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Sample records for thrombin topical recombinant

  1. Topical thrombin preparations and their use in cardiac surgery

    Directory of Open Access Journals (Sweden)

    Brianne L Dunn

    2009-10-01

    Full Text Available Brianne L Dunn1, Walter E Uber1, John S Ikonomidis21Department of Pharmacy Services and 2Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, South Carolina, USAAbstract: Coagulopathic bleeding may lead to increased morbidity and mortality after cardiac surgery. Topical bovine thrombin has been used to promote hemostasis after surgical procedures for over 60 years and is used frequently as a topical hemostatic agent in cardiac surgery. Recently, use of bovine thrombin has been reported to be associated with increased risk for anaphylaxis, thrombosis, and immune-mediated coagulopathy thought secondary to the production of antifactor V and antithrombin antibodies. In patients who develop bovine thrombin-induced immune-mediated coagulopathy, clinical manifestations may range from asymptomatic alterations in coagulation tests to severe hemorrhage and death. Patients undergoing cardiac surgical procedures may be at increased risk for development of antibodies to bovine thrombin products and associated complications. This adverse immunologic profile has led to the development of alternative preparations including a human and a recombinant thrombin which have been shown to be equally efficacious to bovine thrombin and have reduced antigenicity. However, the potential benefit associated with reduced antigenicity is not truly known secondary to the lack of long-term experience with these products. Given the potentially higher margin of safety and less stringent storage concerns compared to human thrombin, recombinant thrombin may be the most reasonable approach in cardiac surgery.Keywords: bovine thrombin, human thrombin, recombinant thrombin, immune-mediated coagulopathy, topical hemostatic agents, thrombin 

  2. Assessment of thrombus imaging potency of thrombin-targeting recombinant hirudin in vitro and in vivo

    International Nuclear Information System (INIS)

    The purpose of this study is to evaluate the effect of recombinant hirudin HV2 (rHHV2) as a thrombus imaging agent. 125I-rHHV2 and 125I-Th were prepared with Chloramine method, the labeling rate were 86.64% and 62.20%, with the radioactive purity of 89.70% and 91.22%, with the specific activity of 22.4 TBq/mmol and 94.43 TBq/mmol respectively. The competitive radioassay showed that the Th-fibrin complex formation did not affect the ability of rHHV2 binding with Th. In the complex, the molecular binding ratio of rHHV2 to Th and fibrinogen was 14:14:1. 99mTc-rHHV2 was prepared by 2-iminothiolane modified method, the labeled rate was 94%, with the radioactive purity of 93.90%, with the specific activity of 2.30 TBq/mmol. It was used to image fresh thrombi on arteries and veins of dog or rabbit (30 ?g/kg). In SPECT images, all thrombin were clearly visible, arterial thrombosis imaging can be seen clearly within 45 min after injection and fade away slowly, venous thrombosis imaging also can be seen within 30 min after injection and quantitative imaging ratios between the thrombus and opposite vessel increased following the time. Biodistribution studies in mouse demonstrated that rHHV2 was excreted from kidneys. These data indicate that Th in Th-fibrin complex could be a potent target for diagnosis of thrombus and 99mTc-rHHV2 could be a new thrombotic imaging agent. (authors)

  3. [New anticoagulants - direct thrombin inhibitors].

    Science.gov (United States)

    Brand, B; Graf, L

    2012-11-01

    Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other. PMID:23117667

  4. Thrombin and vascular inflammation.

    Science.gov (United States)

    Popovi?, Milan; Smiljani?, Katarina; Dobutovi?, Branislava; Syrovets, Tatiana; Simmet, Thomas; Isenovi?, Esma R

    2012-01-01

    Vascular endothelium is a key regulator of homeostasis. In physiological conditions it mediates vascular dilatation, prevents platelet adhesion, and inhibits thrombin generation. However, endothelial dysfunction caused by physical injury of the vascular wall, for example during balloon angioplasty, acute or chronic inflammation, such as in atherothrombosis, creates a proinflammatory environment which supports leukocyte transmigration toward inflammatory sites. At the same time, the dysfunction promotes thrombin generation, fibrin deposition, and coagulation. The serine protease thrombin plays a pivotal role in the coagulation cascade. However, thrombin is not only the key effector of coagulation cascade; it also plays a significant role in inflammatory diseases. It shows an array of effects on endothelial cells, vascular smooth muscle cells, monocytes, and platelets, all of which participate in the vascular pathophysiology such as atherothrombosis. Therefore, thrombin can be considered as an important modulatory molecule of vascular homeostasis. This review summarizes the existing evidence on the role of thrombin in vascular inflammation. PMID:21858738

  5. The use of thrombin in the radiology department.

    LENUS (Irish Health Repository)

    Ward, E

    2009-03-01

    Thrombin is a naturally occurring coagulation protein that converts soluble fibrinogen into insoluble fibrin and plays a vital role in the coagulation cascade and in turn haemostasis. Thrombin also promotes platelet activation. In the last few years, there has been a rapid increase in the use of thrombin by radiologists in a variety of clinical circumstances. It is best known for its use in the treatment of pseudoaneurysms following angiography. However, there are now a variety of cases in the literature describing the treatment of traumatic, inflammatory and infected aneurysms with thrombin in a variety of locations within the human body. There have even been recent reports describing the use of thrombin in conventional aneurysms as well as ruptured aneurysms. Its use has also been described in the treatment of endoleaks (type II) following aneurysm repair. In nearly all of these cases, treatment with thrombin requires imaging guidance. Recently, thrombin has also been used as a topical treatment post-percutaneous intervention to reduce or stop bleeding. Most radiologists have only a limited knowledge of the pharmacodynamics of thrombin, its wide range of utilisation and its limitations. Apart from a few case reports and case series, there is little in the radiological literature encompassing the wide range of applications that thrombin may have in the radiology department. In this review article, we comprehensively describe the role and pathophysiology of thrombin, describing with examples many of its potential uses. Techniques of usage as well as pitfalls and limitations are also described.

  6. Topics in cosmology: Structure formation, dark energy and recombination

    Science.gov (United States)

    Alizadeh, Esfandiar

    The field of theoretical cosmology consists of numerous, inter-related branches, whose ambitious goal is to uncover the history of the universe from its beginning to its future. Achieving this, no doubt, requires a deep understanding of many areas of physics. In this thesis I touch upon a few of these areas in which I worked during my PhD studies. Chapter (2) describes our work in finding the accretion and merger history of dark matter halos. Dark matter halos are the collapsed dark matter structures in the late time evolution of the universe, whose existence is vital for the formation of galaxies in the Universe as they act as the potential wells where normal matter (collectively called Baryons) can accumulate, cool, and form stars. It is then no surprise that the properties of galaxies depends on the properties of the dark matter halo in which it resides, including its merger history, i.e. the number of times it merged with other halos. Even though these merger rates can be calculated theoretically for infinitesimal time steps, in order to find the merger history over an extended period of time one had to use either Monte-Carlo simulations to build up the total rates of merging and accreting from the infinitesimal rates or use N-body simulations. In chapter (2) we show how we used random walk formalism to write down an analytical (integral) equation for the merger history of halos. We have solved this equation numerically and find very good agreement with Monte-Carlo simulations. This work can be used in theories of galaxy formation and evolution. We then switch from the overdense regions of the Universe, halos, to the underdense ones, voids. These structures have not attracted as much attention from cosmologists as their overdense counterparts in probing the cosmological models. We show here that the shapes of voids as a probe can be of use for future surveys to pin down the equation of state of the dark energy, i.e. the ratio of its pressure to its energy density. As first approximation, voids can be considered to be ellipsoids whose axis ratio evolution depends on the cosmological parameters. This, together with the fact that the initial distribution of the axis ratios is known (because the intial density field is Gaussian) can be used to infer the equation of state of the dark energy statistically from the observation of voids at different redshifts and with different sizes. The standard method of Fisher matrices is then used to forecast how well a future survey can measure the equation of state. We find promising results with constraints coming from void ellipticity measurements comparable to those of other standard methods. Chapter (4) goes farther back in the history of the Universe. During the recombination era, when the Universe was around a thousandth of its present size, it became cool enough that free electrons got captured by free protons to make hydrogen atoms. Consequently, the Thompson scattering of photons off of free electrons dropped dramatically and the Universe became transparent to photon propagation. The Cosmic Microwave Background (CMB) is a remnant from this epoch, consisting of photons last scattered off of a free electron. A wealth of information is contained in the statistical properties of the CMB field. However, in order to take full advantage of this probe one needs to know the recombination history, i.e. the evolution of the number density of free electrons as a function of time, to sub-percent level accuracy during this era. There are a plethora of phenomena, from radiative transfer effects to atomic and molecular ones, that have the potential to change the recombination history to this level. Our work was to calculate the effect that the formation of hydrogen molecules will have on the recombination history. Even though the abundance of hydrogen molecules is very small, they still have the potential to change the recombination history by reshuffling photons from the blue side of the Ly-alpha line to its red side and vise-versa. To find the magnitude of the effect, we solve the appropri

  7. Recombinant snake venom prothrombin activators

    OpenAIRE

    Lövgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  8. DABIGATRAN ETEXILATE: NEW DIRECT THROMBIN INHIBITORS ANTICOAGULANTS

    OpenAIRE

    Patel Kinjal B; Galani Varsha; Patel Paresh B; Mehta Hiren R

    2011-01-01

    Thrombin plays a key role in thrombotic events, and therefore thrombin inhibition represents a therapeutic target for numerous thromboembolic diseases. Thrombin is responsible for the conversion of soluble fibrinogen to fibrin; clot stabilization through activation of factor XIII and the formation of cross-linkage among fibrin molecules; and the generation of additional thrombin through activation of factors V, VIII, and XI. Direct thrombin inhibitors are an innovative class of anticoagulant...

  9. Aptamer Based Microsphere Biosensor for Thrombin Detection

    OpenAIRE

    Xudong Fan; White, Ian M.; Suter, Jonathan D.; Hongying Zhu

    2006-01-01

    We have developed an optical microsphere resonator biosensor using aptamer as receptor for the measurement of the important biomolecule thrombin. The sphere surface is modified with anti-thrombin aptamer, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the sphere surface is monitored by the spectral position of the microsphere's whispering gallery mode resonances. A detection limit on the order of 1 NIH Unit/mL is demonstrated. Control experiments...

  10. Multiple inhibitory kinetics reveal an allosteric interplay among thrombin functional sites.

    Science.gov (United States)

    Zavyalova, Elena; Kopylov, Alexey

    2015-01-01

    Thrombin is a key blood clotting enzyme; therefore, developing of its inhibitors has become a mainstream in antithrombotic pharmacology. As a result, a wide variety of proteins, peptides, peptidomimetics, DNA, RNA, and carbohydrates were reported to be effective inhibitors of thrombin activities. The majority of described inhibitors were characterized kinetically with amidolytic assay only; though some of them inhibit fibrinogen binding rather than amidolytic activity, e.g. hirugen and nucleic acid aptamers. Per contra, studying the inhibition kinetics of fibrinogen hydrolysis might reveal essential peculiarities of mechanism of action of thrombin inhibitors. In this paper the effect of thrombin inhibitors on fibrinogen hydrolysis has been investigated using improved turbidimetric assay. This technique is highly productive versus fibrinopeptide determination allowing elucidation of inhibition type and apparent constant for different types of thrombin inhibitors. The protein (recombinant hirudin, antithrombin III), peptide (bivalirudin, hirugen), and peptidomimetic (argatroban, PPACK) inhibitors were characterized in terms of inhibition types for the first time. Unexpectedly, for others: heparin, RNA aptamer Toggle-25t, partial inhibition has been shown indicating allosteric interplay between exosites. Improved turbidimetric assay is also applicable for studying the fibrin association inhibitors. Hence, GPRP-peptide was characterized kinetically for the first time. The kinetic study revealed a repertoire of different inhibition types and also close allosteric interplay within the thrombin. The results are undoubtedly important for understanding the enzyme activity regulation, as well as for the rational development of new antithrombotic substances. PMID:25467079

  11. Thrombin inhibitory activity of some polyphenolic compounds

    OpenAIRE

    Bijak, M.; Ziewiecki, R.; Saluk, J.; Ponczek, M.; Pawlaczyk, I.; Krotkiewski, H.; Wachowicz, B.; Nowak, P.

    2013-01-01

    Thrombin, also known as an active plasma coagulation factor II, belongs to the family of serine proteases and plays a crucial role in blood coagulation process. The process of thrombin generation is the central event of the hemostatic process and regulates blood coagulant activity. For this reason, thrombin inhibition is key to successful novel antithrombotic pharmacotherapy. The aim of our present study was to examine the effects of the well-known polyphenolic compounds on the activity of th...

  12. DABIGATRAN ETEXILATE: NEW DIRECT THROMBIN INHIBITORS ANTICOAGULANTS

    Directory of Open Access Journals (Sweden)

    Patel Kinjal B

    2011-04-01

    Full Text Available Thrombin plays a key role in thrombotic events, and therefore thrombin inhibition represents a therapeutic target for numerous thromboembolic diseases. Thrombin is responsible for the conversion of soluble fibrinogen to fibrin; clot stabilization through activation of factor XIII and the formation of cross-linkage among fibrin molecules; and the generation of additional thrombin through activation of factors V, VIII, and XI. Direct thrombin inhibitors are an innovative class of anticoagulants that bind directly to thrombin to inhibit its actions and impede the clotting process. Dabigatran is the first direct thrombin inhibitor, orally available first approval by US Food and Drugs Administration in 2010. Specifically and reversibly inhibits thrombin, so the duration of action is predictable. The anticoagulant effect correlates well with plasma drug concentrations, which implies an effective anticoagulation with low bleeding risk without major problems of interactions with other drugs. The predictable pharmacokinetics and pharmacodynamics characteristics of dabigatran may facilitate dental management of patients who until now have been in treatment with traditional anticoagulants, given that it doesn’t require routine laboratory monitoring in the vast majority of patients treated. They also present a profile of drug interactions very favorable.

  13. Aptamer Based Microsphere Biosensor for Thrombin Detection

    Directory of Open Access Journals (Sweden)

    Xudong Fan

    2006-08-01

    Full Text Available We have developed an optical microsphere resonator biosensor using aptamer asreceptor for the measurement of the important biomolecule thrombin. The sphere surface ismodified with anti-thrombin aptamer, which has excellent binding affinity and selectivityfor thrombin. Binding of the thrombin at the sphere surface is monitored by the spectralposition of the microsphere’s whispering gallery mode resonances. A detection limit on theorder of 1 NIH Unit/mL is demonstrated. Control experiments with non-aptameroligonucleotide and BSA are also carried out to confirm the specific binding betweenaptamer and thrombin. We expect that this demonstration will lead to the development ofhighly sensitive biomarker sensors based on aptamer with lower cost and higher throughputthan current technology.

  14. Thrombin-reactive polypeptides of platelets may regulate inhibition of thrombin by antithrombin.

    Science.gov (United States)

    Chelladurai, M; Ganguly, P

    1986-03-28

    The central enzyme involved in blood coagulation and activation of platelets is the serine proteinase thrombin. The principal inhibitor of this proteinase in plasma is antithrombin. The mechanism of regulation of the thrombin-antithrombin reaction remains unknown. Two polypeptides of 74 and 55 kDa present on the platelet surface and in plasma are known to specifically enhance the activity of thrombin on different substrates. This study was undertaken to assess the effects of these platelet proteins on thrombin-antithrombin interaction. Direct measurements of residual thrombin activity in mixtures of thrombin and antithrombin, in the presence or absence of the platelet proteins, were made utilizing a specific chromogenic substrate. Under these conditions, when 60% of thrombin activity was inhibited by antithrombin in controls, 100% of enzyme activity was retained in the presence of the platelet proteins. When heparin was used in these assays, the rate of inhibition of thrombin by antithrombin was much more rapid and 62% of thrombin activity remained after 1 min. Under these conditions, the platelet proteins continued to protect thrombin from inactivation with 98% activity remaining at 1 min and 85% activity at 5 min. In contrast, the inhibition of trypsin by antithrombin was not affected by the platelet proteins. Additional studies in platelet aggregation showed that the platelet polypeptides have two effects on thrombin: (i) protection of the enzyme inhibition by antithrombin and (ii) stabilization of thrombin from loss of activity due to aging. The results suggest a novel role for the platelet proteins in hemostasis - regulation of the inhibition of thrombin by antithrombin. PMID:3955055

  15. Thrombin–aptamer recognition: a revealed ambiguity

    OpenAIRE

    Russo Krauss, Irene; MERLINO, ANTONELLO; Giancola, Concetta; Randazzo, Antonio; Mazzarella, Lelio; Sica, Filomena

    2011-01-01

    Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human ?-thrombin was solved at 2.9-Å resolution, but did not provide details of the...

  16. Correcting thrombin generation ex vivo using different haemostatic agents following cardiac surgery requiring the use of cardiopulmonary bypass.

    Science.gov (United States)

    Percy, Charles L; Hartmann, Rudolf; Jones, Rhidian M; Balachandran, Subramaniam; Mehta, Dheeraj; Dockal, Michael; Scheiflinger, Friedrich; O'Donnell, Valerie B; Hall, Judith E; Collins, Peter W

    2015-06-01

    Recently, lower thrombin generation has been associated with excess bleeding post-cardiopulmonary bypass (CPB). Therefore, treatment to correct thrombin generation is a potentially important aspect of management of bleeding in this group of patients. The objective of the present study was to investigate the effects of fresh frozen plasma (FFP), recombinant factor VIIa (rFVIIa), prothrombin complex concentrate (PCC) and tissue factor pathway inhibitor (TFPI) inhibition on thrombin generation when added ex vivo to the plasma of patients who had undergone cardiac surgery requiring CPB. Patients undergoing elective cardiac surgery were recruited. Blood samples were collected before administration of heparin and 30?min after its reversal. Thrombin generation was measured in the presence and absence of different concentrations of FFP, rFVIIa, PCC and an anti-TFPI antibody. A total of 102 patients were recruited. Thrombin generation following CPB was lower compared with pre-CPB (median endogenous thrombin potential pre-CPB 339?nmol/l per min, post-CPB 155?nmol/l per min, P?patients who have undergone surgery requiring CPB. Inhibition of TFPI may be a further potential therapeutic strategy for managing bleeding in this group of patients. PMID:25928274

  17. The unresolved safety concerns of bovine thrombin

    OpenAIRE

    Javidroozi Mazyar; Shander Aryeh

    2008-01-01

    Abstract A recent review has suggested that bovine thrombin is not associated with an increased risk of bleeding in surgical populations. In spite of extremely limited evidence available, many valuable resources (e.g. safety surveillance and post-marketing programs, case reports) were excluded in reaching this conclusion. While waiting for the adequately powered, controlled clinical trials to address the effects of bovine thrombin on bleeding and thrombotic events, the potential risk cannot b...

  18. Aptamer-based SERRS Sensor for Thrombin Detection

    OpenAIRE

    Cho, Hansang; Baker, Brian R; Wachsmann-Hogiu, Sebastian; Pagba, Cynthia V.; Laurence, Ted A.; Stephen M. Lane; Lee, Luke P.; Tok, Jeffrey B.-H.

    2008-01-01

    We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human ?-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other prot...

  19. Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis.

    Science.gov (United States)

    Liao, Min; Zhou, Jinlin; Gong, Haiyan; Boldbaatar, Damdinsuren; Shirafuji, Rika; Battur, Banzragch; Nishikawa, Yoshifumi; Fujisaki, Kozo

    2009-02-01

    A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding. PMID:19061894

  20. Fibrinogen and thrombin concentrations are critical for fibrin glue adherence in rat high-risk colon anastomoses

    Scientific Electronic Library Online (English)

    Eliseo Portilla-de, Buen; Abel, Orozco-Mosqueda; Caridad, Leal-Cortés; Gonzalo, Vázquez-Camacho; Clotilde, Fuentes-Orozco; Andrea Socorro, Alvarez-Villaseñor; Michel Dassaejv, Macías-Amezcua; Alejandro, González-Ojeda.

    2014-04-01

    Full Text Available OBJECTIVE: Fibrin glues have not been consistently successful in preventing the dehiscence of high-risk colonic anastomoses. Fibrinogen and thrombin concentrations in glues determine their ability to function as sealants, healers, and/or adhesives. The objective of the current study was to compare [...] the effects of different concentrations of fibrinogen and thrombin on bursting pressure, leaks, dehiscence, and morphology of high-risk ischemic colonic anastomoses using fibrin glue in rats. METHODS: Colonic anastomoses in adult female Sprague-Dawley rats (weight, 250-350 g) treated with fibrin glue containing different concentrations of fibrinogen and thrombin were evaluated at post-operative day 5. The interventions were low-risk (normal) or high-risk (ischemic) end-to-end colonic anastomoses using polypropylene sutures and topical application of fibrinogen at high (120 mg/mL) or low (40 mg/mL) concentrations and thrombin at high (1000 IU/mL) or low (500 IU/mL) concentrations. RESULTS: Ischemia alone, anastomosis alone, or both together reduced the bursting pressure. Glues containing a low fibrinogen concentration improved this parameter in all cases. High thrombin in combination with low fibrinogen also improved adherence exclusively in low-risk anastomoses. No differences were detected with respect to macroscopic parameters, histopathology, or hydroxyproline content at 5 days post-anastomosis. CONCLUSIONS: Fibrin glue with a low fibrinogen content normalizes the bursting pressure of high-risk ischemic left-colon anastomoses in rats at day 5 after surgery.

  1. Thrombin regulates the function of human blood dendritic cells

    International Nuclear Information System (INIS)

    Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions

  2. Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration.

    Science.gov (United States)

    Popovic, Milan; Laumonnier, Yves; Burysek, Ladislav; Syrovets, Tatiana; Simmet, Thomas

    2008-07-01

    CX3CL1 (fractalkine, neurotactin) is the sole CX3C chemokine. It induces monocyte locomotion in its cleaved form, but in its membrane-anchored form, it also acts as an adhesion molecule. The expression of CX3CL1 is up-regulated in endothelial cells by proinflammatory cytokines such as IL-1 or TNF-alpha. Here, we studied the effect of the serine protease thrombin on endothelial CX3CL1 induction and its putative relevance for monocyte function. In HUVEC, thrombin triggered a time- and concentration-dependent expression of CX3CL1 at the mRNA and the protein level as shown by RT-PCR, Western immunoblotting, and flow cytometric analysis. Thrombin induced CX3CL1 by activating protease-activated receptor 1 (PAR1) as demonstrated by the use of PAR1-activating peptide and the PAR1-specific antagonist SCH 79797. The thrombin-induced CX3CL1 expression was NF-kappaB-dependent, as shown by EMSA, ELISA, and by inhibition of the NF-kappaB signaling pathway by the IkappaB kinase inhibitor acety-11-keto-beta-boswellic acid or by transient overexpression of a transdominant-negative form of IkappaBalpha. Upon cocultivation of human monocytes with HUVEC, the thrombin-dependent induction of membrane-anchored CX3CL1 in HUVEC triggered monocyte adhesion and an enhanced release of the MCP-1/CCL2 by monocytes and potentiated the monocyte transendothelial migration. Accordingly, the recombinant extracellular domain of CX3CL1 induced CCL2 release by monocytes. Thus, the thrombin-induced monocyte/endothelial cell cross-talk mediated by increased CX3CL1 expression potentiates the CCL2 chemokine generation that might contribute to the recruitment of monocytes into inflamed areas. PMID:18436581

  3. Thrombin Receptor Antagonism in Antiplatelet Therapy

    OpenAIRE

    Olivier, C.; Diehl, P.; Bode, C.; Moser, M.

    2013-01-01

    Activated platelets play a crucial role in the pathogenesis of atherothrombotic disease and its complications. Even under treatment of antiplatelet drugs, such as acetylsalicylic acid and P2Y12 antagonists, morbidity and mortality rates of thromboembolic complications remain high. Hence, the therapeutic inhibition of protease-activated receptor (PAR)-1, which is activated by thrombin, is a novel promising approach in antiplatelet therapy. Recent data suggest that PAR-1 is mainly involved in p...

  4. Thrombin induces endothelial arginase through AP-1 activation

    OpenAIRE

    Zhu, Weifei; Chandrasekharan, Unni M; Bandyopadhyay, Smarajit; Morris, Sidney M.; DiCorleto, Paul E.; Kashyap, Vikram S.

    2009-01-01

    Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin s...

  5. Influence of Aromatic and Aliphatic Moieties on Thrombin Inhibitors Potency

    OpenAIRE

    Poyarkov, Alexey; Rocabayera, Xavier; Poyarkova, Svetlana; Kukhar, Valery

    2008-01-01

    Thrombin is a plasma serine protease that plays a key role in coagulation and hemostasis but also in thromboembolic diseases. Direct thrombin inhibitors could be beneficial for future anticoagulant therapy in the prophylaxis of venous and arterial thrombosis as well as myocardial infarction. To design the efficient thrombin inhibitors we have synthesized and studied peptide-based inhibitors resistant to enzymatic degradation. Compounds with general formula X-DArg-D-Phe-OMe, where X = residue ...

  6. Targeting Platelet Thrombin Receptor Signaling to Prevent Thrombosis

    OpenAIRE

    Susan S. Smyth; Wallace, Eric L.

    2013-01-01

    Platelets contribute fundamentally to ischemic heart disease, and antiplatelet therapy has been critical to reducing acute thrombotic complications of atherosclerotic disease. Thrombin, by acting on protease activated receptors (PAR), is one of the most potent platelet activators. PAR-1 antagonists may therefore provide more comprehensive antithrombotic effects. We review the pathophysiology of atherothrombosis, platelet activation by thrombin, the role of platelet protease activated receptor...

  7. Bare magnetic nanoparticles as fluorescence quenchers for detection of thrombin.

    Science.gov (United States)

    Yu, Jiemiao; Yang, Liangrong; Liang, Xiangfeng; Dong, Tingting; Liu, Huizhou

    2015-06-21

    Rapid and sensitive detection of thrombin has very important significance in clinical diagnosis. In this work, bare magnetic iron oxide nanoparticles (magnetic nanoparticles) without any modification were used as fluorescence quenchers. In the absence of thrombin, a fluorescent dye (CY3) labeled thrombin aptamer (named CY3-aptamer) was adsorbed on the surface of magnetic nanoparticles through interaction between a phosphate backbone of the CY3-aptamer and hydroxyl groups on the bare magnetic nanoparticles in binding solution, leading to fluorescence quenching. Once thrombin was introduced, the CY3-aptamer formed a G-quartet structure and combined with thrombin, which resulted in the CY3-aptamer being separated from the magnetic nanoparticles and restoration of fluorescence. This proposed assay took advantage of binding affinity between the CY3-aptamer and thrombin for specificity, and bare magnetic nanoparticles for fluorescence quenching. The fluorescence signal had a good linear relationship with thrombin concentration in the range of 1-60 nM, and the limit of detection for thrombin was estimated as low as 0.5 nM. Furthermore, this method could be applied for other target detection using the corresponding fluorescence labeled aptamer. PMID:25894923

  8. Dabigatran abrogates brain endothelial cell permeability in response to thrombin.

    Science.gov (United States)

    Hawkins, Brian Thomas; Gu, Yu-Huan; Izawa, Yoshikane; Del Zoppo, Gregory John

    2015-06-01

    Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell ?1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge. PMID:25669912

  9. Thrombin inhibitor reduces bleeds in older coronary patients.

    Science.gov (United States)

    2015-03-01

    Bivalirudin has been approved as part of the anticoagulation routine in patients with acute coronary syndrome. It is a thrombin inhibitor acting on circulating and clot-bound thrombin. Several studies have suggested that it is associated with lower rates of major bleeding in patients who have invasive procedures associated with acute coronary syndrome. PMID:25727630

  10. Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay

    Directory of Open Access Journals (Sweden)

    Leflem Lena

    2005-10-01

    Full Text Available Abstract Background Thrombin generation assay was developed several years ago to mimic physiological coagulation mechanisms but it had important limitations. Thrombogram-Thrombinoscope assay using a fluorogenic substrate, allows obtaining thrombin generation curves in non-defibrinated platelet rich plasma (PRP in a fully automated manner. Methods We standardised the methodology of Thrombogram-Thrombinoscope and we evaluated the precision of thrombin generation parameters (lag-time, maximum concentration of thrombin [Cmax], time required to reach Cmax [Tmax] and endogenous thrombin potential ETP using different concentrations of recombinant human tissue factor, platelets or phospholipids. Normal values of thrombin generation assay were established in optimal experimental conditions. Results In the presence of low TF concentrations (final dilution of thromboplastin in plasma: 1/1000–1/2000 the Thrombogram assay showed intra-assay and inter-assay coefficients of variation lower than 9%. Thrombin generation parameters showed an important inter-individual variability and the coefficients of variation ranged from 18% to 50%. In PRP the lag-time, Cmax and Tmax but not the ETP, were influenced by TF concentration. Thrombin generation parameters were not influenced by variations of platelet concentration from 50 × 109/l to 400 × 109/l. The addition of synthetic procoagulant phospholipids in PPP strongly influenced all the parameters of thrombogram. For all the parameters of thrombogram a threshold effect was observed in the presence of phspholipid concentrations equal or higher to 4 ?M. In frozen-thawed PRP the lag-time and the Tmax were significantly reduced and the Cmax was increased compared to the fresh PRP, but the ETP, the intra assay and the inter-assay coefficients of variation were similar in both test-systems. Conclusion Thrombogram-Thrombinoscope assay performed in fresh or in frozen-thawed PRP has an acceptable precision, with low inter-assay and intra-assay coefficient of variations. The concentration of TF is determinant for the normal values of the studied parameters of thrombin generation. When the assay is performed in PPP, thrombin generation parameters are influenced by the concentration of procoagulant synthetic phospholipids. The optimal experimental conditions were obtained in the presence of 1/1000 final dilution of thromboplastin, a platelet count higher than 50 × 109/l and a synthetic phospholipid concentration higher than 4 ?M.

  11. Injury to cultured endothelial cells by thrombin-stimulated platelets

    International Nuclear Information System (INIS)

    In vivo, stimulated platelets may injure the endothelium. We have used cultured endothelial cells to assess endothelial cell damage caused by platelet stimulation with thrombin. Endothelial cells were cultured from umbilical veins and semiconfluent cultures were labeled with Na251CrO4. Twenty four hours later washed human platelets (final concentration 200,000 platelets/microliters) and thrombin (final concentration 4 units/ml) were added to the medium and the culture dish was shaken for 15 minutes. The percentage of cells detached from the culture dish and the percentage of 51Cr lost from the endothelial cells into the ambient fluid during the shaking were determined and used as indicators of cell injury. Increased percentages of loosened cells and 51Cr in the ambient fluid were observed with platelet suspension and thrombin compared to controls with neither platelet suspension nor thrombin and controls with either platelet suspension or thrombin. The platelet-free supernatant obtained after reaction of the platelets with thrombin also increased the percentage of loosened cells, but it did not increase the percentage of 51Cr in the ambient fluid to a significant degree. Thrombin alone caused a moderate loss of 51Cr, but no increased loosening of cells. Treatment of the platelets with acetylsalicylic acid prior to the experiment depressed the detachment effect of thrombin-stimulated plateletsnt effect of thrombin-stimulated platelets, but did not alter the effect on the release of 51Cr into the ambient fluid. Scanning and transmission electron microscopy of cultured endothelial cells exposed to thrombin-stimulated platelets confirmed the presence of loosening and injury to the endothelial cells

  12. Reduced surface expression and binding of fibronectin by thrombin-stimulated thrombasthenic platelets.

    OpenAIRE

    Ginsberg, M.H.; Forsyth, J.; Lightsey, A; Chediak, J; Plow, E F

    1983-01-01

    Thrombin stimulation results in increased surface expression of endogeneous fibronectin and binding of plasma fibronectin to human platelets. Platelets of patients with Glanzmann's thrombasthenia, a bleeding disorder, exhibit reduced thrombin-induced platelet aggregation, little or no clot retraction, and abnormal platelet spreading on glass surfaces. Thrombin stimulation of patient platelets from four thrombasthenic kindreds resulted in little fibronectin binding. Nevertheless, thrombin did ...

  13. Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium: POSSIBLE ROLE IN THE INACTIVATION OF THROMBIN BY ANTITHROMBIN III

    OpenAIRE

    Lollar, Pete; Owen, Whyte G.

    1980-01-01

    The clearance of 125I-thrombin and diisopropylphosphoryl-125I-thrombin (DIP-thrombin) from the circulation in rabbits was studied. When given either intraarterially or intravenously, DIP-thrombin, which is active-site blocked, was ?90% cleared from the circulation by 1 min, the time of earliest sampling, indicating a large first-pass effect. DIP-thrombin given intravenously is found predominantly in the lungs, whereas DIP-thrombin injected into the aortic arch is distributed diffusely in ap...

  14. APTAMER-BASED SERRS SENSOR FOR THROMBIN DETECTION

    Energy Technology Data Exchange (ETDEWEB)

    Cho, H; Baker, B R; Wachsmann-Hogiu, S; Pagba, C V; Laurence, T A; Lane, S M; Lee, L P; Tok, J B

    2008-07-02

    We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human a-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5{prime}-capped, 3{prime}-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.

  15. Label-free impedimetric biosensor for thrombin using the thrombin-binding aptamer as receptor

    International Nuclear Information System (INIS)

    This study presents the further establishment of impedimetric biosensors with aptamers as receptors. Aptamers are short single-stranded oligonucleotides which bind analytes with a specific region of their 3D structure. Electrical impedance spectroscopy is a sensitive method for analyzing changes on the electrode surface, e.g. caused by receptor-ligand-interactions. Fast and inexpensive prototyping of electrodes on the basis of commercially available compact discs having a 24 carat gold reflective layer was investigated. Electrode structures (CDtrodes [1]) in the range from few millimetres down to 100 microns were realized. The well-studied thrombin-binding aptamer (TBA) was used as receptor for characterizing these micro- and macro-electrodes. The impedance signal showed a linear correlation for concentrations of thrombin between 1.0 nM to 100 nM. This range corresponds well with most of the references and may be useful for the point-of-care testing (POCT).

  16. Increased thrombin generation after acute versus chronic coronary disease as assessed by the thrombin generation test

    OpenAIRE

    Orbe, J.; Zudaire, M.; Serrano, R.; Coma-canella, I.; Martinez-de-sizarrondo, S.; Rodriguez, J. A.; Paramo, J. A.

    2008-01-01

    Atherosclerosis is the most common pathophysiologic substrate of coronary artery disease (CAD). Whereas plaque progression and arterial remodeling are critical components in chronic CAD, intracoronary thrombosis over plaque disruption is causally related to acute CAD. It was the objective of this study to investigate the differences between prior acute CAD and chronic CAD by a simple global coagulation assay measuring thrombin generation. A cross-sectional study involving 15 healthy controls,...

  17. Disparate temporal expression of the prothrombin and thrombin receptor genes during mouse development.

    OpenAIRE

    Soifer, S. J.; Peters, K. G.; O Keefe, J.; Coughlin, S. R.

    1994-01-01

    The protease thrombin is a potent agonist for platelet aggregation, mesenchymal cell proliferation, and endothelial production of growth factors and adhesion molecules. Thrombin also modulates neurite outgrowth in neuronal cultures. These apparently disparate responses to thrombin appear to be largely mediated by the recently cloned thrombin receptor. In the adult, thrombin is generated from its zymogen prothrombin at sites of vascular injury when circulating coagulation factors meet extravas...

  18. Preparation of fibrin des-AA by thrombin.

    Science.gov (United States)

    Lougovskoi, E V; Gogolinskaya, G K

    1999-01-01

    The active thrombin is formed in the blood stream when the blood coagulation system is activated. It attacks fibrinogen, splits off two fibrinopeptides A and fibrinogen is transformed into des-AA fibrin which is able to polymerize spontaneously forming protofibrils. At high thrombin concentration the enzyme splits off two fibrinopeptides B and des-AA fibrin units are transformed into des-AABB fibrin. These two forms of fibrin are widely used in the biological experiments. However des-AA fibrin is obtained usually from fibrinogen using the snake poisons (such as reptilase). Des-AA fibrin was obtained also by physiological enzyme thrombin, but that des-AA fibrin samples had the contamination of des-AABB fibrin. At the present paper we have described the method of the des-AA fibrin preparation by thrombin without any contamination of des-AABB fibrin. PMID:10791069

  19. Interaction of radiocontrast agents with the thrombin-antithrombin system

    International Nuclear Information System (INIS)

    This paper presents a precise investigation of the interaction of different contrast media (CM) with the thrombin-antithrombin system. Experimental methods and materials are described and results are analyzed

  20. Antithrombotic effects of bromophenol, an alga-derived thrombin inhibitor

    Science.gov (United States)

    Shi, Dayong; Li, Xiaohong; Li, Jing; Guo, Shuju; Su, Hua; Fan, Xiao

    2010-01-01

    Thrombin, the ultimate proteinase of the coagulation cascade, is an attractive target for the treatment of a variety of cardiovascular diseases. A bromophenol derivative named (+)-3-(2,3-dibromo-4, 5-dihydroxy-phenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroiso-benzofuran 1, isolated from the brown alga Leathesia nana exhibited significant thrombin inhibitory activity. In this study, we investigated the inhibition of human thrombin in vitro with this bromophenol derivative, and its antithrombotic efficacy in vivo using the arteriovenous shunt model and the ferric chloride-induced arterial thrombosis model in rats. The results show that the bromophenol derivative is a potential inhibitor of thrombin (IC50=1.03 nmol/L). In antithrombotic experiments in vivo, the bromophenol derivative also shows good effect comparing with the control group. These data indicate that the bromophenol derivative is a potential drug for prophylaxis and the treatment of thrombotic diseases.

  1. Development and Optimization of a Thrombin Sandwich Aptamer Microarray

    OpenAIRE

    Anna Meneghello; Alice Sosic; Agnese Antognoli; Erica Cretaio; Barbara Gatto

    2012-01-01

    A sandwich microarray employing two distinct aptamers for human thrombin has been optimized for the detection of subnanomolar concentrations of the protein. The aptamer microarray demonstrates high specificity for thrombin, proving that a two-site binding assay with the TBA1 aptamer as capture layer and the TBA2 aptamer as detection layer can ensure great specificity at times and conditions compatible with standard routine analysis of biological samples. Aptamer microarray sensitivity was eva...

  2. Mutant N143P Reveals How Na+ Activates Thrombin*

    OpenAIRE

    Niu, Weiling; Chen, Zhiwei; Bush-pelc, Leslie A.; Bah, Alaji; Gandhi, Prafull S.; Di Cera, Enrico

    2009-01-01

    The molecular mechanism of thrombin activation by Na+ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na+ forms. The extended scheme establishes that analysis of kcat unequivocally identifies allosteric transduction of Na+ binding into enhanced catalytic activity. The thrombin mutant N143P features no Na+-dependent enhancement of kcat yet bi...

  3. Stability and Binding Properties of a Modified Thrombin Binding Aptamer

    OpenAIRE

    Pagano, Bruno; Martino, Luigi; Randazzo, Antonio; Giancola, Concetta

    2007-01-01

    Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), 3?GGT5?-5?TGGTGTGGTTGG3?, which also folds into a quadruplex ...

  4. Fibrinogen Substrate Recognition by Staphylocoagulase·(Pro)thrombin Complexes*

    OpenAIRE

    Panizzi, Peter; Friedrich, Rainer; Fuentes-Prior, Pablo; Richter, Klaus; Bock, Paul E.; Bode, Wolfram

    2005-01-01

    Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates prothrombin (ProT) without proteolysis. The SC·(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC fragment containing residues 1–325 (SC-(1–325)) bound to human prethrombin 2 showed prev...

  5. A novel thrombin-reactive protein complex in human platelets.

    Science.gov (United States)

    Chelladurai, M; Fossett, N G; Ganguly, P

    1983-02-10

    Attempts were made to isolate a 74,000-dalton protein which specifically and competitively blocked platelet aggregation by thrombin (Ganguly, P., and Fossett, N. G. (1981) Blood 57, 343-352) by two independent affinity chromatography methods. The protein isolates showed a main band migrating slightly faster than albumin in gel electrophoresis under nondenaturing conditions. In the presence of sodium dodecyl sulfate, electrophoresis of protein preparations consistently revealed the presence of four polypeptides of 74,000, 55,000, 27,000, and 20,000 daltons. The results suggest that these polypeptides are probably present as a multiprotein complex in platelets. The 55,000-dalton protein, like the 74,000-dalton protein, inhibited thrombin-induced platelet aggregation, while aggregation induced by adenosine diphosphate or trypsin was not significantly affected. However, incubation of the 55,000-dalton protein with the 74,000-dalton protein prior to the addition of thrombin did not inhibit platelet aggregation by thrombin, while the individual components under the same conditions did. In fact, the two proteins in combination could significantly enhance platelet aggregation by thrombin. These results suggest the existence of a multiprotein complex in human platelets, the components of which may modulate the action of thrombin on platelet aggregation. PMID:6822519

  6. Automatic and Integrated Micro-Enzyme Assay (AI?EA) Platform for Highly Sensitive Thrombin Analysis via an Engineered Fluorescence Protein-Functionalized Monolithic Capillary Column.

    Science.gov (United States)

    Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

    2015-04-21

    Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AI?EA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AI?EA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AI?EA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AI?EA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AI?EA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening. PMID:25811786

  7. Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI

    Directory of Open Access Journals (Sweden)

    Kristensen Torsten

    2009-05-01

    Full Text Available Abstract Background TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI, and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. Results The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. Conclusion The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

  8. Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)

    DEFF Research Database (Denmark)

    Valnickova, Zuzana; Thaysen-Andersen, Morten

    2009-01-01

    BACKGROUND: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. RESULTS: The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. CONCLUSION: The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

  9. The structure-function relationship of thrombin-like enzymes from the green pit viper (Trimeresurus albolabris).

    Science.gov (United States)

    Pradniwat, Paweena; Rojnuckarin, Ponlapat

    2015-06-15

    Pit viper venoms can decrease fibrinogen levels in snakebite patients. Studies have shown that the hypofibrinogenemia is a consequence of snake venom thrombin-like enzymes (TLEs), the serine proteases that have the potential to be both diagnostic and therapeutic agents. Exosites of thrombin are the molecular regions that determine the substrate specificities, but its presence and significance in TLEs are unclear. Therefore, the putative exosites of recombinant TLEs derived from Green pit viper (Trimeresurus albolabris), GPV-TL1 and GPV-TL2, were mutated in a Pichia pastoris system. In a previous report, GPV-TL1 showed a strong fibrinogenolytic activity on the A? and B? chains of fibrinogen, as well as a plasma clotting activity. Compared with GPV-TL1, the GPV-TL1m mutated in the putative exosite (TRN to RRR at residues 60-62) showed a weaker fibrinogenolytic activity with a similar clotting activity of 207.1 thrombin units/mg. GPV-TL2 contained two-residue differences from GPV-TL1 in the putative exosite (N73M and V74Y). GPV-TL2 selectively cleaved only the A? chain of fibrinogen without detectable clotting activity. The mutated GPV-TL2 (GPV-TL2m) showed a weaker fibrinogenolytic activity compared with that of the wild type. These results support the important roles of the putative exosite in snake venom TLE activities. This information is helpful for future protein engineering. PMID:25912946

  10. Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia

    Directory of Open Access Journals (Sweden)

    PaulaGrammas

    2013-05-01

    Full Text Available Considerable evidence implicates hypoxia and vascular inflammation in Alzheimer’s disease (AD. Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1? (HIF-1?, a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of transgenic AD mice and to explore the role of thrombin as a mediator of cerebrovascular inflammation and oxidative stress in AD and in hypoxia-induced changes in brain endothelial cells. Immunofluorescent analysis of the cerebrovasculature in AD mice demonstrates significant (p<0.01-0.001 increases in thrombin, HIF-1?, interleukin-6 (IL-6, monocyte chemoattractant protein-1 (MCP-1, matrix metalloproteinases (MMPs, and reactive oxygen species (ROS compared to controls. Administration of the thrombin inhibitor dabigatran (100 mg/kg to AD mice for 34 wks significantly decreases expression of inflammatory proteins and ROS. Exposure of cultured brain endothelial cells to hypoxia for 6 h causes an upregulation of thrombin, HIF-1?, MCP-1, IL-6 and MMP2 and ROS. Treatment of endothelial cells with the dabigatran (1 nM reduces ROS generation and inflammatory protein expression (p<0.01-0.001. The data demonstrate that inhibition of thrombin in culture blocks the increase in inflammatory protein expression and ROS generation evoked by hypoxia. Also, administration of dabigatran to transgenic AD mice diminishes expression of inflammatory proteins and ROS in the cerebromicrovasculature. Taken together, these results suggest that inhibiting thrombin generation could have therapeutic value in AD and other disorders where hypoxia, inflammation and oxidative stress are involved.

  11. Recombinant activated factor VII: 30 years of research and innovation.

    Science.gov (United States)

    Hedner, Ulla

    2015-06-01

    Recombinant activated factor VII (rFVIIa) was initially developed to treat bleeding episodes in patients with congenital haemophilia and inhibitors. The story of its development began in the 1970s, when FVIIa was identified as one of the activated coagulation factors that has minimal potential for inducing thromboembolic side-effects. Extensive research over the last 30 years has greatly increased our knowledge of the characteristics of FVII, its activation, and the mechanisms by which rFVIIa restores haemostasis. In haemophilia, the haemostatic effect of rFVIIa is mediated via binding to thrombin-activated platelets at the site of injury, thereby enhancing thrombin generation also in the absence of factor (F) VIII or FIX. The mechanism of action of rFVIIa has also allowed its successful use in other clinical scenarios characterised by impaired thrombin generation, and its licensed uses have now been extended to acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia. PMID:26073368

  12. Subconjunctival and topical application of recombinant tissue plasminogen activator in rabbits / Uso tópico e subconjuntival de ativador de plasminogênio tecidual recombinante em coelhos

    Scientific Electronic Library Online (English)

    José Ricardo de Abreu, Reggi; Richard Yudi, Hida; Milton Massato, Hida; Maria Cristina, Nishiwaki-Dantas; Hisashi, Suzuki.

    2015-02-01

    Full Text Available Objetivo: Quantificar produtos de degradação de fibrina (PDF) após uso tópico e subconjunctival de ativador de plasminogênio tecidual recombinante (r-TPA) em coelhos. Métodos: Formação de fibrina foi induzida na câmara anterior em 25 coelhos. Cinco coelhos foram submetidos a injeção intracameral de [...] r-TPA (controle positivo). Dez coelhos foram submetidos a injeção subconjuntival de r-TPA e dez coelhos foram submetidos a instilação tópica de r-TPA. Amostras de humor aquoso foram coletados e uma análise quantitativa dos produtos de degradação de fibrina foi realizada. Resultados: Não foi observado diferença estatisticamente significativa na degradação de fibrina em nenhum dos momentos estudados quando comparados com o controle. Porém foi observado diferença estatisticamente significante na quantificação do produtos de degradação de fibrina no grupo controle e no grupo subconjuntival. Conclusão: Produtos de degradação de fibrina foi observado nas amostras do grupo subconjunctival, porém, provavelmente não foi suficiente para degradar a fibrin presente. r-TPA tópico não foi efetivo em absorver fibrina na câmara anterior. Abstract in english Purpose: To quantify fibrin degradation products after topical and subconjunctival administration of recombinant tissue plasminogen activator in rabbits. Methods: Fibrin formation was induced in the anterior chamber in 25 rabbits. Subsequently, five rabbits received an injection of r-TPA (positive [...] control) in the anterior chamber, another 10 received a subconjunctival injection of r-TPA, and the remaining 10 received instillations of topical r-TPA. Afterwards, samples of aqueous humor were collected and semi-quantitative analysis of fibrin degradation products (FDP) was performed. Results: No statistical differences were noted between the treatment and control groups at any time point. Fibrin degradation products semi-quantification showed statistical improvement in the control group and the subconjunctival group. Conclusion: Fibrin degradation products were observed in the anterior chamber after subconjunctival administration of r-TPA. However, it was probably not sufficient to cause fibrin degradation. Topical r-TPA did not effectively absorb anterior chamber fibrin.

  13. Identification of the thrombin receptor on human platelets by chemical crosslinking.

    OpenAIRE

    Takamatsu, J.; Horne, M. K.; Gralnick, H. R.

    1986-01-01

    To identify the molecular site of thrombin binding to the platelet membrane, we covalently linked 125I-thrombin to platelets by using the bifunctional chemical cross-linking agents disuccinimidyl suberate and dithiobis(succinimidyl propionate). The proteins cross-linked to 125I-thrombin by this method were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by autoradiography. Two radiolabeled thrombin complexes were identified, a major species of Mr approximate...

  14. A novel thrombin enhancement factor in human plasma.

    Science.gov (United States)

    Ganguly, P; Chelladurai, M; Fossett, N; Jefferson, W

    1983-10-14

    A protein has been isolated from human plasma by gel filtration followed by affinity chromatography with a derivative of wheat germ agglutinin and ion exchange chromatography. This protein showed one peak in high performance liquid chromatography but in gel electrophoresis, in the presence of sodium dodecyl sulfate and beta-mercaptoethanol, revealed two major components of 74 kDa and 55 kDa. These results indicate that the protein probably exists as a complex of the two polypeptides. This protein complex enhanced platelet aggregation by thrombin while aggregation induced by ADP was not significantly affected. Similarly, the rate of thrombin action on fibrinogen and N-benzoylarginine ethyl ester as measured in a spectrophotometer was increased in the presence of this plasma protein. These results suggest the presence of a protein complex in human plasma which can directly interact with thrombin and enhance its reactivity. PMID:6639657

  15. Development and Optimization of a Thrombin Sandwich Aptamer Microarray

    Directory of Open Access Journals (Sweden)

    Anna Meneghello

    2012-08-01

    Full Text Available A sandwich microarray employing two distinct aptamers for human thrombin has been optimized for the detection of subnanomolar concentrations of the protein. The aptamer microarray demonstrates high specificity for thrombin, proving that a two-site binding assay with the TBA1 aptamer as capture layer and the TBA2 aptamer as detection layer can ensure great specificity at times and conditions compatible with standard routine analysis of biological samples. Aptamer microarray sensitivity was evaluated directly by fluorescent analysis employing Cy5-labeled TBA2 and indirectly by the use of TBA2-biotin followed by detection with fluorescent streptavidin. Sub-nanomolar LODs were reached in all cases and in the presence of serum, demonstrating that the optimized aptamer microarray can identify thrombin by a low-cost, sensitive and specific method.

  16. Activation of human factor V by factor Xa and thrombin

    International Nuclear Information System (INIS)

    The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polycarylamide gel electrophoresis followed by either autoradiography of 125I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of Mr 220,000 and 105,000. Although thrombin cleaved the Mr 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the Mr 220,000 peptide. The factor Xa dependent functional assessment of 125I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the Mr 220,000he appearance of the Mr 220,000 peptide. The data indicate that factor Xa is as efficient an enzyme toward factor V as thrombin

  17. Intravitreal thrombin activity is elevated in retinal vein occlusion.

    Science.gov (United States)

    Bertelmann, Thomas; Stief, Thomas; Sekundo, Walter; Mennel, Stefan; Nguyen, Nauke; Koss, Michael J

    2014-10-01

    To evaluate whether intravitreal thrombin activity is elevated in eyes with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) in comparison to healthy controls. Prospective clinical case series of 19 patients with BRVO, 13 patients suffering from CRVO and nine participants serving as controls. Vitreous taps were extracted from the central vitreous body, 200??l frozen/thawed sample was immediately stabilized with 200??l 5% human albumin, and 200??l mixture thereof was stabilized with 200??l 2.5?mol/l arginine, pH 8.6. Thrombin activity was determined chromogenically. Intravitreal levels of vascular endothelial growth factor (VEGF) as a marker for blood-retina barrier (BRB) breakdown were measured by a commercial chemiluminescent enzyme immuno assay (R&D). Intravitreal thrombin activity and VEGF levels were 1.6?±?1.2?mIU/ml (mean value?±?SD; range: 0.2-4.2?mIU/ml) and 554?±?568?pg/ml (range: 20-2005?pg/ml) in BRVO-affected eyes, 2.6?±?1.2?mIU/ml (range: 0.8-5.2?mIU/ml) and 1332?±?1350?pg/ml (range: 58-3943?pg/ml) in eyes suffering from CRVO as well as 0.8?±?0.8?mIU/ml (range: 0.2-2.7?mIU/ml) and 115?±?120?pg/ml (range: 32-431?pg/ml) in controls. There are significant differences of intravitreal thrombin activity and intravitreal VEGF levels between eyes with BRVO, CRVO, and controls (P?=?0.007 and P?=?0.003, Kruskal-Wallis test). Intravitreal thrombin activity is significantly correlated with intravitreal VEGF levels (r?=?0656; P?thrombin activity might serve as a new marker for BRB breakdown or macular fibrin deposition in ophthalmology. Significant differences of intravitreal thrombin activity between eyes with BRVO, CRVO, and healthy controls might offer new therapeutic strategies for RVO-affected eyes. The effect of oral and intravitrealy injected direct thrombin inhibitors needs to be evaluated in further investigations. PMID:24625582

  18. Cell biology of mitotic recombination.

    Science.gov (United States)

    Lisby, Michael; Rothstein, Rodney

    2015-03-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination. PMID:25731763

  19. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.

  20. Recombinant Programming

    OpenAIRE

    Pawlak, Renaud; Cuesta, Carlos; Younessi, Houman

    2004-01-01

    This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and allows the programmer to recombine these sequences through the definition of cohesive code units called extensions. The output of such recombination is a mesh that can be used by the interpretation la...

