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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. Development of gel-forming lyophilized formulation with recombinant human thrombin.

    Science.gov (United States)

    Murányi, Andrej; Bartoš, Peter; Tichý, Eduard; Lazová, Jana; Pšenková, Jana; Žabka, Marián

    2015-09-01

    The objective of this work was development and evaluation of gel-forming lyophilized formulation with recombinant human thrombin for topical administration. The influence of pH, ionic strength and buffer type on protein stability was evaluated as part of the pre-formulation screening studies. Results indicated an optimal pH from 6.0 to 7.0 and increased stability with increasing content of sodium chloride. The tested buffer types had no significant effect on thrombin stability. For further development, thermosensitive Pluronic® F-127 was employed as a bulking and gelling agent. Physical and mechanical characterization and viscosity measurement confirmed the gel-forming properties of the formulation at the application temperature of 32?°C. Several techniques (addition of well-soluble polyols, different freezing protocols and reconstitution under vacuum) were tested to decrease the reconstitution time. The obtained results revealed that a vacuum in the vial headspace is crucial for acceptable reconstitution. The freeze drying process has no negative impact on recombinant thrombin stability, and this was confirmed by reverse-phase-HPLC, activity assay and optical density measurements. PMID:25347143

  3. Clinical use of topical thrombin as a surgical hemostat

    OpenAIRE

    Lew, Wesley K.; Weaver, Fred A.

    2008-01-01

    Wesley K Lew1, Fred A Weaver21University of Southern California, Department of Surgery, Los Angeles, CA, USA; 2CardioVascular Thoracic Institute at the University of Southern California, Los Angeles, CA, USAAbstract: When surgical ligation of bleeding fails, or is not possible, surgeons rely on a number of hemostatic aids, including thrombin. This review discusses the history, pharmacology and clinical application of thrombin as a surgical hemostat. The initial thrombin was bovine in origin, ...

  4. Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of?thrombopoietin

    OpenAIRE

    Kato, Takashi; Oda, Atsushi; Inagaki, Yoshimasa; Ohashi, Hideya; MATSUMOTO, ATSUSHI; Ozaki, Katsutoshi; Miyakawa, Yoshitaka; Watarai, Hiroshi; Fuju, Kazumi; Kokubo, Atsuko; Kadoya, Toshihiko; IKEDA, YASUO; Miyazaki, Hiroshi

    1997-01-01

    A heterogeneity in the molecular weight (Mr) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low Mr peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the pr...

  5. Equilibrium binding of thrombin to recombinant human thrombomodulin: Effect of hirudin, fibrinogen, factor Va, and peptide analogues

    Energy Technology Data Exchange (ETDEWEB)

    Tsiang, Manuel; Lentz, S.R.; Dittman, W.A.; Wen, D.; Scarpati, E.M.; Sadler, J.E. (Howard Hughes Medical Institute, St. Louis, MO (USA))

    1990-11-01

    Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomudlin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had M{sub r} {approximately} 100 000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar K{sub d}'s for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The K{sub d} for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of {sup 125}I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir{sup 53-64} and the thrombomodulin fifth-EGF-domain peptide Tm{sup 426-444} displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV{sup 30-43} which is similar in composition and charge to Hir{sup 53-64} showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.

  6. Equilibrium binding of thrombin to recombinant human thrombomodulin: Effect of hirudin, fibrinogen, factor Va, and peptide analogues

    International Nuclear Information System (INIS)

    Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomudlin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr ? 100 000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site

  7. Efficacy of a topical bovine-derived thrombin solution as a hemostatic agent in a rodent model of hepatic injury.

    Science.gov (United States)

    Rosselli, Desiree D; Brainard, Benjamin M; Schmiedt, Chad W

    2015-10-01

    Hemorrhage is a major concern in patients undergoing hepatic surgery or in those with hepatic trauma. In these cases, employing traditional hemostatic strategies can be problematic due to the diffuse nature of hepatic hemorrhage and limited opportunities for direct hemostasis. This study assessed the efficacy of a bovine-derived thrombin solution, (BT), as a topical liquid agent to augment hemostasis and survival following severe hepatic hemorrhage in a rat model. Heart rate (HR), arterial blood pressure (ABP), packed cell volume (PCV), and overall survival were evaluated in 54 rats randomly assigned to receive topical application of BT, saline, or suture ligation applied immediately to a liver lobe following controlled laceration. Six additional rats received liver laceration with no applied treatment. Intravenous fluid resuscitation was initiated and HR and ABP were recorded for 60 min, after which survivors were recovered from anesthesia. Rats were then monitored for 72 h, after which survivors were euthanized. There was no significant difference in survival time, percentage survival, intra-operative ABP or HR, or post-operative PCV between treatment groups. There is insufficient evidence to recommend BT as the sole therapy using this delivery method for mitigating severe hemorrhage from liver injury. PMID:26424911

  8. Nanostructured bioluminescent sensor for rapidly detecting thrombin.

    Science.gov (United States)

    Chen, Longyan; Bao, Yige; Denstedt, John; Zhang, Jin

    2016-03-15

    Thrombin plays a key role in thrombosis and hemostasis. The abnormal level of thrombin in body fluids may lead to different diseases, such as rheumatoid arthritis, glomerulonephritis, etc. Detection of thrombin level in blood and/or urine is one of important methods for medical diagnosis. Here, a bioluminescent sensor is developed for non-invasively and rapidly detecting thrombin in urine. The sensor is assembled through conjugating gold nanoparticles (Au NPs) and a recombinant protein containing Renilla luciferase (pRluc) by a peptide, which is thrombin specific substrate. The luciferase-catalyzed bioluminescence can be quenched by peptide-conjugating Au NPs. In the presence of thrombin, the short peptide conjugating luciferase and Au NPs is digested and cut off, which results in the recovery of bioluminescence due to the release of luciferase from Au NPs. The bioluminescence intensity at 470nm is observed, and increases with increasing concentration of thrombin. The bioluminescence intensity of this designed sensor is significantly recovered when the thrombin digestion time lasts for 10min. In addition, a similar linear relationship between luminescence intensity and the concentration of thrombin is found in the range of 8nM to 8?M in both buffer and human urine spiked samples. The limit of detection is as low as 80pM. It is anticipated that our nanosensor could be a promising tool for clinical diagnosis of thrombin in human urine. PMID:26397418

  9. Recombinant DNA Technology. A Topics Course for Undergraduates.

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    Parson, Kathleen A.

    1988-01-01

    Describes the development of a topics course offered jointly by the chemistry and biology departments at Macalester College (Minnesota). Outlines the syllabus for the course. Discusses teaching and laboratory methods used. (CW)

  10. Interaction of thrombin with PAR1 and PAR4 at the thrombin cleavage site†

    OpenAIRE

    Nieman, Marvin T.; Schmaier, Alvin H.

    2007-01-01

    Investigations determined the critical amino acids for ?-thrombin’s interaction with protease activated receptors 1 and 4 (PAR1 and PAR4) at the thrombin cleavage site. Recombinant PAR1-wild type (wt) exodomain was cleaved by ?-thrombin with a Km of 28 ?M, a kcat of 340 s-1 and kcat/Km of 1.2×107. When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the Km was unchanged, 29 or 23 ?M, respectively; however the kcat and kcat/Km were reduced in each case. In c...

  11. Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity.

    Science.gov (United States)

    Gierczak, Richard F; Bhakta, Varsha; Xie, Michael; Sheffield, William P

    2015-08-20

    Serpins are a widely distributed family of serine proteases. A key determinant of their specificity is the reactive centre loop (RCL), a surface motif of ?20 amino acids in length. Expression libraries of variant serpins could be rapidly probed with proteases to develop novel inhibitors if optimal systems were available. The serpin variant alpha-1 proteinase inhibitor M358R (API M358R) inhibits the coagulation protease thrombin, but at sub-maximal rates compared to other serpins. Here we compared two approaches to isolate functional API variants from serpin expression libraries, using the same small library of API randomized at residue 358 (M358X): flow cytometry of transfected HEK 293 cells expressing membrane-displayed API; and a thrombin capture assay (TCA) performed on pools of bacterial lysates expressing soluble API. No enrichment for specific P1 residues was observed when the RCL codons of the 1% of sorted transfected 293 cells with the highest fluorescent thrombin-binding signals were subcloned and sequenced. In contrast, screening of 16 pools of bacterial API-expressing transformants led to the facile identification of API M358R and M358K as functional variants. Kinetic characterization showed that API M358R inhibited thrombin 17-fold more rapidly than API M358K. Reducing the incubation time with immobilized thrombin improved the sensitivity of TCA to detect supra-active API M358R variants and was used to screen a hypervariable library of API variants expressing 16 different amino acids at residues 352-357. The most active variant isolated, with TLSATP substituted for FLEAI, inhibited thrombin 2.9-fold more rapidly than API M358R. Our results indicate that flow cytometric approaches used in protein engineering of antibodies are not appropriate for serpins, and highlight the utility of the optimized TCA for serpin protein engineering. PMID:26043905

  12. Subconjunctival and topical application of recombinant tissue plasminogen activator in rabbits

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    José Ricardo de Abreu Reggi

    2015-02-01

    Full Text Available 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. Whole Blood Thrombin: Development of a Process for Intra-Operative Production of Human Thrombin

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    Kumar, Vijay; Chapman, John R.

    2007-01-01

    Abstract: Thrombin-based clotting agents currently used for topical hemostasis with absorbable sponges, fibrin sealants, and platelet gels are primarily derived from bovine or pooled human plasma sources. Autologous thrombin has important safety advantages in that it does not carry the same safety concerns as pooled plasma-derived products and it avoids exposure to risks associated with bovine-derived proteins. The goal of our research was to develop a rapid, reliable, and simple to perform process to generate autologous human thrombin in the intraoperative setting, from patient whole blood as the starting source material. Using whole blood instead of plasma as the starting material, it is possible to avoid the inherent delay in thrombin availability associated with a primary step of plasma isolation. In this study, we varied several key processing parameters to maximize thrombin production, reproducibility and stability. Autologous thrombin production was generated using a dedicated, single use disposable with a sterile reagent. The disposable consists of a tubular reaction chamber containing glass microsphere beads to activate the alternative pathway of the coagulation cascade. At the end of the process, thrombin-activated serum was harvested from the reaction chamber. The average activity of the thrombin produced at room temperature by this system was 82.8 ± 15.9 IU/mL. The total processing time was <30 minutes. The system was compatible with Anticoagulant Citrate Dextrose-Solution A (ACD-A) (8%–12%). The average volume of thrombin harvested from each aliquot of blood was 7.0 ± 0.3 mL, and the stability of thrombin was observed to be temperature dependent, with cold storage better preserving thrombin activity. Clot times with platelet concentrates at 1:4.3 and 1:11 ratios (thrombin to platelet concentrate) were <10 and 20 seconds, respectively. A process for the preparation of thrombin from whole blood, under conditions compatible with the resources of an operating room, has been developed. The device is simple to use, requires 30 minutes, and can consistently produce thrombin solutions that achieve rapid clotting of platelet concentrates, plasma, and fibrinogen concentrates even when mixed at thrombin to blood product ratios of 1:11. PMID:17486869

  14. The use of thrombin in the radiology department

    Energy Technology Data Exchange (ETDEWEB)

    Ward, E.; Buckley, O.; Browne, R.F. [Adelaide and Meath Hospitals incorporating the National Children' s Hospital (AMNCH), Department of Radiology, Dublin 24 (Ireland); Collins, A. [Royal Victoria Hospital, Department of Radiology, Belfast (United Kingdom); Torreggiani, W.C. [Adelaide and Meath Hospitals incorporating the National Children' s Hospital (AMNCH), Department of Radiology, Dublin 24 (Ireland)]|[Adelaide and Meath Hospital, Department of Radiology, Dublin 24 (Ireland)

    2009-03-15

    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. (orig.)

  15. 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.

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

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    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

  17. Effect of Electronic Polarization to Human ?-Thrombin

    Science.gov (United States)

    Duan, Li-Li; Li, Zong-Chao; He, Xiang; Zhang, Qing-Gang

    2014-04-01

    The polarized protein-specific charges (PPC) of human ?-thrombin (thrombin) and its inhibitor (L86) are made possible by employing the recently developed molecular fractionation with conjugate caps approach incorporated the Poisson—Boltzmann model. Molecular dynamics (MD) simulations of thrombin have been carried out to investigate the dynamics and stability of the thrombin-inhibitor using PPC and AMBER charges respectively. Detailed analysis and comparison of MD results show that the PPC can correctly describe the polarized state of the thrombin and L86. Especially, the root-mean-square deviation of backbone atoms and the hydrogen bonds using PPC are more stable than the AMBER charge. The present results indicate that protein polarization plays critical roles in maintaining the compact structure of thrombin.

  18. Thrombin receptor peptide inhibits thrombin-induced increase in endothelial permeability by receptor desensitization

    OpenAIRE

    1993-01-01

    Thrombin, a potent activator of cellular responses, proteolytically cleaves, and thereby activates its receptor. In the present study, we compared the effects of the thrombin receptor 14-amino acid peptide (TRP-14; SFLLRNPNDKYEPF), which comprises the NH2 terminus after cleavage of the thrombin receptor, and of the native alpha-thrombin on endothelial monolayer permeability. Addition of TRP-14 (1-200 microM) to bovine pulmonary artery endothelial cells increased [Ca2+]i in a dose-dependent ma...

  19. Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation.

    OpenAIRE

    Hung, D T; Vu, T K; Wheaton, V I; Ishii, K.; Coughlin, S.R.

    1992-01-01

    Platelet activation by thrombin is critical for hemostasis and thrombosis. Structure-function studies with a recently cloned platelet thrombin receptor suggest that a hirudin-like domain in the receptor's extracellular amino terminal extension is a thrombin-binding determinant important for receptor activation. We now report that a peptide antiserum to this domain is a potent and specific antagonist of thrombin-induced platelet activation. This study demonstrates that the cloned platelet thro...

  20. Thrombin inhibitory activity of some polyphenolic compounds

    OpenAIRE

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

    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...

  1. Autologous Thrombin: Intraoperative Production From Whole Blood

    Science.gov (United States)

    Kumar, Vijay; Chapman, John R.

    2008-01-01

    Abstract: Thrombin is routinely combined in surgical practice with a fibrinogen source to prepare fibrin sealant to promote hemostasis or with platelet concentrates to prepare platelet gels to enhance wound healing. The purpose of this study was to evaluate the robustness and reproducibility of a new sterile handheld disposable thrombin-processing device (TPD) to generate autologous human thrombin in the intraoperative setting, using whole blood as the starting source material. By using whole blood instead of plasma as the starting material, it is possible to eliminate the plasma separation step from whole blood and reduce the thrombin production time and increase its availability to the surgical team intraoperatively. Active thrombin was prepared by combining 4 mL of thrombin reagent (a mixture of calcium chloride and ethanol) to 11 mL of blood in a reaction chamber containing negatively charged particles. The whole blood, reagent and particle mixture was incubated for 25 minutes at either 18°C or 24°C (n = 25/group) to assess stability of the thrombin activity. The mean activity of the thrombin produced at 18°C and 24°C was 52 ± 14 (n = 25) and 61 ± 12.2 IU/mL (n = 25), respectively. The average volume of thrombin harvested from each aliquot of blood at 18°C and 24°C was 10 ± 0.4 and 9 ± 0.6 mL, respectively. The thrombin concentration generated was shown to rapidly (<5 seconds) coagulate fibrinogen concentrate and retained clotting activity for 1 hour at room temperature (18–26°C) and up to 4 hours when stored on ice. The results show that the TPD is able to consistently generate high thrombin activity from human whole blood. The device offers a robust and rapid approach for preparing active thrombin from whole blood. PMID:18705544

  2. Investigation of the selectivity of thrombin-binding aptamers for thrombin titration in murine plasma.

    Science.gov (United States)

    Trapaidze, Ana; Hérault, Jean-Pascal; Herbert, Jean-Marc; Bancaud, Aurélien; Gué, Anne-Marie

    2016-04-15

    Detection of thrombin in plasma raises timely challenges to enable therapeutic management of thrombosis in patients under vital threat. Thrombin binding aptamers represent promising candidates as sensing elements for the development of real-time thrombin biosensors; however implementation of such biosensor requires the clear understanding of thrombin-aptamer interaction properties in real-like environment. In this study, we used Surface Plasmon Resonance technique to answer the questions of specificity and sensitivity of thrombin detection by the thrombin-binding aptamers HD1, NU172 and HD22. We systematically characterized their properties in the presence of thrombin, as well as interfering molecular species such as the thrombin precursor prothrombin, thrombin in complex with some of its natural inhibitors, nonspecific serum proteins, and diluted plasma. Kinetic experiments show the multiple binding modes of HD1 and NU172, which both interact with multiple sites of thrombin with low nanomolar affinities and show little specificity of interaction for prothrombin vs. thrombin. HD22, on the other hand, binds specifically to thrombin exosite II and has no affinity to prothrombin at all. While thrombin in complex with some of its inhibitors could not be recognized by any aptamer, the binding of HD1 and NU172 properties is compromised by thrombin inhibitors alone, as well as with serum albumin. Finally, the complex nature of plasma was overwhelming for HD1, but we define conditions for the thrombin detection at 10nM range in 100-fold diluted plasma by HD22. Consequently HD22 showed key advantage over HD1 and NU172, and appears as the only alternative to design an aptasensor. PMID:26594887

  3. Studies on thrombin-induced platelet agglutination.

    Science.gov (United States)

