Academic literature on the topic 'In vitro antithrombotic properties'
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Journal articles on the topic "In vitro antithrombotic properties"
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Vasconcelos, Ariana, Isabela Sucupira, Alessandra Guedes, Ismael Queiroz, Flavia Frattani, Roberto Fonseca, and Vitor Pomin. "Anticoagulant and Antithrombotic Properties of Three Structurally Correlated Sea Urchin Sulfated Glycans and Their Low-Molecular-Weight Derivatives." Marine Drugs 16, no. 9 (August 30, 2018): 304. http://dx.doi.org/10.3390/md16090304.
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The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from Strongylocentrotus franciscanus was observed to be poorly active in most assays, the 4-sulfated fucan from Lytechinus variegatus, the 2-sulfated galactan from Echinometra lucunter and their derivatives showed multiple effects. All marine compounds showed no capacity to activate factor XII and similar low bleeding tendencies regardless of the dose concentrations used to achieve the highest antithrombotic effect observed. The 2-sulfated galactan showed the best combination of results. Our work improves the background about the structure-function relationship of the marine sulfated glycans in anticoagulation and antithrombosis. Besides confirming the negative effect of the 2-sulfated fucose and the positive effect of the 2-sulfated galactose on anticoagulation in vitro, our results also demonstrate the importance of this set of structural requirements on antithrombosis in vivo, and further support the involvement of high-molecular weight and 4-sulfated fucose in both activities.
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Tsoupras, Alexandros, Ronan Lordan, Katie Shiels, Sushanta Saha, Constantina Nasopoulou, and Ioannis Zabetakis. "In Vitro Antithrombotic Properties of Salmon (Salmo salar) Phospholipids in a Novel Food-Grade Extract." Marine Drugs 17, no. 1 (January 18, 2019): 62. http://dx.doi.org/10.3390/md17010062.
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Marine and salmon polar lipids (PLs) extracted by conventional extractions with non-food-grade solvents (CE-salmon-PLs) possess antithrombotic bioactivities against platelet-activating factor (PAF) and thrombin. Similar effects of food-grade-extracted (FGE) marine PLs have not yet been reported. In this study, food-grade solvents were used to extract PLs from Irish organic farmed salmon (Salmo salar) fillets (FGE-salmon-PLs), while their antithrombotic bioactivities were assessed in human platelets induced by platelet aggregation agonists (PAF/thrombin). FGE-salmon-PLs were further separated by thin layer chromatography (TLC) into lipid subclasses, and the antithrombotic bioactivities of each subclass were also assessed. LC-MS was utilized to elucidate the structure-activity relationships. FGE-salmon-PLs strongly inhibited PAF-induced platelet aggregation, while their relevant anti-thrombin effects were at least three times more potent than the previously reported activities of CE-salmon-PLs. TLC-derived lipid fractions corresponding to phosphatidylcholines (PC) and phosphatidylethanolamines (PE) were the most bioactive lipid subclasses obtained, especially against thrombin. Their LC-MS analysis elucidated that they are diacyl- or alkyl-acyl- PC and PE moieties baring ω3 polyunsaturated fatty acids (PUFA) at their sn-2 position, such as eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Our results concerning the potent antithrombotic effects of FGE-salmon-PLs against both PAF and thrombin pathways strongly suggest that such food-grade extracts are putative candidates for the development of novel cardioprotective supplements and nutraceuticals.
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Merton, R. E., and D. P. Thomas. "Experimental Studies on the Relative Efficacy of Dermatan Sulphate and Heparin as Antithrombotic Agents." Thrombosis and Haemostasis 58, no. 03 (1987): 839–42. http://dx.doi.org/10.1055/s-0038-1646001.
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SummaryIn this study, the anticoagulant and antithrombotic properties of unfractionated heparin (UFH) and dermatan sulphate (DS) were compared. The ability of UFH and DS to impair thrombin generation in vitro and in ex vivo plasma samples was also studied. DS has minimal anticoagulant activity by conventional assays but impairs thrombin generation both in vitro and in ex vivo plasma samples. However, thrombin generation could not be suppressed below about 35% of control values at all doses of DS studied. While this was sufficient to impair experimental venous thrombosis during 10 minutes’ stasis, DS was ineffective in preventing thrombosis following 20 minutes’ stasis in doses up to 1.25 mg/kg. In contrast, 1 μg/ml of UFH completely suppressed thrombin generation in vitro, and 150 μg/kg prevented throm- bogenesis over a period of 20 minutes’ stasis. Neither drug prolonged the bleeding time (BT) at effective antithrombotic doses, but 2.5 mg/kg UFH significantly increased the BT, whereas DS did not. While DS has antithrombotic activity, it is less effective than UFH in inhibiting thrombin generation, and as an antithrombotic agent.
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Lordan, Ronan, Eoin O’Keeffe, Darren Dowling, Michael Mullally, Hannah Heffernan, Alexandros Tsoupras, and Ioannis Zabetakis. "The in vitro antithrombotic properties of ale, lager, and stout beers." Food Bioscience 28 (April 2019): 83–88. http://dx.doi.org/10.1016/j.fbio.2019.01.012.
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Nestele, Jeremy A., Anne-Katrin Rohlfing, Valerie Dicenta, Alexander Bild, Daniela Eißler, Frederic Emschermann, Marcel Kremser, et al. "Characterization of GPVI- or GPVI-CD39-Coated Nanoparticles and Their Impact on In Vitro Thrombus Formation." International Journal of Molecular Sciences 23, no. 1 (December 21, 2021): 11. http://dx.doi.org/10.3390/ijms23010011.
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Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.
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Chung, Amy, Frederick A. Ofosu, and Morris A. Blajchman. "The Antithrombotic Properties of Human Prothrombin Fragment 1.2 in Mice." Thrombosis and Haemostasis 63, no. 03 (1990): 413–16. http://dx.doi.org/10.1055/s-0038-1645057.
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SummaryWe have investigated the antithrombotic properties of prothrombin fragment 1.2 (F1.2) in this study. To do this, we established the minimum concentration of human placental tissue factor or human a-thrombin that was lethal in mice within 5 min after intravenous injection. Prothrombin F1.2 protected the mice from the lethal effect of tissue factor or α-thrombin in a dose dependent manner, with 500 μg (14 nmoles) of prothrombin F1.2 per mouse being the minimum amount required to protect all mice from the lethal effect of either thrombogenic stimulus. The minimum dose of heparin which protected mice from the lethal effect of thrombin or tissue factor was 6 units or ∼3.3 nmoles. The observation that prothrombin F1.2 has antithrombotic properties suggests prothrombin F1.2 can modulate coagulation in vivo, as it has previously been shown to do in vitro.
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Schwarz, Richard P., Jean-Claude P. Becker, R. Lynn Brooks, Marcie J. Hursting, James L. Joffrion, Gary D. Knappenberger, Timothy P. Kogan, Patricia W. Kogan, and Anthony A. McKinney. "State-of-the-Art Review: The Preclinical and Clinical Pharmacology of Novastan (Argatroban): A Small-Molecule, Direct Thrombin Inhibitor." Clinical and Applied Thrombosis/Hemostasis 3, no. 1 (January 1997): 1–15. http://dx.doi.org/10.1177/107602969700300101.
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Because of the unsatisfactory options available for safe and effective antithrombotic therapy, recent, intense research and development efforts have been focused on direct thrombin inhibitors, also known as site-directed thrombin inhibitors. The intravenous agent Novastan (argatroban) is a small-molecule, reversible, direct thrombin inhibitor that is selective for the catalytic site of the thrombin molecule. Argatroban's molecular properties (small molecule; fast, selective, and reversible inhibition of the thrombin catalytic site; and similar in vitro potency for inhibiting both clot-bound and soluble thrombin) offer the potential for significant antithrombotic efficacy with minimal systemic anticoagulant ef fects. Its clinical pharmacologic properties offer the potential for minimal risk of bleeding, very rapid achievement of therapeutic antithrombotic efficacy, predictable dose-response, and rapid restoration of the hemostatic systems to normal upon termination of intravenous infusion. Argatroban is currently approved for clinical use in Japan for the treatment of peripheral arterial occlusive disease. It is in advanced clinical development in North America, South America, and Western Europe for several clinical indications, including (1) adjunctive therapy to thrombolytic agents in the treatment of acute myocardial infarction and (2) antithrombotic therapy for patients with heparin-induced thrombocytopenia and heparin-induced thrombocytopenia and thrombosis syndrome. Key Words: Molecular properties—Novastan (argatroban)—Pharmacology—Thrombin inhibitor.
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Boulaftali, Yacine, Frédéric Adam, Laurence Venisse, Véronique Ollivier, Benjamin Richard, Sabrina Taieb, Denis Monard, et al. "Anticoagulant and antithrombotic properties of platelet protease nexin-1." Blood 115, no. 1 (January 7, 2010): 97–106. http://dx.doi.org/10.1182/blood-2009-04-217240.
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AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.
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Sie, P., D. Dupouy, C. Caranobe, M. Petitou, and B. Boneu. "Antithrombotic properties of a dermatan sulfate hexadecasaccharide fractionated by affinity for heparin cofactor II." Blood 81, no. 7 (April 1, 1993): 1771–77. http://dx.doi.org/10.1182/blood.v81.7.1771.1771.