  1. Thrombin induces rapid PAR1-mediated non-classical FGF1 release

    International Nuclear Information System (INIS)

    Thrombin induces cell proliferation and migration during vascular injury. We report that thrombin rapidly stimulated expression and release of the pro-angiogenic polypeptide fibroblast growth factor 1 (FGF1). Thrombin failed to induce FGF1 release from protease-activated receptor 1 (PAR1) null fibroblasts, indicating that this effect was dependent on PAR1. Similarly to thrombin, FGF1 expression and release were induced by TRAP, a specific oligopeptide agonist of PAR1. These results identify a novel aspect of the crosstalk between FGF and thrombin signaling pathways which both play important roles in tissue repair and angiogenesis

  2. Crystal structure of thrombin in complex with S-variegin: insights of a novel mechanism of inhibition and design of tunable thrombin inhibitors.

    OpenAIRE

    Koh, Cy; Kumar, S.; Kazimirova, M.; Nuttall, Pa; Radhakrishnan, Up; Kim, S.; Jagadeeswaran, P.; Imamura, T.; Mizuguchi, J.; Iwanaga, S.; Swaminathan, K.; Kini, Rm

    2011-01-01

    The inhibition of thrombin is one of the important treatments of pathological blood clot formation. Variegin, isolated from the tropical bont tick, is a novel molecule exhibiting a unique ‘two-modes’ inhibitory property on thrombin active site (competitive before cleavage, noncompetitive after cleavage). For the better understanding of its function, we have determined the crystal structure of the human ?-thrombin:synthetic-variegin complex at 2.4 Å resolution. The structure reveals a ne...

  3. Thrombin injection for the treatment of mycotic gluteal aneurysm.

    Science.gov (United States)

    Rubinstein, Chen; Endean, Eric D; Minion, David J; Sorial, Ehab E; O'Keeffe, Shane D; Xenos, Eleftherios S

    2012-01-01

    Gluteal aneurysms are rare entity, whose surgical or endovascular management is traditionally challenging. Infectious source being increasingly more common as the underlying etiology. We herein describe successful implementation of direct thrombin injection as another therapeutic option for these patients. PMID:21890561

  4. A Guided Mode Resonance Aptasensor for Thrombin Detection

    Directory of Open Access Journals (Sweden)

    Wen-Yih Chen

    2011-09-01

    Full Text Available Recent developments in aptamers have led to their widespread use in analytical and diagnostic applications, particularly for biosensing. Previous studies have combined aptamers as ligands with various sensors for numerous applications. However, merging the aptamer developments with guided mode resonance (GMR devices has not been attempted. This study reports an aptasensor based home built GMR device. The 29-mer thrombin aptamer was immobilized on the surface of a GMR device as a recognizing ligand for thrombin detection. The sensitivity reported in this first trial study is 0.04 nm/?M for thrombin detection in the concentration range from 0.25 to 1 ?M and the limit of detection (LOD is 0.19 ?M. Furthermore, the binding affinity constant (Ka measured is in the range of 106 M?1. The investigation has demonstrated that such a GMR aptasensor has the required sensitivity for the real time, label-free, in situ detection of thrombin and provides kinetic information related to the binding.

  5. The thrombin binding aptamer GGTTGGTGTGGTTGG forms a bimolecular guanine tetraplex.

    Czech Academy of Sciences Publication Activity Database

    Fialová, Markéta; Kypr, Jaroslav; Vorlí?ková, Michaela

    2006-01-01

    Ro?. 344, 1 (2006), s. 50-54. ISSN 0006-291X R&D Projects: GA AV ?R(CZ) IAA4004201 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA tetraplex * thrombin binding aptamer * CD spectroscopy Subject RIV: BO - Biophysics Impact factor: 2.855, year: 2006

  6. An aptamer-gated silica mesoporous material for thrombin detection

    OpenAIRE

    Oroval Cucarella, María del Mar; Climent Terol, Estela; Coll Merino, Mª Carmen; Eritja, Ramon; Aviñó, Anna; Marcos Martínez, María Dolores; Sancenón Galarza, Félix; Martínez Mañez, Ramón; AMOROS DEL TORO, PEDRO JOSE

    2013-01-01

    Oroval Cucarella, MDM.; Climent Terol, E.; Coll Merino, MC.; Eritja, R.; Aviñó, A.; Marcos Martínez, MD.; Sancenón Galarza, F.... (2013). An aptamer-gated silica mesoporous material for thrombin detection. Chemical Communications. 49(48):5480-5482. doi:10.1039/c3cc42157k.

  7. VACCINATION OF CATTLE WITH RECOMBINANT SALIVARY PROTEINS OF HORN FLIES (HAEMATOBIA IRRITANS IRRITANS).

    Science.gov (United States)

    Adult horn flies are ectoparasites of cattle that require multiple daily bloodmeals for basic nutrition and reproduction. We demonstrated earlier that vaccination of cattle with a recombinant form of an anti-thrombin protein in horn fly saliva, Thrombostasin (TB8), caused a reduction in blood uptake...

  8. Thrombin generation using the calibrated automated thrombinoscope to assess reversibility of dabigatran and rivaroxaban.

    Science.gov (United States)

    Herrmann, Richard; Thom, James; Wood, Alicia; Phillips, Michael; Muhammad, Shoaib; Baker, Ross

    2014-05-01

    The new direct-acting anticoagulants such as dabigatran and rivaroxaban are usually not monitored but may be associated with haemorrhage, particularly where renal impairment occurs. They have no effective "antidotes". We studied 17 patients receiving dabigatran 150 mg twice daily for non-valvular atrial fibrillation and 15 patients receiving rivaroxaban 10 mg daily for the prevention of deep venous thrombosis after hip or knee replacement surgery. We assessed the effect of these drugs on commonly used laboratory tests and Calibrated Automated Thrombogram (CAT) using plasma samples. We also assessed effects in fresh whole blood citrated patient samples using thromboelastography on the TEG and the ROTEM. The efficacy of nonspecific haemostatic agents prothrombin complex concentrate (PCC), Factor VIII Inhibitor By-passing Activity (FEIBA) and recombinant activated factor VII (rVIIa) were tested by reversal of abnormal thrombin generation using the CAT. Concentrations added ex vivo were chosen to reflect doses normally given in vivo. Dabigatran significantly increased the dynamic parameters of the TEG and ROTEM and the lag time of the CAT. It significantly reduced the endogenous thrombin potential (ETP) and reduced the peak height of the CAT. Rivaroxaban did not affect the TEG and ROTEM parameters but did increase the lag time and reduce ETP and peak height of the CAT. For both drugs, these parameters were significantly and meaningfully corrected by PCC and FEIBA and to a lesser but still significant extent by rFVIIa. These results may be useful in devising a reversal strategy in patients but clinical experience will be needed to verify them. PMID:24352511

  9. The NMR solution structure of recombinant RGD-hirudin

    International Nuclear Information System (INIS)

    The solution structure of a new recombinant RGD-hirudin, which has the activities of anti-thrombin and anti-platelet aggregation, was determined by 1H nuclear magnetic resonance spectroscopy and compared with the conformations of recombinant wild-type hirudin and hirudin (variant 2, Lys47) of the hirudin thrombin complex. On the basis of total 1284 distance and dihedral angle constraints derived from a series of NMR spectra, 20 conformers were computed with ARIA/CNS programs. The structure of residues 3-30 and 37-48 form a molecular core with two antiparallel ?-sheets as the other two hirudins. However, significant differences were found in the surface electrostatic charge distributions among the three hirudins, especially in the RGD segment of recombinant RGD-hirudin. This difference may be greatly beneficial to its additional function of anti-platelet aggregation. The difference in extended C-terminal makes its both ionic and hydrophobic interactions with the fibrinogen recognition exosite of thrombin more effective

  10. Skin regeneration in deep second-degree scald injuries either by infusion pumping or topical application of recombinant human erythropoietin gel

    Directory of Open Access Journals (Sweden)

    Giri P

    2015-05-01

    Full Text Available Priya Giri,1 Sabine Ebert,1 Ulf-Dietrich Braumann,2 Mathias Kremer,3 Shibashish Giri,1 Hans-Günther Machens,4 Augustinus Bader1 1Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ, Faculty of Medicine, University of Leipzig, Leipzig, Germany; 2Interdisciplinary Center for Bioinformatics (IZBI, University of Leipzig, Leipzig, Germany; 3Department of Plastic and Hand Surgery, University of Lübeck, Lübeck, Germany; 4Department of Plastic and Hand Surgery, Technical University of Munich, Munich, Germany Abstract: Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application were applied to deliver recombinant human erythropoietin (rHuEPO for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 µm thickness, which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin, which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds. Keywords: re-epithelialization, scald wound, skin regeneration, neovascularization, vascularization, segmentation

  11. Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents

    International Nuclear Information System (INIS)

    Two DNA aptamers directed against two separate exosites on human ?-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis

  12. Thrombin generation by activated factor VII on platelet activated by different agonists. Extending the cell-based model of hemostasis

    Directory of Open Access Journals (Sweden)

    Herrera Maria

    2006-04-01

    Full Text Available Abstract Background Platelet activation is crucial in normal hemostasis. Using a clotting system free of external tissue factor, we investigated whether activated Factor VII in combination with platelet agonists increased thrombin generation (TG in vitro. Methods and results TG was quantified by time parameters: lag time (LT and time to peak (TTP, and by amount of TG: peak of TG (PTG and area under thrombin formation curve after 35 minutes (AUC?35min in plasma from 29 healthy volunteers using the calibrated automated thrombography (CAT technique. TG parameters were measured at basal conditions and after platelet stimulation by sodium arachidonate (AA, ADP, and collagen (Col. In addition, the effects of recombinant activated FVII (rFVIIa alone or combined with the other platelet agonists on TG parameters were investigated. We found that LT and TTP were significantly decreased (p 35min were significantly increased (p 35min (but not PTG when compared to platelet rich plasma activated with agonists in the absence of rFVIIa. Conclusion Platelets activated by AA, ADP, Col or rFVIIa triggered TG. This effect was increased by combining rFVIIa with other agonists. Our intrinsic coagulation system produced a burst in TG independent of external tissue factor activity an apparent hemostatic effect with little thrombotic capacity. Thus we suggest a modification in the cell-based model of hemostasis.

  13. Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia

    OpenAIRE

    PaulaGrammas

    2013-01-01

    Considerable evidence implicates hypoxia and vascular inflammation in Alzheimer's disease (AD). Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1? (HIF-1?), a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of t...

  14. Thrombin-Binding Aptamer Quadruplex Formation: AFM and Voltammetric Characterization

    OpenAIRE

    Ana Maria Oliveira-Brett; Ramon Eritja; Ana-Maria Chiorcea-Paquim; Victor Constantin Diculescu

    2010-01-01

    The adsorption and the redox behaviour of thrombin-binding aptamer (TBA) and extended TBA (eTBA) were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K+, and voltammetric behaviour of TBA and eTBA. In the presence of K+, only a few single-stranded sequences present adsorption, while the majority ...

  15. Metabolic Plasticity in Resting and Thrombin Activated Platelets

    Science.gov (United States)

    Ravi, Saranya; Chacko, Balu; Sawada, Hirotaka; Kramer, Philip A.; Johnson, Michelle S.; Benavides, Gloria A.; O’Donnell, Valerie; Marques, Marisa B.; Darley-Usmar, Victor M.

    2015-01-01

    Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand. PMID:25875958

  16. Recombination in nuclear collisions

    OpenAIRE

    Rudolph C. Hwa

    2010-01-01

    Recombination is a hadronization process that converts partons to hadrons at late time, but the description has no quantitative significance without some meaningful input on the parton distributions at earlier time. Thus observations of particle spectra and correlations have definitive implications on the partonic processes at all transverse momenta. After presenting a general review of the subject at the Workshop, I selected two topics in nuclear collisions for more detaile...

  17. Nonradiative recombination in semiconductors

    CERN Document Server

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

  18. Combined administration of aspirin and a specific thrombin inhibitor in man.

    OpenAIRE

    Clarke, Rj; Mayo, G.; Fitzgerald, Ga; Fitzgerald, Dj

    1991-01-01

    BACKGROUND: Heparin is of limited value as an antithrombotic drug in the presence of platelet activation and residual thrombus. Greater anticoagulant activity can be achieved in vivo with more specific thrombin inhibitors. Heparin may also increase the risk of bleeding by an effect on platelets that is independent of its thrombin inhibitory activity. METHODS AND RESULTS: The pharmacodynamic and pharmacokinetic effects of a novel thrombin inhibitor, argatroban, were examined alone and in combi...

  19. Evaluation of Antithrombotic Activity of Thrombin DNA Aptamers by a Murine Thrombosis Model

    OpenAIRE

    Zavyalova, Elena; Samoylenkova, Nadezhda; Revishchin, Alexander; Golovin, Andrey; Pavlova, Galina; Kopylov, Alexey

    2014-01-01

    Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the react...

  20. Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase

    OpenAIRE

    Alice Sosic; Erica Cretaio; Barbara Gatto; Anna Meneghello

    2011-01-01

    We have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whet...

  1. The cleaved peptide of the thrombin receptor is a strong platelet?agonist

    OpenAIRE

    Furman, Mark I.; Liu, Longbin; Benoit, Stephen E.; Becker, Richard C; Barnard, Marc R; Michelson, Alan D.

    1998-01-01

    Thrombin cleaves its G-protein-linked seven-transmembrane domain receptor, thereby releasing a 41-aa peptide and generating a new amino terminus that acts as a tethered ligand for the receptor. Peptides corresponding to the new amino terminal end of the proteolyzed seven-transmembrane domain thrombin receptor [TR42–55, SFLLRNPNDKYEPF, also known as TRAP (thrombin receptor-activating peptide)], previously have been demonstrated to activate the receptor. In this study, we demonstrate that the 4...

  2. Aptamer-Crosslinked Microbubbles: Smart Contrast Agents for Thrombin-Activated Ultrasound Imaging

    OpenAIRE

    Nakatsuka, Matthew A.; Mattrey, Robert F.; Esener, Sadik C.; Cha, Jennifer N.; Goodwin, Andrew P.

    2012-01-01

    Thrombosis, or malignant blood clotting, is associated with numerous cardiovascular diseases and cancers. This report describes a microbubble contrast agent that produces ultrasound harmonic signal only when exposed to elevated thrombin levels. Silenced initially, microbubbles activated in the presence of both thrombin-spiked and freshly clotting blood in three minutes with detection limits of 20 nM thrombin and 2 aM microbubbles.

  3. Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells.

    Science.gov (United States)

    Levin, E G; Marzec, U; Anderson, J; Harker, L A

    1984-01-01

    The effect of thrombin on the release of tissue plasminogen activator from endothelial cells was studied in primary cultures of human umbilical vein endothelial cells. Tissue plasminogen activator concentration in conditioned medium was measured by a two-site radioimmunometric assay. The addition of increasing concentrations (0.01 to 10 U/ml) of thrombin to confluent cultures produced a saturable, dose-dependent increase in the rate of release of tissue plasminogen activator. A sixfold increase in tissue plasminogen activator concentration (from 2 to 12 ng/ml) occurred after the addition of 1 U/ml thrombin (8 X 10(-9) M) to cultures containing 5 X 10(4) cells/cm2. Enhanced release was not observed until 6 h after thrombin addition, reached a maximum rate of 1.3 ng/ml per h between 8 and 16 h, and then declined to 0.52 ng/ml per h after 16 h. The 6-h lag period before increased tPA release was reproducible and independent of thrombin concentration. Thrombin inactivated with diisopropylfluorophosphate or hirudin did not induce an increase in tissue plasminogen activator levels. A 50-fold excess of diisopropylfluorophosphate-treated thrombin, which inhibits binding of active thrombin to endothelial cell high affinity binding sites, did not inhibit the thrombin-induced increase. It is concluded that proteolitically active thrombin causes an increase in the rate of release of tissue plasminogen activator from cultured human endothelial cells. The 6-h interval between thrombin treatment and enhanced tissue plasminogen activator release may reflect a delaying mechanism that transiently protects hemostatic plugs from the sudden increase in the local concentration of this fibrinolytic enzyme. Images PMID:6542570

  4. Recombination instability

    DEFF Research Database (Denmark)

    D'Angelo, N.

    1967-01-01

    A recombination instability is considered which may arise in a plasma if the temperature dependence of the volume recombination coefficient, alpha, is sufficiently strong. Two cases are analyzed: (a) a steady-state plasma produced in a neutral gas by X-rays or high energy electrons; and (b) an afterglow plasma.

  5. Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

    OpenAIRE

    Vouret-craviari, V.; Obberghen-schilling, E.; Rasmussen, U. B.; Pavirani, A.; Lecocq, J. P.; Pouysse?gur, J.

    1992-01-01

    alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl ...

  6. A plasmonic aptasensor for ultrasensitive detection of thrombin via arrested rolling circle amplification.

    Science.gov (United States)

    Wang, Sai; Bi, Sai; Wang, Zonghua; Xia, Jianfei; Zhang, Feifei; Yang, Min; Gui, Rijun; Li, Yanhui; Xia, Yanzhi

    2015-05-01

    A sensitive signal generation mechanism for gold nanoparticle growth by reducing gold ions with hydrogen peroxide is applied in a plasmonic aptasensor, achieving naked-eye detection of thrombin at the single-molecule level based on the specific interaction of aptamer-thrombin via an arrested rolling circle amplification to yield horseradish peroxidase (HRP)-mimicking DNAzymes as biocatalysts. PMID:25864665

  7. Thrombin receptor activating peptide (TRAP) stimulates mitogenesis, c-fos and PDGF-A gene expression in human vascular smooth muscle cells.

    Science.gov (United States)

    Kanthou, C; Benzakour, O; Patel, G; Deadman, J; Kakkar, V V; Lupu, F

    1995-11-01

    The synthetic peptide SFLLRNPNDKYEPF, identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage of thrombin, acts a thrombin receptor agonist/activating peptide (TRAP). In this study, Northern blot analysis showed that cultured human vascular smooth muscle cells (HVSMC) express a thrombin receptor transcript. TRAP, in contrast to thrombin was shown to be a weak mitogen for HVSMC. A combination of TRAP and enzymatically-inactivated thrombin (PPACK-thrombin) which provides receptor occupancy, did not potentiate TRAP-induced mitogenesis, indicating that TRAP and PPACK-thrombin do not reproduce the mitogenic effect of enzymatically-active thrombin. Both thrombin and TRAP, induced the expression of c-fos and the PDGF-A gene in a pertussis toxin (PTX)-insensitive manner. Examination of thrombin and TRAP-treated cells by immunofluorescence staining followed by computer assisted image analysis revealed that thrombin and to a lesser extent TRAP induced PDGF-AA protein expression. Antibodies to PDGF-AA partially inhibited thrombin but not TRAP-induced mitogenesis in HVSMC. This study indicates that in addition to the common signalling pathways utilised by thrombin and TRAP, enzymatically-active thrombin activates other signalling pathways and hence TRAP does not mimic fully the biological effect of thrombin on HVSMC. PMID:8607120

  8. Thrombin binds to murine bone marrow-derived macrophages and enhances colony-stimulating factor-1-driven mitogenesis

    International Nuclear Information System (INIS)

    The binding and mitogenic properties of thrombin have been established in various transformed cell lines. In such systems, thrombin induces cell division in the absence of exogenous growth factors, and the enzyme is considered to act directly as a mitogen. This study explores thrombin's interaction with nontransformed, growth factor-dependent cells. Binding of 125I-alpha-thrombin to colony-stimulating factor (CSF)-1-dependent bone marrow-derived macrophages is saturable, time-dependent, and displaceable by both unlabeled alpha-thrombin, and esterolytically inactive thrombin. Both dissociation studies of pre-bound radio-labeled thrombin and Scatchard analysis assisted by the program Ligand suggest adherence of thrombin-binding data to a multi-site model. There are an estimated 2 x 10(4) high affinity sites (Kd = 7 x 10(-9)M) and 2 x 10(6) low affinity sites (Kd = 9 x 10(-7)M) per cell. Quiescent bone marrow-derived macrophages were cultured with either 10(-8)M thrombin, 1000 units of CSF-1/ml, or both and [3H]thymidine incorporation was determined. Thrombin alone did not induce mitogenesis. CSF-1 induced mitogenesis with peak [3H] thymidine incorporation occurring 24 h after addition of the mitogen. This CSF-1-dependent mitogenic influence was enhanced greater than 2-fold by treatment with thrombin

  9. Hyperbranched rolling circle amplification based electrochemiluminescence aptasensor for ultrasensitive detection of thrombin.

    Science.gov (United States)

    Jin, Guixiao; Wang, Chunmei; Yang, Linlin; Li, Xiaojuan; Guo, Longhua; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

    2015-01-15

    An ultrasensitive electrochemiluminescence (ECL) aptamer sensor for protein (thrombin as an example) detection based on hyperbranched rolling circle amplification (HRCA) had been developed. A complementary single-strand DNA (CDNA) of the thrombin aptamer had been modified on the gold electrode firstly, and then hybridized with thrombin aptamer to make the aptamer immobilized on the electrode surface, in the presence of thrombin, aptamer-thrombin bioaffinity complexes formed and made thrombin aptamer leave the electrode surface. Thus, the linear padlock probe hybridized with the free CDNA on the electrode surface and circularized by Escherichia coli DNA ligase. Subsequently, the linear padlock probe was served as a template for the initiation of HRCA reaction, and a lot of dsDNA modified on the electrode surface. Then Ru(phen)?²? (acted as the ECL indicator) intercalates specifically into double-stranded DNA (dsDNA) grooves to generate ECL signal. The ECL intensity of the system has a linear relationship with thrombin concentration in the range of 3.0-300 aM with a detection limit of 1.2 aM (S/N=3). The proposed method combines the high sensitivity of ECL, exponential ampli?cation of HRCA for signal enhancement and high selectivity of aptamer. PMID:25086328

  10. Platelet activation and aggregation : the importance of thrombin activity--a laboratory model

    DEFF Research Database (Denmark)

    Jensen, Maria Sander; Larsen, O H

    2013-01-01

    This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated by tissue factor evaluated by means of impedance aggregometry. Citrated whole blood from healthy volunteers and haemophilia A patients with the addition of inhibitors of the contact pathway and fibrin polymerization was evaluated. In healthy persons, a second wave of platelet aggregation was found to coincide with the thrombin burst and to be abolished by thrombin inhibitors. In this system, platelet aggregation in severe haemophilia A (n = 10) was found to be significantly decreased as compared with healthy individuals (912 ± 294 vs. 1917 ± 793 AU × min, P = 0.003), most probably due to the weak level of thrombin generation. For the first time, analysis of platelet aggregation as induced by endogenously generated thrombin was demonstrated. The new method makes it possible to explore the influence of the coagulation system on platelet function. In contrast to the general understanding, the data suggest that the impaired thrombin generation in haemophilia may affect platelet activation. Future studies will address whether our results may contribute to understanding differences in bleeding phenotypes and response to haemostatic substitution observed among patients.

  11. OLIGONUCLEOTIDE-HIRULOG18 CONJUGATED BY CLICK CHEMISTRY AND ITS INHIBITION ON THROMBIN

    Directory of Open Access Journals (Sweden)

    JIE CHAO, JIAN-NING LIU?SHOU-JUN XIAO AND YAN-CHUN TANG

    2011-10-01

    Full Text Available We have shown previously that oligonucleotide conjugated hirulog (bivalirudin has a higher inhibitory activityagainst thrombin compared with hirulog. The negative charged oligonucleotide was considered to be responsible for theadditive activity. To further investigate the related effect of oligonucleotide, the oligonucleotide conjugated hirulog18 wasprepared in the present work and the activity of the conjugate against thrombin was measured. Hirulog18, a peptide lack oftwo N-terminal amino acid residues of hirulog, has little inhibitory activity against thrombin compared with hirulog. Theconjugate oligonucleotide-hiruglog18 was successfully synthesized using click chemistry and validated by MALDI-MS andgel electrophoresis. The activity of the conjugate against thrombin was measured by the chromogenic assay usingChromozym TH as substrate. It was found surprisingly that oligonucleotide-hirulog18 had an inhibitory effect better thanhirulog18 and hirulog. Strong negative charged heparin was used to study the binding mode betweenoligonucleotide-hirulog18 and thrombin. The results suggested that the negative charged oligonucleotide could be helpful forthe conjugate’s binding to the anion-binding exosite of thrombin via the Coulomb force. The highly inhibitory effect of theoligonucleotide-hirulog18 conjugate against thrombin may present a new strategy to generate a novel class of directthrombin inhibitors.

  12. Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation.

    LENUS (Irish Health Repository)

    Ni Ainle, Fionnuala

    2009-08-20

    Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB\\/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% +\\/- 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

  13. Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces

    Science.gov (United States)

    Wheatley Myerson, Jacob; He, Li; Allen, John Stacy; Williams, Todd; Lanza, Gregory; Tollefsen, Douglas; Caruthers, Shelton; Wickline, Samuel

    2014-09-01

    Restoring an antithrombotic surface to suppress ongoing thrombosis is an appealing strategy for treatment of acute cardiovascular disorders such as erosion of atherosclerotic plaque. An antithrombotic surface would present an alternative to systemic anticoagulation with attendant risks of bleeding. We have designed thrombin-targeted nanoparticles (NPs) that bind to sites of active clotting to extinguish local thrombin activity and inhibit platelet deposition while exhibiting only transient systemic anticoagulant effects. Perfluorocarbon nanoparticles (PFC NP) were functionalized with thrombin inhibitors (either D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone or bivalirudin) by covalent attachment of more than 15 000 inhibitors to each PFC NP. Fibrinopeptide A (FPA) ELISA demonstrated that thrombin-inhibiting NPs prevented cleavage of fibrinogen by both free and clot-bound thrombin. Magnetic resonance imaging (MRI) confirmed that a layer of thrombin-inhibiting NPs prevented growth of clots in vitro. Thrombin-inhibiting NPs were administered in vivo to C57BL6 mice subjected to laser injury of the carotid artery. NPs significantly delayed thrombotic occlusion of the artery, whereas an equivalent bolus of free inhibitor was ineffective. For thrombin-inhibiting NPs, only a short-lived (˜10 min) systemic effect on bleeding time was observed, despite prolonged clot inhibition. Imaging and quantification of in vivo antithrombotic NP layers was demonstrated by MRI of the PFC NP. 19F MRI confirmed colocalization of particles with arterial thrombi, and quantitative 19F spectroscopy demonstrated specific binding and retention of thrombin-inhibiting NPs in injured arteries. The ability to rapidly form and image a new antithrombotic surface in acute vascular syndromes while minimizing risks of bleeding would permit a safer method of passivating active lesions than current systemic anticoagulant regimes.

  14. Pseudoaneurysm After Spontaneous Rupture of Renal Angiomyolipoma in Tuberous Sclerosis: Successful Treatment with Percutaneous Thrombin Injection

    International Nuclear Information System (INIS)

    We report a case of a large perinephric pseudoaneurysm due to spontaneous rupture of renal angiomyolipoma, occluded by percutaneous thrombin injection under ultrasound guidance in a young woman affected by tuberous sclerosis

  15. Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

    DEFF Research Database (Denmark)

    Rangnekar, Abhijit; Zhang, Alex M.

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over blood coagulation.

  16. Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase

    Directory of Open Access Journals (Sweden)

    Alice Sosic

    2011-10-01

    Full Text Available We have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1 binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2 binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA, in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized.

  17. Fibrinolytic action of an enzyme preparation covalently bound with modified thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Maksimenko, A.V.; Rusetskii, A.N.; Torchilin, V.P.

    1987-06-01

    It was pointed out previously that modification of thrombin at the tryptophan, tyrosine, arginine and lysine residues impairs its affinity for fibrinogen. Since a long binding site of macromolecular substrate in the thrombin molecule is responsible for binding of the enzyme with the platelet membrane also, it is probably preferable to carry out the modification at other amino acid residues. The purpose of this paper was to confirm experimentally the validity of this approach to the targeted modification of alpha-thrombin in order to obtain a protein polymer matrix with affinity for centers of thrombus formation. Technetium 99m was used as a label in assessing the ability of the modified thrombin to destroy the fibrin clot.

  18. A fluorescent sandwich assay for thrombin using aptamer modified magnetic beads and quantum dots

    International Nuclear Information System (INIS)

    We describe an aptamer-based sandwich assay for thrombin by using a pair of thrombin-binding aptamers, namely one 15-mer aptamer (denoted as Apt15) and one 29-mer aptamer (denoted as Apt29). Either Apt29 or Apt15 can be used as capture aptamers on magnetic beads or reporter aptamers on the quantum dots to form the sandwich complex. Detection of thrombin is achieved by the fluorescent measurement of quantum dots in the sandwich complex. The choice of capture aptamers and reporter aptamers, and the effect of the addition order of the aptamers modified magnetic beads and the aptamers modified quantum dots were investigated. Detection of 0.05 nM thrombin was accomplished. The proteins hemoglobin, lysozyme, and transferrin did not interfere in this assay. (author)

  19. Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis.

    Science.gov (United States)

    Liu, Yanan; Liu, Nan; Ma, Xinhua; Li, Xiaoli; Ma, Jia; Li, Ya; Zhou, Zhijiang; Gao, Zhixian

    2015-04-21

    A novel aptamer-based suspension array detection platform was designed for the sensitive, specific and rapid detection of human ?-thrombin as a model. Thrombin was first recognized by a 29-mer biotinylated thrombin-binding aptamer (TBA) in solution. Then 15-mer TBA modified magnetic beads (MBs) captured the former TBA-thrombin to form an aptamer-thrombin-aptamer sandwich complex. The median fluorescence intensity obtained via suspension array technology was positively correlated with the thrombin concentration. The interactions between TBAs and thrombin were analyzed using microscale thermophoresis (MST). The dissociation constants could be respectively achieved to be 44.2 ± 1.36 nM (TBA1-thrombin) and 15.5 ± 0.637 nM (TBA2-thrombin), which demonstrated the high affinities of TBA-thrombin and greatly coincided with previous reports. Interaction conditions such as temperature, reaction time, and coupling protocol were optimized. The dynamic quantitative working range of the aptamer-based suspension array was 18.37-554.31 nM, and the coefficients of determination R(2) were greater than 0.9975. The lowest detection limit of thrombin was 5.4 nM. This method was highly specific for thrombin without being affected by other analogs and interfering proteins. The recoveries of thrombin spiked in diluted human serum were in the range 82.6-114.2%. This innovative aptamer-based suspension array detection platform not only exhibits good sensitivity based on MBs facilitating highly efficient separation and amplification, but also suggests high specificity by the selective aptamer binding, thereby suggesting the expansive application prospects in research and clinical fields. PMID:25710359

  20. Thrombin related peptide TP508 promoted fracture repair in a mouse high energy fracture model

    OpenAIRE

    Pan Xiao-Hua; Ryaby James T; Hanratty Brain M; Li Gang(School of Physics and Material Science, Anhui University, Hefei, Anhui 230039, P.R. China)

    2009-01-01

    Abstract Background Thrombin related peptide (TP508) is a 23 amino-acid synthetic peptide that represents a portion of the receptor-binding domain of thrombin molecule. Previous studies have shown that TP508 can accelerate musculoskeletal tissue repair including fracture healing. Objectives The aim of this study was to investigate the effect of TP508 on fracture healing in a murine fracture model representing high energy fracture situation. Methods Eighty CD 1 mice underwent controlled quadri...

  1. Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI

    OpenAIRE

    Tucker, Erik I.; Marzec, Ulla M.; White, Tara C.; Hurst, Sawan; Rugonyi, Sandra; Mccarty, Owen J. T.; Gailani, David; Gruber, Andra?s; Hanson, Stephen R.

    2009-01-01

    The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)–dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti–human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and ?-thromboglobulin (?TG...

  2. OLIGONUCLEOTIDE-HIRULOG18 CONJUGATED BY CLICK CHEMISTRY AND ITS INHIBITION ON THROMBIN

    OpenAIRE

    Jie Chao, Jian-ning Liu?shou-jun Xiao And Yan-chun Tang

    2011-01-01

    We have shown previously that oligonucleotide conjugated hirulog (bivalirudin) has a higher inhibitory activityagainst thrombin compared with hirulog. The negative charged oligonucleotide was considered to be responsible for theadditive activity. To further investigate the related effect of oligonucleotide, the oligonucleotide conjugated hirulog18 wasprepared in the present work and the activity of the conjugate against thrombin was measured. Hirulog18, a peptide lack oftwo N-terminal amino a...

  3. Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide.

    Science.gov (United States)

    Wang, Jie; Morita, Ikuo; Onodera, Mitsue; Murota, Sei-Itsu

    2002-02-01

    Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved. PMID:11807828

  4. Rapid purification of high purity thrombin and preparation of a novel hemostat for clinical purposes

    OpenAIRE

    Turaga, Krishna Kumar; Chakradhara Rao, P.; Sripad, G.

    2008-01-01

    Thrombin was prepared from crude prothrombin enriched plasma by activation using Russell’s viper venom. Prothrombin was prepared by barium sulphate adsorption and elution of prothrombin enriched fraction using high concentrations of sodium citrate. This fraction was directly activated with venom and thrombin was purified by SP Sepharose ion-exchange chromatography and subsequently over Phenyl-sepharose column. This product exhibits a purity of >98% with an activity of at least 6000U/mg or h...

  5. A label-free electrochemical aptasensor for sensitive thrombin detection in whole blood

    International Nuclear Information System (INIS)

    In this paper, we reported a novel label-free electrochemical aptasensors for thrombin detection in whole blood using self-assembled multilayers with carboxymethyl-PEG-carboxymethyl (CM-PEG-CM) and thrombin-binding aptamer (TBA). In the sensing strategy, CM-PEG-CM and TBA were assembled on the electrode surface via covalent binding. In the presence of target, the TBA on the outermost layer of the self-assembled multilayer would catch the target on the electrode interface, which makes a barrier for electrons and inhibits the electro-transfer, resulting in the decreased DPV signals. Using this strategy, a wide detection range (1 pM–160 nM) for target thrombin was obtained, with a low detection limit of 1.56 × 10?14 M. The control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The results showed that the aptasensors had good specificity, stability and reproducibility to thrombin. Moreover, the aptasensors could be used for detection of thrombin in whole blood which could provide a promising platform for fabrication of aptamer based biosensors in clinical application

  6. Pulsatile equibiaxial stretch inhibits thrombin-induced RhoA and NF-?B activation

    International Nuclear Information System (INIS)

    This study investigated interactions between the effects of mechanical stretch and thrombin on RhoA activation in rat aortic smooth muscle cells (RASMC). Equibiaxial, pulsatile stretch, or thrombin produced a significant increase in RhoA activation. Surprisingly, in combination, 30 min of stretch inhibited the ability of thrombin to activate RhoA. NO donors and 8-bromo-cGMP significantly inhibited thrombin-induced RhoA activation. Interestingly, the nitric oxide synthase (NOS) inhibitor L-NAME increased basal RhoA activity, suggesting that NOS activity exerts a tonic inhibition on RhoA. Stretching RASMC increases nitrite production, consistent with the idea that NO contributes to the inhibitory effects of stretch. Thrombin stimulates MAP kinase and NF-?B pathways through Rho and these responses were blocked by 8-bromo-cGMP or stretch and restored by L-NAME. These data suggest that stretch, acting through NO and cGMP, can prevent the ability of thrombin to stimulate Rho signaling pathways that contribute to pathophysiological proliferative and inflammatory responses

  7. The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation.

    Science.gov (United States)

    Aslan, Joseph E; Baker, Sandra M; Loren, Cassandra P; Haley, Kristina M; Itakura, Asako; Pang, Jiaqing; Greenberg, Daniel L; David, Larry L; Manser, Ed; Chernoff, Jonathan; McCarty, Owen J T

    2013-09-01

    Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, ?PIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets. PMID:23784547

  8. Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis

    Directory of Open Access Journals (Sweden)

    Nicolette Prevost

    2011-12-01

    Full Text Available Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improves wound healing and examine the effects of charge in cotton/cellulosic dressing design on thrombin production and elastase sequestration (uptake by the wound dressing. Thrombin is central to the initiation and propagation of coagulation, and elastase is released from neutrophils that can function detrimentally in a stalled inflammatory phase characteristic of chronic wounds. Electrokinetic fiber surface properties of the biomaterials of this study were determined to correlate material charge and polarity with function relative to thrombin production and elastase sequestration. Human neutrophil elastase sequestration was assessed with an assay representative of chronic wound concentration with cotton gauze cross-linked with three types of polycarboxylic acids and one phosphorylation finish; thrombin production, which was assessed in a plasma-based assay via a fluorogenic peptide substrate, was determined for cotton, cotton-grafted chitosan, chitosan, rayon/polyester, and two kaolin-treated materials including a commercial hemorrhage control dressing (QuickClot Combat Gauze. A correlation in thrombin production to zeta potential was found. Two polycarboxylic acid cross linked and a phosphorylated cotton dressing gave high elastase sequestration.

  9. Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

    OpenAIRE

    Postenka Carl O; Goodale David; Schulze Erika B; Beausoleil Michel S; Allan Alison L

    2011-01-01

    Abstract Background Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. Methods To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-?TC) or ...

  10. Thrombin generation as a predictor of radiotherapy induced skin erythema

    International Nuclear Information System (INIS)

    Background and purpose: Biological mechanisms underlying radiation induced erythema remain largely unknown, with no simple way to accurately predict or prevent extreme cases. Based on the recent findings in patients suffering from chronic urticaria, we sought to determine if similar mechanisms of hypercoagulation contributed to comparable skin reactions during radiotherapy. Materials and methods: Plasma levels of prothrombin factor 1+2 (F1+2), D-dimers and plasminogen activator inhibitor-1 (Pai-1) were tested in 32 women undergoing irradiation following breast conserving surgery for early breast cancer. Reflectance spectrophotometry was used to objectively assess erythema throughout the treatment by measuring the amount of light reflected from the skin surface as a function of wavelength. Correlations between peak levels of erythema and plasma biomarkers were then assessed. Results: Individual peak reflectance readings generally occurred between day 29 of treatment and 2 weeks post radiotherapy, and represented a median increase of 66% (range: 11-146%; p < 0.001) from baseline. Peak reflectance correlated with F1+2 and Pai-1 levels measured both at baseline and day 29 of treatment, and multivariate analysis indicated that these two baseline measurements were the best predictors of peak reflectance, accounting for 59% of the variability in erythema (p = 0.000004). Conclusions: Patients with signs of intravascular thrombin generation are at higher risk of radiotherapy-iation are at higher risk of radiotherapy-induced skin reactions, providing a new therapeutic avenue for possibly predicting and preventing this side effect of cancer treatment

  11. Construction of photoelectrochemical thrombin aptasensor via assembling multilayer of graphene-CdS nanocomposites.

    Science.gov (United States)

    Shangguan, Li; Zhu, Wei; Xue, Yanchun; Liu, Songqin

    2015-02-15

    A photoelectrochemical (PEC) aptasensor for highly sensitive and specific detection of thrombin was developed by using graphene–CdS nanocomposites multilayer as photoactive species and electroactive mediator hexaammineruthenium(III) chloride (Ru(NH(3))(6)(3+)) as signal enhancer. Graphene–CdS nanocomposites (G–CdS) were synthesized by one-pot reduction of oxide graphene and CdCl2 with thioacetamide. The photoactive multilayer was prepared by alternative assembly of the negatively charged 3-mercaptopropionic acid modified graphene–CdS nanocomposites (MPA-G–CdS) and the positively charged polyethylenimine (PEI) on ITO electrode. This layer-by-layer assembly method enhanced the stability and homogeneity of the photocurrent readout of G–CdS. Thrombin aptamer was covalently bound to the multilayer by using glutaraldehyde as cross-linking. Electroactive mediator (Ru(NH(3))(6)(3+)) could interact with the DNA phosphate backbone and thus facilitated the electron transfer between G–CdS multilayer and electrode and enhanced the photocurrent. Hybridizing of a long complementary DNA with thrombin aptamer could increase the adsorption amount of (Ru(NH(3))(6)(3+)), which in turn boosted the signal readout. In the presence of target thrombin, the affinity interaction between thrombin and its aptamer resulted in the long complementary DNA releasing from the G–CdS multilayer and decreasing of photocurrent signal. On the basis of G–CdS multilayer as the photoactive species, (Ru (NH(3))(6)(3+)) as an electroactive mediator, and aptamer as a recognition module, a high sensitive PEC aptasensor for thrombin detection was proposed. The thrombin aptasensor displayed a linear range from 2.0 pM to 600.0 pM and a detection limit of 1.0 pM. The present strategy provided a promising ideology for the future development of PEC biosensor. PMID:25314620

  12. Nanochannels for diagnostic of thrombin-related diseases in human blood.

    Science.gov (United States)

    de la Escosura-Muñiz, Alfredo; Chunglok, Wilanee; Surareungchai, Werasak; Merkoçi, Arben

    2013-02-15

    A high sensitive voltammetric method for rapid determination of thrombin spiked in whole blood by taking advantage of both aptamer-based recognition and the use of a nanoporous membrane has been developed. The nanoporous membrane not only acts as platform for the thrombin recognition but also as filter of the micrometric components such as white and red blood cells, consequently minimizing matrix effects. The protocol involves a sandwich format in the inner walls (200 nm diameter) of an anodized alumina oxide filter membrane (AAO). The analytical signal, by DPV oxidation of [Fe(CN)(6)](4-), is based on the blockage in the pores which affects the diffusion of [Fe(CN)(6)](4-) to the screen-printed carbon electrotransducer (SPCEs) modified with the membrane. By labeling the anti-thrombin IgG with AuNPs followed by silver enhancement a greater passive signal enhancement in comparison to the membrane blockage has been observed. The contribution of both electrostatic/steric effects in this blockage due to the subsequent formation of the aptamer-thrombin complex and the final sandwich assay is investigated. The efficiency of the system is also monitored by microscopic techniques. The resulted biosensing system allows detecting thrombin spiked in whole blood at very low levels (LOD 1.8 ng mL(-1)) which are within the range of clinical interest for the diagnostic of coagulation abnormalities as well as pulmonary metastasis. PMID:22704840

  13. Normal thrombin generation in neonates in spite of prolonged conventional coagulation tests.

    Science.gov (United States)

    Tripodi, Armando; Ramenghi, Luca A; Chantarangkul, Veena; De Carli, Agnese; Clerici, Marigrazia; Groppo, Michela; Mosca, Fabio; Mannucci, Pier Mannuccio

    2008-08-01

    Conventional coagulation tests might be inadequate to explore mechanisms regulating thrombin generation in neonates, because they do not allow full activation of the reduced levels of protein C. Therefore, they do not reflect the action of pro- and anti-coagulants as does the endogenous thrombin potential assessed in the presence of thrombomodulin. Endogenous thrombin potential measured without thrombomodulin was greater than the lower-limit of the adult reference interval in 30% of 109 full-term and 49% of 55 pre-term neonates, a finding consistent with the reduced levels of procoagulants in this setting. When the test was modified adding thrombomodulin, endogenous thrombin potential reverted into the adult reference interval in 97% and 100% full-term and pre-term neonates. In conclusion, the coagulation balance in neonates is restored by the concomitant reduction of pro- and anticoagulants. The restored balance can be shown in vitro by the endogenous thrombin potential test that includes thrombomodulin, but not by conventional coagulation tests. PMID:18403390

  14. Thrombin induces IL-6 but not TNFalpha secretion by mouse mast cells: threshold-level thrombin receptor and very low level FcepsilonRI signaling synergistically enhance IL-6 secretion.

    Science.gov (United States)

    Gordon, J R; Zhang, X; Stevenson, K; Cosford, K

    2000-11-01

    Mast cells become activated in multiple diseases wherein thrombin generation is often clinically apparent, but the effect of thrombin on cytokine release by mast cells remains unexplored. Thus, we examined IL-6 and TNFalpha release by thrombin-challenged mast cells. Thrombin and the protease-activated receptor (PAR)-1 peptide TRAP(14) induced these cells to secrete IL-6 in a dose-dependent fashion. Mast cells secreted > or =2800 pg IL-6/10(6) cells over 24 h, but only low levels of serotonin and no significant TNFalpha. Furthermore, at near-background levels of allergen, threshold doses of alpha-thrombin synergistically enhanced the IL-6 response (by up to 100-fold), but high-dose costimulation led to a simple additive response. Both the PI(3)- and sphingosine-kinase signaling pathways contributed importantly to the thrombin response. Our data thus clearly demonstrate that low-level thrombin and FcepsilonRI signaling can synergize to augment mast cell IL-6 responses, and that thrombin also differentially induces cytokine secretion by mast cells. PMID:11104585

  15. Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators.

    Science.gov (United States)

    Lau, L F; Pumiglia, K; Côté, Y P; Feinstein, M B

    1994-10-15

    Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors. PMID:7526841

  16. Thrombin and PAF stimulate formation of inositol triphosphate from similar pools of phosphatidylinositol-4,5-bisphosphate in platelets

    International Nuclear Information System (INIS)

    During platelet activation phospholipase C cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) to generate diacylglycerol and inositol triphosphate (IP3). Production of IP3 was therefore used as a probe to differentiate between thrombin and platelet activating factor (PAF) sensitive pools of PIP2. Stimulation of [3H]inositol labelled rabbit platelets (0.5 x 109 cells/ml containing 0.1 mM EGTA and 2 mM LiCl) with thrombin (1 U/ml) caused a maximum 3 to 10 fold increase in [3H]IP3 in 5 min. Treatment with PAF (1 x 10-9 M) led to a maximum 3 to 5 fold increase in [3H]IP3 in 5 s followed by a decrease to basal level in 30 s. Addition of thrombin for 5 min followed by PAF for 5 s showed no further increase in [3H]IP3 over that obtained with thrombin alone. Various other sequential treatments with thrombin and PAF showed negligible cumulative effect. Simultaneous addition of thrombin and PAF showed a partial additive production of [3H]IP3 at short time periods but these levels never significantly exceeded those obtained by thrombin at 5 min or PAF at 5 s. It is concluded that in platelets thrombin and PAF (a) differentially activate PIP2-phosphodiesterase and (b) stimulate production of IP3 from similar pools of PIP2

  17. Evaluation of Antithrombotic Activity of Thrombin DNA Aptamers by a Murine Thrombosis Model

    Science.gov (United States)

    Zavyalova, Elena; Samoylenkova, Nadezhda; Revishchin, Alexander; Golovin, Andrey; Pavlova, Galina; Kopylov, Alexey

    2014-01-01

    Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4–7.1 µmol/kg (14–70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors. PMID:25192011

  18. Arg-Gly-Asp peptide increases endothelial hydraulic conductivity: comparison with thrombin response.

    Science.gov (United States)

    Qiao, R L; Yan, W; Lum, H; Malik, A B

    1995-07-01

    The contribution of integrin receptors to the regulation of endothelial permeability was studied using cultured bovine pulmonary microvascular endothelial cell (BPMVEC) monolayers by the measurement of hydraulic conductivity (Lp). Treatment of monolayers with a peptide containing the sequence Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) (0.85 mM) to compete for the RGD sequence of extracellular matrix (ECM) proteins increased endothelial Lp threefold, whereas the control peptide Gly-Arg-Gly-Glu-Ser-Pro had no effect on Lp. This action of GRGDSP on Lp was not significantly altered by dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP; 0.5 mM). Endothelial Lp increased twofold when the monolayers were challenged with alpha-thrombin (5 x 10(-8) M for 10 min), and this response was completely reversed by DBcAMP. The strength of adhesion of endothelial cells was estimated by evaluating the ability of endothelial cells to remain attached to ECM after treating the monolayers with 0.05% trypsin plus 0.5 mM EDTA. Exposure of the monolayers to either GRGDSP or alpha-thrombin significantly reduced the strength of adhesion to the ECM. DBcAMP prevented the antiadhesive effect of alpha-thrombin but not that of GRGDSP. Treatment of the monolayers with either alpha-thrombin or GRGDSP caused formation of intercellular gaps, but only the thrombin-induced intercellular gaps were accompanied by reorganization of actin filaments. These results indicate that integrin binding to ECM proteins regulates an important determinant of endothelial permeability and that alpha-thrombin and GRGDSP increase endothelial cell monolayer permeability by different mechanisms. PMID:7631737

  19. The effect of the REG2 Anticoagulation System on thrombin generation kinetics: a pharmacodynamic and pharmacokinetic first-in-human study.

    Science.gov (United States)

    Vavalle, J P; Rusconi, C P; Zelenkofske, S; Wargin, W A; Ortel, T L; Alexander, J H; Povsic, T J; Becker, R C

    2014-10-01

    The REG2 Anticoagulation System consists of pegnivacogin, a subcutaneously administered aptamer factor IXa inhibitor, and its intravenous active control agent, anivamersen. Its effect on thrombin generation is unknown. A prospectively designed thrombin generation study was conducted within the phase 1 ascending dose study of REG2 to assess the effect of REG2 on thrombin generation kinetics. A total of 32 healthy volunteers were recruited into four cohorts of ascending dose pegnivacogin for the phase 1 study. In this pre-specified substudy, blood samples were drawn in the presence or absence of corn trypsin inhibitor at specified times within each dosing cohort. Thrombin generation was initiated with tissue factor and thrombin generation kinetics were measured using the Calibrated Automated Thrombogram (CAT). REG2 attenuated thrombin generation in a dose-dependent manner. All parameters of the CAT assay, except for lag time, showed a dose and concentration-dependent response to pegnivacogin [time to peak thrombin generation (PTm), endogenous thrombin potential, peak thrombin generation, and velocity index (VIx)]. Reversal of the effect of pegnivacogin with anivamersen demonstrated restoration of thrombin generation without rebound effect. This first-in-human study of the effect of the REG2 Anticoagulation System on thrombin generation demonstrates concentration-dependent suppression of thrombin generation that is reversible without rebound effect, as measured by the CAT assay. PMID:24880800

  20. Thrombin-reactive polypeptides of human blood. Some biochemical and immunological properties.

    Science.gov (United States)

    Chelladurai, M; Fossett, N G; Ganguly, P

    1985-06-18

    Two polypeptides of 74 kDa and 55 kDa have been isolated from human platelets by immunoaffinity and lectin affinity chromatography and their effects on thrombin reactivity have been examined. These proteins in combination enhanced the aggregation of platelets by thrombin while aggregation induced by trypsin, collagen and adenosine diphosphate was not significantly affected. An enhancement in the action of thrombin on fibrinogen, N-benzoylarginine ethyl ester and H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride was also observed in the presence of the platelet proteins. Under similar conditions, the proteins did not influence the esterolytic activity of trypsin or plasmin. Studies at different thrombin and protein concentrations showed maximum enhancement of enzyme reactivity when the ratio between the peptides and thrombin was optimal. In the presence of these proteins, the affinity of thrombin for N-benzoylarginine ethyl ester was about twofold higher than in the control. Two polypeptides with properties similar to those described above have also been isolated from human plasma. Antibodies to the above proteins isolated from either platelets or plasma were raised in rabbits. Intact platelets solubilized in Triton X-100 or plasma showed two precipitin lines in immunoelectrophoresis against both of the above antisera and a similar pattern was observed with the isolated polypeptides. The polypeptides did not interact in immunoelectrophoresis with antisera to whole serum, antithrombin, C4 binding protein or protein S. These 74-kDa and 55-kDa polypeptides contained radioactivity when radioiodinated platelets were used suggesting that they are located on the cell surface. Fresh plasma was analyzed by gel electrophoresis under nondenaturing and denaturing conditions and the proteins were transferred to nitrocellulose sheets. Staining with antibody to these thrombin-reactive proteins and 125I-protein A showed several reactive plasma proteins under nondenaturing conditions with the major band migrating in the albumin area. In plasma treated with sodium dodecyl sulfate, the 74-kDa and 55-kDa components were observed. A prominent 74-kDa band and a fainter 55-kDa component were again observed when platelets solubilized in sodium dodecyl sulfate were analysed by the above procedure. It is proposed that human platelets and plasma contain polypeptides which may directly modulate thrombin reactivity. PMID:4006939

  1. Longitudinal assessment of thrombin generation potential in response to alteration of antiplatelet therapy after TIA or ischaemic stroke.

    LENUS (Irish Health Repository)

    Tobin, W O

    2013-02-01

    The impact of changing antiplatelet therapy on thrombin generation potential in patients with ischaemic cerebrovascular disease (CVD) is unclear. We assessed patients within 4 weeks of TIA or ischaemic stroke (baseline), and then 14 days (14d) and >90 days (90d) after altering antiplatelet therapy. Thrombin generation was assessed in platelet poor plasma. Ninety-one patients were recruited. Twenty-four were initially assessed on no antiplatelet therapy, and then after 14d (N = 23) and 90d (N = 8) on aspirin monotherapy; 52 were assessed on aspirin monotherapy, and after 14 and 90 days on aspirin and dipyridamole combination therapy; 21 patients were assessed on aspirin and after 14 days (N = 21) and 90 days (N = 19) on clopidogrel. Peak thrombin generation and endogenous thrombin potential were reduced at 14 and 90 days (p ? 0.04) in the overall cohort. We assessed the impact of individual antiplatelet regimens on thrombin generation parameters to investigate the cause of this effect. Lag time and time-to-peak thrombin generation were unchanged at 14 days, but reduced 90 days after commencing aspirin (p ? 0.009). Lag time, peak thrombin generation and endogenous thrombin potential were reduced at both 14 and 90 days after adding dipyridamole to aspirin (p ? 0.01). Lag time was reduced 14 days after changing from aspirin to clopidogrel (p = 0.045), but this effect was not maintained at 90 days (p = 0.2). This pilot study did not show any consistent effects of commencing aspirin, or of changing from aspirin to clopidogrel on thrombin generation potential during follow-up. The addition of dipyridamole to aspirin led to a persistent reduction in peak and total thrombin generation ex vivo, and illustrates the diverse, potentially beneficial, newly recognised \\'anti-coagulant\\' effects of dipyridamole in ischaemic CVD.