    SHERMER, R W; MASON, R G; WAGNER, R H; BRINKHOUS, K M

    1961-12-01

    A one-stage macroscopic test for platelet agglutination was used to study the effect of thrombin and thrombin-cation mixtures on washed platelets. Conclusions regarding platelet agglutination are as follows: (a) Canine, bovine, or human thrombin alone does not cause agglutination of canine or human platelets. (b) Thrombin with calcium or magnesium causes rapid platelet agglutination. Both calcium and magnesium are active at physiologic concentrations. Divalent manganese or cadmium ions can be substituted for calcium or magnesium. (c) The agglutination reaction is affected but little by the species of origin of thrombin or platelets, or by variations in ionic strength or pH over a broad range. (d) Temperature at which the reaction is carried out is critical; optimal temperature for the test is 28 degrees C. (e) Agglutination is inhibited by high ionic strength, by pH values outside the range 6.4-8.6, and by temperatures outside the range 25-28 degrees C. High concentrations of calcium have a specific inhibitory effect. (f) Platelet agglutination time is as sensitive an index of thrombin concentration as is the fibrinogen clotting time. A comparison is made between divalent cations which influence platelet agglutination induced by thrombin, TAg', and TAg. A similar comparison is made of cations influencing the action of thrombin on the "substrates," fibrinogen, TAMe, and platelets. PMID:13911732

  4. Acetazolamide Attenuates Thrombin-Induced Hydrocephalus.

    Science.gov (United States)

    Gao, Feng; Zheng, Mingzhe; Hua, Ya; Keep, Richard F; Xi, Guohua

    2016-01-01

    Our previous studies demonstrated that thrombin is an important factor in brain injury after intracerebral and intraventricular hemorrhage. This study examined the effect of acetazolamide, a carbonic anhydrase inhibitor, on thrombin-induced hydrocephalus. There were two parts in this study. First, rats had an injection of either 50 ?l saline or 3 U thrombin into the right lateral ventricle. Second, rats had an injection of 3 U thrombin into the right lateral ventricle and were treated with either vehicle or acetazolamide (30 mg/kg, intraperitoneally (IP)) at 1 h after thrombin infusion. Lateral ventricle volumes were measured in magnetic resonance imaging T2 images and the brains were used for histology analysis at 24 h later. Intraventricular injection of thrombin induced significantly larger ventricle volume (27.8?±?3.7 vs 8.5?±?1.3 mm(3), n?=?6, p?damage (the breakdown of the ependymal layer, 20.2?±?3.1 vs 2.4?±?0.8 %, n?=?6, p?thrombin-induced hydrocephalus (16.1?±?4.2 mm(3) vs 29.5?±?5.3 mm(3), n?=?6, p?thrombin-induced hydrocephalus in rats. PMID:26463977

  5. Nanocomplexation of thrombin with cationic amylose derivative for improved stability and hemostatic efficacy

    Directory of Open Access Journals (Sweden)

    Zhuang B

    2015-01-01

    Full Text Available Baoxiong Zhuang,1,* Zhihua Li,1,* Jiadong Pang,2,* Wenbin Li,1 Pinbo Huang,1 Jie Wang,1 Yu Zhou,1 Qing Lin,1 Quanbo Zhou,1 Xiao Ye,1 Huilin Ye,1 Yimin Liu,1 Li-Ming Zhang,2 Rufu Chen1 1Department of Hepato-Pancreato-Billiary Surgery, Department of Medical Oncology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China; 2DSAPM Lab and PCFM Lab, Institute of Polymer Science, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, People’s Republic of China *Authors share co-first authorship Abstract: As a topical hemostatic agent, thrombin has wide application for many surgical treatments. However, native thrombin always suffers from its physical and chemical instabilities. In this work, a nanocomplexation strategy was developed for modifying the stability and hemostatic efficacy of thrombin, in which a water-soluble cationic amylose derivative containing poly(l-lysine dendrons was prepared by a click reaction and then used to complex thrombin in an aqueous system. For resultant thrombin nanocomplexes, their morphology and particle size distribution were investigated. Their stabilities were studied in terms of activity retention percentages under different storage time, pH values, and illumination time. In addition, their ability to achieve in vitro fibrinogen and blood coagulation were evaluated. Via a rat hepatic hemorrhage model and a rat iliac artery hemorrhage model, these thrombin nanocomplexes were confirmed to have good tissue biocompatibility and in vivo hemostatic effectiveness. Keywords: thrombin, nanoparticles, amylose derivative, complexation, stability, hemostatic activity

  6. Percutaneous thrombin injection for pseudoaneurysm treatment

    Directory of Open Access Journals (Sweden)

    Gareth Bydawell

    2013-03-01

    Full Text Available Pseudoaneurysm of the femoral artery is a well-documented complication following arterial access. There are several treatment options, but percutaneous injection of thrombin, using ultrasound guidance, has become increasingly popular worldwide and is the therapy of choice in most vascular centres. This short review highlights the procedure and provides step-by-step guidance for radiologists who are interested in performing thrombin injection.

  7. Thrombin Inhibition by Serpins Disrupts Exosite II*

    OpenAIRE

    Li, Wei; Johnson, Daniel J D; Adams, Ty E.; Pozzi, Nicola; FILIPPIS, Vincenzo DE; Huntington, James A.

    2010-01-01

    Thrombin uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite I and the exposure of exosite II. The physiological inhibitors of thrombin are all serpins, whose mechanism involves significant conformational change in both serpin and protease. It has been...

  8. Chemotactic response of monocytes to thrombin

    OpenAIRE

    1983-01-01

    Human alpha-thrombin, the procoagulant activation product of prothrombin, elicits chemotaxis in human peripheral blood monocytes and several macrophagelike continuous cell lines, most notably J-774.2, but not in human peripheral blood granulocytes. alpha-Thrombin is effective in stimulating cell movement at concentrations ranging from 10(-10) to 10(-6) M but is optimally active at 10(-8) M. At the latter concentration, the degree of response is equivalent, on a molar b...

  9. How the protease thrombin talks to cells

    OpenAIRE

    COUGHLIN, SHAUN R.

    1999-01-01

    How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the ...

  10. 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.

  11. Test of hirudin activity by tracking the binding of hirudin to thrombin in the presence of BS3 cross-linking.

    Science.gov (United States)

    Liu, Yanfang; Yang, Jian; Wang, Jiangmin; Huang, Qingmei; Yang, Xiaohong; Zhang, Jianhua

    2015-10-01

    Hirudin has a great potential in inhibiting thrombin, and its antithrombin activity has direct bearing on its clinical application. Using bovine alpha-thrombin and recombinant hirudin of Poecilobdella javanica purified from Phichia pastoris as materials, this study introduced a novel method to testing antithrombin activity of hirudin visually and dynamically by tracking the binding of hirudin to thrombin. After incubating the mixture of thrombin and hirudin at 37?°C for 5?min, the binding of hirudin to thrombin was cross-linked by bis[sulfosuccinimidyl] suberate for 30?min and visualized by SDS-polyacrylamide gel electrophoresis. With the aid of image analysis on the basis of INRA-Noésis E1D analysis software, antithrombin activity of hirudin was calculated through intensity variations of protein bands of either thrombin-hirudin compound, unbound thrombin, or unbound hirudin. In this regard, activity of the given hirudin was tested to be 5625?ATU/mg based on a single reaction, and 5675.3?ATU/mg based on a series of reactions in a stepwise manner, close to the result of 6000?ATU/mg concluded by titration method. The superiorities of the method include good accuracy (the minimum testable concentration of hirudin is 1.5??g/ml) and little sample consumption (sample consumption of hirudin is generally 1-11.5??l using the apparatus of Mini Protean 3 Cell). Easy operation, low input, and equipment requirement also grant it as an effective way. PMID:26332983

  12. Mode of action of thrombin in the rabbit aorta.

    OpenAIRE

    Godin, D.; Rioux, F.; Marceau, F; Drapeau, G.

    1995-01-01

    1. Thrombin is a vasoactive protease that elicits the contraction of the rabbit aorta by activating a G-protein coupled receptor through cleavage of its N-terminal extracellular domain. Synthetic peptides corresponding to the newly exposed N-terminus, following thrombin cleavage, have been shown to reproduce some of the activities of thrombin in the rabbit aorta. 2. Intracellular pathways involved in the contractile response of the rabbit aorta to thrombin and synthetic peptides were examined...

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

    Science.gov (United States)

    Giri, Priya; Ebert, Sabine; Braumann, Ulf-Dietrich; Kremer, Mathias; Giri, Shibashish; Machens, Hans-Günther; Bader, Augustinus

    2015-01-01

    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 ?m 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. PMID:26005333

  14. Effects of thrombin and thrombin receptor activation on cardiac function after acute myocardial infarction

    OpenAIRE

    Gu, Xinyuan; Zhang, Xiaorong; Lu, Guihua; Li, Yanhui; Li, Xiujuan; Huang, He; Zeng, Jianping; Tang, Lilong

    2015-01-01

    Thrombin and thrombin receptor activation impact cardiomyocyte contraction and ventricular remodeling. However, there is some controversy regarding their effects in cardiac function, especially in cardiac dysfunction after acute myocardial infarction (AMI). A rat AMI model was created by left coronary artery ligation (LCA). Cardiac functional parameters, including the maximum left ventricular (LV) systolic pressure (LVSPmax), LV end-diastolic pressure (LVEDP), and the rise and fall rates in L...

  15. The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin.

    Science.gov (United States)

    Chahal, Gurdeep; Thorpe, Michael; Hellman, Lars

    2015-01-01

    Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4' positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance. PMID:26110612

  16. 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

  17. 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.

  18. 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

  19. GpIb? Interacts Exclusively with Exosite II of Thrombin?

    OpenAIRE

    Lechtenberg, Bernhard C.; Freund, Stefan M V; Huntington, James A.

    2014-01-01

    Activation of platelets by the serine protease thrombin is a critical event in haemostasis. This process involves the binding of thrombin to glycoprotein Ib? (GpIb?) and cleavage of protease-activated receptors (PARs). The N-terminal extracellular domain of GpIb? contains an acidic peptide stretch that has been identified as the main thrombin binding site, and both anion binding exosites of thrombin have been implicated in GpIb? binding, but it remains unclear how they are involved. This issu...

  20. Investigation of a thrombin-complexing protein associated with platelets

    International Nuclear Information System (INIS)

    A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77k Da complex with a platelet factor. This platelet factor is in several respects similar to protease nexin I (PNI), a fibroblasts thrombin inhibitor. The complexes are of the right size, bind to agarose that has been derivatized with either anti-PNI antibody or heparin, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. The interaction with the platelet surface may modulate the conformation and function of this platelet form of protease nexin I (PNIp) because: (i) an antibody against protease nexi I inhibited released PNIp, but not platelet-bound PNIp from complexing 125I-thrombin, and (ii) whereas PNIp extracted from platelets bound both thrombin and urokinase, platelet-bound PNIp bound only thrombin. In experiments employing several different platelet isolation methods, PNIp accounted for a large fraction of the rapid high affinity binding of 125I-thrombin to platelets. However, platelets isolated and maintained in the presence of metabolic inhibitors failed to take added thrombin into 125I-thrombine-PNIp complexes

  1. Thrombin generation in a patient with an acquired high-titre factor V inhibitor.

    Science.gov (United States)

    Schmidt, David E; Steinhagen, Friederike; Schnabel, Claudia; Spath, Brigitte; Holstein, Katharina; Fiedler, Walter; Bokemeyer, Carsten; Renné, Thomas; Langer, Florian

    2015-01-01

    The management of patients with acquired factor V inhibitors is challenging, because their bleeding risk is highly variable and only poorly correlated with routine coagulation tests. Furthermore, there is no standardized treatment for bleeding control or inhibitor eradication. An 84-year-old white man underwent uneventful surgery for a ruptured intracerebral haemangioma. There were no perioperative coagulation abnormalities. Eight weeks after surgery, however, the prothrombin and the activated partial thromboplastin times were found to be maximally prolonged without signs of acute haemorrhage. A factor V inhibitor of 212 Bethesda units was diagnosed. We used a fluorogenic real-time thrombin generation assay with low concentrations of tissue factor (TF) to analyse the factor V inhibitor for interference with coagulation in platelet-poor plasma. Compared with three bleeding patients with acquired haemophilia A and severely deficient thrombin generation, total thrombin generation capacity was similar in the patient and healthy controls. However, the lag phase was significantly prolonged, suggesting a defect in the initiation/amplification, but not in the propagation phase of TF-triggered thrombin generation. This defect could be fully reproduced by purified patient IgG and largely corrected by ex-vivo addition of activated prothrombin complex concentrate, but not recombinant human FVIIa. Addition of normal platelets to the patient's plasma resulted in a pronounced shortening of the lag phase, suggesting that platelet-derived factor V can escape the inhibitor. Our findings offer an explanation for the absence of spontaneous bleeding in this patient and support the concept of platelet transfusions for the management of acute haemorrhages in patients with acquired factor V inhibitors. PMID:25158984

  2. In vitro pharmacological characterization of vorapaxar, a novel platelet thrombin receptor antagonist.

    Science.gov (United States)

    Hawes, Brian E; Zhai, Ying; Hesk, David; Wirth, Mark; Wei, Huijun; Chintala, Madhu; Seiffert, Dietmar

    2015-09-01

    Vorapaxar is a novel protease-activated receptor-1 (PAR1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. The present study provides a comprehensive in vitro pharmacological characterization of vorapaxar interaction with the PAR1 receptor on human platelets. Similar studies were performed with a metabolite of vorapaxar (M20). Vorapaxar and M20 were competitive PAR1 antagonists that demonstrated concentration-dependent, saturable, specific, and slowly reversible binding to the receptor present on intact human platelets. The affinities of vorapaxar and M20 for the PAR1 receptor were in the low nanomolar range, as determined by saturation-, kinetic- and competitive binding studies. The calculated Kd and Ki values for vorapaxar increased in the presence of plasma, indicating a decrease in the free fraction available for binding to the PAR1 receptor on human platelets. Vorapaxar was also evaluated in functional assays using thrombin or a PAR1 agonist peptide (SFLLRN). Vorapaxar and M20 completely blocked thrombin-stimulated PAR1/?-arrestin association in recombinant cells and abolished thrombin-stimulated calcium influx in washed human platelets and vascular smooth muscle cells. Moreover, vorapaxar and M20 inhibited PAR1 agonist peptide-mediated platelet aggregation in human platelet rich plasma with a steep concentration response relationship. Vorapaxar exhibited high selectivity for inhibition of PAR1 over other platelet GPCRs. In conclusion, vorapaxar is a potent PAR1 antagonist exhibiting saturable, reversible, selective binding with slow off-rate kinetics and effectively inhibits thrombin's PAR1-mediated actions on human platelets. PMID:26022529

  3. Thrombin functions as an inflammatory mediator through activation of its receptor

    OpenAIRE

    1996-01-01

    A rat model of inflammation was used to investigate the biological effects of thrombin. The thrombin-specific inhibitor Hirulog markedly attentuated the carrageenin-induced edema of the paw of the rat. Injection of thrombin into the paw also produced edema. The effect of thrombin was due to activation of its receptor; a thrombin receptor activating peptide (TRAP) reproduced the effects of thrombin in causing edema. TRAP also increased vascular permeability as demonstrated by extravasation of ...

  4. Role of thrombin signalling in platelets in haemostasis and thrombosis

    Science.gov (United States)

    Sambrano, Gilberto R.; Weiss, Ethan J.; Zheng, Yao-Wu; Huang, Wei; Coughlin, Shaun R.

    2001-09-01

    Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.

  5. Acidosis, magnesium and acetylsalicylic acid: Effects on thrombin

    Science.gov (United States)

    Borisevich, Nikolaj; Loznikova, Svetlana; Sukhodola, Aleksandr; Halets, Inessa; Bryszewska, Maria; Shcharbin, Dzmitry

    2013-03-01

    Thrombin, an enzyme from the hydrolase family, is the main component of the blood coagulation system. In ischemic stroke it acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin forming blood clots in the brain. It has been found to phosphoresce at room temperature in the millisecond and microsecond ranges. The phosphorescence of thrombin was studied under physiological conditions, in acidosis (decrease of pH from 8.0 to 5.0) and on the addition of salts (magnesium sulfate and sodium chloride) and of acetylsalicylic acid, and its connection with thrombin function is discussed. Acidosis significantly increased the internal dynamics of thrombin. We propose that lactate-acidosis plays a protective role in stroke, preventing the formation of clots. The addition of NaCl and MgSO4 in different concentrations increased the internal dynamics of thrombin. Also, the addition of MgSO4 decreased thrombin-induced platelet aggregation. However, magnesium sulfate and acetylsalicylic acid in the therapeutic concentrations used for treatment of ischemic stroke had no effect on thrombin internal dynamics. The data obtained will help to elucidate the conformational stability of thrombin under conditions modulating lactate-acidosis and in the presence of magnesium sulfate.