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Abstract The relationship between the antithrombotic activity of dermatan sulfate (DS) in vivo and its catalytic effect on the inhibition of thrombin by heparin cofactor II (HC II) in vitro was investigated. DS was depolymerized by Smith degradation and the fragments obtained were separated by gel filtration. The fragment of minimal size with full catalytic activity was a hexadecasaccharide, which was further fractionated by affinity for immobilized HC II. Only a small proportion by weight (6.7%) was recovered in the high-affinity fraction, which had about 10 times more catalytic activity than the unfractionated oligosaccharide; the change in activity was primarily caused by the removal of inert materials, recovered in the low-affinity fraction. 1H- NMR spectra indicated strengthening of the signal given by Ido A (2S04) in the high-affinity fraction compared with that of the low-affinity fraction. The anticoagulant activity of the high-affinity fraction was exclusively HC II-dependent. The antithrombotic potency was evaluated in rabbits using the Wessler-thromboplastin model. Half-maximal prevention of thrombosis was obtained after injection of 250 micrograms/kg DS, of 500 micrograms/kg hexadecasaccharide, or of 60 micrograms/kg of its high-affinity fraction. The low-affinity fraction was ineffective at the highest dose tested (1,200 micrograms/kg) and did not potentiate the effect of the high-affinity fraction. These results show that the antithrombotic effect of DS is essentially dependent on HC II binding and activation and that HC II is therefore a suitable target for antithrombotic drugs.
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Sie, P., D. Dupouy, C. Caranobe, M. Petitou, and B. Boneu. "Antithrombotic properties of a dermatan sulfate hexadecasaccharide fractionated by affinity for heparin cofactor II." Blood 81, no. 7 (April 1, 1993): 1771–77. http://dx.doi.org/10.1182/blood.v81.7.1771.bloodjournal8171771.
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The relationship between the antithrombotic activity of dermatan sulfate (DS) in vivo and its catalytic effect on the inhibition of thrombin by heparin cofactor II (HC II) in vitro was investigated. DS was depolymerized by Smith degradation and the fragments obtained were separated by gel filtration. The fragment of minimal size with full catalytic activity was a hexadecasaccharide, which was further fractionated by affinity for immobilized HC II. Only a small proportion by weight (6.7%) was recovered in the high-affinity fraction, which had about 10 times more catalytic activity than the unfractionated oligosaccharide; the change in activity was primarily caused by the removal of inert materials, recovered in the low-affinity fraction. 1H- NMR spectra indicated strengthening of the signal given by Ido A (2S04) in the high-affinity fraction compared with that of the low-affinity fraction. The anticoagulant activity of the high-affinity fraction was exclusively HC II-dependent. The antithrombotic potency was evaluated in rabbits using the Wessler-thromboplastin model. Half-maximal prevention of thrombosis was obtained after injection of 250 micrograms/kg DS, of 500 micrograms/kg hexadecasaccharide, or of 60 micrograms/kg of its high-affinity fraction. The low-affinity fraction was ineffective at the highest dose tested (1,200 micrograms/kg) and did not potentiate the effect of the high-affinity fraction. These results show that the antithrombotic effect of DS is essentially dependent on HC II binding and activation and that HC II is therefore a suitable target for antithrombotic drugs.
Dissertations / Theses on the topic "In vitro antithrombotic properties"
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Aggarwal, Rajesh Kumar. "The antithrombotic properties of polymer-coated, drug eluting coronary stents." Thesis, University of Leicester, 1996. http://hdl.handle.net/2381/34296.
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Mnonopi, Nandipha Olivia. "In vitro testing to investigate the anticoagulant/antithrombotic and antidiabetic biological activity of Leonotis Leonurus." Thesis, Nelson Mandela Metropolitan University, 2007. http://hdl.handle.net/10948/693.
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The rising costs of prescription drugs in the maintenance of personal health and wellbeing have increased the interest in medicinal plants. The World Health Organization estimates that 65 percent-80 percent of the world’s population use traditional medicine as their primary form of health care. In this project the focus has been on the use of Leonotis leonurus extracts as a traditional medicine. The major chemical constituent of this plant is marrubiin, which is a diterpenoid labdane lactone formed from a precursor called premarrubiin. Aqueous and acetone extract (AL and OL extract, respectively) of this plant has been found to have an antithrombotic effect, with IC50 values of 3mg/ml and 6mg/ml, respectively. The extracts also have an effect on fibrinolysis, where the lysis time was decreased by more than 50 percent by the organic extract and standard marrubiin. In whole blood ADP-induced platelet aggregation, the organic extract inhibited aggregation by 68 percent at a final concentration of 138μg/ml (equivalent to 7.2μg/ml marrubiin). Marrubiin has also been screened for antithrombotic/anticoagulant activity; no antithrombotic activity has been observed but it increased the rate of fibrinolysis, by decreasing lysis time by 64 percent and also decreasing fibrin formation. From these findings it can be concluded that marrubiin has a fibrinolytic effect and antiplatelet aggregation effect. In the diabetic studies, in hyperglycemic condition, the OL (10μg/ml) extract and standard marrubiin significantly increased insulin secretion by 200 percent (2-fold) and 400 percent (4-fold), respectively, with respect to the control. The OL extract and standard marrubiin stimulated the release of insulin, the stimulatory index was significantly increased by 450 percent (4.5-fold) and 500 percent (5-fold), respectively, with respect to the control. In the apoptotic studies, in the normoglycemic and hyperglycemic conditions, the OL extract decreased the occurrence of apoptosis, in a dose-dependent manner, with the lower concentrations inducing apoptosis significantly higher than the relevant controls. Standard marrubiin did not have an effect on apoptosis in hyperglycemic condition, but it decreased the occurrence of apoptosis by 200 percent (2-fold) under normoglycemic conditions. The OL extract increased proliferation by 148 percent (1.48- fold) and 155 percent (1.55-fold) in normoglycemic and hyperglycemic conditions, respectively. The same effect was observed for standard marrubiin, where, proliferation was increased by 180 percent (1.8-fold) and 200 percent (2.0-fold) in normoglycemic and hyperglycemic conditions, respectively. RT-PCR displayed that standard marrubiin inhibited the expression of insulin by 50 percent under normoglycemic conditions.
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Hartley, Richard William. "The development of an in-vivo method for assessing the antithrombotic properties of pharmaceutical compounds." Thesis, Loughborough University, 1989. https://dspace.lboro.ac.uk/2134/12084.
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The formation of a thrombus stems from the malfunction of a normal physiological function referred to as haemostasis and the activity of blood platelets; such thrombi give rise to debilitating and often fatal strokes. Consequently much effort is associated with the search for pharmacological compounds capable of their prevention or dispersion. · Most of the primary screens associated with such work rely on in-vitro tests and in separating the blood from it's vasculature, the influence and results associated with several naturally occuring moderators may be lost. There therefore exists the incentive to develop more representative in-vivo screening methods. Following an introduction to the underlying physiology and pharmacology and a review of established screening methods, this thesis proceeds to describe the development of a novel technique suitable for such in-vivo studies. It's inception is shown to be a consequence of an amalgamation of ultrasonic methods associated with the clinical detection of occlusions and laser Doppler velocimetry. Both topics are individually surveyed and then brought together through a concept whereby the efficacy of compounds might be evaluated in animal models by measuring the velocity of blood in the fluid jet formed distal to an induced thrombus.The main underlying assumption is that the jet velocity will reflect the degree of encroachment of the thrombus into the vasculature. In accord with the evolved measurement rationale there then follows a description of a specific laser Doppler velocimeter and some associated experiments, designed to qualitatively appraise the validity of the underlying assumptions. The ensuing results in turn give rise to the design of a laser Doppler microscope, an analyser for extracting the required velocity information from the Doppler shift spectrum and an additional series of experiments. Central to this latter stage of validation is the use of a thrombus analogue in a narrow bored glass flow tube. Finally, some preliminary in-vivo experiments and results are presented.
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Lai, Benjamin Fook Lun. "Bioactive polymers : a comparative study on the antithrombotic properties of soluble polymers and surface grafted polymers." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/20871.
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Use of synthetic materials in medical applications is one of the most common practices in modern medicine. Yet occurrence of surface-induced thrombus formation on these materials, especially those associated with cardiovascular applications, generates a need for surface modifications. Limiting thrombus formation on a biomaterial surface represents the ultimate success for blood contacting devices. One interesting approach is to enhance fibrinolysis before the blood clot becomes stabilized. Herein, two synthetic polymers, poly-N- [(2, 2-dimethyl-1, 2-dioxolane) methyl] acrylamide (PDMDOMA) and poly- (N-isopropylacrylamide) (PNIPAm), were tested for this particular antithrombotic property. Surface-grafted PNIPAm samples, brush-PNIPAm and star-PNIPAm, were also tested for the biological activity.
We evaluated the influence of these synthetic polymers on blood hemostasis by studying the fibrin polymerization process, the three-dimensional clot structure, and the mechanical properties of blood clot such as its clot strength, clot elasticity and clot fibrinolysis. Both linear PDMDOMA and PNIPAm altered the normal fibrin polymerization by changing the rate of protofibril aggregation and resulting in a 5-fold increase in the overall turbidity. Fibrin clots formed in presence of these synthetic polymers exhibited thinner fibers with less branching and resulted in a more porous and heterogeneous clot structure in scanning electron micrographs. The structural changes in these clots led to significant difference to their mechanical properties. Lower clot strength and clot elasticity were recorded from the thromboelastography study. More interestingly, enhanced clot lysis was measured by thromboelastography when whole blood was clotted in presence of PDMDOMA or PNIPAm. Further evidence of the altered clot structure and clot cross-linking was obtained from the significant decrease in D-dimer levels measured from degraded plasma clot. Similar results were obtained when star-form of PNIPAm was used but not for brush-form PNIPAm.