  2. Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

    Science.gov (United States)

    Vouret-Craviari, V; Van Obberghen-Schilling, E; Rasmussen, U B; Pavirani, A; Lecocq, J P; Pouysségur, J

    1992-01-01

    alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential. PMID:1312881

  3. (-)-Epigallocatechin-3-gallate decreases thrombin/paclitaxel-induced endothelial tissue factor expression via the inhibition of c-Jun terminal NH2 kinase phosphorylation

    International Nuclear Information System (INIS)

    Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (-)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6 h up to a concentration of 25 ?mol/L. At a concentration of 25 ?mol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease in TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60 min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-?B nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.

  4. Recombinant Technology and Probiotics

    Directory of Open Access Journals (Sweden)

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  5. Recombinant Technology and Probiotics

    OpenAIRE

    Icy D’Silva

    2011-01-01

    Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecule...

  6. Placental vascular pathology and increased thrombin generation as mechanisms of disease in obstetrical syndromes

    OpenAIRE

    Mastrolia, Salvatore Andrea; Mazor, Moshe; Loverro, Giuseppe; Klaitman, Vered; Erez, Offer

    2014-01-01

    Obstetrical complications including preeclampsia, fetal growth restriction, preterm labor, preterm prelabor rupture of membranes and fetal demise are all the clinical endpoint of several underlying mechanisms (i.e., infection, inflammation, thrombosis, endocrine disorder, immunologic rejection, genetic, and environmental), therefore, they may be regarded as syndromes. Placental vascular pathology and increased thrombin generation were reported in all of these obstetrical syndromes. Moreover, ...

  7. Fibrin formation is more impaired than thrombin generation and platelets immediately following cardiac surgery

    DEFF Research Database (Denmark)

    Solomon, Cristina; Rahe-Meyer, Niels

    2011-01-01

    Cardiac surgery performed on cardio-pulmonary bypass (CPB) may be complicated by coagulopathy and bleeding. This prospective observational study investigated the CPB-induced changes in thrombin generation, fibrin formation, and in the platelet component of the whole blood clot elasticity. The effects of haemostatic therapy with fresh frozen plasma (FFP) and platelet concentrate on these parameters were also evaluated.

  8. Increased thrombin generation measured in the presence of thrombomodulin in women with early pregnancy loss.

    Science.gov (United States)

    de Saint Martin, Luc; Duchemin, Jérôme; Bohec, Caroline; Couturaud, Francis; Mottier, Dominique; Collet, Michel; Blouch, Marie-Thérèse; Pasquier, Elisabeth

    2011-04-01

    Compared with 537 parous controls with no history of pregnancy loss, a lower thrombomodulin-related inhibition of the endogenous thrombin potential was measured in 264 cases with previous unexplained pregnancy loss, especially when losses occurred between 9 and 12 weeks of gestation. Adjusting age, protein S, factor VIII, factor V Leiden, and prothrombin G20210A did not change the results. PMID:21130429

  9. Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

    DEFF Research Database (Denmark)

    Pasternak, Anna; Hernandez, Frank J

    2011-01-01

    A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15-0.50?kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by T(m) versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation.

  10. Direct thrombin inhibitors: novel antithrombotics on the horizon in the thromboprophylactic management of atrial fibrillation

    OpenAIRE

    Katira, R.; Chauhan, A.; More, R.

    2005-01-01

    Antithrombotic agents have verified efficacy in reducing the thromboembolic risk associated with atrial fibrillation. This article focuses on the emergence of a new oral direct thrombin inhibitor, ximelagatran, into the arena of atrial fibrillation thromboprophylaxis. This review does not cover atrial fibrillation in the context of valvular heart disease. The efficacy of aspirin and warfarin will be discussed briefly.

  11. Rac inhibits thrombin-induced Rho activation: evidence of a Pak-dependent GTPase crosstalk

    Directory of Open Access Journals (Sweden)

    Rosenfeldt Hans

    2006-12-01

    Full Text Available Abstract The strict spatio-temporal control of Rho GTPases is critical for many cellular functions, including cell motility, contractility, and growth. In this regard, the prototypical Rho family GTPases, Rho, Rac, and Cdc42 regulate the activity of each other by a still poorly understood mechanism. Indeed, we found that constitutively active forms of Rac inhibit stress fiber formation and Rho stimulation by thrombin. Surprisingly, a mutant of Rac that is unable to activate Pak1 failed to inhibit thrombin signaling to Rho. To explore the underlying mechanism, we investigated whether Pak1 could regulate guanine nucleotide exchange factors (GEFs for Rho. We found that Pak1 associates with P115-RhoGEF but not with PDZ-RhoGEF or LARG, and knock down experiments revealed that P115-RhoGEF plays a major role in signaling from thrombin receptors to Rho in HEK293T cells. Pak1 binds the DH-PH domain of P115-RhoGEF, thus suggesting a mechanism by which Rac stimulation of Pak1 may disrupt receptor-dependent Rho signaling. In agreement, expression of a dominant-negative Pak-Inhibitory Domain potentiated the activation of Rho by thrombin, and prevented the inhibition of Rho by Rac. These findings indicate that Rac interferes with receptor-dependent Rho stimulation through Pak1, thus providing a mechanism for cross-talk between these two small-GTPases.

  12. A Reusable Impedimetric Aptasensor for Detection of Thrombin Employing a Graphite-Epoxy Composite Electrode

    Directory of Open Access Journals (Sweden)

    Manel del Valle

    2012-03-01

    Full Text Available Here, we report the application of a label-free electrochemical aptasensor based on a graphite-epoxy composite electrode for the detection of thrombin; in this work, aptamers were immobilized onto the electrodes surface using wet physical adsorption. The detection principle is based on the changes of the interfacial properties of the electrode; these were probed in the presence of the reversible redox couple [Fe(CN6]3?/[Fe(CN6]4? using impedance measurements. The electrode surface was partially blocked due to formation of aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by Electrochemical Impedance Spectroscopy (EIS. The aptasensor showed a linear response for thrombin in the range of 7.5 pM to 75 pM and a detection limit of 4.5 pM. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution at 42 °C, showing its operation for different cycles. The interference response caused by main proteins in serum has been characterized.

  13. Development of a Multiplex Sandwich Aptamer Microarray for the Detection of VEGF165 and Thrombin

    Directory of Open Access Journals (Sweden)

    Anna Meneghello

    2013-10-01

    Full Text Available In this work we have developed a multiplex microarray system capable of detecting VEGF165 and thrombin. We recently described a Sandwich Aptamer Microarray (SAM for thrombin detection feasible for use in multiplex microarrays; here we describe a new aptasensor for VEGF165 detection employing Vap7 and VEa5, two DNA aptamers recognizing different sites of the protein. The aptamers were modified to be adapted to the solid phase platform of SAM and their capability to simultaneously recognize VEGF165 by forming a ternary complex was analyzed in solution. Having so defined the best tandem arrangement of modified aptamers, we set up the aptasensor for VEGF165, and finally analyzed the multiplex system with the two aptasensors for the simultaneous detection of VEGF165 and thrombin. The results indicate that each sandwich is specific, even when the two proteins are mixed. The system performance is consistent with the behavior evidenced by the biochemical analysis, which proves to be valuable to drive the evaluation and refinement of aptamers prior to or along the development of a detection platform. Since thrombin upregulates VEGF expression, the simultaneous recognition of these two proteins could be useful in the analysis of biomarkers in pathologies characterized by neo-angiogenesis.

  14. Role of thrombin in the proliferative response of T-47D mammary tumor cells

    International Nuclear Information System (INIS)

    The growth of the human metastatic cell line (T-47D) in a chemically defined medium (DM) is shown to be dependent on the presence of three traditional growth factors: epidermal growth factor, insulin, and transferrin. The addition of thrombin further stimulates its growth. The mitogenic action on a human mammary tumor cell lines from epithelial origin is a novel action of thrombin. Cells in the DM show striking morphological changes which are dramatically enhanced by the addition of thrombin. These observations are part of a pleiotropic response to the growth factors: the protein content of the cells increases in the defined medium; the 2DG gels of the 35S- and 35P-labeled proteins show important changes in spots, several of which are probably of cytoskeletal origin. It is also shown that cells in a semisolid growth factor-supplemented medium have growth advantages over their counterparts grown with serum. All the phenotypic changes mentioned above reveal the important role of growth factors in the growth and behavior of this mammary cell line. The results obtained with thrombin indicate a new site of action of this enzyme which may be important in the metastatic spread of human mammary tumor cells

  15. Label-free aptamer biosensor for thrombin detection based on functionalized graphene nanocomposites.

    Science.gov (United States)

    Wang, Qingqing; Zhou, Zhixue; Zhai, Yanling; Zhang, Lingling; Hong, Wei; Zhang, Zhiquan; Dong, Shaojun

    2015-08-15

    A label-free and amplified electrochemical impedimetric aptasensor based on functionalized graphene nanocomposites (rGO-AuNPs) was developed for the detection of thrombin, which played a vital role in thrombosis and hemostasis. The thiolated aptamer and dithiothreitol (TBA15-DTT) were firstly immobilized on the gold electrode to capture the thrombin molecules, and then aptamer functionalized graphene nanocomposites (rGO-TBA29) were used to fabricate a sandwich sensing platform for amplifying the impedimetric signals. As numerous negative charges of TBA29 on the electrode repelled to the [Fe(CN)6](4-/3-) anions, resulting in an obvious amplified charge-transfer resistance (Rct) signal. The Rct increase was linearly proportional to the thrombin concentration from 0.3 to 50nM and a detection limit of 0.01nM thrombin was achieved. In addition, graphene could also be labeled with other probes via electrostatic or ?-? stacking interactions to produce signals, therefore different detection methods expanding wide application could be used in this model. PMID:25966410

  16. Thrombin binds to a high-affinity approximately 900,000-dalton site on human platelets

    International Nuclear Information System (INIS)

    The functional sizes of the binding sites for thrombin on human platelets and isolated membranes have been determined by the technique of radiation inactivation: similar results were obtained. Independent studies using different radiation doses (0, 3, and 48 Mrad) and different thrombin concentrations (10(-10), 10(-8), and 10(-6) M) confirmed the presence of three binding sites with functional sizes of 900,000, 30,000, and 4000 daltons. The binding site of lowest apparent size (4000 daltons) probably corresponds to what has been termed nonspecific binding since its dissociation constant (2900 nM) is well outside the physiological range. The site of intermediate size (30,000 daltons) is also probably not involved in platelet activation since its dissociation constant (11 nM) is also beyond the concentration range required for activation, although it may be involved in other aspects of platelet-thrombin interaction. The sites with the largest functional size are probably important in platelet function since their dissociation constant (0.3 nM) is in the range required for platelet activation. The functional size of these sites (900,000 daltons) suggests that the high-affinity site for thrombin binding to platelets may involve a multimolecular complex of membrane components

  17. Thrombin inhibition with dabigatran protects against high-fat diet-induced fatty liver disease in mice.

    Science.gov (United States)

    Kopec, Anna K; Joshi, Nikita; Towery, Keara L; Kassel, Karen M; Sullivan, Bradley P; Flick, Matthew J; Luyendyk, James P

    2014-11-01

    Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. Robust coagulation cascade activation is common in obese patients with NAFLD. We identified a critical temporal relationship between thrombin generation and the manifestation of hepatic steatosis, inflammation, and injury in C57BL/6J mice fed a high-fat diet (HFD) for 1, 2, and 3 months. Mice fed a HFD exhibited dramatic increases in hepatocellular injury and inflammation over time. Hepatic fibrin deposition preceded an increase in serum alanine aminotransferase, and the most dramatic changes in liver histopathology occurred in conjunction with a detectable increase in plasma thrombin-antithrombin levels at 3 months. To directly determine whether thrombin activity promotes NAFLD pathogenesis, mice were fed a HFD and simultaneously treated with the direct thrombin inhibitor dabigatran etexilate for 3 months. Notably, dabigatran treatment significantly reduced hepatic fibrin deposition, hepatic inflammation, hepatocellular injury, and steatosis in mice fed a HFD. Of interest, dabigatran treatment also significantly attenuated HFD-induced body weight gain. Gene expression analysis suggested that thrombin potentially drives NAFLD pathogenesis by altering the expression of genes associated with lipid metabolism and bile acid synthesis. Collectively, the results suggest that thrombin activity is central to HFD-induced body weight gain, liver injury, and inflammation and provide the proof-of-principle evidence that pharmacological thrombin inhibition could be effective in limiting NAFLD and associated pathologies. PMID:25138021

  18. Multimerin 1 binds factor V and activated factor V with high affinity and inhibits thrombin generation.

    Science.gov (United States)

    Jeimy, Samira B; Fuller, Nola; Tasneem, Subia; Segers, Kenneth; Stafford, Alan R; Weitz, Jeffrey I; Camire, Rodney M; Nicolaes, Gerry A F; Hayward, Catherine P M

    2008-12-01

    Multimerin 1 (MMRN1) is a polymeric, factor V (FV) binding protein that is stored in platelet and endothelial cell secretion granules but is undetectable in normal plasma. In human platelet alpha-granules, FV is stored complexed to MMRN1, predominantly by noncovalent binding interactions. The FV binding site for MMRN1 is located in the light chain, where it overlaps the C1 and C2 domain membrane binding sites essential for activated FV (FVa) procoagulant function. Surface plasmon resonance (SPR), circular dichroism (CD) and thrombin generation assays were used to study the binding of FV and FVa to MMRN1, and the functional consequences. FV and FVa bound MMRN1 with high affinities (K(D): 2 and 7 nM, respectively). FV dissociated more slowly from MMRN1 than FVa in SPR experiments, and CD analyses suggested greater conformational changes in mixtures of FV and MMRN1 than in mixtures of FVa and MMRN1. SPR analyses indicated that soluble phosphatidylserine (1,2-Dicaproylsn-glycero-3-phospho-L-serine) competitively inhibited both FV-MMRN1 and FVa-MMRN1 binding. Furthermore, exogenous MMRN1 delayed and reduced thrombin generation by plasma and platelets, and it reduced thrombin generation by preformed FVa. Exogenous MMRN1 also delayed FV activation, triggered by adding tissue factor to plasma, or by adding purified thrombin or factor Xa to purified FV. The high affinity binding of FV to MMRN1 may facilitate the costorage of the two proteins in platelet alpha-granules. As a consequence, MMRN1 release during platelet activation may limit platelet dependent thrombin generation in vivo. PMID:19132231

  19. A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development.

    Science.gov (United States)

    Ellery, Paul E R; Maroney, Susan A; Cooley, Brian C; Luyendyk, James P; Zogg, Mark; Weiler, Hartmut; Mast, Alan E

    2015-06-25

    Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi(-/-)) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi(+/-) mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4(-/-)), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi(-/-) embryos, but >40% of expected Tfpi(-/-):Par4(-/-) offspring survived to adulthood. Adult Tfpi(-/-):Par4(-/-) mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi(-/-):Par4(-/-) mice have platelet and fibrin accumulation similar to Par4(-/-) mice following venous electrolytic injury but were more susceptible than Par4(-/-) mice to TF-induced pulmonary embolism. In addition, ?30% of the Tfpi(-/-):Par4(-/-) mice were born with short tails. Tfpi(-/-):Par4(-/-) mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation. PMID:25954015

  20. Autocrine production of basic fibroblast growth factor translated from novel synthesized mRNA mediates thrombin-induced mitogenesis in smooth muscle cells.

    Science.gov (United States)

    Cucina, Alessandra; Borrelli, Valeria; Lucarelli, Marco; Sterpetti, Antonio V; Cavallaro, Antonino; Strom, Roberto; Santoro-D'Angelo, Luciana; Scarpa, Sigfrido

    2002-03-01

    Thrombin is known to stimulate smooth muscle cell (SMC) growth in culture but the mechanisms underlying growth stimulation remain unclear. Previous works have observed a significant increase in platelet-derived growth factor AA and basic fibroblast growth factor (bFGF) release by bovine aortic SMC after addition of thrombin. The aim of this study was to clarify the link between thrombin, bFGF and SMC proliferation by examining the kinetics of autocrine production of bFGF by thrombin-stimulated SMC and its contribution to thrombin-induced mitogenesis. Experiments were performed to assess the dynamics of thrombin-induced bFGF mRNA transcription and to distinguish, following thrombin stimulus, between the activation of 'old' bFGF protein and/or bFGF mRNA, or novel mRNA synthesis and subsequent translation. Bovine aortic SMCs were stimulated with thrombin in serum-free culture. bFGF mRNA expression was determined by RT-PCR. Mitogenic activity of thrombin was determined by 3H-thymidine uptake. Our results demonstrate that the peak of bFGF mRNA expression occurred 24 h after thrombin stimulation. Experiments performed with cycloheximide, a translation inhibitor, revealed a translation peak later than 24 h after thrombin stimulation. Thrombin-induced mitogenic activity in SMCs was partially inhibited by the addition of anti-bFGF antibody (p<0.001) and of hirudin (p<0.001). When hirudin was added 24 h after stimulation, thrombin-induced mitogenic activity was not inhibited. In conclusion, thrombin-induced mitogenesis was partially mediated by the autocrine production of bFGF, mainly due to protein synthesis by novel mRNA with a transcription peak at 24 h and a later translation peak. PMID:11835269

  1. Inositol cyclic triphosphate [inositol 1,2-(cyclic)-4,5-triphosphate] is formed upon thrombin stimulation of human platelets.

    OpenAIRE

    Ishii, H.; Connolly, T. M.; Bross, T. E.; Majerus, P. W.

    1986-01-01

    Cleavage of polyphosphoinositides in vitro by phospholipase C results in formation of both cyclic and noncyclic inositol phosphates. We have now isolated the cyclic product of phosphatidylinositol 4,5-bisphosphate cleavage, inositol 1,2(cyclic)-4,5-triphosphate [cIns(1:2,4,5)P3], from thrombin-treated platelets. We found 0.2-0.4 nmol of cIns-(1:2,4,5)P3 per 10(9) platelets at 10 sec after thrombin; none was found in unstimulated platelets or in platelets 10 min after thrombin addition. We con...

  2. MicroRNA-146a Decreases High Glucose/Thrombin-Induced Endothelial Inflammation by Inhibiting NAPDH Oxidase 4 Expression

    OpenAIRE

    Huang-Joe Wang; Yuan-Li Huang; Ya-Yun Shih; Hsing-Yu Wu; Ching-Tien Peng; Wan-Yu Lo

    2014-01-01

    Diabetes is associated with hyperglycemia and increased thrombin production. However, it is unknown whether a combination of high glucose and thrombin can modulate the expression of NAPDH oxidase (Nox) subtypes in human aortic endothelial cells (HAECs). Moreover, we investigated the role of a diabetes-associated microRNA (miR-146a) in a diabetic atherothrombosis model. We showed that high glucose (HG) exerted a synergistic effect with thrombin to induce a 10.69-fold increase in Nox4 mRNA leve...

  3. Cardiovascular and biochemical studies on the effects of thrombin and dabigatran and the interaction with vasopressor molecules

    Directory of Open Access Journals (Sweden)

    R. Anand

    2014-10-01

    Conclusion: The thrombin inhibitor, dabigatran reduces vascular oxidative stress and inflammation, improves endothelial function, and decreases atherosclerosis in rodents. [Int J Basic Clin Pharmacol 2014; 3(5.000: 874-878

  4. Improving baculovirus recombination

    OpenAIRE

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

  5. Balloon-assisted ultrasound-guided thrombin injection of a pseudoaneurysm in the posterior tibial artery: A case report

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Taeg Ki; Jeon, Yong Sun; Hong, Kee Chun; Cho, Soon Gu; Kim, Eu Gene [Inha University School of Medicine, Incheon (Korea, Republic of)

    2014-05-15

    An ultrasound-guided direct injection of thrombin is currently the first choice of treatment for the postcatheterization pseudoaneurysm, mainly in the femoral artery. A pseudoaneurysm in the posterior tibial artery is very rare, so there are not enough reports about proper treatment yet. We report a case of a balloon-assisted injection of thrombin under ultrasonography-guidance to manage a pseudoaneurysm in the posterior tibial artery and concurrently to prevent a distal artery embolization.

  6. Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of the qacA Locus

    OpenAIRE

    Kupferwasser, Leon Iri; Skurray, Ronald A.; Brown, Melissa H.; Firth, Neville; Yeaman, Michael R.; Bayer, Arnold S.

    1999-01-01

    Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded st...

  7. Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

    OpenAIRE

    Vouret-craviari, Vale?rie; Boquet, Patrice; Pouysse?gur, Jacques; Obberghen-schilling, Ellen

    1998-01-01

    Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin ...

  8. Balloon-assisted ultrasound-guided thrombin injection of a pseudoaneurysm in the posterior tibial artery: A case report

    International Nuclear Information System (INIS)

    An ultrasound-guided direct injection of thrombin is currently the first choice of treatment for the postcatheterization pseudoaneurysm, mainly in the femoral artery. A pseudoaneurysm in the posterior tibial artery is very rare, so there are not enough reports about proper treatment yet. We report a case of a balloon-assisted injection of thrombin under ultrasonography-guidance to manage a pseudoaneurysm in the posterior tibial artery and concurrently to prevent a distal artery embolization.

  9. Cardiovascular and biochemical studies on the effects of thrombin and dabigatran and the interaction with vasopressor molecules

    OpenAIRE

    Anand, R.; Arumugasamy, K.; Tyagi, Manoj G.

    2014-01-01

    Background: The effect of serine protease thrombin and its directly acting inhibitor dabigatran were evaluated on the heart rate, blood pressure, and phospholipase C (PLC) enzyme activity and the intracellular calcium levels in the platelets. Methods: Heart rate and blood pressure were estimated using electrophysiology equipment. Results: While thrombin was unable to significantly affect the heart rate and blood pressure, the inhibitor dabigatran was able to reduce the heart rate apprec...

  10. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  11. Inhibition of the tissue factor-thrombin pathway limits infarct size after myocardial ischemia-reperfusion injury by reducing inflammation.

    Science.gov (United States)

    Erlich, J H; Boyle, E M; Labriola, J; Kovacich, J C; Santucci, R A; Fearns, C; Morgan, E N; Yun, W; Luther, T; Kojikawa, O; Martin, T R; Pohlman, T H; Verrier, E D; Mackman, N

    2000-12-01

    Functional inhibition of tissue factor (TF) has been shown to improve coronary blood flow after myocardial ischemia/reperfusion (I/R) injury. TF initiates the coagulation protease cascade, resulting in the generation of the serine protease thrombin and fibrin deposition. Thrombin can also contribute to an inflammatory response by activating various cell types, including vascular endothelial cells. We used a rabbit coronary ligation model to investigate the role of TF in acute myocardial I/R injury. At-risk areas of myocardium showed increased TF expression in the sarcolemma of cardiomyocytes, which was associated with a low level of extravascular fibrin deposition. Functional inhibition of TF activity with an anti-rabbit TF monoclonal antibody administered either 15 minutes before or 30 minutes after coronary ligation reduced infarct size by 61% (P = 0.004) and 44% (P = 0.014), respectively. Similarly, we found that inhibition of thrombin with hirudin reduced infarct size by 59% (P = 0.014). In contrast, defibrinogenating the rabbits with ancrod had no effect on infarct size, suggesting that fibrin deposition does not significantly contribute to infarct size. Functional inhibition of thrombin reduced chemokine expression and inhibition of either TF or thrombin reduced leukocyte infiltration. We propose that cardiomyocyte TF initiates extravascular thrombin generation, which enhances inflammation and injury during myocardial I/R. PMID:11106558

  12. Spectroscopic and Electrochemical Detection of Thrombin/5'-SH or 3'-SH Aptamer Immobilized on (porous) Gold Substrates

    International Nuclear Information System (INIS)

    Thrombin is a serine protease that catalyzes the conversion of soluble fibrinogen to insoluble fibrin, and thus induces physiological and pathological blood coagulation. Therefore, it is important to detect thrombin in blood serum for purposes of diagnosis. To achieve this goal, it has been suggested that a 15-mer aptamer strongly binds with thrombin to form a G-quartet structure of the aptamer. Generally, 5'-end thiol-functionalized aptamer has been used as an anti-thrombin binder. Herein, we evaluate the possibility of utilizing a 3'-SH aptasensor for thrombin detection using SPR spectroscopy, and compare the enhancement of the electrochemical signal of the thrombin-aptamer bound on a porous gold substrate. Although the two aptamers have similar configurations, in SPR analysis, the 3'-SH aptamer was a effective aptasensor as well as 5'-SH aptamer. Results from electrochemical analysis showed that the porous gold substrate acted as a good substrate for an aptasensor and demonstrated 5-fold enhancement of current change, as compared to gold thin film

  13. Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

    Directory of Open Access Journals (Sweden)

    Postenka Carl O

    2011-01-01

    Full Text Available Abstract Background Osteopontin (OPN is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. Methods To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN, mutant OPN lacking the thrombin cleavage domain (468-?TC or an empty vector (468-CON and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior. Results All three cell lines were found to equivalently express thrombin, tissue factor, CD44, ?v?5 integrin and ?1 integrin. Relative to 468-OPN and 468-CON cells, 468-?TC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p in vitro. Furthermore, injection of 468-?TC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p Conclusions The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer.

  14. Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin.

    OpenAIRE

    Kelly, A B; Maraganore, J M; Bourdon, P.; Hanson, S R; Harker, L. A.

    1992-01-01

    To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thr...

  15. Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates.

    OpenAIRE

    Hanson, S R; Griffin, J H; Harker, L. A.; Kelly, A B; ESMON, C.T.; A. Gruber

    1993-01-01

    The effects on thrombosis and hemostasis of thrombin-induced activation of endogenous protein C (PC) were evaluated in baboons. Thrombosis was induced by placing into arteriovenous shunts a segment of Dacron vascular graft, which generated arterial platelet-rich thrombus, followed by an expansion region of low-shear blood flow, which in turn accumulated fibrin-rich venous-type thrombus. Thrombosis was quantified by 111In-platelet imaging and 125I-fibrinogen accumulation. Intravenous infusion ...

  16. Crystal structures of thrombin with thiazole-containing inhibitors: probes of the S1' binding site.

    OpenAIRE

    Matthews, J. H.; Krishnan, R.; Costanzo, M J; Maryanoff, B E; Tulinsky, A.

    1996-01-01

    Structures of the blood clotting enzyme thrombin complexed with hirugen and two active site inhibitors, RWJ-50353 10080(N-methyl-D-phenylalanyl-N-[5-[(aminoiminomethyl)amino]-1- [[(2-benzothiazolyl)carbonyl]butyl]-L-prolinamide trifluoroacetate hydrate) and RWJ-50215 (N-[4-(aminoiminomethyl)amino-1-[2- (thiazol-2-ylcarbonylethyl)piperidin- 1-ylcarbonyl]butyl]-5-(dimethylamino)naphthalenesulfonamide trifluoroacetate hydrate), were determined by x-ray crystallography. The refinements converged ...

  17. Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

    OpenAIRE

    Moy, A. B.; Engelenhoven, J.; Bodmer, J.; Kamath, J.; Keese, C.; Giaever, I.; Shasby, S.; Shasby, D. M.

    1996-01-01

    We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did ...

  18. Rac inhibits thrombin-induced Rho activation: evidence of a Pak-dependent GTPase crosstalk

    OpenAIRE

    Rosenfeldt Hans; Castellone Maria; Randazzo Paul A; Silvio, Gutkind J.

    2006-01-01

    Abstract The strict spatio-temporal control of Rho GTPases is critical for many cellular functions, including cell motility, contractility, and growth. In this regard, the prototypical Rho family GTPases, Rho, Rac, and Cdc42 regulate the activity of each other by a still poorly understood mechanism. Indeed, we found that constitutively active forms of Rac inhibit stress fiber formation and Rho stimulation by thrombin. Surprisingly, a mutant of Rac that is unable to activate Pak1 failed to inh...

  19. Successful endovascular treatment of a hemodialysis graft pseudoaneurysm by covered stent and direct percutaneous thrombin injection.

    LENUS (Irish Health Repository)

    Keeling, Aoife N

    2011-07-25

    Vascular access for hemodialysis remains a challenge for nephrologists, vascular surgeons, and interventional radiologists alike. Arteriovenous fistula and synthetic grafts remain the access of choice for long-term hemodialysis; however, they are subject to complications from infection and repeated needle cannulation. Pseudoaneurysms are an increasingly recognized adverse event. At present, there are many minimally invasive methods to repair these wall defects. We present a graft pseudoaneurysm, which required a combination of endovascular stent graft placement and percutaneous thrombin injection for successful occlusion.

  20. Spirostanol saponins and esculin from Rusci rhizoma reduce the thrombin-induced hyperpermeability of endothelial cells.

    Science.gov (United States)

    Barbi?, M; Willer, E A; Rothenhöfer, M; Heilmann, J; Fürst, R; Jürgenliemk, G

    2013-06-01

    Rusci rhizoma extracts are traditionally used against chronic venous disorders (CVD). To determine the effect of its secondary plant metabolites on the endothelium, phenolic compounds and saponins from Butcher's broom were isolated from a methanolic extract, and their activity on the thrombin-induced hyperpermeability of human microvascular endothelial cells (HMEC-1) was investigated in vitro. In addition to the six known spirostanol saponins deglucoruscin (5), 22-O-methyl-deglucoruscoside (6), deglucoruscoside (7), ruscin (8), ruscogenin-1-O-(?-l-rhamnopyranosyl-(1?2)-?-d-galactopyranoside (9) and 1-O-sulpho-ruscogenin (10), three new spirostanol derivatives were isolated and identified: 3'-O-acetyl-4'-O-sulphodeglucoruscin (1), 4'-O-(2-hydroxy-3-methylpentanoyl)-deglucoruscin (2) and 4'-O-acetyl-deglucoruscin (3). Furthermore, the coumarin esculin (4), which is also prominently present in other medicinal plants used in the treatment of CVD, was isolated for the first time from Rusci rhizoma. Five of the isolated steroid derivatives (2, 5, 8, 9 and 10) and esculin (4) were tested for their ability to reduce the thrombin-induced hyperpermeability of endothelial cells in vitro, and the results were compared to those of the aglycone neoruscogenin (11). The latter compound showed a slight but concentration-dependent reduction in hyperpermeability to 71.8% at 100?M. The highest activities were observed for the spirostanol saponins 5 and 8 and for esculin (4) at 10?M, and these compounds resulted in a reduction of the thrombin-induced hyperpermeability to 41.9%, 42.6% and 53.3%, respectively. For 2, 5 and 8, the highest concentration tested (100?M) resulted in a drastic increase of the thrombin effect. The effect of esculin observed at a concentration of 10?M was diminished at 100?M. These in vitro data provide insight into the pharmacological mechanism by which the genuine spirostanol saponins and esculin can contribute to the efficacy of Butcher's broom against chronic venous disorders. PMID:23499166

  1. Effects of the oral, direct thrombin inhibitor dabigatran on five common coagulation assays

    OpenAIRE

    Lindahl, Tomas; Baghaei, Fariba; Fagerberg Blixter, Inger; Gustafsson, Kerstin; Stigendal, Lennart; Sten-linder, Margareta; Strandberg, Karin; Hillarp, Andreas

    2011-01-01

    Dabigatran is an oral, reversible thrombin inhibitor that has shown promising results in large clinical trials. Laboratory monitoring is not needed but the effects on common coagulation assays are incompletely known. Dabigatran was added to plasma from healthy subjects in the concentration range 0-1,000 mu g/l and analysed using several reagents for activated thromboplastin time (APTT), prothrombin time (PT), fibrinogen, antithrombin, and activated protein C resistance. Typical trough concent...

  2. Theory of dissociative recombination

    International Nuclear Information System (INIS)

    An analytic expression is derived for the cross section for the dissociative recombination of an electron with a molecular ion. A Morse potential is adopted as the internuclear potential for both the electronic ground state of the molecular ion and the resonance state of the molecule. The recombination coefficient is found by taking the average of the dissociative-recombination cross section over a Maxwellian distribution. This yields the temperature dependence of the recombination coefficient alpha approx.T/sup -1/2/. The temperature dependence of the coefficient for the dissociative recombination e+NO+ is calculated as an example. The results agree well with experiment

  3. Treatment of Iatrogenic femoral artery pseudoaneurysms using ultrasound-guided injection of thrombin

    International Nuclear Information System (INIS)

    AIM: To evaluate the use of ultrasound-guided percutaneous injection of thrombin for treatment of femoral artery pseudoaneurysms. METHOD: Nine patients with a confirmed femoral false aneurysm were included in the study. 0.5-1 ml of a 2000 U/ml solution of activated bovine thrombin was injected under ultrasound visualization into the neck of the aneurysm to induce thrombosis. The parent artery and adjacent major vessels were checked during and after the procedure to exclude propagation of thrombus. A check ultrasound examination was undertaken on the following day. RESULTS: Eight patients were successfully treated by a single injection. One patient required a second injection due to recurrence of their pseudoaneurysm 4 days after the initial treatment. The procedure was well tolerated in all cases and no complications were encountered. CONCLUSION: This small series provides further evidence that ultrasound-guided thrombin injection is a promising new method for the treatment of femoral false aneurysms. Hughes, M.J. et al. (2000)

  4. Electrochemiluminescence biosensor based on CdSe quantum dots for the detection of thrombin

    International Nuclear Information System (INIS)

    A novel QDs electrochemiluminescence (ECL) biosensor for the determination of thrombin was described. The CdSe QDs solution was dripped onto the clear surface of the ITO and then immersed in PBS which contained EDC and NHS as a coupling agent to activate the carboxyl-terminated surface of the CdSe QDs. The ITO electrode was immersed in the PBS containing 0.4 ?M aptamer, followed by rinsing with PBS and dried with N2 again, then dipped in the BSA solution for 30 min to decrease the non-specific binding. After that, the aptamer modified ITO was soaked in PBS to remove unbound aptamer. Under optimal conditions, the linear range was obtained from 0 to 64 ?g mL?1 with a correlation coefficient of 0.9986 (n = 16). The control experiment was also carried out by using BSA, lysozyme and IgG in the absence of thrombin. The results showed that the aptasensor had good specificity, stability and reproducibility to the thrombin. Moreover, the aptasensor could be used for detection of real sample with consistent results in comparison with those obtained by electrochemical method which could provide a promising platform for fabrication of aptamer based biosensors.

  5. Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI

    Science.gov (United States)

    Tucker, Erik I.; Marzec, Ulla M.; White, Tara C.; Hurst, Sawan; Rugonyi, Sandra; McCarty, Owen J. T.; Gailani, David; Hanson, Stephen R.

    2009-01-01

    The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)–dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti–human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and ?-thromboglobulin (?TG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface. PMID:18945968

  6. Direct thrombin inhibitor-bivalirudin functionalized plasma polymerized allylamine coating for improved biocompatibility of vascular devices.

    Science.gov (United States)

    Yang, Zhilu; Tu, Qiufen; Maitz, Manfred F; Zhou, Shuo; Wang, Jin; Huang, Nan

    2012-11-01

    The direct thrombin inhibitor of bivalirudin (BVLD), a short peptide derived from hirudin, has drawn an increasing attention in clinical application because it is safer and more effective than heparin for diabetic patients with moderate- or high-risk for acute coronary syndromes (ACS). In this study, BVLD was covalently conjugated on plasma polymerized allylamine (PPAam) coated 316L stainless steel (SS) to develop an anticoagulant surface. QCM-D real time monitoring result shows that 565±20 ng/cm2 of BVLD was bound to the PPAam surface. Infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of BVLD. The conjugation of BVLD onto the PPAam coating led to enhanced binding of thrombin, and the activity of the thrombin adsorbed on its surface was effectively inhibited. As a result, the BVLD immobilized PPAam (BVLD-PPAam) substrate prolonged the clotting times, and exhibited inhibition in adhesion and activation of platelets and fibrinogen. We also found that the BVLD-PPAam coating significantly enhanced endothelial cell adhesion, proliferation, migration and release of nitric oxide (NO) and secretion of prostaglandin I2 (PGI2). In vivo results indicate that the BVLD-PPAam surface restrained thrombus formation by rapidly growing a homogeneous and intact endothelium on its surface. These data suggest the potential of this multifunctional BVLD-PPAam coating for the application not only in general vascular devices such as catheters, tubes, oxygenator, hemodialysis membranes but also vascular grafts and stents. PMID:22877639

  7. Elevated Cytokines, Thrombin and PAI-1 in Severe HCPS Patients Due to Sin Nombre Virus

    Directory of Open Access Journals (Sweden)

    Virginie Bondu

    2015-02-01

    Full Text Available Sin Nombre Hantavirus (SNV, Bunyaviridae Hantavirus is a Category A pathogen that causes Hantavirus Cardiopulmonary Syndrome (HCPS with case fatality ratios generally ranging from 30% to 50%. HCPS is characterized by vascular leakage due to dysregulation of the endothelial barrier function. The loss of vascular integrity results in non-cardiogenic pulmonary edema, shock, multi-organ failure and death. Using Electric Cell-substrate Impedance Sensing (ECIS measurements, we found that plasma samples drawn from University of New Mexico Hospital patients with serologically-confirmed HCPS, induce loss of cell-cell adhesion in confluent epithelial and endothelial cell monolayers grown in ECIS cultureware. We show that the loss of cell-cell adhesion is sensitive to both thrombin and plasmin inhibitors in mild cases, and to thrombin only inhibition in severe cases, suggesting an increasing prothrombotic state with disease severity. A proteomic profile (2D gel electrophoresis and mass spectrometry of HCPS plasma samples in our cohort revealed robust antifibrinolytic activity among terminal case patients. The prothrombotic activity is highlighted by acute ?30 to >100 fold increases in active plasminogen activator inhibitor (PAI-1 which, preceded death of the subjects within 48 h. Taken together, this suggests that PAI-1 might be a response to the severe pathology as it is expected to reduce plasmin activity and possibly thrombin activity in the terminal patients.

  8. Elevated cytokines, thrombin and PAI-1 in severe HCPS patients due to Sin Nombre virus.

    Science.gov (United States)

    Bondu, Virginie; Schrader, Ron; Gawinowicz, Mary Ann; McGuire, Paul; Lawrence, Daniel A; Hjelle, Brian; Buranda, Tione

    2015-02-01

    Sin Nombre Hantavirus (SNV, Bunyaviridae Hantavirus) is a Category A pathogen that causes Hantavirus Cardiopulmonary Syndrome (HCPS) with case fatality ratios generally ranging from 30% to 50%. HCPS is characterized by vascular leakage due to dysregulation of the endothelial barrier function. The loss of vascular integrity results in non-cardiogenic pulmonary edema, shock, multi-organ failure and death. Using Electric Cell-substrate Impedance Sensing (ECIS) measurements, we found that plasma samples drawn from University of New Mexico Hospital patients with serologically-confirmed HCPS, induce loss of cell-cell adhesion in confluent epithelial and endothelial cell monolayers grown in ECIS cultureware. We show that the loss of cell-cell adhesion is sensitive to both thrombin and plasmin inhibitors in mild cases, and to thrombin only inhibition in severe cases, suggesting an increasing prothrombotic state with disease severity. A proteomic profile (2D gel electrophoresis and mass spectrometry) of HCPS plasma samples in our cohort revealed robust antifibrinolytic activity among terminal case patients. The prothrombotic activity is highlighted by acute ?30 to >100 fold increases in active plasminogen activator inhibitor (PAI-1) which, preceded death of the subjects within 48 h. Taken together, this suggests that PAI-1 might be a response to the severe pathology as it is expected to reduce plasmin activity and possibly thrombin activity in the terminal patients. PMID:25674766

  9. Molecular modeling studies of novel retro-binding tripeptide active-site inhibitors of thrombin.

    Science.gov (United States)

    Lau, W F; Tabernero, L; Sack, J S; Iwanowicz, E J

    1995-08-01

    A novel series of retro-binding tripeptide thrombin active-site inhibitors was recently developed (Iwanowicz, E. I. et al. J. Med. Chem. 1994, 37, 2111(1)). It was hypothesized that the binding mode for these inhibitors is similar to that of the first three N-terminal residues of hirudin. This binding hypothesis was subsequently verified when the crystal structure of a member of this series, BMS-183,507 (N-[N-[N-[4-(Aminoiminomethyl)amino[-1-oxobutyl]-L- phenylalanyl]-L-allo-threonyl]-L-phenylalanine, methyl ester), was determined (Taberno, L.J. Mol. Biol. 1995, 246, 14). The methodology for developing the binding models of these inhibitors, the structure-activity relationships (SAR) and modeling studies that led to the elucidation of the proposed binding mode is described. The crystal structure of BMS-183,507/human alpha-thrombin is compared with the crystal structure of hirudin/human alpha-thrombin (Rydel, T.J. et al. Science 1990, 249,227; Rydel, T.J. et al. J. Mol Biol. 1991, 221, 583; Grutter, M.G. et al. EMBO J. 1990, 9, 2361) and with the computational binding model of BMS-183,507. PMID:7582978

  10. Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release

    OpenAIRE

    He Shaoheng; Luo Jianmin; Wang Li

    2007-01-01

    Abstract Background It has been recognized that dermal fibroblasts and matrix metalloproteases (MMP) play crucial roles in wound healing process in skin. Thrombin was found to stimulate IL-8 release from human dermal fibroblasts (HDFs). However, little is known of the effect of thrombin on secretion of MMPs from dermal fibroblasts. In the present study, the influence of thrombin on proMMP-2 and proMMP-9 activity release from primary cultured HDFs, and its potential signaling pathways were inv...

  11. Effect of Locked-Nucleic Acid on a Biologically Active G-Quadruplex. A Structure-Activity Relationship of the Thrombin Aptamer

    OpenAIRE

    Jarstfer, Michael B.; Church, Frank C.; Laura Bonifacio

    2008-01-01

    Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG), by using locked nucleic acid (LNA) to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. In...

  12. Fundamental study of recombination and recombineering in Escherichia coli

    OpenAIRE

    Sun, Xiaohang; Huang, Yang

    2008-01-01

    Recombination and recombineering systems have been used in Escherichia coli to recombinant DNA sequences. With endonuclease and DNA lipase the bacterial plasmid and target DNA fragment can bind together and recombinant for a new DNA sequences. Red Proteins have been used in recombineering system to perform the function as the enzymes in recombination system, and faster and easier than the other way of recombinant new DNA sequences in E.coli. In this report we get to know the pr...

  13. Investigation of the thrombin-generating capacity, evaluated by thrombogram, and clot formation evaluated by thrombelastography of platelets stored in the blood bank for up to 7 days

    DEFF Research Database (Denmark)

    Johansson, Per Ingemar; Svendsen, M.S.

    2008-01-01

    BACKGROUND AND OBJECTIVES: Transfusion based on the Thrombelastograph (TEG) results reduces transfusion requirements in cardiac surgery and in liver transplantation. Taking the pivotal role of thrombin generation in the coagulation process into consideration, the clinical utility of the TEG may, in part, depend on its reflection of the dynamics of thrombin generation. MATERIAL AND METHODS: The kinetics of thrombin generation of platelets stored for 2 and 7 days, respectively, was assessed by calibrated automated thrombogram (CAT) and the lag time (min), time to peak (ttPeak; min), peak (nm thrombin) and endogenous thrombin potential (ETP; nm thrombin*min) were registered. Clot formation was evaluated by TEG and the R time (min), maxial amplitude (MA; mm), time to maximum thrombus generation (TMG; min) and maximum thrombus generation (MTG; dynes cm(-2) s(-1)) and total thrombus generation (TTG; dyne cm(-2)) were registered. RESULTS: Platelets become more procoagulant, evaluated both by TEG and CAT during storage. The reduction in CAT lag time and the ttPeak correlated with a decrease in the TEG R time and TMG (P < 0.0001) as did the CAT peak thrombin generation and the TEG MTG (P = 0.0035). No correlation between ETP and TTG was found (P = 0.65). CONCLUSION: The kinetics of thrombin generation, as evaluated by CAT, correlates with the thrombus generation, as evaluated by thrombelastography and this may in part explain the clinical utility of the TEG in identifying clinically relevant coagulopathies, secondary to impaired thrombin generation Udgivelsesdato: 2008/2

  14. Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/pulmonary and activation-regulated chemokine induction.

    Science.gov (United States)

    Li, Xuehua; Syrovets, Tatiana; Paskas, Svetlana; Laumonnier, Yves; Simmet, Thomas

    2008-07-15

    Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) is scarce. Monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-alpha or CD40 ligand, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicits actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-alpha-matured DC. The thrombin-induced migration is a true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase as well as subsequent phosphorylation of the regulatory myosin L chain 2. The ERK1/2- and Rho kinase 1-mediated phosphorylation of myosin L chain 2 was indispensable for the PAR-mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/pulmonary and activation-regulated chemokine, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83(+) DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. The thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism. PMID:18606675

  15. Transformation and DNA repair: linkage by DNA recombination.

    Science.gov (United States)

    Tønjum, Tone; Håvarstein, Leiv S; Koomey, Michael; Seeberg, Erling

    2004-01-01

    The stability of microbial genomes is constantly challenged by horizontal gene transfer, recombination and DNA damage. Mechanisms for rapid genome variation, adaptation and maintenance are a necessity to ensure microbial fitness and survival in changing environments. Indeed, genome sequences reveal that most, if not all, bacterial species have numerous gene functions for DNA repair and recombination. These important topics were addressed at the Second Genome Maintenance Meeting (GMM2). PMID:14700543

  16. DNA recombination during PCR.

    OpenAIRE

    Meyerhans, A.; Vartanian, J P; Wain-Hobson, S.

    1990-01-01

    PCR co-amplification of two distinct HIV1 tat gene sequences lead to the formation of recombinant DNA molecules. The frequency of such recombinants, up to 5.4% of all amplified molecules, could be decreased 2.7 fold by a 6 fold increase in Taq DNA polymerase elongation time. Crossover sites mapped essentially to three discrete regions suggesting specific Taq DNA polymerase pause or termination sites. PCR mediated recombination may be a problem when studying heterogeneous genetic material such...

  17. Recombination and Genetic Diversity

    Scientific Electronic Library Online (English)

    T. C., Coutinho; T.T.da, Silva; G.L., Toledo.

    2012-12-01

    Full Text Available In this paper we present a spatial stochastic model for genetic recombination, that answers if diversity is preserved in an infinite population of recombinat-ing individuals distributed spatially. We show that, for finite times, recombination may maintain all the various potential different types, b [...] ut when time grows infinitely, the diversity of individuals extinguishes off. So under the model premisses, recombination and spatial localization alone are not enough to explain diversity in a population. Further we discuss an application of the model to a controversy regarding the diversity of "Major Histocompatibility Complex" (MHC).

  18. Magnetic relaxation switch and colorimetric detection of thrombin using aptamer-functionalized gold-coated iron oxide nanoparticles

    International Nuclear Information System (INIS)

    We describe a sensitive biosensing system combining magnetic relaxation switch diagnosis and colorimetric detection of human ?-thrombin, based on the aptamer-protein interaction induced aggregation of Fe3O4-Au nanoparticles. To demonstrate the concept, gold-coated iron oxide nanoparticle was synthesized by iterative reduction of HAuCl4 onto the dextran-coated Fe3O4 nanoparticles. The resulting core-shell structure had a flowerlike shape with pretty narrow size distribution (referred to as 'nanorose'). The two aptamers corresponding to human ?-thrombin were conjugated separately to two distinct nanorose populations. Once a solution containing human ?-thrombin was introduced, the nanoroses switched from a well dispersed state to an aggregated one, leading to a change in the spin-spin relaxation time (T2) as well as the UV-Vis absorption spectra of the solution. Thus the qualitative and quantitative detection method for human ?-thrombin was established. The dual-mode detection is clearly advantageous in obtaining a more reliable result; the detection range is widened as well. By using the dual-mode detection method, a detectable T2 change is observed with 1.0 nM human ?-thrombin, and the detection range is from 1.6 nM to 30.4 nM.

  19. Fluorescence correlation spectroscopy of gold nanoparticles, and its application to an aptamer-based homogeneous thrombin assay

    International Nuclear Information System (INIS)

    We have studied the fluorescence properties and diffusion behaviors of gold nanoparticles (GNPs) in solution by using fluorescence correlation spectroscopy (FCS) at single molecule level. The GNPs display a high photo-saturation feature. Under illumination with strong laser light, they display higher brightness per particle (BPP) despite their low quantum yields. Based on the unique fluorescence properties and diffusion behaviors of GNPs, we have developed a sensitive and homogenous thrombin assay. It is based on a sandwich strategy and is making use of GNPs to which two different aptamers are conjugated. When the differently aptamer-labeled GNPs are mixed with solutions containing thrombin, the affinity reaction causes the GNPs to form dimers or oligomers. This leads to an increase in the diffusion time of the GNPs in the detection volume that is seen in FCS. The FCS method enables sensitive detection of the change in the characteristic diffusion time of the GNPs before and after the affinity reaction. Quantitative analysis of thrombin is based on the measurement of the change in the diffusion time. Under optimal conditions, the calibration plot is linear in the 0.5 nM to 110 nM thrombin concentration range, and the detection limit is 0.5 nM. The method was successfully applied to the direct determination of thrombin in human plasma. (author)

  20. Effects of thrombin, PAR-1 activating peptide and a PAR-1 antagonist on umbilical artery resistance in vitro

    Directory of Open Access Journals (Sweden)

    Elliott John T

    2005-02-01

    Full Text Available Abstract Background The non-thrombotic effects of thrombin in cardiovascular tissues, as mediated via the protease activated receptors (PARs, and particularly PAR-1, have been the focus of much recent research. The aims of this study were to evaluate the effects of thrombin, a specific PAR-1 activating peptide (PAR1-AP, and a PAR-1 antagonist on human umbilical artery tone in vitro. Methods Human umbilical artery samples were obtained from 17 women at term. Arterial rings were suspended under physiologic conditions for isometric recording. The in vitro effects of thrombin (0.5 units/mL to 3 units/mL, PAR1-AP TFLLR-NH2 [10(-9 to 10(-6 M], and PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH2 [10(-9 M to 10(-5 M] on umbilical artery tone were measured. Results Both thrombin and TFLLR-NH2 exerted a potent cumulative vasodilatory effect on human umbilical artery resistance (P 0.05. Conclusion These findings highlight a potential role for thrombin and PAR-1 receptors in vascular regulation of feto-placental blood flow in normal pregnancy, and in association with the vascular lesions associated with IUGR and pre-eclampsia.

  1. Anti-Thrombin Is Expressed in the Benign Prostatic Epithelium and in Prostate Cancer and Is Capable of Forming Complexes with Prostate-Specific Antigen and Human Glandular Kallikrein 2

    OpenAIRE

    Cao, Y.; Lundwall, A.; Gadaleanu, V.; Lilja, H.; Bjartell, A.

    2002-01-01

    Anti-thrombin, a member of the serpin family and an inhibitor of thrombin and blood coagulation factor Xa, was recently shown to inhibit angiogenesis and tumor growth. In the present study, we examined the expression of anti-thrombin in benign and malignant prostate gland. Using immunohistochemistry, anti-thrombin was found in prostate epithelium and stroma cells. Tissue microarrays of tumors (n = 112) and three different prostate cancer cell lines (PC-3, LNCaP, and DU-145) were all positive ...

  2. Thrombin related peptide TP508 promoted fracture repair in a mouse high energy fracture model

    Directory of Open Access Journals (Sweden)

    Pan Xiao-Hua

    2009-01-01

    Full Text Available Abstract Background Thrombin related peptide (TP508 is a 23 amino-acid synthetic peptide that represents a portion of the receptor-binding domain of thrombin molecule. Previous studies have shown that TP508 can accelerate musculoskeletal tissue repair including fracture healing. Objectives The aim of this study was to investigate the effect of TP508 on fracture healing in a murine fracture model representing high energy fracture situation. Methods Eighty CD 1 mice underwent controlled quadriceps muscle crush and open transverse mid diaphyseal femoral fracture that was then fixed with an external fixator. Animals were randomised into four groups to receive an intra-operative dose of either 100 ?g TP508 into the fracture gap; 100 ?g TP508 into the surrounding damaged muscle tissues; 10 ?g TP508 into the fracture gap, or control equal amount of saline into the fracture gap. Radiographic assessment was performed weekly for 5 weeks; histological analysis was at 3 and 5 weeks post fracture and biomechanical testing of the fractured bone was performed at 5 weeks post fracture. Results Mechanical testing data showed that the fracture stiffness was significantly higher in the group receiving 100 ?g TP508 into the fracture gap than other groups. Histological and radiographic analysis revealed a trend of increase in bone formation in the 100 ?g TP508 injected into the fracture gap group compared to the saline control group. It was noted that the scar tissues was significantly less in Group II comparing with the saline control group and there was increased blood vessel formation in the crushed muscles and fracture gap areas in the groups receiving TP508 comparing to the saline control group. Conclusion The results from this study demonstrated the use of thrombin related peptide TP508 in the situation of a high energy fracture can promote fracture healing and reduce the potential complications such as muscle fibrosis and fracture delayed or non-union.

