and were increased with degree of albuminuria...

  5. Specific identification of Lachesis muta muta snake venom using antibodies against the plasminogen activator enzyme, LV-PA.

    Felicori, Liza F; Chvez-Olrtegui, Carlos; Snchez, Eladio F

    2005-05-01

    Sandwich-type enzyme linked immunosorbent assays (ELISA) were developed to detect Lachesis muta muta (bushmaster) snake venom using antibodies against the plasminogen activator enzyme (LV-PA). Antibodies to LV-PA were obtained by immunization of one rabbit with the purified enzyme. The IgG fraction was purified from rabbit blood in a single step on a column of Sepharose-L. m. muta venom and used to coat the microtiter plates. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with L. m. muta venom from those in mice inoculated with venoms from Bothrops atrox, B. brazili, B. castelnaudi, Bothriopsis taeniata, B. bilineata, Crotalus durissus ruruima and the antigenic Bothrops (AgB) and Crotalus (AgC) pools venoms used to produce Bothropic and Crotalic antivenoms at Fundacao Ezequiel Dias (FUNED). Measurable absorbance signals were obtained with 1.5 ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice. PMID:15804530

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

    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)

  7. 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

  8. 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.

  9. 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, Ole Nrregaard; Bugge, T H; Lund, L. R.; Ploug, M; Dan, K; Behrendt, N

    2000-01-01

    potency of the plasmin, that is generated during the progress of the cascade. Using gene-targeted mice deficient in plasminogen (Plg -/- mice) [Bugge, T. H., Flick, M. J., Daugherty, C. C., and Degen, J. L. (1995) Genes Dev. 9, 794-807], we have now demonstrated and identified a component capable of...

  10. 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

  11. 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

  12. Effect of varying days of tissue plasminogen activator therapy on the prevention of postsurgical adhesions in a rabbit model.

    Dunn, R C; Mohler, M

    1993-03-01

    The depression of focal fibrinolytic activity in mesothelial or serosal tissues which results from a surgical ischemic insult can lead to the formation of permanent fibrinous adhesions. To assess the time course for the prevention of adhesion formation by recombinant tissue plasminogen activator (rt-PA), infusions of rt-PA were administered to rabbits every 12 hr for a total of 1, 2, 4, or 8 days. Animals in each rt-PA treatment group received a total cumulative dose of 16 mg. Control animals received saline with the same corresponding dosing regimens as the treated groups. Prior to dosing, all animals had a devascularizing injury to the uterine horns performed bilaterally by bipolar cauterization of the uterine mesenteric vascular arcade below the horns. A catheter was then placed from a subcutaneous port to the intraperitoneal space. rt-PA, or saline, was instilled intraperitoneally via the subcutaneous port. Adhesions were scored at the second laparotomy by assessing the percentage of the involvement of the uterine horns in adhesions. Mean score for adhesion formation in each placebo group was 35 to 40%. Mean score (+/- standard error of the mean) for rt-PA after 1 day of therapy was 13.1% +/- 3.89; after 2 days of therapy, 6.3% +/- 1.57; after 4 days of therapy, 3.8% +/- 1.57; and after 8 days of therapy, 6.9% +/- 2.3 (P < 0.01 compared to the control grouped mean for each day of treatment).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8474240

  13. Isolation and interrelationships of the multiple molecular tissue-type and urokinase-type plasminogen activator forms produced by cultured human umbilical vein endothelial cells.

    Booyse, F M; Scheinbuks, J; Lin, P H; Traylor, M; Bruce, R

    1988-10-15

    Primary and early subcultures (1st- to 3rd passage) of human umbilical vein endothelial cells produce tissue-type plasminogen activator (t-PA) antigen, consisting only of a major Mr 110,000 t-PA form. Later subcultures (greater than 4th passage) produce increasing amounts of t-PA antigen, consisting of a major Mr 110,000 and a minor Mr 68,000 form as well as increasing amounts of urokinase-type plasminogen activator (u-PA) antigen, consisting of a minor Mr 95,000 and major Mr 54,000 form. All of the major plasminogen activator forms were purified to homogeneity from 72 h serum-free conditioned media (3 liters, 1-1.8 x 10(9) cells) by a combination of immunoaffinity and gel filtration chromatography. Typically, 4th to 6th passage cultures produced/secreted t-PA-type proteins consisting of an inactive Mr 110,000 (220 IU/mg) and active Mr 68,000 (76,500 IU/mg) form representing about 39 and 8%, respectively, of the total starting sodium dodecyl sulfate stable t-PA activity, and u-PA-type proteins consisting of an inactive Mr 95,000 (700 IU/mg) and active Mr 54,000 (81,000 IU/mg) form representing about 9 and 38%, respectively, of the total starting sodium dodecyl sulfate stable u-PA activity. The isolated Mr 68,000 t-PA and Mr 54,000 u-PA proteins, exist only as two-chain forms in the absence of aprotinin and as mixtures of single- and two-chain proteins in the presence of aprotinin. Treatment with nucleophilic agents completely dissociated the Mr 110,000 t-PA and Mr 95,000 u-PA proteins into their respective Mr 68,000 t-PA and Mr 54,000 u-PA activity forms and a common Mr 46,000 protein, confirming the enzyme-inhibitor complex nature of these inactive plasminogen activator forms. PMID:3139666

  14. Tissue plasminogen activator induces microglial inflammation via a noncatalytic molecular mechanism involving activation of mitogen-activated protein kinases and Akt signaling pathways and AnnexinA2 and Galectin-1 receptors

    Serratosa, Joan; Tusell, Josep Maria; Saura, Josep; Planas, Anna M.; Navarro, Pilar

    2012-01-01

    Inflammatory responses mediated by glial cells play a critical role in many pathological situations related to neurodegeneration such as Alzheimer's disease. Tissue plasminogen activator (tPA) is a serine protease which best-known function is fibrinolysis, but it is also involved in many other physiological and pathological events as microglial activation. Here, we found that tPA is required for Aβ-mediated microglial inflammatory response and tumor necrosis factor-α release. We further inv...

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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 01 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

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

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C; Chen, Zhuo; Blouse, Grant E; Jensen, Janni M; Mortensen, Kim; Huang, Mingdong; Deryugina, Elena; Quigley, James P; Declerck, Paul J; Andreasen, Peter A

    2011-01-01

    high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found...... 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......, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active u...

  1. Plasminogen activator activity in lung and alveolar macrophages of rats exposed to graded single doses of γ rays to the right hemithorax

    Male rats were sacrificed 2 or 6 months after a single dose of 0-30 Gy of 60Co γ rays to the right hemithorax. At autopsy, macrophages were lavaged from the right lung, counted, and frozen. The right (irradiated) and the left (shielded) lungs were frozen, then assayed for plasminogen activator (PLA) activity by the fibrin plate lysis method. Freeze-thawed macrophages were assayed for both PLA activity (125I-fibrin clot lysis method) and fibrinolytic inhibitor activity (inhibition of urokinase-induced fibrin lysis). There was a linear, dose-dependent decrease in right lung PLA activity over the dose range of 10-30 Gy at 2 and 6 months postirradiation, reductions of 3.1 and 2.6% per Gy, respectively. PLA activity at all radiation doses was 10-15% higher at 6 months than at 2 months indicative of a partial recovery of this endothelial function in the irradiated lung. PLA activity per 106 macrophages decreased with increasing radiation dose at both autopsy times, closely paralleling lung PLA activity. This radiation-induced decrease in macrophage PLA activity was not due to increased fibrinolytic inhibitor activity in the irradiated macrophages. These data quantitate the dose response and time course of radiation-induced fibrinolytic defects in rat lung and suggest that information obtained from a minimally invasive procedure such as bronchoalveolar lavage may serve as an index of the degree of pulmonary fibrinolytic dysfunction after irradiation

  2. Enhanced poly(ADP-ribose) polymerase-1 activation contributes to recombinant tissue plasminogen activator-induced aggravation of ischemic brain injury in vivo.

    Crome, Olaf; Doeppner, Thorsten R; Schwarting, Snke; Mller, Barbara; Bhr, Mathias; Weise, Jens

    2007-06-01

    Recombinant tissue plasminogen activator (rt-PA) treatment improves functional outcome after acute ischemic stroke, inducing reperfusion by its thrombolytic activity. Conversely, there is evidence that rt-PA can mediate neuronal damage after ischemic brain injury in vivo. In addition to other mechanisms, enhancement of N-methyl-D-aspartate (NMDA) receptor signalling has been proposed to underlie rt-PA-mediated neurotoxicity. However, the role of poly(ADP-ribose) polymerase-1 (PARP-1) activation, which mediates postischemic excitotoxic cell death, in rt-PA-mediated aggravation of ischemic brain injury has not been established and was therefore addressed in this study. After permanent focal cerebral ischemia, intravenous rt-PA application significantly increased early postischemic PARP-1 activation within ischemic hemispheres and infarct volumes compared with control mice without affecting cerebral blood flow. Rt-PA induced increase in PARP-1 activation, and infarct volumes could be blocked by the PARP inhibitor 3-aminobenzamide. Moreover, the rt-PA-induced increase in PARP-1 activation was also prevented by the NMDA antagonist MK-801. In summary, we demonstrate that rt-PA treatment enhances postischemic PARP-1 activation, which contributes to rt-PA induced aggravation of ischemic brain injury in vivo. Furthermore, we provide evidence that NMDA receptor activation is required for rt-PA-mediated effects on postischemic PARP-1 activation. PMID:17455321

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

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R; Jensen, Jens-Erik B; Mrak, Emanuela; Rubinacci, Alessandro; Talotta, Francesco; Verde, Pasquale; Blasi, Francesco

    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...

  4. 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

  5. Jet and ultrasonic nebulization of single chain urokinase plasminogen activator (scu-PA)

    Münster, Anna-Marie; Bendstrup, E; Jensen, J.I.; Gram, Jørgen Brodersen

    2000-01-01

    'AM DP-100 ultrasonic nebulizer (Pulmolink, Kent, UK). The particle size distribution was measured with a laser diffraction method and the drug output was determined by collection on filters. The amount of protein on the filters was determined with the Lowry method, and the enzymatic activity after...... nebulization was measured with a microtiter fibrin plate assay. The mass median diameter (MMD) of the scu-PA aerosol generated with the ultrasonic nebulizer was 3.69 (3.53-3.83) microm and with the jet nebulizer 2.96 (2.91-3.03) microm (p < 0.001). The drug output from the two nebulizers did not differ between...... nebulizers (p = 0.054). Fibrinolytically active scu-PA was generated with both nebulizers, but in contrast to jet nebulization, ultrasonic nebulization caused partial inactivation of scu-PA (p < 0.001). In conclusion, nebulization of scu-PA with the jet nebulizer is superior to ultrasonic nebulization in...

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

    Ohki, Makiko; Ohki, Yuichi; Ishihara, Makoto; Nishida, Chiemi; Tashiro, Yoshihiko; Akiyama, Haruyo; Komiyama, Hiromitsu; Lund, Leif R; Nitta, Atsumi; Yamada, Kiyofumi; Zhu, Zhenping; Ogawa, Hideoki; Yagita, Hideo; Okumura, Ko; Nakauchi, Hiromitsu; Werb, Zena; Heissig, Beate; Hattori, Koichi

    2010-01-01

    tissue regeneration is not well understood. Bone marrow (BM)-derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool and...... mobilizes CD45(+)CD11b(+) proangiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signaling. tPA improves the incorporation of CD11b(+) cells into ischemic tissues and increases expression of neoangiogenesis-related genes, including VEGF......-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b(+) cells and VEGFR-1(+) cells, but not carrier-mobilized cells or CD11b(-) cells, accelerates neovascularization and ischemic tissue regeneration. Inhibition of VEGF signaling suppresses tPA-induced neovascularization in a model of hind limb...

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

    Song, Xiaoling

    2011-12-01

    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.

  8. 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 diabetes (more than 10 years indicated a gene-environment interaction in determining the risk of diabetic retinopathy.

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

    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

  10. 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

  11. 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

  12. 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.

  13. VEGF increases the fibrinolytic activity of endothelial cells within fibrin matrices: involvement of VEGFR-2, tissue type plasminogen activator and matrix metalloproteinases.

    Ratel, David; Mihoubi, Samira; Beaulieu, Edith; Durocher, Yves; Rivard, Georges-Etienne; Gingras, Denis; Béliveau, Richard

    2007-01-01

    Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation was completely abrogated by inhibitors of either the plasminogen activator/plasmin or matrix metalloproteinases (MMP) proteolytic systems, suggesting the involvement of both classes of proteases in fibrin degradation. Accordingly, VEGF-induced fibrinolysis correlated with an increase in the expression of tPA and of some MMPs, such as MT2-MMP and was completely blocked by a neutralizing antibody against tPA. Overall, these results indicate that efficient proteolysis of three dimensional fibrin matrices during VEGF-mediated angiogenesis involves a complex interplay between the MMP and plasmin-mediated proteolytic systems. PMID:17512973

  14. Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase.

    Jiao, J; Yu, M; Ru, B

    2001-01-01

    A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required. PMID:11879733

  15. 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, Salom; Clemente, Alda; Malcata, F.Xavier

    2001-01-01

    The rates and extents of hydrolysis of RS- and -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...

  16. Biochemical, thrombolytic and pharmacokinetic properties of rt-PA P47G, K49N, a substitution variant of human tissue-type plasminogen activator.

    Nelles, L; Li, X K; Vanlinthout, I; De Cock, F; Lijnen, H R; Collen, D

    1992-04-01

    rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahern et al. (J Biol Chem 1990; 265:5540-5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse). The specific fibrinolytic activities on fibrin plates were 160,000 +/- 17,000, 210,000 +/- 88,000 and 460,000 +/- 72,000 IU/mg (mean +/- SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k2/Km) in the absence of fibrin were comparable (1.1 to 1.7 x 10(-3) microM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 +/- 0.14 micrograms/ml rt-PA P47G, K49N, 0.36 +/- 0.01 micrograms/ml rt-PA and 0.17 +/- 0.01 micrograms/ml alteplase, respectively (mean +/- SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1631793

  17. Protein kinase G inhibits basal and stimulated nitric oxide synthase activity in neonatal ovine lung microvascular endothelial cells.

    John, T A

    2010-06-01

    Protein kinase G (PKG) is abundant in neonatal ovine lung microvascular endothelial cells (LMVECs) but its various functions are not known. To test the hypothesis that PKG plays a role in feedback regulation of nitric oxide (NO) synthesis, we studied the effects of modulators of PKG signaling on real time NO release from LMVECs microcultures in 96-well clusters. We used 0.5 - 2.43 microM DAF FM and DAF-FM diacetate to measure amount of NO present in the cells and in the cell bathing medium. We found a dose-response relationship between 8-Br-cGMP (0.02 - 2 microM), a stimulator of PKG activity, and inhibition of basal NO production. The time-course of the effect of 2 microM 8-Br-cGMP on NO production exhibited a parallel shift downwards in the presence of PKG receptor inhibitor, 100 ng/ml DT-2, indicating that 8-Br-cGMP acts through PKG to inhibit NOS. PKG also decreased stimulated NO production: acetylcholine produced raw fluorescence of 59999 +/- 702 and in the presence of 1 mM 8-Br-cGMP the value was 20645 +/- 292 (p < 0.0001) while carbachol produced raw fluorescence of 60600 +/- 890 and in the presence of 1 mM 8-Br-cGMP was 30442 +/- 2000 (p < 0.01). The PKG inhibitor 125 nM guanosine 3'-5'-cyclic-monophosphorothionate-8-Br-Rp isomer increased basal NO production (p < 0.01). NO synthase inhibitor, L-NNA reversed this effect (p < 0.05) as well as that of another PKG inhibitor 25 microM Rp-8-Br-PET-cGMPS but enhanced the effect of 25 microM 8-Br-cGMP. Both basal and stimulated NO production is regulated by the downstream activation of PKG by NO-induced 8-Br-cGMP production in endothelial cells. PMID:21117404

  18. Schistosoma bovis: plasminogen binding in adults and the identification of plasminogen-binding proteins from the worm tegument.

    Ramajo-Hernndez, Alicia; Prez-Snchez, Ricardo; Ramajo-Martn, Vicente; Oleaga, Ana

    2007-01-01

    Schistosoma bovis is a ruminant haematic parasite that lives for years in the mesenteric vessels of the host. The aim of this work was to investigate the ability of adult S. bovis worms to interact with plasminogen, a central component in the host fibrinolytic system. Confocal microscopy analysis revealed that plasminogen bound to the tegument surface of the male-but not female-S. bovis worms and that this binding was strongly dependent on lysine residues. It was also observed that a protein extract of the worm tegument (TG) had the capacity to generate plasmin and to enhance the plasmin generation by the tissue-type plasminogen activator. Proteomic analysis of the TG extract identified 10 plasminogen-binding proteins, among which the major ones were enolase, glyceraldehyde-3-phosphate dehydrogenase and actin. This study represents the first report about the binding of plasminogen to Schistosoma sp. proteins. PMID:16962583

  19. In vitro and in vivo antiangiogenic activity of a novel deca-peptide derived from human tissue-type plasminogen activator kringle 2

    A synthetic deca-peptide corresponding to the amino acid sequence Arg54-Trp63 of human tissue-type plasminogen activator (t-PA) kringle 2 domain, named TKII-10, is produced and tested for its ability to inhibit endothelial cell proliferation, migration, tube formation in vitro, and angiogenesis in vivo. At the same time, another peptide TKII-10S composed of the same 10 amino acids as TKII-10, but in a different sequence, is also produced and tested. The results show that TKII-10 potently inhibits VEGF-stimulated endothelial cell migration and tube formation in a dose-dependent, as well as sequence-dependent, manner in vitro while it is inactive in inhibiting endothelial cell proliferation. Furthermore, TKII-10 potently inhibits angiogenesis in chick chorioallantoic membrane and mouse cornea. The middle four amino acids DGDA in their sequence play an important role in TKII-10 angiogenesis inhibition. These results suggest that TKII-10 is a novel angiogenesis inhibitor that may serve as a prototype for antiangiogenic drug development.

  20. In vitro and in vivo antiangiogenic activity of a novel deca-peptide derived from human tissue-type plasminogen activator kringle 2

    Su, Li; Xu, Xun; Zhao, Hui; Gu, Qing [Department of Ophthalmology, Shanghai First People' s Hospital, Affiliate of Shanghai Jiaotong University, No. 100 Haining Road, Shanghai 200080 (China); Zou, Haidong, E-mail: zouhaidong@hotmail.com [Department of Ophthalmology, Shanghai First People' s Hospital, Affiliate of Shanghai Jiaotong University, No. 100 Haining Road, Shanghai 200080 (China)

    2010-06-11

    A synthetic deca-peptide corresponding to the amino acid sequence Arg{sup 54}-Trp{sup 63} of human tissue-type plasminogen activator (t-PA) kringle 2 domain, named TKII-10, is produced and tested for its ability to inhibit endothelial cell proliferation, migration, tube formation in vitro, and angiogenesis in vivo. At the same time, another peptide TKII-10S composed of the same 10 amino acids as TKII-10, but in a different sequence, is also produced and tested. The results show that TKII-10 potently inhibits VEGF-stimulated endothelial cell migration and tube formation in a dose-dependent, as well as sequence-dependent, manner in vitro while it is inactive in inhibiting endothelial cell proliferation. Furthermore, TKII-10 potently inhibits angiogenesis in chick chorioallantoic membrane and mouse cornea. The middle four amino acids DGDA in their sequence play an important role in TKII-10 angiogenesis inhibition{sub .} These results suggest that TKII-10 is a novel angiogenesis inhibitor that may serve as a prototype for antiangiogenic drug development.

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

    Marzia Albenzio; Rosaria Marino; Mariangela Caroprese; Antonella Santillo

    2010-01-01

    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 chees...

  2. Formyl-methyl-methylenetetrahydrofolate synthetase-(combined). An ovine protein with multiple catalytic activities.

    Paukert, J L; Straus, L D; Rabinowitz, J C

    1976-08-25

    Formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclohydrolase, and methylenetetrahydrofolate dehydrogenase in sheep liver co-purify 200-fold to yield a homogeneous preparation containing a single protein species observed on discontinuous polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The synthetase and dehydrogenase activities migrate with the single protein band observed on polyacrylamide gel electrophoresis in two discontinuous buffer systems near pH 8. The protein sediments with a single symmetrical boundary with sedimentation coefficient (S20,w) = 8.30 S and elutes from molecular exclusion chromatography columns at a position corresponding to a diffusion coefficient (D20,w) = 3.99 X 10(-7) cm2 sec-1. Dodecyl sulfate-polyacrylamide gel electrophoresis of the protein intramolecularly cross-linked with dimethylsuberimidate shows one protein species of Mr = 218,000 in addition to the species of Mr = 108,500 characteristic of the unmodified protein. NH2-terminal analysis of the protein using 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chloride in the presence of dodecyl sulfate results in the recovery of a single dansyl amino acid, alanine. The three activities are thus shown to reside in a protein composed of two apparently identical subunits, and the trivial name formyl-methenyl-methylenetetrahydrofolate synthetase(combined) is proposed for this enzyme to suggest the multiple catalytic activities associated with the single protein species. PMID:956178

  3. Expression and characterization of clustered charge-to-alanine mutants of low M(r) single-chain urokinase-type plasminogen activator.

    Ueshima, S; Holvoet, P; Lijnen, H R; Nelles, L; Seghers, V; Collen, D

    1994-01-01

    In an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M(r) single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala). The rscu-PA-32k moieties were expressed in High Five Trichoplasiani cells, and purified to homogeneity from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/l. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion to two-chain moieties by plasmin were comparable for mutant and wild-type rscu-PA-32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 microliters 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 microgram/ml of wild-type or mutant rscu-PA-32k, except with LUK-5 (no significant lysis with 16 micrograms/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8165632

  4. 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

  5. Treatment of neonatal fungal infective endocarditis with recombinant tissue plasminogen: activator in a low birth weight infant case report and review of the literature.

    Babayigit, Aslan; Cebeci, Burcu; Buyukkale, Gokhan; Semerci, Seda Yılmaz; Bornaun, Helen; Oztarhan, Kazim; Gokce, Muge; Cetinkaya, Merih

    2015-10-01

    With advances in medical sciences, an increase in survival rates of low birth weight; increased incidence in use of catheter and antibiotics, and total parenteral nutrition are reported, therefore, the rate of fungal infections in late and very late onset neonatal sepsis have increased. Although fungal endocarditis rarely occur in newborns, it has a high morbidity and mortality. Antifungal therapy is often insufficient in cases who develop fungal endocarditis and surgical treatment is not preferred due to its difficulty and high mortality. Herein, fungal endocarditis in a preterm newborn treated with single-dose recombinant tissue plasminogen activator in addition to antifungal therapy is presented and relevant literature has been reviewed. The vegetation completely disappeared following treatment and no complication was observed. PMID:26214750

  6. 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; Andreasen, Peter A; Jørgensen, Thomas J D

    2014-01-01

    RNA aptamers, selected from large synthetic libraries, are attracting increasing interest as protein ligands, with potential uses as prototype pharmaceuticals, conformational probes, and reagents for specific quantification of protein levels in biological samples. Very little is known, however......, 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...... aptamers to PAI-1 is associated with substantial and widespread protection against deuterium uptake in PAI-1. The aptamers induce protection against exchange with the solvent both in the protein-aptamer interface as well as in other specific areas. Interestingly, the aptamers induce substantial protection...