The antithrombotic activity of soluble PDMDOMA and PNIPAm could potentially lead to the development of novel antithrombotic agents that could enhance endogenous fibrinolytic activity by modulating the fibrin clot structure. In the exploratory analysis of surface grafted PNIPAm (brush-PINPAm), brush-PNIPAm showed that the biological activity of attached chains is quite different from soluble polymers and several parameters need to be optimized to generate an antithrombotic coating for biomaterials.
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Travers, M. "In vitro and clinical investigation of blood-membrane interactions : Influence on platelets and the immune system of membrane structure and antithrombotic agents." Thesis, University of Strathclyde, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382445.
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Huddie, P. L. "The properties of pituitary cells in vitro." Thesis, University of Newcastle Upon Tyne, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379245.
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Sousa, Hiran Reis. "Investigação da atividade anti-agregante plaquetária in vitro de peptídeos inibidores da dissulfeto isomerase protéica - etapa 2." Universidade Federal do Maranhão, 2016. http://tedebc.ufma.br:8080/jspui/handle/tede/1632.
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ)
Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA)
Recent researches have emphasized the importance of redox mechanisms for platelet function modulation. The platelet surface contains a large variety of integrin receptors and other molecules presenting functional thiol groups in their structures, which are potential targets for redox regulation. Among these various thiol-containing proteins, integrin αIIbβ3 stands out for being the convergence path of platelet activation induced by various agonists. Activation of αIIbβ3 integrin is catalyzed by protein disulfide isomerase (PDI) through an essential conformational change leading to the exposure of fibrinogen-binding site. Thus, PDI has been shown to be an important target for the development of antiplatelet drugs. In recent years, many studies have described substances from plan (DE A. PAES et al., 2011), as well as synthetics that are capable of inhibiting PDI. In a previous study of our research group has shown that the synthetic peptide CxxC, which contains the redox motif of PDI in its original sequence CGHC, inhibited reductase activity of this enzyme, effect not observed with AxxA peptide, whose cysteines were replaced with alanine and Scr peptide, which contains the same aminoacids from CxxC peptide, but under random sequence. It has been also demonstrated that CxxC peptide was the only to reduce by 30% ADP-induced aggregation (5μM) in platelet rich plasma, an effect apparently mediated by the association of CxxC and PDI at platelet surface. Thus, in this work, we further assessed the effects of CxxC and its control peptides on platelet aggregation. Washed human platelets were incubated with CxxC peptide at concentrations of 3, 6 and 10 μM, resulting in a dose-dependent inhibition of maximum aggregation activated by thrombin (0.02 U/mL) at 25, 60 and 74%, respectively with IC50 of 6.13 ± 1.09 μM. The presence of control peptides did not produce any inhibitory effect. CxxC peptide also reduced the activation of αIIbβ3 integrin at platelet surface, but did not affect the expression of the markers CD 62-P and CD 63. Control peptides did not alter the expression of these markers. Analysis by mass spectrometry of the interaction of recombinant human PDI with the peptide showed that only CxxC peptide associated with the redox Cys400 of a’ motif of PDI, which has been considered essential for platelet aggregation. Together, these results demonstrate that CxxC peptide reduces platelet aggregation by association with PDI and can be further used as a model for the development of new antithrombotic drugs.
Investigações recentes têm enfatizado a importância de mecanismos redox na modulação da função plaquetária. A superfície da plaqueta contém grande variedade de integrinas e outras moléculas receptoras que possuem tióis funcionais em sua estrutura, os quais são alvos potenciais de regulação redox. Dentre estas várias proteínas tiólicas, a integrina αIIbβ3 destaca-se por ser a via de convergência da ativação plaquetária induzida por diversos agonistas. A ativação da integrina αIIbβ3 é catalisada pela proteína dissulfeto isomerase (PDI), essencial à mudança de conformação que leva à exposição do sitio de ligação ao fibrinogênio. Sendo assim, a PDI tem se mostrado como um alvo importante para o desenvolvimento de fármacos reguladores da agregação plaquetária. Nos últimos anos, diversos estudos têm descrito substâncias de origem vegetal, animal e sintéticas que são capazes de inibir a PDI. Em trabalho do nosso grupo de pesquisa (DE A. PAES et al., 2011), demonstrou que o peptídeo sintético CxxC, o qual contém o motivo redox da PDI na sua sequência original CGHC, inibiu a atividade redutase desta enzima; efeito não observado com os peptídeos AxxA, que possui as cisteínas substituídas por alanina e Scr, peptídeo controle contendo os mesmos aminoácidos do peptídeo CxxC, porém com sequência aleatória sem formação de ditiol. Demonstrou-se, também, que apenas o peptídeo CxxC reduziu em 30% a agregação induzida por ADP (5M) em plasma rico em plaquetas, efeito aparentemente mediado pela associação do CxxC com a PDI na superfície plaquetária. Sendo assim, neste trabalho continuamos a avaliação dos efeitos do peptídeo CxxC e seus controles sobre a agregação plaquetária. Para tanto, incubamos lavado de plaquetas humanas com o peptídeo CxxC nas concentrações de 3, 6 e 10 μM, resultando em inibição concentração-dependente da agregação ativada por trombina (0,02 U/mL) em 25, 60 e 74 %, respectivamente, com IC50 de 6,13 ± 1,09 μM. A presença dos peptídeos controle não produziu quaisquer efeitos inibitórios. O peptídeo CxxC reduziu a ativação da integrina αIIbβ3 na superfície da plaqueta, porém não impactou a expressão dos antígenos CD 62-P e CD 63. Os peptídeos controle não alteraram a expressão desses marcadores. A análise por espectrometria de massas da interação da PDI recombinante humana com os peptídeos, mostrou que apenas o peptídeo CxxC associa-se com a Cys400 do motivo redox a’ da hPDI, o qual tem sido considerado fundamental para a agregação plaquetária. Em conjunto, estes resultados demonstram que o peptídeo CxxC reduz a agregação plaquetária via associação com a PDI, podendo ser empregado como modelo para o desenvolvimento de fármacos novos antitrombogênicos.
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Hammad, Mohammad. "In vitro properties of novel root canal filling materials." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.502267.
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The three dimensional obturation of root canals is vital and cannot be overstated. The attainment of a fluid-tight seal in a root canal system is very important for the longterm success of endodontic treatment. A number of different materials have been used for obturation. Gutta percha is the most common root filling material in use today. It is used as the core material in combination with sealers of different compositions. Despite gutta percha has enjoyed a long history of use and his success rate; it does not offer all of the properties of an ideal root canal filling materials. New root canal filling materials have been developed. These include resin-based (EndoRez and RealSeal systems) and silicone-based (Guttaflow') filling materials. This study aimed to investigate properties of these new root canal filling materials and compare it to gutta percha and a zinc oxide based sealer (TubliSeal).
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Mould, A. P. "The aggregation properties of type I procollagen in vitro." Thesis, University of Manchester, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377670.
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Zhang, Qing. "Role of Polymer Physicochemical Properties on in vitro Mucoadhesion." Scholarly Commons, 2020. https://scholarlycommons.pacific.edu/uop_etds/3698.
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Polymers with mucoadhesive properties are universally used in the development of mucoadhesive drug delivery system. Their physicochemical properties as well as the mechanisms related to their adhesive actions draw great attention for the modification of mucoadhesive properties.
In this study, relationships between physicochemical properties of hydroxypropyl methylcellulose (HPMC) compacts and mucoadhesive performance were investigated. Different commercial grades of HPMC (K3, E3, E5, E50, K4M, E4M and K15M) were prepared into compacts, and their surface hydrophilicity and hydration behavior were characterized. The in vitro mucoadhesive performance was determined by the tension strength between the compacts and different regions of mucous membrane (buccal, sublingual, stomach, and intestine). Positive correlations were found between: (1) viscosity of HPMC compacts and contact angle values measured by different simulated body fluids; (2) viscosity of HPMC compacts and in vitro mucoadhesive force; (3) contact angle values and in vitro mucoadhesive force. The hydration behavior exhibited improvement with the increasing viscosity of HPMC compacts. Moreover, the polar lipid content of each mucosa was likely an important factor affecting the mucoadhesion phenomenon.
Different ratios of ethyl cellulose (EC) was mixed with HPMC grade K15M to form combination compacts for the purpose of modifying the surface property. The mucoadhesive mechanism of both different grades of HPMC compacts and combination compacts were studied via the thermodynamic analysis of Lifshiz-van der Waals interaction and Lewis acid-base interaction. The total free energy of adhesion (〖∆G〗^TOT) provided a prediction of an overall tendency of mucoadhesion, however, the results were showing disagreement with the measured mucoadhesive force. In general, the involving of EC in the combination compacts did not give a boost to the whole mucoadhesive performance.
Books on the topic "In vitro antithrombotic properties"
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Pickering, Anthony Edward. Integrative properties of rat sympathetic preganglionic neurones in vitro. Birmingham: University of Birmingham, 1993.