  6. Effects of thrombin and thrombin receptor activation on cardiac function after acute myocardial infarction.

    Science.gov (United States)

    Gu, Xinyuan; Zhang, Xiaorong; Lu, Guihua; Li, Yanhui; Li, Xiujuan; Huang, He; Zeng, Jianping; Tang, Lilong

    2015-01-01

    Thrombin and thrombin receptor activation impact cardiomyocyte contraction and ventricular remodeling. However, there is some controversy regarding their effects in cardiac function, especially in cardiac dysfunction after acute myocardial infarction (AMI). A rat AMI model was created by left coronary artery ligation (LCA). Cardiac functional parameters, including the maximum left ventricular (LV) systolic pressure (LVSPmax), LV end-diastolic pressure (LVEDP), and the rise and fall rates in LV pressure (dp/dt max and dp/dt min, respectively), were measured. Hirudin decreased cardiac function within 120 minutes after AMI, whereas treatment with thrombin receptor-activating peptide (TRAP) reversed this hirudin-induced decrease in cardiac function. The mRNA and protein expression levels of inositol 1,4,5-trisphosphate receptor (IP3R) subtypes in infarct area tissues were analyzed by reverse transcription-polymerase chain reaction and immunoreaction. Hirudin decreased the expression levels of IP3R-1, -2, and -3 in the infarct area for up to 40 minutes after AMI, whereas TRAP treatment reversed these hirudin-induced effects. Treatment with the IP3R antagonist 2-aminoethoxydiphenyl borate (2.5 mg/kg) eliminated the effect of TRAP on the hirudin-induced decrease in cardiac function after AMI. Finally, TRAP increased the maximum binding capacity of the three IP3R subtypes, but only enhanced the affinity of IP3R-2. Thrombin and thrombin receptor activation improved cardiac function after AMI by an IP3R-mediated pathway, probably through the IP3R-2 subtype. PMID:26064435

  7. Facile fabrication of an aptasensor for thrombin based on graphitic carbon nitride/TiO2 with high visible-light photoelectrochemical activity.

    Science.gov (United States)

    Fan, Dawei; Guo, Cuijuan; Ma, Hongmin; Zhao, Di; Li, Yina; Wu, Dan; Wei, Qin

    2016-01-15

    A novel aptasensor for thrombin with high visible-light activity was facilely fabricated based on graphitic carbon nitride/TiO2 (g-C3N4/TiO2) photoelectrochemical (PEC) composite. Crystallization of TiO2 nanoparticles (NPs) and their strong interaction with g-C3N4 sheet were confirmed by high-resolution transmission electron microscope (HR-TEM), both of which contributed to the high photocurrent intensity under visible-light irradiation. Carboxyl functionalized thrombin aptamers were first successfully bound to the g-C3N4/TiO2 modified electrode as proven by photoelectrochemical test and electrochemical impedance spectroscopy (EIS) analysis. Ascorbic acid was utilized as the electron donor for scavenging photo-generated holes and inhibiting light driven electron-hole pair recombination. The specific recognition between thrombin aptamer and thrombin led to the linear decrease of photocurrent with the increase of logarithm of thrombin concentration in the range of 5.0×10(-13)molL(-1) to 5.0×10(-9)molL(-1) with a detection limit of 1.2×10(-13)molL(-1). This proposed low-cost, convenient and sensitive aptasensor showed promising applications in biosensor and photoelectrochemical analysis. PMID:26301999

  8. 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.

  9. Altered Flow Changes Thrombin Generation Rate of Circulating Platelets.

    Science.gov (United States)

    Yin, Wei; Bond, Kyle; Rouf, Farzana; Rubenstein, David A

    2015-12-01

    Shear stress affects platelet participation in coagulation. Many numerical models have been developed to describe coagulation kinetics. However, most of those models used rate constants determined under static conditions. Little is known about the effects of flow on coagulation rate constants. In the present study, platelets were exposed to constant or pulsatile shear stress/rate, with or without prothrombin, factor Xa, and factor Va. Thrombin generation was measured using a modified prothrombinase assay, and the overall thrombin generation rate was solved using typical Michaelis-Menten kinetics. Platelet surface P-selectin and phosphatidylserine (PS) expression was measured using flow cytometry. The results demonstrated that the concentration of factor Va had a dominant effect on thrombin generation rate under flow. In comparison, the expression of PS was less sensitive to altered flow. The lumped overall rate constant for prothrombin conversion to thrombin was significantly affected by the shear forces that were applied to the coagulation complex. Constant shear stress/rate induced faster thrombin generation compared to pulsatile shear stress/rate, but elevated shear stress/rate did not necessarily enhance thrombin generation. Therefore, the overall thrombin generation rate is dynamic and must be described as a function of shear stress/rate, shear exposure time and the immediate availability of coagulation proteins. PMID:26036336

  10. Thrombin Preconditioning in Surgical Brain Injury in Rats.

    Science.gov (United States)

    Benggon, Michael; Chen, Hank; Applegate, Richard L; Zhang, John

    2016-01-01

    The surgical brain injury model replicates neurosurgical brain parenchymal damage. Postsurgical brain edema correlates with postoperative neurological dysfunction. Intranasal administration is a proven method of delivering therapies to brain tissue. Thrombin preconditioning decreased brain edema and improved neurological outcomes in models of ischemic brain injury. We hypothesized thrombin preconditioning in surgical brain injury may improve postoperative brain edema and neurological outcomes. Adult male Sprague-Dawley rats (n?=?78) weighing 285-355 g were randomly assigned to sham or pre-injury treatment: one-time pretreatment 1 day prior, one-time pretreatment 5 days prior, and daily preconditioning for 5 days prior. Treatment arms were divided into vehicle or thrombin therapies, and subdivided into intranasal (thrombin 5 units/50 ?L 0.9 % saline) or intracerebral ventricular (thrombin 0.1 unit/10 ?L 0.9 % saline) administration. Blinded observers performed neurological testing 24 h after brain injury followed immediately by measurement of brain water content. There was a significant difference in ipsilateral brain water content and neurological outcomes between all treatment groups and the sham group. However, there was no change in brain water content or neurological outcomes between thrombin- and vehicle-treated animals. Thrombin preconditioning did not significantly improve brain edema or neurological function in surgical brain injury in rats. PMID:26463965

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

    OpenAIRE

    Giri, Priya; Ebert, Sabine; Braumann, Ulf-Dietrich; Kremer, Mathias; Giri, Shibashish; Machens, Hans-Günther; Bader, Augustinus

    2015-01-01

    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 topica...

  12. Red blood cells and thrombin generation in sickle cell disease

    OpenAIRE

    Whelihan, Matthew F; Lim, Ming Y; Key, Nigel S

    2014-01-01

    The prothrombotic nature of sickle cell disease (SCD) is evidenced by the chronically elevated levels of almost all coagulation activation biomarkers, and an increased incidence of certain thrombotic events, including venous thromboembolism. Numerous studies have attempted to define the extent and elucidate the mechanism of the observed increase in thrombin generation in SCD patients in vivo. In general, these studies were performed using thrombin generation assays in platelet poor or platele...

  13. Amelioration of collagen-induced arthritis by thrombin inhibition

    OpenAIRE

    Marty, Ingrid; Péclat, Veronique; Kirdaite, Gailute; Salvi, Roberto; So, Alexander; Busso, Nathalie

    2001-01-01

    The deleterious role of fibrin deposition in arthritic joints prompted us to explore the effect of the thrombin inhibition on the course of collagen-induced arthritis (CIA) in the mouse. CIA was induced in male DBA/1J mice using native chicken type II collagen. The thrombin inhibitor polyethyleneglycol-hirudin (PEG-hirudin) was given for 16 days, starting 20 days after the first immunization (preventive treatment) or at the onset of clinical signs of arthritis (curative treatment). All the mi...

  14. Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups

    Science.gov (United States)

    Hindawi, Salwa; Hemker, H. Coenraad; de Laat, H. Bas; Huskens, Dana; Al Dieri, Raed

    2015-01-01

    Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and ?2Macroglobulin (?2M) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and ?2M levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, ?2M plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of ?2M to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII. PMID:26509437

  15. Active-site-dependent, thrombin-induced release of adenine nucleotides from cultured human endothelial cells

    International Nuclear Information System (INIS)

    The effect of thrombin on release of adenine nucleotides is studied in cultured cell monolayers from the human umbilical vein. Thrombin-induced release of radioactivity from endothelial cells is dose-dependent and saturable with maximal response seen at 1 x 10(-8) M thrombin. The products are identified by thin-layer chromatography as adenine nucleotides. Diisopropylphosphoryl thrombin, which is enzymatically inactive, does not cause release of tritium. A 50-fold excess of diisopropylphosphoryl-thrombin, despite causing 98 percent inhibition of binding of 125I-thrombin to its high-affinity binding sites, does not inhibit thrombin-induced release. We conclude that (1) thrombin causes release from endothelial cells of adenine nucleotides and that (2) high-affinity, active-site-independent binding of thrombin is not involved in this process

  16. 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

  17. Aptamer RA36 inhibits of human, rabbit, and rat plasma coagulation activated with thrombin or snake venom coagulases.

    Science.gov (United States)

    Savchik, E Yu; Kalinina, T B; Drozd, N N; Makarov, V A; Zav'yalova, E G; Lapsheva, E N; Mudrik, N N; Babij, A V; Pavlova, G V; Golovin, A V; Kopylov, A M

    2013-11-01

    RA36 DNA aptamer is a direct anticoagulant prolonging clotting time of human, rabbit, and rat plasma in the thrombin time test. Anticoagulant activity of RA36 is lower than that of recombinant hirudin. During inhibition of human plasma clotting activated with echitox (coagulase from Echis multisquamatus venom), the aptamer presumably binds to meisothrombin exosite I. The sensitivity of human plasma to the aptamer 5-fold surpasses that of rat plasma. Analysis of RA36 binding to coagulase of Agkistrodon halys venom (ancistron) is required for proving the effect of aptamer on polymerization of human fibrinogen. PMID:24319726

  18. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

    Science.gov (United States)

    Parker, William H; Qu, Zhi-Chao; May, James M

    2015-08-28

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729

  19. Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers

    OpenAIRE

    1992-01-01

    The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby activates its own seven-transmembrane- domain receptor which couples to G proteins. Thrombin also can inhibit neuronal differentiation, supposedly by degrading components of the extracellular matrix. Here we report that active thrombin induces immediate cell rounding and neurite retraction in differentiating N1E- 115 and NG108-15 neural cells in serum-free culture. Serum (0.5-5% vol/vol) evokes sim...

  20. Multiple active forms of thrombin. IV. Relative activities of meizothrombins

    International Nuclear Information System (INIS)

    The prothrombin activation intermediates meizothrombin and meizothrombin(desF1) (meizothrombin that has been autoproteolyzed to remove fragment 1) have been obtained in a relatively pure, active form with minimal autolysis, making them suitable for enzymatic characterization. When compared at equimolar concentrations, alpha-thrombin, fragment 1.2+ alpha-thrombin, meizothrombin(desF1), and meizothrombin have approximately 100, 100, 10, and 1% activity, respectively, toward the macromolecular substrates factor V, fibrinogen, and platelets. The difference in activity of these four enzymes cannot be attributed to alterations in the catalytic triad, as all four enzymes have nearly identical catalytic efficiency toward the chromogenic substrate S2238. Further, the ability of meizothrombin and meizothrombin(desF1) to activate protein C was 75% of the activity exhibited by alpha-thrombin or fragment 1.2+ alpha-thrombin. All four enzymes bind to thrombomodulin, as judged by the enhanced rate of protein C activation upon preincubation of the enzymes with thrombomodulin. The extent of rate enhancement varied, with meizothrombin/thrombomodulin exhibiting only 50% of the alpha-thrombin/thrombomodulin rate. This difference in rate is not due to a decreased affinity of the meizothrombin for thrombomodulin since the apparent dissociation constants for the alpha-thrombin-thrombomodulin complex and the meizothrombin-thrombomodulin complex are virtually identical. The difference in the observed rate is due in part to the higher Km for protein C exhibited by the meizothrombin-thrombomodulin complex. Incubation of the thrombomodulin-enzyme complex with phospholipid vesicles caused an increase in the protein C activation rates. The kinetic constants for protein C activation in the presence of phospholipid are virtually identical for these enzyme-thrombomodulin complexes

  1. Thrombin mutant W215A/E217A treatment improves neurological outcome and attenuates central nervous system damage in experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Verbout, Norah G; Yu, Xiaolin; Healy, Laura D; Phillips, Kevin G; Tucker, Erik I; Gruber, András; McCarty, Owen J T; Offner, Halina

    2015-02-01

    Multiple sclerosis (MS) is a neuroinflammatory disease characterized by demyelination and axonal damage of the central nervous system. The pathogenesis of MS has also been linked to vascular inflammation and local activation of the coagulation system, resulting in perivascular fibrin deposition. Treatment of experimental autoimmune encephalomyelitis (EAE), a model of human MS, with antithrombotic and antiinflammatory activated protein C (APC) reduces disease severity. Since recombinant APC (Drotecogin alfa), originally approved for the treatment of severe sepsis, is not available for human MS studies, we tested the hypothesis that pharmacologic activation of endogenous protein C could likewise improve the outcome of EAE. Mice were immunized with murine myelin oligodendrocyte glycoprotein (MOG) peptides and at the onset of EAE symptoms, were treated every other day with either WE thrombin (25 ?g/kg; i.v.), a selective recombinant protein C activator thrombin analog, or saline control. Mice were monitored for changes in disease score until euthanized for ex vivo analysis of inflammation. Administration of WE thrombin significantly ameliorated clinical severity of EAE, reduced inflammatory cell infiltration and demyelination, suppressed the activation of macrophages comprising the CD11b + population and reduced accumulation of fibrin (ogen) in the spinal cord. These data suggest that symptomatic MS may respond to a treatment strategy that involves temporal pharmacological enhancement of endogenous APC generation. PMID:24810631

  2. High-affinity RNA ligands to human alpha-thrombin.

    OpenAIRE

    Kubik, M F; Stephens, A W; Schneider, D; Marlar, R A; Tasset, D

    1994-01-01

    Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to isolate from a population of 10(13) RNA molecules two classes of high affinity RNAs that bind specifically to human alpha-thrombin. Class I RNAs are represented by a 24-nucleotide RNA (RNA 16.24), and class II RNAs are represented by a 33-nucleotide RNA (RNA 27.33). RNA 16.24 inhibits thrombin-catalyzed fibrin clot formation in vitro. Secondary structures are proposed for these RNAs, revealing a novel stem-loop stru...

  3. Human thrombin for the treatment of gastric and ectopic varices

    Directory of Open Access Journals (Sweden)

    Norma C McAvoy

    2012-01-01

    Full Text Available AIM: To evaluate the efficacy of human thrombin in the treatment of bleeding gastric and ectopic varices. METHODS: Retrospective observational study in a Tertiary Referral Centre. Between January 1999-October 2005, we identified 37 patients who were endoscopically treated with human thrombin injection therapy for bleeding gastric and ectopic varices. Patient details including age, gender and aetiology of liver disease/segmental portal hypertension were documented. The thrombin was obtained from the Scottish National Blood Transfusion Service and prepared to give a solution of 250 IU/mL which was injected via a standard injection needle. All patient case notes were reviewed and the total dose of thrombin given along with the number of endoscopy sessions was recorded. Initial haemostasis rates, rebleeding rates and mortality were catalogued along with the incidence of any immediate complications which could be attributable to the thrombin therapy. The duration of follow up was also listed. The study was conducted according to the United Kingdom research ethics guidelines. RESULTS: Thirty-seven patients were included. 33 patients (89% had thrombin (250 U/mL for gastric varices, 2 (5.4% for duodenal varices, 1 for rectal varices and 1 for gastric and rectal varices. (1 Gastric varices, an average of 15.2 mL of thrombin was used per patient. Re-bleeding occurred in 4 patients (10.8%, managed in 2 by a transjugular intrahepatic portosystemic shunt (TIPSS (one unsuccessfully who died and in other 2 by a distal splenorenal shunt; (2 Duodenal varices (or type 2 isolated gastric varices, an average of 12.5 mL was used per patient over 2-3 endoscopy sessions. Re-bleeding occurred in one patient, which was treated by TIPSS; and (3 Rectal varices, an average of 18.3 mL was used per patient over 3 endoscopy sessions. No re-bleeding occurred in this group. CONCLUSION: Human thrombin is a safe, easy to use and effective therapeutic option to control haemorrhage from gastric and ectopic varices.