  7. Preoperative plasma plasminogen activator inhibitor type-1 and serum C-reactive protein levels in patients with colorectal cancer. The RANX05 Colorectal Cancer Study Group

    Nielsen, Hans Jrgen; Christensen, Ib Jarle; Srensen, Steen; Moesgaard, F; Brnner, N

    2000-01-01

    statistical analysis including Dukes classification, gender, age, tumor location, perioperative blood transfusion, PAI-1 and CRP, plasma PAI-1 was a dependent prognostic variable, while serum CRP (P <.0001; HR: 1.4; 95% CI: 1.3-1.5) was found to be a Dukes independent prognostic variable. Similar analyses......BACKGROUND: Preoperative plasma plasminogen activator inhibitor-1 (PAI-1) is a prognostic variable in patients with colorectal cancer. It has been suggested, however, that plasma PAI-1 is a nonspecific prognostic parameter similar to the acute-phase reactant C-reactive protein (CRP). In the present...... study we analyzed the association between plasma PAI-1 and serum CRP in patients scheduled for elective resection of colorectal cancer. In addition, the prognostic value of PAI-1 and CRP was studied in this patient cohort. METHODS: PAI-1 and CRP were analyzed in citrated plasma and serum, respectively...

  8. Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist by microPET

    Li, Z.B.; Niu, G.; Wang, H.; He, L.; Yang, L.; Ploug, M.; Chen, X.

    2008-01-01

    PURPOSE: Malignant tumors are capable of degrading the surrounding extracellular matrix, resulting in local invasion or metastasis. Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) are central molecules in one of the major protease systems involved in extracellular...... matrix degradation. Noninvasive imaging of this receptor in vivo with radiolabeled peptides that specifically target uPAR may therefore be useful to decipher the potential invasiveness of malignant lesions. EXPERIMENTAL DESIGN: In this study, we developed a (64)Cu-labeled uPAR-binding peptide for...... positron emission tomography (PET) imaging. A linear, high-affinity uPAR-binding peptide antagonist AE105 was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled with (64)Cu for microPET imaging of mice bearing U87MG human glioblastoma (uPAR positive) and MDA-MB-435...

  9. 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; Sørensen, Hans Peter; Jensen, Jan K; Christensen, Anni; Hosseini, Masood; Nielsen, Niels Christian; Jensen, Knud J; Andreasen, Peter A.; Huang, Mingdong

    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...

  10. Preoperative plasma plasminogen activator inhibitor type-1 and serum C-reactive protein levels in patients with colorectal cancer. The RANX05 Colorectal Cancer Study Group

    Nielsen, Hans Jørgen; Christensen, Ib Jarle; Sørensen, Steen; Moesgaard, F; Brünner, N

    2000-01-01

    BACKGROUND: Preoperative plasma plasminogen activator inhibitor-1 (PAI-1) is a prognostic variable in patients with colorectal cancer. It has been suggested, however, that plasma PAI-1 is a nonspecific prognostic parameter similar to the acute-phase reactant C-reactive protein (CRP). In the present...... study we analyzed the association between plasma PAI-1 and serum CRP in patients scheduled for elective resection of colorectal cancer. In addition, the prognostic value of PAI-1 and CRP was studied in this patient cohort. METHODS: PAI-1 and CRP were analyzed in citrated plasma and serum, respectively......, obtained preoperatively from 594 patients. Patients who required preoperative blood transfusion received SAGM blood, in which soluble PAI-1 is not present. None of the patients received pre- or postoperative adjuvant chemotherapy, and all were followed in the outpatient clinic for at least 5 years or until...

  11. Cyclic AMP-mediated cytoskeletal effects in adrenal cells are modified by serum, insulin, insulin-like growth factor-I, and an antibody against urokinase plasminogen activator.

    Hornsby, P J; Maghsoudlou, S S; Cheng, V; Cheng, C Y

    1989-12-01

    In adrenocortical cells in culture, increased intracellular cyclic AMP resulting from exposure to agents such as ACTH and cholera toxin causes a change in cell morphology termed 'retraction' or 'rounding'. The breakdown of actin-containing stress fibers in rounding suggested a role for microfilaments in steroidogenesis. Previously, we showed that cultured bovine adrenal cells under standard conditions (medium with 10% fetal bovine serum) do not round in response to intracellular cyclic AMP. Here, we show that these cells do round in defined, serum-free medium. Rounding was maximal within 1 h of addition of 1 nM cholera toxin and after 10 h most cells remained rounded. Cycloheximide at 100 micrograms/ml did not inhibit the response to cholera toxin. The rounding response was abolished when 10% fetal bovine serum, horse serum, or ether-extracted fetal bovine serum was included in the medium. The inhibitory effect of serum was not mimicked by growth factors with the exception that insulin and insulin-like growth factor-I (IGF-I), while not preventing rounding, accelerated the return of cells to a flattened morphology. A monoclonal antibody against urokinase plasminogen activator completely prevented rounding whereas a monoclonal antibody against tissue plasminogen activator had only a slight effect. Fluorescence visualization of F-actin with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin showed that rounding in cultured bovine adrenocortical cells resembles that defined earlier for human and rat adrenocortical cells and includes depolymerization of actin microfilaments. These cytoskeletal changes in adrenal cells are unlikely to play a role in steroidogenesis; however, they may be involved in tissue remodeling occurring as part of the indirect mitogenic effects of ACTH. PMID:2558936

  12. Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPA alpha 1 expressed in Chinese hamster ovary cells.

    Gohlke, M; Nuck, R; Kannicht, C; Grunow, D; Baude, G; Donner, P; Reutter, W

    1997-02-01

    The recombinant plasminogen activator (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fluorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was alpha 2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA alpha 1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA alpha 1 is structurally closely related. PMID:9061366

  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. Characteristics of ovine cytotoxic lymphocytes

    Experiments were conducted to examine characteristics of the effector cells responsible for cell-mediated cytotoxicity in the sheep. Conditions for the production and assay of ovine T cell growth factor (TCGF) activity were evaluated. Peripheral blood leukocytes (PBL) were stimulated with concanavalin A (Con A) in the presence of 2% autologous serum or serum-free media. A 28 h proliferation assay with 2.5 x 104 h Con A blasts per well was optimal for detection of TCGF. Peak TCGF activity occurred with a 30-37kD molecular weight fraction. Ovine PBL were used for in vitro generation of genetically-restricted cytotoxic T lymphocytes (CTL). Peripheral blood leukocytes from sheep that had been previously inoculated with live vaccinia virus were stimulated by being cultured in vitro on glutaraldehyde-fixed vaccinia-infected autologous skin fibroblasts. Cytotoxic T lymphocyte activity was assessed in a 6 h 51Cr-release assay on autologous and allogeneic fibroblasts targets. Killing was restricted to virus-infected autologous targets. In vitro generation of both anti-vaccinia and anti-TNP CTL activity could be enhanced by the addition of TCGF containing media from ConA-stimulated PBL

  15. 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 treated with nicotine displayed enhanced invasiveness. ► Nicotine induces uPAR expression and, in turn, stimulates invasiveness. ► MAPK/AP-1 and ROS/NF-κB signals are involved in nicotine-induced uPAR.

  16. 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 displayed enhanced invasiveness. ► Nicotine induces uPAR expression and, in turn, stimulates invasiveness. ► MAPK/AP-1 and ROS/NF-κB signals are involved in nicotine-induced uPAR.

  17. Three are better than one: plasminogen receptors as cancer theranostic targets.

    Ceruti, Patrizia; Principe, Moitza; Capello, Michela; Cappello, Paola; Novelli, Francesco

    2013-01-01

    Activation of plasminogen on the cell surface initiates a cascade of protease activity with important implications for several physiological and pathological events. In particular, components of the plasminogen system participate in tumor growth, invasion and metastasis. Plasminogen receptors are in fact expressed on the cell surface of most tumors, and their expression frequently correlates with cancer diagnosis, survival and prognosis. Notably, they can trigger multiple specific immune resp...

  18. 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

  19. Tissue plasminogen activator followed by antioxidant-loaded nanoparticle delivery promotes activation/mobilization of progenitor cells in infarcted rat brain.

    Petro, Marianne; Jaffer, Hayder; Yang, Jun; Kabu, Shushi; Morris, Viola B; Labhasetwar, Vinod

    2016-03-01

    Inherent neuronal and circulating progenitor cells play important roles in facilitating neuronal and functional recovery post stroke. However, this endogenous repair process is rather limited, primarily due to unfavorable conditions in the infarcted brain involving reactive oxygen species (ROS)-mediated oxidative stress and inflammation following ischemia/reperfusion injury. We hypothesized that during reperfusion, effective delivery of antioxidants to ischemic brain would create an environment without such oxidative stress and inflammation, thus promoting activation and mobilization of progenitor cells in the infarcted brain. We administered recombinant human tissue-type plasminogen activator (tPA) via carotid artery at 3h post stroke in a thromboembolic rat model, followed by sequential administration of the antioxidants catalase (CAT) and superoxide dismutase (SOD), encapsulated in biodegradable nanoparticles (nano-CAT/SOD). Brains were harvested at 48h post stroke for immunohistochemical analysis. Ipsilateral brain slices from animals that had received tPA+nano-CAT/SOD showed a widespread distribution of glial fibrillary acidic protein-positive cells (with morphology resembling radial glia-like neural precursor cells) and nestin-positive cells (indicating the presence of immature neurons); such cells were considerably fewer in untreated animals or those treated with tPA alone. Brain sections from animals receiving tPA+nano-CAT/SOD also showed much greater numbers of SOX2- and nestin-positive progenitor cells migrating from subventricular zone of the lateral ventricle and entering the rostral migratory stream than in t-PA alone treated group or untreated control. Further, animals treated with tPA+nano-CAT/SOD showed far fewer caspase-positive cells and fewer neutrophils than did other groups, as well as an inhibition of hippocampal swelling. These results suggest that the antioxidants mitigated the inflammatory response, protected neuronal cells from undergoing apoptosis, and inhibited edema formation by protecting the blood-brain barrier from ROS-mediated reperfusion injury. A longer-term study would enable us to determine if our approach would assist progenitor cells to undergo neurogenesis and to facilitate neurological and functional recovery following stroke and reperfusion injury. PMID:26735970

  20. Moraxella catarrhalis Binds Plasminogen To Evade Host Innate Immunity.

    Singh, Birendra; Al-Jubair, Tamim; Voraganti, Chandrashekar; Andersson, Tobias; Mukherjee, Oindrilla; Su, Yu-Ching; Zipfel, Peter; Riesbeck, Kristian

    2015-09-01

    Several bacterial species recruit the complement regulators C4b-binding protein, factor H, and vitronectin, resulting in resistance against the bactericidal activity of human serum. It was recently demonstrated that bacteria also bind plasminogen, which is converted to plasmin that degrades C3b and C5. In this study, we found that a series of clinical isolates (n = 58) of the respiratory pathogen Moraxella catarrhalis, which is commonly isolated from preschool children and adults with chronic obstructive pulmonary disease (COPD), significantly binds human plasminogen. Ubiquitous surface protein A2 (UspA2) and hybrid UspA2 (UspA2H) were identified as the plasminogen-binding factors in the outer membrane proteome of Moraxella. Furthermore, expression of a series of truncated recombinant UspA2 and UspA2H proteins followed by a detailed analysis of protein-protein interactions suggested that the N-terminal head domains bound to the kringle domains of plasminogen. The binding affinity constant (KD) values of full-length UspA2(30-539) (amino acids 30 to 539 of UspA2) and full-length UspA2H(50-720) for immobilized plasminogen were 4.8 × 10(-8) M and 3.13 × 10(-8) M, respectively, as measured by biolayer interferometry. Plasminogen bound to intact M. catarrhalis or to recombinant UspA2/UspA2H was readily accessible for a urokinase plasminogen activator that converted the zymogen into active plasmin, as verified by the specific substrate S-2251 and a degradation assay with fibrinogen. Importantly, plasmin bound at the bacterial surface also degraded C3b and C5, which consequently may contribute to reduced bacterial killing. Our findings suggest that binding of plasminogen to M. catarrhalis may lead to increased virulence and, hence, more efficient colonization of the host. PMID:26099590

  1. Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients.

    Sloand, Elaine M; Pfannes, Loretta; Scheinberg, Phillip; More, Kenneth; Wu, Colin O; Horne, McDonald; Young, Neal S

    2008-12-01

    Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients' plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells. PMID:18954937

  2. Ovine TRIM5 Can Restrict Visna/Maedi Virus

    P. Juregui; Crespo, H.; Glaria, I.; A. Contreras; De Andrs, D.; Amorena, B.; Towers, G. J.; Reina, R.

    2012-01-01

    The restrictive properties of tripartite motif-containing 5 alpha (TRIM5?) from small ruminant species have not been explored. Here, we identify highly similar TRIM5? sequences in sheep and goats. Cells transduced with ovine TRIM5? effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5? restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5? active molecular sp...

  3. 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 abstinence.Keywords: B428, CPP, two-bottle choice

  4. Expression of Met protein and urokinase-type plasminogen activator receptor (uPA-R) in papillary carcinoma of the thyroid.

    Zanetti, A; Stoppacciaro, A; Marzullo, A; Ciabatta, M; Fazioli, F; Prat, M; Comoglio, P M; Baroni, C D; Ruco, L P

    1998-11-01

    Met protein encoded by MET oncogene is the high affinity receptor for hepatocyte growth factor (HGF)/scatter factor (SF). HGF/SF has to be cleaved in its heterodimeric form by the urokinase-type plasminogen activator (uPA) to become active as a ligand for Met receptor. The expression of Met protein and of the high affinity receptor for uPA (uPA-R) was investigated in 39 samples of papillary carcinoma using immunohistochemistry. Reactivity for Met protein was present in 33 of 34 tumours, mostly with a diffuse pattern of staining. Reactivity for uPA-R was present in 78 per cent of papillary tumours and exhibited a pattern of staining similar to that of Met protein. Staining for uPA-R was present in 23 of 25 cases (92 per cent) of papillary carcinoma with prominent sclerosis, and in only 1 of 7 cases (14 per cent) without sclerosis. Peritumoural normal thyroid, follicular adenomas, and follicular carcinomas were negative for Met protein and for uPA-R. Hyperfunctioning tall thyroid cells showed weak membrane reactivity for uPA-R and for Met protein. The findings of immunohistochemistry were confirmed at the mRNA level using in situ hybridization, since the signal for uPA-R and Met RNAs was detected in most tumour cells of five cases of papillary carcinoma. PMID:10211118

  5. Binding of human plasminogen to Borrelia burgdorferi.

    Hu, L T; Perides, G; Noring, R; Klempner, M S

    1995-01-01

    We studied the binding of plasminogen to Borrelia burgdorferi, a spirochete which causes Lyme disease and produces no endogenous proteases which digest extracellular matrix proteins. Using 125I-labeled plasminogen, we demonstrated that B. burgdorferi bound human plasminogen and that this binding was inhibitable with unlabeled plasminogen. 125I-labeled plasminogen binding by B. burgdorferi was also inhibited by the lysine analog epsilon-aminocaproic acid. There was no significant difference in...

  6. 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

  7. 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; Eugen-Olsen, Jesper; Nielsen, Olav W; Blyme, Adam; Willenheimer, Ronnie; Wachtell, Kristian; Olsen, Michael H

    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<0.001) and suPAR (βyear0 = 0.05, p...

  8. 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.

  9. Gene Therapy to Promote Thromboresistance: Local Overexpression of Tissue Plasminogen Activator to Prevent Arterial Thrombosis in an in vivo Rabbit Model

    Waugh, J. M.; Kattash, M.; Li, J.; Yuksel, E.; Kuo, M. D.; Lussier, M.; Weinfeld, A. B.; Saxena, R.; Rabinovsky, E. D.; Thung, S.; Woo, S. L. C.; Shenaq, S. M.

    1999-02-01

    Tissue-type plasminogen activator (tPA) catalyzes the rate-limiting initial step in the fibrinolytic cascade. Systemic infusion of tPA has become the standard of care for acute myocardial infarction. However, even the relatively short-duration protocols currently employed have encountered significant hemorrhagic complications, as well as complications from rebound thrombosis. Gene therapy offers a method of local high-level tPA expression over a prolonged time period to avoid both systemic hemorrhage and local rebound thrombosis. To examine the impact of local tPA overexpression, an adenoviral vector expressing tPA was created. The construct was characterized functionally in vitro, and the function of the vector was confirmed in vivo by delivery to the rabbit common femoral artery. Systemic coagulation parameters were not perturbed at any of the doses examined. The impact of local overexpression of tPA on in vivo thrombus formation was examined subsequently in a stasis/injury model of arterial thrombosis. The construct effectively prevented arterial thrombosis in treated animals, whereas viral and nonviral controls typically developed occluding thrombi. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery.

  10. Comparison of bezafibrate versus lovastatin for lowering plasma insulin, fibrinogen, and plasminogen activator inhibitor-1 concentrations in hyperlipemic heart transplant patients.

    Zambrana, J L; Velasco, F; Castro, P; Concha, M; Valls, F; Montilla, P; Jimenz-Pereprez, J A; Lpez-Miranda, J; Prez-Jimnez, F

    1997-10-01

    Accelerated coronary artery disease is the most serious obstacle to long-term survival in heart transplant recipients. Hyperlipemia, hyperinsulinism, and changes in endothelial cell hemostatic function have been implicated in cardiac allograft vascular disease. Both lovastatin and bezafibrate are safe, effective, and well tolerated therapies for hyperlipidemia. Our study compares the effect of these lipid-lowering drugs in 21 patients with post-heart transplantation hyperlipidemia on different risk factors related to insulin resistance syndrome. Patients were given the same diet for 3 months, then randomized to lovastatin or bezafibrate for a period of 8 weeks, and crossed over to an additional 8 weeks of either bezafibrate or lovastatin. Baseline parameters were also compared with those of a control group of healthy subjects and after both periods of pharmacologic treatment. Transplant patients had higher insulin (35 +/- 3 vs 24 +/- 3 microIU/L), fibrinogen (298 +/- 15 vs 261 +/- 14 mg/dl), and plasminogen activator inhibitor-1 (PAI-1) (17 +/- 2 vs 11.7 +/- 2 arbitrary units/ml) plasma levels than controls. Significant decreases in insulin (-37 +/- 3%), fibrinogen (-12 +/- 4%), and PAI-1 plasma levels (-18 +/- 12%) were only observed after bezafibrate treatment. In conclusion, bezafibrate decreases plasma insulin, fibrinogen, and PAI-1 in hyperlipidemic heart transplant recipients. PMID:9381994

  11. 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.

  12. 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.

  13. Preparation of excipient-free recombinant human tissue-type plasminogen activator by lyophilization from ammonium bicarbonate solution: an investigation of the two-stage sublimation phenomenon.

    Overcashier, D E; Brooks, D A; Costantino, H R; Hsu, C C

    1997-04-01

    Dry, excipient-free recombinant human tissue-type plasminogen activator (tPA) powder was prepared by lyophilization from ammonium bicarbonate solution. Ammonium bicarbonate sublimes into ammonia, water, and carbon dioxide upon lyophilization, without causing measurable harm to the protein. There were approximately 4 mol of residual ammonium ion per mole of lyophilized tPA. Under certain lyophilization conditions, a large pressure increase in the lyophilizer chamber occurred, presenting a pressure control problem. Microscopy and sublimation rate measurements on the frozen matrix revealed that ice sublimation occurred first, followed by the sublimation of ammonium bicarbonate. Analysis of the sectioned frozen matrix indicated that the bicarbonate salt was evenly distributed throughout the vial, suggesting that the delay of ammonium bicarbonate sublimation was not due to hindrance by ice. In the two-stage process, ice sublimation proceeded according to zero-order kinetics, whereas ammonium bicarbonate sublimation followed a grain-burning (2/ 3-order) model and was governed by a higher activation enthalpy. In most cases, the sublimation rate of ammonium bicarbonate in the presence of tPA was lower than that in the absence of the protein. Sublimation activation enthalpy for ammonium bicarbonate in the presence of tPA was 26.1 +/- 3.8 kcal/mol, which was approximately 10 kcal/mol greater than that for the tPA-free system. Consistent with a prediction from our kinetic modeling, a 6-h extension of primary drying enabled us to conduct lyophilization while maintaining pressure control. PMID:9109048

  14. 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

  15. An Anti-Urokinase Plasminogen Activator Receptor Antibody (ATN-658 Blocks Prostate Cancer Invasion, Migration, Growth, and Experimental Skeletal Metastasis In Vitro and In Vivo

    Shafaat A. Rabbani

    2010-10-01

    Full Text Available Urokinase plasminogen activator receptor (uPAR is a multidomain protein that plays important roles in the growth, invasion, and metastasis of a number of cancers. In the present study, we examined the effects of administration of a monoclonal anti-uPAR antibody (ATN-658 on prostate cancer progression in vitro and in vivo. We examined the effect of treatment of ATN-658 on human prostate cancer cell invasion, migration, proliferation, and regulation of intracellular signaling pathways. For in vivo studies, PC-3 cells (1 x 106 were inoculated into the right flank of male Balb C nu/nu mice through subcutaneous or through intratibial route (2 x 105 of male Fox Chase severe combined immunodeficient mice to monitor the effect on tumor growth and skeletal metastasis. Treatment with ATN-658 resulted in a significant dose-dependent decrease in PC-3 cell invasion and migration without affecting cell doubling time. Western blot analysis showed that ATN-658 treatment decreased the phosphorylation of serine/threonine protein kinase B (AKT, mitogen-activated protein kinase (MAPK, and focal adhesion kinase (FAK without affecting AKT, MAPK, and FAK total protein expression. In in vivo studies, ATN-658 caused a significant decrease in tumor volume and a marked reduction in skeletal lesions as determined by Faxitron x-ray and micro-computed tomography. Immunohistochemical analysis of subcutaneous and tibial tumors showed a marked decrease in the levels of expression of pAKT, pMAPK, and pFAK, consistent with the in vitro observations. Results from these studies provide compelling evidence for the continued development of ATN-658 as a potential therapeutic agent for the treatment of prostate and other cancers expressing uPAR.

  16. 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 phosphorylation of FHKRL1 that is required for PS-mediated neuronal protection after tPA/NMDA-induced injury. Conclusions PS blocks the extrinsic apoptotic cascade through a novel mechanism mediated by Tyro3-dependent FKHRL1 phosphorylation which inhibits FasL-dependent caspase-8 activation and can control tPA-induced neurotoxicity associated with pathologic activation of NMDA receptors. The present findings should encourage future studies in animal stroke models to determine whether PS can increase the therapeutic window of tPA by reducing its post-ischemic neuronal toxicity.