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Reska, Anna. Elektrophysiologische Charakterisierung neuronaler Netzwerke von Insekten in vitro. Jülich: Forschungszentrum Jülich, 2006.
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Randall, Robert Lawrence Merrill. Sequential dependence of in vitro freeze-drying and irradiation on the biomechanical properties of rat bone. [s.l: s.n.], 1992.
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Wells, Jeffrey Donald. The processing and in vitro degradation properties of gravity sintered calcium polyphosphate powders. Ottawa: National Library of Canada = Bibliothèque nationale du Canada, 1999.
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Lnenicek-Allen, Mirna. Studies of the DNA binding and bending properties of HMG boxes using in vitro mutagenesis. Portsmouth: University of Portsmouth, School of Biological Sciences, 1998.
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Larochelle, Elaine. Comparative "in vitro" study of chemical properties of cavity liners: PH level, calcium and fluoride release measurements. [Toronto: Faculty of Dentistry, University of Toronto], 1989.
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Celotti, Carlo Emilio. The effects of prior heat shock on in vitro muscle contractile properties at 20°C amd 42°C. Ottawa: National Library of Canada, 2003.
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Liu, Siyu. Antithrombotic/antiplatelet properties and mechanism of action of tetramethylpyrazine, including structure-activity relationship studies. 1991.
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Hartley, Richard William. The development of an in-vivo method for assessing the antithrombotic properties of pharmaceutical compounds. 1989.
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Omelon, Sidney Jennifer. Material properties, and in vitro and in vivo degradation of calcium polyphosphate. 2006.
Find full textBook chapters on the topic "In vitro antithrombotic properties"
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Caen, J. P., Y. Legrand, A. Karniguian, D. Leger, C. Soria, J. Soria, and J. Fareed. "In Vitro and in Vivo Antithrombotic Effect of a Collagen-Derived Octapeptide." In Cardiovascular Disease, 277–81. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4684-5296-9_32.
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Osol, George. "Myogenic Properties of Blood Vessels In Vitro." In The Resistance Vasculature, 143–57. Totowa, NJ: Humana Press, 1991. http://dx.doi.org/10.1007/978-1-4612-0403-9_9.
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Traub, Roger D., and Richard Miles. "Some Collective Phenomena in the Hippocampus in Vitro." In Self-Organization, Emerging Properties, and Learning, 97–112. Boston, MA: Springer US, 1991. http://dx.doi.org/10.1007/978-1-4615-3778-6_7.
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Yamagami, Masahito, Shohei Sawada, Toshiyuki Tamagaki, Kyoichiro Kobayashi, Kazuharu Kato, Katsumi Yamamoto, Kaoru Shirai, et al. "Effects of Intracellular pH on the Antithrombotic Properties of Human Vascular Endothelial Cells." In Current Aspects of Blood Coagulation, Fibrinolysis, and Platelets, 175–82. Tokyo: Springer Japan, 1993. http://dx.doi.org/10.1007/978-4-431-68323-0_30.
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Naiki, Hironobu, Keiichi Higuchi, Kazuya Nakakuki, and Toshio Takeda. "Kinetic Properties of Amyloid Fibril Polymerization in Vitro." In Amyloid and Amyloidosis 1990, 493–96. Dordrecht: Springer Netherlands, 1991. http://dx.doi.org/10.1007/978-94-011-3284-8_122.
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Gokcekaya, Ozkan, Kyosuke Ueda, Takayuki Narushima, Kouetsu Ogasawara, and Hiroyasu Kanetaka. "In Vitro Properties of Ag-Containing Calcium Phosphates." In Ceramic Engineering and Science Proceedings, 85–93. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2017. http://dx.doi.org/10.1002/9781119321682.ch10.
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Moore, Anna R., Wen-Liang Zhou, Igor Jakovcevski, Nada Zecevic, and Srdjan D. Antic. "Physiological Properties of Human Fetal Cortex In Vitro." In Isolated Central Nervous System Circuits, 125–58. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-62703-020-5_3.
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Kay, S. A. "In Vitro Protein-DNA Interactions in the Rice Phytochrome Promoter." In Phytochrome Properties and Biological Action, 129–40. Berlin, Heidelberg: Springer Berlin Heidelberg, 1991. http://dx.doi.org/10.1007/978-3-642-75130-1_8.
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Bruch, J., and J. Rosmanith. "Properties of Mixed Mine Dusts and Their Relationship to Pneumoconiosis." In In Vitro Effects of Mineral Dusts, 433–40. Berlin, Heidelberg: Springer Berlin Heidelberg, 1985. http://dx.doi.org/10.1007/978-3-642-70630-1_55.
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Chandy, Thomas, and Chandra P. Sharma. "Urokinase:AT-III:PGE1:Methyl Dopa Complex Immobilized Albumin-Blended Chitosan Membranes — Antithrombotic and Permeability Properties." In Applied Bioactive Polymeric Materials, 297–311. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4684-5610-3_22.
Full textConference papers on the topic "In vitro antithrombotic properties"
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Rehse, K., U. Lukens, S. Leibring, V. Schein, and A. Kesselhut. "ANTITHROMBOTIC OLIGOAMINES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643439.
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We have found that oligoamines of the general formula R2−3X(R=-(CH2)m-NH-(CH2)n-Y) in which X and Y may be aliphatic, alicyc-lic, aromatic or even heterocyclic moieties are a new class of compounds which exhibit platelet aggregation inhibiting and anticoagulant activities in vitro and have antithrombotic properties in vivo. The compound RE 1492 (N,N’,N1’-Tris-4-phenylbutylbenzene-1,3,5-trimethanamine) is chosen as example to demonstrate these effects. In PRP the following IC50 of RE 1492 (inductor in brackets) were measured: 3 ¼mol/L (Collagen), 1 ¼mol/L (ADP, 2ndphase), 7.5 μmol/L (ADP, lstphase), 2,5 μmol/L (A 23187, 2ndphase), 7,5 μmol/L (Ionophor A 23187, lstphase), 30 ¼mol/L (Thrombin). The inhibition of the aggregation Induced by ADP could as well be demonstrated in whole blood. The formation of fibrin was inhibited as shown by the prolongation of the thromboplastin time (Quick) and the partial thromboplastin time (PTT) the first being more sensitive (25% of normal at 50 ymol/L) than the latter (25% of normal at 100 ymol/L). The reason was the inhibition of coagulation factors in the following order: VII (25% of normal at 12.5 ¼mol/L) >WErwnr/IX (25 ¼mol/L) »X (200 μmol/L). The thrombin time remains normal. The antithrombotic properties of RE 1492 were investigated in an in vivo thrombosis model. The formation of platelet thrombi in mesenteric arterioles and venoles of rats (diameterM5 ym) was induced by a laser beam. In controls 1,76±1,14 (SD) shots (50 msec, 50 mW) on the arterioles were necessary for thrombus formation. Twenty minutes after i.v. application of RE 1492 this number rose to 3,18±2,08 (3 mg/kg, p ≤ 0,01, X2-test) and 4,59±1,93 (10 mg/kg, p ≤ 0,01) in arterioles. In venoles of the control animals 1,29±0,45 shots were necessary for thrombus formation. This number rose to 2,11±1,62 (p ≤ 0,05) after 3 mg/kg and 3,28±2,03 (p0,01) after 10 mg/kg. As the number of shots applied was limited to five an average shot number of 5± SD would indicate that no thrombus formation takes place at all. As RE 1492 does neither influence the metabolic pattern of arachidonic acid in platelets nor the activity of phosphodiesterase or adenylatcyclase it is supposed that the oligoamines exert their effects by interaction with phospholipids (PL) resulting in a “membrane stabilization” in platelets and inhibition of PL dependent coagulation factors during fibrin formation.
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Casu, B., L. Marchese, A. Naggi, G. Torri, J. Fareed, A. Racanelli, and J. M. Walenga. "INFLUENCE OF THE SULFATION PATTERN ON CERTAIN BIOLOGICAL PROPERTIES OF GALACTOSAMINOGLYCANS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643251.
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In order to investigate the influence of charge distribution and chain length on the biological properties of sulfated polysaccharides, additional sulfate groups were introduced into the galactosaminoglycans, chondriotin sulfate and dermatan sulfate. Using a flexible method (with sulfuric acid and chlorosulfonic acid) for concurrent sulfation and controlled depolymerization, numerous products were obtained and characterized by chemical, enzymatic and nuclear magnetic resonance spectroscopic methods. The biologic actions of these products were profiled in both in vitro and in vivo assays for antithrombotic activity. Despite a weaker in vitro anticoagulant activity, low molecular weight over sulfated galactosaminoglycans produced significant dose-dependent antithrombotic actions in animal models which were similar to the actions observed with oversulfated low molecular weight heparins. These results suggest that a significant antithrombotic activity can be elicited through non-specific interactions of polysulfates with cellular and plasma components, and that clusters of sulfate groups such as the 4-6 disulfate group on D-galactosaminoglycan residues may be important for these interactions. Furthermore, these results, also suggest that supersulfation of glycosaminogly-cans results in products with biologic activity distinct from the native material.
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Dhall, D. P., A. G. Shah, and C. H. Nair. "ANTITHROMBOTIC PROPHYLAXIS THROUGH MODIFICATION OF FIBRIN NETWORK STRUCTURE:A NEW APPROACH." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643313.