  4. Thrombin regulates components of the fibrinolytic system in human mesangial cells

    International Nuclear Information System (INIS)

    Besides its procoagulant activity, thrombin has been shown to stimulate cell proliferation and to regulate the fibrinolytic pathway. We report here the effect of purified human alpha thrombin on the synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) by cultured human mesangial cells. Thrombin (0 to 2.5 U/ml) increased in a time- and dose-dependent manner the production of t-PA and PAI-1 (2- to 3-fold increase of secreted t-PA and PAI-1 release during a 24 hour incubation). This effect was associated with a twofold increase in DNA synthesis measured by 3H-thymidine incorporation. Zymographic analysis and reverse fibrin autography showed that thrombin also increased the level of the 110 Kd t-PA-PAI-1 complex, whereas PAI-1 was present as a free 50 Kd form in the culture medium conditioned by unstimulated and thrombin-stimulated cells. Free t-PA was never observed. Both membrane binding and catalytic activity of thrombin were required since the effects of 1 U/ml thrombin were inhibited by addition 2 U/ml hirudin, which inhibits the membrane binding and catalytic activity of thrombin, and since DFP-inactivated thrombin, which has the ability to bind but which has no enzymatic activity, did not induce t-PA or PAI-1. Gamma thrombin, which does not bind to thrombin receptor, did not increase t-PA and PAI-1 releases. The effects of thrombin were probably mediated by protein kinase C activation since H7, an inhibitor of protein kinases, inhibited significantly thrombin effects on t-PA and PAI-1 production, and since addition of an activator of protein kinase A, 8-bromocyclic AMP (100 microM), induced a significant inhibition of the thrombin effect. The effects of thrombin were also suppressed by 1.25 micrograms/ml alpha amanitin, suggesting a requirement of de novo RNA synthesis

  5. Thrombin regulates components of the fibrinolytic system in human mesangial cells

    Energy Technology Data Exchange (ETDEWEB)

    Villamediana, L.M.; Rondeau, E.; He, C.J.; Medcalf, R.L.; Peraldi, M.N.; Lacave, R.; Delarue, F.; Sraer, J.D. (INSERM Unite 64, Paris (France))

    1990-11-01

    Besides its procoagulant activity, thrombin has been shown to stimulate cell proliferation and to regulate the fibrinolytic pathway. We report here the effect of purified human alpha thrombin on the synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) by cultured human mesangial cells. Thrombin (0 to 2.5 U/ml) increased in a time- and dose-dependent manner the production of t-PA and PAI-1 (2- to 3-fold increase of secreted t-PA and PAI-1 release during a 24 hour incubation). This effect was associated with a twofold increase in DNA synthesis measured by 3H-thymidine incorporation. Zymographic analysis and reverse fibrin autography showed that thrombin also increased the level of the 110 Kd t-PA-PAI-1 complex, whereas PAI-1 was present as a free 50 Kd form in the culture medium conditioned by unstimulated and thrombin-stimulated cells. Free t-PA was never observed. Both membrane binding and catalytic activity of thrombin were required since the effects of 1 U/ml thrombin were inhibited by addition 2 U/ml hirudin, which inhibits the membrane binding and catalytic activity of thrombin, and since DFP-inactivated thrombin, which has the ability to bind but which has no enzymatic activity, did not induce t-PA or PAI-1. Gamma thrombin, which does not bind to thrombin receptor, did not increase t-PA and PAI-1 releases. The effects of thrombin were probably mediated by protein kinase C activation since H7, an inhibitor of protein kinases, inhibited significantly thrombin effects on t-PA and PAI-1 production, and since addition of an activator of protein kinase A, 8-bromocyclic AMP (100 microM), induced a significant inhibition of the thrombin effect. The effects of thrombin were also suppressed by 1.25 micrograms/ml alpha amanitin, suggesting a requirement of de novo RNA synthesis.

  6. Thrombin as a multi-functional enzyme. Focus on in vitro and in vivo effects.

    Science.gov (United States)

    Siller-Matula, Jolanta M; Schwameis, Michael; Blann, Andrew; Mannhalter, Christine; Jilma, Bernd

    2011-12-01

    Thrombin is the central protease in the coagulation cascade and one of the most extensively studied of all enzymes. In addition to its recognised role in the coagulation cascade and haemostasis, thrombin is known to have multiple pleiotropic effects, which mostly have been shown only in in vitro studies: it plays a role in inflammation and cellular proliferation and displays a mitogen activity on smooth muscle cells and endothelial cells, predominantly by activation of angiogenesis. In vivo , thrombin effects were examined in animal models of intravenous or intraarterial thrombin infusion. An extensive literature search regarding in vivo data showed that i) thrombin administered as a bolus causes microembolism, ii) thrombin infused slowly at steady-state conditions (up to 1.6 U/kg/min) leads to bleeds but not to intravascular clotting, iii) large quantity of thrombin infused at low rates (0.05 U/kg/min) does not have any measurable effect, and iv) thrombin increases vascular permeability leading to tissue damage. Although several decades of research on thrombin functions have provided a framework for understanding the biology of thrombin, animal and human studies with use of newer laboratory techniques are still needed to confirm the pleiotropic thrombin functions shown in in vitro studies. PMID:21979864

  7. 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.

  8. Stimulation of phosphoinositide hydrolysis by thrombin in embryonic chick heart cells

    International Nuclear Information System (INIS)

    The authors have shown that muscarinic receptor stimulation results in phosphoinositide (PI) hydrolysis in dissociated embryonic chick heart cells, with maximal stimulation produced by 1 mM carbachol. In an effort to identify other stimulators of PI turnover, they studied the effects of thrombin in primary cultures of 13-day embryonic chick heart cells. Thrombin increased PI hydrolysis, as measured by the accumulation of [3H]inositol 1-phosphate in the presence of 10 mM LiCl. Stimulation occurred in a dose-dependent manner with half-maximal stimulation obtained at 0.07 U/ml and maximal stimulation at 0.3 U/ml thrombin. The effects of maximal doses of carbachol and thrombin were additive, suggesting different mechanisms of action or different populations of cells. Effects of both hormones were inhibited by pretreatment of the monolayer cultures with 4?-phorbol 12 ?-myristate 13?-acetate. The simultaneous addition of hirudin with thrombin blocked thrombin-stimulated PI turnover. An active-site-blocked derivative of thrombin was ineffective in stimulating IP formation. These data indicate a proteolytic action of thrombin in stimulation of the PI response. To their knowledge, these are the first data suggesting that there may be thrombin receptors or effects of thrombin on heart cells

  9. Iron levels found in hemochromatosis patients inhibit ?-thrombin-induced platelet aggregation.

    Science.gov (United States)

    Lynch, Stephen; Soslau, Gerald

    2012-01-01

    Hemochromatosis is a common genetic disorder of iron metabolism. The increase in systemic iron associated with hemochromatosis can negatively affect every system in the body, with potentially fatal implications. Little is known about the effects of iron overload on hemostasis and platelet activation. While performing platelet aggregation studies on hemochromatotic blood samples, it was discovered that the increased serum iron levels associated with this disease almost completely inhibited ?-thrombin-induced platelet aggregation. Further studies were conducted with samples derived from control and hemochromatotic individuals to determine the effects of iron on both ?- and ?-thrombin-induced platelet aggregations. It was found that ?-thrombin-induced platelet aggregations were strongly inhibited by the direct binding of iron to these enzymes. ?-Thrombin activates platelets at multiple receptor sites including the protease-activated receptor-1 (PAR-1), and at high concentrations, at protease-activated receptor-4 (PAR-4), while ?-thrombin selectively activates platelets at PAR-4. ?-Thrombin activity was significantly more sensitive to toxic levels of iron than ?-thrombin in the activation of the PAR receptors. Also, the hydrolysis of a synthetic peptide by ?-thrombin was unaffected by iron while ?-thrombin was strongly inhibited. These results indicate that the iron-thrombin complex strongly regulates the activity of ?-thrombin while has little or no effect on ?-thrombin-induced platelet aggregation or hydrolytic activity. It is unknown, at this time, what specific clinical implications iron inhibition of ?-thrombin may have, but it is very possible that other conditions caused by hemochromatosis could be exacerbated by the inability of platelets to aggregate normally. PMID:22111666

  10. 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

  11. Efinaconazole Topical

    Science.gov (United States)

    Efinaconazole topical solution is used to treat fungal toenail infections (infections that may cause nail discoloration, splitting, or pain). Efinaconazole topical solution is in a class of medications ...

  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 Receptor and Ventricular Arrhythmias after Acute Myocardial Infarction

    OpenAIRE

    Tang, Lilong; Deng, Chunyu; Long, Ming; Tang, Anli; Wu, Shulin; Dong, Yugang; Saravolatz, Louis D.; Gardin, Julius M

    2008-01-01

    The mechanism mediating the development of ventricular arrhythmia (VA) after acute myocardial infarction (AMI) is still uncertain. Thrombin receptor (TR) activation has been proven to be arrhythmogenic in many other situations, and we hypothesize that it may participate in the genesis of post-AMI VA. Using a left coronary artery ligation rat model of AMI, we found that a local injection of hirudin into the left ventricle (LV) significantly reduced the ratio of VA durations to infarction sizin...

  14. 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.

  15. 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...

  16. Atherosclerosis proceeds independently of thrombin-induced platelet activation in ApoE-/- mice

    OpenAIRE

    Hamilton, J.R.; Cornelissen, I.; Mountford, J.K.; Coughlin, S.R.

    2009-01-01

    Platelet activation has long been postulated to contribute to the development of atherosclerotic plaques, although the mechanism by which this might occur remains unknown. Thrombin is a potent platelet activator and transfusion of thrombin-activated platelets into mice increases plaque formation, suggesting that thrombin-induced platelet activation might contribute to platelet-dependent atherosclerosis. Platelets from protease-activated receptor 4-deficient (Par4-/-) mice fail to respond to t...

  17. TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features

    OpenAIRE

    Fusté, Berta; Díaz-Ricart, Maribel; Jensen, Morten Krogh; Ordinas, Antonio; Escolar, Ginés; White, James G.

    2002-01-01

    We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptor-activating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, throm...

  18. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor.

    OpenAIRE

    McNamara, C A; Sarembock, I J; Gimple, L. W.; Fenton, J. W.; Coughlin, S.R.; Owens, G. K.

    1993-01-01

    Thrombin has been implicated in the stimulation of smooth muscle cell (SMC) proliferation that contributes to post angioplasty restenosis. The present studies demonstrated that human alpha-thrombin was a potent and efficacious mitogen for cultured rat aortic SMC, stimulating an increase in 3H-thymidine incorporation, as well as an increase in cell number at 1 to 10 nM concentration. gamma-Thrombin, which is enzymatically active but lacks fibrinogen clotting activity, stimulated SMC mitogenesi...

  19. 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...

  20. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin

    OpenAIRE

    Kahn, Mark L.; Nakanishi-Matsui, Mayumi; Michael J. Shapiro; Ishihara, Hiroaki; COUGHLIN, SHAUN R.

    1999-01-01

    Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin — PAR1, PAR3, and PAR4 — are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 accoun...

  1. [Peculiarities of thrombin generation and prognosis of unfavourable outcome in patients with severe pneumonia and pneumogenic sepsis].

    Science.gov (United States)

    Martynenko, T I; Momot, A P; Balatskaia, I V; Sho?khet, Ia N; Grebeniuk, A A; Rusakova, O V; Kapitulin, S Iu

    2014-01-01

    The study of thrombin production included 68 patients with severe pneumonia (SP) undergoing monitoring plasma thrombin potential in the thrombin generation test. Thrombin production was found to decrease in the patients who died compared with those alive on days 3-5 and 7-10 after onset of the disease. Endogenous thrombin potential decreased progressively during the first 7-10 days among the patients with the fatal outcome of SP. This trend in thrombin generation can be used to predict the unfavourable outcome of SP. PMID:25799829

  2. Percutaneous Ultrasound-Guided Thrombin Injection in Iatrogenic Arterial Pseudoaneurysms: Effectiveness and Complications

    Energy Technology Data Exchange (ETDEWEB)

    Koh, Young Hwan [Boramae Hospital, Seoul (Korea, Republic of); Kim, Hak Soo; Kim, Hyung Sik; Min, Seung Kee [Gachon Medical School, Incheon (Korea, Republic of)

    2005-09-15

    To evaluate and describe the efficacy and side effects of a percutaneous thrombin injection under ultrasonography guidance for the treatment of iatrogenic pseudo aneurysms Eighteen consecutive iatrogenic pseudo aneurysm cases were treated with a thrombin injection. The thrombin was injected into the pseudo aneurysm cavity using a 22-gauge needle under ultrasonographic guidance. The causes of the pseudo aneurysms are as follows: post coronary angiography (9 cases), percutaneous coronary balloon angioplasty (5 cases), cerebral angiography (1 case), transhepatic chemo embolization (1 case), percutaneous trans femoral arterial stent insertion (1 case) and bone marrow aspiration for a marrow transplant (1 case). Only one case required a secondary thrombin injection due to recurrent flow in the pseudo aneurysm lumen, which was detected at the follow up Doppler ultrasound. Other seventeen cases were successfully treated on the first trial. There were no technical failures or complication related to the procedure. The average amount of thrombin injected was 733 IU. Nine out of 18 treated patients (50%) showed mild reactions to the thrombin including mild fever (4 cases), chilling sensation (3 cases), a chilling sensation with mild dyspnea (1 case), mild chest discomfort (1 case) after the thrombin injection. All these side effects were transient and improved several hours later. All the iatrogenic pseudo aneurysms were treated successfully with an ultrasound-guided percutaneous thrombin injection. There was a high rate of hypersensitivity to the bovine thrombin, which precaution should be taken to prevent more serious side effects

  3. Effects of Aerobic Capacity on Thrombin-Induced Hydrocephalus and White Matter Injury.

    Science.gov (United States)

    Ni, Wei; Gao, Feng; Zheng, Mingzhe; Koch, Lauren G; Britton, Steven L; Keep, Richard F; Xi, Guohua; Hua, Ya

    2016-01-01

    We have previously shown that intracerebral hemorrhage-induced brain injury is less in rats bred for high aerobic capacity (high capacity runners; HCR) compared with those bred for low aerobic capacity (low capacity runners; LCRs). Thrombin, an essential component in the coagulation cascade, is produced after cerebral hemorrhage. Intraventricular injection of thrombin causes significant hydrocephalus and white matter damage. In the present study, we examined the effect of exercise capacity on thrombin-induced hydrocephalus and white matter damage. Mid-aged (13-month-old) female LCRs (n?=?13) and HCRs (n?=?12) rats were used in this study. Rats received an intraventricular injection of thrombin (3 U, 50 ?l). All rats underwent magnetic resonance imaging (MRI) at 24 h and were then euthanized for brain histology and Western blot. The mortalities were 20 % in LCRs and 33 % in HCRs after thrombin injection (p?>?0.05). No rats died after saline injection. Intraventricular thrombin injection resulted in hydrocephalus and periventricular white matter damage as determined on MRI. In LCR rats, thrombin induced significant ventricle enlargement (23.0?±?2.3 vs12.8?±?1.9 mm(3) in LCR saline group; p?stress marker, and microglial activation in the periventricular white matter. These changes were significantly reduced in HCR rats. Intraventricular injection of thrombin caused more white matter damage and hydrocephalus in rats with low aerobic capacity. A differential effect of thrombin may contribute to differences in the effects of cerebral hemorrhage with aerobic capacity. PMID:26463978

  4. Estradiol Topical

    Science.gov (United States)

    Estradiol topical gel and emulsion (lotion type mixture) are used to treat and prevent hot flushes (hot ... of life; the end of monthly menstrual periods). Estradiol topical gel is also used to treat vaginal ...

  5. Thrombin Induces Tumor Invasion through the Induction and Association of Matrix Metalloproteinase-9 and ?1-Integrin on the Cell Surface*

    OpenAIRE

    Radjabi, A. Reza; Sawada, Kenjiro; Jagadeeswaran, Sujatha; Eichbichler, Alfred; Kenny, Hilary A; Montag, Anthony; Bruno, Katharina; Lengyel, Ernst

    2007-01-01

    The procoagulatory serine protease, thrombin, is known to induce invasion and metastasis in various cancers, but the mechanisms by which it promotes tumorigenesis are poorly understood. Because the 92-kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion, we sought to determine whether and how thrombin regulates MMP-9. The thrombin receptor, PAR-1, and MMP-9 are expressed in osteosarcomas, as determined by immunohistochemistry. Stimulation of U2-OS osteosarcoma cells with thrombin...

  6. Kinetic characterization of inhibition of human thrombin with DNA aptamers by turbidimetric assay.

    Science.gov (United States)

    Zavyalova, Elena G; Protopopova, Anna D; Yaminsky, Igor V; Kopylov, Aleksey M

    2012-02-01

    A sensitive turbidimetric method for detecting fibrin association was used to study the kinetics of fibrinogen hydrolysis with thrombin. The data were complemented by high-performance liquid chromatography (HPLC) measurements of the peptide products, fibrinopeptides released during hydrolysis. Atomic force microscopy (AFM) data showed that the fibril diameter is the main geometric parameter influencing the turbidity. The turbidimetric assay was validated using thrombin with the standard activity. To study thrombin inhibitors, a kinetic model that allows estimating the inhibition constants and the type of inhibition was proposed. The kinetic model was used to study the inhibitory activity of the two DNA aptamers 15-TBA (thrombin-binding aptamer) and 31-TBA, which bind to thrombin exosites. For the first time, 31-TBA was shown to possess the competitive inhibition type, whereas the shortened aptamer 15-TBA has the noncompetitive inhibition type. PMID:22056408

  7. Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface

    Science.gov (United States)

    Gerszten, Robert E.; Chen, Ji; Ishli, Maki; Ishil, Kenji; Wang, Ling; Nanevicz, Tania; Turck, Christoph W.; Vu, Thien-Khai H.; Coughlin, Shaun R.

    1994-04-01

    G-PROTEIN-COUPLED receptors for catecholamines and some other small ligands are activated when agonists bind to the transmem-brane region of the receptor1. The docking interactions through which peptide agonists activate their receptors are less well characterized2-7. The thrombin receptor is a specialized peptide receptor. It is activated by binding its tethered ligand domain, which is unmasked upon receptor cleavage by thrombin8,9. Human and Xenopus thrombin receptor homologues are each selectively activated by the agonist peptide representing their respective tethered ligand domains. Here we identify receptor domains that confer this agonist specificity by replacing the Xenopus receptor's amino-terminal exodomain and three extracellular loops with the corresponding human structures. This switches receptor specificity from Xenopus to human. The specificity of these thrombin receptors for their respective peptide agonists is thus determined by their extracellular surfaces. Our results indicate that agonist interaction with extracellular domains is important for thrombin receptor activation.

  8. Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor

    OpenAIRE

    1992-01-01

    We recently isolated a cDNA clone encoding a functional platelet thrombin receptor that defined a unique mechanism of receptor activation. Thrombin cleaves its receptor's extracellular amino terminal extension, unmasking a new amino terminus that functions as a tethered peptide ligand and activates the receptor. A novel peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, suggesting that this unusual mechanism accounts for platelet activation by...

  9. 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.

  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. 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.