  3. Enzyme-guided plasmonic biosensor based on dual-functional nanohybrid for sensitive detection of thrombin.

    Science.gov (United States)

    Yan, Jing; Wang, Lida; Tang, Longhua; Lin, Lei; Liu, Yang; Li, Jinghong

    2015-08-15

    Rapid and sensitive methodologies for the detection of protein are in urgent requirement for clinic diagnostics. Localized surface plasmon resonance (LSPR) of metal nanostructures has the potential to circumvent this problem due to its sensitive optical properties and strong electromagnetic near-field enhancements. In this work, an enzyme mediated plasmonic biosensor on the basis of a dual-functional nanohybrid was developed for the detection of thrombin. By utilizing LSPR-responsive nanohybrid and anaptamer-enzyme conjugated reporting probe, the sensing platform brings enhanced signal, stability as well as simplicity. Enzymatic reaction catalyzed the reduction of Au(3+) to Au° in situ, further leading to the rapid crystal growth of gold nanoparticles (AuNPs). The LSPR absorbance band and color changed company with the nanoparticle generation, which can be real-time monitoring by UV-visible spectrophotometer and naked eye. Nanohybrid constructed by gold and magnetic nanoparticles acts as a dual functional plasmonic unit, which not only plays the role of signal production, but also endows the sensor with the function of magnetic separation. Simultaneously, the introduction of enzyme effectively regulates the programming crystal growth of AuNPs. In addition, enzyme also serves as signal amplifier owing to its high catalysis efficiency. The response of the plasmonic sensor varies linearly with the logarithmic thrombin concentration up to 10nM with a limit of detection of 200pM. The as-proposed strategy shows good analytical performance for thrombin determination. This simple, disposable method is promising in developing universal platforms for protein monitoring, drug discovery and point-of-care diagnostics. PMID:25845332

  4. Monitoring of dabigatran therapy using Hemoclot(®) Thrombin Inhibitor assay in patients with atrial fibrillation.

    Science.gov (United States)

    Samoš, Matej; Stan?iaková, Lucia; Ivanková, Jela; Staško, Ján; Ková?, František; Dobrotová, Miroslava; Galajda, Peter; Kubisz, Peter; Moká?, Marián

    2015-01-01

    Dabigatran, a new direct thrombin inhibitor, achieves strong anticoagulation that is more predictable than warfarin. Nevertheless, a patient on dabigatran therapy (DT) may suffer from thrombotic or bleeding events. The routine monitoring of DT is not recommended, and standard coagulation tests are not sensitive enough for the assessment of DT activity. The aim of this study was to examine the clinical usefulness of the Hemoclot(®) Thrombin Inhibitor (HTI) assay in the assessment of dabigatran plasma levels in patients with non-valvular AF. Nineteen patients (12 men, 7 women) on DT were included in this preliminary prospective observational study. Dabigatran was administrated twice daily in a two dose regimens: 150 mg (5 patients) and 110 mg (14 patients). Blood samples were taken for the assessment of trough and peak levels of dabigatran. Dabigatran concentrations were measured with the HTI assay. The average dabigatran trough level was 69.3 ± 55.5 ng/ml and the average dabigatran peak level was 112.7 ± 66.6 ng/ml. The dabigatran trough plasma concentration was in the established reference range in 15 patients and the dabigatran peak plasma concentration was in the established reference range in 9 patients, respectively. Despite the fact that the activated partial thromboplastin and thrombin times were generally changed (prolonged), these tests failed to identify the patients with too low or too high dabigatran concentrations. The study confirmed the high sensitivity of the HTI assay for the assessment of dabigatran plasma levels. When compared to standard coagulation tests, the HTI is a more suitable assay for the monitoring of patients treated with dabigatran. Monitoring of DT may be beneficial in selected patients; however, further studies will be needed for the final clarification of this issue. PMID:25103614

  5. Minimally Invasive Therapy of Pseudoaneurysms of the Trunk: Application of Thrombin

    International Nuclear Information System (INIS)

    Thrombin injection has been proven to be successful in postcatheterization pseudoaneurysms. However, there are only a few reports on the treatment of pseudoaneurysms of the trunk. We report our first experiences using a percutaneous as well as an endovascular access. Eight iatrogenic pseudoaneurysms of the trunk (aorta, n = 4; pulmonary artery, n = 1; gastroduodenal artery, n = 1; left gastric artery, n = 1, renal artery, n = 1) were treated either percutaneously using CT guidance (n = 3) or via an endovascular access (n = 5). Noninvasive control angiograms were performed at day 1 and weeks 1 and 3 by either CT or MR angiography. The total volume of the pseudoaneurysms was 31.2 ± 23.1 ml on average, with a mean volume of the perfused aneurysmal lumen of 12.9 ± 7.2 ml. The maximum diameter was 4.1 ± 1.39 cm on average. In each case, the aneurysmal neck was not wider than 2 mm. One pseudoaneurysm occluded spontaneously following selective catheterization. The remaining pseudoaneurysms were successfully treated by injection of 765 ± 438.1 IU thrombin. In one individual, a nontarget embolization occurred, as well as an intervention-associated rupture of a pseudoaneurysm. High-grade stenoses of the donor artery were found in a different case. Only once was the endoluminal access converted to a percutaneous one. Thrombin injection might be a future first-line treatment of vascular lesions such as pseudoaneurysms of the trunk. In our experience both percutanous and endol experience both percutanous and endoluminal access are technically feasible and safe. However, further experiences are mandatory, especially concerning the question of dosage and long-term results

  6. Thrombin generation and factor X assays for the assessment of warfarin anticoagulation in thrombotic antiphospholipid syndrome.

    Science.gov (United States)

    Efthymiou, Maria; Lawrie, Andrew S; Mackie, Ian; Arachchillage, Deepa; Lane, Philip J; Machin, Samuel; Cohen, Hannah

    2015-06-01

    Monitoring warfarin anticoagulation in patients with thrombotic antiphospholipid syndrome (APS) may be complicated by the sensitivity of different thromboplastins to lupus anticoagulant. The aim of this study was to compare the degree of anticoagulation intensity in thrombotic APS and non-APS patients (50 in each group) on long-term warfarin, by measurement of the INR with two widely available thromboplastins with instrument-specific ISI values, and to investigate the potential role of amidolytic FX levels and thrombin generation (TG) testing in the assessment of anticoagulant intensity in thrombotic APS patients. There were no overall differences in INR between reagents or patient groups, but 20% (10/50) of APS patients showed ?0.5 INR unit difference between reagents, which would have resulted in altered clinical management in some patients. FX levels were useful in assessing anticoagulation intensity for INR 2.0-3.0, but showed poor utility at INR ?3.5 where the lowest measured FX level was 12IU/dL. In contrast, ETP and peak thrombin showed significant inverse correlations with the INR, suggesting that TG testing may be helpful in the determination of true anticoagulant intensity in APS patients, including those with ?3.5 INR. TG testing also highlighted a subgroup of APS patients with increased peak thrombin relative to the intensity of anticoagulation as assessed by INR and FX, suggesting that TG testing may be useful in identifying an ongoing prothrombotic state in patients with apparently adequate anticoagulation intensity as assessed by INR. PMID:25895847

  7. Low frequency recombination lines

    International Nuclear Information System (INIS)

    Low frequency recombination lines are those transitions with frequencies significantly less than 1 GHz. The authors discuss observations of extended low brightness HII regions, conventional HII regions, line enhancement caused by background continum sources and low frequency carbon recombination lines. (Auth.)

  8. Label-free aptasensor for thrombin using a glassy carbon electrode modified with a graphene-porphyrin composite

    International Nuclear Information System (INIS)

    We report on an electrochemical aptasensor for the ultrasensitive determination of thrombin. A glassy carbon electrode modified with a graphene-porphyrin nanocomposite exhibits excellent electrochemical activity and can be used as a redox probe in differential pulse voltammetry of the porphyrin on its surface. The thrombin aptamer is then immobilized via p-stacking interactions between aptamer and graphene and ?-? stacking with porphyrin simultaneously. The resulting electrochemical aptasensor displays a linear response to thrombin in the 5–1,500 nM concentration range and with a limit of detection of 0.2 nM (at an S/N of 3). The sensor benefits from the synergetic effects of graphene (with its high conductivity and high surface area), of the porphyrin (possessing excellent electrochemical activity), and of the aptamer (with its high affinity and specificity). This kind of aptasensor conceivably represents a promising tool for bioanalytical applications. (author)

  9. Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance.

    Science.gov (United States)

    Ostatná, Veronika; Vaisocherová, Hana; Homola, Jirí; Hianik, Tibor

    2008-07-01

    We report a multichannel surface plasmon resonance (SPR) sensor for detection of thrombin via DNA aptamers immobilized on the SPR sensor surface. A detailed investigation of the effect of the immobilisation method on the interaction between thrombin and DNA aptamers is presented. Three basic approaches to the immobilisation of aptamers on the surface of the SPR sensor are examined: (i) immobilisation based on chemisorption of aptamers modified with SH groups, (ii) immobilisation of biotin-tagged aptamers via previously immobilized avidin, neutravidin or streptavidin molecular linkers, and (iii) immobilisation employing dendrimers as a support layer for subsequent immobilisation of aptamers. A level of nonspecific binding of thrombin to immobilized human serum albumin (HSA) for each of the immobilisation methods is determined. Immobilisation of aptamers by means of the streptavidin-biotin system yields the best results both in terms of sensor specificity and sensitivity. PMID:18481050

  10. Glanzmann thrombasthenia: deficient binding of von Willebrand factor to thrombin-stimulated platelets.

    OpenAIRE

    Ruggeri, Z M; R. Bader; L. De Marco

    1982-01-01

    Glanzmann thrombasthenia is an inherited bleeding disorder characterized by the failure of platelets to aggregate in response to almost all stimuli. However, thrombasthenic platelets will aggregate with bovine and porcine von Willebrand factor (vWF) and will show normal ristocetin-induced binding and aggregation in the presence of human vWF. In contrast, we now report that the specific binding of vWF to the thrombin-stimulated platelets was less than 20% of normal in three patients with Glanz...

  11. Thrombin effectuates therapeutic arteriogenesis in the rabbit hindlimb ischemia model: A quantitative analysis by computerized in vivo imaging

    International Nuclear Information System (INIS)

    We report on an experimental mammalian controlled study that documents arteriogenic capacity of thrombin and utilizes computerized algorithms to quantify the newly formed vessels. Hindlimb ischemia was surgically invoked in 10 New Zealand white rabbits. After quiescence of endogenous angiogenesis heterologous bovine thrombin was intramuscularly injected (1500 units) in one hindlimb per rabbit (Group T). Contralateral limbs were infused with normal saline (Group C). Digital subtraction angiography (DSA) of both limbs was performed after thrombin infusion by selective cannulation of the abdominal aorta and digital images were post-processed with computerized algorithms in order to enhance newly formed vessels. Total vessel area and total vessel length were quantified. In vivo functional evaluation included measurements of blood flow volume at the level of the external iliac artery by Doppler ultrasonography both at baseline and at 20 days after thrombin infusion. Total vessel area and length (in pixels) were 14,713+/-1023 and 5466+/-1327 in group T versus 12,015+/-2557 and 4598+/-1269 in group C (p=0.0062 and 0.1526, respectively). Blood flow volumes (ml/min) at baseline and at 20 days after thrombin infusion were 25.87+/-11.09 and 38.06+/-11.72 in group T versus 26.57+/-11.19 and 20.35+/-7.20 in group C (p=0.8898 and 0.0007, respectively). Intramuscular thrombin effectuates an arteriogenic response in the rabbit hindlimb ischemia model. Computerized algorithms may enabia model. Computerized algorithms may enable accurate quantification of the neovascularization outcome

  12. Vascular actions of thrombin receptor-derived polypeptides: structure-activity profiles for contractile and relaxant effects in rat aorta.

    OpenAIRE

    Laniyonu, A. A.; Hollenberg, M. D.

    1995-01-01

    1. Using endothelium-denuded and intact rat aortic rings, we have determined the contractile and relaxant structure-activity profile for a series of thrombin receptor-derived polypeptides (TRPs) based on the human and rat receptor sequences: SFLLR (P5), SFLLR-NH2 (P5-NH2) SFFLR (Rat P5), SFFLR-NH2 (Rat P5-NH2), SFLLRNP (P7), SFLLRNP-NH2 (P7-NH2), SFFLRNP (Rat P7), SFFLRNP-NH2 (Rat P7-NH2), and SFLLRNPNDKYEPF (P14). 2. A contractile response to thrombin and the TRPs in the endothelium-denuded ...

  13. Acidic Residues C-Terminal to the A2 Domain Facilitate Thrombin-Catalyzed Activation of Factor VIII

    OpenAIRE

    Newell, Jennifer L.; Fay, Philip J.

    2008-01-01

    Factor VIII is activated by thrombin through proteolysis at Arg740, Arg372, and Arg1689. One region implicated in this exosite-dependent interaction is the factor VIII a2 segment (residues 711-740) separating the A2 and B domains. Residues 717-725 (DYYEDSYED) within this region consist of five acidic residues and three sulfo-Tyr residues, thus representing a high density of negative charge potential. The contributions of these residues to thrombin-catalyzed activation of factor VIII were asse...

  14. Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction.

    Science.gov (United States)

    Wang, Zhen; Ginnan, Roman; Abdullaev, Iskandar F; Trebak, Mohamed; Vincent, Peter A; Singer, Harold A

    2010-07-01

    Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration. PMID:20442409

  15. A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

    International Nuclear Information System (INIS)

    To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

  16. The role of structural information in the discovery of direct thrombin and factor Xa inhibitors.

    Science.gov (United States)

    Nar, Herbert

    2012-05-01

    The quest for novel medications to treat thromboembolic disorders such as venous thrombosis, pulmonary embolism and stroke received a boost when the 3D structures of two major players in the blood coagulation cascade were determined in 1989 and 1993. Structure-guided design of inhibitors of thrombin (factor IIa, fIIa) and factor Xa (fXa) eventually led to the discovery of potent, selective, efficacious, orally active and safe compounds that proved successful in clinical studies. In 2008, the direct thrombin inhibitor dabigatran etexilate developed by Boehringer Ingelheim became the first novel antithrombotic molecular entity to enter the market in 50 years. Additional compounds targeting factor Xa were subsequently granted marketing authorization or are in late-stage clinical studies. In this review, I use selected case studies to describe the discovery of novel fIIa and fXa inhibitors, with a particular emphasis on the pre-eminent role that structural information played in this process. PMID:22503439

  17. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, Andre?; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  18. Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance.

    Czech Academy of Sciences Publication Activity Database

    Hianik, T.; Ostatná, V.; Vaisocherová, Hana; Homola, Ji?í

    2008-01-01

    Ro?. 391, ?. 5 (2008), s. 1861-1869. ISSN 1618-2642 R&D Projects: GA AV ?R KAN200670701 Institutional research plan: CEZ:AV0Z20670512 Keywords : DNA aptamer * thrombin * dendrimers Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.328, year: 2008

  19. Rolling-circle amplification detection of thrombin using surface-enhanced Raman spectroscopy with core-shell nanoparticle probe.

    Science.gov (United States)

    Li, Xuemei; Wang, Linlin; Li, Chunxiang

    2015-04-27

    An ultrasensitive surface-enhanced Raman spectroscopy (SERS) sensor based on rolling-circle amplification (RCA)-increased "hot-spot" was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO2 core-shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer-by-layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single-stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10(-12) to 1.0×10(-8) ?M and a detection limit of 4.2×10(-13) ?M, and showed good performance in real serum samples. PMID:25766032

  20. Thrombin-induced platelet aggregation is affected by external Na+ independently of the Na+/H+ exchange.

    Science.gov (United States)

    Agam, G; Argaman, A; Livne, A

    1989-02-13

    Thrombin affects blood platelets by activation of Na+/H+ exchange and induction of aggregation, but the relationship between these effects is under debate. The present study attempts to clarify whether the activation of the exchanger activity is required for platelet aggregation. In apparent support of such a requirement, thrombin-induced aggregation is higher in Na+ medium than in N-methylglucamine+ medium and is inhibited by sphingosine, an inhibitor of protein kinase C known to regulate the Na+/H+ exchanger. However, the inhibition of aggregation by sphingosine occurs in both Na+-containing and Na+-free media, the aggregation is identical in Na+ and K+-containing media, and is not inhibited by 5-N-(3-aminophenyl)amiloride, at a concentration 10-fold higher than its Ki for platelet Na+/H+ exchange. Furthermore, at low concentration (0.005 U/ml) thrombin induces aggregation but does not activate the exchange. It is concluded that the activation of Na+/H+ exchange is not required for thrombin-induced platelet aggregation and that the apparent augmentation of aggregation by Na+ is due to an inhibitory effect of N-methylglucamine+. PMID:2538349

  1. Aptamer-based organic-silica hybrid affinity monolith prepared via "thiol-ene" click reaction for extraction of thrombin.

    Science.gov (United States)

    Wang, Zheng; Zhao, Jin-Cheng; Lian, Hong-Zhen; Chen, Hong-Yuan

    2015-06-01

    A novel strategy for preparing aptamer-based organic-silica hybrid monolithic column was developed via "thiol-ene" click chemistry. Due to the large specific surface area of the hybrid matrix and the simplicity, rapidness and high efficiency of "thiol-ene" click reaction, the average coverage density of aptamer on the organic-silica hybrid monolith reached 420pmol?L(-1). Human ?-thrombin can be captured on the prepared affinity monolithic column with high specificity and eluted by NaClO4 solution. N-p-tosyl-Gly-Pro-Arg p-nitroanilide acetate was used as the sensitive chromogenic substrate of thrombin. The thrombin enriched by this affinity column was detected with a detection of limit of 0.01?M by spectrophotometry. Furthermore, the extraction recovery of thrombin at 0.15?M in human serum was 91.8% with a relative standard deviation of 4.0%. These results indicated that "thiol-ene" click chemistry provided a promising technique to immobilize aptamer on organic-inorganic hybrid monolith and the easily-assembled affinity monolithic material could be used to realize highly selective recognition of trace proteins. PMID:25863371

  2. GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

    International Nuclear Information System (INIS)

    Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with [3H]inositol. Ca2+ has opposite effects on the formation of [3H]inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of [3H]inositol phosphates is inhibited by Ca2+, action of thrombin is stimulated by Ca2+. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca2+ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca2+ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein

  3. Effect of Locked-Nucleic Acid on a Biologically Active G-Quadruplex. A Structure-Activity Relationship of the Thrombin Aptamer

    Directory of Open Access Journals (Sweden)

    Michael B. Jarstfer

    2008-03-01

    Full Text Available Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG, by using locked nucleic acid (LNA to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. Interestingly, the thermal stability of the substituted aptamers does not correlate to activity – the more stable aptamers with LNA in position G5, T7, or G8 showed the least thrombin inhibition, while a less stable aptamer with LNA at G2 was as active as the un-substituted aptamer. These results suggest that LNA substitution at sites G5, T7, and G8 directly perturbs aptamer-thrombin affinity. This further implies that for the thrombin aptamer, activity is not dictated solely by the stability of the G-quadruplex structure, but by specific interactions between the central TGT loop and thrombin and that LNA can be tolerated in a biologically active nucleic acid structure albeit in a position dependent fashion.

  4. A electrochemiluminescence aptasensor for detection of thrombin incorporating the capture aptamer labeled with gold nanoparticles immobilized onto the thio-silanized ITO electrode

    International Nuclear Information System (INIS)

    A novel electrochemiluminescence (ECL) aptasensor was proposed for sensitive and cost-effective detection of the target thrombin adopted an aptamer-based sandwich format. To detect thrombin, capture aptamers labeled with gold nanoparticles (AuNPs) were first immobilized onto the thio-silanized ITO electrode surface through strong Au-S bonds. After catching the target thrombin, signal aptamers tagged with ECL labels were attached to the assembled electrode surface. As a result, an AuNPs-capture-aptamer/thrombin/ECL-tagged-signal-aptamer sandwich type was formed. Treating the resulting electrode surface with tri-n-propylamine (TPA) and applying a swept potential to the electrode, ECL response was generated which realized the detection of target protein. Spectroscopy and electrochemical impedance techniques were used to characterize and confirm the fabrication of the ECL aptasensor. AuNPs amplification and smart sensor fabrication art were implemented for the sensitive and cost-effective detection purpose. Signal-to-dose curve excellently followed a sandwich format equation and could be used to quantify the protein, and the detection limit was estimated to be 10 nM. Other forms of thrombin such as ?- and ?-thrombins had negligible response, which indicated a high specificity of ?-thrombin detection. The aptasensor opened up new fields of aptamer applications in ECL domain, a highly sensitive technique, and had a promising perspective to be applied in microarray analystive to be applied in microarray analysis

  5. Topical haemostatic agents for skin wounds: a systematic review

    Directory of Open Access Journals (Sweden)

    Ubbink Dirk T

    2011-07-01

    Full Text Available Abstract Background Various agents and techniques have been introduced to limit intra-operative blood loss from skin lesions. No uniformity regarding the type of haemostasis exists and this is generally based on the surgeon's preference. To study the effectiveness of haemostatic agents, standardized wounds like donor site wounds after split skin grafting (SSG appear particularly suitable. Thus, we performed a systematic review to assess the effectiveness of haemostatic agents in donor site wounds. Methods We searched all randomized clinical trials (RCTs on haemostasis after SSG in Medline, Embase and the Cochrane Library until January 2011. Two reviewers independently assessed trial relevance and quality and performed data analysis. Primary endpoint was effectiveness regarding haemostasis. Secondary endpoints were wound healing, adverse effects, and costs. Results Nine relevant RCTs with a fair methodological quality were found, comparing epinephrine, thrombin, fibrin sealant, alginate dressings, saline, and mineral oil. Epinephrine achieved haemostasis significantly faster than thrombin (difference up to 2.5 minutes, saline or mineral oil (up to 6.5 minutes. Fibrin sealant also resulted in an up to 1 minute quicker haemostasis than thrombin and up to 3 minutes quicker than placebo, but was not directly challenged against epinephrine. Adverse effects appeared negligible. Due to lack of clinical homogeneity, meta-analysis was impossible. Conclusion According to best available evidence, epinephrine and fibrin sealant appear superior to achieve haemostasis when substantial topical blood loss is anticipated, particularly in case of (larger SSGs and burn debridement.

  6. Thrombin is an extracellular signal that activates intracellular death protease pathways inducing apoptosis in model motor neurons.

    Science.gov (United States)

    Smirnova, I V; Zhang, S X; Citron, B A; Arnold, P M; Festoff, B W

    1998-07-01

    Apoptosis, often also termed "programmed cell death", occurs in normal development in the brain and spinal cord. Important to concepts of disease and potential intervention is the exciting finding that apoptosis is also found after neurotrauma and in a number of neurodegenerative diseases. Although the precise mechanism of neuronal cell loss remains unknown, much emphasis has been placed recently on the activation of cell death protease cascades within the cell. How these cascades may be activated, especially from extracellular influences, is currently poorly understood. Thrombin, the multifunctional coagulation protease, is an early phase modulator at sites of tissue injury and has been shown to induce cell death in neurons by an apoptotic mechanism by activating its receptor, PAR-1. Using a model motor neuronal cell line, NSC19, which we have shown undergoes apoptosis after treatment with classic apoptosis inducers such as the topoisomerase inhibitors camptothecin and etoposide, we unambiguously found that nanomolar thrombin induced characteristic signs of apoptosis. Strikingly, endonucleolysis was accompanied by an increase in caspase-3-like activity in cellular extracts, which correlated with both detection of caspase-induced signature cleavage of the cortical cytoskeleton component nonerythroid spectrin (alpha-fodrin) and identification of increased accessibility of a caspase cleavage domain, using an antibody (Ab127) made against a synthetic peptide KGDEVD. Demonstrating that thrombin activation of death proteases was linked to cell death, we were able to inhibit thrombin-induced apoptosis by using a caspase family inhibitor, benzyloxycarbonyl-Asp-(oMe)-fluoromethyl ketone (Boc-D-FMK). These novel results demonstrate that thrombin serves as an extracellular "death signal" to activate intracellular protease pathways. These pathways lead to apoptotic cell death and can be modulated by inhibiting caspase activity downstream to PAR-1. PMID:9658339

  7. Thrombin generation in first-degree relatives of patients with venous thromboembolism who have factor V Leiden. A pilot study.

    Science.gov (United States)

    Couturaud, Francis; Duchemin, Jérôme; Leroyer, Christophe; Delahousse, Bénédicte; Abgrall, Jean François; Mottier, Dominique

    2008-01-01

    The thrombin generation test appears to be a highly sensitive and specific test in the detection of thrombophilia in patients with venous thromboembolism. We aimed to determine the accuracy of the thrombin generation test to detect factor V Leiden and/or other prothrombotic states in first-degree relatives of patients with venous thromboembolism and factor V Leiden. Sixty-two first-degree relatives of 21 index cases were tested for factor V Leiden, the G20210A prothrombin gene mutation and thrombin generation. Information about oestrogen therapy and previous VTE was also collected. The normalized Thrombomodulin sensitivity ratio (n-TMsr) was defined as the ratio of endogenous thrombin potential determined in the presence and absence of thrombomodulin which was normalized against the same ratio determined in normal control plasma. The mean n-TMsr was 1.37 (+/- 0.33) in the 45 relatives with one or more prothrombotic state (factor V Leiden, G20210A prothrombin mutation, oestrogen therapy or hormonal therapy) and 1.02 (+/- 0.34) in the 17 relatives without prothrombotic state (p = 0.001). The positive predictive value was 90.3 (95%CI, 73.1-97.4). In relatives with an abnormal n-TMsr, the adjusted odds ratio for having a prothrombotic state was 8.3 (95%CI, 1.9-36.9) and the adjusted odds ratio for having the factor V Leiden was 14.3 (95%CI, 2.9-71.2). An abnormal thrombin generation test appears highly predictive for having factor V Leiden and/or other prothrombotic states in first-degree relatives of patients with venous thromboembolism and factor V Leiden. PMID:18217158

  8. Thermodynamics of substrates and reversible inhibitors binding to the active site cleft of human alpha-thrombin.

    Science.gov (United States)

    De Cristofaro, R; Landolfi, R

    1994-06-17

    The study of the temperature effect on the binding to the active site of human alpha-thrombin for ten different ligands, i.e. nine peptide substrates and the tight binding inhibitor N alpha -(naphthalene-sulphonyl-glycyl)-4-amidino-DL-phenyl-alanine-piperidine (alpha-NAPAP), showed that the enthalpy is constant over the temperature range spanning from 10 to 40 degrees C. It was found that the values of the binding enthalpy are linearly correlated to those of entropy, and that this correlation arises from a real phenomenon of chemical compensation. On the other hand, no compensatory chemical effect was found for the process of thrombin acylation. Additional experiments showed that binding to thrombin of two competitive thrombin inhibitors, i.e. proflavin and p-aminobenzamidine, is characterized by a change in the standard heat capacity change (delta Cp), approximately equal to -1 kcal/mol K. By analogy with model compound transfer studies and protein folding investigations, it is proposed that a burial of a large surface area of non-polar residues, roughly equal to 3000 A2, brings about the observed heat capacity change. Altogether, the observed phenomena of the chemical compensation and heat capacity change, although qualitatively different, are interpreted as expressions of the same property of the enzyme, i.e. the capacity to undergo conformational transitions upon ligation of the catalytic domain. These structural transitions are strictly ligand-linked and could play a central role for setting the rules which regulate the specificity of substrates and inhibitors binding to the catalytic groove of human alpha-thrombin. PMID:8006969

  9. cAMP controls the restoration of endothelial barrier function after thrombin-induced hyperpermeability via Rac1 activation.

    Science.gov (United States)

    Aslam, Muhammad; Tanislav, Christian; Troidl, Christian; Schulz, Rainer; Hamm, Christian; Gündüz, Dursun

    2014-10-01

    Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier integrity leading to oedema formation followed by resealing of AJs and a slow recovery of the barrier function. The molecular mechanisms of this process have not yet been fully delineated. The aim of the present study was to analyse the molecular mechanism of endothelial barrier recovery and thrombin was used as model inflammatory mediator. Thrombin caused a strong increase in endothelial permeability within 10 min accompanied by loss of Rac1 but not cdc42 activity, drop in cellular cAMP contents, and a strong activation of the endothelial contractile machinery mainly via RhoA/Rock signalling. Activation of RhoA/Rock signalling precedes and is dependent upon a rise in the cytosolic Ca(2+) concentration. Inhibition of cytosolic Ca(2+) rise but not MLCK or Rock enhances the recovery of endothelial barrier function. The cellular cAMP contents increased gradually during the barrier recovery phase (30-60 min after thrombin challenge) accompanied by an increase in Rac1 activity. Inhibition of Rac1 activity using a specific pharmacological inhibitor (NSC23766) abrogated the endothelial barrier recovery process, suggesting a Rac1-dependent phenomenon. Likewise, inhibition of either adenylyl cyclase or the cAMP-effectors PKA and Epac (with PKI and ESI-09, respectively) caused an abrogation of Rac1 activation, resealing of endothelial AJs and recovery of endothelial barrier function. The data demonstrate that endothelial barrier recovery after thrombin challenge is regulated by Rac1 GTPase activation. This Rac1 activation is due to increased levels of cellular cAMP and activation of downstream signalling during the barrier recovery phase. PMID:25344477

  10. Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

    Directory of Open Access Journals (Sweden)

    Thiel Gerald

    2009-05-01

    Full Text Available Abstract Background The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts. Results Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK. Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M1 muscarinic acetylcholine receptors required the transactivation of the EGF receptor, while thrombin signaling did not. Conclusion This study shows that stimulus-transcription coupling in thrombin-treated lung fibroblasts relies on the elevation of the intracellular Ca2+-concentration and the activation of PKC and ERK. In the nucleus, ternary complex factors function as key proteins linking the intracellular signaling cascade with enhanced transcription of the Egr-1 gene. This study further shows that the dominant-negative Elk-1 mutant is a valuable tool to study Elk-1-mediated gene transcription.

  11. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system for assaying recombination using tetrad analysis in a higher eukaryotic system (6). This system enabled the measurement of the frequency and distribution of recombination events at a genome wide level in wild type Arabidopsis (7), construction of genetic linkage maps which include positions for each centromere (8), and modeling of the strength and pattern of interference (9). This proposal extends the use of tetrad analysis in Arabidopsis by using it as the basis for assessing the phenotypes of mutants in genes important for recombination and the regulation of crossover interference and performing a novel genetic screen. In addition to broadening our knowledge of a classic genetic problem - the regulation of recombination by crossover interference - this proposal also provides broader impact by: generating pedagogical tools for use in hands-on classroom experience with genetics, building interdisciplinary collegial partnerships, and creating a platform for participation by junior scientists from underrepresented groups. There are three specific aims: (1) Isolate mutants in Arabidopsis MUS81 homologs using T-DNA and TILLING (2) Characterize recombination levels and interference in mus81 mutants (3) Execute a novel genetic screen, based on tetrad analysis, for genes that regulate meiotic recombination

  12. A study of the conditions and accuracy of the thrombin time assay of plasma fibrinogen

    DEFF Research Database (Denmark)

    Jespersen, J; Sidelmann, Johannes Jakobsen

    1982-01-01

    The conditions, accuracy, precision and possible error of the thrombin time assay of plasma fibrinogen are determined. Comparison with an estimation of clottable protein by absorbance at 280 nm gave a correlation coefficient of 0.96 and the regression line y = 1.00 x + 0.56 (n = 34). Comparison with a radial immunodiffusion method yielded the correlation coefficient 0.97 and the regression line y = 1.18 x = 2.47 (n = 26). The presence of heparin in clinically applied concentrations produced a slight shortening of the clotting times. The resulting error in the estimated concentrations of fibrinogen was too small to affect the clinical usefulness of the determinations. The influence of fibrin(ogen) degradation products was significant only in excessive amounts in samples containing low levels of fibrinogen.

  13. Cyclodextrin functionalized graphene-gold nanoparticle hybrids with strong supramolecular capability for electrochemical thrombin aptasensor.

    Science.gov (United States)

    Xue, Qiong; Liu, Zhiguang; Guo, Yujing; Guo, Shaojun

    2015-06-15

    We demonstrate a facile one-pot synthetic strategy for controlled synthesis of thio-?-cyclodextrin functionalized graphene/gold nanoparticles (SH-?-CD-Gr/AuNPs) composites using SH-?-CD as both the dispersant and linker. The obtained SH-?-CD-Gr/AuNPs integrate the excellent electrical properties and large surface area of graphene and AuNPs with supramolecular recognition ability of CD, which show more effective electron transfer and higher enriched ability for the ferrocene probe via the host-guest interaction between CD and ferrocene than SH-?-CD-Gr. In the presence of target, the stronger interaction between aptamer and target makes the ferrocene move closer to the electrode surface, thus facilitating the electron transfer. Based on this sensing mechanism, a new and highly sensitive biosensing concept by the use of SH-?-CD-Gr/AuNPs as enhancing materials is demonstrated for "signal-on" detection of targets (thrombin as a model target). This biosensor exhibits a wide linear range for thrombin from 1.6×10(-17) M to 8.0×10(-15) M and a very low limit of detection 5.2×10(-18) M, which is two-order magnitude better than those of SH-?-CD-Gr (the detection linear range from 1.6×10(-15) M to 8.0×10(-13) M and detection limit of 1.0×10(-15) M). Our proposed electrochemical aptasensor based on SH-?-CD-Gr/AuNPs shows good selectivity against other proteins such as human serum albumin, lysozyme and insulin. To the best of our knowledge, the present SH-?-CD-Gr/AuNPs hybrids are the most efficient graphene-based electrochemical active probes ever reported for biosensors. PMID:25618374

  14. Resolution of radiolabeled molecular species of phospholipid in human platelets: effect of thrombin

    International Nuclear Information System (INIS)

    Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [3H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [3H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[3H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containingogy indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate

  15. Recent Developments in Dissociative Recombination

    International Nuclear Information System (INIS)

    There have been a number of recent developments in dissociative recombination research as it relates to ITER, that should be highlighted. These concern primarily experimental and modelling issues and this document will not touch upon the topics of the other scientists involved in DR studies that are present at the meeting. The topic of branching ratios in general is a topic fundamental to DR especially how it influences the formation of radical and stable neutral molecules that again might play a role in particle formation. It should be remembered that the reactions of neutral radicals to form cyclic compounds are responsible for the formation of soot in combustion, though the role played by ions in flames is at best uncertain. In the near wall plasma environment, ion processes may well be more important since neutral species are rarer. Modelling studies by Pernot and collaborators at the Universite de Paris-Sud have shown that if one compares the yields of individual neutral species in ion-chemistry models (in this particular case, the ionosphere of Titan), and if one assumes that DR reactions of hydrocarbon ions primarily decay via the ejection of a hydrogen ion (which is assumed by most Titan ionospheric models) and if one compares these predictions with those coming from a model where actual measured branching ratios are used, differences of up to 5 orders of magnitude are found. This shows very clearly the need for branching ratio studies. In early merged beam sting ratio studies. In early merged beam studies of DR performed in Canada in the 1970's, it was noticed that cross sections for polyatomic species typically displayed a sharp fall-off above 0.1 eV. This has since been seen in many storage ring studies and clearly this has important consequences for ITER chemistry where plasma temperatures are likely to be well above ambient. In a recent analysis, Jungen and Pratt have explained this phenomenon on the basis that the recombination is dominated by the indirect process (initial capture into a vibrationally excited, neutral Rydberg state) in which the propensity rule (+?v=1) dominates the capture. When the electron energy exceeds that between the v'=0 and v'=1 levels of the ion, where the capture must now involve a ?v=2 transition, this will be much less effective and so the cross section drops precipitously. This assumes of course that the recombining ion is primarily in the ground v=0 level. H3+ continues to be an active subject of research and a very recent experiment at the TSR ring in Heidelberg has examined the influence of rotational excitation on the rate of the recombination. This is a very beautiful study but an important outcome is that even though a cryogenically cooled storage trap was used to produce the ions, the internal rotational temperature of the ions was never found to be below 150K. This suggests that ion cooling by storage in the ring leads eventually to an equilibrium value for the internal energy of the ions as they are de-excited/re-excited by passage through the electron cooler. As observed in earlier merged beam experiments in Canada, the extraction field in the ion source plays an important role in determining the excitation state of the ions as collisions outside the source can lead to re-heating. Indeed in the TSR experiments using a conventional Penning source and a normal extraction field, the ions were found to have a rotational temperature of several thousands of degrees. This clearly has important significance for earlier measurements taken in storage rings. Finally, the world will soon have a new storage ring facility for dissociative recombination research and this will be in Langzhou in China. This machine will have a higher magnetic rigidity that previous rings used for DR and so heavier ions and higher mass resolution experiments can be performed there. Experimental operation of this new ring is expected to commence in 2012/2013. (author)

  16. Dual-colored graphene quantum dots-labeled nanoprobes/graphene oxide: functional carbon materials for respective and simultaneous detection of DNA and thrombin

    Science.gov (United States)

    Qian, Zhao Sheng; Shan, Xiao Yue; Chai, Lu Jing; Chen, Jian Rong; Feng, Hui

    2014-10-01

    Convenient and simultaneous detection of multiple biomarkers such as DNA and proteins with biocompatible materials and good analytical performance still remains a challenge. Herein, we report the respective and simultaneous detection of DNA and bovine ?-thrombin (thrombin) entirely based on biocompatible carbon materials through a specially designed fluorescence on-off-on process. Colorful fluorescence, high emission efficiency, good photostability and excellent compatibility enables graphene quantum dots (GQDs) as the best choice for fluorophores in bioprobes, and thus two-colored GQDs as labeling fluorophores were chemically bonded with specific oligonucleotide sequence and aptamer to prepare two probes targeting the DNA and thrombin, respectively. Each probe can be assembled on the graphene oxide (GO) platform spontaneously by ?–? stacking and electrostatic attraction; as a result, fast electron transfer in the assembly efficiently quenches the fluorescence of probe. The presence of DNA or thrombin can trigger the self-recognition between capturing a nucleotide sequence and its target DNA or between thrombin and its aptamer due to their specific hybridization and duplex DNA structures or the formation of apatamer–substrate complex, which is taken advantage of in order to achieve a separate quantitative analysis of DNA and thrombin. A dual-functional biosensor for simultaneous detection of DNA and thrombin was also constructed by self-assembly of two probes with distinct colors and GO platform, and was further evaluated with the presence of various concentrations of DNA and thrombin. Both biosensors serving as a general detection model for multiple species exhibit outstanding analytical performance, and are expected to be applied in vivo because of the excellent biocompatibility of their used materials.

  17. Dabigatran, a direct thrombin inhibitor, blocks differentiation of normal fibroblasts to a myofibroblast phenotype and demonstrates anti-fibrotic effects on scleroderma lung fibroblasts

    OpenAIRE

    Bogatkevich, Galina S.; Ludwicka-bradley, Anna; Silver, Richard M.

    2009-01-01

    Myofibroblasts are the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of lung parenchyma associated with pulmonary fibrosis. We previously reported that thrombin activates protease-activated receptor (PAR)-1 thereby inducing normal lung fibroblasts to differentiate to a myofibroblast phenotype resembling scleroderma lung myofibroblasts. Here we demonstrate that the thrombin inhibitor dabigatran inhibits in a dose-dependant manner...

  18. Thrombin Activates AMP-Activated Protein Kinase in Endothelial Cells via a Pathway Involving Ca2+/Calmodulin-Dependent Protein Kinase Kinase ?

    OpenAIRE

    Stahmann, Nadine; Woods, Angela; Carling, David; Heller, Regine

    2006-01-01

    AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2+/calmodulin-dependent protein kinase kinase ? (CaMKK?). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca2+-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C...

  19. Testing for recombinant human erythropoietin

    Science.gov (United States)

    Joris R Delanghe (Ghent University Hospital Clinical Chemistry)

    2008-08-10

    ERYTHROPOIETIN (Epo) may have effects on exercise capacity and physiological regulation beyond a simple increase in red cell mass and the associated improvement in oxygen transport (4). In the context of a larger study on this topic, Lundby and colleagues (11) also asked questions about the reliability of urine testing for recombinant human Epo (rHuEpo). They studied eight healthy male subjects during a 4-wk "loading" and 2-wk "boosting" phase of Epo use followed by a 2-wk maintenance phase. In the parent study they showed that the effects of Epo on exercise performance were confined to its impact on red cell mass and not to other physiological effects of the hormone. These results were consistent with ideas about the relationship between maximal oxygen uptake and red cell mass or total body hemoglobin that emerged in the 1950s. The findings are timely and have implications for public policy relating to the control of doping practices. In this short report a number of challenges related to urine testing for Epo are highlighted.

  20. Recombineering Pseudomonas syringae

    Science.gov (United States)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  1. Recombineering linear BACs.

    Science.gov (United States)

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells. PMID:25239740

  2. Recombinant hormones in osteoporosis

    DEFF Research Database (Denmark)

    Rejnmark, Lars; Rejnmark, Lars

    2013-01-01

    For the last 10 years, bone anabolic therapy with the recombinant human parathyroid hormone (rhPTH) analogue, teriparatide (rhPTH[1 - 34]), or full-length rhPTH(1 - 84) has been an option in the treatment of osteoporosis. Both drugs are given as a daily subcutaneous injection. In the USA, only teriparatide is marketed.

  3. ?-thrombin-induced inositol phosphate formation in G0-arrested and cycling hamster lung fibroblasts: evidence for a protein kinase C-mediated desensitization response

    International Nuclear Information System (INIS)

    In resting Chinese hamster fibroblasts (CCL39) ?-thrombin rapidly induces the breakdown of phosphoinositides. Accumulation of inositol phosphates (IP), measured in the presence of Li+, is detectable within 5s (seconds) of thrombin stimulation. Formation of inositol tris- and bisphosphates slightly precedes that of inositol monophosphate, indicating that thrombin activates primarily the phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate. Initial rates of IP production increase with thrombin concentration, with no apparent saturability over the range 10-4-10 U/ml. Thrombin-induced phosphoinositide hydrolysis rapidly desensitizes (t/sub 1/2/ > 5 min), but a residual activity, corresponding to about 10% of the initial stimulation is sustained for at least 9 h, in contrast with the undetectable activity of G0-arrested cells. This apparent desensitization may be due to a feedback regulation by protein kinase C, since pretreatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly inhibits (by up to 70%) subsequent thrombin-induced inositol phosphate formation. This up regulation was found maximal in A51, a very well growth-arrested CCL39 derivative,and reduced or virtually abolished in two tumoral and growth factor-relaxed derivatives of CCL39. Although preliminary, this observation suggests that a persistent activation of phosphatidyl inositol breakdown might operate in vayl inositol breakdown might operate in variants selected for autonomous growth

  4. Secretory products from thrombin-stimulated human platelets exert an inhibitory effect on NK-cytotoxic activity.

    DEFF Research Database (Denmark)

    Skov Madsen, P; Hokland, P

    1987-01-01

    We have investigated the interaction between human platelets and the NK-system, with special emphasis on the action of secretory products from platelets in an NK assay with 51Cr-labelled K562 as target cells. Supernatants from thrombin-stimulated platelets added to the NK assay consistently decreased the NK-cytotoxicity by 40% +/- 4.3%, indicating the existence of secreted products from platelets as a source of NK-inhibiting substances. In contrast, no direct cytotoxic effect of these secretory products on the target cells (K562) was seen. Thus, normal human platelets, when stimulated with thrombin, are capable of secreting different, yet undefined factors, which significantly inhibit NK activity in vitro. The results also suggest that the role of products from contaminating in vitro activated platelets should be borne in mind when performing conventional NK assays. Udgivelsesdato: 1986-Oct

  5. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

    OpenAIRE

    Semeraro, Fabrizio; Ammollo, Concetta T.; Morrissey, James H.; Dale, George L; Friese, Paul; Esmon, Naomi L.; Esmon, Charles T

    2011-01-01

    The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regard...

  6. Successful direct thrombin injection of a femoral artery pseudoaneurysm in an anticoagulated patient with a mechanical mitral valve

    OpenAIRE

    Gelwix, Christopher; Harrison, Melissa; Berman, Lorraine; Prinz, Andreas W; Ali, Asghar; Jovin, Ion S.

    2010-01-01

    Femoral pseudoaneurysm is a complication of cardiac catheterization and may be related to the use of anticoagulants, antiplatelet agents, larger diameter sheaths and prolonged duration of sheath insertion. The treatment ranges from direct compression with or without direct thrombin injection to surgical repair. The present report describes a unique scenario of postcardiac catheterization femoral artery pseudoaneursym in a patient with a pre-existing mechanical mitral valve requiring anticoagu...

  7. Antithrombotic actions of the thrombin inhibitor, argatroban, in a canine model of coronary cyclic flow: comparison with heparin.

    OpenAIRE

    Duval, N; Lunven, C.; O'Brien, D. P.; Grosset, A.; O'Connor, S E; Berry, C N

    1996-01-01

    1. The antithrombotic action of argatroban, a synthetic thrombin inhibitor, was studied in a canine model of coronary cyclic flow having some of the characteristics of acute unstable angina. Heparin was studied as a reference anticoagulant. 2. Localized endothelial damage was induced in the circumflex coronary artery of anaesthetized open-chest foxhounds and a critical stenosis was applied by use of a Lexan constrictor placed around the artery at the site of endothelial damage. An electro-mag...

  8. Aptamer-functionalized solid phase microextraction-liquid chromatography/tandem mass spectrometry for selective enrichment and determination of thrombin.

    Science.gov (United States)

    Du, Fuyou; Alam, Md Nazmul; Pawliszyn, Janusz

    2014-10-01

    In this publication, a novel solid phase microextraction (SPME) coating functionalized with a DNA aptamer for selective enrichment of a low abundance protein from diluted human plasma is described. This approach is based on the covalent immobilization of an aptamer ligand on electrospun microfibers made with the hydrophilic polymer poly(acrylonitrile-co-maleic acid) (PANCMA) on stainless steel rods. A plasma protein, human ?-thrombin, was employed as a model protein for selective extraction by the developed Apt-SPME probe, and the detection was carried out with liquid chromatography/tandem mass spectrometry (LC-MS/MS). The SPME probe exhibited highly selective capture, good binding capacity, high stability and good repeatability for the extraction of thrombin. The protein selective probe was employed for direct extraction of thrombin from 20-fold diluted human plasma samples without any other purification. The Apt-SPME method coupled with LC-MS/MS provided a good linear dynamic range of 0.5-50 nM in diluted human plasma with a good correlation coefficient (R(2)=0.9923), and the detection limit of the proposed method was found to be 0.30 nM. Finally, the Apt-SPME coupled with LC-MS/MS method was successfully utilized for the determination of thrombin in clinical human plasma samples. One shortcoming of the method is its reduced efficiency in undiluted human plasma compared to the standard solution. Nevertheless, this new aptamer affinity-based SPME probe opens up the possibility of selective enrichment of a given targeted protein from complex sample either in vivo or ex vivo. PMID:25201271

  9. Effects of the direct thrombin inhibitor dabigatran on ex vivo coagulation time in orthopaedic surgery patients: a population model analysis

    OpenAIRE

    Liesenfeld, K. H.; Scha?fer, G. H.; Troconiz, I. F.; Tillmann, C. C.; Eriksson, B. I.; Stangier, J.

    2006-01-01

    Aims To describe the pharmacokinetic–pharmacodynamic (PK–PD) characteristics of the direct thrombin inhibitor dabigatran in hip replacement patients by assessing coagu- lation parameters activated partial thromboplastin time (aPTT) and ecarin clotting time (ECT), interindividual variability and factors affecting PD responses. Methods BISTRO I patients received oral dabigatran etexilate postsurgery for 6–10 days. Dabig- atran plasma concentrations and aPTT/ECT were measured on t...

  10. Increased thrombin generation in women with polycystic ovary syndrome. A pilot study on the effect of metformin and oral contraceptives.

    DEFF Research Database (Denmark)

    Glintborg, Dorte; Sidelmann, Johannes Jakobsen

    2015-01-01

    Objective. Polycystic ovary syndrome (PCOS) is associated with risk factors for cardiovascular disease (CVD) which may be modified by the use of metformin and oral contraceptives (OC). Thrombin generation (TG) measures are risk markers of CVD and address the composite of multiple factors that influence blood coagulation. This prospective, randomized, intervention study evaluated the potential influence of PCOS on TG measures and the effect of OC and/or metformin on TG measures in women with PCOS. Material and methods. Ninety patients with PCOS and 35 controls were included. Patients were randomized to 12 months treatment with metformin, metformin + OC or OC alone. C-reactive protein (CRP), fibrinogen, total cholesterol, trunk fat mass, body mass index, estradiol, testosterone, sex hormone binding globulin (SHBG) as well as TG measures, i.e. the lag time for formation of thrombin, the endogenous thrombin potential (ETP), peak thrombin concentration (peak) and time to peak were determined at baseline and after 12 months of treatment. Results. CRP and total testosterone were significantly higher and SHBG significantly lower in PCOS women than in controls (P=0.012, P0.01). ETP (P=0.006), peak (P=0.003) and lag time (P=0.023) remained increased after adjustment for these potential confounders. Treatment with OC and metformin +OC further increased ETP (P<0.001) and peak (P<0.005) and reduced time to peak (P<0.04). The increase in ETP was significantly lower in the metformin+OC group than in the OC group (P<0.05). Metformin alone did not affect TG significantly. Conclusions. PCOS is associated with increase in TG measures independent of other risk factors of CVD. OC increases TG measures further and may thus add to the increased risk of CVD already present in women with PCOS.

  11. A signal amplification strategy using the cascade catalysis of gold nanoclusters and glucose dehydrogenase for ultrasensitive detection of thrombin.

    Science.gov (United States)

    Han, Jing; Zhuo, Ying; Chai, Yaqin; Gui, Guofeng; Zhao, Min; Zhu, Qiang; Yuan, Ruo

    2013-12-15

    This work reports a novel signal amplification strategy for ultrasensitive detection of thrombin by cascade catalysis of gold nanoclusters (AuNCs) and glucose dehydrogenase (GDH). Herein, the AuNCs prepared by using polyamidoamine dendrimer as template were constructed not only as nanocarriers for anchoring the large amounts of secondary thrombin aptamers but also as nanocatalysts to catalyze the oxidation of NADH efficiently. Moreover, a large amount of GDH was loaded through the immobilization technology of DNA hybridization and a large amount of toluidine blue (Tb) was intercalated into the DNA grooves via electrostatic interaction. Significantly, the electrochemical signal was greatly enhanced based on cascade catalysis: firstly, GDH catalyzed the oxidation of glucose to gluconolactone with the concomitant generation of NADH in the presence of NAD(+). Then, AuNCs as nanocatalysts could effectively catalyze NADH to produce NAD(+) with the help of Tb as redox probe. Under the optimal conditions, the proposed aptasensor exhibits a linear range of 1.0×10(-14)-5×10(-9) M with a low detection limit of 3.3×10(-15) M for thrombin detection and shows high sensitivity and good specificity. PMID:23850783

  12. A sensitive electrochemical aptasensor based on water soluble CdSe quantum dots (QDs) for thrombin determination

    International Nuclear Information System (INIS)

    A novel aptamer biosensor with easy operation and good sensitivity, specificity, stability and reproducibility was developed by immobilizing the aptamer on water soluble CdSe quantum dots (QDs) modified on the top of the glassy carbon electrode (GCE). Methylene blue (MB) was intercalated into the aptamer sequence and used as an electrochemical marker. CdSe QDs improved the electrochemical signal because of their larger surface area and ion centers of CdSe QDs may also had a major role on amplifying the signal. The higher ion concentration caused more combination of aptamer which caused larger signal. The thrombin was detected by differential pulse voltammetry (DPV) quantitatively. Under optimal conditions, the two linear ranges were obtained from 3 to 13 ?g mL-1 and from 14 to 31 ?g mL-1, respectively. The detection limit was 0.08 ?g mL-1 at 3?. The constructed biosensor had better responses compared with that in the absence of the CdSe QDs immobilizing. The control experiment was also carried out by using BSA, casein and IgG in the absence of thrombin. The results showed that the aptasensor had good specificity, stability and reproducibility to the thrombin. Moreover, the aptasensor could be used for detection of real sample with consistent results in comparison with those obtained by fluorescence method which could provide a promising platform for fabrication of aptamer based biosensors.

  13. Clinical and pharmacological properties of new oral anticoagulants for the prevention of cerebral thromboembolism: Factor Xa and thrombin inhibitors

    Directory of Open Access Journals (Sweden)

    Wolfgang H. Oertel

    2012-02-01

    Full Text Available Vitamin K antagonists, such as warfarin and phen-procoumon, are the first-line oral anticoagulants for primary and secondary prevention of cerebral embo-lism in patients with atrial fibrillation. Although vitamin K antagonists can significantly decrease the risk of stroke, their use is limited by several important drawbacks, such as a narrow therapeutic window, the risk of intracranial and gastrointestinal bleeding, interactions with a number of drugs and nutrients, and the need for regular laboratory tests for therapy adjustment. Currently, new oral anticoagulants, such as direct thrombin inhibitors (e.g., dabigatran and direct factor Xa inhibitors (e.g., apixaban, rivaroxaban, are being developed and tested in clinical trials. Dabigatran and rivaroxaban were recently approved for prevention of cerebral embolism in patients with atrial fibrillation. The ad-vantages of dabigatran in comparison to warfarin are a lower rate of major bleedings with dabigatran 110mg bid, a better efficacy with dabigatran 150mg bid, no clinically relevant interactions with other drugs and no need for routine coagulation monitoring. The disadvantages are the absence of antidote and the absence of routine laboratory tests for precise mea-surements of anticoagulant effect of direct thrombin/ factor Xa inhibitors. This review will focus on throm-bin and factor Xa inhibitors, which are new and promising oral anticoagulants for the prevention of cerebral embolism. We will discuss their pharmacol-ogical and clinical properties and provide the most recent updates on their clinical trials.