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Morphometric analysis of electron micrographs have demonstrated that diameters of fibrin strands follow a bimodal distribution comprising a major network of thicker fibres with a minor network interspersed and made up of very fine fibres. The relationship between the two networks is not fixed and invariant. It will be shown that the two networks are highly responsive to changes in the physiological conditions of clotting. Such a system has biological adaptability and tends to result in fibrin which is more suited to serve its varying roles in haemostasis, in limiting sepsis, in promoting neovascularization and in acting as a scaffolding for wound repair.The response of the two network system to dextran, an agent widely used and well known for its antithrombotic properties, has been examined. It will be shown that in vitro dextran increases fibrin fibre thickness, reduces permeability and tensile strength of networks developed in plasma. Similar changes were found to follow when dextran is infused in clinical dosage in man. It was found that the increase in the thickness of major network fibre is at the expense of protein in the minor network. Such means of modulating distribution of fibrin fibre diameter within fibrin networks provide a new approach to antithrombotic prophylaxis.
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Ofosu, F. A., G. J. Modi, M. A. Blajchman, M. R. Buchanan, and E. A. Johnson. "INCREASED SULFATION IMPROVES THE ABILITY OF VESSEL WALL GLYCOSA-MINOGLYCANS TO REGULATE THROMBIN ACTIVITY AND PROTHROMBIN ACTIVATION IN PLASMA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643252.
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Studies have shown that dermatan sulfate (DS), heparan sulfate (HS) and chondroitin-4-sulfate (C4S), have antithrombotic properties. The sulfate to carboxylate ratios of these three glycosaminoglycans (GAGs) are approximately half that of heparin (HEP) and the gravimetric dose of each of the three GAGs required to achieve antithrombotic effects in vivo comparable to HEP can be 10 times or more than that of HEPT Since antithrombotic effects depend on the ability of a GAG to catalyse thrombin inhibition and/or to inhibit prothrombin activation, we determined the relationship between the extent of sulfation of various GAGs and their effects on these two reactions in normal plasma. In addition to the three GAGs, DS, HS and C4S were resulfated in vitro to yield DS-S, HS-S and C4S-S, each with a sulfate to carboxylate ratio comparable to that of heparin. As summarized below, increased sulfation improved the ability of a GAG to catalyse thrombin inhibition and to inhibit prothrombin activation. Increasing the degree of sulfation primarily improved the ability of a GAG to accelerate the inhibition of thrombin by heparin cofactor II. The degree of sulfation, therefore, appears to be an important functional attribute of the ability of vessel wall GAGs to regulate the formation and activity of thrombin in plasma.
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de Agostini, A., J. Marcum, and R. Rosenberg. "THE BINDING OF ANTITHROMBIN TO CAPILLARY ENDOTHELIAL CELLS GROWN IN VITRO." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643343.
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Cloned endothelial cells from rat epididymal fat pads synthesize anticoagulantly active heparan sulfate proteoglycans containing the disaccharide, GlcA→ AMN-3,6-O-SO3, which is a marker for the antithrombin-binding domain of heparin. To demonstrate that antithrombin (AT) binds to cell surface heparan sulfate, a binding assay employing 125I-AT and cell monolayers has been developed. Post-confluent endothelial cells (7 days) were incubated with radiolabeled AT for 1 h at 4° and washed with PBS. Bound radioactivity was quantitated after solubilizing whole cells. Under these conditions, ∼1% (2174±50 cpm/5x104 cells) of the 125I-AT bound to the endothelial cell monolayer, whereas none of the radiolabeled protein bound to CHO cells or bovine smooth muscle cells. Utilization of unlabeled AT (1 μM) in experiments conducted as described above resulted in a reduction (73%) of the binding of the labeled species to endothelial cells. To assess whether heparan sulfate was responsible for AT binding, cell monolayers were incubated for 1 h at 37° with purified Flavobacterium heparinase (0.2 units). Over 90% of 125I-AT binding to these cellular elements was suppressed with the bacterial enzyme. Internalization of radiolabeled AT by endothelial cells was examined by incubating the protease inhibitor and cells at 4° and 37 . An initial rapid binding was observed at both temperatures. At 4° AT binding plateaued within 15 min, whereas at 37° binding did not plateau until 60 min and was 30% greater than that observed at 4. These data suggest that surface-associated AT can be internalized by endothelial cells. In addition, AT binding was shown to increase with the length of endothelial cell postconfluence, indicating an accumulation of heparan sulfate by these cells during quiescence. In conclusion, our studies support the hypothesis that the vascular endothelium is coated with heparan sulfate-bound AT, which is responsible for the antithrombotic properties of these natural surfaces.
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Merton, R. E., T. W. Barrowcliffe, and D. P. Thomas. "A COMPARISON OF DERMATAN SULPHATE AND HEPARIN AS ANTITHROMBOTIC DRUGS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642933.
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Dermatan sulphate (DS) has been shown to accelerate thrcmbin inhibition by its action on heparin cofactor II (HCII) but has no effect on anti thrombin III (Tollefson et al., 1983). In this study, we have examined the in vitro anticoagulant effect of a purified preparation of DS (free from heparin and heparan sulphate), m comparison with that of unfractionated heparin (UEH). We have also studied the effect of DS and UEH in preventing experimental venous thrombosis in rabbits and in inhibiting thrombin generation, both in vitro and in ex vivo plasma samples.Dermatan had low activity in vitro by APTT and anti-Xa assays (< 5 iu/ mg). When thrombin generation was measured in vitro, 1 μg/ml UEH was sufficient to inhibit thrombin formation. Although 1 μg/ml DS reduced thrombin generation to 40% of control values, there was no further reduction when the concentration of DS was increased to 8 μg/ml.When DS was injected into rabbits (n = 10), a dose of 150 μg/kg inpaired thrombogenesis m a Wessler stasis model. The mean thrombus score was reduced to 25% of the control values, although thrombosis could not be completely prevented, even after an eight-fold increase m dose (1250 μg/kg). When the duration of stasis was extended from 10 to 20 minutes, there was no impairment of thrombosis (mean thrombus score 100%) following 1250 μg/kg of DS. Thrombin generation measured in ex vivo plasma after 150 μg/kg of DS was 72% (s.e.m. 63-81) of that measured in pre-injection plasma. In contrast, 150 μg/kg of heparin prevented thrombosis after both 10 and 20 minutes stasis (mean score 0%) and thrombin generation was reduced to 17% (s.e.m. 12-23) of control values m ex vivo plasma samples.Unlike heparin, DS does not completely abolish thrcmbin generation in vitro and is not as potent as UEH in inhibiting thrombin generation m ex vivo plasma. While DS has demonstrable antithrombotic activity under defined conditions, it is less effective than heparin and increasing doses of DS do not improve antithrombotic effectiveness in this model.
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Hoppensteadt, D., A. Kumar, J. Fareed, and J. Mardigian. "STUDIES ON THE ANTICOAGULANT AND ANTITHROMBOTIC ACTIONS OF DERMATANS AND THEIR SULFATED DERIVATIVES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643241.
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Non-antithrombin III mediated effects such as interaction with heparin cofactor II, modulation of endothelium and polymorphonuclear leukocytes contribute to the overall antithrombotic effects of glycosaminoglycans. In order to study the role of these dermatans, we investigated their in vitro anticoagulant effects using the clot based (PT, APTT, TT, and Heptest), antiprotease (anti IIa and anti Xa) and Thromboplastin C activated fibrinopeptide A generation test. The in vivo antithrombotic actions were investigated, against activated and non activated prothrombin complex concentrates, and in combination with Russells viper venom in jugular and femoral vein stasis thrombosis models (rabbit). The dermatans studied consisted of a standard dermatan of porcine intestinal origin and four sulfated dermatans with varying degrees of sulfation. All of the dermatans studied showed weak anticoagulant effects on the routinely performed clot based assays. Marked variability was seen on the protease inhibition (anti Xa and anti IIa) assays. In the in vivo studies all dermatans studied showed varying degrees of antithrombotic actions against various thrombogenic agents in a modified stasis thrombosis model. Sulfation appeared to produce stronger anticoagulant effects as determined by in vitro assays, whereas the intravenous antithrombotic actions of native dermatan were stronger than sulfated derivatives. This data suggests that dermatans produce their antithrombotic actions via non-antithrombin III mediated pathways. Furthermore, in vitro testing methods are of limited value in the evaluation of the biologic actions of dermatans and their derivatives.
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Cornelli, U., J. M. Welena, J. Fareed, X. Huan, and D. Hoppensteadt. "ANTITHROMBOTIC ACTIONS OF A SULFOMUCOPOLYSACCHARIDE MIXTURE (ATERIOD) IN ANIMAL MODELS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644160.