  12. 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.

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

    International Nuclear Information System (INIS)

    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. (paper)

  14. Thrombostatin FM compounds: direct thrombin inhibitors ? mechanism of action in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Nieman, M.T.; Burke, F.; Warnock, M.; Zhou, Y.; Sweigart, J.; Chen, A.; Ricketts, D.; Lucchesi, B.R.; Chen, Z.; Cera, E.Di; Hilfinger, J.; Kim, J.S.; Mosberg, H.I.; Schmaier, A.H. (Case Western); (Michigan); (TSRL); (WU-MED)

    2008-04-29

    Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin - RPPGF. These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at {>=}0.78, 1.6, and 1.6 {mu}m, respectively. They competitively inhibit {alpha}-thrombin-induced cleavage of a chromogenic substrate at 4.4--8.2 {mu}m. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks {alpha}-thrombin-induced calcium flux in fibroblasts with an IC{sub 50} of 6.9 {+-} 1.2 {mu}m. FM19 achieved 100% inhibition of threshold {alpha}- or {gamma}-thrombin-induced platelet aggregation at 8.4 {+-} 4.7 {mu}m and 16 {+-} 4 {mu}m, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.

  15. Conserved structure and adjacent location of the thrombin receptor and protease-activated receptor 2 genes define a protease-activated receptor gene cluster.

    OpenAIRE

    Kahn, M.; Ishii, K.; Kuo, W. L.; Piper, M.; Connolly, A.; Shi, Y.P.; Wu, R.; Lin, C. C.; Coughlin, S.R.

    1996-01-01

    BACKGROUND: Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism. Recently, a second protease-activated receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin receptor, PAR2 can be act...

  16. Detection of a new heparin-dependent inhibitor of thrombin in human plasma.

    OpenAIRE

    Tollefsen, D.M.; Blank, M K

    1981-01-01

    We have demonstrated that human plasma contains a heparin-dependent inhibitor of thrombin that is distinguishable from antithrombin III (AT III). When a 1:50 dilution of plasma was incubated with greater than or equal to 0.01 U/ml heparin and 1 U/ml 125I-thrombin, the labeled thrombin B-chains became incorporated into two complexes of Mr-96,000 and Mr-85,000 that were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Neither co...

  17. Clindamycin Topical

    Science.gov (United States)

    ... slowing or stopping the growth of bacteria that cause acne and by decreasing swelling. ... or bloody stools stomach cramps Topical clindamycin may cause other side ... the Food and Drug Administration's (FDA) MedWatch Adverse Event Reporting ...

  18. 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 ...

  19. 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.

  20. Behaviour of homologous 125I fibrinogen after thrombin and ancrod infusion in rabbits

    International Nuclear Information System (INIS)

    The behaviour of radioactively labelled fibrinogen after infusion of thrombin or ancrod is investigated. Common factors and differences in the behaviour of fibrinogen after infusion of these two enzymes, which act proteolytically on the fibrinogen, are dealt with. Rabbits received an i.v. injection of homologous 125I-fibrinogen 3 days before ancrod or thrombin infusion. On the day of the experiments, one group of animals received an ancrod infusion (1.5 U/kg body weight for 30 minutes), the other a thrombin infusion (600 U/kg body weight for 60 minutes). Intravenous ancrod and thrombin infusions lowered the fibrinogen level to 30% or 50% of the initial value due to intravascular coagulation. About 50% of the 125I fibrinogen was transformed after ancrod exposure into a non-coagulating fraction of fibrinogen derivatives which produces no fibrinolytic decomposition products. (orig./AJ)

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

    International Nuclear Information System (INIS)

    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

  2. Thrombin inhibits the anti-myeloperoxidase and ferroxidase functions of ceruloplasmin: relevance in rheumatoid arthritis.

    Science.gov (United States)

    Sokolov, Alexej V; Acquasaliente, Laura; Kostevich, Valeria A; Frasson, Roberta; Zakharova, Elena T; Pontarollo, Giulia; Vasilyev, Vadim B; De Filippis, Vincenzo

    2015-09-01

    Human ceruloplasmin (CP) is a multifunctional copper-binding protein produced in the liver. CP oxidizes Fe(2+) to Fe(3+), decreasing the concentration of Fe(2+) available for generating harmful oxidant species. CP is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (Kd=130nM), a major source of oxidants in vivo. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting flexible joints and characterized by activation of both inflammatory and coagulation processes. Indeed, the levels of CP, MPO, and thrombin are markedly increased in the synovial fluid of RA patients. Here we show that thrombin cleaves CP in vitro at (481)Arg-Ser(482) and (887)Lys-Val(888) bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the MPO inhibitory function of CP is abrogated. Analysis of the synovial fluid of 24 RA patients reveals that CP is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with thrombin in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. Using independent biophysical techniques, we show that thrombin has intrinsic affinity for CP (Kd=60-270nM), independent of proteolysis, and inhibits CP ferroxidase activity (KI=220±20nM). Mapping of thrombin binding sites with specific exosite-directed ligands (i.e., hirugen, fibrinogen ?'-peptide) and thrombin analogues having the exosites variably compromised (i.e., prothrombin, prethrombin-2, ?T-thrombin) reveals that the positively charged exosite-II of thrombin binds to the negatively charged upper region of CP, while the protease active site and exosite-I remain accessible. These results suggest that thrombin can exacerbate inflammation in RA by impairing the MPO inhibitory function of CP via proteolysis and by competitively inhibiting CP ferroxidase activity. Notably, local administration of hirudin, a highly potent and specifc thrombin inhibitor, reduces the concentration of active MPO in the synovial fluid of RA patients and has a beneficial effect on the clinical symptoms of the disease. PMID:26001728

  3. Percutaneous ultrasound-guided thrombin injection for the treatment of iatrogenic femoral artery pseudoaneurysms

    Energy Technology Data Exchange (ETDEWEB)

    Vlachou, Paraskevi A. [Department of Radiology, Leicester Royal Infirmary, Leicester (United Kingdom); Karkos, Christos D., E-mail: ckarkos@hotmail.com [Department of Vascular and Endovascular Surgery, Leicester Royal Infirmary, Leicester (United Kingdom); Bains, Salena; McCarthy, Mark J. [Department of Vascular and Endovascular Surgery, Leicester Royal Infirmary, Leicester (United Kingdom); Fishwick, Guy; Bolia, Amman [Department of Radiology, Leicester Royal Infirmary, Leicester (United Kingdom)

    2011-01-15

    Purpose: To audit our experience with ultrasound-guided thrombin injection for the treatment of iatrogenic femoral artery pseudoaneurysms. Methods: A retrospective study of 85 consecutive patients undergoing percutaneous ultrasound-guided thrombin injection of post-catheterization femoral pseudoaneurysms during the period January 2002 to May 2007. Results: Pseudoaneurysms had a mean maximum diameter of 3.3 cm (range 1.0-7.6 cm) and a mean neck width of 3.4 mm (range 1.0-7.0 mm). No statistically significant correlation existed between maximum diameter and neck width (Kendall's rank correlation tau b = -0.09, p = 0.5). The median dose of thrombin injected was 425 U (range 100-1500 U). The procedure resulted in complete sac thrombosis in 81 (95%) patients. Seventy-nine pseudoaneurysms thrombosed immediately after one injection, whereas two required a second thrombin injection. There were no procedural complications. The maximum diameter of the pseudoaneurysm was predictive of procedural success (Wilcoxon's rank sum test, p = 0.001) and of the 5 patients with a pseudoaneurysm measuring {>=}6 cm, ultrasound-guided thrombin injection was unsuccessful in 4 (4/5 versus 0/80, p < 0.0001, Fisher's exact test). Three of these necessitated implantation of a stent-graft, whereas one required repeated thrombin injection and coil placement. In contrast, the pseudoaneurysm neck width did not seem to relate to the success of the procedure. Conclusion: Percutaneous ultrasound-guided thrombin injection of is a quick, effective and safe treatment for iatrogenic femoral pseudoaneurysms. For larger pseudoaneurysms, although it is worth attempting more than one thrombin injection, endovascular repair may eventually be required.

  4. 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

    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...

  5. A novel purification method for histidine-tagged proteins containing a thrombin cleavage site

    OpenAIRE

    Hefti, M.H.; Vugt-Toorn, C.J., van der; Dixon, R.; Vervoort, J. J. M.

    2001-01-01

    A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified protein. The advantage of this method is that thrombin is used instead of imidazole in the final purification step. Imidazole can influence NMR experiments, competition studies, or crystallographic tri...

  6. Thrombin receptor signalling in platelets: PAR1, but not PAR4, is rapidly desensitized

    OpenAIRE

    Haglund, Linda

    2009-01-01

    Platelets play a key role in primary haemostasis but are also related to the pathogenesis of arterial thrombosis. Thrombin is the most effective agonist inducing platelet activation. Human platelets express two G-protein coupled thrombin receptors (GPCRs), called protease activated receptor (PAR)1 and PAR4. The aim of this study was to clarify differences in the activities of PAR1 and PAR4, especially focusing on their resistance towards the platelet inhibitor nitric oxide (NO) and their abil...

  7. The role of platelet thrombin receptors PAR1 and PAR4 in platelet activation

    OpenAIRE

    Vretenbrant Öberg, Karin

    2009-01-01

    Platelets play a pivotal role in coagulation and haemostasis. Their most prominent task is to seal damaged blood vessels by the formation of a platelet plug at the damaged area. Once the injury is covered, platelets retract the coagulum to close the wound and allow the blood to flow freely in the vessel. Platelets are strongly activated by the essential enzyme thrombin, formed in the coagulation cascade. Activation of the platelet thrombin receptors PAR1 and PAR4 leads to shape change, secret...

  8. Factor Xa and thrombin evoke additive calcium and proinflammatory responses in endothelial cells subjected to coagulation

    OpenAIRE

    Daubie, Valéry; Cauwenberghs, Sandra; Senden, Nicole; Pochet, Roland; Lindhout, Théo; Buurman, Wim; Heemskerk, Johan

    2006-01-01

    Endothelial cells react to factor Xa and thrombin by proinflammatory responses. It is unclear how these cells respond under physiological conditions, where the serine proteases factor VIIa, factor Xa and thrombin are all simultaneously generated, as in tissue factor-driven blood coagulation. We studied the Ca2+ signaling and downstream release of interleukins (ILs), induced by these proteases in monolayers of human umbilical vein endothelial cells. In single cells, factor Xa, but not factor V...

  9. Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin.

    OpenAIRE

    Colotta, F; SCIACCA, F. L.; Sironi, M.; Luini, W.; Rabiet, M J; A. Mantovani

    1994-01-01

    Thrombin, in addition to being a key enzyme in hemostasis, affects a series of endothelial and leukocyte functions and thus may be involved in the regulation of inflammatory reactions. Because leukocyte recruitment and activation are important events in inflammatory and thrombotic processes, in this study we have examined the possibility that thrombin induces the production of a cytokine chemotactic for mononuclear phagocytes. Human peripheral blood mononuclear cells (PBMC) exposed in vitro t...

  10. Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells.

    OpenAIRE

    Gelehrter, T. D.; Sznycer-Laszuk, R

    1986-01-01

    We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h c...

  11. Identification of a thrombin sequence with growth factor activity on macrophages

    International Nuclear Information System (INIS)

    In contrast to fibroblasts, the exposure of G0/G1-arrested J774 cells, a murine macrophage-like tumor cell line, with either active or esterolytically inactive diisopropyl phosphorofluoridate-conjugated ?-thrombin results in a mitogenic response as measured by increased [3H]thymidine incorporation. This response to thrombin is optimal at 10 nM and is specifically blocked by hirudin, a high-affinity thrombin inhibitor. When prethrombin 1 is cleaved with cyanogen bromide, a fragment (peptide CB67-129) is produced that, like the parent thrombin molecule, is mitogenic for J774 cells but not for fibroblasts. Limited tryptic digests of this fragment retain the ability to stimulate macrophages - a function that can be mimicked by a synthetic tetradecapeptide homologue of CB67-129 but not by any of a series of well-known growth promoters. The mitogenic effects of this peptide are not limited to J774 cells but can be expressed in other macrophage-like tumor cells lines. In addition to increased [3H]thymidine incorporation, the synthetic B chain peptide stimulates cell proliferation as evidenced by a dose-dependent increase in total protein per culture well and cell number. The authors conclude that the thrombin molecule contains a macrophage growth factor domain that is separate and distinct from its active center. Thus, thrombin, in addition to its major role in hemostasis and thrombosis, may also have important functions in such basic processes as the inflammatory response and monocytopoiesis

  12. Regulation of fibrin-mediated tumor cell adhesion to the endothelium using anti-thrombin aptamer.

    Science.gov (United States)

    Gaddes, Erin R; Lee, Deborah; Gydush, Gregory; Wang, Yong; Dong, Cheng

    2015-12-10

    Molecular intervention during transient stages of various metastatic pathways may lead to development of promising therapeutic technologies. One of such involves soluble fibrin (sFn) that has been implicated as a cross-linker between circulating blood or tumor cells and endothelial cell receptors, promoting cell arrest on the endothelium during circulation. sFn generation is a result of thrombin-mediated fibrinogen (Fg) cleavage due to either vascular injuries or a tumor microenvironment. For cancer therapy, thrombin-mediated conversions of Fg to sFn thus serve as potential intervention points to decrease circulating tumor cell adhesion to the endothelium and subsequent metastatic events. The purpose of this work was to investigate the function of an anti-thrombin oligonucleotide aptamer in reducing tumor cell arrest. Both molecular and cellular interactions were examined to demonstrate the binding and inhibitory effects of anti-thrombin aptamer. The results show that the aptamer is capable of inhibiting thrombin-mediated Fg conversion, thereby reducing sFn-mediated tumor cell adhesion in a concentration-dependent manner. Notably, the aptamer is able to bind thrombin under dynamic flow conditions and reduce tumor cell adhesive events at various physiological shear rates. This study further indicates that oligonucleotide aptamers hold great promise as therapeutic regulators of tumor cell adhesion, and consequently, metastatic activity. PMID:26481421

  13. 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.

  14. Active but inoperable thrombin is accumulated in a plasma protein layer surrounding Streptococcus pyogenes.

    Science.gov (United States)

    Naudin, C; Hurley, S M; Malmström, E; Plug, T; Shannon, O; Meijers, J C M; Mörgelin, M; Björck, L; Herwald, H

    2015-09-30

    Activation of thrombin is a critical determinant in many physiological and pathological processes including haemostasis and inflammation. Under physiological conditions many of these functions are involved in wound healing or eradication of an invading pathogen. However, when activated systemically, thrombin can contribute to severe and life-threatening conditions by causing complications such as multiple multi-organ failure and disseminated intravascular coagulation. In the present study we investigated how the activity of thrombin is modulated when it is bound to the surface of Streptococcus pyogenes. Our data show that S. pyogenes bacteria become covered with a proteinaceous layer when incubated with human plasma, and that thrombin is a constituent of this layer. Though the coagulation factor is found attached to the bacteria with a functional active site, thrombin has lost its capacity to interact with its natural substrates and inhibitors. Thus, the interaction of bacteria with human plasma renders thrombin completely inoperable at the streptococcal surface. This could represent a host defense mechanism to avoid systemic activation of coagulation which could be otherwise induced when bacteria enter the circulation and cause systemic infection. PMID:25994766

  15. Design, synthesis and evaluation of graftable thrombin inhibitors for the preparation of blood-compatible polymer materials.

    OpenAIRE

    Salvagnini, Claudio; Michaux, Catherine; Remiche, Julie; Wouters, Johan; Charlier, Paulette; Marchand-Brynaert, Jacqueline

    2005-01-01

    Piperazinyl-amide derivatives of N-alpha-(3-trifluoromethyl-benzenesulfonyl)-L-arginine (1) were synthesized as graftable thrombin inhibitors. The possible disturbance of biological activity due to a variable spacer-arm fixed on the N-4 piperazinyl position was evaluated in vitro, against human alpha-thrombin, and in blood coagulation assay. Molecular modelling (in silico analysis) and X-ray diffraction studies of thrombin-inhibitor complexes were also performed. The fixation of bioactive mol...

  16. Effects of alpha(2)-macroglobulin and antithrombin on thrombin generation and inhibition in cord and adult plasma.

    Science.gov (United States)

    Cvirn, G; Gallistl, S; Muntean, W

    2001-02-01

    Thromboembolic complications rarely occur during infancy and childhood. It has been reported that increased capacity of cord plasma to inhibit thrombin due to elevated alpha(2)-macroglobulin (alpha(2)-M) levels may in part provide protection from thrombosis. In antithrombin (AT)-deficient plasma, alpha(2)-M exhibits anticoagulant action by complexing substantial amounts of generated free thrombin. It has been suggested that alpha(2)-M has the same impact on thrombin inhibition as AT, the most important thrombin inhibitor in adult plasma. The aim of our study was to examine this assumption by determining time-courses of free thrombin generation and prothrombin activation. Additionally, the amount of thrombin complexed to alpha(2)-M was assessed by comparing the heights of the end-level of amidolytic activity curves (AACs) after extrinsic activation of platelet poor plasma in the presence of different concentrations of AT or alpha(2)-M. Increasing the AT content by 30% resulted in significantly suppressed generation of free thrombin and prothrombin fragment 1+2 (F1+2) in cord and adult plasma. In contrast, increasing the alpha(2)-M content in plasma containing physiologic amounts of AT by the same percentage had no effect on free thrombin generation and on F1+2 generation in both cord and adult plasma. In addition, the effect of AT supplementation on the end-level of the AACs was significantly higher compared to the effect of alpha(2)-M supplementation. Since alpha(2)-M, in contrast to AT, had no effect on free thrombin generation and prothrombin activation, our study suggests that the action between alpha(2)-M and thrombin might not be fast enough to prevent thrombin from its feedback activation in both cord and adult plasma and, therefore, in cord and adult plasma containing physiological amounts of AT alterations of the alpha(2)-M content had no effect on thrombin generation and inhibition. PMID:11228341

  17. Protease-Activated Receptor 1 Mediates Thrombin-Dependent, Cell-Mediated Renal Inflammation in Crescentic Glomerulonephritis

    OpenAIRE

    Cunningham, Malcolm A.; Rondeau, Eric; Chen, Xin; COUGHLIN, SHAUN R.; Holdsworth, Stephen R; Tipping, Peter G.