  17. Soluble urokinase-type plasminogen activator receptor (suPAR) as an independent factor predicting worse prognosis and extra-bone marrow involvement in multiple myeloma patients.

    Rigolin, Gian Matteo; Tieghi, Alessia; Ciccone, Maria; Bragotti, Letizia Zenone; Cavazzini, Francesco; Della Porta, Matteo; Castagnari, Barbara; Carroccia, Rosanna; Guerra, Giovanni; Cuneo, Antonio; Castoldi, Gianluigi

    2003-03-01

    The urokinase-type plasminogen activator (uPA) system, which consists of a proteinase (uPA), a receptor (uPAR or CD87) and inhibitors, is involved in proteolysis, cell migration, tissue remodelling, angiogenesis and cell adhesion. Recent findings suggest that malignant plasma cells express uPA and uPAR. The expression of these factors could represent a process by which myeloma plasma cells interact with the bone marrow (BM) environment and influence important biological events such as bone matrix degradation, plasma cell invasion and homing and, possibly, clinical evolution. We evaluated uPAR (CD87) and its soluble form (suPAR) in 49 multiple myeloma (MM) patients and correlated their expression and levels with clinico-biological characteristics of the disease. Flow cytometric analysis demonstrated that CD87 was expressed in all MM patients. High CD87 expression was associated with higher intensity of expression of CD56 (P = 0.038), CD38 (P = 0.058) and CD138 (P = 0.054) and CD45bright positivity (P = 0.014). suPAR levels correlated positively with soluble serum CD138 (P = 0.001), creatinine (P = 0.001), beta2-microglobulin (P < 0.001), disease stage (P = 0.017) and extra-BM involvement (P = 0.002). In the 46 evaluable patients, multivariate analysis showed that high levels of suPAR (P = 0.0214) and disease stage (P = 0.0064) were predictive of extra-BM involvement. In multivariate Cox analysis, 13q deletion (P = 0.0278), high soluble serum CD138 (P = 0.0201) and high suPAR (P = 0.0229) were the only parameters that independently affected survival. We conclude that CD87 is expressed on myeloma plasma cells and that suPAR, which predicts extra-BM involvement and poor prognosis, possibly represents a molecule with a relevant role in the biology of MM. PMID:12648064

  18. 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

  19. 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 invivo. Invitro, 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

  20. 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

  1. Stereotactic fibrinolysis of spontaneous intracerebral hematoma using infusion of recombinant tissue plasminogen activator Fibrinlise com infuso de rtPA e drenagem estereotxica de hematoma intracerebral espontneo profundo

    Jos Augusto Nasser

    2002-06-01

    Full Text Available PURPOSE: The authors present a prospective study on 10 patients with stereotactic infusion of tissue plasminogen activator (rtPA intraparenchimal hemorrhage. METHODS: Between 1999 and 2000, 10 patients with deep seated hematomas in the basal ganglia were selected for stereotactic infusion of rtPA and spontaneous clot drainage. RESULTS: All cases had about 80% reduction of the hematoma volume in the CT scan at the third day. The intracranial pressure was normalized by the third day too. There were no local or systemic complications with the use of this trombolitic. The results were shown by the Glasgow Outcome Scale with six patients in V, three in IV and one in III after 3 months. CONCLUSION: Early treatment and drainage with minimally invasive neurosurgery , can make these patients with deep-seated hematomas recover the consciousness and they can be rehabilitated earlier avoiding secondary complications.OBJETIVO: Estudo prospectivo em 10 pacientes com infuso de tromboltico (rtPA dentro do hematoma cerebral profundo supratentorial e drenagem estereotxica. MTODO: Entre 1999 e 2000 10 pacientes com hematomas de profundidade foram selecionados para infuso de rtPA e drenagem do cogulo espontnea. RESULTADO: Todos os casos obtiveram 80% de reduo do volume do hematoma medidos por TC no terceiro dia. A presso intracraniana estava normalizada no terceiro dia. No houve complicaes locais ou sistmicas relacionadas com o uso deste tromboltico. Os resultados comparados foram mostrados pela Escala de Prognstico de Glasgow com 6 pacientes em GrauV, 3 pacientes em Grau IV e 1 paciente em Grau III aps trs meses. CONCLUSO: Tratamento precoce e drenagem com tcnica neurocirrgica minimamente invasiva pode fazer estes pacientes terem uma recuperao da conscincia mais rpida e assim serem reabilitados mais precocemente evitando complicaes secundrias.

  2. Results of phase III clinical trial of 99mTc-labelled recombinant tissue plasminogen activator in the detection of deep venous thrombosis

    Full text: The purpose of this study was to compare the accuracy of this new technique against the accepted ''gold standard'' of contrast venography in 79 patients suspected of DVT. A kit formulation has been devised in which 99mTc is labelled to rt- PA where the plasminogen binding site has been permanently inhibited but the fibrin binding site remained active. Kit preparation takes five minutes. Scintigraphic imaging is performed at four hours post-injection (10 min/scan for thighs and calves). The results of scintigraphic imaging were then compared to those of contrast venography. Mean thrombus age was 5.4 days. 58% patients were receiving intravenous heparin. Mean time interval between contrast venography and scanning was 20 hours. For the purpose of analysis, the leg was divided into proximal and distal segments for both the scintigraphic study and the contrast venography. Of the 14 thrombosed proximal segments, 13 had positive scans; in the 53 non-thrombosed proximal segments, 49 had negative scans. Thus in proximal vein thrombosis, scanning had a sensitivity of 93% and a specificity of 92%. Of the 36 thrombosed calf vein segments, 31 had positive scans; in the 30 non-thrombosed calf segments, 28 had negative scans. Thus in calf vein thrombosis, scanning has a sensitivity of 86% and a specificity of 93%. Scintigraphic scanning with this new radiopharmaceutical permits accurate detection of thrombus in both proximal and calf veins. The technique detects both fresh and aged thrombi and is unaffected by heparin administration. Further work in different patient groups will need to be performed to define its clinical usefulness

  3. Plasma levels of soluble urokinase-type plasminogen activator receptor (suPAR and early mortality risk among patients enrolling for antiretroviral treatment in South Africa

    Bangani Nonzwakazi

    2007-05-01

    Full Text Available Abstract Background Serum concentrations of soluble urokinase-type plasminogen activator receptor (suPAR have a strong independent association with HIV-1-related mortality. The practical utility of plasma suPAR in assessing short-term all-cause mortality risk was evaluated in patients with advanced immunodeficiency enrolling in an antiretroviral treatment (ART programme in South Africa. Methods An enzyme-linked immunosorbent assay (ELISA was used to measure plasma concentrations of suPAR in patients at the time of enrolment to the ART programme. The association between plasma suPAR concentrations, baseline patient characteristics and cohort outcomes after 4 months of ART were determined. Results Patients (n = 293, 70% female had a median age of 33 years and were followed up for a median of 5 months from enrolment. The median CD4 cell count was 47 cells/?l (IQR = 2272 and 38% of patients had WHO stage 4 disease. 218 (74% patients remained alive after 4 months of ART; 39 (13% died and 36 (12% were lost to the programme for other reasons. Patients who died had significantly higher plasma suPAR concentrations compared to those who either survived (P 10 suPAR concentrations were significantly associated with lower CD4 cell counts, WHO clinical stage 4 disease and male sex. In multivariate analysis to identify factors associated with death, log10 suPAR concentration was the most strongly associated variable (P Conclusion Plasma suPAR concentration was the strongest independent predictor of short-term mortality risk among patients with advanced immunodeficiency enrolling in this ART programme. However, lack of a discriminatory threshold did not permit this marker to be used to triage patients according to short-term mortality risk.

  4. 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

  5. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

    Gately, S; Twardowski, P; Stack, M S; Cundiff, D L; Grella, D; Castellino, F J; Enghild, J; Kwaan, H C; Lee, F; Kramer, R A; Volpert, O; Bouck, N; Soff, G A

    1997-01-01

    FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant...

  6. 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 p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR. Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels

  7. The Role of Plasminogen Activator Inhibitor-1 and Angiotensin-Converting Enzyme Gene Polymorphisms in Bronchopulmonary Dysplasia

    Ince, Deniz Anuk; Atac, Fatma Belgin; Ozkiraz, Servet; Dilmen, Ugur; Gulcan, Hande; Tarcan, Aylin; Ozbek, Namik

    2010-01-01

    Background: Bronchopulmonary dysplasia (BPD) is a multifactorial disease of preterm infants that is characterized by airway injury, inflammation, and parencymal remodeling. Activation of the coagulation cascade leads to intraalveolar fibrin deposition in many inflammatory pulmonary disorders. Increased fibrin formation or decreased fibrinolysis may cause extravascular fibrin deposition. Extravascular fibrin deposits in septae and alveoli due to the altered fibrin turnover are the pathological...

  8. Plasmodium ookinetes coopt mammalian plasminogen to invade the mosquito midgut

    Ghosh, Anil K; Coppens, Isabelle; Gårdsvoll, Henrik; Ploug, Michael; Jacobs-Lorena, Marcelo

    2011-01-01

    inhibits oocyst development of both Plasmodium berghei and Plasmodium falciparum, suggesting that enolase may act as an invasion ligand. Importantly, we demonstrate that surface enolase captures plasminogen from the mammalian blood meal via its lysine motif (DKSLVK) and that this interaction is essential...... for ookinete invasion. The results support the hypothesis that enolase on the surface of Plasmodium ookinetes plays a dual role in midgut invasion: by acting as a ligand that interacts with the midgut epithelium and, further, by capturing plasminogen, whose conversion to active plasmin promotes the...

  9. 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 were reduced in the cells treated with desmopressin and docetaxel. • uPA-MMP pathway mediates the desmopressin-induced migration and invasion of PC3 cells. • Desmopressin in combination with docetaxel inhibits PC-3 cell growth in a xenograft model

  10. 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.

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

    2012-01-01

    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. PMID:22672326

  12. 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 were reduced in the cells treated with desmopressin and docetaxel. • uPA-MMP pathway mediates the desmopressin-induced migration and invasion of PC3 cells. • Desmopressin in combination with docetaxel inhibits PC-3 cell growth in a xenograft model.

  13. Synthesis of 99Tcm-HYNIC-AE105 and its application for imaging urokinase plasminogen activator receptors in human pancreatic cancer xenografts by gamma imaging

    Objective: To synthesize 99Tcm-labeled urokinase plasminogen activator receptor (uPAR)-binding peptide D-Cha-F-s-r-Y-L-W-S (AE105) (99Tcm-AE105), and evaluate its value as an uPAR imaging agent in pancreatic cancer xenograft models. Methods: 99Tcm-AE105 and 99Tcm-D-cha-F-s-r-Y-L-E-S (AE105mut) were synthesized by using Tricine as the coligand and SnCl2 as the reductant. Gamma imaging was performed on 2 groups of pancreatic cancer (bxpc-3 cells) xenograft mice models (30 mice in each group, by completely randomized design) post injection of 18.5 MBq (0.2 ml) 99Tcm-AE105 and 99Tcm-AE105mut, respectively. The tumor uptake (%ID/g) of the 2 groups was calculated and compared by two-sample t test. The uPAR expression was measured by immunohistochemistry. Correlation between uPAR expression and tumor uptake was analyzed by Pearson correlation analysis. Results: The labeling efficiency of 99Tcm-AE105 was (94.640.72)%. The radiochemical purity was (97.72 1.73)% after purification and the stability was acceptable. The tumor could be visualized on gamma imaging at 2 h post injection, and better visualized at 4 h and 6 h in the 99Tcm-AE105 group. However, the tumor could not be visualized on gamma imaging in the 99Tcm-AE105mut group. The tumor uptake of 99Tcm-AE105 was higher than that of 99Tcm-AE105mut (4 h:(3.120.27) vs (1.650.53) %ID/g, 6 h: (2.980.15) vs (1.410.38) %ID/g; t=4.496, 6.787, both P<0.01). Significant correlation between tumor uptake of 99Tcm-AE105 and uPAR expression was found in 4-6 h (r =0.791, P<0.01). There was no correlation between 99Tcm-AE105mut uptake and uPAR expression in 4-6 h (r=-0.415, P>0.05). Conclusion: 99Tcm-AE105 could be synthesized with high radiochemical purity. The specific tumor uptake of 99Tcm-AE105, which correlates with uPAR expression in pancreatic cancer, makes it a potential clinical gamma imaging tracer for cancer detection and therapy monitoring. (authors)

  14. 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. Venous blood t-PA and D-dimer contents were tested before and 1, 2, 4, and 8 weeks after the operation.Results: The high expression of t-PA could be seen in myocardium with increases in blood t-PA and D-dimer contents and thrombosis was prevented 8 weeks after operation.Conclusion: We successfully fabricated an albumin nano-t-PA gene ultrasound-targeted agent that could prevent dog thrombosis after mechanical heart valve replacement. Our study provides an experimental basis for prevention of human thrombosis-related diseases.Keywords: albumin nanoparticles, ultrasonic microbubbles, valve replacement

  15. EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression

    Lescaille Géraldine

    2012-03-01

    Full Text Available Abstract Backgrounds 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. Methods 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. Results 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. Conclusions Our results suggest that the upregulation of uPA contributes to EMMPRIN's effect in promoting oral tumor invasion.

  16. 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

  17. 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 in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. PMID:9548184

  18. 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...

  19. 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 may protect against certain cancers.

  20. 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

  1. Gestational Hypoxia Up-regulates Protein Kinase C and Inhibits Calcium-Activated Potassium Channels in Ovine Uterine Arteries

    Xiao, DaLiao; Zhu, Ronghui; Zhang, Lubo

    2014-01-01

    Objective: The present study tested the hypothesis that gestational hypoxia up-regulates protein kinase C (PKC) and inhibits calcium-activated potassium channels (KCa)-mediated relaxations of uterine arteries in pregnancy. Study design: Uterine arteries were isolated from nonpregnant (NPUA) and pregnant (PUA) (~140 day gestation) sheep maintained at either sea level or high altitude (3,820 m for 110 days, PaO2: 60 mmHg). Contractions of uterine arteries were determined. Key findings: In normo...

  2. Cloning and characterization of a plasminogen-binding surface-associated enolase from Schistosoma bovis.

    de la Torre-Escudero, Eduardo; Manzano-Romn, Ral; Prez-Snchez, Ricardo; Siles-Lucas, Mar; Oleaga, Ana

    2010-10-11

    Schistosoma bovis is a ruminant parasite able to survive prolonged periods in the vasculature of its host without either being cleared by the host defensive systems or inducing thrombotic or coagulation disturbances. This suggests that the parasite modulates both the immune and haemostatic host responses. Previous studies have shown that host plasminogen binds to the surface of S. bovis adult worms, and that a tegument extract from S. bovis fixes and activates host plasminogen, generating plasmin, which in turn could both inhibit blood clotting and dissolve clots. Enolase has been identified among the tegumental proteins that bind plasminogen. The aim of the present study is to determine the physiological role of the enolase found in the tegument of S. bovis adult worms as regards plasminogen-binding and activation, and to confirm its surface exposure on the parasite. The study included the cloning and sequencing of S. bovis enolase cDNA, collection of the corresponding recombinant protein and evaluation of its plasminogen-binding and activation activity, and an exploration of the expression and localization of native enolase in adult worms and lung schistosomulae. Here we show that S. bovis male adult worms express enolase on their tegumental surface and that this protein binds host plasminogen and increases its activation in the presence of host tissue plasminogen activator (t-PA). This suggests that the surface-associated enolase found here is a physiological receptor of plasminogen that plays a role in the activation of the host fibrinolytic system, most probably to avoid blood clot formation on the worm's surface. PMID:20609522

  3. 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

  4. 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.

  5. Use of plasma C-reactive protein, procalcitonin, neutrophils,macrophage migration inhibitory factor, soluble urokinase-type plasminogen activator receptor, and soluble triggering receptor expressed on myeloid cells-1 in combination to diagnose infections: a prospective study

    Kofoed, Kristian; Andersen, Ove; Kronborg, Gitte; Tvede, Michael; Petersen, Janne; Eugen-Olsen, Jesper; Larsen, Klaus

    2007-01-01

    Introduction: Accurate and timely diagnosis of community acquired bacterial infections in patients with systemic inflammation remains challenging both for clinician and laboratory. Combinations of markers, as opposed to single ones, may improve diagnosis and thereby survival. We therefore compared...... the diagnostic characteristics of novel and routinely used biomarkers of sepsis alone and in combination. Methods: This prospective cohort study included patients with systemic inflammatory response syndrome who were suspected of having community-acquired infections. It was conducted in a medical...... emergency department and department of infectious diseases at a university hospital. A multiplex immunoassay measuring soluble urokinase-type plasminogen activator (suPAR) and soluble triggering receptor expressed on myeloid cells (sTREM)-1 and macrophage migration inhibitory factor (MIF) was used in...

  6. Role of Protein Kinase C Isozymes in the Regulation of alpha1-Adrenergic Receptor-Mediated Contractions in Ovine Uterine Arteries1

    Zhang, Hongying; Zhang, Lubo

    2007-01-01

    Previously, we demonstrated that activation of protein kinase C (PRKC) enhanced alpha1-adrenergic receptor-induced contractions in nonpregnant ovine uterine arteries but inhibited the contractions in pregnant ovine uterine arteries. The present study tested the hypothesis that differential regulation of PRKC isozyme activities contributes to the different effects of phorbol 12, 13-dibutyrate (PDBu) on alpha1-adrenergic receptor-mediated contractions between the pregnant and nonpregnant ovine ...

  7. Angiotensin I-converting-enzyme (ACE)-inhibitory activity of tryptic peptides of ovine $\\beta$-lactoglobulin and of milk yoghurts obtained by using different starters

    Chobert, Jean-Marc; El-Zahar, Khaled; Sitohy, Mahmoud; Dalgalarrondo, Michle; Mtro, Franois; Choiset, Yvan; Haertl, Thomas

    2005-01-01

    Activit inhibitrice de l'enzyme de conversion de l'angiotensine I (ECA) de peptides trypsiques de la$\\beta$-lactoglobuline du lait de brebis et de yoghourts obtenus partir de diffrents levains. Une activit inhibitrice de l'enzyme de conversion de l'angiotensine I (ECA) a t recherche dans les peptides trypsiques de la $\\beta$-lactoglobuline du lait de brebis et dans des fractions issues de yoghourts fabriqus partir de diffrents ferments lactiques. La $\\beta$-lactoglobuline ovine (u...

  8. 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

  9. 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

  10. 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

  11. Human plasminogen binding protein tetranectin

    Kastrup, J S; Rasmussen, H; Nielsen, B B; Larsen, I K; Holtet, T L; Graversen, Jonas Heilskov; Etzerodt, M; Thøgersen, H C

    diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract to......The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...

  12. 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...

  13. 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.

  14. VEGF increases the fibrinolytic activity of endothelial cells within fibrin matrices: involvement of VEGFR-2, tissue type plasminogen activator and matrix metalloproteinases.

    Ratel, David; Mihoubi, Samira; Beaulieu, Edith; Durocher, Yves; Rivard, Georges-Etienne; Gingras, Denis; Béliveau, Richard

    2007-01-01

    Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation wa...

  15. Primary structure of corticotropin-releasing factor from ovine hypothalamus.

    Spiess, J.; Rivier, J.; Rivier, C; Vale, W

    1981-01-01

    Sequence analysis was performed of an ovine hypothalamic 41-residue polypeptide that had been postulated to be a putative corticotropin-releasing factor (CRF) because of its high intrinsic corticotropin releasing activity. The NH2-terminal 39 residues of CRF were determined by Edman degradation of 0.6-3.5 nmol of peptide in a Wittmann-Liebold modified Beckman 890C spinning cup sequencer with reverse-phase high-pressure liquid chromatography for the identification of amino acid phenylthiohydan...

  16. Human urokinase-type plasminogen activator gene-modified bone marrow-derived mesenchymal stem cells attenuate liver fibrosis in rats by down-regulating the Wnt signaling pathway

    Ma, Zhi-Gang; Lv, Xiao-Dan; Zhan, Ling-Ling; Chen, Lan; Zou, Qi-Yuan; Xiang, Ji-Qiao; Qin, Jiao-Li; Zhang, Wei-Wei; Zeng, Zhao-Jing; Jin, Hui; Jiang, Hai-Xing; Lv, Xiao-Ping

    2016-01-01

    AIM: To evaluate the therapeutic effects of bone marrow-derived mesenchymal stem cells (BMSCs) with human urokinase-type plasminogen activator (uPA) on liver fibrosis, and to investigate the mechanism of gene therapy. METHODS: BMSCs transfected with adenovirus-mediated human urokinase plasminogen activator (Ad-uPA) were transplanted into rats with CCl4-induced liver fibrosis. All rats were sacrificed after 8 wk, and their serum and liver tissue were collected for biochemical, histopathologic, and molecular analyzes. The degree of liver fibrosis was assessed by hematoxylin and eosin or Masson’s staining. Western blot and quantitative reverse transcription-polymerase chain reaction were used to determine protein and mRNA expression levels. RESULTS: Serum levels of alanine aminotransferase, aminotransferase, total bilirubin, hyaluronic acid, laminin, and procollagen type III were markedly decreased, whereas the levels of serum albumin were increased by uPA gene modified BMSCs treatment. Histopathology revealed that chronic CCl4-treatment resulted in significant fibrosis while uPA gene modified BMSCs treatment significantly reversed fibrosis. By quantitatively analysing the fibrosis area of liver tissue using Masson staining in different groups of animals, we found that model animals with CCl4-induced liver fibrosis had the largest fibrotic area (16.69% ± 1.30%), while fibrotic area was significantly decreased by BMSCs treatment (12.38% ± 2.27%) and was further reduced by uPA-BMSCs treatment (8.31% ± 1.21%). Both protein and mRNA expression of β-catenin, Wnt4 and Wnt5a was down-regulated in liver tissues following uPA gene modified BMSCs treatment when compared with the model animals. CONCLUSION: Transplantation of uPA gene modified BMSCs suppressed liver fibrosis and ameliorated liver function and may be a new approach to treating liver fibrosis. Furthermore, treatment with uPA gene modified BMSCs also resulted in a decrease in expression of molecules of the Wnt signaling pathway. PMID:26877613

  17. Identification of peptides in functional Scamorza ovine milk cheese.

    Albenzio, M; Santillo, A; Marino, R; Della Malva, A; Caroprese, M; Sevi, A

    2015-12-01

    Ovine bulk milk was used to produce Scamorza cheese with probiotics: either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus as the probiotic strains. Peptides obtained from reverse phase-HPLC water-soluble extract of Scamorza cheeses were analyzed using a quadrupole time-of-flight liquid chromatography-mass spectrometry system. Identified fragments were derived from casein hydrolysis or probiotic bacterial enzymes; some of the fragments showed encrypted peptide sequences that shared structural homology with previously described bioactive peptides in ovine milk and dairy products. Bifidobacterium longum and B. lactis showed greater proteolytic potential both in terms of level of pH 4.6 water-soluble nitrogen extract and ability to generate peptides with potential biofunctionality. Fragments deriving from microbial enzymes may be regarded as tracing fragments useful for monitoring probiotic activity in functional Scamorza cheese. PMID:26409967

  18. Cancer cells overexpress mRNA of urokinase-type plasminogen activator, its receptor and inhibitors in human non-small-cell lung cancer tissue: analysis by Northern blotting and in situ hybridization.