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Ateriod obtained from beef mucosal lining is a sulfomuco-polysaccharide mixture of various glycosaminoglycans which contains derma tans, heparatans and traces of heparin. It has been used in the treatment ofatherosclerosis and related vaso-oclusive disorders. Ateriod is standardized in terms of its lipoprotein lipase activation actions. Ateriod contains signfi-cant in vitro anticoagulant and antiprotease (anti-factor Xa and anti-factor Ila) activities as measured by clot-based and chromr ogenic substrate methods. However, this in vitro activity is 7-10 times lesser than heparin. In order to study the antithrombotic actions of this agent in subcutaneous, intravenous and oral routes, we utilized a rabbit stasis thrombosis model with a prothrombin complex concentrate/Russell's viper venom thrombogenic challenge and prolonged stasis. The apparent ED50 for the antithrombotic action were found to be: IV (75-100 ug/ kg), SC (0.8-1.3 mg/kg) and oral (20-30 mg/kg). In both the IV- and SC studies, sustained anticoagulant and antiprotease actions were evident. The observed antithrombotic actions did not relate to the anti-factor IIa or anti-factor Xa actions. Pretreatment of Ateriod with equigravimetric amounts of protamine and platelet factor 4 did not neutralize the antithrombotic actions of this agent in the rabbit model. In a primate (Macaca mulatta) model of pharmacokinetics, ex vivo analysis following subcutaneously administered Ateriod showed sustained anticoagulant and antiprotease effects. The time course of the subcutaneously administered Ateriod was markedly different than heparin and a low molecular weight heparin. Treated animals were shown to resist induced hypercoagulability following injection of homologous serum as measured by FPA generation for extended periods. These studies suggest that Ateriod produces a strong antithrombotic action and that it has highly sustained pharmacokinetics. The antithrombotic activity appears to be primarily mediated via non-antithrombin - HI dependent events which may be related to heparin cofactor II and vascular/ cellular modifications.
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Witt, W., B. Baldus, and P. Donner. "ANTITHROMBOTIC EFFECTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR AT PHYSIOLOGICAL BLOOD LEVELS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643575.
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Effective thrombolysis in human patients and experimental animals by tissue-type plasminogen activator (t-PA) usually requires t-PA plasma levels in the microgram range. Compared to that physiological plasma levels of t-PA are about 100 - 1000 times lower. To investigate the effects of t-PA at physiological blood levels rat studies were performed in vitro and in vivo employing highly purified recombinant single-chain t-PA (sct-PA: 500,000 IU/mg).t-PA activity in rat whole blood as assessed by dilute blood clot-lysis time (DBC-LT) was increased by addition of sct-PA as low as 3 ng/ml (20 % decrease in DBC-LT). Injection of brady-kinin 10, 100 and 1000 μg/kg i.v. shortened DBC-LT to 54, 23, and 10 % of controls corresponding to the effect of about 10, 30, and 100 ng/ml sct-PA added in vitro. Infusion of sct-PA 15 - 450 μg/kg/h i.v. shortened DBC-LT ex vivo dose-dependently by 20 - 90 % at steady state levels (n = 5). In the same dose range sct-PA inhibited thrombus formation along a silk thread introduced into an arteriovenous shunt in anaesthetized rats. The reduction in thrombus dry weight was dose-dependent amounting to 33 - 67 % of preapplication values (n = 5 - 8) at 15 - 450 μg/kg/h i.v. sct-PA. Already 50 μg/kg/h sct-PA corresponding to a sct-PA activity of about 15 ng/ml displayed a significant (a = 0.05) effect in this model.The results of this study suggest that t-PA present at physiological resting or activation (bradykinin) levels during acute clot formation may have potent antithrombotic efficacy. This study provides further evidence for the importance of a balance coagulation-fibrinolysis which can be influenced on both sides towards thrombophilia as well as to achieve antithrombotic therapy, e.g. by elevating plasma fibrinolytic activity with low-dose t-PA treatment or with drugs which stimulate the endogenous fibrinolytic potential.
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Müller, T. H., K. Rühr, H. H. Callisen, and W. G. Eisert. "MODULATION OF ANTITHROMBOTIC EFFECTS OF CULTURED HUMAN ENDOTHELIAL CELLS BY INHIBITORS OF CYCLOOXIGENASE OR PHOSPHODIESTERASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643364.
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Intact endothelial cells are known to form a non-thrombogenic surface and to actively restrict the extent of thrombus formation on denuded vessel walls via such mechanisms as the binding of thrombin and activation of protein C, or the synthesis and release of prostacyclin. In an in vitro system, we have investigated how platelet inhibitors modulate the antithrombotic effects of human endothelial cells. Human endothelial cells isolated from umbilical veins were plated on one half of a subendothelial matrix (SEM) harvested from bovine cornea endothelial cells. The endothelial cells were preincubated with a drug and then exposed to anticoagulated whole blood from human donors in the presence or absence of the same drug and agitated for 15 min. The number and size of platelets interacting with the SEM were quantified by morphometric analysis.In our in vitro system, platelet aggregates on SEM that was partially covered with human endothelial cells were significantly smaller than on uncovered SEM. No difference in platelet adhesion was observed. In the absence of endothelial cells, the cyclooxigenase inhibitors acetylsalicylic acid (ASA) and flurbiprofen strongly reduced the size of aggregates formed on the SEM. Pretreatment of only the endothelial cells with ASA increased the size of the aggregates, while ASA treatment of endothelial cells as well as the whole blood did not reduce the mean aggregate size below that of controls. in contrast, the platelet phosphodiesterase inhibitors AHP 719 and UDCG 212 strongly decreased platelet aggregation without reducing platelet adhesion not only in the absence but also in the presence of endothelial cells pretreated with the inhibitors.Our results demonstrate that this in vitro model of a partially injured vessel wall is well suited to study the effects of endothelial cells on platelet function. Moreover, inhibitors of phosphodiesterase in contrast to ASA have profound antithrombotic effects in this model.
Reports on the topic "In vitro antithrombotic properties"
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Davidson, Irit, Hsing-Jien Kung, and Richard L. Witter. Molecular Interactions between Herpes and Retroviruses in Dually Infected Chickens and Turkeys. United States Department of Agriculture, January 2002. http://dx.doi.org/10.32747/2002.7575275.bard.
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Tumors in commercial poultry are caused mainly by infection with avian herpes and retroviruses, the herpesvirus Marek's disease virus (MDV) and the retroviruses, reticuloendotheliosis (REV), lymphoid leukosis, subgroups A-I and J (ALV and ALV-J) in chickens, or Iymphoprolipherative disease (LPDV) in turkeys. Infection with one virus aggravates the clinical outcome of birds that are already infected by another oncogenic virus. As these viruses do not interfere for infection, MDV and one or more retroviruses can infect the same flock, the same bird and the same cell. While infecting the same cell, herpes and retroviruses might interact in at least three ways: a) Integration of retrovirus genomes, or genomic fragments (mainly the LTR) into MDV;b) alteration of LTR-driven expression of retroviral genes by MDV immediate- early genes, and c) by herpesvirus induced cellular transcriptional factors. The first type of molecular interaction have been demonstrated to happen efficiently in vitro by Dr. Kung, in cases multiple infection of cell cultures with MDV and REV or MDV and ALV. Moreover, Dr. Witter showed that an in vitro-created recombinant, RM1, had altered in vitro replication and in vivo biological properties. A more comprehensive characterization of RM1 was carried out in the present project. We sought to highlight whether events of such integrations occur also in the bird, in vivo. For that, we had first to determine the prevalence of dually-infected individual birds in commercial flocks, as no systematic survey has been yet reported. Surprisingly, about 25% of the commercial flocks infected with avian oncogenic viruses had a multiple virus infection and 5% of the total samples ana lysed had multiple virus sequences. Then, we aimed to evaluate and characterize biologically and molecularly the resulting recombinants, if formed, and to analyse the factors that affect these events (virus strains, type and age of birds and time interval between the infection with both viruses). The perception of retrovirus insertions into herpesviruses in vivo is not banal, as the in vivo and in vitro systems differ in the viral-target cells, lymphocytes or fibroblasts, in the MDV-replicative type, transforming or productive, and the immune system presence. We realized that previous methods employed to study in vitro created recombinant viruses were not adequate for the study of samples taken directly from the bird. Therefore, the Hot Spot-combined PCR was developed based on the molecularly known RM1 virus. Also, the PFGE that was used for tissue cultured-MDV separation was inefficient for separating MDV from organs, but useful with feather tips as a source of bird original MDV. Much attention was dedicated now to feathers, because if a recombinant virus would be formed in vivo, its biological significance would be evident by horizontal dissemination through the feathers. Major findings were: a) not only in vitro, but also in vivo MDV and retrovirus co-infections lead to LTR integrations into MDV. That was shown by the detection of chimeric molecules. These appeared in low quantities and as quasispecies, thus interfering with sequence analysis of cloned gel-purified chimeric molecules. Mainly inserts were located in the repeat long MDV fragments. In field birds chimeric molecules were detected at a lower frequency (2.5%) than in experimentally infected birds (30-50%). These could be transmitted experimentally to another birds by inoculation with chimeric molecules containing blood. Several types of chimeric molecules were formed, and same types were detected in birds infected by a second round. To reproduce viral integrations, in vivo infection trials were done with field inoculate that contained both viruses, but the chimeric molecule yield was undetectable.
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Schuster, Gadi, and David Stern. Integrated Studies of Chloroplast Ribonucleases. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7697125.bard.