  14. Anti-thrombin III, Protein C, and Protein S deficiency in acute coronary syndrome

    Directory of Open Access Journals (Sweden)

    Dasnan Ismail

    2002-05-01

    Full Text Available The final most common pathway for the majority of coronary artery disease is occlusion of a coronary vessel. Under normal conditions, antithrombin III (AT III, protein C, and protein S as an active protein C cofactor, are natural anticoagulants (hemostatic control that balances procoagulant activity (thrombin antithrombin complex balance to prevent thrombosis. If the condition becomes unbalanced, natural anticoagulants and the procoagulants can lead to thrombosis. Thirty subjects with acute coronary syndrome (ACS were studied for the incidence of antithrombin III (AT III, protein C, and protein S deficiencies, and the result were compare to the control group. Among patients with ACS, the frequency of distribution of AT-III with activity < 75% were 23,3% (7 of 30, and only 6,7% ( 2 of 30 in control subject. No one of the 30 control subject have protein C activity deficient, in ACS with activity < 70% were 13,3% (4 of 30. Fifteen out of the 30 (50% control subjects had protein S activity deficiency, while protein S deficiency activity < 70% was found 73.3.% (22 out of 30. On linear regression, the deterministic coefficient of AT-III activity deficiency to the development ACS was 13,25 %, and the deterministic coefficient of protein C activity deficient to the development of ACS was 9,06 %. The cut-off point for AT-III without protein S deficiency expected to contribute to the development of vessel disease was 45%. On discriminant analysis, protein C activity deficiency posed a risk for ACS of 4,5 greater than non deficient subjects, and AT-III activity deficiency posed a risk for ACS of 3,5 times greater than non deficient subjects. On binary logistic regression, protein S activity acted only as a reinforcing factor of AT-III activity deficiency in the development of ACS. Protein C and AT III deficiency can trigger ACS, with determinant coefficients of 9,06% and 13,25% respectively. Low levels of protein C posed a greater risk of ACS than low levels of AT III. Protein S deficiency was a reinforcing factor on AT-III deficient to development of ACS. The cut-off point of AT-III without protein S deficiency expected to give single vessel disease was 45%, and 9,5% for the development of triple vessel disease. (Med J Indones 2002; 11: 87-92Keywords: acute coronary syndrome, Anti-thrombin III, Protein C, Protein S

  15. A Differential Recombination Chamber

    International Nuclear Information System (INIS)

    Tbe recombination chambers previously described by the authors, i.e. ionization chambers providing conditions for columnar recombination of ions, are principally used to determine the quality factor (QF) of mixed penetrating radiation. A measure of the QF is the efficiency of ion collection in the chamber at a given voltage of the electric field. The present paper describes a modification to the recombination chamber making it suitable for direct measurement of the dose-equivalent (DE). For this purpose, the measuring electrode or system of electrodes divides the operating volume of the chamber into two parts with a fixed ratio between the mass of gas contained between the measuring electrode and the input electrodes on either side. The voltage of the electric field or the gas pressure differ on either side of the measuring electrode, and are so selected that the efficiency of ion collection in one of the parts, as a result of the columnar recombination occurring in the gas, changes linearly with QF. The other part operates in conditions close to saturation. The directions of the electric field in relation to the measuring electrode are opposite in the two parts of the chamber. Thus, the measuring current is the difference in the charges collected per unit of time from the operating volumes of the chamber. In view of the differences in ion collection efficiency, this differential current is proportional to QF. The proportionality of the current to absorbed dose-rate iity of the current to absorbed dose-rate is also obvious. As a result, the current measured is proportional to the product of dose-rate and QF, i.e. to the DE rate. The differential recombination chamber can be used for continuous recording of the degree of radiation hazard from mixed penetrating radiation of any composition and spectrum which changes quantitatively and qualitatively with time. A further advantage of the differential chamber over the normal recombination chamber is the reduced need for stability of the electrometer. Thus it can be used with a conventional small electrometer as a portable instrument for dosimetric control. The authors have constructed one of the first models of such a differential recombination chamber. It can be used both for dosimetric control measurements and for laboratory investigations. The chamber contains 25 flat electrodes made of a tissue-equivalent material. The distance between the electrodes is fixed by spacers. Appropriate connection of the external leads of the electrodes permits the instrument to be used both as a differential chamber and as a double differential-saturated chamber, mainly for determining the QF of radiation. From the data obtained in investigations of ion recombination characteristics in gases, we can choose optimum values for the distances between the electrodes, the pressure and the composition of the gas mixture, depending on the use to which the chamber is to be put. (author)

  16. AECL passive autocatalytic recombiners

    International Nuclear Information System (INIS)

    Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

  17. Exploding foil recombination laser

    International Nuclear Information System (INIS)

    In addition to experiments using the collisional-excitation approach to a soft x-ray laser, the authors tested a recombination-pumped laser scheme. Intense (I/sub L/ approx. 2 x 1014 W/cm2) 0.53-?m light of 100- to 200-ps duration from the Novette laser was used to fully ionize a magnesium exploding-foil target. After the peak of the laser pulse, the plasma cooled rapidly due to expansion, electron conduction, and radiation. In the regime of high electron density (approx. 1020 cm-3) and low electron temperature (approx. 100 eV), three-body recombination preferentially populates the upper levels of the hydrogen-like magnesium ion. The population of the lower levels is depleted by fast radiative decay. This process can result in a population inversion on the n = 4 to 3, 130-A transition

  18. Relativistic dielectronic recombination theory

    International Nuclear Information System (INIS)

    Dielectronic recombination (DR) is an inverse Auger process in which a free electron is captured by a recombining ion to form a doubly excited autoionizing state. The subsequent decay of the autoionizing state to a stabilized bound state by emitting photons completes the recombination process. DR is an important recombination process for high temperature plasmas. It can affect the ionization balance and level kinetics of the hot plasmas. In addition, the dielectronic satellite lines observed in the emission spectra are frequently used as plasmas diagnostic tools. In the past decade, intense theoretical and experimental studies on the DR process have been carried out. Most of the earlier theoretical calculations on the DR rate coefficients were done either by using a term average approximation or in LS coupling without including the effects of relativity and configuration interaction. The early experimental investigations were concentrated on few times ionized low-Z ions. Recently, the development of electron beam ion trap (EBIT), electron beam ion source (EBIS) and heavy ion storage ring has become possible to produce very highly-charged heavy ions (e.g. U82+ and Xe53+)and to study the interaction between electrons and these ions. For highly-charged heavy ions, one excepts that the nonrelativistic method would be inadequate and a relativistic treatment is necessary. To meet this challenge we have developed a relativistic package based on the multiconfiguration Dirac-Fock method and have carried out systematic relativistic calculations of DR cross sections and rate coefficients and resonant transfer and excitation cross sections in ion-atom collisions. In this paper, we will briefly discuss the relativistic calculations of atomic structure and transition rates and will focus for attention on the effects of relativity and intermediate coupling on the DR cross sections and rate coefficients

  19. Demystified… recombinant antibodies

    OpenAIRE

    Smith, K. A.; Nelson, P N; Warren, P.; Astley, S J; Murray, P G; Greenman, J.

    2004-01-01

    Recombinant antibodies are important tools for biomedical research and are increasingly being used as clinical diagnostic/therapeutic reagents. In this article, a background to humanised antibodies is given, together with details of the generation of antibody fragments—for example, single chain Fv fragments. Phage antibody fragments are fast becoming popular and can be generated by simple established methods of affinity enrichment from libraries derived from immune cells. Phage display method...

  20. Exposure- response for biomarkers of anticoagulant effects by the oral direct thrombin inhibitor AZD0837 in patients with atrial fibrillation

    DEFF Research Database (Denmark)

    Lip, Gregory Y H; Rasmussen, Lars H

    2015-01-01

    BACKGROUND: AZD0837 is a novel oral anticoagulant investigated in clinical studies for stroke prevention in patients with atrial fibrillation (AF). It is bioconverted to its active form, AR-H067637, a potent, specific and reversible thrombin inhibitor. OBJECTIVES: A population pharmacokinetic (PK) analysis was performed and the effect of AZD0837 therapy on fibrin D-dimer levels was correlated to the PK exposure of AR-H067637, as well as the effect on thrombin generation measured ex vivo, to guide selection of the effective dose regimen for a confirmatory efficacy study in AF patients. PATIENTS AND METHODS: Blood samples were obtained from 601 AF patients randomized to receive 1 of 4 doses of AZD0837 (blinded treatment) or dose-adjusted vitamin K antagonists (VKA, open treatment) for 3-9?months. A pharmacodynamic model was developed to describe time course of the AR-H067637 exposure dependent effects and the effect of VKA on fibrin D-dimer. The concentration-effect relationship for thrombin generation measured ex vivo in venous plasma was also investigated. RESULTS: AZD0837 was rapidly bioconverted to AR-H067637, showing stable exposure with an estimated interindividual variability of 33% with no or only minor influence of patient demographics or comedications. For all dose groups of AZD0837, D-dimer levels decreased with more rapid onset of effect compared to VKA. The decrease in D-dimer levels correlated to the steady state plasma concentrations (Css) of AR-H067637, with a maximum decrease of baseline D-dimer levels estimated to approximately 60% for both AZD0837 and VKA therapy. Anticoagulant effect measured ex vivo as decreased thrombin generation correlated closely with the plasma concentration of AR-H067637. CONCLUSIONS: Following oral therapy with AZD0837, inhibitory effects on thrombin generation and fibrin D-dimer levels were correlated to the plasma concentration of its active form and provides comparable effects as well-controlled VKA therapy at an exposure at least corresponding to the 300?mg qd dose AZD0837.

  1. Dielectronic recombination theory

    Science.gov (United States)

    Lagattuta, K. J.

    A theory now in wide use for the calculation of dielectronic recombination cross sections (sigma(sup DR)) and rate coefficients (alpha(sup DR)) was one introduced originally by Feshbach for nuclear physics applications and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of sigma(sup DR) have been described by Fano and by Seaton. We will not consider those theories here. Calculations of alpha(sup DR) have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently by a variety of direct laboratory measurements of sigma(sup DR). While the measurements of sigma(sup DR) for delta n not = 0 excitations have tended to agree very well with calculations; the case of delta n = 0 has been problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.

  2. Dielectronic recombination theory

    International Nuclear Information System (INIS)

    A theory now in wide use for the calculation of dielectronic recombination cross sections (?DR) and rate coefficients (?DR) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of ?DR have been described by Fano and by Seaton. We will not consider those theories here. Calculations of ?DR have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of ?DR. While the measurements of ?DR for ?n ? 0 excitations have tended to agree very well with calculations, the case of ?n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain

  3. Effect of thrombin on purine metabolism in the guinea pig heart

    International Nuclear Information System (INIS)

    In vitro coronary endothelial cells (EC) release adenosine (ADO) in response to thrombin (THR). The authors tested the hypothesis that THR causes the release of ADO from in situ EC of isolated guinea pig hearts. The authors preferentially labelled the EC by infusing 2,8-3H-ADO (ADO; 5 x 10-8 M) into the heart for 30 minutes. Then THR (1 U/ml) or THR plus allopurinol (2.4 x 10-/sup 4M) was infused into the heart. Venous effluent samples analyzed for ADO, ADO and 3H-H2O. THR increased the release of ADO from 32 +/- 11 pm/min/g to 175 +/- 47 after 4 minutes (p 2O increased from 10.1 +/- 103 dpm/min/g to 23.5 +/- 5.7 x 103 at 4 minutes. In the presence of the xanthine oxidase/dehydrogenase inhibitor allopurinol, H2O release in response to THR fell to 3.6 +/- 1.2 x 10 dpm/min/gm. The authors conclude that the labelled EC are not the source of ADO released in response to THR. Instead the ADO released by THR comes from an unlabelled compartment, most likely the myocytes. This suggests that ADO release from myocytes may be a mechanism to regulate thrombogenesis. The THR-induced increase in H2O release and it's inhibition by allopurinol indicates that THR enhances purine catabolism in EC

  4. Thrombin antithrombin complex and IL-18 serum levels in stroke patients

    Directory of Open Access Journals (Sweden)

    Rosalba Tufano

    2010-06-01

    Full Text Available The complex picture of inflammation and coagulation alterations comes to life in acute stroke phases. Increasing evidence points to a strong interaction and extensive crosstalk between the inflammation and coagulation systems: the interest towards this relationship has increased since recent experimental research showed that the early administration of antithrombin III (ATIII decreases the volume of ischemia in mice and might be neuroprotective, playing an antiinflammatory role. We aimed to establish the extent of the relationship among markers of inflammation (S100B and IL-18 and procoagulant and fibrinolytic markers (ATIII, thrombin-antithrombin III complex (TAT, Fibrin Degradation Products (FDP, D-dimer in 13 comatose patients affected by focal cerebral ischemia. Plasma levels of TAT, D-dimer and FDP, IL18 and S100B were increased. IL-18 and S100B high serum levels in ischemic patients suggest an early activation of the inflammatory cascade in acute ischemic injury. The basic principles of the interaction between inflammatory and coagulation systems are revised, from the perspective that simultaneous modulation of both coagulation and inflammation, rather than specific therapies aimed at one of these systems could be more successful in stroke therapy.

  5. Selective deacylation of 1-acyl-2-arachidonoyl PC and PE in thrombin-stimulated human platelets

    International Nuclear Information System (INIS)

    Previously the authors have shown that uptake and stimulated release of 3H-arachidonate (AA) in human platelets involves mainly 1-acyl-2-arachidonoyl phospholipids. To determine deacylation of molecular species of 1-acyl-2-arachidonoyl phosphatidylcholine (PC) and phosphatidylethanolamine (PE), phospholipid was extracted by the method of Bligh and Dyer from cells (109/ml) stimulated or not by thrombin (THR, 5 U/ml .370C, 5 min). Total PC and PE were isolated by thin-layer chromatography (TLC) and converted to 1-radyl-2-acyl glycerobenzoates. The 1,2 diacyl glycerobenzoates were separated from other subclasses by TLC. Individual molecular species of 1,2 diacyl glycerobenzoates were resolved by reverse phase HPLC. Mass (O.D.230) and 3H-AA radioactivity (i.e. specific activity) were determined on-line, and changes in individual molecular species could be deduced. Significant deacylation of all 1-acyl-2-arachidonoyl PC and PE molecular species occurred with no apparent deacylation in any non-AA-containing molecular species of 1,2 diacyl PC/PE. At 5 minutes the net deacylation of 1-acyl-2-arachidonoyl PC and PE was approximately 15 and 5 nanomoles, respectively/109 cells. These results indicate that selective deacylation of arachidonoyl-containing molecular species compared to non-arachidonoyl-containing molecular species of PC/PE occurs in THR-stimulated cells. This suggests certain AA-containing phospholipids are compartmentalized with and susceptible to, the action of phospholipase A2

  6. Two related thrombin-like enzymes present in Bothrops atrox venom

    Directory of Open Access Journals (Sweden)

    J.H. Petretski

    2000-11-01

    Full Text Available This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

  7. Two related thrombin-like enzymes present in Bothrops atrox venom

    Scientific Electronic Library Online (English)

    J.H., Petretski; M., Kanashiro; C.P., Silva; E.W., Alves; T.L., Kipnis.

    1293-13-01

    Full Text Available This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal ami [...] no acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

  8. The hemostatic profile of recombinant activated factor VII. Can low concentrations stop bleeding in off-label indications?

    Directory of Open Access Journals (Sweden)

    de Lourdes Herrera Maria

    2010-05-01

    Full Text Available Abstract Background High concentrations of recombinant activated factor VII (rFVIIa can stop bleeding in hemophilic patients. However the rFVIIa dose needed for stopping haemhorrage in off-label indications is unknown. Since thrombin is the main hemostatic agent, this study investigated the effect of rFVIIa and tissue factor (TF on thrombin generation (TG in vitro. Methods Lag time (LT, time to peak (TTP, peak TG (PTG, and area under the curve after 35 min (AUCo-35 min with the calibrated automated thrombography was used to evaluate TG. TG was assayed in platelet-rich plasma (PRP samples from 29 healthy volunteers under basal conditions and after platelet stimulation with 5.0 ?g/ml, 2.6 ?g/ml, 0.5 ?g/ml, 0.25 ?g/ml, and 0.125 ?g/ml rFVIIa alone and in normal platelet-poor plasma (PPP samples from 22 healthy volunteers, rFVIIa in combination with various concentrations of TF (5.0, 2.5, 1.25 and 0.5 pM. Results In PRP activated by rFVIIa, there was a statistically significant increase in TG compared to basal values. A significant TF dose-dependent shortening of LT and increased PTG and AUCo?35 min were obtained in PPP. The addition of rFVIIa increased the effect of TF in shorting the LT and increasing the AUCo?35 min with no effect on PTG but were independent of rFVIIa concentration. Conclusion Low concentrations of rFVIIa were sufficient to form enough thrombin in normal PRP or in PPP when combined with TF, and suggest low concentrations for normalizing hemostasis in off-label indications.

  9. Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106).

    Science.gov (United States)

    Kaplanski, G; Marin, V; Fabrigoule, M; Boulay, V; Benoliel, A M; Bongrand, P; Kaplanski, S; Farnarier, C

    1998-08-15

    Thrombin, a central molecule in coagulation, is also involved in inflammation. Notably, thrombin induces endothelial neutrophil adhesion, P- and E-selectin expression, and chemokine production. We show here that thrombin induces expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106) on human umbilical vein endothelial cells (HUVECs) associated with increased adhesion of monocytes. Thrombin increased mRNA steady-state levels and expression of ICAM-1 over 24 hours. Thrombin-induced VCAM-1 expression exhibited unusual kinetics, reaching maximum levels after 6 to 12 hours, but decreasing to near baseline after 24 hours. Thrombin activity on HUVECs was mediated through interaction with its specific receptor, because ICAM-1 and VCAM-1 expression were similarly induced by the 14-amino acid thrombin receptor-activating peptide. Thrombin-induced ICAM-1 and VCAM-1 expression was significantly inhibited by hirudin, but not by interleukin-1 receptor antagonist or anti-tumor necrosis factor alpha monoclonal antibody (MoAb). Thrombin-activated HUVECs significantly increased greater numbers of adhering THP-1 macrophagic cells, peripheral blood mononuclear cells, or purified monocytes than unstimulated HUVECs. This adhesion was inhibited by anti-CD18 and anti-CD49d MoAb, demonstrating that thrombin-induced ICAM-1 and VCAM-1 were functional. These results show that, in addition to selectins, thrombin directly induces a cytokine-independent expression of adhesion molecules of the Ig superfamily on HUVECs that may support firm leukocyte attachment during inflammation. PMID:9694714

  10. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  11. Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2?-C-piperazino-UNA monomer

    DEFF Research Database (Denmark)

    Jensen, Troels B.; Henriksen, Jonas Rosager

    2011-01-01

    Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2?-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2?-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28–0.44kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (??G37°=?1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.

  12. Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer

    DEFF Research Database (Denmark)

    Jensen, Troels B; Henriksen, Jonas R

    2011-01-01

    Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (??G(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.

  13. Detection of thrombin using electrogenerated chemiluminescence based on Ru(bpy)32+-doped silica nanoparticle aptasensor via target protein-induced strand displacement

    International Nuclear Information System (INIS)

    A sensitive and selective aptasensor using tri(2,2'-bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)32+-doped SNPs) as DNA tags for detection of thrombin is developed based on the target protein-induced strand displacement of the DNA probe. For the proposed aptasensor, the aptamer was assembled on the surface of the Au electrode through Au-S binding. The hybridization event between the DNA probe labeled by the Ru(bpy)32+-doped SNPs and the aptamer was evaluated by electrogenerated chemiluminescence (ECL) measurements. Then, the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again. The difference of ECL intensity (?IECL) of the two events could be used to quantify the thrombin. Other proteins, such as bovine serum albumin and bovine hemoglobin, had almost negligible ?IECL. Under the optimal conditions, the ?IECL was linearly related to the concentration of the thrombin in the range of 10 fM to 10 pM and the detection limit was down to 1.0 fM since SNPs containing a large number of Ru(bpy)32+ molecules were labeled on the DNA probe

  14. Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome.

    Science.gov (United States)

    Della Corte, Anna; Maugeri, Norma; Pampuch, Agnieszka; Cerletti, Chiara; de Gaetano, Giovanni; Rotilio, Domenico

    2008-02-01

    Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets. PMID:18231937

  15. Primordial magnetogenesis before recombination

    CERN Document Server

    Fabre, Ophélia

    2015-01-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order $10^{-49}$ G. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications.

  16. Effect of thrombin on purine metabolism in the guinea pig heart

    Energy Technology Data Exchange (ETDEWEB)

    Whelton, B.K.; Thompson, C.I.; Sparks, H.V.

    1986-03-01

    In vitro coronary endothelial cells (EC) release adenosine (ADO) in response to thrombin (THR). The authors tested the hypothesis that THR causes the release of ADO from in situ EC of isolated guinea pig hearts. The authors preferentially labelled the EC by infusing 2,8-/sup 3/H-ADO (ADO; 5 x 10/sup -8/ M) into the heart for 30 minutes. Then THR (1 U/ml) or THR plus allopurinol (2.4 x 10-/sup 4M) was infused into the heart. Venous effluent samples analyzed for ADO, ADO and /sup 3/H-H/sub 2/O. THR increased the release of ADO from 32 +/- 11 pm/min/g to 175 +/- 47 after 4 minutes (p < 0.02, n = 5). Despite the increase in total ADO release, ADO did not increase. Release of H/sub 2/O increased from 10.1 +/- 10/sup 3/ dpm/min/g to 23.5 +/- 5.7 x 10/sup 3/ at 4 minutes. In the presence of the xanthine oxidase/dehydrogenase inhibitor allopurinol, H/sub 2/O release in response to THR fell to 3.6 +/- 1.2 x 10 dpm/min/gm. The authors conclude that the labelled EC are not the source of ADO released in response to THR. Instead the ADO released by THR comes from an unlabelled compartment, most likely the myocytes. This suggests that ADO release from myocytes may be a mechanism to regulate thrombogenesis. The THR-induced increase in H/sub 2/O release and it's inhibition by allopurinol indicates that THR enhances purine catabolism in EC.

  17. Effects of normoxic and hypoxic exercise regimens on monocyte-mediated thrombin generation in sedentary men.

    Science.gov (United States)

    Wang, Jong-Shyan; Chang, Ya-Lun; Chen, Yi-Ching; Tsai, Hsing-Hua; Fu, Tieh-Cheng

    2015-08-01

    Exercise and hypoxia paradoxically modulate vascular thrombotic risks. The shedding of procoagulant-rich microparticles from monocytes may accelerate the pathogenesis of atherothrombosis. The present study explores the manner in which normoxic and hypoxic exercise regimens affect procoagulant monocyte-derived microparticle (MDMP) formation and monocyte-promoted thrombin generation (TG). Forty sedentary healthy males were randomized to perform either normoxic (NET; 21% O2, n=20) or hypoxic (HET; 15% O2, n=20) exercise training (60% VO2max) for 30 min/day, 5 days/week for 5 weeks. At rest and immediately after HET (100 W under 12% O2 for 30 min), the MDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. The results demonstrated that acute 12% O2 exercise (i) increased the release of coagulant factor V (FV)/FVIII-rich, phosphatidylserine (PS)-exposed and tissue factor (TF)-expressed microparticles from monocytes, (ii) enhanced the peak height and rate of TG in monocyte-rich plasma (MRP) and (iii) elevated concentrations of norepinephrine/epinephrine, myeloperoxidase (MPO) and interleukin-6 (IL-6) in plasma. Following the 5-week intervention, HET exhibited higher enhancements of peak work-rate and cardiopulmonary fitness than NET did. Moreover, both NET and HET decreased the FV/FVIII-rich, PS-exposed and TF-expressed MDMP counts and the peak height and rate of TG in MRP following the HET. However, HET elicited more suppression for the HE (hypoxic exercise)-enhanced procoagulant MDMP formation and dynamic TG in MPR and catecholamine/peroxide/pro-inflammatory cytokine levels in plasma than NET. Hence, we conclude that HET is superior to NET for enhancing aerobic capacity. Furthermore, HET effectively suppresses procoagulant MDMP formation and monocyte-mediated TG under severe hypoxic stress, compared with NET. PMID:25826125

  18. Thrombin-activatable fibrinolysis inhibitor activity in healthy and diseased dogs

    DEFF Research Database (Denmark)

    Jessen, Lisbeth Rem; Wiinberg, Bo

    2010-01-01

    Background: In people, increased thrombin-activatable fibrinolysis inhibitor (TAFI) antigen has been associated with increased risk of thrombosis, and decreased TAFI may contribute to bleeding diathesis. TAFI activity in dogs has been described in experimental models, but not in dogs with spontaneous disease. Objective: The aim of this study was to compare TAFI activity in healthy dogs with TAFI activity in dogs with spontaneous disease. Methods: Plasma samples from 20 clinically healthy Beagles and from 35 dogs with various diseases were analyzed using a commercial chromogenic assay that measured TAFI activity relative to activity in standardized pooled human plasma. Results: Median TAFI activity for the 20 Beagles was 46.1% (range 32.2-70.8%) compared with 62.6% (29.1-250%) for the 35 diseased dogs, and 14/35 (40%) had TAFI activities >the upper limit for controls. The highest individual activities (>225%) were in 3 dogs with malignant neoplasms and 1 dog with thrombocytopenia. For data grouped by diagnosis, median TAFI activity was 61.7% for benign neoplasia (n=5), 64.9% for malignant neoplasia (n=8), 75.5% for Angiostrongylus vasorum infection (n=4), 68.8% for bacterial sepsis (n=7), and 58.7% for miscellaneous diseases (n=11). Compared with TAFI activity in control dogs, median TAFI activity was significantly increased only in the group of dogs with bacterial sepsis. Conclusion: Bacterial sepsis was associated with significantly increased TAFI activity, and individual dogs with increased TAFI activities were found in all disease groups. The role of TAFI in the pathogenesis of hemostatic disorders in dogs and its value as a prognostic indicator deserve further investigation.

  19. In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia.

    Science.gov (United States)

    Augustsson, Cecilia; Persson, Egon

    2014-11-13

    Successful competition of activated factor VII (FVIIa) with zymogen factor VII (FVII) for tissue factor (TF) and loading of the platelet surface with FVIIa are plausible driving forces behind the pharmacological effect of recombinant FVIIa (rFVIIa) in hemophilia patients. Thrombin generation measurements in platelet-rich hemophilia A plasma revealed competition for TF, which potentially could reduce the effective (r)FVIIa:TF complex concentration and thereby attenuate factor Xa production. However, (auto)activation of FVII apparently counteracted the negative effect of zymogen binding; a small impact was observed at endogenous concentrations of FVII and FVIIa but was virtually absent at pharmacological amounts of rFVIIa. Moreover, corrections of the propagation phase in hemophilia A required rFVIIa concentrations above the range where a physiological level of FVII was capable to downregulate thrombin generation. These data strongly suggest that rFVIIa acts independently of TF in hemophilia therapy and that FVII displacement by rFVIIa is a negligible mechanistic component. PMID:25232061

  20. Unraveling recombination rate evolution using ancestral recombination maps

    DEFF Research Database (Denmark)

    Munch, Kasper; Schierup, Mikkel H

    2014-01-01

    Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it.

  1. Plasma centrifugation does not influence thrombin-antithrombin and plasmin-antiplasmin levels but determines platelet microparticles count

    Science.gov (United States)

    Gruszczy?ski, Krzysztof; Kapusta, Przemys?aw; Kowalik, Artur; Wybra?ska, Iwona

    2015-01-01

    Introduction Centrifugation is an essential step for plasma preparation to remove residual elements in plasma, especially platelets and platelet-derived microparticles (PMPs). Our working hypothesis was that centrifugation as a preanalytical step may influence some coagulation parameters. Materials and methods Healthy young men were recruited (N = 17). For centrifugation, two protocols were applied: (A) the first centrifugation at 2500 x g for 15 min and (B) at 2500 x g for 20 min at room temperature with a light brake. In protocol (A), the second centrifugation was carried out at 2500 x g for 15 min, whereas in protocol (B), the second centrifugation involved a 10 min spin at 13,000 x g. Thrombin-antithrombin (TAT) and plasmin-antiplasmin (PAP) complexes concentrations were determined by enzyme-linked immunosorbent assays. PMPs were stained with CD41 antibody and annexin V, and analyzed by flow cytometry method. Procoagulant activity was assayed by the Calibrated Automated Thrombogram method as a slope of thrombin formation (CAT velocity). Results Median TAT and PAP concentrations did not differ between the centrifugation protocols. The high speed centrifugation reduced the median (IQR) PMP count in plasma from 1291 (841-1975) to 573 (391-1010) PMP/µL (P = 0.001), and CAT velocity from 2.01 (1.31-2.88) to 0.97 (0.82-1.73) nM/min (P = 0.049). Spearman’s rank correlation analysis showed correlation between TAT and PMPs in the protocol A plasma which was (rho = 0.52, P < 0.050) and between PMPs and CAT for protocol A (rho = 0.74, P < 0.050) and protocol B (rho = 0.78, P < 0.050). Conclusion Centrifugation protocols do not influence the markers of plasminogen (PAP) and thrombin (TAT) generation but they do affect the PMP count and procoagulant activity.

  2. Anticoagulant profile of iopamidol and meglumine amidotrizoate and their lack of thrombin generation: an in vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Gritli, N.; Nsiri, B.; Mazigh, C.; Ghazouani, E.; M`henni, H.; Machghoul, S.; Gueddiche, M. [Hopital Militaire Principal d`Instruction de Tunis (Tunisia)

    1998-01-01

    The aim of this in vitro study was to sketch the subtle anticoagulant profile of iopamidol 300 mg l/ml (low osmolality non ionic contrast medium) and meglumine amidotrizoate 370 mg l/ml (high osmolality ionic contrast medium) in situations where variable amounts of clotting factors are observed and to check whether thrombin-generation significantly occurred in non anti-coagulated blood-contrast materials mixtures. In the first experiment, mixtures of deficient plasmas with a routine plasma pool provided different ranges with a variable amounts of clotting factor II, V, VIII, X, XI and XII. For each clotting factor level studied within these ranges, an activated partial thromboplastin time was determined with either contrast material loaded thromboplastin (5% v/v) used as a control. In the second experiment fibrino-peptide A (FpA) or modified anti-thrombin III (ATM) assays were performed in either (9:1) non anti-coagulated blood contrast materials mixtures or blood-glucose mixtures (control). Differing aPTT prolongation profiles were observed when clotting factors V, VIII, XI and XII were lowered in the plasma. However, neither iopamidol not amidotrizoate induced an aPTT prolongation with decreasing clotting factor II. In the second experiment no significant thrombin generation was observed as both blood - contrast materials mixtures showed significantly lower FpA and ATM levels (p < 0.001) than glucose control after 5 minutes and 10 minutes incubation at room temperature. These findings provide evidence that the use of iopamidol in angiographic procedures does not increase risk of clotting or hemorrhage. (author)

  3. Novel magnetic fibrin hydrogel scaffolds containing thrombin and growth factors conjugated iron oxide nanoparticles for tissue engineering

    Directory of Open Access Journals (Sweden)

    Ziv-Polat O

    2012-03-01

    Full Text Available Ofra Ziv-Polat1, Hadas Skaat1, Abraham Shahar2, Shlomo Margel11Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan 52900, Israel; 2NVR Research Ltd, Nes-Ziona 74031, IsraelAbstract: Novel tissue-engineered magnetic fibrin hydrogel scaffolds were prepared by the interaction of thrombin-conjugated iron oxide magnetic nanoparticles with fibrinogen. In addition, stabilization of basal fibroblast growth factor (bFGF was achieved by the covalent and physical conjugation of the growth factor to the magnetic nanoparticles. Adult nasal olfactory mucosa (NOM cells were seeded in the transparent fibrin scaffolds in the absence or presence of the free or conjugated bFGF-iron oxide nanoparticles. The conjugated bFGF enhanced significantly the growth and differentiation of the NOM cells in the fibrin scaffolds, compared to the same or even five times higher concentration of the free bFGF. In the presence of the bFGF-conjugated magnetic nanoparticles, the cultured NOM cells proliferated and formed a three-dimensional interconnected network composed mainly of tapered bipolar cells. The magnetic properties of these matrices are due to the integration of the thrombin- and bFGF-conjugated magnetic nanoparticles within the scaffolds. The magnetic properties of these scaffolds may be used in future work for various applications, such as magnetic resonance visualization of the scaffolds after implantation and reloading the scaffolds via magnetic forces with bioactive agents, eg, growth factors bound to the iron oxide magnetic nanoparticles.Keywords: thrombin, fibroblast growth factor, fibrin scaffold, iron oxide nanoparticles, tissue engineering, magnetism, bioactive nanoparticle

  4. Anticoagulant profile of iopamidol and meglumine amidotrizoate and their lack of thrombin generation: an in vitro study

    International Nuclear Information System (INIS)

    The aim of this in vitro study was to sketch the subtle anticoagulant profile of iopamidol 300 mg l/ml (low osmolality non ionic contrast medium) and meglumine amidotrizoate 370 mg l/ml (high osmolality ionic contrast medium) in situations where variable amounts of clotting factors are observed and to check whether thrombin-generation significantly occurred in non anti-coagulated blood-contrast materials mixtures. In the first experiment, mixtures of deficient plasmas with a routine plasma pool provided different ranges with a variable amounts of clotting factor II, V, VIII, X, XI and XII. For each clotting factor level studied within these ranges, an activated partial thromboplastin time was determined with either contrast material loaded thromboplastin (5% v/v) used as a control. In the second experiment fibrino-peptide A (FpA) or modified anti-thrombin III (ATM) assays were performed in either (9:1) non anti-coagulated blood contrast materials mixtures or blood-glucose mixtures (control). Differing aPTT prolongation profiles were observed when clotting factors V, VIII, XI and XII were lowered in the plasma. However, neither iopamidol not amidotrizoate induced an aPTT prolongation with decreasing clotting factor II. In the second experiment no significant thrombin generation was observed as both blood - contrast materials mixtures showed significantly lower FpA and ATM levels (p < 0.001) than glucose control after 5 minutes and 10 minutes incubation at room temperatand 10 minutes incubation at room temperature. These findings provide evidence that the use of iopamidol in angiographic procedures does not increase risk of clotting or hemorrhage. (author)

  5. Percutaneous Thrombin Injection of a Femoral Artery Pseudoaneurysm with Simultaneous Venous Balloon Occlusion of a Communicating Arteriovenous Fistula

    International Nuclear Information System (INIS)

    An 82-year-old woman developed acute occlusion of her right coronary artery. She underwent percutaneous coronary stent placement and aortic balloon pump installation. In the postprocedural period, she developed a common femoral artery pseudoaneurysm (PSA) that communicated with the common femoral vein via an arteriovenous fistula (AVF). After unsuccessful ultrasound-guided compression, ultrasound-guided thrombin injection of the PSA was performed, with simultaneous balloon occlusion of the common femoral vein at the level of the AVF. There was complete thrombosis of the PSA and AVF.

  6. Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

    OpenAIRE

    A.V Pérez; A Rucavado; Sanz, L; Calvete, J. J.; Gutiérrez, J. M.

    2008-01-01

    A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence ...

  7. Differential effects of formoterol on thrombin- and PDGF-induced proliferation of human pulmonary arterial vascular smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Goncharova Elena A

    2012-11-01

    Full Text Available Abstract Background Increased pulmonary arterial vascular smooth muscle (PAVSM cell proliferation is a key pathophysiological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PH. The long-acting ?2-adrenergic receptor (?2AR agonist formoterol, a racemate comprised of (R,R- and (S,S-enantiomers, is commonly used as a vasodilator in chronic obstructive pulmonary disease (COPD. PH, a common complication of COPD, increases patients’ morbidity and reduces survival. Recent studies demonstrate that formoterol has anti-proliferative effects on airway smooth muscle cells and bronchial fibroblasts. The effects of formoterol and its enantiomers on PAVSM cell proliferation are not determined. The goals of this study were to examine effects of racemic formoterol and its enantiomers on PAVSM cell proliferation as it relates to COPD-associated PH. Methods Basal, thrombin-, PDGF- and chronic hypoxia-induced proliferation of primary human PAVSM cells was examined by DNA synthesis analysis using BrdU incorporation assay. ERK1/2, mTORC1 and mTORC2 activation were determined by phosphorylation levels of ERK1/2, ribosomal protein S6 and S473-Akt using immunoblot analysis. Results We found that (R,R and racemic formoterol inhibited basal, thrombin- and chronic hypoxia-induced proliferation of human PAVSM cells while (S,S formoterol had lesser inhibitory effect. The ?2AR blocker propranolol abrogated the growth inhibitory effect of formoterol. (R,R, but not (S,S formoterol attenuated basal, thrombin- and chronic hypoxia-induced ERK1/2 phosphorylation, but had little effect on Akt and S6 phosphorylation levels. Formoterol and its enantiomers did not significantly affect PDGF-induced DNA synthesis and PDGF-dependent ERK1/2, S473-Akt and S6 phosphorylation in human PAVSM cells. Conclusions Formoterol inhibits basal, thrombin-, and chronic hypoxia-, but not PDGF-induced human PAVSM cell proliferation and ERK1/2, but has little effect on mTORC1 and mTORC2 signaling. Anti-proliferative effects of formoterol depend predominantly on its (R,R enantiomer and require the binding with ?2AR. These data suggest that (R,R formoterol may be considered as potential adjuvant therapy to inhibit PAVSM cell proliferation in COPD-associated PH.

  8. Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets.

    OpenAIRE

    E.A. Clark; Brugge, J S

    1993-01-01

    Thrombin stimulation of platelets induces a transient increase in the specific activity of pp60c-src followed by a redistribution of pp60c-src to the Triton X-100-insoluble, cytoskeleton-rich fraction. Concomitant with the observed increase in pp60c-src activity was a rapid dephosphorylation of tyrosine 527 in 10 to 15% of pp60c-src molecules. In addition, we found that pp60c-src from the Triton-insoluble fraction was phosphorylated on tyrosine 416, the autophosphorylation site which is phosp...

  9. The effects of hormone replacement therapy on thrombin generation, fibrinolysis inhibition, and resistance to activated protein C: prospective cohort study and review of literature.

    Science.gov (United States)

    Douketis, J D; Gordon, M; Johnston, M; Julian, J A; Adachi, J R; Ginsberg, J S

    2000-07-01

    Recent studies have found that hormone replacement therapy (HRT) is associated with a two- to fourfold increased risk of venous thromboembolism, but the thrombogenic mechanism of HRT remains unclear. To investigate whether HRT use induces a procoagulant state, we undertook a prospective cohort study in postmenopausal women to investigate the effects of 3 months of treatment with oral HRT (conjugated equine estrogen 0.625 mg daily and medroxyprogesterone 2.5 mg daily) on markers of thrombin generation (prothrombin fragment 1+2, thrombin-antithrombin complexes), fibrinolytic potential (plasminogen activator inhibitor-1 (PAI-1) activity), and activated protein C (APC) resistance. In addition, we reviewed the literature for studies investigating the effects of HRT on markers of thrombin generation and fibrinolytic potential. In 12 patients who received HRT for a mean of 3.8 months, there was no significant effect of HRT on levels of F1+2, thrombin-antithrombin complexes, or the APC ratio. HRT use had the greatest effect on PAI-1 activity (mean difference = -3.75 UI/mL; 95% confidence interval: - 8.9, 1.1) compared to other coagulation parameters, but this did not attain statistical significance (p = 0.12). In the literature review, the effects of HRT on markers of thrombin generation were inconsistent across studies. There was a consistent pattern of increased fibrinolytic potential with HRT use associated with one marker (PAI-1), but not with another marker (tissue plasminogen activator antigen). We conclude that there is a lack of consistent evidence that the increased risk of venous thromboembolism associated with HRT use is due to a procoagulant state related to increased thrombin generation, decreased fibrinolytic potential, or acquired APC resistance. PMID:11012376

  10. Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)

    International Nuclear Information System (INIS)

    The effects of thrombin and GTP?S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [3H]inositol-labeled membranes or with lipid vesicles containing either [3H]phosphatidylinositol or [3H]phosphatidylinositol 4,5-bisphosphate. GTP?S (1 ?M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP3), inositol bisphosphate (IP2), or inositol phosphate (IP) from [3H]inositol-labeled membranes. IP2 and IP3, but not IP, from [3H]inositol-labeled membranes were, however, stimulated 3-fold by GTP?S (1 ?M) plus thrombin (1 unit/mL). A higher concentration of GTP?S (100 ?M) alone also stimulated IP2 and IP3, but not IP, release. In the presence of 1 mM calcium, release of IP2 and IP3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) by platelet membrane associated PLC was also markedly enhanced by GTP?S (100 ?M) or GTP?S (1 ?M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysiium. Thrombin-induced hydrolysis of PIP2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP?S (100 ?M) or calcium (1 mM) dependent PIP2 breakdown, while TPA inhibited GTP?S-dependent but not calcium-dependent phospholipase C activity

  11. Hadron Correlations and Parton Recombination

    OpenAIRE

    Fries, Rainer J.

    2007-01-01

    Parton recombination has been found to be an extremely useful model to understand hadron production at the Relativistic Heavy Ion Collider. It is particularly important to explore its connections with hard processes. This article reviews some of the aspects of the quark recombination model and places particular emphasis on hadron correlations.

  12. Urokinase-mediated fibrinolysis in the synovial fluid of rheumatoid arthritis patients may be affected by the inactivation of single chain urokinase type plasminogen activator by thrombin

    OpenAIRE

    Braat, E; Jie, A; Ronday, H; Beekman, B.; Rijken, D

    2000-01-01

    BACKGROUND—Excessive fibrin deposition within the inflamed joints of rheumatoid arthritis (RA) patients suggests that local fibrinolysis is inefficient, which seems to be in contrast with the observed increased levels of urokinase type plasminogen activator (u-PA). Thrombin-mediated inactivation of single chain u-PA (scu-PA) into an inactive form called thrombin-cleaved two chain u-PA (tcu-PA/T) may provide a possible explanation for this contradiction.?AIM—To assess the occurrence of t...

  13. CT-Guided Thrombin Injection to Control Rapid Expansion of Ascending Aortic False Aneurysm 15 Months After Bentall–Bono Operation

    International Nuclear Information System (INIS)

    We report a case of 57-year-old man treated emergently with CT-guided local thrombin injection as the first, life-saving step for control rapid expansion of the aortic pseudoaneurysm. Fifteen months earlier, he was operated on for ascending aortic true aneurysm and coronary artery disease. Upon admission, he had an anterior thoracic wall pulsatile tumor. Due to critical status, definite surgery was postponed and thrombin was injected close to the origin of pseudoaneurysm. It controlled successfully, bleeding from the ascending aorta and enabled the patient to survive the acute phase.

  14. Topical anesthesia.

    OpenAIRE

    Keyes, P. D.; Tallon, J. M.; Rizos, J.

    1998-01-01

    OBJECTIVE: To consider topical anesthetic options available to primary care physicians, indications for their use, and efficacy and safety of these agents as supported by the literature. QUALITY OF EVIDENCE: Five randomized controlled trials were retrieved that compared various topical anesthetics as well as topical anesthetics versus infiltrative anesthesia. MAIN FINDINGS: A combination of lidocaine, epinephrine, and tetracaine (LET) is currently the topical anesthetic of choice for repair o...

  15. Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

    Scientific Electronic Library Online (English)

    A.V, Pérez; A, Rucavado; L, Sanz; J.J, Calvete; J.M, Gutiérrez.

    2008-01-01

    Full Text Available A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for a [...] pproximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

  16. Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

    Directory of Open Access Journals (Sweden)

    A.V Pérez

    2008-01-01

    Full Text Available A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg and fibrinogen (minimum coagulant dose = 4.2 µg in vitro, and promotes defibrin(ogenation in vivo (minimum defibrin(ogenating dose = 1.0 µg. In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

  17. A label-free electrochemical aptasensor based on the catalysis of manganese porphyrins for detection of thrombin.

    Science.gov (United States)

    Zheng, Yingning; Yuan, Yali; Chai, Yaqin; Yuan, Ruo

    2015-04-15

    A novel manganese porphyrin (MnPP)-catalyzed aerobic oxidation of l-cysteine to disulfides (RSSR) was firstly found and applied into electrochemical aptasensor with a label-free technique for signal amplification. The possible catalytic mechanism of the catalytic reaction where MnPP catalyzed l-cysteine with thiol (RSH) structure to RSSR was discussed in detail. For fabrication of the aptasensor, thionine (Thi), which served as an electron mediator, was mixed with MnPP and immobilized on the nafion coated carbon electrode through ion exchange adsorption. Gold nanoparticle (nano-Au) was assembled on the Thi for immobilizing thrombin binding aptamer (TBA). In the presence of thrombin (TB), TBA will capture TB and form TBA-TB composite thus perturbed electron transfer, leading to decrease of the current for quantitatively detecting TB. Under optimal condition, the electrochemical aptasensor exhibited a linear range of 0.1-25nM with a detection limit of 0.02nM. This work opens a novel way for signal amplification study about porphyrins that served as mimetic enzyme to thiol in electrochemical aptasensor. PMID:25530538

  18. Application of Europium Multiwalled Carbon Nanotubes as Novel Luminophores in an Electrochemiluminescent Aptasensor for Thrombin Using Multiple Amplification Strategies.

    Science.gov (United States)

    Wu, Dan; Xin, Xia; Pang, Xuehui; Pietraszkiewicz, Marek; Hozyst, Robert; Sun, Xian Ge; Wei, Qin

    2015-06-17

    A novel electrochemiluminescent (ECL) aptasensor was proposed for the determination of thrombin (TB) using exonuclease-catalyzed target recycling and hybridization chain reaction (HCR) to amplify the signal. The capture probe was immobilized on an Au-GS-modified electrode through a Au-S bond. Subsequently, the hybrid between the capture probe and the complementary thrombin binding aptamer (TBA) was aimed at obtaining double-stranded DNA (dsDNA). The interaction between TB and its aptamer led to the dissociation of dsDNA because TB has a higher affinity to TBA than the complementary strands. In the presence of exonuclease, aptamer was selectively digested and TB could be released for target recycling. Extended dsDNA was formed through HCR of the capture probe and two hairpin DNA strands (NH2-DNA1 and NH2-DNA1). Then, numerous europium multiwalled carbon nanotubes (Eu-MWCNTs) could be introduced through amidation reaction between NH2-terminated DNA strands and carboxyl groups on the Eu-MWCNTs, resulting in an increased ECL signal. The multiple amplification strategies, including the amplification of analyte recycling and HCR, and high ECL efficiency of Eu-MWCNTs lead to a wide linear range (1.0 × 10(-12)-5.0 × 10(-9) mol/L) and a low detection limit (0.23 pmol/L). The method was applied to serum sample analysis with satisfactory results. PMID:26005759

  19. Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

    International Nuclear Information System (INIS)

    The thrombin-like enzyme saxthrombin has been purified from G. saxatilis snake venom. Crystallization conditions were found and a data set was obtained to 1.43 Å. The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatography procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, ? = 96.72°, and the Matthews coefficient (VM) was calculated to be 2.13 Å3 Da?1 with one molecule in the asymmetric unit

  20. Delayed recombination and standard rulers

    International Nuclear Information System (INIS)

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  1. Recombineering: genetic engineering in bacteria using homologous recombination.

    Science.gov (United States)

    Thomason, Lynn C; Sawitzke, James A; Li, Xintian; Costantino, Nina; Court, Donald L

    2014-01-01

    The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques. Curr. Protoc. Mol. Biol. 106:1.16.1-1.16.39. © 2014 by John Wiley & Sons, Inc. PMID:24733238

  2. The Anopheles gambiae cE5, a tight- and fast-binding thrombin inhibitor with post-transcriptionally regulated salivary-restricted expression.

    Czech Academy of Sciences Publication Activity Database

    Ronca, R.; Kotsyfakis, Michalis; Lombardo, F.; Rizzo, C.; Currà, C.; Ponzi, M.; Fiorentino, G.; Ribeiro, J.M.C.; Arcà, B.

    2012-01-01

    Ro?. 42, ?. 9 (2012), s. 610-620. ISSN 0965-1748 R&D Projects: GA ?R GAP502/12/2409 Institutional research plan: CEZ:AV0Z60220518 Keywords : Anopheles * Saliva ry protein * Anti-thrombin * Anophelin * Hematophagy * Post-transcriptional regulation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2012

  3. Controlled release from recombinant polymers.

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  4. Hydrogen recombiner development at AECL

    International Nuclear Information System (INIS)

    Catalytic recombiners have been developed at AECL for the purpose of hydrogen removal in post-accident nuclear containment buildings. The recombiners are based on a particular catalyst designed by AECL which has extraordinary resistance to fouling from water and water vapour and a large thermodynamic range of operation. The catalysts were developed, originally, for the purpose of heavy water manufacturing by way of a catalytic exchange process. Application of these catalyst materials in recombiners for containment applications began in the late 1980's. The first application was a passive recombiner, qualified for use in control of radiolytic hydrogen in the headspace of a pool-type experimental reactor of AECL design in 1988. The passive, or natural convection recombiner concept has continued development to commercial stage for application in power reactor containments. This paper reviews the AECL recombiner development, describes the current model and shows results from tests of full-scale recombiners in the Large Scale Vented Combustion Test Facility at AECL-WL. The AECL recombiner is designed for compactness and ease of engineering into containment. The design is a simple, open-ended rectangular enclosure with catalyst elements arranged inside to promote optimum convective flow driven by heat of recombination at the catalyst surface. Self start, as evidenced by catalyst heating and initiation of flow, is achieved in less than 1% hydrogen, with available oxygen, at room temperature and 100% relative humidity. This low temperature start-up in condensing atmospheres is viewed as the most challenging condition for wet-proofing effectiveness. Cold start-up is a vital performance requirement in containments, such as CANDU, where engineered air-cooling systems are operating and where long-term hydrogen control is required, after containment atmospheres have cooled. Once started, the removal capacity scales linearly with the inlet cross-section area and the partial pressure of hydrogen. The recombiner also reacts carbon monoxide, in the presence of hydrogen, at approximately the same rate as the hydrogen. The catalyst materials and wet-proofing are unaffected by radiation or high temperatures. Large scale tests confirm self-start behavior and demonstrate strong mixing, irrespective of recombiner placement. (author)

  5. Progenitors of Recombining Supernova Remnants

    OpenAIRE

    Moriya, Takashi J.

    2012-01-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with the ionization temperature higher than the electron temperature, is recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the t...

  6. Inhomogeneous recombinations during cosmic reionization

    OpenAIRE

    Sobacchi, Emanuele; Mesinger, Andrei

    2014-01-01

    By depleting the ionizing photon budget available to expand cosmic HII regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a sub-grid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parameterizing p...

  7. Dielectronic recombination on heliumlike argon

    International Nuclear Information System (INIS)

    We have used the electron-energy dependence of yields of heliumlike and lithiumlike argon ions from the Kansas State University electron-beam ions source (EBIS) to measure the ratio of the cross section for ? n=1 dielectronic recombination of heliumlike argon to that for electron ionization of lithiumlike argon. By normalizing to the latter cross section we obtain absolute dielectronic recombination cross sections and find good agreement with theoretical calculations for the lower-energy resonances

  8. Eukaryotes arose after genetic recombination

    Directory of Open Access Journals (Sweden)

    Stupar Milanko R.

    2006-01-01

    Full Text Available Division of ancestral prokaryotic pragenome into two circular double-stranded DNA molecules by genetic recombination, is a base for future separate evolution of nuclear and mitochondrial gene compartment. This suggests monophyletic origin of both, mitochondrion and nucleus. Presumed organism which genome undergoes genetic recombination has to be searched among an aerobic, oxygen nonproducing, archaeon with no rigid cell wall, but a plasma membrane. Plastid evolves from an aerobic, oxygen producing protoeukaryote, after mitoplastid genome duplication and subsequent functional segregation.

  9. Tunnel recombinations in yttrium orthophosphate

    International Nuclear Information System (INIS)

    The tunnel luminescence induced by recombination of radiative centres is investigated in synthetic crystals of YPO4. Its spectrum, kinetics, effects of accumulation and connection with thermostimulated processes are studied in the temperature range from 80 to 220 K. It is shown that tunnel recombinations occur only in the genetically associated electron-hole centres. The paramagnetic centres (Zr3+, O-, SiO43-, Eu2+, ASO44-) are interpreted; their thermal stability and connection with the luminescence are investigated

  10. Ethanol production by recombinant hosts

    Science.gov (United States)

    Fowler, David E. (Gainesville, FL); Horton, Philip G. (Gainesville, FL); Ben-Bassat, Arie (Gainesville, FL)

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  11. Delayed recombination and cosmic parameters

    International Nuclear Information System (INIS)

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, ns, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z*=1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1? to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: ??i<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  12. Interchromatid and Interhomolog Recombination in Arabidopsis thaliana

    Science.gov (United States)

    Molinier, Jean; Ries, Gerhard; Bonhoeffer, Sebastian; Hohn, Barbara

    2004-01-01

    Intermolecular recombination events were monitored in Arabidopsis thaliana lines using specially designed recombination traps consisting of tandem disrupted ?-glucuronidase or luciferase reporter genes in direct repeat orientation. Recombination frequencies (RFs) varied between the different lines, indicating possible position effects influencing intermolecular recombination processes. The RFs between sister chromatids and between homologous chromosomes were measured in plants either hemizygous or homozygous for a transgene locus. The RFs in homozygous plants exceeded those of hemizygous plants by a factor of >2, implying that in somatic plant cells both sister chromatid recombination and recombination between homologous chromosomes exist for recombinational DNA repair. In addition, different DNA-damaging agents stimulated recombination in homozygous and hemizygous plants to different extents in a manner dependent on the type of DNA damage and on the genomic region. The genetic and molecular analysis of recombination events showed that most of the somatic recombination events result from gene conversion, although a pop-out event has also been characterized. PMID:14729918

  13. Percutaneous treatment of femoral pseudoaneurysms: comparison of fibrin sealant against thrombin / Tratamento percutaneo do pseudoaneurisma femoral: comparacao entre selante de fibrina e trombina

    Scientific Electronic Library Online (English)

    Daniel Mendes, Pinto; Paulo, Bastianetto.