    Morita, S; Sato, A; Hayakawa, H; Ihara, H; Urano, T; Takada, Y; Takada, A

    1998-10-29

    The transcriptional localizations of urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitors (PAI-1 and PAI-2), which are possibly involved in cancer metastasis, have not been determined in human lung cancer. To identify their regulation in primary non-small-cell lung cancer, we assayed mRNA levels by Northern blot analysis in 25 cases and determined the localizations of mRNA by in situ hybridization in 10 cases. The amounts of uPA and PAI-2 mRNA were significantly higher in cancerous relative to normal lung tissues. However, no significant difference was observed in uPAR and PAI-1 mRNA levels. All transcripts were present in cancer cells and were predominantly located in tumor edges in several cases. In addition, PAI-1 transcripts were more abundant in poorly and moderately differentiated carcinomas relative to well-differentiated carcinomas and PAI-2 transcripts were more abundant in squamous cell carcinomas than in adenocarcinomas. Thus, PAIs may be involved in modulation of malignant potency. Our results indicate that human non-small-cell lung cancer cells can autonomously express the mRNAs of uPA, uPAR and PAIs, which are possibly involved in metastasis. PMID:9766559

  19. 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

  20. A serial fluoroscopy-guided thrombolytic therapy of a mechanical tricuspid prosthetic valve thrombosis with low-dose and ultra-slow infusion of tissue-type plasminogen activator.

    Kal?k, Macit; Grsoy, Ozan Mustafa; Astarc?o?lu, Mehmet Ali; zkan, Mehmet

    2014-07-01

    Prosthetic valve thrombosis is a life-threatening complication that is seen most commonly in patients with left-sided prosthetic valves. However, mechanical tricuspid valves carry the highest risk of thrombosis of any cardiac valve. Thrombolysis has been performed successfully in right-sided prosthetic valve thrombosis and has been recommended as the first-line treatment in these patients. Although two-dimensional and real-time three-dimensional transesophageal echocardiography are the gold standard imaging modalities for the diagnosis of prosthetic valve thrombosis, right-sided prosthetic valves may not be evaluated precisely. This is a serious problem during the follow-up of patients who receive thrombolytic therapy for tricuspid valve thrombosis. Fluoroscopy is an alternative noninvasive imaging method that provides valuable information about leaflet motion and may be used for such cases with restricted leaflets. Herein, we report a case of tricuspid valve thrombosis who was managed with low-dose (25 mg) and ultra-slow (25 hours) infusion of tissue-type plasminogen activator under the guidance of serial fluoroscopy. PMID:25080958

  1. 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.

  2. Thrombolytic and pharmacokinetic properties of human tissue-type plasminogen activator variants, obtained by deletion and/or duplication of structural/functional domains, in a hamster pulmonary embolism model.

    Collen, D; Lijnen, H R; Vanlinthout, I; Kieckens, L; Nelles, L; Stassen, J M

    1991-02-12

    A pulmonary embolism model in hamsters was used for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of variants of tissue-type plasminogen activator (t-PA). A 25 microliters 125I-fibrin labeled human plasma clot was made in vitro and injected into the jugular vein of heparinized hamsters. The extent of thrombolysis within 90 min was determined as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs. Recombinant t-PA (home-made rt-PA or Activase) infused intravenously over 60 min caused dose-dependent progressive thrombolysis. The results of thrombolytic potency (clot lysis in percent versus dose administered in mg/kg) and of specific thrombolytic activity (clot lysis in percent versus steady state plasma level in microgram/ml) were fitted with an exponentially transformed sigmoidal function y = 100 c/(1 + e-a(ax-eh] and the maximal percent lysis (c), the dose or plasma level at which maximal rate of lysis is achieved (b) and the maximal rate of lysis (z = 1/4 ac.eb) were determined. With rt-PA, these parameters were c = 72 +/- 6% (mean +/- SEM), b = 0.19 +/- 0.08 mg/kg, z = 68 +/- 25% lysis per mg/kg, with corresponding values of 87 +/- 5%, 0.07 +/- 0.03 mg/kg and 150 +/- 38% lysis per mg/kg for Activase (p = NS). Deletion of the finger and growth factor domains in rt-PA (rt-PA-delta FE) was not associated with marked alteration of the thrombolytic potency (c = 90 +/- 30%, b = 0.34 +/- 0.35 mg/kg, and z = 54 +/- 14% per mg/kg), but was associated with a significant reduction of the specific thrombolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1905070

  3. A complete DNA sequence map of the ovine Major Histocompatibility Complex

    Gao Jianfeng

    2010-08-01

    Full Text Available Abstract Background The ovine Major Histocompatibility Complex (MHC harbors clusters of genes involved in overall resistance/susceptibility of an animal to infectious pathogens. However, only a limited number of ovine MHC genes have been identified and no adequate sequence information is available, as compared to those of swine and bovine. We previously constructed a BAC clone-based physical map that covers entire class I, class II and class III region of ovine MHC. Here we describe the assembling of a complete DNA sequence map for the ovine MHC by shotgun sequencing of 26 overlapping BAC clones. Results DNA shotgun sequencing generated approximately 8-fold genome equivalent data that were successfully assembled into a finished sequence map of the ovine MHC. The sequence map spans approximately 2,434,000 nucleotides in length, covering almost all of the MHC loci currently known in the sheep and cattle. Gene annotation resulted in the identification of 177 protein-coding genes/ORFs, among which 145 were not previously reported in the sheep, and 10 were ovine species specific, absent in cattle or other mammals. A comparative sequence analyses among human, sheep and cattle revealed a high conservation in the MHC structure and loci order except for the class II, which were divided into IIa and IIb subregions in the sheep and cattle, separated by a large piece of non-MHC autosome of approximately 18.5 Mb. In addition, a total of 18 non-protein-coding microRNAs were predicted in the ovine MHC region for the first time. Conclusion An ovine MHC DNA sequence map was successfully assembled by shotgun sequencing of 26 overlapping BAC clone. This makes the sheep the second ruminant species for which the complete MHC sequence information is available for evolution and functional studies, following that of the bovine. The results of the comparative analysis support a hypothesis that an inversion of the ancestral chromosome containing the MHC has shaped the MHC structures of ruminants, as we currently observed in the sheep and cattle. Identification of relative large numbers of microRNAs in the ovine MHC region helps to provide evidence that microRNAs are actively involved in the regulation of MHC gene expression and function.

  4. 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

  5. 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.

  6. 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

  7. [The role of the sympathetic cholinergic pathway in the neurogenic control of the release of tissue-type plasminogen activator into the blood].

    Bashkov, G B; Sergeev, I Iu; Medvedeva, N A; Makarov, V A

    1992-05-01

    In anesthetized cats, electric stimulation of the sympathetic chain induced vasoconstriction and release of t-PA from the vascular wall into the blood. Efferent stimulation of the sympathetic chain against the background of alpha-adrenoceptor blockade increased the blood flow and the t-PA activity. Atropin suppressed both the vascular relaxation and the t-PA release into the blood. A neurogenous mechanism controlling t-PA release from the vascular wall caused by activation of sympathetic cholinergic pathway and associated with excitation of vascular M-cholinoreceptors by acetylcholine, has been suggested. PMID:1334882

  8. 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 M; Gårdsvoll, Henrik; Bekes, Erin M; Thuesen, Cathrine K; Chen, Zhou; Ploug, Michael; Quigley, James P; Andreasen, Peter

    2012-01-01

    , 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...... catalyzed by two-chain uPA. The aptamer also inhibited the binding of pro-uPA to uPAR and the binding of vitronectin to the preformed pro-uPA/uPAR complex, both in cell-free systems and on cell surfaces. Furthermore, upanap-126 inhibited human tumor cell invasion in vitro in the Matrigel assay and in vivo...

  9. 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

  10. 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.

  11. Seeking for binding determinants of the prion protein to human plasminogen

    Menziani, M. C.; de Benedetti, P. G.; Langella, E.; Barone, V.

    Plasminogen (Pg), a pro-protease implicated in neuronal excitotoxicity, has recently been identified as binding to prion protein (PrP) from several species. Although the precise effect of the binding of PrP to plasminogen in the course of prion-caused diseases has not yet been demonstrated, the implications of this important finding for diagnostic applications are straightforward. In this paper we have investigated the possible modes of interaction of PrP with plasminogen, by means of molecular modelling and computational simulation techniques. To this goal, we first exploited the information available for the mK2Pg/VEK-30 complex in order to identify the PrP residues which satisfy the specific electronic and geometric requirements needed to interact with the kringle lysine binding site, we compared the relevant mK2Pg/VEK-30 and mK2Pg/PrP interactions obtained from the simulated protein-protein complexes and we assessed the docking hypothesis utilized for the mK2Pg/PrP complex by simulating the interaction of PrP with the multi-kringle angiostatin, a more realistic model of the physiological target. The results obtained will be instrumental for planning experiments tailored to clarify the role of the plasminogen activator system in prion-related diseases and, eventually, for mimicking dominant binding determinants through structure-based drug design.

  12. 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...

  13. 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...

  14. 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...

  15. 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...... 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...

  16. Sensory and microbiological evaluation of traditional ovine ricotta cheese in modified atmosphere packaging

    Isabella Mancuso

    2014-06-01

    Full Text Available Ovine ricotta cheese is a traditional Sicilian dairy product characterised by high humidity and a short shelf life (2-4 days when refrigerated. The increasing demand for fresh food has prompted manufacturers to develop special packaging techniques, such as modified atmosphere packaging (MAP, that can extend the shelf life and maintain the organoleptic characteristics of ovine ricotta cheese. The aim of the present study was to evaluate the shelf life of fresh MAP-packed ovine ricotta cheese by monitoring the microbiological, chemical, physical and organoleptic characteristics of the product. Samples of a single batch were packed in MAP or vacuum packed and stored at 4C for 24 and 7 days, respectively. Water activity, pH, physicochemical parameters and microbiological characteristics were examined. A sensory panel rated the products main organoleptic characteristics (colour, odour, flavour and texture. Results showed that MAP controlled the development of any unwanted microflora, but did not affect the development of intrinsic lactic floras or chemical parameters. Sensory analysis revealed that overall the MAP-packed ricotta remained acceptable for up to 15 days of storage. The vacuum-packed ricotta cheese, however, showed a progressive deterioration in organoleptic characteristics from day 5 onward and therefore had a shorter shelf life. In conclusion, the ability of MAP to extend the shelf life of a traditional regional product (such as fresh ovine ricotta cheese guarantees consumers a quality product and provides opportunities for manufacturers to expand their markets beyond national boundaries.

  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. 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... evidence found of vesicular disease. (f) The meat consists only of bovine parts and ovine parts that are... of Ovine Meat From Uruguay AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION:...

  19. 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...

  20. Genetic architecture of gene expression in ovine skeletal muscle

    Kogelman, Lisette Johanna Antonia; Byrne, Keren; Vuocolo, Tony; Watson-Haigh, Nathan S.; Kadarmideen, Haja; Kijas, James W.; Oddy, Hutton V.; Gardiner, Graham E.; Gondro, Cedric; Tellam, Ross L.

    2011-01-01

    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 ovine longissimus lumborum muscle. The balance between muscle protein synthesis, at the levels of both transcription and translation control, and protein...... 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...

  1. The anti-invasive activity of synthetic alkaloid ethoxyfagaronine on L1210 leukemia cells is mediated by down-regulation of plasminogen activators and MT1-MMP expression and activity.

    Devy, Jérôme; Ouchani, Farid; Oudot, Christelle; Helesbeux, Jean Jacques; Vanquelef, Enguerran; Salesse, Stéphanie; Rabenoelina, Fanja; Al-Khara, Siana; Letinois, Isabelle; Duval, Olivier; Martiny, Laurent; Charpentier, Emmanuelle

    2011-10-01

    Quaternary benzo[c]phenanthridines such as fagaronine are natural substances which have been reported to exhibit anticancer and anti-leukemic properties. However, the therapeutic use of these molecules is limited due to the high dose required to exhibit anti-tumor activity and subsequent toxicity. In this study, we describe the therapeutic potential of a new derivative of fagaronine, Ethoxyfagaronine (N-methyl-12-ethoxy-2hydroxy-3, 8, 9-trimethoxybenzo[c]-phenanthridiniumchlorhydrate) as an anti-leukemic agent. Cytotoxic activity and cell growth inhibition of Ethoxyfagaronine (Etxfag) was tested on murine L1210 leukemia cells using trypan blue assay and MTT assay. At the concentration of 10(-7) M, Etxfag induced less than 10% of cell death. Etxfag (10(-7) M) was tested on L1210 cell invasiveness using matrigel™ precoated transwell chambers and efficiently reduces the invasive potential of L1210 cells by more than 50% as compared with untreated cells. Western blot and immunofluorescence experiments showed that Etxfag decreased both MT1-MMP expression and activation at the cell surface, decreased plasmin activity by down-regulating u-PAR and uPA expression at the cell surface and increasing PAI-1 secretion in conditioned media. The set of our findings underscore the therapeutic potential of ethoxyfagaronine as a new potential anticancer agent able to prevent leukemic cell dissemination. PMID:20349265

  2. 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...

  3. A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species.

    Pereyra-Bonnet, F; Fernndez-Martn, R; Olivera, R; Jarazo, J; Vichera, G; Gibbons, A; Salamone, D

    2008-01-01

    Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos. PMID:18842176

  4. CipA of Acinetobacter baumannii Is a Novel Plasminogen Binding and Complement Inhibitory Protein.

    Koenigs, Arno; Stahl, Julia; Averhoff, Beate; Göttig, Stephan; Wichelhaus, Thomas A; Wallich, Reinhard; Zipfel, Peter F; Kraiczy, Peter

    2016-05-01

    Acinetobacter baumanniiis an emerging opportunistic pathogen, responsible for up to 10% of gram-negative, nosocomial infections. The global increase of multidrug-resistant and pan-resistantAcinetobacterisolates presents clinicians with formidable challenges. To establish a persistent infection,A. baumanniimust overcome the detrimental effects of complement as the first line of defense against invading microorganisms. However, the immune evasion principles underlying serum resistance inA. baumanniiremain elusive. Here, we identified a novel plasminogen-binding protein, termed CipA. Bound plasminogen, upon conversion to active plasmin, degraded fibrinogen and complement C3b and contributed to serum resistance. Furthermore, CipA directly inhibited the alternative pathway of complement in vitro, irrespective of its ability to bind plasminogen. A CipA-deficient mutant was efficiently killed by human serum and showed a defect in the penetration of endothelial monolayers, demonstrating that CipA is a novel multifunctional protein that contributes to the pathogenesis ofA. baumannii. PMID:26681776

  5. Developmental Expression and Glucocorticoid Control of the Leptin Receptor in Fetal Ovine Lung.

    De Blasio, Miles J; Boije, Maria; Vaughan, Owen R; Bernstein, Brett S; Davies, Katie L; Plein, Alice; Kempster, Sarah L; Smith, Gordon C S; Charnock-Jones, D Stephen; Blache, Dominique; Wooding, F B Peter; Giussani, Dino A; Fowden, Abigail L; Forhead, Alison J

    2015-01-01

    The effects of endogenous and synthetic glucocorticoids on fetal lung maturation are well-established, although the role of leptin in lung development before birth is unclear. This study examined mRNA and protein levels of the signalling long-form leptin receptor (Ob-Rb) in fetal ovine lungs towards term, and after experimental manipulation of glucocorticoid levels in utero by fetal cortisol infusion or maternal dexamethasone treatment. In fetal ovine lungs, Ob-Rb protein was localised to bronchiolar epithelium, bronchial cartilage, vascular endothelium, alveolar macrophages and type II pneumocytes. Pulmonary Ob-Rb mRNA abundance increased between 100 (0.69 fractional gestational age) and 144 days (0.99) of gestation, and by 2-4-fold in response to fetal cortisol infusion and maternal dexamethasone treatment. In contrast, pulmonary Ob-Rb protein levels decreased near term and were halved by glucocorticoid treatment, without any significant change in phosphorylated signal transducer and activator of transcription-3 (pSTAT3) at Ser727, total STAT3 or the pulmonary pSTAT3:STAT3 ratio. Leptin mRNA was undetectable in fetal ovine lungs at the gestational ages studied. These findings demonstrate differential control of pulmonary Ob-Rb transcript abundance and protein translation, and/or post-translational processing, by glucocorticoids in utero. Localisation of Ob-Rb in the fetal ovine lungs, including alveolar type II pneumocytes, suggests a role for leptin signalling in the control of lung growth and maturation before birth. PMID:26287800

  6. Enrichment and Short Term Culture of the Ovine Gonocyte

    Rongfeng Li

    2011-01-01

    Full Text Available The aim of this study was to investigate the effects of two types of ovine testis cells population as feeder cell on in vitro culture of the enriched ovine gonocytes. The feeder cell populations were prepared from 5-6 months old ovine testis. The 1-2 months old neonatal rams were used to isolate germ cells through a two step enzymatic digestion followed by differential plating for SSCs enrichment. Isolated and enriched cells were characterized by using PLZF and VASA antibody. During the 1st week of culture, gonocyte formed pairs and chains of type A spermatogonia. After 1 week, colonies started to increase in size. About 2 weeks later, more colonies in type II feeder group kept undifferentiated and looks more effective regarding colony formation of spermatogonia compared with type I feeder group in the ovine spermatogonial stem cell culture system.

  7. 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.

  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. The ovine mammary gland as an experimental model to determine the virulence of animal ureaplasmas.

    Ball, H. J.; Mackie, D. P.

    1985-01-01

    As an estimate of their virulence, the ability of ovine, bovine, canine, feline and simian ureaplasma strains to cause mastitis in the ovine mammary gland was investigated. Five ovine ureaplasmas produced a clinical mastitis. Broth cultures of seven bovine ureaplasmas were unable to infect the ovine gland, but two of these strains plus one other were able to do so following passage through the bovine udder. One of two canine strains and a feline strain both caused mastitis, but the simian str...

  10. 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 ovine longissimus lumborum muscle. The balance between muscle protein synthesis, at the levels of both transcription and translation control, and protein catabolism mediated by regulated proteolysis is likely to be the primary determinant of the genetic merit for the muscling trait in this sheep population. There is also evidence that high genetic merit for muscling is associated with a fibre type shift toward fast glycolytic fibres. This study provides insight into mechanisms, presumably subject to strong artificial selection, that underpin enhanced muscling in sheep populations.

  11. Schistosoma bovis: Plasminogen binding in adults and the identification of plasminogen-binding proteins from the worm tegument

    Ramajo Hernndez, Alicia; Prez Snchez, Ricardo; Ramajo Martn, Vicente; Oleaga, Ana

    2007-01-01

    Schistosoma bovis is a ruminant haematic parasite that lives for years in the mesenteric vessels of the host. The aim of this work was to investigate the ability of adult S. bovis worms to interact with plasminogen, a central component in the host fibrinolytic system. Confocal microscopy analysis revealed that plasminogen bound to the tegument surface of the malebut not femaleS. bovis worms and that this binding was strongly dependent on lysine residues. It was also observed that a protein ...

  12. 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.

  13. Characterization and Isolation of Ovine Spermatogonial Stem Cells

    Muren Herrid

    2012-01-01

    Full Text Available Identification and isolation of Spermatogonial Stem Cells (SSCs are a prerequisite for culture, genetic manipulation and transplantation research. In this study, the appropriate age of the donor ram for SSCs isolation was investigated. Spermatogonial stem cell specific marker protein Promyelocytic Leukaemia Zinc-Finger (PLZF and male germ cell marker VASA double staining were checked on the paraffin sections of ovine testis. Type A spermatogonial marker PGP9.5 and sertoli cell marker Vimentin double staining were also used to confirm testes development. Results indicated that testes from neonatal rams had only one type of undifferentiated germ cell gonocyte and should be suitable for ovine SSCs isolation. VASA and Vimentin double staining of the isolated testis cells confirm the efficiency of ovine SSCs isolation.

  14. Global distribution and diversity of ovine-associated Staphylococcus aureus.

    Smith, Edward M; Needs, Polly F; Manley, Grace; Green, Laura E

    2014-03-01

    Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe (n=153), Africa (n=28), South America (n=14) and Australia (n=1); 172 bovine, 68 caprine and 433 human S. aureus profiles. Overall there were 59 STs and 87 spa types in the 293 ovine isolates; in the 97 new ovine isolates there were 22 STs and 37 spa types, including three novel MLST alleles, four novel STs and eight novel spa types. Three main CCs (CC133, CC522 and CC700) were detected in sheep and these contained 61% of all isolates. Four spa types (t002, t1534, t2678 and t3576) contained 31% of all isolates and were associated with CC5, CC522, CC133 and CC522 respectively. spa types were consistent with MLST CCs, only one spa type (t1403) was present in multiple CCs. The three main ovine CCs have different but overlapping patterns of geographical dissemination that appear to match the location and timing of sheep domestication and selection for meat and wool production. CC133, CC522 and CC700 remained ovine-associated following the inclusion of additional host species. Ovine isolates clustered separately from human and bovine isolates and those from sheep cheeses, but closely with caprine isolates. As with cattle isolates, patterns of clonal diversification of sheep isolates differ from humans, indicative of their relatively recent host-jump. PMID:24035790

  15. 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

  16. 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.

  17. Application of ovine luteinizing hormone (LH) radioimmunoassay in the quantitation of LH in different mammalian species

    A sensitive double antibody radioimmunoassay has been developed for measuring luteinizing hormone (LH) in various African mammalian species, using rabbit anti-ovine LH serum (GDN 15) and radioiodinated rat LH or ovine LH. Serum and pituitary homogenates from some African mammals (hyrax, reedbuck, sable, impala, tsessebe, thar, spring-hare, ground squirrel and cheetah, as well as the domestic sheep, cow and horse and laboratory rat and hamster) produced displacement curves parallel to that of the ovine LH standards. The specificity of the assay was examined in detail for one species, the rock hyrax. Radioimmunoassay and bioassay estimates of LH in hyrax pituitaries containing widely differing quantities of pituitary hormones were similar. In sexually active male hyrax mean plasma LH was 12.1 ng/ml and pituitary LH 194 μg/gland, but in sexually quiescent hyrax mean plasma LH was 2.4 ng/ml and mean pituitary LH 76 μg/gland. Intravenous injection of 10 μg of luteinizing hormone releasing hormone increased mean LH levels in hyrax from 0.9 ng/ml to 23.2 ng/ml by 30 min. Conversely, im injection of 250 μg testosterone induced a fall in LH levels in male hyrax from 1.7 ng/ml to 0.7 ng/ml 6 h after administration. Although the specificity of the assay for quantitating plasma LH in other species was not categorically established, there was a good correlation between plasma LH concentration and reproductive state in the bontebok, impala, spring-hare, thar, cheetah, domestic horse and laboratory rat, suggesting the potential use of the antiserum in quantitating LH in a variety of mammalian species

  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; 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

  19. Experimental infection of cattle with ovine Dichelobacter nodosus isolates

    Knappe-Poindecker, Maren; Jørgensen, Hannah Joan; Jensen, Tim Kåre; Tesfamichael, Bereket; Ulvund, Martha Jakobsen; Hektoen, Lisbeth; Fjeldaas, Terje

    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 ...