Full textAbstract:
Gene regulation at the RNA level encompasses multiple mechanisms in prokaryotes and eukaryotes, including splicing, editing, endo- and exonucleolytic cleavage, and various phenomena related to small or interfering RNAs. Ribonucleases are key players in nearly all of these post-transcriptional mechanisms, as the catalytic agents. This proposal continued BARD-funded research into ribonuclease activities in the chloroplast, where RNase mutation or deficiency can cause metabolic defects and is often associated with plant chlorosis, embryo or seedling lethality, and/or failure to tolerate nutrient stress. The first objective of this proposal was to examined a series of point mutations in the PNPase enzyme of Arabidopsis both in vivo and in vitro. This goal is related to structure-function analysis of an enzyme whose importance in many cellular processes in prokaryotes and eukaryotes has only begun to be uncovered. PNPase substrates are mostly generated by endonucleolytic cleavages for which the catalytic enzymes remain poorly described. The second objective of the proposal was to examine two candidate enzymes, RNase E and RNase J. RNase E is well-described in bacteria but its function in plants was still unknown. We hypothesized it catalyzes endonucleolytic cleavages in both RNA maturation and decay. RNase J was recently discovered in bacteria but like RNase E, its function in plants had yet to be explored. The results of this work are described in the scientific manuscripts attached to this report. We have completed the first objective of characterizing in detail TILLING mutants of PNPase Arabidopsis plants and in parallel introducing the same amino acids changes in the protein and characterize the properties of the modified proteins in vitro. This study defined the roles for both RNase PH core domains in polyadenylation, RNA 3’-end maturation and intron degradation. The results are described in the collaborative scientific manuscript (Germain et al 2011). The second part of the project aimed at the characterization of the two endoribonucleases, RNase E and RNase J, also in this case, in vivo and in vitro. Our results described the limited role of RNase E as compared to the pronounced one of RNase J in the elimination of antisense transcripts in the chloroplast (Schein et al 2008; Sharwood et al 2011). In addition, we characterized polyadenylation in the chloroplast of the green alga Chlamydomonas reinhardtii, and in Arabidopsis (Zimmer et al 2009). Our long term collaboration enabling in vivo and in vitro analysis, capturing the expertise of the two collaborating laboratories, has resulted in a biologically significant correlation of biochemical and in planta results for conserved and indispensable ribonucleases. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture.
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Galili, Gad, and Alan Bennett. Role of Molecular Chaperone in Wheat Storage Protein Assembly. United States Department of Agriculture, April 1995. http://dx.doi.org/10.32747/1995.7604926.bard.
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Following sequestration into the ER, wheat gliadins assemble into complexes that initiate the formation of protein bodies. In the present work we have characterized the DNA sequence and regulation of expression of a plant BiP and also studied its interaction with wheat storage proteins as well as its role in the maturation of these storage proteins. In the Israeli lab, immunoprecipitation studies were made using anti BiP and anti storage proteins sera, both in wheat and in transgenic tobacco plants expressing a wheat gliadin storage proteins. In both cases, we could show that BiP interacts with the gliadin storage proteins. In addition, we could show that BiP also played an important role in the initial assembly of the gliadins. In the American lab, the complexity, structure and properties of tomato BiP was characterized at the molecular and biochemical levels. In addition, tomato BiP was also overexpressed in bacteria and the overexpressed protein was found to be active. The cooperative findings of the Israeli and American labs clearly improves our understanding of the structure and expression of a plant BiP as well as its role in the maturation of storage proteins in plants seeds. In addition, it will serve as a foundation for future studies of the mechanisms of BiP function in in vitro studies using purified storage proteins and purified recombinant active BiP.
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Evans, Donald L., Avigdor Eldar, Liliana Jaso-Friedmann, and Herve Bercovier. Streptococcus Iniae Infection in Trout and Tilapia: Host-Pathogen Interactions, the Immune Response Towards the Pathogen and Vaccine Formulation. United States Department of Agriculture, February 2005. http://dx.doi.org/10.32747/2005.7586538.bard.
Full textAbstract:
The objectives of the BARD proposal were to determine the mechanisms of nonspecific cytotoxic cells (NCC) that are necessary to provide heightened innate resistance to infection and to identify the antigenic determinants in Streptococcus iniae that are best suited for vaccine development. Our central hypothesis was that anti-bacterial immunity in trout and tilapia can only be acquired by combining "innate" NCC responses with antibody responses to polysaccharide antigens. These Objectives were accomplished by experiments delineated by the following Specific Aims: Specific aim (SA) #1 (USA) "Clone and Identify the Apoptosis Regulatory Genes in NCC"; Specific aim #2 (USA)"Identify Regulatory Factors that Control NCC Responses to S. iniae"; Specific aim #3 (Israel) "Characterize the Biological Properties of the S. iniae Capsular Polysaccharide"; and Specific aim #4 (Israel) "Development of an Acellular Vaccine". Our model of S. iniae pathogenesis encompassed two approaches, identify apoptosis regulatory genes and proteins in tilapia that affected NCC activities (USA group) and determine the participation of S.iniae capsular polysaccharides as potential immunogens for the development of an acellular vaccine (Israel group). We previously established that it was possible to immunize tilapia and trout against experimental S. difficile/iniaeinfections. However these studies indicated that antibody responses in protected fish were short lived (3-4 months). Thus available vaccines were useful for short-term protection only. To address the issues of regulation of pathogenesis and immunogens of S. iniae, we have emphasized the role of the innate immune response regarding activation of NCC and mechanisms of invasiveness. Considerable progress was made toward accomplishing SA #1. We have cloned the cDNA of the following tilapia genes: cellular apoptosis susceptibility (CAS/AF547173»; tumor necrosis factor alpha (TNF / A Y 428948); and nascent polypeptide-associated complex alpha polypeptide (NACA/ A Y168640). Similar attempts were made to sequence the tilapia FasLgene/cDNA, however these experiments were not successful. Aim #2 was to "Identify Regulatory Factors that Control NCC Responses to S. iniae." To accomplish this, a new membrane receptor has been identified that may control innate responses (including apoptosis) of NCC to S. iniae. The receptor is a membrane protein on teleost NCC. This protein (NCC cationic antimicrobial protein-1/ncamp-1/AAQ99138) has been sequenced and the cDNA cloned (A Y324398). In recombinant form, ncamp-l kills S. iniae in vitro. Specific aim 3 ("Characterize the Biological Properties of the S.iniae Capsular Polysaccharide") utilized an in- vitro model using rainbow trout primary skin epithelial cell mono layers. These experiments demonstrated colonization into epithelial cells followed by a rapid decline of viable intracellular bacteria and translocation out of the cell. This pathogenesis model suggested that the bacterium escapes the endosome and translocates through the rainbow trout skin barrier to further invade and infect the host. Specific aim #4 ("Development of an Acellular Vaccine") was not specifically addressed. These studies demonstrated that several different apoptotic regulatory genes/proteins are expressed by tilapia NCC. These are the first studies demonstrating that such factors exist in tilapia. Because tilapia NCC bind to and are activated by S. iniae bacterial DNA, we predict that the apoptotic regulatory activity of S. iniae previously demonstrated by our group may be associated with innate antibacterial responses in tilapia.
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Shoseyov, Oded, Steven A. Weinbaum, Raphael Goren, and Abhaya M. Dandekar. Biological Thinning of Fruit Set by RNAase in Deciduous Fruit Trees. United States Department of Agriculture, August 1993. http://dx.doi.org/10.32747/1993.7568110.bard.
Full textAbstract:
Fruit thinning is a common and necessary practice for commercial fruit production in many deciduous tree fruit species. Fruit thinning in apple may be accomplished with a variety of chemical thinning agents, but the use of these chemicals is a subject of environmental concern. It has been shown recently that RNase enzyme, secreted from the stigma and the style, inhibits pollen germination and pollen tube elongation. In this study we have been able to show that Aspergillus niger B-1 RNase can effectively inhibit peach and apple pollen germination, and tube elongation in-vitro, as well as thin fruit in peach and apple, and reduce the number of seeds in citrus. The objectives of the research were to detrmine the conditions for effective thinning of (USA and Israel), develop fermentation process for cost effective production of RNase from A. niger. (Israel), and clone apple S-RNase cDNA (USA). All the objectives of the research were addressed. We have determined the optimal fermentation conditions for cost effective production of the A. niger at a 20,000 liters scale. TheA. niger B1 RNase was isolated to homogeneity and its kinetic and biochemical properties including its N-terminal sequence were fully characterized. The field test results both in Israel and California have shown variability in effectiveness and more work is needed to define the RNase concentration necessary to completely inhibit pollen development. Plant transformation vectors expressing anti-sense apple S-RNase genes were constructed (USA) with an attempt to produce self compatible transgenic apple trees. Bovine S-Protein cDNA was cloned and successfully expressed in E. coli (Israel). Plant transformation vector expressing the S-Protein gene was constructed (USA) with an attempt to produce transgenic plants expressing S-protein in the style. Exogenous application of S-peptide to these plants will result in active RNase and consequently prevention of fertilization.
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Sessa, Guido, and Gregory Martin. role of FLS3 and BSK830 in pattern-triggered immunity in tomato. United States Department of Agriculture, January 2016. http://dx.doi.org/10.32747/2016.7604270.bard.