    2000-01-01

    Protease-activated receptor (PAR)-1 is a cellular receptor for thrombin that is activated after proteolytic cleavage. The contribution of PAR-1 to inflammatory cell–mediated renal injury was assessed in murine crescentic glomerulonephritis (GN). A pivotal role for thrombin in this model was demonstrated by the capacity of hirudin, a selective thrombin antagonist, to attenuate renal injury. Compared with control treatment, hirudin significantly reduced glomerular crescent formation, T cell and...

  18. 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.

  19. Tretinoin Topical

    Science.gov (United States)

    ... required before improvement is seen.Use only nonmedicated cosmetics on cleansed skin. Do not use topical preparations with a lot ... not swallow it. Do not apply dressings, bandages, cosmetics, lotions, or other skin medications to the area being treated unless your ...

  20. Testosterone Topical

    Science.gov (United States)

    ... to use testosterone topical.Testosterone is in a class of medications called hormones. Testosterone is a hormone ... decrease in the number of sperm (male reproductive cells) produced, ... online (http://www.fda.gov/Safety/MedWatch) or by phone (1-800-332-1088).

  1. Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin.

    OpenAIRE

    de Munk, G A; Groeneveld, E; Rijken, D.C.

    1991-01-01

    The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50% inactivation of scu-PA occurred in 45 min at 160 ng/ml thrombin in the absence of thrombomodulin and at 4.6 ng/ml thrombin in the presence of thrombomodulin. No difference was found in either the absence or ...

  2. Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin

    International Nuclear Information System (INIS)

    A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of ?-thrombin. These peptides, called hirulogs, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 angstrom in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 [(D-Phe)-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu] inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with Ki = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir53-64, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with Ki = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir53-64, was found to inhibit the incorporation of [14C]diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir53-64 showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir53-64 in increasing the activated partial thromboplastin time of normal human plasma

  3. Percutaneous treatment of femoral pseudoaneurysms: comparison of fibrin sealant against thrombin

    Directory of Open Access Journals (Sweden)

    Daniel Mendes Pinto

    2013-12-01

    Full Text Available 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<0.01. The three cases of failure in group T+F needed surgery and in one of these cases the complication was embolization to the femoral bifurcation. The pseudoaneurysms that were treated with fibrin sealant were larger (25 cm3 in Group T and 57.7 cm3 in Group T+F, p=0.02 and required larger volumes of thrombin (0.5 mL in Group T and 1.0 mL in Group T+F, p<0.01. There was one complication in Group T and two complications in Group T+F (p<0.01. CONCLUSIONS: Irrespective of the small number of cases reviewed, treatment with thrombin alone was superior to treating with fibrin sealant, since it caused few complications and was more effective at correcting pseudoaneurysms.

  4. How does association process affect fibrinogen hydrolysis by thrombin?

    Science.gov (United States)

    Zavyalova, Elena; Kopylov, Alexey

    2014-12-01

    Thrombin, a key enzyme in the blood coagulation cascade, hydrolyzes fibrinogen into fibrin, which specifically associates into the fibers that build up a thrombus scaffold. The assembly of fibrin involves a set of stepwise reactions, for which a complete and detailed kinetic portrait is needed. Existing kinetic models focus on particular parts of the process, for example the mechanism of enzyme action itself or the kinetics of formation of fibrin assemblies. The current study considers a thorough model of the process from fibrinogen hydrolysis to the assembly of fibrin. Composing the model requires taking into account several reaction intermediates, stepwise removal of fibrinopeptides, and association of partially hydrolyzed fibrin, in particular desAA fibrin. The model is versatile enough to adopt new data both on fibrinogen hydrolysis and fibrin association. In addition, the model could be considered as an example of a kinetic description of other complex enzyme systems having several intermediates and feedbacks, such as the blood coagulation cascade and signal transduction. PMID:25239831

  5. 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-induced skin reactions, providing a new therapeutic avenue for possibly predicting and preventing this side effect of cancer treatment

  6. PAR-1-dependent and PAR-independent pro-inflammatory signaling in human lung fibroblasts exposed to thrombin.

    Science.gov (United States)

    Ortiz-Stern, Alejandro; Deng, Xiaoling; Smoktunowicz, Natalia; Mercer, Paul F; Chambers, Rachel C

    2012-11-01

    Proteinase-activated receptors (PARs) are crucial in orchestrating cellular responses to coagulation proteinases, such as thrombin and FXa. Four PARs have been characterized and have been shown to be differentially expressed in mice and humans and between tissues. We have previously shown that in murine lung fibroblasts, PAR-1 is solely responsible for all cellular responses to thrombin and FXa. In contrast, we report here that in primary human lung fibroblasts (pHLFs), known PARs fail to account for all of the cellular responses to thrombin, in particular in the presence of high, but physiologically achievable concentrations of thrombin. We report that pHLFs secrete CCL2 in a PAR-1-dependent manner at low thrombin concentration (?0.3 nM). At or above 10 nM thrombin, pharmacological antagonism (RWJ-58259) fails to block thrombin-induced CCL2 release; whereas PAR-1 cleavage-blocking monoclonal antibodies (ATAP2 and WEDE15) only partially inhibit thrombin-induced CCL2 secretion. In addition, activation of PAR-3, PAR-4, and transactivation of either PAR-2 or EGFR were ruled out as being responsible for thrombin-mediated CCL2 secretion at high yet standard concentrations of the proteinase. We further provide evidence that PAR-1-dependent and PAR-independent signaling involves the rapid phosphorylation of ERK, which in turn is absolutely required for thrombin-induced CCL2 secretion at both low and standard concentration of the proteinase. Our findings suggest the existence of a PAR-independent signaling mechanism in human lung fibroblasts and have important implications for the design of therapeutic strategies aimed at blocking pro-inflammatory signaling responses associated with excessive thrombin generation. PMID:22278285

  7. Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger.

    Science.gov (United States)

    Andrikopoulos, Petros; Kieswich, Julius; Harwood, Steven M; Baba, Akemichi; Matsuda, Toshio; Barbeau, Olivier; Jones, Keith; Eccles, Suzanne A; Yaqoob, Muhammad M

    2015-07-24

    Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling. PMID:25979335

  8. Ultrasensitive electrochemical detection for thrombin using hybridization chain reaction with enzyme-amplification.

    Science.gov (United States)

    Song, Weiling; Xie, Xuxu; Sun, Wenbo; Zhang, Ningbo; Li, Chunxiang

    2015-02-20

    In this work, a new electrochemical aptasensor using hybridization chain reaction (HCR) for signal amplification was developed for highly sensitive detection of thrombin. The sandwich system of aptamer/thrombin/aptamer-primer complex was fabricated as the sensing platform. As the initiator strands, aptamer-primer complex could propagate a chain reaction of hybridization events between the two hairpin probes, and whether long nicked DNA polymers could be formed on the modified electrode. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled horseradish peroxidase (HRP), resulting in significantly amplified electrochemical signal through the electrocatalysis of HRP. On the basis of the enzymatic oxidization of Fe(2+) by H2O2 to yield Fe(3+), the imaging of thrombin was detected by the reduction current of Fe(3+) with the scanning electrochemical microscopic tip. The electrochemical signals had a good linear with logarithm of thrombin concentration in the range from 1.0 fM to 100 fM, reaching a detection limit of thrombin as low as 0.04 fM. In addition, the proposed strategy exhibited excellent specificity and was successfully applied in real sample assay which demonstrated the potential application in clinical diagnostics. PMID:25682250

  9. Antibodies against thrombin in dengue patients contain both anti-thrombotic and pro-fibrinolytic activities.

    Science.gov (United States)

    Chuang, Yung-Chun; Lin, Yee-Shin; Liu, Hsiao-Sheng; Wang, Jen-Reng; Yeh, Trai-Ming

    2013-08-01

    Dengue virus (DENV) infection may result in severe life-threatening Dengue haemorrhagic fever (DHF). The mechanisms causing haemorrhage in those with DHF are unclear. In this study, we demonstrated that antibodies against human thrombin were increased in the sera of Dengue patients but not in that of patients infected with other viruses. To further characterise the properties of these antibodies, affinity-purified anti-thrombin antibodies (ATAs) were collected from Dengue patient sera by thrombin and protein A/L affinity columns. Most of the ATAs belonged to the IgG class and recognized DENV nonstructural protein 1 (NS1). In addition, we found that dengue patient ATAs also cross-reacted with human plasminogen (Plg). Functional studies in vitro indicated that Dengue patient ATAs could inhibit thrombin activity and enhance Plg activation. Taken together, these results suggest that DENV NS1-induced thrombin and Plg cross-reactive antibodies may contribute to the development of haemorrhage in patients with DHF by interfering with coagulation and fibrinolysis. PMID:23740201

  10. 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.

  11. Development of Orally Active Thrombin Inhibitors for the Treatment of Thrombotic Disorder Diseases

    Directory of Open Access Journals (Sweden)

    Li-Wei He

    2015-06-01

    Full Text Available Thrombotic disorders represent the major share of the various cardiovascular diseases, and significant progress has been made in the development of synthetic thrombin inhibitors as new anticoagulants. In addition to the development of highly potent and selective inhibitors with improved safety and suitable half-life, several allosteric inhibitors have been designed and synthesized, that did not fully nullify the procoagulant signal and thus could result in reduced bleeding complications. Furthermore, natural products with thrombin inhibitory activity have been isolated, and some natural products have been modified in order to improve their inhibitory activity and metabolic stability. This review summarizes the development of orally active thrombin inhibitors for the treatment of thrombotic disorder diseases, which could serve as a reference for the interested researchers.

  12. Discovery and evaluation of potent P1 aryl heterocycle-based thrombin inhibitors.

    Science.gov (United States)

    Young, Mary Beth; Barrow, James C; Glass, Kristen L; Lundell, George F; Newton, Christina L; Pellicore, Janetta M; Rittle, Kenneth E; Selnick, Harold G; Stauffer, Kenneth J; Vacca, Joseph P; Williams, Peter D; Bohn, Dennis; Clayton, Franklin C; Cook, Jacquelynn J; Krueger, Julie A; Kuo, Lawrence C; Lewis, S Dale; Lucas, Bobby J; McMasters, Daniel R; Miller-Stein, Cynthia; Pietrak, Beth L; Wallace, Audrey A; White, Rebecca B; Wong, Bradley; Yan, Youwei; Nantermet, Philippe G

    2004-06-01

    In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.84 nM. A study of ortho-substituted five-membered-ring heterocycles was undertaken and subsequently demonstrated that the o-triazole and tetrazole rings were optimal. Combination of these potent P(1) aryl heterocycles with a variety of P(2)-P(3) groups produced a compound with an extraordinary thrombin inhibitory activity of 1.4 pM. It is hoped that this potency enhancement in P(1) will allow for more diversification in the P(2)-P(3) region to ultimately address additional pharmacological concerns. PMID:15163182

  13. Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I.

    Science.gov (United States)

    Myles, T; Le Bonniec, B F; Betz, A; Stone, S R

    2001-04-24

    Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering. PMID:11305913

  14. 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

  15. 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...

  16. Saccular aneurysm of superior vena cava treated with percutaneous, transcatheter thrombin injection.

    Science.gov (United States)

    Jargiello, Tomasz; Durakiewicz, Marek; Sojka, Micha?; Czekajska-Chehab, Elzbieta; Szczerbo-Trojanowska, Malgorzata

    2014-04-01

    We report the case of successful endovascular treatment of large saccular aneurysm of SVC in a patient with vascular malformation of right hand and chest. Considering the high risk of surgery, the patient was referred for percutaneous intervention. Venography showed communication between the aneurysm and SVC, just below brachiocephalic confluence. That is why the decision of balloon-protected transcatheter thrombin injection was made. Selective catheter was placed in the aneurysm and balloon occlusion catheter in SVC. Both catheters were withdrawn right after thrombin injection. During follow-up, aneurysm slightly enlarged in early observation and after a year shrinkage was observed. PMID:23737024

  17. Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release

    OpenAIRE

    Hecquet, Claudie; Biyashev, Dauren; Tan, Fulong; Erdös, Ervin G.

    2005-01-01

    Bradykinin (BK) or kallikreins activate B2 receptors (R) which couple G?i and G?q proteins to release arachidonic acid (AA) and elevate [Ca2+]i. Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples G?i, G?q and G?12/13 proteins. In CHO cells stably transfected with human B2R, thrombin liberated little AA, but it significantly potentiated AA release by B2R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B2R. We also examined human e...

  18. 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.

  19. Topical corticosteroids

    Directory of Open Access Journals (Sweden)

    Laleh Vaziri

    2015-07-01

    Full Text Available Topical corticosteroids (TCs are very useful in the treatment of some skin disorders such as eczema and psoriasis. TCs exhibit potent anti-inflammatory and anti-proliferative effects responsible for their efficacy in the treatment of skin disorders. TCs can be classified into different groups according to their potency; that depends on the intrinsic activity of the corticosteroid molecule, the characteristics of the vehicle (cream, ointment, lotion that directly influence patient’s compliance and may modify penetration through the skin. British National Formulary classified them into four potency groups with Class I grouping the most potent molecules (e.g. clobetasol propionate and Class IV the least potent (e.g. hydrocortisone. In contrast, the American classification considers seven potency groups, with super potent, potent, upper mid-strength, mid-strength, lower mid-strength, mild and least potent.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Huang-Joe [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China); Division of Cardiology, Department of Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Lo, Wan-Yu [Department of Medical Research, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Graduate Integration of Chinese and Western Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Lu, Te-Ling [School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Huang, Haimei, E-mail: hmhuang@life.nthu.edu.tw [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China)

    2010-01-01

    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 {mu}mol/L. At a concentration of 25 {mu}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-{kappa}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.

  1. (-)-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.

  2. Prolyl endopeptidase and thrombin inhibitory diterpenoids from the bark of Xylopia aethiopica.

    Science.gov (United States)

    Diderot, Noungoue Tchamo; Silvere, Ngouela; Yasin, Amsha; Zareen, Seema; Fabien, Zelefack; Etienne, Tsamo; Choudhary, M Iqbal; Atta-Ur-Rahman

    2005-09-01

    The inhibitory effects of seven diterpenes, belonging to three different structural classes and isolated from the bark of Xylopia aethiopica, were investigated against the enzymes prolyl endopeptidase (PEP) and alpha-thrombin. Five compounds exhibited inhibitory activity against them. PMID:16195597

  3. Aptamer modified organic-inorganic hybrid silica monolithic capillary columns for highly selective recognition of thrombin.

    Science.gov (United States)

    Deng, Nan; Liang, Zhen; Liang, Yu; Sui, Zhigang; Zhang, Liyuan; Wu, Qi; Yang, Kaiguang; Zhang, Lihua; Zhang, Yukui

    2012-12-01

    A novel kind of aptamer modified organic-inorganic hybrid silica monolithic capillary column has been developed, via the covalent bonding of 5'-NH(2)-modified aptamer for human ?-thrombin on hybrid silica monolith, prepared by sol-gel method, with tetraethoxysilane and 3-aminopropyltriethoxysilane as precursors. Due to the large specific surface area of the hybrid matrix, the average coverage density of aptamer reached 568 pmol/?L, and the thrombin binding capacity was 1.15 ?g/?L, 14 times higher than that of aptamer modified open tubular capillaries. By such an affinity capillary column, the limit of detection of thrombin was decreased to 3.4 nM with a UV detector. Furthermore, even when thrombin was mixed with 1000 times more concentrated human serum, it could be selectively enriched and detected with the signal-to-noise ratio as ca.10. These results indicate that the developed preparation strategy for aptamer based hybrid silica monolithic capillary column might provide an effective method to achieve highly selective recognition of trace targets. PMID:23137349

  4. Reversible regulation of thrombin adsorption and desorption based on photoresponsive-aptamer modified gold nanoparticles.

    Science.gov (United States)

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

    2015-11-01

    In the protein separation, adsorption and desorption of target protein have been using different buffer condition. Different buffer will change the structure and activity of target protein in some cases. This work describes the use of different wavelength light for remote regulation of adsorption and desorption of target protein in the same buffer solutions. A dynamic system that captured and released protein in response to light is reported. Matrix gold nanoparticles and light-responsive affinity ligand comprising thrombin aptamer (APT15), polyethylene glycol linker, and azobenzene-modified complementary sequence were used. UV light induced a trans-cis isomerization of the azobenzene that destabilized the duplex of aptamer and azobenzene-modified complementary sequence, resulting in thrombin binding to aptamer sequence. Visible light irradiation resulted in DNA duplex rehybridization and thrombin released. Our work demonstrates that different light wavelengths effectively regulated the adsorption and desorption of thrombin in the same buffer, and this system also can capture and release prothrombin from plasma with different wavelength light. Furthermore, this method can be widely applied to a variety of different protein separation process. PMID:26452827

  5. Crystallization and preliminary crystallographic characterization of three peptidic inhibitors in complex with ?-thrombin

    OpenAIRE

    Carvalho Figueiredo, Ana; Clement, Cristina C.; Philipp, Manfred; Barbosa Pereira, Pedro José

    2010-01-01

    Human ?-thrombin was crystallized in complex with specific peptide inhibitors of general sequence d-Phe-Pro-d-Arg-P1?-CONH2. The crystals belonged to the orthorhombic space group P212121 and diffracted to beyond 1.3?Å resolution.