  20. 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

  1. 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...

  2. Detection of deep vein thrombosis with I-123 plasminogen

    An experience with I-123 plasminogen used to investigate patients with clinically suspected deep vein thrombosis is reported. The test is efficient, not invasive and produces no discomfort on the contrary to venography. The test is not sensitive to heparine therapy since 5 thrombosis were detected in patients under heparine. Certain false positive results such as those due to Erysipelas can be foreseen by careful clinical investigation. False negative cases were due to very old thrombi or superficial thrombosis without clinical consequences

  3. 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...

  4. 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

  5. Effect of melatonin on the in vitro secretion of progesterone and estradiol 17 beta by ovine granulosa cells.

    Baratta, M; Tamanini, C

    1992-10-01

    This study was undertaken to determine the effect of melatonin on steroid hormone production by ovine granulosa and luteal cells in vitro. Granulosa and luteal cells from ovine ovaries were cultured for nine days either in D-MEM only or in the presence of melatonin (0.86, 8.6, 86 nmol/l), ovine luteinizing hormone (oLH, 2 micrograms/l) or a combination of both these hormones. Progesterone (P4) and estradiol 17 beta (E2) were determined by validated RIAs. Melatonin stimulation began at either day 1 or day 5 of culture. Melatonin (0.86 nmol/l) significantly increased (p granulosa cells both when administered alone and when administered in combination with oLH; the more marked response was observed in the latter case. When the stimulation began at day 5, at a more advanced degree of differentiation of the cells, higher levels of P4 were observed. Higher concentrations of melatonin did not further increase progesterone production. Melatonin alone did not have a significant effect on the production of estradiol 17 beta; neither did melatonin stimulate progesterone production in either long-term cultured luteal cells or in short-term (1-2 h) cultured luteal and granulosa cells. The results of this study document a direct effect of melatonin in stimulating granulosa cells to produce progesterone, a synergistic activity between melatonin and luteinizing hormone and a different ability of granulosa cells to secrete P4 depending on the degree of differentiation. PMID:1449047

  6. Enhanced degradation of amyloid AA proteins by enzyme activation: a possible model for a therapeutic approach

    The capacity of plasminogen from human serum to degrade amyloid AA protein was tested using radiolabelled protein AA coupled to cyanogen bromide activated Sepharose 6 MB as substrate. Protein AA degrading activity was determined in fractions of normal human serum separated by Sephadex G 150. Each fraction was tested in the presence and absence of the plasminogen activator streptokinase. The AA degrading activity was markedly increased in fractions in which plasminogen activation had occurred. These fractions were also the same as those showing the presence of plasminogen as demonstrated by reaction with a specific anti-plasminogen antiserum. Moreover, the increase in AA degrading activity could be inhibited with antibodies to plasminogen. AA degrading activity could also be enhanced in whole human plasma by streptokinase activation

  7. Molecular cloning and characterisation of ovine dual oxidase 2.

    Lees, M S; H Nagaraj, Shivashankar; Piedrafita, D M; Kotze, A C; Ingham, A B

    2012-05-25

    The dual oxidases (DUOX1 and DUOX2) are NADPH-dependent hydrogen peroxide-producing enzymes that are reported to function in a physiological capacity and as a component of the mucosal immune response. We have previously reported increased expression of the DUOX2 gene in the gut mucosa of sheep in response to gastrointestinal nematode (GIN) challenge. In this paper, we report the cloning of the full-length ovine DUOX2 transcript, using a PCR based strategy. The ovine DUOX2 transcript includes an ORF of 4644 bases, and encodes a protein with 97% identity to the bovine sequence. We also cloned a fragment of DUOX1 (encompassing nucleotides 2692-2829), and the proximal promoter sequence of DUOX2. Through analysis of sequence data we have confirmed that DUOX1 and DUOX2 are co-located in a head to tail arrangement conserved across many species. Alignment of the sequences to the ovine genome predicts a location of this gene cluster on ovine chromosome 7. We quantified the expression of ovine DUOX1 and DUOX2 transcripts in 24 different sheep tissues, and discovered tissue specific expression signatures. DUOX2 was found to be most highly expressed in tissues of the gastrointestinal tract, while expression of DUOX1 predominated in the bladder. Rapid amplification of cDNA ends (RACE) analysis identified the existence of multiple 5' UTR variants in DUOX2, ranging in size from 32 to 242 nucleotides, with 3 distinct transcribed regions. Real time PCR quantification of the DUOX2 UTR variants revealed that these were differentially expressed between tissues, and at various stages of the response to GIN parasite infection. The collective evidence suggested a complex regulation of DUOX2, prompting a bioinformatic analysis of the proximal promoter regions of ovine DUOX2 to identify potential transcription factor binding sites (TFBS) that may explain the differences in the observed expression of the transcript variants of DUOX2. Possible transcription factor families that may regulate this process were identified as Kruppel-like factors (KLF), ETS-factors, erythroid growth receptor factors (EGRF) and myogenic differentiation factors (MYOD). PMID:22465529

  8. 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

  9. Experimental infection of cattle with ovine Dichelobacter nodosus isolates

    Knappe-Poindecker, Maren; Jørgensen, Hannah Joan; Jensen, Tim Kåre; Tesfamichael, Bereket; Ulvund, Martha Jakobsen; Hektoen, Lisbeth; Fjeldaas, Terje

    2015-01-01

    Background 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 transferred to the interdigital skin of cattle under experimental conditions. Further, we wanted to observe the impact of such infection on bovine foot health, and test the effect of topica...

  10. Treatment of ulcerated necrobiosis lipoidica with ovine forestomach matrix.

    Simcock, J W; Than, M; Ward, B R; May, B C H

    2013-07-01

    Necrobiosis lipoidica (NL) results from degradation of the collagen extracellular matrix; these recurring ulcerated lesions are an especially challenging condition to treat. Ovine forestomach matrix (OFM) is a decellularised extracellular matrix and was used to successfully close a pretibial ulcer resulting from NL. Complete closure of the wound was achieved in 22 weeks, after four applications of OFM. This suggests OFM may be considered for the treatment of these challenging wounds. PMID:24159661

  11. Ovine fetal immune response to Cache Valley virus infection.

    Rodrigues Hoffmann, Aline; Dorniak, Piotr; Filant, Justyna; Dunlap, Kathrin A; Bazer, Fuller W; de la Concha-Bermejillo, Andres; Welsh, Christabel Jane; Varner, Patricia; Edwards, John Francis

    2013-05-01

    Cache Valley virus (CVV)-induced malformations have been previously reproduced in ovine fetuses. To evaluate the development of the antiviral response by the early, infected fetus, before the development of immunocompetency, ovine fetuses at 35 days of gestation were inoculated in utero with CVV and euthanized at 7, 10, 14, 21, and 28 days postinfection. The antiviral immune response in immature fetuses infected with CVV was evaluated. Gene expression associated with an innate, immune response was quantified by real-time quantitative PCR. The upregulated genes in infected fetuses included ISG15, Mx1, Mx2, IL-1, IL-6, TNF-?, TLR-7, and TLR-8. The amount of Mx1 protein, an interferon-stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. ISG15 protein expression was significantly increased in target tissues of infected animals. B lymphocytes and immunoglobulin-positive cells were detected in lymphoid tissues and in the meninges of infected animals. These results demonstrated that the infected ovine fetus is able to initiate an innate and adaptive immune response much earlier than previously known, which presumably contributes to viral clearance in infected animals. PMID:23468505

  12. 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 the basis for determining the microbiological quality standards for goat and ovine milk.

  13. Electrically evoked potentials in an ovine model for the evaluation of visual prosthesis efficacy.

    Barriga-Rivera, Alejandro; Eiber, Calvin D; Dodds, Christopher W D; Fung, Adrian T; Tatarinoff, Veronica; Lovell, Nigel H; Suaning, Gregg J

    2015-08-01

    Visual prostheses are becoming a reality as a therapy to restore functional vision to the blind. New stimulation strategies and novel electrode designs are contributing to accelerate the development of such devices triggering the interest of scientists, clinicians and the blind community worldwide. In this scenario, there is a need for large animal models that are suitable for preclinical testing of retinal neuroprostheses. This study presents an electrophysiology assessment of an ovine model for single and simultaneous electrode stimulation from the suprachoroidal space, using symmetric biphasic current pulses with a monopolar return configuration. Visually and electrically evoked potentials were recorded using supradural surface electrodes, showing charge thresholds comparable to those in humans. This model represents an alternative to feline or canine models with analogous activation levels and an eye anatomy similar to that of humans. PMID:26737012

  14. Biomechanical performance of an ovine model of intradural spinal cord stimulation.

    Safayi, S; Jeffery, N D; Fredericks, D C; Viljoen, S; Dalm, B D; Reddy, C G; Wilson, S; Gillies, G T; Howard, M A

    2014-07-01

    The authors are developing a novel type of spinal cord stimulator, designed to be placed directly on the pial surface of the spinal cord, for more selective activation of target tissues within the dorsal columns. For pre-clinical testing of the device components, an ovine model has been implemented which utilizes the agility and flexibility of a sheep's cervical and upper thoracic regions, thus providing an optimal environment of accelerated stress-cycling on small gauge lead wires implanted along the dorsal spinal columns. The results are presented of representative biomechanical measurements of the angles of rotation and the angular velocities and accelerations associated with the relevant head, neck and upper back motions, and these findings are interpreted in terms of their impact on assessing the robustness of the stimulator implant systems. PMID:24841845

  15. Evidence for gene transcription of adenohypophyseal hormones in the ovine pars tuberalis.

    Böckers, T M; Bockmann, J; Fauteck, J D; Wittkowski, W; Sabel, B A; Kreutz, M R

    1996-01-01

    Specific cells of the hypophyseal pars tuberalis (PT) have been associated with the transmission of photoperiodic stimuli to the endocrine system. However, their principal secretory products have not been identified yet. Therefore we studied the expression of several adenohypophyseal hormones and their subunits (TSH, FSH, LH, common alpha-chain, GH, ACTH, PRL, alpha- and gamma-MSH, beta-lipotropin) by immunocytochemistry, Northern blot analysis and in situ hybridization in the sheep pituitary. Only the common alpha-chain of glycoprotein hormones could be detected in ovine PT-specific cells by immunocytochemistry while antibodies directed against the beta-chains of LH, FSH, TSH and beta-lipotropin labeled single cells in the PT but failed to detect these antigens in PT-specific cells. In situ hybridization and Northern blot analysis with antisense oligonucleotides against the common alpha-chain, beta-LH, beta-FSH, beta-TSH, PRL and POMC revealed the expression of these subunits in the ovine PT. The mRNA of the common alpha-chain, beta-TSH and, to a far lower extent, PRL and POMC were found throughout the entire pars tuberalis while beta-FSH and beta-LH could only be detected in cells of the caudal PT. Hence, GH-mRNA and GH immunoreactivity were exclusively found in the pituitary pars distalis. Compared to these results--obtained under the short photoperiod (winter)--we found clear ultrastructural signs of altered secretory activity in PT-specific cells of animals exposed to the long photoperiod (summer); the common alpha-chain immunoreactivity was nearly absent in PT-specific cells of summer animals. However, no seasonal influence on gene transcription or translation for other adenohypophyseal hormone was observed. These findings suggest that ovine PT-specific cells, which are only immunopositive for the common alpha-chain, are capable to express different mRNAs of adenohypophyseal hormones. Although it remains elusive how gene transcription and translation are related in this cell type, the presence of an mRNA pool for hormone subunits leads to the speculation that--at least in the sheep--hormone synthesis is mainly regulated at the translational level and that secretion of hormones may be primarily constitutive. PMID:8839351

  16. Bicyclic peptide inhibitor of urokinase-type plasminogen activator

    Roodbeen, Renée; Jensen, Berit Paaske; Jiang, Longguang; Jensen, Jan Kristian; Christensen, Anni; Nielsen, Jakob T.; Huang, Mingdong; Mulder, Frans; Nielsen, Niels Chr.; Andreasen, Peter; Jensen, Knud Jørgen

    2013-01-01

    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...

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

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

  18. 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...

  19. 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; Overgaard, Jens

    2007-01-01

    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...

  20. 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

  1. Lower apoptosis rate in ovine preantral follicles from ovaries stored in supplemented preservation media.

    Gonalves, R J S; Cavalcante, A Y P; Gouveia, B B; Lins, T L B; Barberino, R S; Menezes, V G; Barros, V R P; Macedo, T J S; Figueiredo, J R; Matos, M H T

    2015-12-01

    The aim of this study was to investigate the effect of ovarian tissue transportation conditions (medium and period of time) on the morphology, apoptosis and development of ovine preantral follicles cultured in vitro. Each ovarian pair was cut into nine slices, with one fragment being fixed immediately (fresh control). The remaining fragments were placed individually in cryotubes containing conservation medium (minimal essential medium (MEM) without supplementation or MEM+ - with supplementation) and stored at 35C for 6 or 12 h without (non-cultured) or with subsequent culture for 5 days. Then, the fragments were processed for histological and terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labelling (TUNEL) examination. Preservation of ovarian slices in MEM or MEM+ (non-cultured) resulted in similar percentages of normal follicles when compared with the fresh control. Nevertheless, compared with the fresh control, a decrease in the percentage of normal follicles was observed in tissues cultured for 5 days. Only for tissues preserved in supplemented medium (MEM+) for 6 h, the percentage of TUNEL positive cells was similar between non-cultured tissues and tissues cultured for 5 days. Follicular activation and growth (follicular and oocyte diameter) were higher in cultured tissues than in fresh control or non-cultured tissues, except those from fragments preserved for 6 h in MEM and then cultured for 5 days in which no growth was observed. In conclusion, ovine ovarian tissue was successfully preserved in supplemented medium (MEM+) at a temperature close to physiological values (35C) for up to 6 h without affecting apoptosis in the ovarian follicles and their ability to develop in vitro. PMID:25626913

  2. Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI.

    Pereyra-Bonnet, Federico; Gibbons, Alejandro; Cueto, Marcela; Sipowicz, Pablo; Fernndez-Martn, Rafael; Salamone, Daniel

    2011-04-01

    Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors. PMID:21079375

  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. 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.

  5. Comparison of some peptidic and proteic ovine pineal fractions with a bovine pineal E5 fraction

    Using rather simple and mild extraction and separation methods, three ovine pineal fractions (XM 300R - PP 7.2, PP 7.2' and PP 7.2S) were obtained, which contain peptidic/proteic substances and which show fluorescence characteristics of indoles. The ovine fractions were compared with the bovine pineal E5-fraction. The ovine fractions are chemically sensitive to normal laboratory light and stable in red light (#betta# > 600 nm). Immunologically, these fractions and the bovine E5 fraction are stable. From the results of radioimmunological experiments it was concluded that the bovine pineal E5 fraction as well as the ovine pineal fraction XM 300R - PP 7.2 and PP 7.2S may contain (a) peptide(s) ending by the same carboxy terminal tripeptide Pro-Arg-Gly(NH2). (Author)

  6. 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)....

  7. 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.

  8. 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

  9. Specific radioimmunoassay for ovine bone gla-protein (osteocalcin)

    We developed a sensitive and specific radioimmunoassay for ovine bone gla-protein (osteocalcin) using a polyclonal rabbit antibody raised against ovine bone gla-protein. Bone from lambs was extracted in 0.5 mol/l EDTA and desalted on Sephadex G-25. Bone gla-protein was purified by gel filtration chromatography over Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-25. The protein, subjected to monodimensional electrophoresis migrated as a single spot in SDS PAGE with the same apparent molecular weight of 12 kD as bovine bone gla-protein. The amino acid composition of pufified bone gla-protein was in agreement with a previous publication. The competitive RIA uses 125I-labelled bone gla-protein as a tracer and a complex of a second antibody and polyethylene glycol to separate free and antibody-bound 125I-labelled bone gla-protein. The intra- and inter-assay variations are less than 6 and 10%, respectively. There is no reactivity of our antisera with dog sera. The cross-reactivity is only partial with calf and human sera and complete with ovine sera. We measured bone gla-protein levels in serum of 96 normal male sheep of different ages. Serum bone gla-protein rapidly and significantly (P<0.001) decreased from 532 ± 169 μg/l at birth, to 240 ± 43 μg/l at 45 days, 152 ± 44 μg/l at 90 days, and 5.9 ± 0.7 μg/l at 7 years age. In addition, bone gla-protein levels at birth were higher in normal birth weight than in hypotrophic lambs with low birth weight (535 ± 169 vs 271 ± 156 μg/l, P<0.0001). Furthermore, lambs raised outside in free conditions tended to have higher serum bone gla-protein levels than lambs raised under shelter (1984 ± 53 vs 137 ± 34 μg/l), suggesting a role of breeding factors such as diet or relative immobilization on bone gla-protein levels. These results emphasize the interest of a RIA for the bone-specific protein bone gla-protein as a potential tool for experimental studies on skeletal growth and bone remodelling in a large animal. (author)

  10. Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen.

    Domingos, Renan F; Fernandes, Luis G; Romero, Eliete C; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2015-04-01

    Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions. PMID:25627443

  11. Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions.

    Sumitomo, Tomoko; Nakata, Masanobu; Higashino, Miharu; Yamaguchi, Masaya; Kawabata, Shigetada

    2016-01-01

    Group A Streptococcus (GAS) is a human-specific pathogen responsible for local suppurative and life-threatening invasive systemic diseases. Interaction of GAS with human plasminogen (PLG) is a salient characteristic for promoting their systemic dissemination. In the present study, a serotype M28 strain was found predominantly localized in tricellular tight junctions of epithelial cells cultured in the presence of PLG. Several lines of evidence indicated that interaction of PLG with tricellulin, a major component of tricellular tight junctions, is crucial for bacterial localization. A site-directed mutagenesis approach revealed that lysine residues at positions 217 and 252 within the extracellular loop of tricellulin play important roles in PLG-binding activity. Additionally, we demonstrated that PLG functions as a molecular bridge between tricellulin and streptococcal surface enolase (SEN). The wild type strain efficiently translocated across the epithelial monolayer, accompanied by cleavage of transmembrane junctional proteins. In contrast, amino acid substitutions in the PLG-binding motif of SEN markedly compromised those activities. Notably, the interaction of PLG with SEN was dependent on PLG species specificity, which influenced the efficiency of bacterial penetration. Our findings provide insight into the mechanism by which GAS exploits host PLG for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions. PMID:26822058

  12. Ontogeny and regulation of the AT1 and AT2 receptors in the ovine fetal adrenal gland.

    Wintour, E M; Moritz, K; Butkus, A; Baird, R; Albiston, A; Tenis, N

    1999-11-25

    The expression and regulation of the receptors for angiotensin II (both AT1 and AT2) were examined in the ovine fetal adrenal gland by RNase protection assay (RPA), in situ hybridisation histochemistry, immunohistochemistry and Western blotting. Both mRNA and protein for the AT1 receptor were present in the zona glomerulosa and zona fasciculata of the cortex, but not in the medulla, from as early as these zonas were distinguishable (60 days of gestation; term is 145-150 days), and even present in the steroidogenic cells of the unzoned gland at 40 days. The mRNA for the AT2 receptor was present in the same locations (but never in the medulla) from 40-130 days, and declined to extremely low levels after 140 days. The infusion of ang II, 1 microg/h, for 3 days, at mid-gestation (76 +/- 2 days) caused a significant decrease in mRNA for AT1 but no change in AT2 levels. Thus, the biologically active receptor (in terms of aldosterone stimulation) is present in the ovine fetal adrenal from very early in development, and can be down-regulated by mid-gestation. PMID:10619407

  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. Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects.

    Muttini, A; Valbonetti, L; Abate, M; Colosimo, A; Curini, V; Mauro, A; Berardinelli, P; Russo, V; Cocciolone, D; Marchisio, M; Mattioli, M; Tosi, U; Podaliri Vulpiani, M; Barboni, B

    2013-02-01

    In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries. PMID:22954787

  15. 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

  16. 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.

  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. 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 DH5? 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.

  19. 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.

  20. Transcellular route as the most probable explanation for the presence of plasminogen in mammal׳s milk.

    Silanikove, Nissim

    2016-04-21

    Paracellular and transcellular route of passage of several milk metabolites, albumin, lactic acid dehydrogenase and plasminogen from the systemic circulation to milk is considered. The shortcoming of prevalence views considering paracellular leakage as the most probable explanation is highlighted. A detailed discussion explaining why transcellular route is the most probable explanation for the presence of plasminogen in milk is presented. PMID:26826486

  1. 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

  2. Covariate selection in multivariate spatial analysis of ovine parasitic infection.

    Musella, V; Catelan, D; Rinaldi, L; Lagazio, C; Cringoli, G; Biggeri, A

    2011-05-01

    Gastrointestinal (GI) strongyle and fluke infections remain one of the main constraints on health and productivity in sheep dairy production. A cross-sectional survey was conducted in 2004-2005 on ovine farms in the Campania region of southern Italy in order to evaluate the prevalence of Haemonchus contortus, Fasciola hepatica, Dicrocoelium dendriticum and Calicophoron daubneyi from among other parasitic infections. In the present work, we focused on the role of the ecological characteristics of the pasture environment while accounting for the underlying long range geographical risk pattern. Bayesian multivariate spatial statistical analysis was used. A systematic grid (10 km×10 km) sampling approach was used. Laboratory procedures were based on the FLOTAC technique to detect and count eggs of helminths. A Geographical Information System (GIS) was constructed by using environmental data layers. Data on each of these layers were then extracted for pasturing areas that were previously digitalized aerial images of the ovine farms. Bayesian multivariate statistical analyses, including improper multivariate conditional autoregressive models, were used to select covariates on a multivariate spatially structured risk surface. Out of the 121 tested farms, 109 were positive for H. contortus, 81 for D. dendriticum, 17 for C. daubneyi and 15 for F. hepatica. The statistical analysis highlighted a north-south long range spatially structured pattern. This geographical pattern is treated here as a confounder, because the main interest was in the causal role of ecological covariates at the level of each pasturing area. A high percentage of pasture and impermeable soil were strong predictors of F. hepatica risk and a high percentage of wood was a strong predictor of C. daubneyi. A high percentage of wood, rocks and arable soil with sparse trees explained the spatial distribution of D. dendriticum. Sparse vegetation, river, mixed soil and permeable soil explained the spatial distribution of the H. contortus. Bayesian multivariate spatial analysis of parasitic infections with covariates from remote sensing at a very small geographical level allowed us to identify relevant risk predictors. All the covariates selected are consistent with the life cycles of the helminths investigated. This research showed the utility of appropriate GIS-driven surveillance systems. Moreover, spatial features can be used to tailor sampling design where the sampling fraction can be a function of remote sensing covariables. PMID:21167615

  3. Ultrastructural characterization of in vivo-produced ovine morulae and blastocysts.

    Bettencourt, E M V; Bettencourt, C M V; Chagas e Silva, J N; Ferreira, P; Oliveira, E; Romão, R; Rocha, A; Sousa, M

    2015-08-01

    The ultrastructure of in vivo-produced ovine embryos, at the morula, early blastocyst and late blastocyst stages, was evaluated using transmission electron microscopy. Embryonic cells were characterized by the presence of intact intercellular junctions, numerous mitochondria, smooth endoplasmic reticulum cisternae and light vesicles. Polyribosomes, rough endoplasmic reticulum cisternae, secondary lysosomes, Golgi complexes and lipid droplets were also observed in the cytoplasm. The nucleus was well defined and organized, with an intact envelope rich in nuclear pore complexes, and one or more reticular nucleoli. Microvilli were present in external blastomeres of morulae and became more abundant in trophectoderm cells of early and late blastocysts. Light vesicles seemed to be associated with small cisternae of Golgi and endoplasmic reticulum origin. These cisternae fused and created light vesicles with engulfed heterogeneous cytosolic structures, small cisternae and vesicles. Their labile membrane enabled them to rapidly coalesce into medium-sized vesicles that began to engulf mitochondria and lipid droplets, forming giant vacuoles mostly filled with fat. Incomplete matured secretory vesicles were observed to exocytose into the perivitelline space of morulae, whereas fully matured secretory vesicles appeared only in trophectoderm cells, being exocytosed into the blastocoelic cavity. These observations suggested that these endoplasmic-/Golgi-derived vesicles behave as active autophagic organelles presenting probably a maturation process from compact morulae to blastocyst. PMID:25076424

  4. 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

  5. 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.