    2013-12-01

    Full Text Available INTRODUÇÃO: O pseudoaneurisma femoral é complicação descrita em até 8% dos procedimentos percutâneos. Dentre os tratamentos, a injeção de trombina guiada por ultrassom tem alta taxa de sucesso e boa tolerância pelos pacientes. O uso da trombina associada ao fibrinogênio, chamado selante de fibrina [...] , forma um coágulo estável que pode ser usado para o tratamento do pseudoaneurisma, principalmente aqueles de anatomia complexa e maiores. OBJETIVO: Comparar os resultados do tratamento do pseudoaneurisma femoral em dois grupos: Grupo T, tratado com trombina isoladamente, e Grupo T+F, tratado com selante de fibrina (trombina+fibrinogênio). MÉTODO: Análise retrospectiva dos casos de pseudoaneurisma femoral tratados entre janeiro/2005 e dezembro/2012. RESULTADOS: Foram tratados 28 pacientes, 21 com trombina isolada e sete com selante de fibrina. Houve sucesso no tratamento de todos os pacientes do grupo T e somente em quatro casos do grupo T+F (57,1% no Grupo T+F, p Abstract in english INTRODUCTION: Femoral pseudoaneurysms are a complication that occurs in connection with up to 8% of percutaneous procedures. Of the available treatments, ultrasound guided thrombin injection has a high success rate and is well-tolerated by patients. The combination of thrombin and fibrinogen known [...] as fibrin sealant forms a stable clot and can be used to treat pseudoaneurysms, particularly those with complex anatomy and larger size. OBJECTIVE: To compare the results of treating femoral pseudoaneurysm in two ways: Group T was treated with thrombin alone and Group T+F was treated with fibrin sealant (thrombin+fibrinogen). METHODS: A retrospective analysis was conducted of femoral pseudoaneurysm cases treated between January 2005 and December 2012. RESULTS: Twenty-eight patients were treated, 21 with thrombin alone and seven with fibrin sealant. All patients in group T were treated successfully, but only four patients in group T+F were treated successfully (57.1% success rate in Group T+F, p

  14. Percutaneous treatment of femoral pseudoaneurysms: comparison of fibrin sealant against thrombin / Tratamento percutaneo do pseudoaneurisma femoral: comparacao entre selante de fibrina e trombina

    Scientific Electronic Library Online (English)

    Daniel Mendes, Pinto; Paulo, Bastianetto.

    2013-10-21

    Full Text Available INTRODUÇÃO: O pseudoaneurisma femoral é complicação descrita em até 8% dos procedimentos percutâneos. Dentre os tratamentos, a injeção de trombina guiada por ultrassom tem alta taxa de sucesso e boa tolerância pelos pacientes. O uso da trombina associada ao fibrinogênio, chamado selante de fibrina [...] , forma um coágulo estável que pode ser usado para o tratamento do pseudoaneurisma, principalmente aqueles de anatomia complexa e maiores. OBJETIVO: Comparar os resultados do tratamento do pseudoaneurisma femoral em dois grupos: Grupo T, tratado com trombina isoladamente, e Grupo T+F, tratado com selante de fibrina (trombina+fibrinogênio). MÉTODO: Análise retrospectiva dos casos de pseudoaneurisma femoral tratados entre janeiro/2005 e dezembro/2012. RESULTADOS: Foram tratados 28 pacientes, 21 com trombina isolada e sete com selante de fibrina. Houve sucesso no tratamento de todos os pacientes do grupo T e somente em quatro casos do grupo T+F (57,1% no Grupo T+F, p Abstract in english INTRODUCTION: Femoral pseudoaneurysms are a complication that occurs in connection with up to 8% of percutaneous procedures. Of the available treatments, ultrasound guided thrombin injection has a high success rate and is well-tolerated by patients. The combination of thrombin and fibrinogen known [...] as fibrin sealant forms a stable clot and can be used to treat pseudoaneurysms, particularly those with complex anatomy and larger size. OBJECTIVE: To compare the results of treating femoral pseudoaneurysm in two ways: Group T was treated with thrombin alone and Group T+F was treated with fibrin sealant (thrombin+fibrinogen). METHODS: A retrospective analysis was conducted of femoral pseudoaneurysm cases treated between January 2005 and December 2012. RESULTS: Twenty-eight patients were treated, 21 with thrombin alone and seven with fibrin sealant. All patients in group T were treated successfully, but only four patients in group T+F were treated successfully (57.1% success rate in Group T+F, p

  15. Epoetin: human recombinant erythropoietin.

    Science.gov (United States)

    Flaharty, K K; Grimm, A M; Vlasses, P H

    1989-11-01

    The chemistry, pharmacology, pharmacokinetics, clinical uses and efficacy, adverse effects, drug interactions, dosage and administration, and formulary considerations of epoetin are described. Erythropoietin, a glycoprotein hormone primarily synthesized in the kidney, is the chief regulator of red blood cell production. Erythropoietin concentrations increase in response to a hypoxic state, resulting in increased red blood cell formation, accelerated hemoglobin production, and premature movement of reticulocytes into the circulation. The human gene responsible for the production of erythropoietin recently was cloned, and the recombinant product--epoetin--has been made available through mass production. The apparent volume of distribution of i.v. epoetin approximates the assumed plasma volume both in healthy volunteers and in patients with chronic renal failure. Little is known about the metabolism and route of elimination of epoetin and erythropoietin. Epoetin recently was approved by the FDA for treatment of anemia associated with chronic renal failure. Clinical trials in patients receiving hemodialysis or peritoneal dialysis and in predialysis patients with renal dysfunction demonstrate epoetin's efficacy. Other potential indications include augmentation of blood production in patients enrolled in autologous blood donation programs and treatment of anemias associated with rheumatoid arthritis, sickle cell disease, acquired immunodeficiency syndrome, cancer, and premature birth. The most frequent adverse effect associated with epoetin therapy is the worsening or development of hypertension. Other adverse effects include thrombocytosis, hyperkalemia, rise in serum urea concentration, iron deficiency, and flu-like symptoms. No drug interactions with epoetin have been reported in humans. The recommended starting epoetin dosage in patients with chronic renal failure is 50-100 IU/kg three times weekly. Epoetin is available only as an injection for i.v. or s.c. administration. Epoetin provides a new therapeutic approach to the treatment of anemia associated with chronic renal failure in hemodialysis, peritoneal dialysis, and predialysis patients. Benefits of epoetin therapy include reduced need for blood transfusions, the amelioration of anemic symptoms, and an improved quality of life. PMID:2680241

  16. Radioiodination and biodistribution study of a thrombin-like enzyme/gyroxin from Lachesis muta muta venom

    Energy Technology Data Exchange (ETDEWEB)

    Pujatti, Priscilla Brunelli; Soares, Marcella Araugio; Silva, Paulo R.O.; Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)]. E-mail: priscillapujatti@yahoo.com.br; Magalhaes, Henrique P.B. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Faculdade de Farmacia

    2007-07-01

    Recently, our group isolated and sequenced a thrombin-like enzyme (TLE) of 40kDa from the snake Lachesis muta muta. This protein hydrolyses synthetic substrates with specificity similar to that of trypsin and may be involved in the haemorrhagic, proteolytic and blood-clotting activities of the Lachesis venom. When injected into the tail veins of mice at levels of 0.015-0.130{mu}g/g mouse, the TLE induce temporary episodes of opisthotonus and rapid rolling around the long axis of the animals and that is the reason why it is also called gyroxin. If this gyroxin activity is caused by direct interaction with the brain or by indirect effect remains to be investigated. We report in this work the radioiodination of TLE and the biodistribution of I-TLE in order to investigate its pharmacokinetics and verify if this enzyme are able to penetrate the blood brain barrier to evoke directly the gyroxin effects. (author)

  17. Investigations for designing catalytic recombiners

    International Nuclear Information System (INIS)

    In case of a severe accident in pressurised water reactors (PWR) a high amount of hydrogen up to about 20,000 m3 might be generated and released into the containments. The mixture consisting of hydrogen and oxygen may either burn or detonate, if ignited. In case of detonation the generated shock wave may endanger the components of the plant or the plant itself. Consequently, effective removal of hydrogen is required. The fact that hydrogen and oxygen react exo-thermally on catalytically acting surfaces already at low temperatures generating steam and heat is made use of in catalytic recombiners. They consist of substrates coated with catalyst (mainly platinum or palladium) which are arranged inside a casing. Being passively acting measures, recombiners do not need any additional energy supply. Experimental investigations on catalytic hydrogen recombination are conducted at FZJ (Forschungszentrum Juelich) using three test facilities. The results yield insight in the development potential of contemporary recombiner systems as well as of innovative systems. Detailed investigations on a recombiner section show strong temperature gradients over the surface of a catalytically coated sample. Dependent on the flow velocity, ignition temperature may be reached at the leading edge already at an inlet hydrogen concentration of about 5 vol.-%. The thermal strain of the substrate leads to considerable detachment of catalyst particles probably causing unintended ignition of the flammable mixture. Temperature peaks can be prevented effectively by leaving the first part of the plate uncoated. In order to avoid overheating of the catalyst elements of a recombiner even at high hydrogen concentrations a modular system of porous substrates is proposed. The metallic substrates are coated with platinum at low catalyst densities thus limiting the activity of the single specimen. A modular arrangement of these elements provides high recombination rates over a large hydrogen concentration range without igniting the mixture

  18. Recombinant protein scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. (topical review)

  19. Thrombin generation assay and transmission electron microscopy: a useful combination to study tissue factor-bearing microvesicles

    Directory of Open Access Journals (Sweden)

    Damien Gheldof

    2013-03-01

    Full Text Available Introduction. Patients with cancer have a 7- to 10-fold increased risk of developing venous thromboembolism. Circulating microvesicles could be a useful predictive biomarker for venous thromboembolism in cancer. Validated and standardised techniques that could be used to determine the complete microvesicle phenotype are required. These were two-fold: a to characterise tissue factor (TF-bearing microvesicles released by cultured breast cancer cells MDA-MB-231 by flow cytometry (FCM, transmission electron microscopy (TEM and thrombin generation assay (TGA; and b to validate the sensitivity and variability intra/inter-assay of TGA as a useful method to study the procoagulant activity (PCA of microvesicles. Methods. Cultured breast cancer cells MDA-MB-231 were incubated for 45 minutes at 37°C. Samples were then centrifuged or not at 4,500 g for 15 minutes, and cells and MVs or MV-containing supernatants were used for TEM, FCM and TGA. In activity assays, microvesicles (i.e. cell-depleted supernatants were incubated with anti-TF antibodies or with annexin V to assess the contribution of TF and phospholipids to the PCA. Alternatively, supernatants were filtered through 0.1, 0.22, 0.45 or 0.65 µm membranes and subjected to TGA. Results. The majority of the PCA was associated with microvesicles smaller than 0.1 µm, and the mean microvesicle size estimated by TEM after 10,000 g centrifugation was 121±54 nm with a majority of vesicles between 100 and 200 nm. Microvesicles derived from 5,000 MDA-MB-231cells/ml were sufficient to significantly increase the thrombin generation of normal pooled plasma. Conclusions. TEM, FCM and filtration coupled to TGA represent a useful combination to study the PCA of TF-bearing microvesicles, whatever their size. And it will be interesting to implement these techniques in patients.

  20. Three months of strictly controlled daily endurance exercise reduces thrombin generation and fibrinolytic risk markers in younger moderately overweight men

    DEFF Research Database (Denmark)

    Gram, Anne Sofie; Bladbjerg, Else-Marie

    2015-01-01

    PURPOSE: Physical activity is associated with a decreased risk of cardiovascular disease, but dose dependency of long-term physical exercise on biomarkers within coagulation and fibrinolysis is unknown. We aimed to investigate effects of two doses of daily endurance exercise on biomarkers of the haemostatic balance in overweight men. METHODS: Haemostatic variables were investigated in 53 healthy, younger (20-40 years), moderately overweight (BMI 25-30 kg/m(2)) men randomly assigned to 3 months of strictly controlled endurance exercise at two different doses corresponding to an energy expenditure of 600 kcal/day (HIGH), 300 kcal/day (MOD), or to maintain their habitual lifestyle (CON). Fasting blood samples were collected before and after the intervention and analysed for thrombin generation (endogenous thrombin potential, ETP) and concentrations of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand factor (vWF). RESULTS: We observed significant within-group decreases in ETP (MOD 7 %; HIGH 6 %) and in t-PA (MOD 22 %; HIGH 21 %) and PAI-1 (MOD 16 %; HIGH 32 %) in both training groups, and no changes in the CON group. At 3 months, between-group differences were observed for ETP (p = 0.016) and t-PA (p = 0.012) due to significantly lower values in MOD and HIGH compared with CON. Borderline significant between-group differences were observed for PAI-1 (p = 0.082). A significant increase was observed in vWF in HIGH, but with no between-group differences. CONCLUSIONS: Our results demonstrate an effect of 3 months of daily endurance exercise on biomarkers of the haemostatic balance in the direction of reduced cardiovascular risk, independent of exercise dose.

  1. Recombining WMAP: Constraints on ionizing and resonance radiation at recombination

    International Nuclear Information System (INIS)

    We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent cosmic microwave background temperature and polarization spectra coming from the Wilkinson Microwave Anisotropy Probe (WMAP). We find that non-standard recombination scenarios are still consistent with the current data. In light of this we study the impact that such models can have on the determination of several cosmological parameters. In particular, the constraints on curvature and baryon density appear to be weakly affected by a modified recombination scheme. However, it may affect the current WMAP constraints on inflationary parameters such as the spectral index ns and its running. Physically motivated models, such as those based on primordial black holes or super heavy dark matter decay, are able to provide a good fit to the current data. Future observations in both temperature and polarization will be needed to more stringently test these models

  2. AI Topics

    OpenAIRE

    Buchanan, Bruce G; Glick, Jonathan

    2002-01-01

    The debut of the AI in the News column elsewhere in this issue of AI Magazine created a good opportunity to introduce the professional community to the AI Topics web site, home of the AI in the news virtual page. Although AI Topics is designed for the lay public, it serves a much larger audience.

  3. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    Energy Technology Data Exchange (ETDEWEB)

    Moriya, Takashi J., E-mail: takashi.moriya@ipmu.jp [Kavli Institute for the Physics and Mathematics of the Universe, Todai Institutes for Advanced Study, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba 277-8583 (Japan)

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  4. Workshop on Radio Recombination Lines

    CERN Document Server

    1980-01-01

    Since their first detection 15 years ago, radio recombination lines from several elements have been observed in a wide variety of objects including HII regions, planetary nebulae, molecular clouds, the diffuse interstellar medium, and recently, other galaxies. The observations span almost the entire range from 0.1 to 100 GHz, and employ both single­ djsh and aperture synthesis techniques. The theory of radio recombination lines has also advanced strongly, to the point where it is perhaps one of the best-understood in astro­ physics. In a parallel development, it has become possible over the last decade to study these same highly-excited atoms in the laboratory; this work provides further confirmation of the theoretical framework. However there has been continuing controversy over the astrophysical interpre­ tation of radio recombination line observations, especially regarding the role of stimulated emission. A workshop was held in Ottawa on 24-25 August, 1979, bringing together many of the active scientist...

  5. Inhomogeneous recombinations during cosmic reionization

    CERN Document Server

    Sobacchi, Emanuele

    2014-01-01

    By depleting the ionizing photon budget available to expand cosmic HII regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a sub-grid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parameterizing photo-heating feedback on star-formation, we present large-scale, semi-numeric reionization simulations which self-consistently track the local (sub-grid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly-evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large HII regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reioniza...

  6. Containment protection with hydrogen recombiners

    International Nuclear Information System (INIS)

    Core meltdown and concrete - melt interactions cause free hydrogen to be produced. Devices protecting against concrete - melt interactions can largely help to avoid hydrogen releases outside the containment. In the hydrogen -steam - air system, the hydrogen concentration should not exceed the limits of deflagration; on no account must it reach the detonation level. Minimum steam fractions can be maintained by means of hydrogen recombiners. The devices should be designed so that the limits of hydrogen concentration can be observed. In the process of catalytic recombination, hydrogen is oxidized at an early stage without producing any flames and before reaching the level of ignitability. (orig.)

  7. Selenium incorporation using recombinant techniques

    International Nuclear Information System (INIS)

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included

  8. Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

    OpenAIRE

    Bayer, A. S.; Kupferwasser, L. I.; Brown, M. H.; Skurray, R. A.; Grkovic, S.; Jones, T.; Mukhopadhay, K.; Yeaman, M. R.

    2006-01-01

    Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized expo...

  9. Functional expression of subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex and reconstitution of recombinant protein with three-subunit core complex.

    Science.gov (United States)

    Chen, Y R; Yu, C A; Yu, L

    1996-01-26

    Subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex was over-expressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-RSIV) using the expression vector, pGEX/RSIV. Maximum yield of soluble active recombinant fusion protein was obtained from cells harvested 3 h after induction of growth at 37 degrees C in LB medium. Subunit IV was released from the fusion protein by proteolytic cleavage with thrombin. When subjected to SDS-polyacrylamide gel electrophoresis, isolated recombinant subunit IV of R. sphaeroides cytochrome b-c1 complex. Although the isolated recombinant subunit IV is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1000 kDa. The addition of detergent deaggregates the isolated protein, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. When the three-subunit core cytochrome b-c1 complex, purified from RS delta IV-adapted chromatophores containing a fraction of the wild type cytochrome b-c1 complex activity, was reacted with varying amounts of recombinant subunit IV, the activity increased as the subunit IV concentration increased. Maximum activity restoration was reached when 1 mol of subunit IV/mol of three-subunit core complex was used. The reconstituted cytochrome b-c1 complex is similar to the wild-type complex in molecular size, apparent Km for Q2H2, and inhibitor sensitivity, indicating that recombinant subunit IV is properly assembled into the active cytochrome b-c1 complex. A tryptophan residue in subunit IV was found to be involved in the interaction with the three-subunit core complex. PMID:8567659

  10. Genetics Home Reference: Recombinant 8 syndrome

    Science.gov (United States)

    ... this population have been found. What are the genetic changes related to recombinant 8 syndrome? Recombinant 8 ... Center . Where can I find general information about genetic conditions? The Handbook provides basic information about genetics ...

  11. Pulsed ion-ion recombination laser

    Science.gov (United States)

    Petrash, Gueorgii G.; Zemskov, K. I.

    2003-11-01

    A new mechanism of pulsed laser using ion-ion recombination is considered. Advantages of using ion-ion recombination in pulsed operation are considered and estimates of possible characteristics of the lasers are presented.

  12. Recombination in immunoglobulin gene loci

    OpenAIRE

    Komisarenko S. V.; Halytskiy V. A.

    2009-01-01

    Gene network of the lymphoid cell differentiation coordinates precisely the recombination process in immunoglobulin gene loci. In our opinion, cellular microRNAs can contribute to the allelic exclusion through microRNA-directed DNA methylation and participate in retargeting recombinases activity from the gene loci of heavy immunoglobulin chains to the gene loci of light chains

  13. Tacrolimus Topical

    Science.gov (United States)

    Tacrolimus ointment is used to treat the symptoms of eczema (atopic dermatitis; a skin disease that causes ... whose eczema has not responded to another medication. Tacrolimus is in a class of medications called topical ...

  14. Estradiol Topical

    Science.gov (United States)

    ... a medication that is applied topically to the vagina. Estradiol is in a class of medications called ... swelling, redness, burning, irritation, or itching of the vagina vaginal discharge Some side effects can be serious. ...

  15. High efficiency recombineering in lactic acid bacteria

    OpenAIRE

    van Pijkeren, Jan-Peter; BRITTON, ROBERT A.

    2012-01-01

    The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to Gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lact...

  16. Population inversion in recombining hydrogen plasma

    International Nuclear Information System (INIS)

    The collisional-radiative model is applied to a recombining hydrogen plasma in order to investigate the plasma condition in which the population inversion between the energy levels of hydrogen can be generated. The population inversion is expected in a plasma where the three body recombination has a large contribution to the recombining processes and the effective recombination rate is beyond a certain value for a given electron density and temperature. Calculated results are presented in figures and tables. (author)

  17. The Recombinational Anatomy of a Mouse Chromosome

    OpenAIRE

    Paigen, Kenneth; Szatkiewicz, Jin P.; Sawyer, Kathryn; Leahy, Nicole; Parvanov, Emil D.; Ng, Siemon H. S.; Graber, Joel H.; Broman, Karl W.; Petkov, Petko M.

    2008-01-01

    Among mammals, genetic recombination occurs at highly delimited sites known as recombination hotspots. They are typically 1–2 kb long and vary as much as a 1,000-fold or more in recombination activity. Although much is known about the molecular details of the recombination process itself, the factors determining the location and relative activity of hotspots are poorly understood. To further our understanding, we have collected and mapped the locations of 5,472 crossover events along mouse ...

  18. Structural dynamics of thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG) quadruplex DNA studied by large-scale explicit solvent simulations.

    Czech Academy of Sciences Publication Activity Database

    Reshetnikov, R.; Golovin, A.; Spiridonova, V.; Kopylov, A.; Šponer, Ji?í

    2010-01-01

    Ro?. 6, ?. 10 (2010), s. 3003-3014. ISSN 1549-9618 R&D Projects: GA AV ?R(CZ) IAA400040802; GA ?R(CZ) GA203/09/1476; GA MŠk(CZ) LC06030 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : molecular dynamics * quadruplex DNA * thrombin Subject RIV: BO - Biophysics Impact factor: 5.138, year: 2010

  19. Comparative Evaluation of Direct Thrombin and Factor Xa Inhibitors with Antiplatelet Agents under Flow and Static Conditions: An In Vitro Flow Chamber Model

    OpenAIRE

    Hosokawa, Kazuya; Ohnishi, Tomoko; Sameshima, Hisayo; Miura, Naoki; Koide, Takehiko; Maruyama, Ikuro; Tanaka, Kenichi A.

    2014-01-01

    Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 2...

  20. Calcium/Calmodulin-dependent Protein Kinase II Delta 6 (CaMKII?6) and RhoA Involvement in Thrombin-induced Endothelial Barrier Dysfunction*

    OpenAIRE

    Wang, Zhen; Ginnan, Roman; Abdullaev, Iskandar F.; Trebak, Mohamed; Vincent, Peter A.; Singer, Harold A.

    2010-01-01

    Multiple Ca2+ release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca2+ signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monol...

  1. Prediction of enzyme binding: human thrombin inhibition study by quantum chemical and artificial intelligence methods based on X-ray structures.

    Science.gov (United States)

    Mlinsek, G; Novic, M; Hodoscek, M; Solmajer, T

    2001-01-01

    Thrombin is a serine protease which plays important roles in the human body, the key one being the control of thrombus formation. The inhibition of thrombin has become a target for new antithrombotics. The aim of our work was to (i) construct a model which would enable us to predict Ki values for the binding of an inhibitor into the active site of thrombin based on a database of known X-ray structures of inhibitor-enzyme complexes and (ii) to identify the structural and electrostatic characteristics of inhibitor molecules crucially important to their effective binding. To retain as much of the 3D structural information of the bound inhibitor as possible, we implemented the quantum mechanical/molecular mechanical (QM/MM) procedure for calculating the molecular electrostatic potential (MEP) at the van der Waals surfaces of atoms in the protein's active site. The inhibitor was treated quantum mechanically, while the rest of the complex was treated by classical means. The obtained MEP values served as inputs into the counter-propagation artificial neural network (CP-ANN), and a genetic algorithm was subsequently used to search for the combination of atoms that predominantly influences the binding. The constructed CP-ANN model yielded Ki values predictions with a correlation coefficient of 0.96, with Ki values extended over 7 orders of magnitude. Our approach also shows the relative importance of the various amino acid residues present in the active site of the enzyme for inhibitor binding. The list of residues selected by our automatic procedure is in good correlation with the current consensus regarding the importance of certain crucial residues in thrombin's active site. PMID:11604028

  2. Dielectronic recombination in laser generated plasmas

    International Nuclear Information System (INIS)

    Dielectronic recombination coefficients have been computed for hydrogenic ions from HeII to FeXVI over a range of conditions typical of laser generated plasma. The results are displayed in a set a graphs together with the corresponding collisional radiative recombination coefficients. A comparison of these results indicates plasma conditions where dielectronic recombination is a significant process. (author)

  3. The effect of a single recombination event

    DEFF Research Database (Denmark)

    Schierup, Mikkel Heide; Jensen, Thomas Mailund

    We investigate the variance in how visible a single recombination event is in a SNP data set as a function of the type of recombination event and its age. Data is simulated under the coalescent with recombination and inference is by the popular composite likelihood methods. The major determinant of the effect of a recombination event is the genealogical type of the event and whether SNP variation is present that can reveal the genealogical consequences of the recombination event. Recombination events that only change some branch lengths in the genealogy have a very small, but detectable, effect. The more lineages left when the recombination event occurs, the larger effect it has, implying that it is mainly young recombination events that we detect when estimating the rate. If the population is growing, though, more lineages are present back in time and relatively more ancient recombination events may leave a stronger effect on data. We also investigate the amount of recombination events expected to be shared by two populations as a function of their separation time and explicitly model the European and African population in at attempt to survey how large an effect recombination events shared by these two populations are expected to contribute compared to the effect of private recombination events

  4. A cascade signal amplification strategy for surface enhanced Raman spectroscopy detection of thrombin based on DNAzyme assistant DNA recycling and rolling circle amplification.

    Science.gov (United States)

    Gao, Fenglei; Du, Lili; Tang, Daoquan; Lu, Yao; Zhang, Yanzhuo; Zhang, Lixian

    2015-04-15

    A sensitive protocol for surface enhanced Raman spectroscopy (SERS) detection of thrombin is designed with R6G-Ag NPs as a signal tag by combining DNAzyme assistant DNA recycling and rolling circle amplification (RCA). Molecular beacon (MB) as recognition probe immobilizes on the glass slides and performs the amplification procedure. After thrombin-induced structure-switching DNA hairpins of probe 1, the DNAzyme is liberated from the caged structure, which hybridizes with the MB for cleavage of the MB in the presence of cofactor Zn(2+) and initiates the DNA recycling process, leading to the cleavage of a large number of MB and the generation of numerous primers for triggering RCA reaction. The long amplified RCA product which contained hundreds of tandem-repeat sequences, which can bind with oligonucleotide functionalized Ag NPs reporters. The attached signal tags can be easily read out by SERS. Because of the cascade signal amplification, these newly designed protocols provides a sensitive SERS detection of thrombin down to the femolar level (2.3fM) with a linear range of 5 orders of magnitude (from 10(-14) to 10(-9)M) and have high selectivity toward its target protein. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease. PMID:25497982

  5. A molecular dynamics study on liquid 1-octanol. Part 3. Evaluating octanol/water partition coefficients of novel thrombin inhibitors via free-energy perturbations

    Science.gov (United States)

    Fernandes de Oliveira, César Augusto; Werneck Guimarães, Cristiano Ruch; de Mello, Heloisa; Echevarria, Aurea; de Alencastro, Ricardo Bicca

    Thrombin inhibition is an important strategy for the treatment of thrombotic and embolic disorders. Despite high in vitro affinity for the enzyme, synthetic thrombin inhibitors have limited oral bioavailability due to low intestinal permeability. In a previous work, we obtained the following order for thrombin binding by using molecular dynamics (MD) simulations and the finite difference thermodynamic integration (FDTI) method: p-(2-oxo-1-propyl)benzamidine (POPBz) > p-ethylbenzamidine (PEBz) > p-(1-propyl)benzamidine (PPBz) > p-methylbenzamidine (PMBz) > benzamidine (Bz). As the octanol/water partition coefficient (log Po/w) is a thermodynamic property that may be related to drug transfer across biological membranes, we now turn our attention to the calculation by the FDTI method of relative log Po/w for the benzamidine derivatives. To examine the reliability of the method, log Po/w values for Bz and PMBz were determined experimentally. Experimental log Po/w of -1.02 and -0.89 were obtained for Bz and PMBz, and theoretical log Po/w of -0.12, -0.01, and -1.00 were obtained for PEBz, PPBz, and POPBz. Thus, we improved the hydrophobicity of Bz by adding the methyl, ethyl, propyl, and 2-oxo-propyl groups, but further substitutions should be conducted to raise log Po/w above zero and into a region where reasonable oral absorption might be expected.

  6. Galpha12/13- and rho-dependent activation of phospholipase C-epsilon by lysophosphatidic acid and thrombin receptors.

    Science.gov (United States)

    Hains, Melinda D; Wing, Michele R; Maddileti, Savitri; Siderovski, David P; Harden, T Kendall

    2006-06-01

    Because phospholipase C epsilon (PLC-epsilon) is activated by Galpha(12/13) and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC-epsilon. Expression of the LPA(1) or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC-epsilon. Both LPA(1) and PAR1 receptor-mediated activation of PLC-epsilon was inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for Galpha(12/13) but not by expression of the RGS domain of GRK2, which inhibits Galpha(q) signaling. In contrast, activation of the G(q)-coupled M1 muscarinic or P2Y(2) purinergic receptor was neither enhanced by coexpression with PLC-epsilon nor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPA- or SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLC-epsilon but completely prevented the PLC-epsilon-dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC-epsilon-dependent stimulatory effects of the LPA(1), LPA(2), LPA(3), or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y(2) receptor. Moreover, PLC-epsilon-dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Ras- but not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via activation of Galpha(12/13) and Rho. PMID:16554409

  7. Detection of Quantitative Trait Loci Influencing Recombination Using Recombinant Inbred Lines

    OpenAIRE

    Dole, Jefferey; Weber, David F.

    2007-01-01

    The genetic basis of variation in recombination in higher plants is polygenic and poorly understood, despite its theoretical and practical importance. Here a method of detecting quantitative trait loci (QTL) influencing recombination in recombinant inbred lines (RILs) is proposed that relies upon the fact that genotype data within RILs carry the signature of past recombination. Behavior of the segregational genetic variance in numbers of chromosomal crossovers (recombination) over generations...

  8. Extended recombinant bacterial ghost system.

    Science.gov (United States)

    Lubitz, W; Witte, A; Eko, F O; Kamal, M; Jechlinger, W; Brand, E; Marchart, J; Haidinger, W; Huter, V; Felnerova, D; Stralis-Alves, N; Lechleitner, S; Melzer, H; Szostak, M P; Resch, S; Mader, H; Kuen, B; Mayr, B; Mayrhofer, P; Geretschläger, R; Haslberger, A; Hensel, A

    1999-08-20

    Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines. PMID:10486935

  9. To the theory of dissociative recombination

    International Nuclear Information System (INIS)

    An analytical expression for cross section of dissociative recombination of electron and molecular ion has been obtained. The Morse potential has been choosen as an internuclear potential of both ground electron state of the molecular ion and resonance molecular state. To obtain the recombination coefficient, averaging of dissociative recombination cross section in the Maxwell distribution has been performed in consequence of which the recombination coefficient temperature dependence, ? approximately Tsup(-1/2), has been determined. The temperature dependence of the dissociative recombination which well agrees with experiment has been calculated for the e+NO+ process as an example

  10. mRNA expression of genes involved in inflammation and haemostasis in equine fibroblast-like synoviocytes followingexposure to lipopolysaccharide, fibrinogen and thrombin

    DEFF Research Database (Denmark)

    Andreassen, Stine Mandrup; Berg, Lise Charlotte

    2015-01-01

    Background: Studies in humans have shown that haemostatic and inflammatory pathways both play important roles in the pathogenesis of joint disease. The aim of this study was to assess mRNA expression of haemostatic and inflammatory factors in cultured equine fibroblast-like synoviocytes exposed to lipopolysaccharide (LPS), fibrinogen and thrombin. Synovial membranes were collected from metacarpo-phalangeal joints of 6 skeletally mature horses euthanized for non-orthopaedic reasons. Passage 4 fibroblast-like synoviocytes were left non-treated or treated with either 0.1 ? g/ml LPS, 5 mg/ml fibrinogen or 5 U/ml thrombin and harvested at time points 0, 6, 24 and 48 h. mRNA expression of serum amyloid A (SAA), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), plasminogen activator inhibitor 1 (PAI-1), urokinase plasminogen activator (uPA), vascular endothelial growth factor (VEGF) and protease activator receptor 1 (PAR-1) was assessed using quantitative real time reverse transcriptase PCR. Results: LPS caused a significant increase in mRNA expression of SAA, IL-6, MCP-1 and uPA, and a decrease in TF, PAI-1 and PAR-1 when compared to non-treated cells. Treatment with thrombin resulted in increased mRNA expression of SAA, IL-6, MCP-1 and PAI-1, and a decreased PAR-1 expression compared to non-treated cells. The fibrinogen-treated synoviocytes showed significantly increased mRNA expression of IL-6, MCP-1, TF and PAI-1, and decreased PAR-1 expression compared to non-treated cells. Conclusion: LPS, fibrinogen and thrombin induced an increased gene expression of inflammatory markers in isolated equine fibroblast-like synoviocytes. LPS caused changes in gene expression promoting increased fibrinolysis, while fibrinogen and thrombin changed the gene expression resulting potentially in reduced fibrinolysis. Overall, it appeared that both inflammatory and haemostatic stimuli affected expression of genes involved in inflammatory and haemostatic pathways, supporting their importance in equine joint diseases.

  11. Homologous Recombination in Negative Sense RNA Viruses

    Directory of Open Access Journals (Sweden)

    Michael Worobey

    2011-08-01

    Full Text Available Recombination is an important process that influences biological evolution at many different levels. More and more homologous recombination events have been reported among negative sense RNA viruses recently. While sporadic authentic examples indicate that homologous recombination does occur, recombination seems to be generally rare or even absent in most negative sense RNA viruses, and most of the homologous recombination events reported in the literature were likely generated artificially due to lab contamination or inappropriate bioinformatics methods. Homologous recombination in negative sense RNA viruses should be reported with caution in the future, and only after stringent quality control efforts. Moreover, co-infection experiments should be performed to confirm whether recombination can occur.

  12. Progenitors of Recombining Supernova Remnants

    CERN Document Server

    Moriya, Takashi J

    2012-01-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with the ionization temperature higher than the electron temperature, is recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling which occurs after the shock wave gets out of the dense circumstellar medium makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium which is dense enough to establis...

  13. Tetraplex structure formation in the thrombin-binding DNA aptamer by metal cations measured by vibrational spectroscopy.

    Science.gov (United States)

    Mondragon-Sanchez, J A; Liquier, J; Shafer, R H; Taillandier, E

    2004-12-01

    Formation of intramolecular tetraplex structures by the thrombin-binding DNA aptamer (TBA) in the presence of K(+), Pb(2+), Ba(2+), Sr(2+) and Mn(2+) has been studied by vibrational spectroscopy. All tetraplex structures contain G-G Hoogsteen type base pairing, both C2'endo/anti and C2'endo/syn deoxyguanosine glycosidic conformations and local B like form DNA phosphate geometries. Addition of Pb(2+) ions modifies the structure by interacting at the level of the guanine carbonyl groups. The very important downshift of the guanine C6=O6 carbonyl vibration mode in the TBA spectrum induced by the addition of one Pb(2+) ion per TBA molecule is in agreement with a localization of the metal ion between both guanine quartets. FTIR melting experiments show an important stabilization of the tetraplex structure upon addition of Pb(2+) ions (DeltaT = 15 degrees C). This strong interaction of lead cations may be correlated with a change in the geometry of the cage formed by the two guanine quartets. A similar but weaker effect is observed for barium and strontium cations. PMID:15473710

  14. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race

    Science.gov (United States)

    Edelstein, Leonard C.; Simon, Lukas M.; Lindsay, Cory R.; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E.; Chen, Edward S.; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A.

    2014-01-01

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists. PMID:25293779

  15. Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study

    International Nuclear Information System (INIS)

    Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K+ and Na+ ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15 mer deoxynucleotide, d(G2T2G2TGTG2T2G2), labeled with 5'-fluorescein and 3'-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K+ ions bind with higher affinity than the Na+ ions. The enthalpy changes, ?H, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strandse association of two strands

  16. International Normalized Ratio (INR), coagulation factor activities and calibrated automated thrombin generation - influence of 24 h storage at ambient temperature

    DEFF Research Database (Denmark)

    Christensen, T D; Jensen, C

    2010-01-01

    International Normalized Ratio (INR) measurements are used to monitor oral anticoagulation therapy with coumarins. Single coagulation factor activities and calibrated automated thrombin (CAT) generation are considered as more advanced methods for evaluating overall haemostatic capacity. The aims were to assess the variability of INR, coagulation factor activities, and CAT, during 24 h of storage of blood samples at ambient temperature. A total of 24 patients on stable coumarin treatment were followed prospectively for 6 weeks. INR was analyzed at 0, 6 and 24 h after blood sampling and 1-stage clotting activity of coagulation factors II, VII, IX, and X as well as CAT generation was recorded after 0 and 24 h respectively. Statistical analyses included Bland-Altman plot, 95% limits of agreement, and a variability test using a mixed effect model. The level of INR remained statistically unchanged from 0 to 6 and 24 h of storage. Coagulation factor activities and CAT revealed no significant difference induced by 24h of storage, although the limits of agreement were wide. Patients' individual INR, coagulation factor activities, and CAT generation were not significantly influenced by 24 h storage of blood samples, but for the CAT generation analyses a trend toward time dependency was detected.

  17. Evaluation of a standardized protocol for thrombin generation measurement using the calibrated automated thrombogram: an international multicentre study.

    Science.gov (United States)

    Dargaud, Yesim; Wolberg, Alisa S; Luddington, Roger; Regnault, Veronique; Spronk, Henri; Baglin, Trevor; Lecompte, Thomas; Ten Cate, Hugo; Negrier, Claude

    2012-12-01

    The thrombin generation test (TGT) has demonstrated utility in evaluating overall hemostatic capacity both in bleeding and thrombotic disorders. Although the test is currently well accepted as a research tool, its role in clinical practice has not yet been defined through large prospective multicenter clinical studies. Such prospective studies have been limited by the lack of official standardization of the assay and its large inter-laboratory variability. This international study assessed the intra- and inter-assay imprecision of TGT as well as the inter-centre variability of results in one US and four European centres. Contact-inhibited plasmas from six healthy volunteers, one mild haemophilia A patient, and five patients with heterozygous prothrombin G20210 mutation were assayed. We demonstrated that, using identical equipement, standardized reagents, a carefully selected reference plasma for normalization of results and the same test procedure as described in our DVD, the assay variability was highly reduced compared to previously published data. Our results emphasize the importance of preheating on TGT results and the variability of the assay. In conclusion, our data demonstrated that the standardized TGT methodology evaluated in this study effectively reduces the variability of the assay to acceptable limits and may be used in clinical trials. PMID:22909826

  18. Inhibited Recombination of Charged Magnetoexcitons

    CERN Document Server

    Okamura, H; Sundaram, M; Gossard, A C

    1998-01-01

    Time-resolved photoluminescence measurements show that the decay time for charged excitons in a GaAs two-dimensional electron gas increases by an order of magnitude at high magnetic fields. Unlike neutral excitons, the charged exciton center-of-mass is spatially confined in a ``magnetically-adjustable quantum dot'' by the cyclotron orbit and the quantum well. The inhibited recombination is explained by a reduced phase coherence volume of the magnetically-confined charged excitons.

  19. Vortex Collisions Crossing or Recombination?

    CERN Document Server

    Bou-Diab, M; Blatter, G; Bou-Diab, Malek; Dodgson, Matthew J. W.; Blatter, Gianni

    2000-01-01

    We investigate the collision of two vortex lines moving with viscous dynamics and driven towards each other by an applied current. Using London theory in the approach phase we observe a non-trivial vortex conformation producing anti-parallel segments; their attractive interaction triggers a violent collision. The collision region is analyzed using the time-dependent Ginzburg-Landau equation. While we find vortices will always recombine through exchange of segments, a crossing channel appears naturally through a double collision process.

  20. Vortex Collisions: Crossing or Recombination?

    OpenAIRE

    Bou-diab, Malek; Dodgson, Matthew J. W.; Blatter, Gianni

    2000-01-01

    We investigate the collision of two vortex lines moving with viscous dynamics and driven towards each other by an applied current. Using London theory in the approach phase we observe a non-trivial vortex conformation producing anti-parallel segments; their attractive interaction triggers a violent collision. The collision region is analyzed using the time-dependent Ginzburg-Landau equation. While we find vortices will always recombine through exchange of segments, a crossin...

  1. Experimental evidence of ternary recombinations

    International Nuclear Information System (INIS)

    Full text: About four decades ago D'Angelo [1] noticed that the measured rate coefficients for the radiative recombination (RR) for a fully stripped ions were orders of magnitude larger than that predicted by early theories. He noted that the discrepancy was, in fact, due to the dominance of another recombination process often called ternary recombination (TR) because in this phenomenon two electrons and one ion get involved. This picture becomes more complicated for projectiles, containing one or more electrons as dielectronic recombinations (DR) also start contributing. Thus, it has been impossible to decouple TR from RR and DR processes yet. It may be worth mentioning that experimental situations allow low lying bound levels through RR and DR processes while TR into high Rydberg states is conceivable at low electron energies. One observes a transition in a time resolved spectroscopy experiment whose upper level is produced by RR and DR. TR may lead to the same transition after a certain time as electron has to cascade down to the same low lying level from a Rydberg state. Thus one may see that RR and DR are prompt and TR is a delayed one. However, separation of these processes was not possible yet. One needs the right situation which may well resolve TR from RR and DR. Such situation was evident in a beam-foil experiment favoring nuclear reactions. Residue nuclei may undergo electron capture processes such as DR and TR. These nuclei being fully striped ions would nse nuclei being fully striped ions would not allow DR to take place. Impact of 170MeV Ni ions on carbon foil in beam-foil experiments led to formation of the residues 62,64Zn, 65Ga, 68Ge, and 68As. X-rays due to M1 transition from such residue H-like ions were observed in the time resolved x-ray spectroscopy. Prompt x-ray produced by RR and the delayed by TR are quite well distinguished in the decay curve. Such results reveals experimental evidence of ternary recombination for the first time. Experimental technique, novel analysis, and salient results will be highlighted

  2. Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

    International Nuclear Information System (INIS)

    Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 ± 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 ± 0.012 to 0.124 ± 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 ± 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1,800 ± 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1,700-2,000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component. Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. The authors have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. This mutant constitutively expressed 38 ± 7% of the activity of the RVV-V-activated protein. These results suggest that one of the functions of the la that one of the functions of the large connecting region in factor V is to inhibit constitutive procoagulant activity

  3. Effects of Recombination on Complex Regulatory Circuits

    Science.gov (United States)

    Martin, Olivier C.; Wagner, Andreas

    2009-01-01

    Mutation and recombination are the two main forces generating genetic variation. Most of this variation may be deleterious. Because recombination can reorganize entire genes and genetic circuits, it may have much greater consequences than point mutations. We here explore the effects of recombination on models of transcriptional regulation circuits that play important roles in embryonic development. We show that recombination has weaker deleterious effects on the expression phenotypes of these circuits than mutations. In addition, if a population of such circuits evolves under the influence of mutation and recombination, we find that three key properties emerge: (1) deleterious effects of mutations are reduced dramatically; (2) the diversity of genotypes in the population is greatly increased, a feature that may be important for phenotypic innovation; and (3) cis-regulatory complexes appear. These are combinations of regulatory interactions that influence the expression of one gene and that mitigate deleterious recombination effects. PMID:19652184

  4. Detecting the cosmological recombination signal from space

    CERN Document Server

    Desjacques, Vincent; Silk, Joseph; de Bernardis, Francesco; Doré, Olivier

    2015-01-01

    Spectral distortions of the CMB have recently experienced an increased interest. One of the inevitable distortion signals of our cosmological concordance model is created by the cosmological recombination process, just a little before photons last scatter at redshift $z\\simeq 1100$. These cosmological recombination lines, emitted by the hydrogen and helium plasma, should still be observable as tiny deviation from the CMB blackbody spectrum in the cm--dm spectral bands. In this paper, we present a forecast for the detectability of the recombination signal with future satellite experiments. We argue that serious consideration for future CMB experiments in space should be given to probing spectral distortions and, in particular, the recombination line signals. The cosmological recombination radiation not only allows determination of standard cosmological parameters, but also provides a direct observational confirmation for one of the key ingredients of our cosmological model: the cosmological recombination histo...

  5. Heterogeneity in recombinant protein production

    DEFF Research Database (Denmark)

    Schalén, Martin; Johanson, Ted

    2012-01-01

    A crucial step in biotechnology is the scale-up process. Normally, lab scale verification and optimization of production processes and strains are performed in small reactors with perfect mixing and hence the cells experience a homogenous environment. The gradients that occur in industrial scale bioreactors are often not taken into consideration in these experiments. Gradients occur due to insufficient mixing in the reactor, and affect the process in a variety of ways. When cells travel through the reactor and encounter different substrate concentrations, oxygen availability, pH, temperature, etc. the cell physiology is affected. Cells are stressed, and this may severely affect growth, by-product accumulation, biomass yield and recombinant product yield. The stress caused by exposure to divergent microenvironments, genetic differences of individual cells, differing cell cycle stage and cell age, all contribute to make a population in a fermenter heterogeneous, resulting in cell-to-cell variation in physiological parameters of the microbial culture. Our study aims at investigating how population heterogeneity and recombinant protein production is affected by environmental gradients in bioreactors. For this purpose, a Saccharomyces cerevisiae strain, that functions as a protein production reporter, has been developed. A heterologous protein has been tagged with a fluorescent protein providing a way to measure the amount of heterologous protein produced by the cells on single cell level. Gradients are simulated in small bioreactors and the population heterogeneity can be visualised by analysing single cells with flow cytometry. This can give new insights to cell physiology and recombinant protein production at the industrial scale.

  6. Recombinant DNA technology in apple.

    Science.gov (United States)

    Gessler, Cesare; Patocchi, Andrea

    2007-01-01

    This review summarizes the achievements of almost 20 years of recombinant DNA technology applied to apple, grouping the research results into the sections: developing the technology, insect resistance, fungal disease resistance, self-incompatibility, herbicide resistance, fire blight resistance, fruit ripening, allergens, rooting ability, and acceptance and risk assessment. The diseases fire blight, caused by Erwinia amylovora, and scab, caused by Venturia inaequalis, were and still are the prime targets. Shelf life improvement and rooting ability of rootstocks are also relevant research areas. The tools to create genetically modified apples of added value to producers, consumers, and the environment are now available. PMID:17522823

  7. Thrombin-activatable fibrinolysis inhibitor Thr325Ile polymorphism and plasma level in breast cancer: A pilot study.

    Science.gov (United States)

    Fawzy, Manal S; Mohammed, Eman A; Ahmed, Amal S; Fakhr-Eldeen, Abeer

    2015-06-01

    This study aimed to investigate thrombin-activatable fibrinolysis inhibitor (TAFI) Thr325Ile polymorphism and TAFI antigen (Ag) levels in breast cancer (BC) in the Egyptian population to clarify their role in relation to BC. A group of 300 females was recruited in this study; of these 150 unrelated patients with different stages of BC and 150 age-matched healthy controls. Plasma TAFI Ag was measured by ELISA and TAFI Thr325Ile (rs1926447) polymorphism was genotyped using TaqMan single nucleotide polymorphism (SNP) genotyping assay. The results showed the genotypes of the minor allele; Thr/Ile (CT) and Ile/Ile (TT) were significantly more frequent in patients compared to control group (50.0% and 22.0% vs. 42.0% and 13.3%, respectively) and were also associated with BC susceptibility [OR = 1.9 and 2.6; 95% CI: (1.1-3.3) and (1.3-5.5), respectively P = 0.01]. Ile325 allele carriers were more frequent in cases than in controls (47.0% vs. 34.0%) [OR = 1.7, (95% CI = 1.2-2.4), P = 0.001]. However, TAFI Thr325Ile polymorphism was not associated with BC stage or other clincopathological characteristics. TAFI Ag levels were correlated with advanced stages of BC, poor prognosis and risk of recurrence (P = 0.02, P = 0.04 and P  < 0.001, respectively) and Thr325Ile SNP was significantly correlated with TAFI antigen levels with the C/C genotype corresponding to the highest and the T/T genotype to the lowest TAFI antigen levels (P < 0.001) in the study groups. In conclusion, this study showed for the first time that TAFI Thr325Ile polymorphism could have a contribution to BC susceptibility in our population. Furthermore, high TAFI plasma levels may serve as a predictor of poor prognosis in patients with BC. PMID:25893174

  8. Meta-analysis of randomized controlled trials on risk of myocardial infarction from the use of oral direct thrombin inhibitors.

    Science.gov (United States)

    Artang, Ramin; Rome, Eric; Nielsen, Jørn Dalsgaard; Vidaillet, Humberto J

    2013-12-15

    Dabigatran has been associated with greater risk of myocardial infarction (MI) than warfarin. It is unknown whether the increased risk is unique to dabigatran, an adverse effect shared by other oral direct thrombin inhibitors (DTIs), or the result of a protective effect of warfarin against MI. To address these questions, we systematically searched MEDLINE and performed a meta-analysis on randomized trials that compared oral DTIs with warfarin for any indication with end point of MIs after randomization. We furthermore performed a secondary meta-analysis on atrial fibrillation stroke prevention trials with alternative anticoagulants compared with warfarin with end point of MIs after randomization. A total of 11 trials (39,357 patients) that compared warfarin to DTIs (dabigatran, ximelagatran, and AZD0837) were identified. In these trials, patients treated with oral DTIs were more likely to experience an MI than their counterparts treated with warfarin (285 of 23,333 vs 133 of 16,024, odds ratio 1.35, 95% confidence interval 1.10 to 1.66, p = 0.005). For secondary analysis, 8 studies (69,615 patients) were identified that compared warfarin with alternative anticoagulant including factor Xa inhibitors, DTIs, aspirin, and clopidogrel. There was no significant advantage in the rate of MIs with the use of warfarin versus comparators (odds ratio 1.06, 95% confidence interval 0.85 to 1.34, p = 0.59). In conclusion, our data suggest that oral DTIs were associated with increased risk of MI. This increased risk appears to be a class effect of these agents, not a specific phenomenon unique to dabigatran or protective effect of warfarin. These findings support the need for enhanced postmarket surveillance of oral DTIs and other novel agents. PMID:24075284

  9. Meta-analysis of randomized controlled trials on risk of myocardial infarction from the use of oral direct thrombin inhibitors

    DEFF Research Database (Denmark)

    Artang, Ramin; Rome, Eric

    2013-01-01

    Dabigatran has been associated with greater risk of myocardial infarction (MI) than warfarin. It is unknown whether the increased risk is unique to dabigatran, an adverse effect shared by other oral direct thrombin inhibitors (DTIs), or the result of a protective effect of warfarin against MI. To address these questions, we systematically searched MEDLINE and performed a meta-analysis on randomized trials that compared oral DTIs with warfarin for any indication with end point of MIs after randomization. We furthermore performed a secondary meta-analysis on atrial fibrillation stroke prevention trials with alternative anticoagulants compared with warfarin with end point of MIs after randomization. A total of 11 trials (39,357 patients) that compared warfarin to DTIs (dabigatran, ximelagatran, and AZD0837) were identified. In these trials, patients treated with oral DTIs were more likely to experience an MI than their counterparts treated with warfarin (285 of 23,333 vs 133 of 16,024, odds ratio 1.35, 95% confidence interval 1.10 to 1.66, p = 0.005). For secondary analysis, 8 studies (69,615 patients) were identified that compared warfarin with alternative anticoagulant including factor Xa inhibitors, DTIs, aspirin, and clopidogrel. There was no significant advantage in the rate of MIs with the use of warfarin versus comparators (odds ratio 1.06, 95% confidence interval 0.85 to 1.34, p = 0.59). In conclusion, our data suggest that oral DTIs were associated with increased risk of MI. This increased risk appears to be a class effect of these agents, not a specific phenomenon unique to dabigatran or protective effect of warfarin. These findings support the need for enhanced postmarket surveillance of oral DTIs and other novel agents.