Full textAbstract:
Pattern-recognition receptors (PRRs) located on the plant cell surface initiate immune responses by perceiving conserved pathogen molecules known as pathogen-associated molecular patterns (PAMPs). PRRs typically function in multiprotein complexes that include transmembrane and cytoplasmickinases and contribute to the initiation and signaling of pattern-triggered immunity (PTI). An important challenge is to identify molecular components of PRR complexes and downstream signaling pathways, and to understand the molecular mechanisms that mediate their function. In research activities supported by BARD-4931, we studied the role of the FLAGELLIN SENSING 3 (FLS3) PRR in the response of tomato leaves to flagellin-derivedPAMPs and PTI. In addition, we investigated molecular properties of the tomato brassinosteroid signaling kinase 830 (BSK830) that physically interacts with FLS3 and is a candidate for acting in the FLS3 signaling pathway. Our investigation refers to the proposal original objectives that were to: 1) Investigate the role of FLS3 and its interacting proteins in PTI; 2) Investigate the role of BSK830 in PTI; 3) Examine molecular and phosphorylation dynamics of the FLS3-BSK830 interaction; 4) Examine the possible interaction of FLS3 and BSK830 with Pstand Xcveffectors. We used CRISPR/Cas9 techniques to develop plants carrying single or combined mutations in the FLS3 gene and in the paralogsFLS2.1 and FLS2.2 genes, which encode the receptor FLAGELLIN SENSING2 (FLS2), and analyzed their function in PTI. Domain swapping analysis of the FLS2 and FLS3 receptors revealed domains of the proteins responsible for PAMP detection and for the different ROS response initiated by flgII-28/FLS3 as compared to flg22/FLS2. In addition, in vitro kinase assays and point mutations analysis identified FLS2 and FLS3 domains required for kinase activity and ATP binding. In research activities on tomato BSK830, we found that it interacts with PRRs and with the co-receptor SERK3A and PAMP treatment affects part of these interactions. CRISPR/Cas9 bsk830 mutant plants displayed enhanced pathogen susceptibility and reduced ROS production upon PAMP treatment. In addition, BSK830 interacted with 8 Xanthomonastype III secreted effectors. Follow up analysis revealed that among these effectors XopAE is part of an operon, is translocated into plant cells, and displays E3 ubiquitinligase activity. Our investigation was also extended to other Arabidopsis and tomato BSK family members. Arabidopsis BSK5 localized to the plant cell periphery, interacted with receptor-like kinases, and it was phosphorylatedin vitro by the PEPR1 and EFRPRRs. bsk5 mutant plants displayed enhanced susceptibility to pathogens and were impaired in several, but not all, PAMP-induced responses. Conversely, BSK5 overexpression conferred enhanced disease resistance and caused stronger PTI responses. Genetic complementation suggested that proper localization, kinase activity, and phosphorylation by PRRs are critical for BSK5 function. BSK7 and BSK8 specifically interacted with the FLS2 PRR, their respective mutant plants were more susceptible to B. cinereaand displayed reduced flg22-induced responses. The tomato BSK Mai1 was found to interact with the M3KMAPKKK, which is involved in activation of cell death associated with effector-triggered immunity. Silencing of Mai1 in N. benthamianaplants compromised cell death induced by a specific class of immune receptors. In addition, co-expression of Mai1 and M3Kin leaves enhanced MAPKphosphorylation and cell death, suggesting that Mai1 acts as a molecular link between pathogen recognition and MAPK signaling. Finally, We identified the PP2C phosphatase Pic1 that acts as a negative regulator of PTI by interacting with and dephosphorylating the receptor-like cytoplasmickinase Pti1, which is a positive regulator of plant immunity. The results of this investigation shed new light on the molecular characteristics and interactions of components of the immune system of crop plants providing new knowledge and tools for development of novel strategies for disease control.
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Levisohn, Sharon, Maricarmen Garcia, David Yogev, and Stanley Kleven. Targeted Molecular Typing of Pathogenic Avian Mycoplasmas. United States Department of Agriculture, January 2006. http://dx.doi.org/10.32747/2006.7695853.bard.
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Intraspecies identification (DNA "fingerprinting") of pathogenic avian mycoplasmas is a powerful tool for epidemiological studies and monitoring strain identity. However the only widely method available for Mycoplasma gallisepticum (MG) and M. synoviae (MS)wasrandom amplified polymorphic DNA (RAPD). This project aimed to develop alternative and supplementary typing methods that will overcome the major constraints of RAPD, such as the need for isolation of the organism in pure culture and the lack of reproducibility intrinsic in the method. Our strategy focussed on recognition of molecular markers enabling identification of MG and MS vaccine strains and, by extension, pathogenic potential of field isolates. Our first aim was to develop PCR-based systems which will allow amplification of specific targeted genes directly from clinical material. For this purpose we evaluated the degree of intraspecies heterogeneity in genes encoding variable surface antigens uniquely found in MG all of which are putative pathogenicity factors. Phylogenic analysis of targeted sequences of selected genes (pvpA, gapA, mgc2, and lp) was employed to determine the relationship among MG strains.. This method, designated gene targeted sequencing (GTS), was successfully employed to identify strains and to establish epidemiologically-linked strain clusters. Diagnostic PCR tests were designed and validated for each of the target genes, allowing amplification of specific nucleotide sequences from clinical samples. An mgc2-PCR-RFLP test was designed for rapid differential diagnosis of MG vaccine strains in Israel. Addressing other project goals, we used transposon mutagenesis and in vivo and in vitro models for pathogenicity to correlated specific changes in target genes with biological properties that may impact the course of infection. An innovative method for specific detection and typing of MS strains was based on the hemagglutinin-encoding gene vlhA, uniquely found in this species. In parallel, we evaluated the application of amplified fragment length polymorphism (AFLP) in avian mycoplasmas. AFLP is a highly discriminatory method that scans the entire genome using infrequent restriction site PCR. As a first step the method was found to be highly correlated with other DNA typing methods for MG species and strain differentiation. The method is highly reproducible and relatively rapid, although it is necessary to isolate the strain to be tested. Both AFLP and GTS are readily to amenable to computer-assisted analysis of similarity and construction of a data-base resource. The availability of improved and diverse tools will help realize the full potential of molecular typing of avian mycoplasmas as an integral and essential part of mycoplasma control programs.
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Sessa, Guido, and Gregory Martin. A functional genomics approach to dissect resistance of tomato to bacterial spot disease. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695876.bard.
Full textAbstract:
The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.
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Tzfira, Tzvi, Michael Elbaum, and Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, December 2005. http://dx.doi.org/10.32747/2005.7695881.bard.
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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.
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Lichter, Amnon, David Obenland, Nirit Bernstein, Jennifer Hashim, and Joseph Smilanick. The role of potassium in quality of grapes after harvest. United States Department of Agriculture, October 2015. http://dx.doi.org/10.32747/2015.7597914.bard.
Full textAbstract:
Objectives: The objectives of the proposal were to study how potassium (K) enters the berry and in what tissues it accumulates, to determine what is the sensitive phenological stage that is responsive to K, to study the influence of K on sugar translocation, to determine if K has effects on expression of genes in source and sink organs and to study applied aspects of the responses to K at the vineyard level. During the research it was realized that K acts externally so a major part of the original objectives had to be deserted and new ones, i.e. the role of K in enhancing water loss from the berry, had to be developed. In addition, the US partners developed practical objectives of understanding the interaction of K application and water deficit as well as application of growth regulators. Background: In our preliminary data we showed that application of K at mid-ripening enhanced sugar accumulation of table grapes. This finding is of major implications to both early and late harvested grapes and it was essential to understand the mode of action of this treatment. Our major hypothesis was that K enters the berry and by that increases sugar translocation into the berry. In addition it was important to cover practical issues of the application which may influence its efficacy and its reproducibility. Conclusions: The major conclusion from the research was that our initial hypothesis was wrong. Mineral analysis of pulp tissue indicated that upon application of K there was a significant increase in most of the major minerals. Subsequently, we developed a new hypothesis that K acts by increasing the water loss from the berry. In vitro studies of K-treated berries corroborated this hypothesis showing greater weight-loss of treated berries. This was not necessarily expressed in the vineyard as in some experiments berry weight remained unchanged, suggesting that the vine compensated for the enhanced water loss. Importantly, we also discovered that the efficacy of different K salts was strongly correlated to the pH of the salt solution: basic K salts had better efficacy than neutral or acidic salts and modifying the pH of the same salt changed its efficacy. It was therefore suggested that K changes the properties of the cuticle making it more susceptible to water loss. Of the practical aspects it was found that application of K to the clusters was sufficient to trigger its affect and that dual application of K had a stronger effect than single application. With regard to timing, it was realized that application of K after veraison was affective and the berries responded also when ripe. While the effect of K application was significant at harvest, it was mostly insignificant one week after application, suggesting that prolonged exposure to K was required. Implications: The scientific implications of the study are that the external mineral composition of the berry may have a significant role in sugar accumulation and that water loss may have an important role in sugar accumulation in grapes. It is not entirely clear how K modulates the cuticle but according to the literature its incorporation into the cuticle may increase its polarity and facilitate generation of "water bridges" between the flesh and the environment. The practical implications of this study are very significant because realizing the mode of action of K can facilitate a much more efficient application strategy. For example, it can be understood that sprays must be directed to the clusters rather than the whole vines and it can be predicted that the length of exposure is important. Also, by increasing the pH of simple K salts, the efficacy of the treatment can be enhanced, saving in the costs of the treatment. Finally, the ability of grape growers to apply K in a safe and knowledgeable way can have significant impact on the length of the season of early grape cultivars and improve the flavor of high grape yields which may otherwise have compromised sugar levels.