  6. Immunisation against ovine caseous lymphadenitis: correlation between Corynebacterium pseudotuberculosis toxoid content and protective efficacy in combined clostridial-corynebacterial vaccines.

    Eggleton, D G; Haynes, J A; Middleton, H D; Cox, J C

    1991-10-01

    Groups of sheep were dosed with vaccines containing Corynebacterium pseudotuberculosis toxoid combined in varying amounts with 5 clostridial antigens. Resistance of the sheep to infection with C pseudotuberculosis was tested at 1, 6 and 12 months after vaccination by infection with pus from ovine lymph glands actively infected with C pseudotuberculosis. The outcome was assessed 3 months after challenge by slaughter and inspection of the sheep for caseous lymphadenitis lesions. Protection was demonstrated by a significant reduction in the proportion of immunised sheep exhibiting lesions compared with control sheep, and by fewer abscesses in affected immunised sheep than in affected control sheep. A positive correlation was found between amount of C pseudotuberculosis toxoid administered and degree of protection obtained. Chromatographically-purified toxoid induced essentially the same protection, suggesting that anti-toxic immunity is the major factor in protection. PMID:1755783

  7. Ovine platelet function is unaffected by extracorporeal membrane oxygenation within the first 24 h.

    Hayes, Rylan A; Foley, Samuel; Shekar, Kiran; Diab, Sara; Dunster, Kimble R; McDonald, Charles; Fraser, John F

    2015-10-01

    This study investigated platelet dysfunction during short-term extracorporeal membrane oxygenation (ECMO) and secondarily to determine if hyperoxaemia contributes to this dysfunction. Healthy sheep were anaesthetized and maintained on ECMO for either 2 or 24 h, with or without induction of smoke inhalation acute lung injury. A specialized animal-operating theatre was used to conduct the experimentation. Forty-three healthy female sheep were randomized into either a test or a control group. Following anaesthesia, test groups received ECMO smoke inhalation acute lung injury (SALI), whereas control groups were maintained with ventilation only SALI. Physiological, biochemical and coagulation data were obtained throughout via continuous monitoring and blood sampling. Platelet function was quantified through whole blood impedance aggregometry using Multiplate. Ovine platelet activity induced by adenosine diphosphate (ADP) and collagen was unaffected during the first 24 h of ECMO. However, progressive divergence of ADP-induced platelet activity was noted at cessation of the experiment. PaO2 was inversely related to ADP-dependent platelet activity in the ECMO groups--a relationship not identified in the control groups. ADP and collagen-dependent platelet activity are not significantly affected within the first 24 h of ECMO in sheep. However, dysfunction in ADP-dependent platelet activity may have continued to develop if observed beyond 24 h. Hyperoxaemia during ECMO does appear to affect how platelets react to ADP and may contribute to this developing dysfunction. Long-term animal models and investigation in clinical animals are warranted to fully investigate platelet function during ECMO. PMID:26196193

  8. Interaction of Corynebacterium pseudotuberculosis with ovine cells in vitro.

    Valdivia, J; Real, F; Acosta, F; Acosta, B; Déniz, S; Ramos-Vivas, J; Elaamri, F; Padilla, D

    2013-03-01

    Caseous lymphadenitis is an infectious and contagious disease caused by Corynebacterium pseudotuberculosis, with a worldwide distribution and high prevalence in small ruminant populations. This disease causes significant economic loss in small ruminants through reduced meat, wool, and milk production. C. pseudotuberculosis can also affect horses, domestic and wild large ruminants, swine, and man. It is considered an occupational zoonosis for humans. As part of in vitro investigations of the pathogenesis of C. pseudotuberculosis, this study analyzed its capacity to adhere to and invade the FLK-BLV-044 cell line, derived from ovine embryonic kidney cells. C. pseudotuberculosis showed a measurable capacity to adhere to and invade this cell line with no significant differences between the four strains assessed. The incubation of the cell line at 4ºC, pre-incubation with sugars, complete and heat inactivated antiserum, and heat-killed and ultraviolet-killed bacteria produced a significant (P pseudotuberculosis survival and play a role in the spread of infection and/or maintenance of a carrier state. PMID:22732360

  9. 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.

  10. Young ovine death during hyperimmunization: crotalic envenomation or copper toxicosis?

    RS Ferreira Junior

    2008-01-01

    Full Text Available The unfavorable evolution of a young ovine during hyperimmunization process with Crotalus durissus terrificus venom was investigated in order to differentiate its origin between ophidic envenomation and copper toxicosis. Clinical, laboratory, necroscopic and histological exams as well as evaluation and measurement of heavy metals (copper in the kidneys and in the liver were carried out. Blood counts revealed anemia and serological tests showed high levels of blood urea nitrogen, creatinine, aspartate aminotransferase, creatine phosphokinase, total bilirubin and indirect bilirubin; which indicates liver, kidney and skeletal muscle damages. At necropsy, the animal presented hepatopathy and nephropathy. Histological examination revealed renal and hepatic features that may imply copper intoxication. Copper levels were 237.8 g/g in the liver and 51.2 g/g in the kidneys. Although the amount of metal found in both organs was below the level that can cause death, according to the literature, anatomopathological signs were suggestive of copper intoxication. Therefore, the hypothesis of metal toxicosis during the hyperimmunization process became more consistent than the crotalic envenomation one.

  11. 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.

  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. 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)

  14. 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

  15. Transcriptomic Determinants of Scrapie Prion Propagation in Cultured Ovine Microglia.

    Muoz-Gutirrez, Juan F; Aguilar Pierl, Sebastin; Schneider, David A; Baszler, Timothy V; Stanton, James B

    2016-01-01

    Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor ?-induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model. PMID:26807844

  16. Ovine reference materials and assays for prion genetic testing

    Smith Timothy PL

    2010-04-01

    Full Text Available Abstract Background Genetic predisposition to scrapie in sheep is associated with several variations in the peptide sequence of the prion protein gene (PRNP. DNA-based tests for scoring PRNP codons are essential tools for eradicating scrapie and for evaluating rare alleles for increased resistance to disease. In addition to those associated with scrapie, there are dozens more PRNP polymorphisms that may occur in various flocks. If not accounted for, these sites may cause base-pair mismatching with oligonucleotides used in DNA testing. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of PRNP polymorphisms in targeted flocks. Results An adaptive DNA sequencing strategy was developed to determine the 771 bp PRNP coding sequence for any sheep and thereby produce a consensus sequence for targeted flocks. The strategy initially accounted for 43 known polymorphisms and facilitates the detection of unknown polymorphisms through an overlapping amplicon design. The strategy was applied to 953 sheep DNAs from multiple breeds in U.S. populations. The samples included two sets of reference sheep: one set for standardizing PRNP genetic testing and another set for discovering polymorphisms, estimating allele frequencies, and determining haplotype phase. DNA sequencing revealed 16 previously unreported polymorphisms, including a L237P variant on the F141 haplotype. Two mass spectrometry multiplex assays were developed to score five codons of interest in U.S. sheep: 112, 136, 141, 154, and 171. Reference tissues, DNA, trace files, and genotypes from this project are publicly available for use without restriction. Conclusion Identifying ovine PRNP polymorphisms in targeted flocks is critical for designing efficient scrapie genetic testing systems. Together with reference DNA panels, this information facilitates training, certification, and development of new tests and knowledge that may expedite the eradication of sheep scrapie.

  17. Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer.

    German, Sergio D; Campbell, Keith H S; Thornton, Elisabeth; McLachlan, Gerry; Sweetman, Dylan; Alberio, Ramiro

    2015-02-01

    Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs. PMID:25513856

  18. [The gene ontology and electro localization of ovine skin derived EST-SSR markers].

    Wang, Zun-Bao; Zhao, Zong-Sheng; Yu, Peng; Wu, Hong-Bin; Ban, Qian; Liang, Yao-Wei; Zheng, Wei

    2011-07-01

    Abstract: In order to study the potential gene function of ovine EST-SSR markers, nine original EST of Ovine Skin Derived polymorphic EST-SSR loci, which were developed in an early study by our lab, were ontology annotated and Electro localized. The results revealed that the original ESTs of the six loci had high homology with known genes and three of them probably played an important role in wool traits. Compared with its cDNA library, 8 loci were located on chromosomes of cattle. The homology of chromosomes between cattle and sheep was estimated based on the similarity coefficients calculated by positioning markers. Additionally, NJ clustering tree was establishedto serve for electro localization of ovine EST-SSR markers. Finally, 8 EST-SSR markers were successfully positioned on ovine chromosomes. The results from this study not only provide references for further studies on genetic mapping, in silico cloning of key genes for wool traits, but also are helpful to the researchs of chromosome evolution in animal. PMID:22049686

  19. Ovine progressive pneumonia virus is transmitted more effectively via aerosol nebulization than oral administration

    A new method of experimental infection of ovine progressive pneumonia virus (OPPV), aerosol nebulization (Nb), was compared to intravenous (IV) and oral (PO) methods of experimental infection. Seven month old lambs were given 3.5 × 107 TCID50 of Dubois OPPV LMH19 isolate using IV, PO, or Nb methods ...

  20. Phylogenetic clades of ovine progressive pneumonia virus (OPPV) associate with sheep TMEM154 genotypes

    Ovine progressive pneumonia virus (OPPV) is a lentivirus within the Retroviridae family that infects sheep. OPPV-induced clinical disease progresses slowly over time and manifests primarily in the lungs and central nervous system. Symptoms include weight loss, respiratory distress, and inevitably ...

  1. Development and Validation of an Ovine Progressive Pneumonia Virus Quantitative PCR

    Ovine progressive pneumonia virus (OPPV) infects at least one sheep in eighty-one percent of U.S. sheep flocks as measured by serological diagnostic tests and can cause viral-induced mastitis, arthritis, dypsnea, and cachexia. Diagnostic tests that quantify OPP proviral load in peripheral blood leu...

  2. Evidence of proviral clearance following postpartum transmission of an ovine lentivirus

    Lentiviral transmission by transfer of infected colostrum and/or milk is considered to be highly efficient. In this study, postpartum transmission of ovine progressive pneumonia virus (OPPV) from 10 naturally infected ewes to their 23 lambs was followed from the perinatal period throughout a four y...

  3. Tracking horizontal transmission of ovine lentivirus by quantitative PCR and envelope sequencing

    Most transmission of ovine lentivirus (OvLV) occurs horizontally since in utero transmission is considered inefficient. However, there is no direct evidence that horizontal transmission occurs from unrelated ewes to other lambs/ewes or from ewes to progeny. One way to assess the source of horizont...

  4. Ovine progressive pneumonia provirus levels associate with breed and Ovar-DRB1

    Previous studies have suggested that host genetics influence both lung histopathology and seroprevalence in sheep infected with ovine progressive pneumonia virus (OPPV). As a first start, a genetic association study was conducted in 383 Idaho sheep of the Columbia, Polypay and Rambouillet breeds ut...

  5. Comparative anatomical measurements of osseous structures in the ovine and human knee.

    Osterhoff, Georg; Löffler, Sabine; Steinke, Hanno; Feja, Christine; Josten, Christoph; Hepp, Pierre

    2011-03-01

    The ovine stifle has been increasingly used as a large animal model for the human knee. Still, comparative anatomical measurements of the knee in sheep and humans are missing. Thus, the purpose of this study was to describe and measure the osseous anatomy of the ovine stifle in comparison to the human knee. Twenty-four stifles of skeletal-mature merino-sheep and 24 human cadaver knees were obtained and distances between selected anatomical structures of the distal femur, the proximal tibia, and the patella were measured digitally and documented. Based on these, intercondylar ratio, tibial aspect ratio, patella aspect ratio and the cortical index were calculated. Regarding epicondylar width, lateral condylar width, medial condylar width and the tibial dimensions, the ovine stifle can be considered as a human knee scaled down by one third. However, sheep have a smaller trochlear width and a narrower femoral intercondylar notch than humans resulting in lower relative values for intercondylar width and intercondylar height. The distal femur's cortical index is the same in both species. In contrast, sheep have a massive bone stock below their tibial plateau and a proximal tibial shaft with remarkably thick cortical bone. The ovine stifle can be regarded as a useful model for the human knee. However, future studies should consider the differences in the femoral intercondylar notch width, the patellofemoral joint's biomechanics and the proximal tibia's cortical bone stock. PMID:20188573

  6. The glycolytic enzyme enolase represents a plasminogen-binding protein on the surface of a wide variety of medically important fungal species.

    Funk, Jana; Schaarschmidt, Barbara; Slesiona, Silvia; Hallstrm, Teresia; Horn, Uwe; Brock, Matthias

    2016-01-01

    Allergies are an increasing issue in human health and can, eventually, cause severe anaphylactic shock. Aspergillus fumigatus and Candida albicans are leading causes of life-threatening invasive fungal infections in immunocompromised patients, but can also cause severe allergic responses in otherwise healthy individuals. The glycolytic enzyme enolase is known as a major allergen despite its function in intracellular metabolism. Therefore, its presentation on surfaces of different fungal species was investigated by using antibodies raised against recombinant enolases from A. fumigatus and C. albicans. Examination of antibody specificity revealed cross-reactivity to cell-free extracts from Aspergillus terreus, Aspergillus flavus, Aspergillus nidulans and Candida glabrata, but not against any of the three human enolases. Antibody specificity was further confirmed by hybridization with other recombinant fungal enolases, where the antibodies recognized different subsets of fungal enolases. When surface presentation of enolase was tested on intact fungal cells, a positive staining was obtained with those antibodies that also recognized the enzyme from the respective cell-free extract. This implies a general surface presentation of this glycolytic enzyme among fungal species and provides hints for its predominant recognition as an allergen. Additionally, A. fumigatus and C. albicans enolase bound to human plasminogen, which remained accessible for the plasminogen activator uPA. This implies a potential role of enolase in the invasion and dissemination process during fungal infections. PMID:26679571

  7. Monocarboxylate transporters 1 and 4: expression and regulation by PPARα in ovine ruminal epithelial cells.

    Benesch, Franziska; Dengler, Franziska; Masur, Franziska; Pfannkuche, Helga; Gäbel, Gotthold

    2014-12-15

    In the intact rumen epithelium, isoforms 1 and 4 of the monocarboxylate transporter (MCT1 and MCT4) are thought to play key roles in mediating transcellular and intracellular permeation of short-chain fatty acids and their metabolites and in maintaining intracellular pH. We examined whether both MCT1 and MCT4 are expressed at mRNA and protein levels in ovine ruminal epithelial cells (REC) maintained in primary culture and whether they are regulated by peroxisome proliferator-activated receptor-α (PPARα). Because both transporters have been characterized to function coupled to protons, the influence of PPARα on the recovery of intracellular pH after l-lactate exposure was evaluated by spectrofluorometry. MCT1 and MCT4 were detected using immunocytochemistry both at the cell margins and intracellularly in cultured REC. To test regulation by PPARα, cells were exposed to WY 14.643, a selective ligand of PPARα, for 48 h. The subsequent qPCR analysis resulted in a dose-dependent upregulation of MCT1 and PPARα target genes, whereas response of MCT4 was not uniform. Protein expression of MCT1 and MCT4 quantified by Western blot analysis was not altered by WY 14.643 treatment. l-Lactate-dependent proton export was blocked almost completely by pHMB, a specific inhibitor of MCT1 and MCT4. However, l-lactate-dependent, pHMB-inhibited proton export in WY 14.643-treated cells was not significantly altered compared with cells not treated with WY 14.643. These data suggest that PPARα is particularly regulating MCT1 but not MCT4 expression. Extent of lactate-coupled proton export indicates that MCT1 is already working on a high level even under unstimulated conditions. PMID:25320343

  8. Development of ovine chorionic somatomammotropin hormone-deficient pregnancies.

    Baker, Callie M; Goetzmann, Lindsey N; Cantlon, Jeremy D; Jeckel, Kimberly M; Winger, Quinton A; Anthony, Russell V

    2016-05-01

    Intrauterine growth restriction (IUGR) is a leading cause of neonatal mortality and morbidity. Chorionic somatomammotropin hormone (CSH), a placenta-specific secretory product found at high concentrations in maternal and fetal circulation throughout gestation, is significantly reduced in human and sheep IUGR pregnancies. The objective of this study was to knock down ovine CSH (oCSH) expression in vivo using lentiviral-mediated short-hairpin RNA to test the hypothesis that oCSH deficiency would result in IUGR of near-term fetal lambs. Three different lentiviral oCSH-targeting constructs were used and compared with pregnancies (n = 8) generated with a scrambled control (SC) lentiviral construct. Pregnancies were harvested at 135 days of gestation. The most effective targeting sequence, "target 6" (tg6; n = 8), yielded pregnancies with significant reductions (P ≤ 0.05) in oCSH mRNA (50%) and protein (38%) concentrations, as well as significant reductions (P ≤ 0.05) in placental (52%) and fetal (32%) weights compared with the SC pregnancies. Fetal liver weights were reduced 41% (P ≤ 0.05), yet fetal liver insulin-like growth factor-I (oIGF1) and -II mRNA concentrations were reduced (P ≤ 0.05) 82 and 71%, respectively, and umbilical artery oIGF1 concentrations were reduced 62% (P ≤ 0.05) in tg6 pregnancies. Additionally, fetal liver oIGF-binding protein (oIGFBP) 2 and oIGFBP3 mRNA concentrations were reduced (P ≤ 0.05), whereas fetal liver oIGFBP1 mRNA concentration was not impacted nor was maternal liver oIGF and oIGFBP mRNA concentrations or uterine artery oIGF1 concentrations (P ≥ 0.10). Based on our results, it appears that oCSH deficiency does result in IUGR, by impacting placental development as well as fetal liver development and function. PMID:26887431

  9. Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

    Tomoko Sumitomo; Masanobu Nakata; Miharu Higashino; Masaya Yamaguchi; Shigetada Kawabata

    2016-01-01

    Group A Streptococcus (GAS) is a human-specific pathogen responsible for local suppurative and life-threatening invasive systemic diseases. Interaction of GAS with human plasminogen (PLG) is a salient characteristic for promoting their systemic dissemination. In the present study, a serotype M28 strain was found predominantly localized in tricellular tight junctions of epithelial cells cultured in the presence of PLG. Several lines of evidence indicated that interaction of PLG with tricelluli...

  10. Colonic lesions, cytokine profiles, and gut microbiota in plasminogen-deficient mice

    Vestergaard, Bill; Krych, Lukasz; Lund, Leif R; Jørgensen, Bettina Merete Pyndt; Hansen, Lars H.; Jensen, Henrik Elvang; Nielsen, Dennis Sandris; Hansen, Axel Kornerup

    2015-01-01

    Plasminogen-deficient (FVB/NPan-plg(tm1Jld), plg(tm1Jld)) mice, which are widely used as a wound-healing model, are prone to spontaneous rectal prolapses. The aims of this study were 1) to evaluate the fecal microbiome of plg(tm1Jld) mice for features that might contribute to the development of r...... composition of the gut microbiota, and none of the clinical or biochemical parameters correlated with the gut microbiota composition....

  11. Sex influence and colect time on the seric levels of thyroid hormone, triiodotryronine, thyronine in ovine Corriedale lineage

    The seric levels of thyroid hormones are stable in ovines blood independing of sex and colect time. The triiodothyronine and thyronine were evaluated. The gamma spectrometry system calibrated for iodine 125 energy was used. (L.M.J.)

  12. Identification of the Target Cells and Sequence of Infection during Experimental Infection of Ovine Fetuses with Cache Valley Virus

    Rodrigues Hoffmann, Aline; Welsh, Christabel Jane; Varner, Patricia Wilcox; de la Concha-Bermejillo, Andres; Ball, Judith Marchand; Ambrus, Andy; Edwards, John Francis

    2012-01-01

    Cache Valley virus-induced malformations have been previously reproduced in ovine fetuses; however, no studies have established the course of infection of cells and tissues with Cache Valley virus. To address these questions, ovine fetuses at 35 days of gestation were inoculated in utero with Cache Valley virus and euthanized at 7, 10, 14, 21, and 28 days postinfection. On postmortem examination, arthrogryposis and oligohydramnios were observed in some infected fetuses. Morphological studies ...

  13. Human-, Ovine-, and Bovine-Specific Viral Source Tracking Tools to Discriminate Between the Major Fecal Sources in Agricultural Waters.