  10. Analysis of blood coagulation in mice: pre-analytical conditions and evaluation of a home-made assay for thrombin-antithrombin complexes

    Directory of Open Access Journals (Sweden)

    Meijers Joost CM

    2005-08-01

    Full Text Available Abstract Background The use of mouse models for the study of thrombotic disorders has gained increasing importance. Methods for measurement of coagulation activation in mice are, however, scarce. The primary aim of this study was to develop a specific mouse thrombin-antithrombin (TAT ELISA for measurement of coagulation activation and to compare it with two commercially available assays for human TAT complexes. In addition, we aimed to improve methods for mouse plasma anticoagulation and preparation. Methods and results First, for the measurement of TAT-complexes in plasma a mouse specific TAT-ELISA was developed using rabbit polyclonal antibodies raised against mouse thrombin and rat antithrombin, respectively. This ELISA detected an increase in TAT levels in a mouse model of endotoxemia. Two commercial human TAT ELISAs appeared to be less specific for mouse thrombin-rat antithrombin complexes. Second, to prevent clotting of mouse blood sodium citrate was either mixed with blood during collection in a syringe or was injected intravenously immediately prior to blood collection. Intravenous sodium citrate completely inhibited blood coagulation resulting in plasma with consistently low TAT levels. Sodium citrate mixed with blood during collection resulted in increased TAT levels in 4 out of 16 plasma samples. Third, heparinase was added to plasma samples after in vivo injection of different heparin doses to test its neutralizing effect. Heparinase neutralized up to a 20 U of heparin/mouse and resulted in accurate APTT and factor VIII determinations. Conclusion These procedures and reagents for plasma preparation and coagulation testing will improve studies on thrombotic disorders in mice.

  11. Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression.

    Science.gov (United States)

    Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

    2014-07-01

    In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation. PMID:24845383

  12. Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain.

    Science.gov (United States)

    Vu, Trang T; Stafford, Alan R; Leslie, Beverly A; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I

    2011-09-01

    Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant ?(A)/?(A)-fibrinogen and the ?-chain elongated isoform, ?(A)/?'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds ?(A)/?(A)- or ?(A)/?'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and ?'-peptide, an analog of the COOH terminus of the ?'-chain that mediates the high affinity interaction of thrombin with ?(A)/?'-fibrin. Thrombin competes with HRG for ?'-peptide binding and displaces (125)I-HRG from ?(A)/?'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for ?(A)/?'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation. PMID:21757718

  13. Recombinant protein production in yeasts.

    Science.gov (United States)

    Mattanovich, Diethard; Branduardi, Paola; Dato, Laura; Gasser, Brigitte; Sauer, Michael; Porro, Danilo

    2012-01-01

    Recombinant protein production is a multibillion-dollar market. The development of a new product begins with the choice of a production host. While one single perfect host for every protein does not exist, several expression systems ranging from bacterial hosts to mammalian cells have been established. Among them, yeast cell factories combine the advantages of being single cells, such as fast growth and easy genetic manipulation, as well as eukaryotic features including a secretory pathway leading to correct protein processing and post-translational modifications. In this respect, especially the engineering of yeast glycosylation to produce glycoproteins of human-like glycan structures is of great interest. Additionally, different attempts of cellular engineering as well as the design of different production processes that are leading to improved productivities are presented. With the advent of cheaper next-generation sequencing techniques, systems biotechnology approaches focusing on genome scale analyses will advance and accelerate yeast cell factories and thus recombinant protein production processes in the near future. In this review we summarize advantages and limitations of the main and most promising yeast hosts, including Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha as those presently used in large scale production of heterologous proteins. PMID:22160907

  14. Charge recombination in undoped cuprates

    Science.gov (United States)

    Lenar?i?, Zala; Prelovšek, Peter

    2014-12-01

    We theoretically analyze the process of charge recombination in the planar Mott-Hubbard insulators with the aim to explain the short picosecond-range lifetimes of photoexcited carriers, experimentally studied via pump-probe experiments on the undoped cuprates. The recombination mechanism consists of two essential ingredients: the formation of a metastable s -type bound holon-doublon pair, i.e., the Mott exciton, and the decay of such an excitonic state via the multimagnon emission. In spite of the large gap that requires many bosons to be emitted, the latter process is fast due to a large exchange scale and strong charge-spin coupling in planar systems. As the starting microscopic model we consider the single-band Hubbard model and then a more realistic three-band model for cuprates, both leading to the same minimal one. The decay rate of the exciton is evaluated numerically via the Fermi golden rule, having consistency also with the direct time-evolution calculation. The decay rate reveals exponential dependence on the ratio of the Mott-Hubbard gap and the exchange coupling, the result qualitatively reproduced also within a toy exciton-boson model.

  15. Recombination chambers for BNCT dosimetry

    International Nuclear Information System (INIS)

    Parallel plate recombination ionization chambers are known as the detectors which can be used for determination of gamma and high-LET dose components and for characterization of radiation quality of mixed radiation fields. Specially designed chambers can operate correctly even at dose rates of therapeutic beams. In this work the investigations were extended to a set of cylindrical chambers including a TE chamber and three graphite chambers filled with different gases - CO2, N2 and 10BF3, in order to determine the thermal neutrons, 14N capture, gamma, and fast neutron dose components. The separation of the dose components is based on differences of the shape of the saturation curve, in dependence on LET spectrum of the investigated radiation. The measurements using all the chambers and a parallel plate recombination chamber were performed in a reactor beam of NRI Rez (Czech Republic). The gamma component was determined with accuracy of about 5%, while the variations of its value could be monitored with accuracy of about 0.5%. Relative changes of the beam components could be detected with accuracy of about 5% using the parallel plate chamber. The use of the chambers filled with different gases considerably improved the resolution of the method. (author)

  16. Thrombin Injection Failure with Subsequent Successful Stent-Graft Placement for the Treatment of an Extracranial Internal Carotid Pseudoaneurysm in a 5-Year-Old Child

    International Nuclear Information System (INIS)

    Internal carotid artery pseudoaneurysm is a rare life-threatening condition that may develop in different clinical situations. We report the case of an extracranial internal carotid artery pseudoaneurysm secondary to a throat infection in a pediatric patient that was initially treated with percutaneous thrombin injection under ultrasound guidance. However, recanalization occurred at 48 h, and definitive treatment was then performed by endovascular stent-graft placement. We briefly review the clinical characteristics of this uncommon clinical condition as well as the treatment options.

  17. Molecular requirements for radiation-activated recombination

    International Nuclear Information System (INIS)

    Purpose/Objective: The major stumbling block to successful gene therapy today is poor gene transfer. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. We further hypothesized that known DNA-damage-repair proteins might also be important in radiation-activated recombination. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human (A549 and 39F) and rodent (NIH/3T3) cell lines. Continuous low dose rate and multiple radiation fractions were also tested. Nuclear extracts were made and the effect of irradiation on inter-plasmid recombination/ligation determined. Multiple DNA damage-repair deficient cell lines were tested for radiation-activated recombination. Results: A significant radiation dose-dependent improvement in stable plasmid transfection (by as much as 1300 fold) is demonstrated in neoplastic and primary cells. An improvement in transient plasmid transfection is also seen, with as much as 85% of cells transiently expressing b-galactosidase (20-50 fold improvement). Stable transfection is only improved for linearized or nicked plasmids. Cells have improved gene transfer for at least 96 hours after irradiation. Both fractionated and continuous low dose rate irradiation are effective at improving stable gene transfer in mammalian cells, thus making relatively high radiation dose delivery clinically feasible. Inter-plasmid recombination is radiation Inter-plasmid recombination is radiation dose dependent in nuclear extract assays, and the type of overhang (3', 5' or blunt end) significantly affects recombination efficiency and the type of product. The most common end-joining activity involves filling-in of the overhang followed by blunt end ligation. Adenovirus is a linear, double stranded DNA virus. We demonstrate that adenoviral infection efficiency is increased by irradiation. The duration of transgene expression is lengthened because the virus integrates with high efficiency (?10% of treated cells) into cellular DNA. The mechanism of radiation enhanced stable gene transfer requires effector proteins to accomplish the recombination. The Ku proteins, which are required for V(D)J recombination, account for at least 90% of radiation induced recombination. There is also an absolute requirement for the Ataxia Telangiectasia gene (ATM) for any radiation induced recombination to occur, although the transfection efficiency in unirradiated cells is unaffected by ATM. Removal of p53 by transfection of E6 (Human Papilloma Virus) significantly inhibits radiation activated recombination, and this is confirmed in nuclear extract recombination assays. Conclusions: Ionizing radiation activates a recombination pathway which may be useful in gene therapy. The molecular mechanism of radiation activated recombination requires a number of DNA-damage-repair proteins

  18. Comparison of device models for organic solar cells: Band-to-band vs. tail states recombination

    Energy Technology Data Exchange (ETDEWEB)

    Soldera, Marcos; Taretto, Kurt [Departamento de Electrotecnia, Universidad Nacional del Comahue, Buenos Aires, Neuquen (Argentina); Kirchartz, Thomas [Department of Physics, Imperial College London, South Kensington (United Kingdom)

    2012-01-15

    The efficiency-limiting recombination mechanism in bulk-heterojunction (BHJ) solar cells is a current topic of investigation and debate in organic photovoltaics. In this work, we simulate state-of-the-art BHJ solar cells using two different models. The first model takes into account band-to-band recombination and field dependent carrier generation. The second model assumes a Shockley-Read-Hall (SRH) recombination mechanism via tail states and field independent carrier generation. Additionally, we include in both cases optical modelling and, thus, position-dependent exciton generation and non-ideal exciton collection. We explore both recombination mechanisms by fitting light and dark current-voltage (JV) characteristics of BHJ cells of five materials: P3HT, MDMO-PPV, MEH-PPV, PCDTBT and PF10TBT, all blended with fullerene derivatives. We show that although main device parameters such as short circuit current, open circuit voltage, fill factor and ideality factor are accurately reproduced by both Langevin and tail recombination, only tail recombination reproduces also the ideality factor of dark characteristics accurately. Nevertheless, the model with SRH recombination via tail states needs the inclusion of external circuitry to account for the heavy shunt present in all the blends, except P3HT:PCBM, when illuminated. Finally, we propose a means to find analytical expressions for the short circuit current by assuming a linear relation between the recombination rate and the concentration of free minority carriers. The model reproduces experimental data of P3HT cells at various thickness values using realistic parameters for this material. Dark JV measurement (circles) of a PCDTBT:PC{sub 70}BM solar cell (Park et al., Nature Photon. 3, 297 (2009) [1]), the fit with the model including recombination via tail states (solid line) and the fit with the model reported by (Koster et al., Phys. Rev. B 72, 085205 (2005) [2]) that includes bimolecular band-to-band recombination and charge transfer state (CTS) dissociation. The inset shows the JV curves under white light. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  19. Telomeric recombination induced by dysfunctional telomeres

    OpenAIRE

    Brault, Marie Eve; Autexier, Chantal

    2011-01-01

    Telomeric recombination has been observed in telomerase-negative alternative lengthening of telomeres in human cancer cells and following telomerase inhibition or gene deletion. This study shows that telomeric recombination mechanisms can also be activated by dysfunctional telomeres without telomerase inhibition in telomerase-positive cells.

  20. Recombinant organisms for production of industrial products

    OpenAIRE

    Adrio, Jose-luis; Demain, Arnold L.

    2009-01-01

    A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding...

  1. High efficiency recombineering in lactic acid bacteria.

    Science.gov (United States)

    van Pijkeren, Jan-Peter; Britton, Robert A

    2012-05-01

    The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lactic acid bacteria. Mutations were incorporated in the chromosome of Lactobacillus reuteri and Lactococcus lactis without selection at frequencies ranging between 0.4% and 19%. Whole genome sequence analysis showed that ssDNA recombineering is specific and not hypermutagenic. To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold. By creating a single amino acid change in the D-Ala-D-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5?µg/ml, well below the clinically relevant minimum inhibitory concentration. Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry. We expect that this work will serve as a blueprint for the adaptation of ssDNA recombineering to other gram-positive bacteria. PMID:22328729

  2. Cell Biology of Homologous Recombination in Yeast

    OpenAIRE

    Eckert-boulet, Nadine; Rothstein, Rodney; Lisby, Michael

    2011-01-01

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single-and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces cerevisiae using fluorescence microscopy.

  3. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Rothstein, Rodney

    2011-01-01

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces cerevisiae using fluorescence microscopy.

  4. Electron-ion recombination at low energy

    International Nuclear Information System (INIS)

    The work is based on results obtained with a merged-beams experiment. A beam of electronics with a well characterized density and energy distribution was merged with a fast, monoenergetic ion beam. Results have been obtained for radiative recombination and dielectronic recombination at low relative energies (0 to ?70eV). The obtained energy resolution was improved by about a factor of 30. High vacuum technology was used to suppress interactions with electrons from the environments. The velocity distribution of the electron beam was determined. State-selective dielectronic-recombination measurements were performable. Recombination processes were studied. The theoretical background for radiative recombination and Kramers' theory are reviewed. The quantum mechanical result and its relation to the semiclassical theory is discussed. Radiative recombination was also measured with several different non-bare ions, and the applicability of the semiclassical theory to non-bare ions was investigated. The use of an effective charge is discussed. For dielectronic recombination, the standard theoretical approach in the isolated resonance and independent-processes approximation is debated. The applicability of this method was tested. The theory was able to reproduce most of the experimental data except when the recombination process was sensitive to couplings between different electronic configurations. The influence of external perturbing electrostatic fields is discussed. (AB) (31 refs.)

  5. Dielectronic recombination of the helium ion

    International Nuclear Information System (INIS)

    Dielectronic recombination of the helium ion under conditions of intersecting beams is observed for the first time. Two peaks are observed in the energy dependence of the cross section of the process. The dielectronic recombination cross section is comparable to the cross section for excitation of the resonant level of the helium ion

  6. Polynomial identities for ternary intermolecular recombination

    OpenAIRE

    Bremner, Murray R.

    2010-01-01

    The operation of binary intermolecular recombination, originating in the theory of DNA computing, permits a natural generalization to n-ary operations which perform simultaneous recombination of n molecules. In the case n = 3, we use computer algebra to determine the polynomial identities of degree

  7. RNAi and heterochromatin repress centromeric meiotic recombination

    DEFF Research Database (Denmark)

    Ellermeier, Chad; Higuchi, Emily C

    2010-01-01

    During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis.

  8. Dissociative recombination of He2+

    International Nuclear Information System (INIS)

    We present calculated cross sections for dissociative recombination of the He2+ molecular ion in the 1 to 15 eV energy region, where the cross section is dominated by a series of resonances, the repulsive Rydberg states converging to the first excited state of the ion. The resonance parameters for this system were obtained from electron scattering calculations using the complex Kohn variational method. The dissociation dynamics were studied using a time-dependent wave packet calculation. The calculated cross section is reported and compared to available experiment. The effects of rotation and vibration on the total cross section is examined, as well as the effect of electronic couplings on the final state distribution

  9. RecTE(Psy)-mediated recombineering in Pseudomonas syringae.

    Science.gov (United States)

    Swingle, Bryan

    2014-01-01

    A recently developed Pseudomonas syringae recombineering system simplifies the procedure for installing specific mutations at a chosen genomic locus. The procedure involves transforming P. syringae cells expressing recombineering functions with a PCR product that contains desired changes flanked by sequences homologous to a target location. Cells transformed with the substrate undergo homologous recombination between the genomic DNA and the recombineering substrate. The recombinants are found by selection for traits carried by the recombineering substrate, usually antibiotic resistance. PMID:24557893

  10. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  11. Fibrinógeno-trombina como tratamiento puente en un caso de hemoptisis masiva / Fibrinogen-thrombin as bridge therapy in massive hemoptysis

    Scientific Electronic Library Online (English)

    Francisco, Cuervo; Luis F, Giraldo; Carlos, Vélez; María R, Forero.

    2013-03-01

    Full Text Available Se presenta el caso de una paciente joven con hemoptisis masiva por tuberculosis que no pudo ser controlada de forma efectiva con la inserción de un catéter Fogarty por un fibrobroncoscopio. Ante esto y el alto riesgo de asfixia o desangramiento, se decidió infundir fibrinógeno-trombina a través de [...] un catéter, introducido por el fibrobroncoscopio; con esto se logró controlar el sangrado, intubarla con un tubo orotraqueal de doble luz y estabilizarla para remitirla a otra institución, donde fue sometida a lobectomía y se le proporcionó tratamiento antituberculoso. La infusión de fibrinógeno-trombina podría considerarse como una opción terapéutica transitoria, de tipo puente, mientras se practica el manejo definitivo. Abstract in english This article presents the case of a young woman with massive hemoptysis (1,000 mL in 6 hours) due to tuberculosis, which could not be controlled by insertion of a Fogarty catheter through a fiber-optic bronchoscope. Because of asphyxia and persistent bleeding risk we instilled fibrinogen-thrombin th [...] rough a fiber-optic bronchoscope inserted catheter, achieving bleeding cessation and permitting the placing of a double-lumen oro-tracheal tube. Later on, the patient underwent lobectomy and anti-tuberculosis treatment. The fibrinogen-thrombin could be considered as a bridge, transitory measure for massive hemoptysis, while definitive treatment could be established.

  12. Health Topic XML File Description

    Science.gov (United States)

    ... topic URL. The URL for the corresponding health topic page on MedlinePlus. Example: url="http://www.nlm.nih. ... related topics shown on the left sidebar of topic pages and other pages. Example: topic title="Abdominal ...

  13. Radio Recombination Lines. Their Physics and Astronomical Applications

    Science.gov (United States)

    Gordon, M. A.; Sorochenko, R. L.

    2002-11-01

    This book is a comprehensive guide to the physics and observations of Radio Recombination Lines from astronomical sources, written for astronomers, physicists, and graduate students. It serves as a graduate-level textbook. It includes the history of RRL detections, the astrophysics underlying their intensities and line shapes including topics like departures from LTE and Stark broadening, the maximum possible size of an atom, as well as detailed descriptions of the astronomical topics for which RRLs have proved to be effective tools. The text includes more than 250 equations and 110 illustrations. It also contains hundreds of specific references to the astronomical literature to enable readers to explore additional details. The appendix includes supplementary information such as the detailed physics underlying the Bohr atomic model, tables of RRL frequencies including fine structure components, techniques for calculating hydrogenic oscillator strengths, FORTRAN code for calculating departure coefficients, and a discussion with formulas for converting observational (telescope) intensity units to astrophysical ones. Link: http://www.wkap.nl/prod/b/1-4020-1016-8

  14. Inference about recombination from haplotype data: lower bounds and recombination hotspots.

    Science.gov (United States)

    Bafna, Vineet; Bansal, Vikas

    2006-03-01

    Recombination is an important evolutionary mechanism responsible for creating the patterns of haplotype variation observable in human populations. Recently, there has been extensive research on understanding the fine-scale variation in recombination across the human genome using DNA polymorphism data. Historical recombination events leave signature patterns in haplotype data. A nonparametric approach for estimating the number of historical recombination events is to compute the minimum number of recombination events in the history of a set of haplotypes. In this paper, we provide new and improved methods for computing lower bounds on the minimum number of recombination events. These methods are shown to detect a higher number of recombination events for a haplotype dataset from a region in the lipoprotein lipase gene than previous lower bounds. We apply our methods to two datasets for which recombination hotspots have been experimentally determined and demonstrate a high density of detectable recombination events in the regions annotated as recombination hotspots. The programs implementing the methods in this paper are available at www.cs.ucsd.edu/users/vibansal/RecBounds/. PMID:16597254

  15. Recombination Every Day: Abundant Recombination in a Virus during a Single Multi-Cellular Host Infection

    Directory of Open Access Journals (Sweden)

    Froissart Remy

    2005-01-01

    Full Text Available Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10-5 to 4 x 10-5. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

  16. Polynomial identities for ternary intermolecular recombination

    CERN Document Server

    Bremner, Murray R

    2010-01-01

    The operation of binary intermolecular recombination, originating in the theory of DNA computing, permits a natural generalization to n-ary operations which perform simultaneous recombination of n molecules. In the case n = 3, we use computer algebra to determine the polynomial identities of degree <= 9 satisfied by this trilinear nonassociative operation. Our approach requires computing a basis for the nullspace of a large integer matrix, and for this we compare two methods: (i) the row canonical form, and (ii) the Hermite normal form with lattice basis reduction. In the conclusion, we formulate some conjectures for the general case of n-ary intermolecular recombination.

  17. Charges recombination in ? particle tracks in argon

    International Nuclear Information System (INIS)

    The creation and evolution of (neutral) excited states and ionized states in ? particle tracks in high pressure argon are studied. The main features of recently published experimental results on the recombination luminescence can be explained and a track model is proposed. Details are given on the track radius, on the electrons thermallization, and on collisions between electrons and triplet excited states. The most important result is that at high pressure and high electron and ion densities a collective electron-ion recombination is possible, that is more efficient that the well known dissociative recombination

  18. Dissociative Recombination without a Curve Crossing

    Science.gov (United States)

    Guberman, Steven L.

    1994-01-01

    Ab initio calculations show that a curve crossing is not always needed for a high dissociative- recombination cross section. For HeH(+), in which no neutral states cross the ion potential curve, dissociative recombination is driven by the nuclear kinetic-energy operator on adiabatic potential curves. The kinetic-energy derivative operator allows for capture into repulsive curves that are outside of the classical turning points for the nuclear motion. The dominant dissociative route is the C (2)Sigma(+) state leading to H(n = 2) atoms. An analogous mechanism is proposed for the dissociative recombination of H3(+).

  19. Recombinant vaccine for canine parvovirus in dogs.

    OpenAIRE

    Lo?pez Turiso, J. A.; Corte?s, E.; Marti?nez, C.; Ruiz Yba?n?ez, R.; Simarro, I.; Vela, C.; Casal, I.

    1992-01-01

    VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-as...

  20. Random recombination and evolution of drug resistance.

    Science.gov (United States)

    Kleinman, Alan

    2015-05-20

    The effects of random recombination on the random mutation and natural selection phenomenon can be understood by considering the mathematical behavior of this phenomenon. This phenomenon operates in a mathematically predicable behavior, which when understood, explains the empirical observations of this phenomenon. The mathematical behavior of random recombination is derived using the principles given by probability theory. The derivation of the equations describing the random recombination phenomenon is done in the context of an empirical example. Copyright © 2015 John Wiley & Sons, Ltd. PMID:25645658

  1. Influence of hirudin and cobra venom factor on the release of 14C-serotonin and 51chromium from human platelets induced by thrombin, collagen, aggregate gammaglobulin and HLA antibody

    International Nuclear Information System (INIS)

    The present work investigates the influence of hirudin and cobra venom factor on thrombin, collagen, aggregate gammaglobulin and HLA-antibody-induced release of 14C-serotonin and 51chromium from human platelets. Besides the platelet-specific release reaction (14C-serotonin) the extent of platelet lysis was determined by measurement of the loss of 51chromium from the platelets. The results showed the thrombin, collagen and aggregate-gammaglobulin-induced platelet alteration to be a non-complement-dependent reaction of the platelets with release of 14C-serotonin. Following long-term incubation small quantities of 51chromium are also released. As this release of 51chromium cannot be inhibited using cobra venom factor and does not occur in washed platelets either, it is most probably a non-complement-dependent reaction. The HLA-antibody-induced, specific platelet alteration is both complement-dependent and complement-independent. Differentiation is possible by inhibition of the complement-dependent lysis. On the other hand thrombin is of no relevance to the collagen, aggregate gammaglobulin, and HLA-antibody-induced platelet alteration as the interactions of these substances with platelets are not inhibited by hirudin. The above results are confirmed by investigation of the 51chromium uptake capacity of washed platelets treated previously with thrombin, collagen and HLA antibody. (orig./MG)

  2. Influence of cardiopulmonary bypass on the interaction of recombinant factor VIIa with activated platelets

    DEFF Research Database (Denmark)

    Kjalke, M.; Runge, M.

    2009-01-01

    Recombinant factor VIIa (rFVIIa) interacts preferentially with coated platelets characterized by a high exposure of phosphatidyl serine (PS), FV, FVIII, FIX, and FX binding, and fibrinogen. Cardiopulmonary bypass (CPB) is known to impair platelet function. In this study, the influence of CPB on formation of coated platelets and the interaction of rFVIIa with the platelets were studied. Blood was either exposed to a closed CPB circuit or obtained from patients undergoing CPB-assisted cardiac surgery, and platelets were analyzed by flow cytometry with and without dual agonist stimulation with thrombin and a GPVI collagen receptor agonist known to induce coated platelet formation. Platelets circulated within a closed CPB circuit did not spontaneously form coated platelets. Dual agonists stimulation caused formation of coated platelets at a reduced level compared to pre-CPB level (51 +/- 21% vs. 80 +/- 17% before CPB, p < .001). The rFVIIa interaction with the coated platelets was not impaired after CPB. Platelets isolated from patients undergoing CPB-assisted cardiac surgery also formed coated platelets only after dual agonist stimulation but to the same level as before surgery (76 +/- 8% vs. 83 +/- 14% before surgery, p = .17, n = 10). rFVIIa interaction with the coated platelets was not impaired after surgery. No spontaneous rFVIIa-binding platelets were found. The data indicate that CPB exposure in vivo does not compromise the platelet-dependent effects of rFVIIa either by spontaneous formation of coated platelets, thereby limiting the risk of systemic coagulation, or by impairing rFVIIa interaction with the agonist-induced coated platelets, thereby retaining the hemostatic potential of rFVIIa after CPB Udgivelsesdato: 2009/6

  3. Ximelagatran: Direct Thrombin Inhibitor

    OpenAIRE

    Ho, Shir-Jing; Brighton, Tim A

    2006-01-01

    Warfarin sodium is an effective oral anticoagulant drug. However, warfarin has a narrow therapeutic window with significant risks of hemorrhage at therapeutic concentrations. Dosing is difficult and requires frequent monitoring. New oral anticoagulant agents are required to improve current anticoagulant therapy. Furthermore, while warfarin is effective in venous disease, it does not provide more than 60% risk reduction compared with placebo in venous thrombosis prophylaxis and considerably lo...

  4. Topic: A Literature Review

    OpenAIRE

    Dandan Zhang

    2009-01-01

    There is a wide discussion for Chinese topic structure and topic-sentence acquisition in Second Language Acquisition since Li & Thompson (1976). This paper reviews the contribution made by Li & Thompson on topic and later researches on the basis of them. The relationship between subject and topic also is concentrated.

  5. Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro.

    OpenAIRE

    Aoki, K.; Barker, C.; Danthinne, X.; Imperiale, M. J.; Nabel, G. J.

    1999-01-01

    BACKGROUND: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consuming. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. MATERIALS AND METHODS: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle plasmid containing a transgene and ade...

  6. Recombination Form and Epidemiology of HIV-1 Unique Recombinant Strains Identified in Yunnan, China

    OpenAIRE

    Li, Lin; Chen, LiLi; Yang, Shaomin; Li, Tianyi; Li, JianJian; Liu, Yongjian; Jia, Lei; Yang, Bihui; Bao, Zuoyi; Li, Hanping; Wang, Xiaolin; Zhuang, Daomin; Liu, Siyang; Li, Jingyun

    2012-01-01

    Several studies identified HIV-1 recombination in some distinct areas in Yunnan, China. However, no comprehensive studies had been fulfilled in the whole province up to now. To illustrate the epidemiology and recombination form of Unique Recombinant Forms (URFs) circulating in Yunnan, 788 HIV-1 positive individuals residing in 15 prefectures of Yunnan were randomly enrolled into the study. Full-length gag and pol genes were amplified and sequenced. Maximum likelihood tree was constructed for ...

  7. Gaussian Process Topic Models

    CERN Document Server

    Agovic, Amrudin

    2012-01-01

    We introduce Gaussian Process Topic Models (GPTMs), a new family of topic models which can leverage a kernel among documents while extracting correlated topics. GPTMs can be considered a systematic generalization of the Correlated Topic Models (CTMs) using ideas from Gaussian Process (GP) based embedding. Since GPTMs work with both a topic covariance matrix and a document kernel matrix, learning GPTMs involves a novel component-solving a suitable Sylvester equation capturing both topic and document dependencies. The efficacy of GPTMs is demonstrated with experiments evaluating the quality of both topic modeling and embedding.

  8. A pulsed ion-ion recombination laser

    Science.gov (United States)

    Petrash, G. G.; Zemskov, K. I.

    2003-01-01

    A new mechanism of pulsed laser oscillation based on ion-ion recombination is proposed. The advantages of utilizing this mechanism in a pulsed mode are considered, and the expected characteristics of the proposed lasers are estimated.

  9. Gene mapping with recombinant inbreds in maize

    International Nuclear Information System (INIS)

    Recombinant inbred lines of maize have been developed for the rapid mapping of molecular probes to chromosomal location. Two recombinant inbred families have been constructed from F2 populations of T232 x CM37 and CO159 x Tx303. A genetic map based largely on isozymes and restriction fragment length polymorphisms has been produced that covers virtually the entire maize genome. In order to map a new gene, an investigator has only to determine its allelic distribution among the recombinant inbred lines and then compare it by computer with the distributions of all previously mapped loci. The availability of the recombinant inbreds and the associated data base constitute an efficient means of mapping new molecular markers in maize

  10. Constraints from jet calculus on quark recombination

    International Nuclear Information System (INIS)

    Within the quantum-chromodynamic jet-calculus formalism, we deduce an equation describing recombination of quarks and antiquarks into mesons within a quark or gluon jet. This equation relates the recombination function R(x1,x2,x) used in current literature to the fragmentation function for producing that same meson out of the parton initiating the jet. We submit currently used recombination functions to our consistency test, taking as input mainly the u-quark fragmentation ''data'' into ?+ mesons. The qq-bar?? recombination functions popular in the literature are consistent with measured fragmentation functions, but they must be supplemented by other contributions to provide the full D?+/sub u/. We also discuss the Q2 dependence of the resulting fragmentation functions

  11. Sturmian theory of three-body recombination

    CERN Document Server

    Forrey, Robert C

    2013-01-01

    A Sturmian theory of three-body recombination is presented which provides a unified treatment of bound states, quasi-bound states, and continuum states. The Sturmian representation provides a numerical quadrature of the two-body continuum which may be used to generate a complete set of states within any desired three-body recombination pathway. Consequently, the dynamical calculation may be conveniently formulated using the simplest energy transfer mechanism, even for reactive systems which allow substantial rearrangement. For a three atom system which is not in thermal equilibrium, the steady-state recombination rate constants are shown to be weakly dependent on tunneling widths and pressure. Numerical results are presented for H2 recombination due to collisions with H and He using quantum mechanical coupled states and infinite order sudden approximations. These results may be used to remove some of the uncertainties that are currently limiting astrophysical simulations of primordial star formation.

  12. Production of recombinant allergens in plants

    OpenAIRE

    Schmidt, Georg; Gadermaier, Gabriele; Pertl, Heidi; Siegert, Marc; Oksman-caldentey, Kirsi-marja; Ritala, Anneli; Himly, Martin; Obermeyer, Gerhard; Ferreira, Fatima

    2008-01-01

    A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens h...

  13. Competition between ion recombination and scavenging

    International Nuclear Information System (INIS)

    Complete text of publication follows. In low permittivity solvents ion recombination is dominated by the effects of the relative drift of the ions caused by the Coulomb attraction. However, such systems are frequently investigated by scavenging methods. Since the work of Tachiya on electric field effects, drift has been known to affect the steady-state scavenging rate constant. However, during the recombination drift depends on the instantaneous distance between the ions, and is therefore inherently transient. This paper describes an investigation of this problem using simulation methods. It is found that, within the constraint of the diffusion approximation, there are conditions where the Smoluchowski time-dependent rate constant underestimates the degree to which scavenging intercepts geminate recombination. For this to be a substantial effect the initial distance between the ions must be relatively small (e.g. 4 nm) compared to the typical thermalisation distance of an electron (e.g. 8 nm). Simulations have been used to generate numerical time-dependent rate constants for scavenging. But these proved barely more successful than the Smoluchowski theory, in spite of having been calculated from the simulation results. Stratification of results by recombination time shows that there is a strong correlation between the recombination time and the scavenging time. It was hypothesised that this correlation arises through the strong transient drift as the ions approach one transient drift as the ions approach one another. This hypothesis was confirmed by the application of a novel simulation method in which the ion trajectories are simulated conditional on the recombination time. It was found that in every case the scavenging rate increases sharply just prior to recombination. This dependence of scavenging rate on recombination time is a fundamental breakdown of the assumptions underlying both the theory of diffusion kinetics and the IRT method. Nonetheless, a path decomposition method has been devised that allows IRT simulations to be corrected for this effect with good accuracy.

  14. SIR epidemics in monogamous populations with recombination

    OpenAIRE

    Zanette, Damián H

    2011-01-01

    We study the propagation of an SIR (susceptible--infectious--recovered) disease over an agent population which, at any instant, is fully divided into couples of agents. Couples are occasionally allowed to exchange their members. This process of couple recombination can compensate the instantaneous disconnection of the interaction pattern and thus allow for the propagation of the infection. We study the incidence of the disease as a function of its infectivity and of the recombination rate of ...

  15. Recombinant DNA production of spider silk proteins

    OpenAIRE

    Tokareva, Olena; Michalczechen-lacerda, Valqui?ria A.; Rech, Eli?bio L.; Kaplan, David L.

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce...

  16. Electron Recombination with Small Molecular Ions

    OpenAIRE

    Brinne Roos, Johanna

    2007-01-01

    In this thesis I have theoretically studied electron recombination processes with small molecular ions. In these kind of processes resonant states are involved. To calculate the potential energy for these states as a function of internuclear distance, structure calculations and scattering calculations have to be performed. So far I have been studying the ion-pair formation with in electron recombination with H3+. The cross section for this process has been calculated using different kind of m...

  17. Algae-based oral recombinant vaccines

    OpenAIRE

    ElizabethASpecht

    2014-01-01

    Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for “molecular pharming” in food crops has waned in the last decade due to difficulty...

  18. Mechanisms of nonhomologous recombination in mammalian cells.

    OpenAIRE

    Roth, D B; Porter, T N; Wilson, J. H.

    1985-01-01

    The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen...

  19. Gaussian Process Topic Models

    OpenAIRE

    Agovic, Amrudin; Banerjee, Arindam

    2012-01-01

    We introduce Gaussian Process Topic Models (GPTMs), a new family of topic models which can leverage a kernel among documents while extracting correlated topics. GPTMs can be considered a systematic generalization of the Correlated Topic Models (CTMs) using ideas from Gaussian Process (GP) based embedding. Since GPTMs work with both a topic covariance matrix and a document kernel matrix, learning GPTMs involves a novel component-solving a suitable Sylvester equation capturing...

  20. Topical report review status

    International Nuclear Information System (INIS)

    This report provides industry with procedures for submitting topical reports, guidance on how the U.S. Nuclear Regulatory Commission (NRC) processes and responds to topical report submittals, and an accounting, with review schedules, of all topical reports currently accepted for review schedules, of all topical reports currently accepted for review by the NRC. This report will be published annually. Each sponsoring organization with one or more topical reports accepted for review copies

  1. Electron thermalization in a recombining plasma

    International Nuclear Information System (INIS)

    Electron temperatures different from neutral and ion temperatures can persist for appreciable periods in a low-density plasma decaying by dissociative recombination. Such temperature differences can be initially due to the process generating the plasma. The energy dependence of the recombination cross section can also result in an elevation of the electron temperature during the recombination process. The time dependence of the electron temperature and recombination rate is calculated for a variety of experimental conditions. Corrections to the observed recombination rate for these effects are important only if the dimensionless parameter C = (16/3)(2m/sub e/kT/sub A//?)1/2 (n/sub A/ sigma/sub MT//m/sub A/ n/sub e/0 ?/sub R//sup A/) is less than unity. (T/sub A/, n/sub A/, and m/subA/ are the neutral particle temperature, number density, and mass, respectively; n/sube/0 and m/sube/ the electron initial number density and mass; sigma/sub MT/ the electron-neutral momentum-transfer cross section; and ?/subR//supA/ the electron-ion dissociative recombination rate constant at T/sub e/ = T/sub A/.) (U.S.)

  2. Recombination analysis based on the complete genome of bocavirus

    Directory of Open Access Journals (Sweden)

    Chen Shengxia

    2011-04-01

    Full Text Available Abstract Bocavirus include bovine parvovirus, minute virus of canine, porcine bocavirus, gorilla bocavirus, and Human bocaviruses 1-4 (HBoVs. Although recent reports showed that recombination happened in bocavirus, no systematical study investigated the recombination of bocavirus. The present study performed the phylogenetic and recombination analysis of bocavirus over the complete genomes available in GenBank. Results confirmed that recombination existed among bocavirus, including the likely inter-genotype recombination between HBoV1 and HBoV4, and intra-genotype recombination among HBoV2 variants. Moreover, it is the first report revealing the recombination that occurred between minute viruses of canine.

  3. Escherichia coli Type-1 topoisomerases: identification, mechanism, and role in recombination

    International Nuclear Information System (INIS)

    This paper is a discussion of the biological function and properties of Escherichia coli topoisomerases. Five topics are considered. First, in the analysis of the electrophoretic mobility of negatively supercoiled topoisomers, we discovered that adjacent DNA bands on a gel need not differ by one in linking number. Second, we identified a new E. coli topoisomerase, topoisomerase III. Third, the mechanism of catenation of duplex DNA rings by E. coli topoisomerase I has been elucidated. Fourth, we describe the involvement of a type-1 topoisomerase, the resolvase of Tn3, in site-specific recombination. In catenation and recombination by type-1 topoisomerases, duplex DNA is topologically rearranged via the breakage and rejoining of DNA a single strand at a time. Fifth, we consider what cellular processes might be particularly suited for the involvement of type-1, but not type-2, topoisomerases. 47 references, 16 figures, 1 table

  4. Workshop on Molecule Assisted Recombination and Other Processes in Fusion Divertor Plasmas, September 8-9, 2000

    International Nuclear Information System (INIS)

    A brief proceedings of the two-day Workshop on Molecule Assisted Recombination and Other Processes in Fusion Divertor Plasmas, organized by the ORNL Controlled Fusion Atomic Data Center on September 8-9, 2000, is presented. The conclusions and recommendations of the workshop regarding the topics discussed and the collaboration of the U.S. fusion research and atomic physics communities are also summarized

  5. Relation between dabigatran concentration, as assessed using the direct thrombin inhibitor assay, and activated clotting time/activated partial thromboplastin time in patients with atrial fibrillation.

    Science.gov (United States)

    Okubo, Kenji; Kuwahara, Taishi; Takagi, Katsumasa; Takigawa, Masateru; Nakajima, Jun; Watari, Yuji; Nakashima, Emiko; Yamao, Kazuya; Fujino, Tadashi; Tsutsui, Hiroyuki; Takahashi, Atsushi

    2015-06-15

    Dabigatran is a direct thrombin inhibitor that has been approved for preventing stroke in patients with atrial fibrillation. In this study, we aimed to assess the associations between the dabigatran concentration (calculated through plasma-diluted thrombin time, as assessed using the Hemoclot assay) and the activated partial thromboplastin time (aPTT) and activated clotting time (ACT). We recruited 137 patients with atrial fibrillation who were receiving a normal dose of dabigatran (300 mg/d) or a reduced dose of dabigatran (220 mg/d, usually administered to patients who were elderly, had moderate renal dysfunction, or who were also receiving verapamil). We then assessed the aPTT, ACT, and Hemoclot results of the patients and calculated the plasma dabigatran concentration. The mean plasma concentration of dabigatran was 127 ± 88 ng/ml, although no significant differences in dabigatran concentration, ACT, or aPTT were observed when we compared the 2 doses of dabigatran (300 or 220 mg/d). The dabigatran concentration was within the therapeutic levels in most patients, although a high value (>300 ng/ml) was observed in several patients, which indicated a high risk of bleeding. The dabigatran concentration was strongly and positively correlated with ACT and aPTT (r = 0.87, p <0.001; and r = 0.76, p <0.001; respectively). Multivariate analysis revealed that verapamil use was independently associated with elevated dabigatran concentrations (p <0.001). Therefore, ACT and aPTT may be useful for bedside assessment of the anticoagulant activity of dabigatran, and verapamil use may be a risk factor for elevated dabigatran concentrations. PMID:25918026

  6. Recombination Form and Epidemiology of HIV-1 Unique Recombinant Strains Identified in Yunnan, China

    Science.gov (United States)

    Li, Lin; Chen, Lili; Yang, Shaomin; Li, Tianyi; Li, Jianjian; Liu, Yongjian; Jia, Lei; Yang, Bihui; Bao, Zuoyi; Li, Hanping; Wang, Xiaolin; Zhuang, Daomin; Liu, Siyang; Li, Jingyun

    2012-01-01

    Several studies identified HIV-1 recombination in some distinct areas in Yunnan, China. However, no comprehensive studies had been fulfilled in the whole province up to now. To illustrate the epidemiology and recombination form of Unique Recombinant Forms (URFs) circulating in Yunnan, 788 HIV-1 positive individuals residing in 15 prefectures of Yunnan were randomly enrolled into the study. Full-length gag and pol genes were amplified and sequenced. Maximum likelihood tree was constructed for phylogenetic analysis. Recombinant breakpoints and genomic schematics were identified with online software jpHMM. 63 (10.2%) unique recombinant strains were identified from 617 strains with subtypes. The URFs distributed significantly differently among prefectures (Pearson chi-square test, P<0.05). IDUs contained more URFs than sexual transmitted population (Pearson chi-square test, P<0.05). Two main recombinant forms were identified by considering the presence of CRF01_AE segments in full length gag-pol genes, which were B?/C and B?/C/CRF01-AE recombinants. Three clusters were identified in the ML tree which contained more than three sequences and supported by high bootstrap values. One CRF was identified. Many of URFs contained identical breakpoints. The results will contribute to our understanding on HIV recombination and provide clues to the identification of potential CRFs in China. PMID:23056447

  7. Recombination of electrons with an anisotropic velocity distribution. Continuation of recombination continuum to series lines

    International Nuclear Information System (INIS)

    For ions in recombination with electrons with directional motion, the recombination continuum to a J = 0 state is ? polarized, and this polarization characteristic should continue across the ionization threshold down to the series lines. A Monte Carlo calculation has been performed for electron collisions on a classical atom in excited states. No evidence is found to support the above conclusion. (author)

  8. DNA RECOMBINATION IN EUCARYOTIC CELLS BY THE BACTERIOPHAGE PHIC31 RECOMBINATION SYSTEM",

    Science.gov (United States)

    This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the ph:C31 integrase, that can mediate recombination between th...

  9. Graded recombination layers for multijunction photovoltaics.

    Science.gov (United States)

    Koleilat, Ghada I; Wang, Xihua; Sargent, Edward H

    2012-06-13

    Multijunction devices consist of a stack of semiconductor junctions having bandgaps tuned across a broad spectrum. In solar cells this concept is used to increase the efficiency of photovoltaic harvesting, while light emitters and detectors use it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron current from the next cell. We recently reported a tandem solar cell in which the recombination layer was implemented using a progression of n-type oxides whose doping densities and work functions serve to connect, with negligible resistive loss at solar current densities, the constituent cells. Here we present the generalized conditions for design of efficient graded recombination layer solar devices. We report the number of interlayers and the requirements on work function and doping of each interlayer, to bridge an work function difference as high as 1.6 eV. We also find solutions that minimize the doping required of the interlayers in order to minimize optical absorption due to free carriers in the graded recombination layer (GRL). We demonstrate a family of new GRL designs experimentally and highlight the benefits of the progression of dopings and work functions in the interlayers. PMID:22554234

  10. Recombination in Eukaryotic Single Stranded DNA Viruses

    Directory of Open Access Journals (Sweden)

    Philippe Roumagnac

    2011-09-01

    Full Text Available Although single stranded (ss DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution.

  11. Polyploidization increases meiotic recombination frequency in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Rehmsmeier Marc

    2011-04-01

    Full Text Available Abstract Background Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

  12. Comparison of recombination models in organic bulk heterojunction solar cells

    International Nuclear Information System (INIS)

    Recombination in bulk-heterojunction (BHJ) organic solar cells is the key loss mechanism, and it directly affects characteristic parameters such as power conversion efficiency, short-circuit current, open-circuit voltage, and fill factor. However, which recombination mechanism dominates the loss in organic materials is unclear at present. In this work, we simulate state-of-art BHJ solar cells using five recombination models, including direct recombination, Langevin recombination, charge transfer state recombination, trap-assisted recombination, and recombination via tail. All processes are strongly dependent on charge carrier mobility and exhibit a similar recombination distribution in active layer. For high mobilities, all models present a similar behavior along with the increased mobilities, whereas, there are slight differences in open-circuit voltage between trap/tail model and other ones at lower mobilities, resulting from the interaction between photo-carriers and dark-carriers

  13. Diclofenac Topical (osteoarthritis pain)

    Science.gov (United States)

    Diclofenac topical gel (Voltaren) is used to relieve pain from osteoarthritis (arthritis caused by a breakdown of ... the knees, ankles, feet, elbows, wrists, and hands. Diclofenac topical liquid (Pennsaid) is used to relieve osteoarthritis ...

  14. Non-gaussianities from perturbing recombination

    International Nuclear Information System (INIS)

    We approximately compute the bispectrum induced on the CMB temperature by fluctuations in the standard recombination epoch. Of all the second order sources that can induce non-Gaussianity during recombination, we concentrate on those proportional to the perturbation in the free electron density, which is about a factor of 5 larger than the other first order perturbations. This term induces some non-Gaussianity by delaying the time of recombination and by changing the photon diffusion scale. We find that the signal is not scale invariant, peaked on squeezed triangles with the smaller multipole around the scale of the first acoustic peak, and that its size corresponds to an effective fNL ? ?3.5, which could be marginally detected by Planck if both temperature and polarization are measured

  15. Recombinant human erythropoietin in sports: a review

    Directory of Open Access Journals (Sweden)

    Rafael Maia de Almeida Bento

    2003-06-01

    Full Text Available Erythropoietin is an endogenous hormone of glicoproteic nature secreted by the kidneys and is the main regulator of the erythropoiesis. An alteration in its production generates a disturbance in the plasmatic concentration giving rise to several types of pathologies related to the hematopoietic system. The recombinant forms of erythropoietin have indiscriminately been used by athletes, mainly in endurance sports, by increasing the erythrocytes concentration, generating a better delivery of oxygen to the muscle tissue. The administration of recombinant erythropoietin was prohibited by the International Olympic Committee and its use considered as doping. This review has the intention to describe the physical, biological and pharmacokinetic properties of the endogenous erythropoietin, as well as its recombinant form, describing also its use in sports and the process of searching methodologies for its detection in doping control.

  16. Recombinant microorganisms for increased production of organic acids

    Science.gov (United States)

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  17. Recombinant microorganisms for increased production of organic acids

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Jian (East Lansing, MI); Kleff, Susanne (East Lansing, MI); Guettler, Michael V. (Holt, MI)

    2012-02-21

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  18. Dissociative recombination of CF+: Experiment and theory

    International Nuclear Information System (INIS)

    We present results from our recent studies of the dissociative recombination of the CF+ cation. On one hand, dissociative recombination was measured with 3 MeV CF+ ions in the heavy-ion Test Storage Ring in Heidelberg, using the twin electron beam configuration with an electron cooler and a separated electron target for collision measurements. In this experiment, the low temperatures of the electron beam provided by a photocathode (temperature in co-moving frame below 1 meV) account for a fast kinetic cooling of the heavy-ion beam and a high resolution in the measured rate coefficients. Fragment imaging measurements show a complete switching of the dissociation route by only a small change of the collision energy and the disappearance of neutral Rydberg product states on crossing the DE threshold. On the other hand, extensive calculations of energy positions and autoionization widths for the doubly excited states of CF between the first and second ionization thresholds have been obtained from electron scattering calculations using the complex Kohn variational method, followed by calculations of the dissociative recombination process with the multichannel quantum defect theory. In preliminary computations, only the first dissociative state in each molecular symmetry, which lies closest in energy to the ion potential at its equilibrium internuclear separation, and thus is dominant for the low-energy dissociative recombination, was included. Althougative recombination, was included. Although only the direct mechanism of dissociative recombination reaction has been considered in this step, the size and the shape of the DR rate coefficient are already well reproduced.

  19. Dielectronic recombination measurements of multicharged ions

    International Nuclear Information System (INIS)

    Dielectronic recombination rates have been measured for several charge states of isoelectronic Li-like, Be-like, B-like, and Na-like ions. The amount of electron capture attending the passage of MeV/nucleon ion beams through a collinear, magnetically confined space-charge-limited electron beam as a function of relative energy has been observed. The experimental rates are consistent in magnitude and shape with rates determined from distorted-wave calculations of the dielectronic-recombination cross sections. 72 refs., 6 figs

  20. Dielectronic recombination measurements of multicharged ions

    Energy Technology Data Exchange (ETDEWEB)

    Dittner, P.F.

    1987-01-01

    Dielectronic recombination rates have been measured for several charge states of isoelectronic Li-like, Be-like, B-like, and Na-like ions. The amount of electron capture attending the passage of MeV/nucleon ion beams through a collinear, magnetically confined space-charge-limited electron beam as a function of relative energy has been observed. The experimental rates are consistent in magnitude and shape with rates determined from distorted-wave calculations of the dielectronic-recombination cross sections. 72 refs., 6 figs.

  1. Strategies of Loop Recombination in Ciliates

    CERN Document Server

    Brijder, R; Muskulus, M; Brijder, Robert; Hoogeboom, Hendrik Jan; Muskulus, Michael

    2006-01-01

    Gene assembly in ciliates is an extremely involved DNA transformation process, which transforms a nucleus, the micronucleus, to another functionally different nucleus, the macronucleus. In this paper we characterize which loop recombination operations (one of the three types of molecular operations that accomplish gene assembly) can possibly be applied in the transformation of a given gene from its micronuclear form to its macronuclear form. We also characterize in which order these loop recombination operations are applicable. This is done in the abstract and more general setting of so-called legal strings.

  2. Size effects on generation recombination noise

    CERN Document Server

    Gomila, G

    2002-01-01

    We carry out an analytical theory of generation-recombination noise for a two level resistor model which goes beyond those presently available by including the effects of both space charge fluctuations and diffusion current. Finite size effects are found responsible for the saturation of the low frequency current spectral density at high enough applied voltages. The saturation behaviour is controlled essentially by the correlations coming from the long range Coulomb interaction. It is suggested that the saturation of the current fluctuations for high voltage bias constitutes a general feature of generation-recombination noise.

  3. Theoretical models for recombination in expanding gas

    International Nuclear Information System (INIS)

    In laser isotope separation of atomic uranium, one is confronted with the theoretical problem of estimating the concentration of thermally ionized uranium atoms. To investigate this problem theoretical models for recombination in an expanding gas and in the absence of local thermal equilibrium have been constructed. The expansion of the gas is described by soluble models of the hydrodynamic equation, and the recombination by rate equations. General results for the freezing effect for the suitable ranges of the gas parameters are obtained. The impossibility of thermal equilibrium in expanding two-component systems is proven

  4. Characterization of Bacillus subtilis recombinational pathways.

    OpenAIRE

    Alonso, J.C.; Lüder, G; Tailor, R H

    1991-01-01

    Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mu...

  5. Freshman Health Topics

    Science.gov (United States)

    Hovde, Karen

    2011-01-01

    This article examines a cluster of health topics that are frequently selected by students in lower division classes. Topics address issues relating to addictive substances, including alcohol and tobacco, eating disorders, obesity, and dieting. Analysis of the topics examines their interrelationships and organization in the reference literature.…

  6. Avoidance of Protein Fold Disruption in Natural Virus Recombinants

    Science.gov (United States)

    Lefeuvre, Pierre; Lett, Jean-Michel; Reynaud, Bernard; Martin, Darren P

    2007-01-01

    With the development of reliable recombination detection tools and an increasing number of available genome sequences, many studies have reported evidence of recombination in a wide range of virus genera. Recombination is apparently a major mechanism in virus evolution, allowing viruses to evolve more quickly by providing immediate direct access to many more areas of a sequence space than are accessible by mutation alone. Recombination has been widely described amongst the insect-transmitted plant viruses in the genus Begomovirus (family Geminiviridae), with potential recombination hot- and cold-spots also having been identified. Nevertheless, because very little is understood about either the biochemical predispositions of different genomic regions to recombine or what makes some recombinants more viable than others, the sources of the evolutionary and biochemical forces shaping distinctive recombination patterns observed in nature remain obscure. Here we present a detailed analysis of unique recombination events detectable in the DNA-A and DNA-A-like genome components of bipartite and monopartite begomoviruses. We demonstrate both that recombination breakpoint hot- and cold-spots are conserved between the two groups of viruses, and that patterns of sequence exchange amongst the genomes are obviously non-random. Using a computational technique designed to predict structural perturbations in chimaeric proteins, we demonstrate that observed recombination events tend to be less disruptive than sets of simulated ones. Purifying selection acting against natural recombinants expressing improperly folded chimaeric proteins is therefore a major determinant of natural recombination patterns in begomoviruses. PMID:18052529

  7. In thrombin stimulated human platelets Citalopram, Promethazine, Risperidone, and Ziprasidone, but not Diazepam, may exert their pharmacological effects also through intercalation in membrane phospholipids in a receptor-independent manner

    OpenAIRE

    Oruch, Ramadhan; Hodneland, Erlend; Pryme, Ian F.; Holmsen, Holm

    2009-01-01

    Intercalation of drugs in the platelet membrane affects phospholipid-requiring enzymatic processes according to the drugs’ intercalation capability. We investigated effects of Promethazine, Citalopram, Ziprasidone, Risperidone, and Diazepam on phospholipase A2 (PLA2) and polyphosphoinositide (PPI) metabolism in thrombin-stimulated human platelets. We also examined effects of the drugs on monolayers of glycerophospholipids using the Langmuir technique. Diazepam did not influence PLA2 activit...