  6. 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

  7. Protein Z efficiently depletes thrombin generation in disseminated intravascular coagulation with poor prognosis.

    Science.gov (United States)

    Lee, Nuri; Kim, Ji-Eun; Gu, Ja-Yoon; Yoo, Hyun Ju; Kim, Inho; Yoon, Sung-Soo; Park, Seonyang; Han, Kyou-Sup; Kim, Hyun Kyung

    2016-01-01

    Disseminated intravascular coagulation (DIC) is characterized by consumption of coagulation factors and anticoagulants. Thrombin generation assay (TGA) gives useful information about global hemostatic status. We developed a new TGA system that anticoagulant addition can deplete thrombin generation in plasma, which may reflect defective anticoagulant system in DIC. TGAs were measured on the calibrated automated thrombogram with and without thrombomodulin or protein Z in 152 patients who were suspected of having DIC, yielding four parameters including lag time, endogenous thrombin potential, peak thrombin and time-to-peak in each experiment. Nonsurvivors showed significantly prolonged lag time and time-to-peak in TGA-protein Z system, which was performed with added protein Z. In multivariate Cox regression analysis, lag time and time-to-peak in TGA system were significant independent prognostic factors. In TGA-protein Z system, lag time and time-to-peak were revealed as independent prognostic factors of DIC. Protein Z addition could potentiate its anticoagulant effect in DIC with poor prognosis, suggesting the presence of defective protein Z system. The prolonged lag time and time-to-peak in both TGA and TGA-protein Z systems are expected to be used as independent prognostic factors of DIC. PMID:26203764

  8. An aptamer-based single particle method for sensitive detection of thrombin using fluorescent quantum dots as labeling probes.

    Science.gov (United States)

    Yin, Jinjin; Zhang, Aidi; Dong, Chaoqing; Ren, Jicun

    2015-11-01

    In this study, an aptamer-based single particle method was developed for the thrombin detection in human serum samples using fluorescence correlation spectroscopy (FCS). In this method, quantum dots (QDs) were used as the fluorescent probes and thrombin-binding aptamer (TBA) was used as molecular recognition unit. When two QDs probes labeled with TBA (QD-TBA1 and QD-TBA2) are mixed in a sample containing thrombin targets, the binding of targets will cause QDs to form dimers (or oligomers) with bigger sizes, which leads to the nearly double increase in the characteristic diffusion time of QDs in the detection volume of FCS. FCS method can detect the change in the characteristic diffusion time of QDs. Firstly, the diffusion and blinking behaviors of QD-TBA probes in the presence of thrombin were investigated by FCS and total internal reflection fluorescence microscopy (TIRFM) imaging system, and the experimental results documented that QD-TBAs were bound together with "one-by-one" structure when thrombin were added into the solution. And then, the assay conditions were optimized in order to improve the sensitivity and specificity of this method. Under the optimized conditions, the linear range of the method is from 5.0nM to 500nM of thrombin, and the limit of detection is about 2.6nM. Finally, this method was applied to homogeneous determination of thrombin in human serum samples. PMID:26452786

  9. 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

  10. Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut.

    Science.gov (United States)

    Soares, Tatiane Sanches; Watanabe, Renata Midori Okuta; Tanaka-Azevedo, Anita Mitico; Torquato, Ricardo José Soares; Lu, Stephen; Figueiredo, Ana Carvalho; Pereira, Pedro José Barbosa; Tanaka, Aparecida S

    2012-07-01

    Rhipicephalus (Boophilus) microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. The large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5mg/L of culture, respectively. Purified boophilin inhibited trypsin (K(i) 0.65 nM), neutrophil elastase (K(i) 21 nM) and bovine thrombin (K(i) 57 pM), while D1 inhibited trypsin and neutrophil elastase (K(i) of 2.0 and 129 nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control. PMID:22341830

  11. Recombinant DNA in Medicine

    OpenAIRE

    Cederbaum, Stephen D.; Fareed, George C.; Lovett, Michael A.; Shapiro, Larry J.

    1984-01-01

    Studies in bacteria and bacterial viruses have led to methods to manipulate and recombine DNA in unique and reproducible ways and to amplify these recombined molecules millions of times. Once properly identified, the recombinant DNA molecules can be used in various ways useful in medicine and human biology. There are many applications for recombinant DNA technology. Cloned complementary DNA has been used to produce various human proteins in microorganisms. Insulin and growth hormone have been...

  12. 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...

  13. Whole blood coagulation in children with thrombocytopenia and the response to platelet replacement, recombinant factor VIIa, and a potent factor VIIa analogue

    DEFF Research Database (Denmark)

    Larsen, Ole Halfdan; Clausen, Niels; Persson, Egon; Ezban, Mirella; Ingerslev, Jørgen; Sørensen, Benny

    2008-01-01

    The present study evaluated dynamic coagulation profiles, platelet aggregation, and thrombin generation in whole blood (WB) from eight children with thrombocytopenia during chemotherapy, and the haemostatic potential of platelets (+60 x 10(9)/l), recombinant factor VIIa (rFVIIa - NovoSeven), and a potent rFVIIa analogue (NN1731) both at 1 and 4 microg/ml. Dynamic WB coagulation profiles were recorded by thrombelastometry employing activation with tissue factor (TF - Innovin) at low concentration...

  14. 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...

  15. 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…

  16. Recombination in RNA.

    Science.gov (United States)

    King, A M; McCahon, D; Slade, W R; Newman, J W

    1982-07-01

    The aphthovirus genome consists of a single molecule of single-stranded RNA that encodes all the virus-induced proteins. We isolated recombinant aphthoviruses from cells simultaneously infected with temperature-sensitive mutants of two different subtype strains. Analysis of the proteins induced by 16 independently generated recombinants revealed two types of protein pattern, which were consistent with single genetic crossovers on the 5' side and 3' side, respectively, of the central P34-coding region. Recombinants invariably inherited all four coat proteins from the same parent, and novel recombinant proteins were not observed. RNAase T1 fingerprints of virus RNA, prepared from representatives of each recombinant type, confirmed the approximate crossover sites that had been deduced from the inheritance of proteins. These fingerprints provide molecular evidence of recombination at the level of RNA and demonstrate the potential of RNA recombination for producing genetic diversity among picornaviruses. PMID:6295637

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

    Directory of Open Access Journals (Sweden)

    He Shaoheng

    2007-05-01

    Full Text Available 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 investigated. Results The results showed that thrombin induced proMMP-9, but not proMMP-2 release from HDFs in a dose dependent manner at 6 h following incubation. Thrombin also upregulated expression of proMMP-9 mRNA in HDFs. Hirudin completely abolished the action of thrombin on HDFs. An agonist peptide of protease-activated receptor-1, SFLLR-NH2 stimulated an enhanced release of proMMP-9 from HDFs. AG490, an inhibitor of STAT3 inhibited basal and thrombin-provoked proMMP-9 release and phosphorylation of STAT3. PD98059, an inhibitor of MAPK and LY294002, an inhibitor PI3K failed to significantly inhibit thrombin induced proMMP-9 release. Conclusion Thrombin is a potent stimulus of proMMP-9 release from HDFs. Thrombin induced proMMP-9 release is most likely through activation of PAR-1. JAK/STAT3 signaling pathway is involved in proMMP-9 release from HDFs.

  18. The protective role of (?)-epigallocatechin-3-gallate in thrombin-induced neuronal cell apoptosis and JNK-MAPK activation

    OpenAIRE

    He, Qianqian; Bao, Lei; Zimering, Jeffrey; Zan, Kun; Zhang, Zuohui; Shi, Hongjuan; Zu, Jie; YANG, XINXIN; Hua, Fang; Ye, Xinchun; Cui, Guiyun

    2015-01-01

    (?)-Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has anti-inflammatory and antioxidant properties and provides neuroprotection against central nervous system diseases. Yet, it is not known whether EGCG may be neuroprotective against intracerebral hemorrhage. In this study, we used a simplified in-vitro model of thrombin neurotoxicity to test whether EGCG provides neuroprotection against thrombin-associated toxicity. Exposure of primary cortical neurons to ...

  19. Percutaneous Thrombin Injection with a Distal Embolic Protection Device for Treatment of a Common Carotid Artery Pseudoaneurysm

    OpenAIRE

    Lee, J.H.; Tseng, I.K.; Siegel, R.L.; Roychowdhury, S.

    2013-01-01

    Carotid artery pseudoaneurysm is a rare complication from placement of an internal jugular triple lumen catheter. Endovascular stenting is the favored treatment option in the setting of traumatic carotid injury. In other parts of the body, specifically the femoral artery, thrombin injection has become the standard of care. We intend to show that effective management of carotid pseudoaneurysms can also be achieved with thrombin injection after placement of a distal embolic protection device.

  20. Evaluation of the Profile of Thrombin Generation during the Process of Whole Blood Clotting as Assessed by Thromboelastography

    OpenAIRE

    Rivard, Georges E; Brummel, Kathleen; Mann, Kenneth G.; Fan, Li; Hofer, Angélique; Cohen, Eli

    2005-01-01

    Thromboelastography is useful for assessment of whole blood coagulation. The objective of this study was to evaluate the possibility of linking the tracing of whole blood clotted in a thromboelastograph TEG with the generation of thrombin assessed by thrombin/antithrombin complex (TAT). Citrated whole blood containing corn trypsin inhibitor from volunteers was clotted in the presence of CaCl2 and tissue factor. Clotting was monitored with 8 channels of a TEG system. At different time points t...

  1. Nafamostat mesilate attenuates neuronal damage in a rat model of transient focal cerebral ischemia through thrombin inhibition

    OpenAIRE

    Tao Chen; Jing Wang; Chenhui Li; Weining Zhang; Luyong Zhang; Lufan An; Tao Pang; Xinzhong Shi; Hong Liao

    2014-01-01

    Evidence suggests that thrombin, a blood coagulation serine protease, mediates neuronal injury in experimental cerebral ischemia. Here, we test the hypothesis that nafamostat mesilate, a serine protease inhibitor, may ameliorate ischemia-induced neuronal damage through thrombin inhibition after ischemic stroke. Focal ischemia was induced in adult Sprague-Dawley rats by occlusion of the middle cerebral artery for 2?hours followed by 22?hours of reperfusion. The administration of nafamostat mes...

  2. Staphylocidal action of thrombin-induced platelet microbicidal protein is influenced by microenvironment and target cell growth phase.

    OpenAIRE

    Koo, S P; Yeaman, M R; Bayer, A S

    1996-01-01

    Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic peptide released from rabbit platelets following exposure to thrombin in vitro. This peptide exerts potent in vitro microbicidal activity against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. It is known that the microbicidal actions of other cationic antimicrobial peptides (e.g., neutrophil defensins) are influenced by environmental factors and target cell growth phase. However, whether t...

  3. 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.

  4. Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development

    OpenAIRE

    Vretenbrant, Karin; Ramström, Sofia; Bjerke, Maria; Lindahl, Tomas

    2007-01-01

    Thrombin is a pivotal enzyme formed in the coagulation cascade and an important and potent platelet activator. The two protease-activated thrombin receptors on human platelets are denoted PARI and PAR4. The physiological relevance of PAR4 is still unclear, as both aggregation and secretion can be accomplished by PAR1 activation alone. In the present study we have investigated the role of PARS in platelet activation, blood coagulation, clot elasticity and fibrinolysis. Flow cytometry, free osc...

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

    OpenAIRE

    Vouret-Craviari, Valérie; Boquet, Patrice; Pouysségur, Jacques; Van 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 ...

  6. Relationship between calcium mobilization and platelet ?- and ?-granule secretion. A role for TRPC6 in thrombin-evoked ?-granule exocytosis.

    Science.gov (United States)

    Lopez, E; Bermejo, N; Berna-Erro, A; Alonso, N; Salido, G M; Redondo, P C; Rosado, J A

    2015-11-01

    Changes in cytosolic Ca(2+) concentration ([Ca(2+)]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca(2+) from distinct intracellular stores, which, in turn, activates store-operated Ca(2+) entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca(2+) mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that ?- and ?-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca(2+), and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca(2+) mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of ?-granules which precedes the secretion of ?-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573-586 of TRPC6, inhibited thrombin-evoked ?-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced ?- and ?-granule secretion show differences in dependency on Ca(2+) mobilization. PMID:26386308

  7. 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

  8. Amperometric aptasensor for thrombin detection using enzyme-mediated direct electrochemistry and DNA-based signal amplification strategy.

    Science.gov (United States)

    Bai, Lijuan; Chai, Yaqin; Yuan, Ruo; Yuan, Yali; Xie, Shunbi; Jiang, Liping

    2013-12-15

    In this work, a new electrochemical aptasensor based on direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) using exonuclease-catalyzed target recycling and hybridization chain reaction (HCR) for signal amplification was developed for highly sensitive detection of thrombin. The electrochemical signal was originated from HRP without the addition or labeling of redox probes. To construct the aptasensor, the capture probe was immobilized on gold nanoparticles (AuNPs) modified electrode for the following hybridization with the complementary thrombin binding aptamer. In the presence of thrombin, the formation of aptamer-thrombin complex would result in the dissociation of aptamer from the double-strand DNA (dsDNA). Subsequently, with the employment of exonuclease, aptamer was selectively digested and thrombin could be released for analyte recycling. The capture probe and two hairpin helper DNAs lead to the formation of extended dsDNA polymers through HCR on the electrode surface. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled HRP, resulting in significantly amplified electrochemical signal through the direct electrochemistry and electrocatalysis of HRP. The proposed strategy combined the amplification of analyte recycling and HCR, as well as the inherent electroactivity and catalytic activity of HRP, which exhibited high sensitivity for thrombin determination with an ultra-low detection limit of 1.2×10(-13) M. Moreover, the detection scheme could be easily extended to the detection of other biomolecules. PMID:23880107

  9. Identification of thrombin-like activity in ovarian cancer associated ascites and modulation of multiple cytokine networks.

    Science.gov (United States)

    Naldini, Antonella; Morena, Emilia; Belotti, Dorina; Carraro, Fabio; Allavena, Paola; Giavazzi, Raffaella

    2011-10-01

    Blood coagulation cascades can be activated by different mechanisms and to different levels in cancer patients. In a study conducted on the transcriptional profile of epithelial ovarian cancer patients a number of possible links between coagulation and inflammation have been suggested and we and others have reported that, in addition to its central role in blood coagulation and haemostasis, thrombin is a powerful regulator of inflammatory responses. Here, we report that thrombin- like activities were present in the malignant ascites of patients with ovarian carcinoma. Malignant ascites significantly enhanced the release of cytokines/chemokines, which have been previously shown to support tumour progression, such as interleukin (IL)-6, IL-1?, CCL2 and CXCL8, in human peripheral blood mononuclear cells of healthy volunteers. Interestingly, ascites enhanced the release of the anti-inflammatory cytokine IL-10 and inhibited the production of interferon-? and IL-12. The presence of the anticoagulant antithrombin reversed IL-12 inhibition induced by ascites in human monocytes. Finally, the use of thrombin and of the specific thrombin receptor (PAR) agonist peptides, TFLLRN and AYGPK, suggests that IL-12 inhibition is thrombin-specific and related to PAR-1, but not to PAR-4. These findings underline the tight relationship between the coagulation pathway, where thrombin is the key enzyme, and cytokine modulation, including IL-12 inhibition, which is a critical feature of the tumour microenvironment, and may represent a powerful strategy used by tumours to escape immune surveillance. PMID:21833453

  10. Endoscopic ultrasound guided thrombin injection of angiographically occult pancreatitis associated visceral artery pseudoaneurysms: Case series

    Science.gov (United States)

    Gamanagatti, Shivanand; Thingujam, Usha; Garg, Pramod; Nongthombam, Surajkumar; Dash, Nihar Ranjan

    2015-01-01

    Pseudoaneurysm is a known complication of pancreatitis associated with significant mortality and morbidity. Imaging plays an important role in the diagnosis and management. Computed tomography (CT) helps localize the lesion and the severity of the background pancreatitis but digital subtraction angiography with coil embolization is recommended to avoid bleeding and inadvertent surgery. However, in cases where angiographic coil embolization is not feasible due to technical reasons, thrombin injection via CT or ultrasound guidance remains a viable option and often described in literature. In this series, effort has been made to highlight the role of endoscopic ultrasound guided thrombin instillation especially in patients with poorly visualized pseudoaneurysm on ultrasound thereby avoiding surgery and the associated mortality and morbidity. PMID:26421108

  11. Effect of resveratrol, a natural polyphenolic compound, on platelet activation induced by endotoxin or thrombin.

    Science.gov (United States)