    Rusiol, Marta; Moriarty, Elaine; Lin, Susan; Bofill-Mas, Slvia; Gilpin, Brent

    2016-03-01

    This study evaluated the sources of fecal contamination in different river catchments, using a combination of microbial source tracking tools, for human, ruminant, ovine and bovine livestock, in order to define appropriate water management strategies. Every source of waterway pollution was evaluated in river water samples from one urban river catchment and two important farming regions in New Zealand. Fecal pollution was initially measured by testing Escherichia coli and evaluating the presence of human- and ruminant-associated DNA markers of Bacteroidales (BiAdo, BacHum-UCD, BacH, and BacR) and human and ruminant fecal sterols/stanols ratios. Then specific fecal pollution sources were assessed with previously reported quantitative PCR assays targeting human-, bovine-, and ovine-specific viruses: human adenoviruses (HAdV), human JC polyomaviruses, bovine polyomaviruses (BPyV), and ovine polyomaviruses (OPyV). High level of ruminant fecal contamination was detected all over the farming areas, whereas no ruminant sources were identified in the urban river sampling sites. BacR was the most frequently observed ruminant marker and OPyV and BPyV allowed the identification of ovine and bovine fecal sources. The human fecal viral marker (HAdV) was the most frequently observed human marker, highly abundant in the urban sites, and also present in farming areas. This is the first study using simultaneously the ovine and the bovine viral markers to identify and quantify both bovine and ovine fecal pollution. PMID:26607578

  14. Characterization of a radioimmunoassay for ovine FSH utilizing an anti-bovine FSH serum

    In the radioimmunoassay for ovine FSH, utilizing a bovine FSH antiserum and 125I-labelled bovine FSH tracer, the sensitivity was shown to be 5 ng NIH-FSH-S6/ml and cross-reactivities of other ovine pituitary glyco-protein and protein hormones (LH,TSH, growth hormone and prolactin) were less than 1.0%. Both pituitary and serum FSH of the sheep cross-reacted in a manner parallel with that of standard NIH-FSH-S6. Serum FSH concentrations in 4 ewes at various times after the onset of oestrous peaked more or less coincidently with the preovulatory increase of serum LH. The serum levels of FSH and LH did not correlate proportionally but fluctuated considerably in individual animals. The radioimmunoassay system appeared to be sensitive and specific for measuring serum concentrations of FSH in rams and ewes in different physiological states. (author)

  15. Effect of surgery to implant motion and force sensors on vertical ground reaction forces in the ovine model.

    Herfat, Safa T; Shearn, Jason T; Bailey, Denis L; Greiwe, R Michael; Galloway, Marc T; Gooch, Cindi; Butler, David L

    2011-02-01

    Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruciate ligament (ACL). While calibration problems preclude direct measurement in patients, ACL forces can conceivably be measured in animals after technical challenges are overcome. For example, motion and force sensors can be implanted in the animal but investigators must determine the extent to which these sensors and surgery affect normal gait. Our objectives in this study were to determine (1) if surgically implanting knee motion sensors and an ACL force sensor significantly alter normal ovine gait and (2) how increasing gait speed and grade on a treadmill affect ovine gait before and after surgery. Ten skeletally mature, female sheep were used to test four hypotheses: (1) surgical implantation of sensors would significantly decrease average and peak vertical ground reaction forces (VGRFs) in the operated limb, (2) surgical implantation would significantly decrease single limb stance duration for the operated limb, (3) increasing treadmill speed would increase VGRFs pre- and post operatively, and (4) increasing treadmill grade would increase the hind limb VGRFs pre- and post operatively. An instrumented treadmill with two force plates was used to record fore and hind limb VGRFs during four combinations of two speeds (1.0 m/s and 1.3 m/s) and two grades (0 deg and 6 deg). Sensor implantation decreased average and peak VGRFs less than 10% and 20%, respectively, across all combinations of speed and grade. Sensor implantation significantly decreased the single limb stance duration in the operated hind limb during inclined walking at 1.3 m/s but had no effect on single limb stance duration in the operated limb during other activities. Increasing treadmill speed increased hind limb peak (but not average) VGRFs before surgery and peak VGRF only in the unoperated hind limb during level walking after surgery. Increasing treadmill grade (at 1 m/s) significantly increased hind limb average and peak VGRFs before surgery but increasing treadmill grade post op did not significantly affect any response measure. Since VGRF values exceeded 80% of presurgery levels, we conclude that animal gait post op is near normal. Thus, we can assume normal gait when conducting experiments following sensor implantation. Ultimately, we seek to measure ACL forces for ADLs to provide design criteria and evaluation benchmarks for traditional and tissue engineered ACL repairs and reconstructions. PMID:21280882

  16. Ultrastructural study of in vivo produced ovine embryos: characterization of secretory vesicles

    Bettencourt, Elisa Maria Varela; Bettencourt, Carlos Manuel Varela; Ferreira, Paulo; Romão, Ricardo Jorge; Rocha, António; de Sousa, Mário

    2013-01-01

    The ultrastructure of in vivo produced ovine embryos, at the morula, early blastocyst and late blastocyst stages, was evaluated using transmission electron microscopy. In vivo produced embryos (n=14) were obtained from Portuguese Black Merino ewes, superovulated during Spring. Embryos were recovered by abdominal laparotomy, under general anesthesia, on day 6 or 7 after sponge removal. Embryos were processed for electronic microscopy observation and ultrathin sections (700A) were observed in a...

  17. Interleukin-1 β Modulates Melatonin Secretion in Ovine Pineal Gland: Ex Vivo Study

    Herman, A. P.; Bochenek, J.(Florida State University, Tallahassee, USA); J. Skipor; Król, K.; Krawczyńska, A.; H. Antushevich; Pawlina, B.; Marciniak, E.; Tomaszewska-Zaremba, D.

    2015-01-01

    The study was designed to determine the effect of proinflammatory cytokine, interleukin- (IL-) 1β, on melatonin release and expression enzymes essential for this hormone synthesis: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) in ovine pineal gland, taking into account the immune status of animals before sacrificing. Ewes were injected by lipopolysaccharide (LPS; 400 ng/kg) or saline, two hours after sunset during short day period (December). Animal...

  18. Defining the differential sensitivity to norepinephrine and angiotensin II in the ovine uterine vasculature

    Rosenfeld, Charles R.; DeSpain, Kevin; Liu, Xiao-tie

    2011-01-01

    The intact ovine uterine vascular bed (UVB) is sensitive to α-agonists and refractory to angiotensin II (ANG II) during pregnancy; the converse occurs in the systemic circulation. The mechanism(s) responsible for these differences in uterine sensitivity are unclear and may reflect predominance of nonconstricting AT2 receptors (AT2R) in uterine vascular smooth muscle (UVSM). The contribution of the placental vasculature also is unclear. Third generation and precaruncular/placental arteries fro...

  19. An assessment of residual ovine nematodes on pasture under maritime conditions.

    Smith, H. J.; Fulton, N R

    1989-01-01

    Residual ovine nematode pasture infections were assessed by grazing groups of ewes and their lambs on permanent sheep and cattle pastures and by the use of tracer lambs. Ostertagia spp., Cooperia oncophora, Nematodirus spp., Chabertia ovina and Trichuris spp. eggs and/or larvae survived on pastures overwinter. Second generation Ostertagia larvae were present in greatest numbers on pasture during the latter part of August and early September. The failure of a significant build-up of Cooperia o...

  20. Detection of Brucella ovis in ovine from Paraba State, in the Northeast region of Brazil

    Clebert Jos Alves; Salomo Moreira de Figueiredo; Srgio Santos de Azevedo; Incio Jos Clementino; Lara Borges Keid; Slvio Arruda Vasconcellos; Carolina de Sousa Amrico Batista; Vivianne Cambu Mesquita Rocha; Severino Silvano Higino

    2010-01-01

    To determine the presence of Brucella ovis in ovine from Paraba State, in the Northeast region of Brazil, 80 animals slaughtered in the public slaughterhouse of Patos city were used. Before slaughter, blood samples were collected by jugular venopuncture from each animal, and after slaughter, testicles, epidydimus and uterus were aseptically collected. For the serological diagnosis of B. ovis and B. abortus infections, the agar gel immunodiffusion (AGID) and Rose Bengal (RBT) tests were carri...

  1. The effect of incubation time on capacitation and acrosome reaction of in vitro ovine spermatozoa

    J Wattimena

    2012-01-01

    The aim of this research was to know the effect of incubation time on capacitation and acrosome reaction of in vitro ovine spermatozoa. Twelve ejaculates from two Garut sheep were collected using artificial vagina and then evaluated macro and microscopically. After semen washing (centrifugation method), semen was diluted in Brackett and Oliphant (BO) medium and then incubated during 6 hours in incubator CO2, temperature 38.5ºC. Evaluation of capacitation and acrosome reaction was conducted on...

  2. Quantification of Biomechanical Interaction of Transcatheter Aortic Valve Stent Deployed in Porcine and Ovine Hearts

    Mummert, Joseph; Sirois, Eric; Sun, Wei

    2012-01-01

    Success of the deployment and function in transcatheter aortic valve replacement (TAVR) is heavily reliant on the tissue-stent interaction. The present study quantified important tissue-stent contact variables of self-expanding transcatheter aortic valve (TAV) stents when deployed into ovine and porcine aortic roots, such as the stent radial expansion force, stent pullout force, the annulus deformation response and the coefficient of friction on the tissue-stent contact interface. Braided Nit...

  3. Global distribution and diversity of ovine-associated Staphylococcus aureus ☆

    Smith, Edward M.; Needs, Polly F.; Manley, Grace; Green, Laura E.

    2014-01-01

    Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe ...

  4. In Vitro Infection of Ovine Cell Lines by Jaagsiekte Sheep Retrovirus

    Palmarini, Massimo; Sharp, J Michael; Lee, Christine; Fan, Hung

    1999-01-01

    Sheep pulmonary adenomatosis (SPA), also known as jaagsiekte or ovine pulmonary carcinoma, is a contagious lung cancer of sheep, originating from type II pneumocytes and Clara cells. Previous studies have implicated a type D retrovirus (jaagsiekte sheep retrovirus [JSRV]) as the causative agent of SPA. We recently isolated a proviral clone of JSRV from an animal with a spontaneous case of SPA (JSRV21) and showed that it harbors an infectious and oncogenic virus. This demonstrated that JSRV is...

  5. Ovine Forestomach Matrix as a Substrate for Single-Stage Split-Thickness Graft Reconstruction

    Simcock, Jeremy; May, Barnaby C. H.

    2013-01-01

    Objective: Split skin graft reconstruction of scalp defects often leaves an obvious contour defect. Here, we aimed to demonstrate the use of a decellularized extracellular matrix biomaterial, termed ovine forestomach matrix (OFM), as a substrate for split-thickness skin grafts (STSGs) for scalp reconstruction. Methods: Following full-thickness tumor excision, OFM was applied directly to skull periosteum, and then an STSG was applied. Participants were monitored for graft take, epithelializati...

  6. Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal.

    Leavitt, W W; Okulicz, W C; McCracken, J A; Schramm, W; Robidoux, W F

    1985-06-01

    We previously showed that progesterone rapidly down regulates nuclear estrogen receptor (Re) in the estrogen-primed rodent uterus. We have now extended these studies to test the response of the Re system in sheep uterus to progesterone withdrawal. Since the estrogen-Re complex is believed to regulate hormone-dependent gene expression, it was of interest to determine whether withdrawal of progesterone under constant estrogen stimulation would lead to the recovery of nuclear Re levels and estrogen action, i.e. oxytocin receptor (ROT) synthesis. Ovariectomized ewes were primed with estradiol-17 beta and serum steroid levels were maintained by constant infusion of estradiol (0.5 microgram/h) and progesterone (500 micrograms/h) for 5 days. The animals were anesthetized with fluothane/O2, and uterine samples were excised 1 h before and 3, 6 and 12 h after progesterone withdrawal. Estradiol infusion was continued during the experiment in order to maintain estrogen levels at a steady state (14 pg/ml plasma). Re, ROT and progesterone receptor (Rp) were measured in endometrium and myometrium using standard 3H-hormone binding assays. Following progesterone withdrawal, the nuclear Re concentration increased in both uterine compartments, and the nuclear Re level was correlated significantly with the ROT concentration in the membrane fraction of both uterine tissues (endometrium, r = 0.79; myometrium, r = 0.86). Although cytosol Re rose between 6 and 12 h in the endometrium, cytosol Re levels remained unchanged in myometrium. Cytosol Rp appeared to increase in endometrium but not in myometrium. Uterine tissue sampled from a control animal before stopping the progesterone infusion revealed that the observed changes in receptor concentration following progesterone withdrawal were not due to regional differences in receptor levels. These results demonstrate that the recovery of nuclear Re in the ovine endometrium and myometrium following progesterone withdrawal represents a selective effect on Re retention in the nucleus rather than on cytosol Re availability or Re activation which was controlled by constant estrogen infusion. Thus, these results are consistent with the hypothesis that progesterone induces an Re regulatory factor which acts to down regulate nuclear Re, and that the activity of this factor diminishes rapidly after progesterone withdrawal. PMID:2991657

  7. Effect of leptin on invitro development of ovine preantral ovarian follicles.

    Kamalamma, P; Kona, S S R; Praveen Chakravarthi, V; Siva Kumar, A V N; Punyakumari, B; Rao, V H

    2016-01-15

    The influence of human or ovine leptin on invitro culture of preantral follicles (PFs) isolated from sheep ovaries was investigated. Among the 12 different concentrations (0-1000ng/mL) of human leptin tested, proportion of PFs exhibiting growth, mean increase in diameter, antrum formation, and maturation of the oocytes to MII stage were the best in 10ng/mL. Culture of sheep ovarian PFs in TCM 199 supplemented with 10ng/mL of human or ovine leptin FSH (2.5?g/mL), thyroxine (1?g/mL), insulinlike growth factor I (10ng/mL), and GH (1 mIU/mL) resulted in significantly (P?0.05) greater average increase in diameter (11 and 9 vs. 6 ?m), better proportions of PFs exhibiting growth (66% and 58% vs. 48%), antrum formation (51% and 51% vs. 34%), and maturation of oocytes to MII stage (24% and 22% vs. 7%) than the control medium. It is concluded that (1) the optimum dose of leptin for the growth of sheep PFs invitro was 10ng/mL, (2) human or ovine leptin supported similar development invitro of PFs in sheep, (3) inclusion of leptin along with FSH, thyroxine, insulinlike growth factor I, and GH resulted in only a marginal further improvements in invitro development of sheep PFs'. PMID:26489908

  8. Sex Determination in Ovine Embryos Using Amelogenin (AMEL Gene by High Resolution Melting Curve Analysis

    Xiao Ping Sun

    2012-01-01

    Full Text Available In the study, researchers have established and tested the reliability of a method for sex determination of ovine embryos using the AMEL gene by melting curve analysis of PCR amplification. It can be carried out in a regular laboratory or under farm conditions within 1.5 h for 96 samples. The PCR amplicons of 99/99 and 99/54 base pairs produced from female and male sheep, respectively are easily distinguished by both melting curve analysis and gel electrophoresis. The specificity of the method was earlier demonstrated by testing 9 blood samples from small-tailed sheep (5 males and 4 females. No amplification failures and very high agreement between genotypic and phenotypic sex was found (9/9. The sensitivity of the AMEL sexing assay was established for values >10 pg ovine genomic DNA. Forty five biopsied embryos were transferred into 22 recipient sheep on the same day that the embryos were collected and sex of the kid was confirmed after parturition. About 17 kids of predicted sex were born. The sex, as determined by PCR corresponded to the anatomical sex in all cases. To the knowledge, this was the first time that sex determination using the amelogenin gene was performed in ovine embryos by melting curve analysis.

  9. The effects of Ostertagia occidentalis somatic antigens on ovine TLR2 and TLR4 expression

    Hassan BORJI

    2015-10-01

    Full Text Available Background: Recognition of helminth-derived pathogen associated molecular patterns (PAMPs by pattern recognition receptors (PRRs, including toll like recep­tors (TLRs is the first step towards initiating anti–helminth immune re­sponses.Methods: Using somatic antigens of Ostertagia occidentalis, an important abomasal parasite of ruminants, the expression of ovine TLR2 and TLR4 in peripheral blood mononuclear cells (PBMCs was analyzed by real-time quatitative reverse-transcrip­tion polymerase chain reaction (qRT-PCR. Somatic antigens of O. occidentalis were prepared to stimulate ovine PBMCs in a time and dose dependent manner.Results: A high expression of TLR2 and TLR4 was observed in PBMCs cultured with somatic antigens of the parasites specially when PBMCs were cultured with 100 µg/ml of somatic antigens and incubated for 2h. Up-regulation of TLR2 expres­sion was more pronounced and evident in our study.Conclsusion: Somatic antigens of O. occidentalis have immunostimulatory and domi­nant role on peripheral immune cells. This study provide for the first time evidence of induction of TLRs in ovine PBMCs by somatic antigen of O. occidentalis

  10. Prevalence of ovine fasciolosis and its economic significance in basona worana district, central Ethiopia

    G. Zeleke

    2013-08-01

    Full Text Available A cross-sectional andretrospective study was conducted from February-May, 2012 to determine theprevalence of ovine fasciolosis. A total of 1528 sheep were selected andsampled by using systematic simple random sampling, Sedimentation technique,haematocrite centrifugation and postmortem examination were employed during thestudy period. Overall prevalence of ovine fasciolosis based on farm, abattoirand retrospective clinical study was 62.7%, 52.6% and 59.1%, respectively.There was no statistically significant variations (p>0.05 by sex and age ofsheep in both farm and abattoir study. The association of ovine faciolosis withdifferent breeds of sheep was statistically significant variation (p<0.05was observed in farm, abattoir and retrospective veterinary clinical dataanalysis. The prevalence of fasciolosis was different among FAMACHA eye-colourscores, body condition, mean PCV value, liver pathology, types of Fasciolaspecies and worm burden (p<0.05. The prevalence of ovine fasciolosis basedon FAMACHA eye-colour scores was highest (100% in pink white and lowest in red(51.9%. The mean PCV value of Fasciola species in infected and uninfectedsheep were 24.04 ± .381 and 25.87 ± .516, respectively. The mean worm burden oflightly, moderately and severely affected livers was 21.00 ± 1.889, 34.30 ±2.662 and 105.09 ± 9.741, respectively. The average mean worm burden per affectedliver was 56.79 ± 4.635. The predominant species identified in the study areawas Fasciola hepatica (68.8%. The mean fluke burden of each Fasciola speciescounted was 47.17 ± 4.737, 25.50 ± 4.912 and 93.02 ± 12.139 for Fasciolahepatica, Fasciola gigantica and mixed Fasciola species infection,respectively. The prevalence of ovine fasciolosis in the study area should bereduced by sustainable and integrated control programmes which includestrategic application of anthelmintics, efficient farm and grazing managementby Veterinarians and sheep owners in the study area.

  11. Incidence of infection in 39-month-old ewes with TMEM154 diplotypes "1 1," "1 3," and "3 3" after natural exposure to ovine progressive pneumonia virus

    Production and well-being of sheep and goats in many countries are harmfully impacted by small ruminant lentiviruses (SRLV) that cause incurable, progressive diseases. Susceptibility to ovine progressive pneumonia virus (OPPV), the North American form of SRLV, is influenced by variants of the ovine...

  12. Comprehensive study into the activation of the plasma enzyme systems during attacks of hereditary angioedema due to C1-inhibitor deficiency

    Csuka, Dorottya; Veszeli, Nóra; Imreh, Éva; Zotter, Zsuzsanna; Skopál, Judit; Prohászka, Zoltán; Varga, Lilian; Farkas, Henriette

    2015-01-01

    Background The activation of plasma enzyme systems contributes to hereditary angioedema attacks. We aimed to study the activation markers of the fibrinolytic, coagulation, and contact systems in a larger number of paired samples obtained from the same C1-INH-HAE patients in symptom-free periods and during attacks. Methods Eleven parameters (Factors XI, XII, and C1-inhibitor activity; the concentrations of the D-dimer, prothrombin fragments 1 + 2, plasminogen, plasminogen activator inhibitor-1...

  13. Gender affects skin wound healing in plasminogen deficient mice

    Rønø, Birgitte; Engelholm, Lars Henning; Lund, Leif Røge; Hald, Andreas

    2013-01-01

    functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency or......The fibrinolytic activity of plasmin plays a fundamental role in resolution of blood clots and clearance of extravascular deposited fibrin in damaged tissues. These vital functions of plasmin are exploited by malignant cells to accelerate tumor growth and facilitate metastases. Mice lacking...... if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin...

  14. The recombinant LIC10508 is a plasma fibronectin, plasminogen, fibrinogen and C4BP-binding protein of Leptospira interrogans.

    Siqueira, Gabriela H; Teixeira, Aline F; Fernandes, Luis G; de Souza, Gisele O; Kirchgatter, Karin; Romero, Eliete C; Vasconcellos, Silvio A; Vieira, Monica L; Nascimento, Ana Lucia T O

    2016-03-01

    Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. In this study, we report that the recombinant proteins LIC10507, LIC10508 and LIC10509 are recognized by confirmed leptospirosis serum samples at both phases of the disease. The recombinant rLIC10508 and rLIC10507 are plasminogen (PLG)-binding proteins, capable of generating plasmin in the presence of a PLG activator. The proteins bind to PLG in a dose-dependent and saturable manner, fulfilling host-ligand interaction. Furthermore, rLIC10508 interacts with fibrinogen (Fg), plasma fibronectin and C4b binding protein (C4BP). The binding of rLIC10508 to Fg decreases the fibrin clotting in a thrombin-catalyzed reaction. The incubation with 4 μM of protein promoted 40% inhibition upon clotting formation. C4BP bound to rLIC10508 retained its cofactor activity for factor I promoting the cleavage of C4b protein, which may reduce the membrane attack complex formation. Although these proteins have high amino acid sequence similarity, rLIC10508 is the most talented of the three, a behavior that might be explained by its unique putative 3D structure, whereas structures of rLIC10507 and rLIC10509 are very similar. Plasmin generation (rLIC10507 and rLIC10508), together with decreasing fibrin clot formation (rLIC10508) and impairment of the complement system (rLIC10508) may help the bacteria to overcome host defense, facilitating the infection process. PMID:26657108

  15. Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells

    Davidson, D.J.; Fraser, M.J.; Castellino, F.J. (Univ. of Notre Dame, IN (USA))

    1990-06-12

    A comparison has been made between the Asn{sup 289}-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, the authors find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate and truncated high-mannose oligosaccharide. Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. The data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, the authors conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.

  16. TGF-beta-induced apoptosis in endothelial cells mediated by M6P/IGFII-R and mini-plasminogen

    Leksa, V.; Godar, S.; Schiller, H.B.; Fuertbauer, E.; Muhammad, A.; Slezakova, K.; Hořejší, Václav; Steinlein, P.; Weidle, U.H.; Binder, B.R.; Stockinger, H.