  8. The efficacy of recombinant versus urinary HCG in ART outcome

    OpenAIRE

    Eftekhar, Maryam; Khalili, Mohammad Ali; Rahmani, Elham

    2012-01-01

    Background: Human chorionic gonadotropin (HCG) has been used as a replacement for the mid-cycle luteinizing hormone (LH) surge for several years. The recent arrival of recombinant DNA technology has made recombinant HCG (rHCG) accessible.

  9. Catalytic hydrogen recombination for nuclear containments

    International Nuclear Information System (INIS)

    Catalytic recombiners appear to be a credible option for hydrogen mitigation in nuclear containments. The passive operation, versatility and ease of back fitting are appealing for existing stations and new designs. Recently, a generation of wet-proofed catalyst materials have been developed at AECL which are highly specific to H2-O2, are active at ambient temperatures and are being evaluated for containment applications. Two types of catalytic recombiners were evaluated for hydrogen removal in containments based on the AECL catalyst. The first is a catalytic combustor for application in existing air streams such as provided by fans or ventilation systems. The second is an autocatalytic recombiner which uses the enthalpy of reaction to produce natural convective flow over the catalyst elements. Intermediate-scale results obtained in 6 m3 and 10 m3 spherical and cylindrical vessels are given to demonstrate self-starting limits, operating limits, removal capacity, scaling parameters, flow resistance, mixing behaviour in the vicinity of an operating recombiner and sensitivity to poisoning, fouling and radiation. (author). 13 refs., 10 figs

  10. Magnetic affinity separation of recombinant fusion proteins.

    Czech Academy of Sciences Publication Activity Database

    Šafa?ík, Ivo; Šafa?íková, Miroslava

    2010-01-01

    Ro?. 38, ?. 1 (2010), s. 1-7. ISSN 1303-5002 R&D Projects: GA MŠk(CZ) OC 157; GA MPO(CZ) 2A-1TP1/094 Institutional research plan: CEZ:AV0Z60870520 Keywords : recombinant fusion proteins * affinity tags * magnetic separation Subject RIV: CE - Biochemistry

  11. Mismatch repair during homologous and homeologous recombination.

    Science.gov (United States)

    Spies, Maria; Fishel, Richard

    2015-03-01

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans. PMID:25731766

  12. Recombination times in germanium under high pressure

    International Nuclear Information System (INIS)

    The influence of pressure on a well defined recombination process was studied. The centres were introduced by ?irradiation and the lifetime determined by the decay time of photoconductivity. An optical pressure vessel is described which allows for a hydrostatic variation of 3000 bars. The diffusion constant and lifetime measurements are presented and analysed. (V.J.C.)

  13. Selected techniques in recombinant DNA technology

    International Nuclear Information System (INIS)

    Recombined DNA technology comprises a complex of techniques in the fields of nucleic acid biochemistry and molecular biology. This presentation gives an introduction, a brief description and example of the procedures of some of the basic techniques in the DNA cloning work currently used. 8 refs

  14. Theory of dielectronic recombination and plasma effects

    International Nuclear Information System (INIS)

    Current status of the various theoretical approaches to calculation of dielectronic recombination rates is summarized, with emphasis on the available data base and on the plasma effects of both the plasma ion (and external) fields and plasma electron collisional effects which seriously affect the rates and complicate compilation of data. (author)

  15. Asthma and Therapeutics: Recombinant Therapies in Asthma

    Directory of Open Access Journals (Sweden)

    Cockcroft Donald W

    2005-03-01

    Full Text Available Abstract Numerous recombinant therapies are being investigated for the treatment of asthma. This report reviews the current status of several of these novel agents. Anti-immunoglobulin (IgE (omalizumab, Xolair markedly inhibits all aspects of the allergen challenge in subjects who have reduction of free serum IgE to undetectable levels. Several clinical studies in atopic asthma have demonstrated benefit by improved symptoms and lung function and a reduction in corticosteroid requirements. Early use in atopic asthmatics may be even more effective. Several approaches target interleukin (IL-4. Soluble IL-4 receptor has been shown to effectively replace inhaled corticosteroid; further studies are under way. Recombinant anti-IL-5 and recombinant IL-12 inhibit blood and sputum eosinophils and allergen-induced eosinophilia without any effect on airway responsiveness, allergen-induced airway responses, or allergen-induced airway hyperresponsiveness. Efalizumab, a recombinant antibody that inhibits lymphocyte trafficking, is effective in psoriasis. A bronchoprovocation study showed a reduction in allergen-induced late asthmatic response and allergen-induced eosinophilia, which suggests that it should be effective in clinical asthma. These exciting novel therapies provide not only promise of new therapies for asthma but also valuable tools for investigation of asthma mechanisms.

  16. Dielectronic recombination cross section for Boron III

    International Nuclear Information System (INIS)

    The dielectronic recombination cross section has been estimated for the B2+ target and projectile electron energies in the range 0.1approx.0.5 Ry, corresponding to the 2s?2p (?n = 0) excitation accompanied by capture of the continuum electron into a high Rydberg state (nl ). Contributions from n-19 cm2

  17. On the radiative recombination of the electrons

    International Nuclear Information System (INIS)

    A process of the radiative recombination of the non-relativistic electron with a hydrogen-like ion is considered. A simple expression is derived for the total cross section of the process with the use of the analytic properties of the electron Green function in a Coulomb field. The dipole approximation is used

  18. Dielectronic recombination cross section for Cl7+

    International Nuclear Information System (INIS)

    The resonant dielectronic recombination cross section is estimated for a Cl7+ target interacting with incident electrons in the 10approx.20-rydberg range. Cross sections averaged over energy bins of size ?E = 0.1 Ry are approximately (1--3) x 10-19 cm2

  19. Atom and ion recombination in spurs

    International Nuclear Information System (INIS)

    The proportion of singlets generated during a recombination of atoms or ions in a radiolytic spur reaction is examined from the viewpoint of a combinational analysis. The results obtained differ from other, already conflicting, published work. The problem is formulated in a way that enables spin relaxation effects to be incorporated. (author)

  20. Algae-based oral recombinant vaccines

    Directory of Open Access Journals (Sweden)

    ElizabethASpecht

    2014-02-01

    Full Text Available Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for “molecular pharming” in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae are poised to become the next candidate in recombinant subunit vaccine production, and they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally-delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and system immune reactivity.

  1. Gas recombination assembly for electrochemical cells

    Science.gov (United States)

    Levy, Isaac (New Fairfield, CT); Charkey, Allen (Brookfield, CT)

    1989-01-01

    An assembly for recombining gases generated in electrochemical cells wherein a catalyst strip is enveloped within a hydrophobic, gas-porous film which, in turn, is encased between gas-porous, metallic layers. The sandwich construction of metallic layers and film is formed into a spiral with a tab for connection to the cell.

  2. Syntactic Topic Models

    CERN Document Server

    Boyd-Graber, Jordan

    2010-01-01

    The syntactic topic model (STM) is a Bayesian nonparametric model of language that discovers latent distributions of words (topics) that are both semantically and syntactically coherent. The STM models dependency parsed corpora where sentences are grouped into documents. It assumes that each word is drawn from a latent topic chosen by combining document-level features and the local syntactic context. Each document has a distribution over latent topics, as in topic models, which provides the semantic consistency. Each element in the dependency parse tree also has a distribution over the topics of its children, as in latent-state syntax models, which provides the syntactic consistency. These distributions are convolved so that the topic of each word is likely under both its document and syntactic context. We derive a fast posterior inference algorithm based on variational methods. We report qualitative and quantitative studies on both synthetic data and hand-parsed documents. We show that the STM is a more pred...

  3. A general theoretical description of N-body recombination

    CERN Document Server

    Mehta, N P; D'Incao, J P; Von Stecher, J; Greene, Chris H

    2009-01-01

    We present a formula for the cross section and event rate constant describing recombination of N particles in terms of general S-matrix elements. Our result immediately yields the generalized Wigner threshold scaling for the recombination of N bosons. We find that four-boson recombination is resonantly enhanced by the presence of metastable states in the entrance channel. Hence, recombination into a trimer-atom channel could be an effective mechanism for the formation of Efimov trimers.

  4. Studies of autoionizing states relevant to dielectronic recombination. Progress report, July 1, 1991--June 30, 1994

    International Nuclear Information System (INIS)

    The goal of this research program supported by grant DE-FG05-85-ER13394 is to study the properties of autoionizing states to understand in detail dielectronic recombination of ions and electrons. During the period of the present grant, July 1, 1991--June 30, 1994, we have put substantial effort into the study of autoionizing states in Mg. The first topic we have investigated is the angular distribution of electrons ejected from the autoionizing Mg 3pns and 3pnd states. Both of these sets of measurements provide more stringent tests of the K matrix calculations than do measurements of total cross sections. The second topic is the effect of static and microwave electric fields on autoionizing states. Our previous measurements in Ba have shown the profound influence of electric fields on autoionization rates and we have made extensive new measurements in both Ba and Mg. Electric fields are of real importance for dielectronic recombination, since they are present as macroscopic fields in beam experiments and as microfields in plasmas. Finally, we have begun time resolved measurements. We have set up a picosecond laser system, and we have just finished the first experiment with it. Specifically we have used picosecond excitation from a bound Mg 3pnd Rydberg wave packet to the autoionizing 3pnd states to show explicitly the correlation between the spatial location of the Rydberg electron and the frequency of the exciting light

  5. Experimental cross sections for electron-impact ionization and electron-ion recombination

    International Nuclear Information System (INIS)

    Full text: Recombination of highly charged ions with free electrons has extensively been studied in experiments at heavy-ion storage rings. Dielectronic recombination of highly charged iron ions is a special topic within the recombination study programme, and is the subject of a long-standing research collaboration project between groups from Giessen University, Max-Planck-Institute for Nuclear Physics in Heidelberg, and Columbia University. The Heidelberg heavy ion storage ring TSR has been employed to measure absolute high resolution cross sections and rate coefficients with uncertainties of the order of 20%. The talk will provide an overview of these experiments, particularly the data on Fe13+ and Fe14+. Electron-impact ionization experiments are carried out at Giessen using a crossed-beams technique. Single and multiple ionization has been investigated for numerous singly and multiply charged ions. Absolute cross sections are being measured for ions in the xenon and tin isonuclear sequences and high-resolution energy scans of normalized ion yields have been recorded to provide the detailed energy dependences of electron-impact ionization cross sections. Both single and multiple ionization processes are addressed. The present status of these experiments will be discussed, particularly the cross section measurements for single ionization of Xe+ through to Xe15+. (author)

  6. A molecular recombination map of Antirrhinum majus

    Directory of Open Access Journals (Sweden)

    Hudson Andrew

    2010-12-01

    Full Text Available Abstract Background Genetic recombination maps provide important frameworks for comparative genomics, identifying gene functions, assembling genome sequences and for breeding. The molecular recombination map currently available for the model eudicot Antirrhinum majus is the result of a cross with Antirrhinum molle, limiting its usefulness within A. majus. Results We created a molecular linkage map of A. majus based on segregation of markers in the F2 population of two inbred lab strains of A. majus. The resulting map consisted of over 300 markers in eight linkage groups, which could be aligned with a classical recombination map and the A. majus karyotype. The distribution of recombination frequencies and distorted transmission of parental alleles differed from those of a previous inter-species hybrid. The differences varied in magnitude and direction between chromosomes, suggesting that they had multiple causes. The map, which covered an estimated of 95% of the genome with an average interval of 2 cM, was used to analyze the distribution of a newly discovered family of MITE transposons and tested for its utility in positioning seven mutations that affect aspects of plant size. Conclusions The current map has an estimated interval of 1.28 Mb between markers. It shows a lower level of transmission ratio distortion and a longer length than the previous inter-species map, making it potentially more useful. The molecular recombination map further indicates that the IDLE MITE transposons are distributed throughout the genome and are relatively stable. The map proved effective in mapping classical morphological mutations of A. majus.

  7. Topic Tracking with Dynamic Topic Model and Topic-based Weighting Method

    OpenAIRE

    Xiaoyan Zhang; Ting Wang

    2010-01-01

    In topic tracking, a topic is usually described by several stories. How to represent a topic is always an issue and a difficult problem in the research on topic tracking. To emphasis the topic in stories, we provide an improved topic-based tf*idf weighting method to measure the topical importance of the features in the representation model. To overcome the topic drift problem and filter the noise existed in the tracked topic description, a dynamic topic model is proposed based on the static m...

  8. Regulation of homologous recombination at telomeres in budding yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Lisby, Michael

    2010-01-01

    Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae.

  9. Trans-Centromere Effects on Meiotic Recombination in the Zebrafish

    OpenAIRE

    Demarest, Bradley L.; Horsley, Wyatt H.; Locke, Erin E.; Boucher, Kenneth; Grunwald, David J.; Trede, Nikolaus S.

    2011-01-01

    We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm's centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.

  10. Oligonucleotide recombination enabled site-specific mutagenesis in bacteria.

    Science.gov (United States)

    Swingle, Bryan M

    2013-01-01

    Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligonucleotide recombination is one type of recombineering that uses ssDNA oligonucleotides to direct chromosomal mutations. Oligo recombination occurs without addition of any exogenous functions, making this approach potentially useful in many different bacteria. Here we describe the basic technique for constructing a site-specific genomic mutation in Pseudomonas syringae. PMID:23423893

  11. Isolation of a recombination-deficient mutant of Rhodopseudomonas capsulata

    Energy Technology Data Exchange (ETDEWEB)

    Genthner, F.J.; Wall, J.D.

    1984-12-01

    To facilitate genetic analysis in the purple photosynthetic bacterium Rhodopseudomonas capsulata, a recombination-deficient derivative was sought. A UV irradiation-sensitive mutant (FG106F) was isolated after mutagenesis, and two procedures were used to determine the recombinational capacity of the mutant. First, recombinants were not detected after transduction of this derivative by the phage-like vector gene transfer agent. Second, an R-prime plasmid containing appropriately marked genes for photosynthesis was introduced by conjugation, and again no recombinants were observed. Additional phenotypes displayed by the mutant that are characteristic of a defect in recombination were an increased sensitivity to DNA-damaging antibiotics and a tendency to filament.

  12. Radiative recombination of hot carriers in narrow-gap semiconductors

    Energy Technology Data Exchange (ETDEWEB)

    Pavlov, N. V.; Zegrya, G. G., E-mail: Zegrya@theory.ioffe.ru [Russian Academy of Sciences, Ioffe Physical Technical Institute (Russian Federation)

    2012-01-15

    The mechanism of the radiative recombination of hot carriers in narrow-gap semiconductors is analyzed using the example of indium antimonide. It is shown that the CHCC Auger recombination process may lead to pronounced carrier heating at high excitation levels. The distribution functions and concentrations of hot carriers are determined. The radiative recombination rate of hot carriers and the radiation gain coefficient are calculated in terms of the Kane model. It is demonstrated that the radiative recombination of hot carriers will make a substantial contribution to the total radiative recombination rate at high carrier concentrations.

  13. Experimental radioimmunoimaging of 99mTc monoclonal anti-activated platelet antibody SZ-51 in dogs with arterial and venous thrombin

    International Nuclear Information System (INIS)

    In experimental canine models of femoral arterial and venous thrombosis, the feasibility of thrombus imaging with 99mTc labeled monoclonal antibody SZ-51 to an ?-granule membrane protein (GMP-140) on the surface of activated platelets was investigated. Both the arterial and venous thrombin were clearly discernible at 2-8h after injection. The optimal imaging time was 4-6h. These results were confirmed by in vivo images of thrombosed arteries and veins followed by killing the dogs at 8h post injection. Quantitative imaging analysis showed that the ratios between the thrombus and the opposite vessel were increased strikingly over time. The ratios of thrombus to blood or surrounding muscle were 57.09 +- 12.24 and 177.46 +-9.00 for arterial thrombosis and 6.43 +- 4.70 and 83.22 +- 68.98 for venous one at the time of sacrifice, respectively. Radiolabeled antibody was cleared from the blood with T1/2? of 16.25 +- 13.39 min T1/2? of 7.28 +- 1.15 h. These results show that it is feasible to noninvasively detect the thrombus formation in vivo using 99mTc labeled monoclonal antibody SZ-51

  14. Platelets from children are hyper-responsive to activation by thrombin receptor activator peptide and adenosine diphosphate compared to platelets from adults.

    Science.gov (United States)

    Yip, Christina; Linden, Matthew D; Attard, Chantal; Monagle, Paul; Ignjatovic, Vera

    2015-02-01

    Platelets are crucial subcellular elements of haemostasis at sites of vascular injury and are also known to be immune mediators in pathological thrombosis. Despite the integral role of platelets in many disease processes, there is very little information available on platelet function and response to agonists in healthy children. We recently reported important differences in the interaction of platelets with monocytes in the circulation, including increased formation of monocyte-platelet aggregates (MPAs) without concomitant increase in P-selectin expression. Our current study investigates parameters of platelet activation (PAC-1 binding and P-selectin expression) and MPA formation in response to a range of physiologically relevant platelet agonists in healthy children compared to healthy adults. All parameters were significantly higher in children in response to sub-maximal concentrations of thrombin receptor activator peptide and adenosine diphosphate, reflecting an age-specific difference in agonist-stimulated platelet reactivity in children. The results of our study challenge the general assumption that platelet reactivity in children is similar to adults. This finding is fundamental to investigating the role of platelets in diseases of childhood and pathogenesis of adult-based diseases that have their origins in childhood. Our findings underscore the need for age-specific reference ranges for platelet function in children rather than extrapolation from adult data. PMID:25266817

  15. An electrochemical aptasensor for thrombin using synergetic catalysis of enzyme and porous Au@Pd core-shell nanostructures for signal amplification.

    Science.gov (United States)

    Xu, Wenju; Yi, Huayu; Yuan, Yali; Jing, Pei; Chai, Yaqin; Yuan, Ruo; Wilson, George S

    2015-02-15

    In this work, a sensitive electrochemical aptasensor for thrombin (TB) based on synergetic catalysis of enzyme and porous Au@Pd core-shell nanostructure has been constructed. With the advantages of large surface area and outstanding catalytic performance, porous Au@Pd core-shell nanostructures were firstly employed as the nanocarrier for the immobilization of electroactive toluidine blue (Tb), hemin/G-quadruplex formed by intercalating hemin into the TB aptamer (TBA) and glucose oxidase (GOx). As a certain amount of glucose was added into the detection cell, GOx rapidly catalyzed the oxidation of glucose, coupling with the local generation of H2O2 in the presence of dissolved O2. Then, porous Au@Pd nanoparticles and hemin/G-quadruplex as the peroxidase mimics efficiently catalyzed the reduction of H2O2, amplifying the electrochemical signal and improving the sensitivity. Finally, a detection limit of 0.037pM for TB was achieved. The excellent performance of the aptasensor indicated its promising prospect as a valuable tool in simple and cost-effective TB detection in clinical application. PMID:25280342

  16. Porous platinum nanotubes labeled with hemin/G-quadruplex based electrochemical aptasensor for sensitive thrombin analysis via the cascade signal amplification.

    Science.gov (United States)

    Sun, Aili; Qi, Qingan; Wang, Xuannian; Bie, Ping

    2014-07-15

    For the first time, a sensitive electrochemical aptasensor for thrombin (TB) was developed by using porous platinum nanotubes (PtNTs) labeled with hemin/G-quadruplex and glucose dehydrogenase (GDH) as labels. Porous PtNTs with large surface area exhibited the peroxidase-like activity. Coupling with GDH and hemin/G-quadruplex as NADH oxidase and HRP-mimicking DNAzyme, the cascade signal amplification was achieved by the following ways: in the presence of glucose and NAD(+) in the working buffer, GDH electrocatalyzed the oxidation of glucose with the production of NADH. Then, hemin/G-quadruplex as NADH oxidase catalyzed the oxidation of NADH to in situ generate H2O2. Based on the corporate electrocatalysis of PtNTs and hemin/G-quadruplex toward H2O2, the electrochemical signal was significantly amplified, allowing the detection limit of TB down to 0.15 pM level. Moreover, the proposed strategy was simple because the intercalated hemin offered the readout signal, avoiding the adding of additional redox mediator as signal donator. Such an electrochemical aptasensor is highly promising for sensitive detection of other proteins in clinical diagnostics. PMID:24534575

  17. Immobilization of the direct thrombin inhibitor-bivalirudin on 316L stainless steel via polydopamine and the resulting effects on hemocompatibility in vitro.

    Science.gov (United States)

    Lu, Lei; Li, Quan-Li; Maitz, Manfred F; Chen, Jia-Long; Huang, Nan

    2012-09-01

    Bivalirudin (BV), a peptidic direct thrombin inhibitor, derived from hirudin, has gained increasing interest in clinical anticoagulant therapy in the recent years. In this work, a hemocompatible surface was prepared by immobilization of BV on 316L stainless steel (SS) using a bonding layer of polydopamine (DA). X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the surfaces to characterize polydopamine intermediate layer and the immobilized BV. The quantity of bound BV was measured by quartz crystal microbalance (QCM). The hemocompatibility in vitro was evaluated by coagulating time of activated partial thromboplastin time (aPTT) and prothrombin time (PT) assay, platelet adhesion and activation, fibrinogen adsorption, and activation and whole blood test. The effect of sterilizing method on the bioactivity of immobilized BV was also evaluated. The results showed that BVs were successfully immobilized on SS surface with the DA interlayer at a density of 98 ng/cm(2) . BV coating surface prolonged aPTT and PT, inhibited the activation of platelet and fibrinogen significantly. Sterilization by ultraviolet radiation was possible with only marginal loss of activity. Thus, the approach described here may provide a basis for the preparation of 316L SS surface modification for use in cardiovascular implants. PMID:22566466

  18. The Thrombin-Derived Host Defense Peptide GKY25 Inhibits Endotoxin-Induced Responses through Interactions with Lipopolysaccharide and Macrophages/Monocytes.

    Science.gov (United States)

    Hansen, Finja C; Kalle-Brune, Martina; van der Plas, Mariena J A; Strömdahl, Ann-Charlotte; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

    2015-06-01

    Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), derived from the C terminus of human thrombin, inhibits proinflammatory responses in vitro and in vivo, but the mode of action is unclear. In this study, we show that GKY25, apart from binding bacterial LPS, also interacts directly with monocytes and macrophages in vitro, ex vivo, and in vivo. Moreover, GKY25 inhibits TLR4- and TLR2-induced NF-?B activation in response to several microbe-derived agonists. Furthermore, GKY25 reduces LPS-induced phosphorylation of MAPKs p38? and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25, based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-?B activity and proinflammatory cytokine production in monocytes and macrophages. PMID:25911750

  19. Heparin affinity of factor VIIa: Implications on the physiological inhibition by antithrombin and clearance of recombinant factor VIIa

    DEFF Research Database (Denmark)

    Martínez-Martínez, I; Ordóñez, A

    2011-01-01

    Factor VIIa (FVIIa), a trypsin-like serine protease, plays an essential role in haemostasis by initiating the coagulation in complex with its cofactor, tissue factor (TF). The TF pathway inhibitor is the main physiological inhibitor of FVIIa-TF complex, but FVIIa can also be inhibited by antithrombin, although little is known about this process. Functional analyses by second order kinetic determination and identification of FVIIa-antithrombin complex by electrophoresis, evaluating the effect of different cofactors: pentasaccharide, low molecular weight heparin (LMWH) and unfractionated heparin (UFH), confirmed that any activation of antithrombin significantly enhanced the inhibition of FVIIa. The analysis of the binding of FVIIa to heparin by surface plasmon resonance identified a high affinity interaction under physiologic conditions (K(D)=3.38?M, with 0.15M of ionic strength) strongly dependent on Ca(2+) and ionic strength. This interaction was verified in cell models, indicating that FVIIa also binds to the surface of endothelial cells with similar requirements. Structural modeling suggests the presence of a potential exosite II in FVIIa. However, the binding of heparin did not display significant changes on both the intrinsic fluorescence and the associated functional consequences of FVIIa. These results indicate that FVIIa binds to exposed glycosaminglycans of the endothelium through an exosite II, structurally similar to that reported for thrombin and suggested for FIXa. This binding may favor its inhibition by antithrombin in the absence of TF, contributing to the physiological control of this protease. This process may also play an important role in the clearance of recombinant FVIIa administered to patients.

  20. Topical report review status

    International Nuclear Information System (INIS)

    A Topical Report Review Status is scheduled to be published semi-annually. The primary purpose of this document is to provide periodic progress reports of on-going topical report reviews, to identify those topical reports for which the Nuclear Regulatory Commission (NRC) staff review has been completed and, to the extent practicable, to provide NRC management with sufficient information regarding the conduct of the topical report program to permit taking whatever actions deemed necessary or appropriate. This document is also intended to be a source of information to NRC Licensing Project Managers and other NRC personnel regarding the status of topical reports which may be referenced in applications for which they have responsibility. This status report is published primarily for internal NRC use in managing the topical report program, but is also used by NRC to advise the industry of report review status

  1. In search of expression bottlenecks in recombinant CHO cell lines--a case study.

    Science.gov (United States)

    Reinhart, David; Sommeregger, Wolfgang; Debreczeny, Monika; Gludovacz, Elisabeth; Kunert, Renate

    2014-07-01

    The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells' quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell's productivity. PMID:24557570

  2. Monitoring homologous recombination in rice (Oryza sativa L.)

    Energy Technology Data Exchange (ETDEWEB)

    Yang Zhuanying; Tang Li [Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631 (China); Li Meiru [South China Botanic Garden, Chinese Academy of Sciences, Guangzhou 510650 (China); Chen Lei; Xu Jie [Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631 (China); Wu Goujiang [South China Botanic Garden, Chinese Academy of Sciences, Guangzhou 510650 (China); Li Hongqing, E-mail: hqli@scnu.edu.cn [Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631 (China)

    2010-09-10

    Here we describe a system to assay homologous recombination during the complete life cycle of rice (Oryza sativa L.). Rice plants were transformed with two copies of non-functional GUS reporter overlap fragments as recombination substrate. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Embryogenic cells exhibited the highest recombination ability with an average of 3 x 10{sup -5} recombination events per genome, which is about 10-fold of that observed in root cells, and two orders of that observed in leaf cells. Histological analysis revealed that recombination events occurred in diverse cell types, but preferentially in cells with small size. Examples of this included embryogenic cells in callus, phloem cells in the leaf vein, and cells located in the root apical meristem. Steady state RNA analysis revealed that the expression levels of rice Rad51 homologs are positively correlated with increased recombination rates in embryogenic calli, roots and anthers. Finally, radiation treatment of plantlets from distinct recombination lines increased the recombination frequency to different extents. These results showed that homologous recombination frequency can be effectively measured in rice using a transgene reporter assay. This system will facilitate the study of DNA damage signaling and homologous recombination in rice, a model monocot.

  3. FASEB Summer Research Conference. Genetic Recombination and Chromosome Rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Jinks-Robertson, Sue

    2002-02-01

    The 2001 meeting entitled ''Genetic Recombination and Genome Rearrangements'' was held July 21-26 in Snowmass, Colorado. The goal of the meeting was to bring together scientists using diverse approaches to study all aspects of genetic recombination. This goal was achieved by integrating talks covering the genetics, biochemistry and structural biology of homologous recombination, site-specific recombination, and nonhomologous recombination. The format of the meeting consisted of a keynote address on the opening evening, two formal plenary sessions on each of the four full meeting days, a single afternoon workshop consisting of short talks chosen from among submitted abstracts, and afternoon poster sessions on each of the four full meeting days. The eight plenary session were entitled: (1) Recombination Mechanisms, (2) Prokaryotic Recombination, (3) Repair and Recombination, (4) Site-specific Recombination and Transposition, (5) Eukaryotic Recombination I, (6) Genome Rearrangements, (7) Meiosis, and (8) Eukaryotic Recombination II. Each session included a mix of genetic, biochemical and structural talks; talks were limited to 20 minutes, followed by 10 minutes of very lively, general discussion. Much of the data presented in the plenary sessions was unpublished, thus providing attendees with the most up-to-date knowledge of this rapidly-moving field.

  4. Monitoring homologous recombination in rice (Oryza sativa L.)

    International Nuclear Information System (INIS)

    Here we describe a system to assay homologous recombination during the complete life cycle of rice (Oryza sativa L.). Rice plants were transformed with two copies of non-functional GUS reporter overlap fragments as recombination substrate. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Embryogenic cells exhibited the highest recombination ability with an average of 3 x 10-5 recombination events per genome, which is about 10-fold of that observed in root cells, and two orders of that observed in leaf cells. Histological analysis revealed that recombination events occurred in diverse cell types, but preferentially in cells with small size. Examples of this included embryogenic cells in callus, phloem cells in the leaf vein, and cells located in the root apical meristem. Steady state RNA analysis revealed that the expression levels of rice Rad51 homologs are positively correlated with increased recombination rates in embryogenic calli, roots and anthers. Finally, radiation treatment of plantlets from distinct recombination lines increased the recombination frequency to different extents. These results showed that homologous recombination frequency can be effectively measured in rice using a transgene reporter assay. This system will facilitate the study of DNA damage signaling and homologous recombination in rice, a model monocot.

  5. Detection of quantitative trait Loci influencing recombination using recombinant inbred lines.

    Science.gov (United States)

    Dole, Jefferey; Weber, David F

    2007-12-01

    The genetic basis of variation in recombination in higher plants is polygenic and poorly understood, despite its theoretical and practical importance. Here a method of detecting quantitative trait loci (QTL) influencing recombination in recombinant inbred lines (RILs) is proposed that relies upon the fact that genotype data within RILs carry the signature of past recombination. Behavior of the segregational genetic variance in numbers of chromosomal crossovers (recombination) over generations is described for self-, full-sib-, and half-sib-generated RILs with no dominance in true crossovers. This genetic variance, which as a fraction of the total phenotypic variance contributes to the statistical power of the method, was asymptotically greatest with half sibbing, less with sibbing, and least with selfing. The statistical power to detect a recombination QTL declined with diminishing QTL effect, genome target size, and marker density. For reasonably tight marker linkage power was greater with less intense inbreeding for later generations and vice versa for early generations. Generational optima for segregation variance and statistical power were found, whose onset and narrowness varied with marker density and mating design, being more pronounced for looser marker linkage. Application of this method to a maize RIL population derived from inbred lines Mo17 and B73 and developed by selfing suggested two putative QTL (LOD > 2.4) affecting certain chromosomes, and using a canonical transformation another putative QTL was detected. However, permutation tests failed to support their presence (experimentwise alpha = 0.05). Other populations with more statistical power and chosen specifically for recombination QTL segregation would be more effective. PMID:17947433

  6. Molecular mechanisms of DNA recombination: testing mitotic and meiotic models

    International Nuclear Information System (INIS)

    A hyperhaploid n + 1 strain of Saccharomyces cerevisiae (LBL1) disomic for chromosome VII was employed to isolate hyper-rec and hypo-rec mutations affecting spontaneous mitotic gene conversion and intergenic recombination. The genotype of LBL1 permits simultaneous and independent identification of rec mutations that enhance or diminish gene conversion and those that enhance or diminish intergenic recombination. Five phenotypic groups of rec mutants were isolated following ultraviolet light mutagenesis. Rec mutations that simultaneously abolish or enhance both classes of recombinational events were detected. These results demonstrate that gene conversion and intergenic recombination are under joint genetic control in mitotic cells. Conversion-specific and intergenic recombination-specific rec mutants were also recovered. Their properties indicate that conversion and intergenic recombination are separable pheonomena dependent upon discrete REC genes. The rec mutants isolated in LBL1 provide a method to test molecular models of mitotic and meiotic recombination

  7. Mechanisms and Factors that Influence High Frequency Retroviral Recombination

    Directory of Open Access Journals (Sweden)

    Krista Delviks-Frankenberry

    2011-09-01

    Full Text Available With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development.

  8. Mechanisms and factors that influence high frequency retroviral recombination

    DEFF Research Database (Denmark)

    Delviks-Frankenberry, Krista; Galli, Andrea

    2011-01-01

    With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development.

  9. Induction of molecular and genetic recombination in eukaryotic cells

    International Nuclear Information System (INIS)

    The purpose of this review was to generally describe mitotic and meiotic recombination and to relate these processes to DNA repair. The results and mechanisms which have been described for genetic recombination in lower eukaryocytes are related to recent observations with mammalian cell systems in an attempt to bridge the gap between these categories of eukaryotes. The types of genetic recombinations which can be identified in lower eukaryotes, the effects of various mutagens and radiations, the similarities between spontaneous mitotic and meiotic recombination, the importance of recombination in DNA repair, and the role of various mechanisms in recombination are described. Molecular recombination is discussed. Gene conversion (informational transfer) is related to biochemical changes and is a very sensitive genetic tool for detecting DNA alterations

  10. TOPICAL TREATMENT OF MELASMA

    OpenAIRE

    Bandyopadhyay Debabrata

    2009-01-01

    Melasma is a common hypermelanotic disorder affecting the face that is associated with considerable psychological impacts. The management of melasma is challenging and requires a long-term treatment plan. In addition to avoidance of aggravating factors like oral pills and ultraviolet exposure, topical therapy has remained the mainstay of treatment. Multiple options for topical treatment are available, of which hydroquinone (HQ) is the most commonly prescribed agent. Besides HQ, other topical ...

  11. Single--crossover recombination in discrete time

    CERN Document Server

    von Wangenheim, Ute; Baake, Michael

    2009-01-01

    Modelling the process of recombination leads to a large coupled nonlinear dynamical system. Here, we consider a particular case of recombination in {\\em discrete} time, allowing only for {\\em single crossovers}. While the analogous dynamics in {\\em continuous} time admits a closed solution, this no longer works for discrete time. A more general model (i.e. without the restriction to single crossovers) has been studied before and was solved algorithmically by means of Haldane linearisation. Using the special formalism introduced by Baake and Baake (2003), we obtain further insight into the single-crossover dynamics and the particular difficulties that arise in discrete time. We then transform the equations to a solvable system in a two-step procedure: linearisation followed by diagonalisation. Still, the coefficients of the second step must be determined in a recursive manner, but once this is done for a given system, they allow for an explicit solution valid for all times.

  12. Regulation of DNA pairing in homologous recombination.

    Science.gov (United States)

    Daley, James M; Gaines, William A; Kwon, YoungHo; Sung, Patrick

    2014-11-01

    Homologous recombination (HR) is a major mechanism for eliminating DNA double-strand breaks from chromosomes. In this process, the break termini are resected nucleolytically to form 3' ssDNA (single-strand DNA) overhangs. A recombinase (i.e., a protein that catalyzes homologous DNA pairing and strand exchange) assembles onto the ssDNA and promotes pairing with a homologous duplex. DNA synthesis then initiates from the 3' end of the invading strand, and the extended DNA joint is resolved via one of several pathways to restore the integrity of the injured chromosome. It is crucial that HR be carefully orchestrated because spurious events can create cytotoxic intermediates or cause genomic rearrangements and loss of gene heterozygosity, which can lead to cell death or contribute to the development of cancer. In this review, we will discuss how DNA motor proteins regulate HR via a dynamic balance of the recombination-promoting and -attenuating activities that they possess. PMID:25190078

  13. SIR epidemics in monogamous populations with recombination

    Directory of Open Access Journals (Sweden)

    Damián H. Zanette

    2011-03-01

    Full Text Available We study the propagation of an SIR (susceptible--infectious--recovered disease over an agent population which, at any instant, is fully divided into couples of agents. Couples are occasionally allowed to exchange their members. This process of couple recombination can compensate the instantaneous disconnection of the interaction pattern and thus allow for the propagation of the infection. We study the incidence of the disease as a function of its infectivity and of the recombination rate of couples, thus characterizing the interplay between the epidemic dynamics and the evolution of the population's interaction pattern.Received: 12 August 2010;Accepted: 18 February 2011; Edited by: G. C. Barker; Reviewed by: B. Blasius, ICBM, University of Oldenburg, Germany; DOI: 10.4279/PIP.030001Cite as: D. H. Zanette, Papers in Physics 3, 030001 (2011

  14. CFD Analysis of Passive Autocatalytic Recombiner

    International Nuclear Information System (INIS)

    In water-cooled nuclear power reactors, significant quantities of hydrogen could be produced following a postulated loss-of-coolant accident (LOCA) along with non availability of emergency core cooling system (ECCS). Passive autocatalytic recombiners (PAR) are implemented in the containment of water-cooled power reactors to mitigate the risk of hydrogen combustion. In the presence of hydrogen with available oxygen, a catalytic reaction occurs spontaneously at the catalyst surfaces below conventional ignition concentration limits and temperature and even in presence of steam. Heat of reaction produces natural convection flow through the enclosure and promotes mixing in the containment. For the assessment of the PAR performance in terms of maximum temperature of catalyst surface and outlet hydrogen concentration an in-house 3D CFD model has been developed. The code has been used to study the mechanism of catalytic recombination and has been tested for two literature-quoted experiments

  15. Scaling and fractal behaviour underlying meiotic recombination.

    Science.gov (United States)

    Waxman, D; Stoletzki, N

    2010-01-01

    In this paper we investigate some of the mathematical properties of meiotic recombination. Working within the framework of a genetic model with n loci, where alpha alleles are possible at each locus, we find that the proportion of all possible diploid parental genotypes that can produce a particular haploid gamete is exp[-n log(alpha(2)/[2alpha-1])]. We show that this proportion connects recombination with a fractal geometry of dimension log(2alpha-1)/log(alpha). The fractal dimension of a geometric object manifests itself when it is measured at increasingly smaller length scales. Decreasing the length scale of a geometric object is found to be directly analogous, in a genetics problem, to specifying a multilocus haplotype at a larger number of loci, and it is here that the fractal dimension reveals itself. PMID:19712721

  16. Single-stranded heteroduplex intermediates in ? Red homologous recombination

    Directory of Open Access Journals (Sweden)

    Zhang Youming

    2010-07-01

    Full Text Available Abstract Background The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined. Results Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Red? exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule. Conclusions Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.

  17. Rapid one-step recombinational cloning

    OpenAIRE

    Fu, Changlin; Wehr, Daniel R.; Edwards, Janice; Hauge, Brian

    2008-01-01

    As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have de...

  18. The landscape of recombination in African Americans.

    OpenAIRE

    Hinch, Ag; Tandon, A.; Patterson, N.; Song, Y.; Rohland, N.; Palmer, Cd; Chen, Gk; Wang, K.; Buxbaum, Sg; Akylbekova, El; Aldrich, Mc; Ambrosone, Cb; Amos, C.; Bandera, Ev; Berndt, Si

    2011-01-01

    Recombination, together with mutation, gives rise to genetic variation in populations. Here we leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and ...

  19. Managing meiotic recombination in plant breeding

    OpenAIRE

    Wijnker, T.G.; Jong, J.H.S.G.M., de

    2008-01-01

    Crossover recombination is a crucial process in plant breeding because it allows plant breeders to create novel allele combnations on chromosomes that can be used for breeding superior F1 hybrids. Gaining control over this process, in terms of increasing crossover incidence, altering crossover positions on chromosomes or silencing crossover formation, is essential for plant breeders to effectively engineer the allelic composition of chromosomes. We review the various means of crossover contro...

  20. Construction of recombinant DNA by exonuclease recession.

    OpenAIRE

    Yang, Y. S.; Watson, W. J.; Tucker, P. W.; Capra, J. D.

    1993-01-01

    We describe a new exonuclease-based method for joining and/or constructing two or more DNA molecules. DNA fragments containing ends complementary to those of a vector or another independent molecules were generated by the polymerase chain reaction. The 3' ends of these molecules as well as the vector DNA were then recessed by exonuclease activity and annealed in an orientation-determined manner via their complementary single-stranded regions. This recombinant DNA can be transformed directly i...

  1. Dissociative recombination of BeH^+

    OpenAIRE

    Roos, J.B.; Larsson, M; Larson, AA.; Orel, A. E.

    2009-01-01

    The cross section for dissociative recombination of BeH^+ is calculated by solution of the time-dependent Schrodinger equation in the local complex potential approximation. The effects of couplings between resonant states and the Rydberg states converging to the ground state of the ion are studied. The relevant potentials, couplings and autoionization widths are extracted using ab initio electron scattering and structure calculations, followed by a diabatization procedure. T...

  2. Domain Recombination: A Workhorse for Evolutionary Innovation

    Science.gov (United States)

    Gordana Apic (UK; Cambridge Cell Networks REV)

    2010-09-14

    Although the combination of modular domains within proteins has been proposed as a determining feature of evolutionary innovation and flexibility, direct evidence for this mechanism of evolution has been sketchy. Two papers, one creating new domain combinations in the yeast mating pathway and another involving a comprehensive analysis of protein function and domain architecture across major organisms, have provided firm evidence that the recombining of domains can lead to evolutionary innovation. The results will guide future studies in synthetic and evolutionary biology.

  3. Cytological studies of recombination in rhesus males

    OpenAIRE

    Hassold, T.; Hansen, T.; Hunt, P.; Vandevoort, C.

    2009-01-01

    An immunofluorescence approach was used to directly examine meiotic recombination events in 483 pachytene spermatocytes from 11 male rhesus monkeys. Specifically, we examined the nuclear localization patterns of the DNA mismatch repair protein MLH1, known from analyses of other mammalian species to be a useful marker of meiotic cross-overs. Our results indicated that rhesus pachytene spermatocytes contain approximately 40 cross-overs per cell, corresponding to about one cross-over per chromos...

  4. Recombinant organisms capable of fermenting cellobiose

    Science.gov (United States)

    Ingram, Lonnie O. (Gainesville, FL); Lai, Xiaokuang (Gainesville, FL); Moniruzzaman, Mohammed (Gainesville, FL); York, Sean W. (Gainesville, FL)

    2000-01-01

    This invention relates to a recombinant microorganism which expresses pyruvate decarboxylase, alcohol dehydrogenase, Klebsiella phospho-.beta.-glucosidase and Klebsiella (phosphoenolpyruvate-dependent phosphotransferase system) cellobiose-utilizing Enzyme II, wherein said phospho-.beta.-glucosidase and said (phosphoenolpyruvate-dependent phosphotransferase) cellobiose-utilizing Enzyme II are heterologous to said microorganism and wherein said microorganism is capable of utilizing both hemicellulose and cellulose, including cellobiose, in the production of ethanol.

  5. Production of recombinant antibodies using bacteriophages

    OpenAIRE

    Shukra, A. M.; Sridevi, N. V.; Dev Chandran,; Kapil Maithal,

    2014-01-01

    Recombinant antibody fragments such as Fab, scFv, diabodies, triabodies, single domain antibodies and minibodies have recently emerged as potential alternatives to monoclonal antibodies, which can be engineered using phage display technology. These antibodies match the strengths of conventionally produced monoclonal antibodies and offer advantages for the development of immunodiagnostic kits and assays. These fragments not only retain the specificity of the whole monoclonal ...

  6. Recombinant CBM-fusion technology : applications overview

    OpenAIRE

    Oliveira, Carla Cristina Marques de; Carvalho, Vera; Domingues, Lucília; Gama, F. M.

    2015-01-01

    Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for...

  7. Designing recombinant Pseudomonas strains to enhance biodesulfurization.

    OpenAIRE

    Gallardo, M.E.; Ferrández, A; De Lorenzo, V.; García, J.L.; Díaz, E.

    1997-01-01

    The dsz biodesulfurization cluster from Rhodococcus erythropolis IGTS8 has been engineered under the control of heterologous broad-host-range regulatory signals to alleviate the mechanism of sulfur repression, and it was stably inserted into the chromosomes of different Pseudomonas strains. The recombinant bacteria were able to desulfurize dibenzothiophene more efficiently than the native host. Furthermore, these new biocatalysts combine relevant industrial and environmental traits, such as p...

  8. Generation and Characterization of Recombinantly Polysialylated Antibody

    OpenAIRE

    Chen, Chen

    2011-01-01

    With high affinity and specificity, antibodies are now proven biotherapuetics for a wide range of diseases, such as cancer and immunological conditions. However, antibody Fc-domain mediated cross reactivity with associated side effects has hindered the development of antibody therapy in a number of applications. The development of engineered recombinant antibody fragments to address the problems seen with whole monoclonal antibodies (mAbs). Their smaller size enables rapid anti...

  9. A Holliday recombination intermediate is twofold symmetric.

    OpenAIRE

    Churchill, M E; Tullius, T. D.; Kallenbach, N. R.; Seeman, N. C.

    1988-01-01

    Four-arm Holliday structures are ephemeral intermediates in genetic recombination. We have used an oligodeoxynucleotide system to form immobile DNA junctions, which are stable analogs of Holliday structures. We have probed the equilibrium structure of a junction by means of hydroxyl radicals generated by the reaction of iron(II)EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern shows twofold symmetry throughout the molecule. Strong protection from hydroxyl radical attack is ev...

  10. Recombinant human erythropoietin in sports: a review

    OpenAIRE

    Rafael Maia de Almeida Bento; Lúcia Menezes Pinto Damasceno; Francisco Radler De Aquino Neto,

    2003-01-01

    Erythropoietin is an endogenous hormone of glicoproteic nature secreted by the kidneys and is the main regulator of the erythropoiesis. An alteration in its production generates a disturbance in the plasmatic concentration giving rise to several types of pathologies related to the hematopoietic system. The recombinant forms of erythropoietin have indiscriminately been used by athletes, mainly in endurance sports, by increasing the erythrocytes concentration, generating a better delivery of ox...

  11. Virus Strain Discrimination Using Recombinant Antibodies

    OpenAIRE

    Boonham, N.; Barker, I.

    2000-01-01

    Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological faciliti...

  12. Modelling of procecces in catalytic recombiners

    International Nuclear Information System (INIS)

    In order to achieve a high degree of safety in nuclear power plants and prevent possible accident scenarios, their consequences are calculated and analysed with numeric codes. One of the most important part of nuclear safety research of hazardous incidents are development and validation of these numeric models, which are implemented into accident codes. The severe hydrogen release during a core meltdown is one of the considered scenario of performed accident analyses. One of the most important measure for the elimination of the hydrogen is catalytic recombiners. Converting the hydrogen with the atmospheric oxygen to water vapor in an exothermic reaction will prevent possible detonation of the hydrogen/air atmosphere. Within the dissertation the recombiner simulation REKO-DIREKT was developed and validated by an extensive experimental database. The performance of recombiners with regard to the conversion of the hydrogen and the temperature development is modelled. The REKO-DIREKT program is unique and has made significant revolution in research of hydrogen safety. For the first time it has been possible to show the performance of the recombiner so great in detail by using REKO-DIREKT. In the future engineers of nuclear power plants will have opportunity to have precise forecasts about the process of the possible accidents with hydrogen release. Also with presence of water vapor or with oxygen depletion which are included in the model. The major discussion of the hydrogen ignition at hot catalyst steel plates can be evaluated in the future with REKO-DIREKT more reliably than the existing used models. (orig.)

  13. Dissociative recombination of small molecular ions

    International Nuclear Information System (INIS)

    In this thesis an analysis is given of merged electron-ion beam experiment and work on dissociative recombination of molecular ions and electrons is described. Chapter II covers a brief introduction of the theory of dissociative recombination. In chapter III, a description is given of the merged electron-ion beam experiment and a method is described which allows the determination of the mean angle between the electron and ion trajectories in a merged electron-ion beam experiment. In chapter IV a paper on the three dominant atmospheric diatomic ions NO+, O2+ and N2+ is presented and in chapter V the dissociative recombination for N2H+ and N2D+ is discussed. In chapter VI two papers on the polyatomic ions of the carbon-containing molecular ions are presented, and in chapter VII a letter with some results of the work presented in more detail in the chapters IV, V and VI is presented. The magnitude and the energy dependence of the cross-section measured by the merged beam technique and by other techniques is compared and discussed. (Auth.)

  14. New recombination methods for Sinorhizobium meliloti genetics.

    Science.gov (United States)

    House, Brent L; Mortimer, Michael W; Kahn, Michael L

    2004-05-01

    The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative alpha-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots. PMID:15128536

  15. Recombinant vaccine for canine parvovirus in dogs.

    Science.gov (United States)

    López de Turiso, J A; Cortés, E; Martínez, C; Ruiz de Ybáñez, R; Simarro, I; Vela, C; Casal, I

    1992-01-01

    VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-assemble, forming viruslike particles similar in size and appearance to CPV virions. These viruslike particles have been used to immunize dogs in different doses and combinations of adjuvants, and the anti-CPV responses have been measured by enzyme-linked immunosorbent assay, monolayer protection assays, and an assay for the inhibition of hemagglutination. A dose of ca. 10 micrograms of VP2 was able to elicit a good protective response, higher than that obtained with a commercially available, inactivated vaccine. The results indicate that these viruslike particles can be used to protect dogs from CPV infection. Images PMID:1313899

  16. Modified recombinant allergens for safer immunotherapy.

    Science.gov (United States)

    Ferreira, Fátima; Briza, Peter; Inführ, Daniela; Schmidt, Georg; Wallner, Michael; Wopfner, Nicole; Thalhamer, Josef; Achatz, Gernot

    2006-01-01

    Molecular cloning and recombinant production of allergens offered new perspectives for the increasing problem of allergies. A variety of preparations are being developed aiming to increase safety and improve efficacy of specific immunotherapy. Recombinant-based approaches are mostly focused on genetic modification of allergens to produce molecules with reduced allergenic activity and conserved antigenicity, i.e. hypoallergens. Studies dealing with genetic modifications of allergen genes reported the production of site-directed mutants, deletion mutants, allergen fragments and oligomers, and allergen chimeras. An alternative to genetic engineering is the chemical modification of pure recombinant allergens. It has been shown that allergens modified with immunostimulatory DNA sequences (allergen-ISS conjugates), which masks IgE epitopes and adds a desirable Th1-inducing character to the allergen molecule. Other chemical modifications include oligomerization by aldehydes (allergoids) and maleylation, which seems to target allergens to particular antigen presenting cells. Several of these modified allergen preparations have been already evaluated for their safety in clinical provocation studies. So far, clinical trials showed the efficacy and safety of immunotherapy with an Amb a 1-ISS conjugate for ragweed pollen-allergic patients. In addition, a preparation consisting of hypoallergenic fragments of Bet v 1 was evaluated for immunotherapy of birch pollen-allergic patients. In parallel, several animal studies have now demonstrated the potential of genetic immunization for allergy treatment in the future. PMID:16613559

  17. Evolutionary Origin of Recombination during Meiosis

    Science.gov (United States)

    Carol Bernstein (University of Arizona; Cell Biology and Anatomy)

    2010-07-14

    Recent evidence indicates that meiosis arose very early in eukaryotic evolution, which suggests that essential features of meiosis were already present in the prokaryotic ancestors of eukaryotes. Furthermore, in extant organisms, proteins with central functions in meiosis are similar in sequence and function to key proteins in bacterial transformation. In particular, RecA recombinaseâ??which performs the central functions of DNA homology search and strand exchange in bacterial transformationâ??has orthologs in eukaryotes that carry out similar functions in meiotic recombination. Both transformation and meiosis (including meiotic recombination) in eukaryotic microorganisms are induced by stressful conditions, such as overcrowding, resource depletion, and DNA-damaging conditions, suggesting that these processes are adaptations for dealing with stress. If such environmental stresses were a persistent challenge to the survival of early microorganisms, then continuity of selection through the prokaryote to eukaryote transition probably would have followed a course in which bacterial transformation naturally gave rise to the recombination process that is central to eukaryote meiosis.

  18. Topical Acne Treatments and Pregnancy

    Science.gov (United States)

    ... visit us online at: www.OTISpregnancy.org . Topical Acne Treatments and Pregnancy In every pregnancy, a woman ... from your health care professional. What are topical acne treatments? Topical acne treatments are medications applied directly ...

  19. The Topic-Prominence Parameter

    OpenAIRE

    Xu, Liejiong

    2000-01-01

    This article aims to recast the properties of topic-prominent languages and their differences from subject-prominent languages as documented in the functionalist literature into the framework of the Principle-and-Parameter approach. It provides a configurational definition of the topic construction called Topic Phrase (TP), with the topic marker as its head. The availablity of TP enables topic prominent languages to develop various topic structures with properties such as morphological markin...

  20. Genetic recombination of ultraviolet-irradiated nonreplicating lambda DNA

    International Nuclear Information System (INIS)

    Genetic recombination of ultraviolet-irradiated, nonreplicating lambda DNA was studied. Escherichia coli homoimmune lysogens were infected with ultraviolet-irradiated lambda phage whose DNA possessed a tandem duplication of the A to V genes. Recombination between duplicated segments produced lambda, DNA molecules possessing only one copy of the A to V region. DNA was extracted from cells and used to transfect recombination-deficient spheroplasts. Transfection lysates were assayed for total lambda phage and recombinant (EDTA-resistant) phage. Ultraviolet-stimulated recombination was shown to be completely RecA-dependent, mostly RecF-dependent, and RecBC and RecE-independent. Experiments with excision repair-deficient (uvr-) bacteria suggested that ultraviolet-stimulated recombination occurred by both Uvr-dependent and Uvr-independent processes. A role for pyrimidine dimers in recombination was indicated by the reduction in recombination frequency subsequent to photoreactivation and by experiments using lambda phase irradiated under conditions that produce almost exclusively pyrimidine dimers. A role for photoproducts other than pyrimidine dimers was suggested by the photo-reactivation-insensitive component of 254nm-stimulated recombination and by the observation that recombination frequencies of 254-irradiated phage were much greater than those of 313 nm/acetophenone-irradiated phage when both types of phage possessed the same number of pyridimidine dimers per lambda duplex