    2005-01-01

    Roč. 118, č. 19 (2005), s. 4577-4586. ISSN 0021-9533 Grant ostatní: Austrian Science Fund(AT) 005 Institutional research plan: CEZ:AV0Z5052915; CEZ:AV0Z50520514 Keywords : apoptosis * TGF-beta * mini-plasminogen Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.543, year: 2005

  17. The use of CR1aa for ovine in vitro embryo production

    Yulnawati

    2006-06-01

    Full Text Available The aim of this study was to investigate the capacity of CR1aa as a simple medium for maturation, fertilization and culture of ovine embryo in vitro. Oocytes were collected by slicing method in Phosphate Buffer Saline (PBS supplemented with 5% Fetal Bovine Serum (FBS and 100 IU/ml penicillin streptomycin. Oocytes were matured in Tissue Culture Medium (TCM-199 as control or CR1aa as treatment medium. Both maturation medium were supplemented with 10% Fetal Bovine Serum (FBS, 10 IU/ml Follicle Stimulating Hormone (FSH, 10 IU/ml Luteinizing Hormone (LH, 1 μg/ml Estradiol and 100 IU/ml penicillin-streptomycin. Oocytes were incubated in 5% CO2 incubator, 38˚C for 24 h. Matured oocytes were fertilized in BO or CR1aa medium, supplemented with 2.5 mM caffeine benzoate and 20 mg /ml heparin. After 18 h in vitro fertilization, oocytes were cultured in TCM-199 or CR1aa medium, both supplemented with 5% FBS, 5 mg/ml insulin and 100 IU/ml penicillin streptomycin. Results showed that the highest maturation rate was found in TCM-199 medium (73.27% and significantly different (P0.05 between cleavage rate of ovine embryos in TCM-199 and CR1aa medium (39.45% vs 50.94%. In conclusion, optimum result on ovine in vitro embryo production can be achieved from a combination of TCM-199 as maturation medium and CR1aa as fertilization and culture medium.

  18. Ovine aquaporin-2: cDNA cloning, ontogeny and control of renal gene expression.

    Butkus, A; Earnest, L; Jeyaseelan, K; Moritz, K; Johnston, H; Tenis, N; Wintour, E M

    1999-06-01

    The aim of this study was to test the hypothesis that the relative insensitivity of the ovine fetal kidney to arginine vasopressin (AVP) is due to low levels of expression of the gene for aquaporin-2 (AQP2) which encodes the AVP-regulated water channel. We report the cloning of the cDNA for the ovine AQP2 which has a major transcript at 4.2 kilobases (kb) and a minor transcript at 1.5 kb, resembling the human gene transcripts. At 40-60 days' (term = 145-150 days'), mRNA levels are very low, detectable only by reverse transcription-polymerase chain reaction (RT-PCR). By Northern blot analysis AQP2 mRNA is detectable at 75 days'. The ratio of AQP2/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA increases approximately 2.4-fold between 100 and 140 days' when it is about 41% of adult values. Both glucocorticoids and the renin-angiotensin system are involved in maturation of renal function. When fetuses at 75 or 85 days of gestation were exposed to high levels of dexamethasone for 2-3 days, mRNAs for both GAPDH and AQP2 doubled, but the ratio was unchanged. Angiotensin I, infused for 3 days at 115-120 days' gestation, increased the AQP2/GAPDH mRNA ratios by twofold (major transcript) and sixfold (minor transcript), which were highly significant (P<0.001). The increasing sensitivity of the ovine fetal kidney to AVP, from 100-140 days of gestation, is largely due to increasing AQP2 gene expression over this period. PMID:10412857

  19. Search for phosphopeptides in the feces of axenic rats fed radioactive ovine casein

    Radioactive ovine casein was obtained by injecting 100 μCi of 14C-Ser into the jugular vein of an ewe. The milk collected 17 and 24 h after this injection contained 12% of the radioactivity injected in protein form. The seryl residues were specificially labelled. This casein was used as the only protein source fed to axenic rats; 0.30% of the tracer ingested was found in the feces of those rats. Since phosphoserine represented 25% of the total casein seryl residues, the phosphopeptides may not be selectively unabsorbable

  20. Characterization of human mucin (MUC15) and identification of ovine and caprine orthologs

    Pallesen, Lone Tjener; Pedersen, Lise Refstrup Linnebjerg; Petersen, Torben Ellebæk; Knudsen, Charlotte Rohde; Rasmussen, Jan Trige

    2008-01-01

    The glycoprotein MUC15 (mucin 15) was initially isolated from the bovine milk fat globule membrane. The present work demonstrates the existence of immunologically similar proteins ( approximately 130 kDa) in ovine, caprine, porcine, and buffalo milk samples. Purification and N-terminal amino acid...... expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions...

  1. Ovine Adenovirus Vectors Overcome Preexisting Humoral Immunity against Human Adenoviruses In Vivo

    Hofmann, Christian; Löser, Peter; Cichon, Günter; Arnold, Wolfgang; Both, Gerald W.; Strauss, Michael

    1999-01-01

    Recombinant human adenoviruses (hAd) have become widely used as tools to achieve efficient gene transfer. However, successful application of hAd-derived vectors in clinical trials is limited due to immunological and potential safety problems inherent in their human origin. In this study, we describe a recombinant ovine adenovirus (OAV) as an alternative vector for gene transfer in vivo. In contrast to an hAd vector, the OAV vector was not neutralized by human sera. An OAV vector which contain...

  2. Selection of ovine oocytes by brilliant cresyl blue staining.

    Wang, Liqin; Lin, Jiapeng; Huang, Juncheng; Wang, Jing; Zhao, Yuncheng; Chen, Tong

    2012-01-01

    Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003), Madison et al. (1992) and De Loos et al. (1992)). BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB-) are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+) due to insuffcient G6PD activity to decolorize the BCB dye. PMID:22675245

  3. Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

    Hansen Peter J

    2008-01-01

    Full Text Available Abstract Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS, inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3 cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes. Results Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 ?g/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin. Conclusion Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

  4. Identification of a novel miRNA from the ovine ovary by a combinatorial approach of bioinformatics and experiments.

    Chang, Weihua; Wang, Juanhong; Tao, Dayong; Zhang, Yong; He, Jianzhong; Shi, Changqing

    2016-01-01

    MicroRNAs (miRNAs) are a class of short endogenous, single-stranded, non-coding small RNA molecules, about 19-25 nucleotides in length that regulate gene expression at the translation level and influence many physiological process, such apoptosis, metabolism, signal transduction, and occurrence and development of diseases. In this study, we constructed a library from the ovine luteal phase ovary by using next-generation sequencing technology (Solexa high-throughput sequencing technique) and identified 267 novel miRNAs by bioinformatics. One of the novel miRNAs (ovis_aries_ovary-m0033_3p), which expressed in the sheep ovary and testis, was confirmed by real time PCR and northern blot. Ovis_aries_ovary-m0033_3p was 21 nucleotides in length and located on chromosome 12, and it had 100% similarity to hsa-miR-214-3p, mmu-miR-214-3p, dre-miR-214and ssc-miR-214. Meanwhile, the pre-miRNA was 82 nucleotides in length and had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_ovary-m0033_3p had a function similar to hsa-miR-214-3p, which is involved in the fine regulation of cell survival, embryonic development, breeding activities and resistance to ovarian cancer, so we defined it as oar-miR-214-3p. These experimental results will enrich the miRNA database for ovis aries and provide the basis for researching the regulation mechanism of miRNA in relation to breeding activities of seasonal breeding animals. PMID:26268666

  5. Identification of a novel miRNA from the ovine ovary by a combinatorial approach of bioinformatics and experiments

    CHANG, Weihua; WANG, Juanhong; TAO, Dayong; ZHANG, Yong; HE, Jianzhong; SHI, Changqing

    2015-01-01

    MicroRNAs (miRNAs) are a class of short endogenous, single-stranded, non-coding small RNA molecules, about 1925 nucleotides in length that regulate gene expression at the translation level and influence many physiological process, such apoptosis, metabolism, signal transduction, and occurrence and development of diseases. In this study, we constructed a library from the ovine luteal phase ovary by using next-generation sequencing technology (Solexa high-throughput sequencing technique) and identified 267 novel miRNAs by bioinformatics. One of the novel miRNAs (ovis_aries_ovary-m0033_3p), which expressed in the sheep ovary and testis, was confirmed by real time PCR and northern blot. Ovis_aries_ovary-m0033_3p was 21 nucleotides in length and located on chromosome 12, and it had 100% similarity to hsa-miR-214-3p, mmu-miR-214-3p, dre-miR-214and ssc-miR-214. Meanwhile, the pre-miRNA was 82 nucleotides in length and had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_ovary-m0033_3p had a function similar to hsa-miR-214-3p, which is involved in the fine regulation of cell survival, embryonic development, breeding activities and resistance to ovarian cancer, so we defined it as oar-miR-214-3p. These experimental results will enrich the miRNA database for ovis aries and provide the basis for researching the regulation mechanism of miRNA in relation to breeding activities of seasonal breeding animals. PMID:26268666

  6. Spontaneous metastasis in congenic mice with transgenic breast cancer is unaffected by plasminogen gene ablation

    Almholt, Kasper; Juncker-Jensen, Anna; Lrum, Ole Didrik; Johnsen, Morten; Rmer, John; Lund, Leif R.

    2013-01-01

    Plasminogen (Plg) plays a central role in tissue remodeling during ontogeny, development, and in pathological tissue remodeling following physical injury, inflammation and cancer. Plg/plasmin is, however, not critical for these processes, as they all occur to a varying extent in its absence, sugg...... effect of a known polymorphic metastasis modifier gene region located adjacent to the Plg gene....... been shown to synergistically inhibit other tissue remodeling events in Plg-deficient mice. While metalloprotease inhibition dramatically reduced metastasis, we found no effect of Plg deficiency on metastasis, either independently or in combination with metalloprotease inhibition. We further show that...... Plg gene deficiency is of no significant consequence in this metastasis model, when analyzed in two different congenic strains: the FVB strain, and a F1 hybrid of the FVB and C57BL/6J strains. We suggest that the extensive backcrossing performed prior to our studies has eliminated the confounding...

  7. High fidelity screening of regulatory sequences in apolipoprotein(a)-plasminogen cluster.

    Lv, Xiang; Shi, Hong-Zhe; Liu, De-Pei; Hao, De-Long; Zhang, Shen; Xin, Li; Li, Xing-Guo; Xu, Hai-Ming; Liang, Chih-Chuan

    2005-09-01

    Identifying networks of gene expression regulation is one of the major tasks in the post-genomic era, this demands firstly high throughput identification of regulatory elements. Apolipoprotein(a)-plasminogen cluster is closely related to both atherosclerosis and thrombosis, and forms a link between the two systems. The mechanism regulating expression of this cluster, through which the balance between atherosclerosis and thrombosis is achieved, is far from been fully understood. Polymerase chain reaction based strategies for screening of regulatory sequences are both simple and efficient yet face severe problems of contamination and sequence mutations. In this work, entity self-competition electrophoresis mobility shift assay and differential library hybridization are introduced to overcome the problems. Cis-element candidates are picked out from a library generated from the screening region, thus avoiding the effect of repetitive amplifications. Sequence fidelity of the selected fragments is guaranteed and one by one identification of each fragment by self-competition is unnecessary. Using this method, 32 potential regulatory elements in apolipoprotein(a)-plasminogen gene cluster were found, including three of the four known DNase I hypersensitive sites in the cluster, attesting the efficiency of our method. We also find an intron-locating element, which is repeatedly screened out. A new in vivo electrophoresis mobility shift assay method based on in situ digestion of crosslinked chromatin DNA and southern hybridization is established for the detection of in vivo protein binding status in the intron region. The result supports our hypothesis that the element may play an important role in expression regulation or evolution of apolipoprotein(a) gene. PMID:15905115

  8. In silico analysis of regulatory and structural motifs of the ovine HSP90AA1 gene.

    González, Carmen; Salces-Ortiz, Judit; Calvo, Jorge H; Serrano, M Magdalena

    2016-05-01

    Gene promoters are essential regions of DNA where the transcriptional molecular machinery to produce RNA molecules is recruited. In this process, DNA epigenetic modifications can acquire a fundamental role in the regulation of gene expression. Recently, in a previous work of our group, functional features and DNA methylation involved in the ovine HSP90AA1 gene expression regulation have been observed. In this work, we report a combination of methylation analysis by bisulfite sequencing in several tissues and at different developmental stages together with in silico bioinformatic analysis of putative regulating factors in order to identify regulative mechanisms both at the promoter and gene body. Our results show a "hybrid structure" (TATA box + CpG island) of the ovine HSP90AA1 gene promoter both in somatic and non-differentiated germ tissues, revealing the ability of the HSP90AA1 gene to be regulated both in an inducible and constitutive fashion. In addition, in silico analysis showed that several putative alternative spliced regulatory motifs, exonic splicing enhancers (ESEs), and G-quadruplex secondary structures were somehow related to the DNA methylation pattern found. The results obtained here could help explain the differences in cell-type transcripts, tissue expression rate, and transcription silencing mechanisms found in this gene. PMID:26810179

  9. The neuropathologic phenotype of experimental ovine BSE is maintained after blood transfusion.

    Sisó, Sílvia; González, Lorenzo; Houston, Fiona; Hunter, Nora; Martin, Stuart; Jeffrey, Martin

    2006-07-15

    Iatrogenic transmission by blood transfusion has been described in cases of human variant Creutzfeldt-Jakob disease (vCJD), experimental ovine bovine spongiform encephalopathy (BSE), and natural sheep scrapie, demonstrating that blood in these prion diseases is infectious. However, the possible effect of the transfusion, derived from differences in the inoculum (blood) and the route of infection (intravenous), on the pathologic phenotype of the disease in the recipients is not known. This study describes the neuropathologic phenotype of PrP(d) accumulation in sheep succumbing to neurologic disease after blood transfusion from donors experimentally infected with BSE; these were either clinically or subclinically affected at the time of donation. We demonstrate that blood can become infectious at early stages of ovine BSE infection and that the PrP(d) immunohistochemical phenotype is maintained after transfusion. This suggests that a change in the pathologic phenotype of vCJD would not be expected as a result of exposure to infected blood. PMID:16569766

  10. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues.

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-05-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism. PMID:26954186

  11. Prevalence and risk factors associated to ovine toxoplasmosis in northeastern Brazil.

    Mendonça, Carlos Eduardo D'Alencar; Barros, Sílvia Letícia Bomfim; Guimarães, Vitor Andrade Accioly; Ferraudo, Antonio Sergio; Munhoz, Alexandre Dias

    2013-01-01

    In this study, we aimed to determine the prevalence and risk factors associated to Toxoplasma gondii infection in sheep from northeastern Brazil. A total of 932 ovine serum samples from 54 properties in 19 municipalities of the state of Sergipe were collected and assayed using indirect fluorescent antibody test. The assay used antibodies against Toxoplasma gondii (IFAT-IgG) with a cutoff point of 1:64. We observed that 28.22% (263/932) of the ovine samples were serum-reactive. In a logistic regression, factors such as consumption of water directly from the source, consumption of water from a deep well, and age below 12 months were associated with protection; whereas factors such as presence of cats on the property, presence of slatted floor, and use of exchanged or borrowed breeding males were associated with infection. The studied area can be considered endemic for toxoplasmosis, so it is necessary to adopt preventive and control measures because this zoonotic infection poses risks to public health. PMID:23856735

  12. Application of small angle X-ray scattering synchrotron technology for measuring ovine meat quality.

    Hoban, J M; Hopkins, D L; Kirby, N; Collins, D; Dunshea, F R; Kerr, M G; Bailes, K; Cottrell, J J; Holman, B W B; Brown, W; Ponnampalam, E N

    2016-07-01

    A small angle X-ray scattering (SAXS) synchrotron was used to evaluate 100 ovine m. longissimus lumborum, representing lamb (n=50) and sheep (n=50). The diffraction of X-rays gives information on muscle myofibril structure and fat content. The linear relationships between SAXS measures with measures such as, shear force, intramuscular fat content (IMF) and collagen content/solubility, were investigated. A relationship was found between the d-spacing of the actin/myosin fibril spacing (SAX1 and SAX2) and the cross sectional area of the rhombohedral unit cell (Cell area) and shear force after 1 and 5day ageing. There was a positive relationship between IMF and a SAXS Fat area measure. There was a muscle site effect on SAX1, SAX2 and Cell area, with the cranial site having a larger distance between myofibrils. The potential of SAXS as a powerful research tool to determine not only the structural components of ovine tenderness, but also the fat content related to IMF is evident. PMID:26971308

  13. Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria.

    Secchi, Nicola; Giunta, Daniela; Pretti, Luca; García, Mónica Ruiz; Roggio, Tonina; Mannazzu, Ilaria; Catzeddu, Pasquale

    2012-01-01

    Scotta is the main by-product in the making of ricotta cheese. It is widely produced in southern Europe and particularly in Italy where it represents a serious environmental pollutant due to its high lactose content. With the aim of evaluating whether scotta bioconversion into lactic acid can be considered as an alternative to its disposal, besides providing it with an added value, here the growth, fermentative performances, and lactic acid productions of pure and mixed cultures of Lactobacillus casei, Lactobacillus helveticus, and Streptococcus thermophilus were evaluated on ovine scotta-based media, without and with the addition of nutritional supplements. The outcomes indicate that ovine scotta can be utilized for the biotechnological production of lactic acid with yields up to 92%, comparable to those obtained on cheese-whey. Indeed, the addition of nutritional supplements generally improves the fermentative performances of lactic acid bacteria leading to about 2 g l(-1) h(-1) of lactic acid. Moreover, the use of mixed cultures for scotta bioconversion reduces the need for nutritional supplements, with no detrimental effects on the productive parameters compared to pure cultures. Finally, by using L. casei and S. thermophilus in pure and mixed cultures, up to 99% optically pure L: -lactic acid can be obtained. PMID:21739193

  14. Determination of imidocarb residues in bovine and ovine liver and milk by immunobiosensor.

    Traynor, I M; Thompson, C S; Armstrong, L; Fodey, T; Danaher, M; Jordan, K; Kennedy, D G; Crooks, S R H

    2013-01-01

    Imidocarb (IMD) is a veterinary drug that has been used for more than 30 years to treat and prevent parasitic diseases. Pharmacokinetic studies have shown that substantial levels of IMD residues are retained in the edible tissues and milk of cattle and sheep for up to 6 months after administration. This has led to concern regarding the potential adverse effects posed through human consumption of edible tissue or milk from treated animals if the recommended withdrawal periods for the drug are not properly implemented. While MRLs have been established by the European Union, it is important that analytical methods are available to monitor food samples for potentially violative levels of IMD residues. A qualitative biosensor-based immunoassay was developed to allow the detection of IMD at less than the European Union MRLs of 50 μg kg(-1) for milk and 2 mg kg(-1) for bovine and ovine liver. Validation of the developed methods provided a detection capability of <25 μg kg(-1) in milk and <0.75 mg kg(-1) in liver. A comparison study was undertaken, with IMD incurred milk and ovine liver samples analysed by the newly developed procedures and results compared with those obtained by LC-MS/MS. The newly developed screening method was applied to both incurred milk and liver samples. This faster, cheaper and reliable screening method has potential use in sample analysis to ensure compliance with legislative requirements. PMID:23656364

  15. Experimental ELISA for diagnosis of ovine dicrocoeliosis and application in a field survey.

    Broglia, A; Heidrich, J; Lanfranchi, P; Nckler, K; Schuster, R

    2009-03-01

    Dicrocoeliosis is a parasitic disease caused by trematodes belonging to the genus Dicrocoelium. It usually produces mild symptoms, and for this reason, dicrocoeliosis often remains undetected. Its diagnosis is mostly based on postmortem examination of the liver or on coprological assays for in vivo diagnosis. However, the latter method has scant sensitivity and because of the long pre-patency of Dicrocoelium spp. only permits late diagnosis. In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed based on excretory/secretory antigen of Dicrocoelium dendriticum. The ELISA detected antibodies (IgG) in experimental infected sheep starting from day 30 post-infection (d.p.i.), whereas coprological samples were positive from 58 d.p.i. The ELISA was used in a field study in order to assess dicrocoeliosis seroprevalence in ovine flocks from the province of Trento (northeastern Italy), and this was the first sero-epidemiological study of ovine dicrocoeliosis in northern Italy. Altogether, 842 sheep sera were tested. In accordance with previous surveys carried out in other regions of Italy, a high prevalence of 80% to 100% was found. PMID:19107521

  16. Different Culture Media Affect Proliferation, Surface Epitope Expression, and Differentiation of Ovine MSC

    Tolba, René; Jahnen-Dechent, Wilhelm; Lethaus, Bernd; Neuss, Sabine

    2013-01-01

    Orthopedic implants including engineered bone tissue are commonly tested in sheep. To avoid rejection of heterologous or xenogeneic cells, autologous cells are preferably used, that is, ovine mesenchymal stem cells (oMSC). Unlike human MSC, ovine MSC are not well studied regarding isolation, expansion, and characterization. Here we investigated the impact of culture media composition on growth characteristics, differentiation, and surface antigen expression of oMSC. The culture media varied in fetal calf serum (FCS) content and in the addition of supplements and/or additional epidermal growth factor (EGF). We found that FCS strongly influenced oMSC proliferation and that specific combinations of supplemental factors (MCDB-201, ITS-plus, dexamethasone, and L-ascorbic acid) determined the expression of surface epitopes. We compared two published protocols for oMSC differentiation towards the osteogenic, adipogenic, and chondrogenic fate and found (i) considerable donor to donor variations, (ii) protocol-dependent variations, and (iii) variations resulting from the preculture medium composition. Our results indicate that the isolation and culture of oMSC in different growth media are highly variable regarding oMSC phenotype and behaviour. Furthermore, variations from donor to donor critically influence growth rate, surface marker expression, and differentiation. PMID:24228035

  17. Peripheral Ovine Progressive Pneumonia Provirus Levels Correlate with and Predict Histological Tissue Lesion Severity in Naturally Infected Sheep

    Studies were undertaken to determine whether host immune responses in the form of serum anti-ovine progressive pneumonia virus (OPPV) antibody responses or virus replication in the form of peripheral OPP provirus levels associate with the degree of histological tissue lesions in naturally OPPV infec...

  18. Ovine progressive pneumonia provirus levels are unaffected by the prion 171R allele in an Idaho sheep flock

    Selective breeding of sheep for arginine (R) at prion gene (PRNP) codon 171 confers resistance to Scrapie. The 171R allele naturally occurs at low frequency, possibly because it imparts a deleterious trait such as impaired disease resistance. Ovine Progressive Pneumonia/Maedi-Visna virus (OPPV) is...

  19. OXIDATION OF GLUCOSE, GLUTAMATE, AND GLUTAMINE BY ISOLATED OVINE ENTEROCYTES IN VITRO IS DECREASED BY PRESENCE OF OTHER METABOLIC FUELS

    The objective of this study was to evaluate oxidative metabolism of glucose, glutamate, and glutamine by isolated ovine enterocytes in presence of other metabolic fuels in vitro. Mucosal enterocytes were isolated from crossbred wether sheep (n=6) fed a mixed forage-concentrate diet, and incubated f...

  20. Activity-dependent release of precursor nerve growth factor, conversion to mature nerve growth factor, and its degradation by a protease cascade

    Bruno, Martin A.; Cuello, A Claudio

    2006-01-01

    In this report, we provide direct demonstration that the neurotrophin nerve growth factor (NGF) is released in the extracellular space in an activity-dependent manner in its precursor form (proNGF) and that it is in this compartment that its maturation and degradation takes place because of the coordinated release and the action of proenzymes and enzyme regulators. This converting protease cascade and its endogenous regulators (including tissue plasminogen activator, plasminogen, neuroserpin,...