    Olas, Beata; Wachowicz, Barbara; Saluk-Juszczak, Joanna; Zieli?ski, Tomasz

    2002-08-15

    Resveratrol (3, 4', 5-trihydroxystilbene), a natural polyphenol, is found in some plants that are used in human nutrition. Grapes are a major source for resveratrol, and a significant amount can also be found in red wine. Several experimental studies have demonstrated biological properties of resveratrol, especially its anti-inflammatory, antioxidant, anti-platelet and antitumor effects. In the present study, we investigated the first step of platelet activation-platelet adhesion stimulated by lipopolysaccharide (LPS) from Proteus mirabilis (weak stimulator) and thrombin (strong activator) in the presence of resveratrol. Our studies show that endotoxin (0.3 microg/10(8) platelets), like thrombin (0.2 U/10(8) platelets), induced the adhesion of platelets (expressed as absorbance of cell attached proteins) to collagen and fibrinogen. Preincubation of washed platelets with resveratrol at physiological plasma concentrations (25-100 microg/ml, 30 min, 37 degrees C) had an inhibitory effect on adhesion of platelets to collagen after activation by LPS alone or LPS with thrombin. The strongest effect on this process was caused by resveratrol at the concentration of 100 microg/ml. Pretreatment of platelets with resveratrol (25-100 microg/ml, 30 min, 37 degrees C) had also inhibitory effects on adhesion of platelets to fibrinogen after stimulation of these cells by LPS alone or by LPS with thrombin at the same concentration. In conclusion, we suggest that resveratrol present in human diet may be an important compound responsible for the reduction of platelet adhesion and changed reactivity of blood platelets in inflammatory process. PMID:12431480

  12. Thrombin Stimulates Human Endothelial Arginase Enzymatic Activity via RhoA/ROCK Pathway

    OpenAIRE

    Ming, Xiu-Fen; Barandier, Christine; Viswambharan, Hema; Kwak, Brenda R.; Mach, François; Mazzolai, Lucia; Hayoz, Daniel; Ruffieux, Jean; Rusconi, Sandro; MONTANI, JEAN-PIERRE; Yang, Zhihong

    2005-01-01

    Background— Arginase competes with endothelial nitric oxide synthase (eNOS) for the substrate L-arginine and decreases NO production. This study investigated regulatory mechanisms of arginase activity in endothelial cells and its role in atherosclerosis. Methods and Results— In human endothelial cells isolated from umbilical veins, thrombin concentration- and time-dependently stimulated arginase enzymatic activity, reaching a 1.9-fold increase (P

  13. Gold nanoparticle enhanced electrochemiluminescence of CdS thin films for ultrasensitive thrombin detection.

    Science.gov (United States)

    Wang, Jing; Shan, Yun; Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2011-06-01

    Interactions between surface plasmons (SP) of metallic surfaces and photoluminescence (PL) of semiconductor nanocrystal (S-NC) surfaces have been extensively investigated, and SP-induced PL enhancement has been used as a sensitive analytical technique. However, this SP induced electrochemiluminescence (ECL) enhancement is rarely studied. In this work, we report greatly enhanced ECL of CdS thin films by gold nanoparticles (Au NPs) for ultrasensitive detection of thrombin. The system was composed of a CdS NC film on glassy carbon electrode (GCE) as ECL emitter attached an aptamer of thrombin. Then, ssDNA-AuNP conjugates hybridized with the aptamer to form a separation length of ca. 12 nm between CdS NCs and Au NPs. The system showed 5-fold enhancement of ECL intensity as compared to that without Au NPs, which might be attributed to the long-distance interaction between the S-NCs and SPR field of noble metal nanoparticles (MNPs).We also found that the enhanced ECL could be influenced by the involving factors such as the separation distance, spectral overlap, and magnetic field. Such enhancement in combination with smart recognition of aptamer and target protein allowed us to construct an ultrasensitive aptasensor for attomolar detection of thrombin. The presence of target protein was reflected by the ECL signal decrease caused by the target-induced removal of ssDNA-AuNP conjugates. The decrease of ECL signal was logarithmically linear with the concentration of thrombin in a wide range from 100 aM to 100 fM. The principle described in this work could be also applied to many other bioassays. PMID:21517100

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

    DEFF Research Database (Denmark)

    Jessen, Lisbeth Rem; Wiinberg, Bo; Kjelgaard-Hansen, Mads; Jensen, Asger Lundorff; Rozanski, Elizabeth; Kristensen, Annemarie Thuri

    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...

  15. 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.

  16. Insights into the molecular inactivation mechanism of human activated thrombin-activatable fibrinolysis inhibitor

    DEFF Research Database (Denmark)

    Sanglas, L; Arolas, J L; Valnickova, Z; Aviles, F X; Enghild, J J; Gomis-Rüth, F X

    2010-01-01

    SUMMARY BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a validated target for thrombotic diseases. TAFI is converted in vivo to activated TAFI (TAFIa) by removal of its pro-domain. Whereas TAFI is stable and persists in the circulation, possibly in complex with plasminogen, TAFIa is unstable and poorly soluble, with a half-life of minutes. OBJECTIVES: In order to study the molecular determinants of this instability, we studied the influence of protein inhibitors on human TAFIa...

  17. Thrombin-stimulated immunoprecipitation of phosphatidylinositol 3-kinase from human platelets.

    OpenAIRE

    Mitchell, C. A.; Jefferson, A B; Bejeck, B E; Brugge, J S; Deuel, T.F.; Majerus, P W

    1990-01-01

    Growth factors and transforming proteins that activate tyrosine phosphorylation have been shown to cause an increased labeling of 3-phosphate-containing phosphatidylinositols. Turnover correlates with the formation of a complex between phosphatidylinositol 3-kinase, the activated protein-tyrosine kinase, and other proteins thought to participate in transmembrane signaling. When human platelets are treated with thrombin, labeling of 3-phosphate-containing phosphatidylinositols is stimulated wi...

  18. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

    OpenAIRE

    Bahou, W F; Coller, B S; Potter, C L; Norton, K J; Kutok, J L; Goligorsky, M S

    1993-01-01

    A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thromb...

  19. Thrombelastograph (TEG®) Analysis of Platelet Gel Formed With Different Thrombin Concentrations

    Science.gov (United States)

    Ellis, William Cory; Cassidy, Linsay K.; Finney, Angela S.; Spiwak, Allison J.; Riley, Jeffrey B.

    2005-01-01

    Abstract: Autologous blood transfusion is the safest and most successful way to decrease transfusion-related risks such as post-operative infections, allo-immunization, and short- and long-term immunosuppression. In addition, these fibrin sealants are known to provide coagulation support at the surgical site and act as an adjunct to the control of postoperative bleeding. The physical formation of autologous platelet fibrin gel clot is dependent on both the common pathway of the coagulation cascade and platelet activation. Platelet gel can help provide control of intraoperative and postoperative bleeding. The Thrombelastograph Hemostasis Analyzer (TEG®) measures the viscoelastic properties of a clot as it forms. Based on the information that the TEG provides, it promises to be a good choice for point of care measurement of the integrity of thrombus formed by platelet gels. Bovine blood from a single donor was sequestered into platelet-rich plasma and was made into platelet gel using calcium and three different concentrations of thrombin. The platelet gel samples were then analyzed with the TEG analyzer. The results for MA, tMA, CI, and angle were recorded and statistical analysis was performed to accept or reject the null hypothesis, which is: There is no difference between TEG parameters when analyzing platelet gels formed with calcium chloride, platelet-rich plasma and three different concentrations of thrombin A one-way analysis of variance test was performed between thrombin concentrations for MA (p = 0.19), tMA (p = 0.443), CI (p = 0.257), and angle (p = 0.323). The results showed that thrombin concentration did not affect the MA, tMA, CI, or angle as measured by the TEG analyzer. The null hypothesis was accepted. Based on a one-way analysis of variance test for MA, tMA, CI, and angle there was no significant statistical difference for the TEG samples in this experiment as reported with a 95% confidence interval. PMID:15804158

  20. (Photoexcited charge pair escape and recombination)

    Energy Technology Data Exchange (ETDEWEB)

    Braun, C.L.

    1990-01-01

    Progress in four research areas on this project are summarized under the following topics: (1) Geminate charge pair recombination in hexane; (2) Fast current measurements resulting from excitation of charge transfer (CT) states; (3) Measurement of the dipole moment of excited states by DC conductivity; and (4) Charge separation at macroscopic interfaces between electron donor and acceptor solids. In a final section, personnel who have contributed to the project during the past budget period are described.

  1. Electrochemical Raduction Potential Shifts of Graphene Oxide Employed in Thrombin Detection

    International Nuclear Information System (INIS)

    We have reported a feasibility study on thrombin detection by the measurement of peak potentials shifts for the electrochemical reduction of GO. Our novel strategy has demonstrated that the shifts are fairly dependent upon the concentration of target thrombin. We are extending this result to determine quantitatively various target material such as proteins, DNA, metal ions. The development of electrochemical biosensor is currently under the intense investigation owing to their great promise for rapid, convenience and low-cost detection of analytes. Especially, graphene oxide (GO), an oxygen-rich carbonaceous layered material, has attracted strong interest as an substrate material because of its own unique characteristics. Based on recent studies GO consists of intact graphitic regions interspersed with sp3-hybridized carbons containing hydroxyl and epoxy functional groups on the top and bottom surfaces of each sheet and sp2-hybridized carbons containing carboxylate and carbonyl groups principally at the sheet edges. The intrinsic oxygen-containing functional groups have been used as initial sites for deposition of biomolecules such as DNA and proteins on the GO sheets and provide the possibility to be an ideal platform for detecting of target materials. In addition, GO is electrochemically reducible, which enables it as an indicator or label for electrochemical sensor applications. Several studies have employed the GO-indicator to determine DNA, Hg2+, and thrombin

  2. Aptamer-conjugated gold functionalized graphene oxide nanocomposites for human ?-thrombin specific recognition.

    Science.gov (United States)

    Deng, Nan; Jiang, Bo; Chen, Yuanbo; Liang, Zhen; Zhang, Lihua; Liang, Yu; Yang, Kaiguang; Zhang, Yukui

    2016-01-01

    The specific recognition toward target proteins from complex biological samples has great potential in clinical diagnostics and therapeutics, receiving more and more attention. Herein, we achieved the specific detection of human ?-thrombin from human serum by aptamer-conjugated gold functionalized graphene oxide nanocomposites (denoted as Apt/Au/PEI/GO nanocomposites). Gold functionalized graphene oxide nanocomposites were synthesized by in situ growth of Au nanoparticles on graphene oxide surface using polyethylenimine as reducing and stabilizing reagents, and then it was used as support for aptamer immobilization through forming an Au-S bonding. The obtained Apt/Au/PEI/GO nanocomposites inherited not only the large surface area which made the immobilizing amount of aptamer up to 36.1nmol/mg, but also the excellent hydrophilicity which showed remarkable selectivity for human ?-thrombin specific recognition, even with the interference of 3000 fold human serum proteins. Furthermore, with its superior properties, Apt/Au/PEI/GO nanocomposites showed advantages of high capture efficiency (>86%) and excellent recognition repeatability. Finally, the Apt/Au/PEI/GO nanocomposites were successfully applied for human ?-thrombin specific recognition in human serum, verifying its great potential in clinical applications. PMID:26689824

  3. Electrochemical Raduction Potential Shifts of Graphene Oxide Employed in Thrombin Detection

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Hanall; Hwang, Shinjae; Kim, Kyuwon [Incheon National Univ., Incheon (Korea, Republic of)

    2014-06-15

    We have reported a feasibility study on thrombin detection by the measurement of peak potentials shifts for the electrochemical reduction of GO. Our novel strategy has demonstrated that the shifts are fairly dependent upon the concentration of target thrombin. We are extending this result to determine quantitatively various target material such as proteins, DNA, metal ions. The development of electrochemical biosensor is currently under the intense investigation owing to their great promise for rapid, convenience and low-cost detection of analytes. Especially, graphene oxide (GO), an oxygen-rich carbonaceous layered material, has attracted strong interest as an substrate material because of its own unique characteristics. Based on recent studies GO consists of intact graphitic regions interspersed with sp{sup 3}-hybridized carbons containing hydroxyl and epoxy functional groups on the top and bottom surfaces of each sheet and sp{sup 2}-hybridized carbons containing carboxylate and carbonyl groups principally at the sheet edges. The intrinsic oxygen-containing functional groups have been used as initial sites for deposition of biomolecules such as DNA and proteins on the GO sheets and provide the possibility to be an ideal platform for detecting of target materials. In addition, GO is electrochemically reducible, which enables it as an indicator or label for electrochemical sensor applications. Several studies have employed the GO-indicator to determine DNA, Hg{sup 2+}, and thrombin.

  4. 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.

  5. The membrane potential modulates thrombin-stimulated Ca²? mobilization and platelet aggregation.

    Science.gov (United States)

    Albarrán, Letizia; Dionisio, Natalia; López, Esther; Salido, Ginés M; Rosado, Juan A

    2013-10-15

    G protein-coupled receptors can be directly modulated by changes in transmembrane voltage in a variety of cell types. Here we show that, while changes in the membrane voltage itself do not induce detectable modifications in the cytosolic Ca(2+) concentration, platelet stimulation with thrombin or the PAR-1 and PAR-4 agonist peptides SFLLRN and AYPGKF, respectively, results in Ca(2+) release from intracellular stores that is sensitive to the membrane depolarisation. Direct activation of G proteins or phospholipase C by AlF4(-) and m-3M3FBS, respectively, leads to Ca(2+) release that is insensitive to changes in the membrane potential. Thapsigargin-, as well as OAG-induced Ca(2+) entry are affected by the membrane voltage, probably as a result of the modification in the driving force for Ca(2+) influx; however, hyperpolarisation does not enhance thrombin- or OAG-evoked Ca(2+) entry probably revealing the presence of a voltage-sensitive regulatory mechanism. Transmembrane voltage also modulates the activity of the plasma membrane Ca(2+)-ATPase (PMCA) most likely due to a decrease in the phosphotyrosine content of the pump. Thrombin-stimulated platelet aggregation is modulated by membrane depolarisation by a mechanism that is, at least partially, independent of Ca(2+). These observations indicate that PAR-1 and PAR-4 receptors are modulated by the membrane voltage in human platelets. PMID:23988350

  6. 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).

  7. Can autologous thrombin with a rest fraction of ethanol be used safely for activation of concentrated autologous platelets applied on nerves?

    OpenAIRE

    de Somer, Filip; De Brauwer, Veerle; Vandekerckhove, Maxence; Ducatelle, Richard; Uyttendaele, Dirk; Nooten, Guido Van

    2005-01-01

    The use of concentrated platelet-rich plasma (cPRP) as a source of growth factors is reported to be beneficial for an enhanced osteogenesis in spine surgery. Today both bovine and autologous thrombin is used for activating the platelets and thus releasing the growth factors. In order to prevent transmission of organisms and the development of antibodies, autologous thrombin seems to be the logical choice. However, the preparation of autologous thrombin is cumbersome and consumes a part of the...

  8. 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...

  9. Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

    Science.gov (United States)

    Schmidbauer, Stefan; Witzel, Reinhild; Robbel, Lars; Sebastian, Petra; Grammel, Nicolas; Metzner, Hubert J; Schulte, Stefan

    2015-08-01

    rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant ?-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on activation, setting the basis for optimal efficacy in the patient. PMID:26037285

  10. A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

    International Nuclear Information System (INIS)

    Highlights: • A label-free thrombin aptamer biosensor applied in whole blood has been developed. • The aptamer biosensor showed a wide detection range and a low detection limit. • The antibiofouling idea utilized for biosensor is significant for diagnostics. - Abstract: In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN)6]3?/4?. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health

  11. A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Chong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Han, Qiaorong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Wang, Daoying; Xu, Weimin [Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Wang, Weijuan [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhao, Wenbo, E-mail: zhaowenbo@njnu.edu.cn [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhou, Min, E-mail: zhouminnju@126.com [Department of Vascular Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008 (China)

    2014-11-19

    Highlights: • A label-free thrombin aptamer biosensor applied in whole blood has been developed. • The aptamer biosensor showed a wide detection range and a low detection limit. • The antibiofouling idea utilized for biosensor is significant for diagnostics. - Abstract: In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN){sub 6}]{sup 3?/4?}. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.

  12. Topical report review status

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-08-01

    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.

  13. 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)

  14. Design, synthesis and antithrombotic evaluation of novel dabigatran etexilate analogs, a new series of non-peptides thrombin inhibitors.

    Science.gov (United States)

    Chen, Dongxing; Wang, Shaochi; Diao, Xiaojuan; Zhu, Qihua; Shen, Huiliang; Han, Xueqing; Wang, Yiwei; Gong, Guoqing; Xu, Yungen

    2015-12-01

    Thrombin is a serine protease that plays a key role in blood clotting, which makes it a promising target for the treatment of thrombotic diseases. Dabigatran is direct potent thrombin inhibitor. Based on bioisosteric and scaffold hopping principle, two dabigatran mimics (I-1 and II-1) in which the benzamidine moiety of dabigatran was replaced by a tricyclic fused scaffold were designed, synthesized and evaluated for their in vitro activities for inhibiting thrombin. The results reveal that compounds I-1 (IC50=9.20nM) and II-1 (IC50=7.48nM) are potent direct thrombin inhibitors and the activity is in the range of reference drug. On this basis, twenty-two ester and carbamate derivatives of I-1 or II-1 were prepared and evaluated for their anticoagulant activity. Prodrugs I-4a (IC50=0.73?M), I-4b (IC50=0.75?M), II-2a (IC50=1.44?M) and II-2b (IC50=0.91?M) display excellent effects of inhibiting thrombin induced-platelet aggregation. Moreover, compounds I-9 and II-4, which contain a cleavable moiety with anti-platelet activity, show the best anticoagulant efficacy among the tested compounds in the rat venous thrombosis model. The compounds which have better in vitro and in vivo activity were subjected to rat tail bleeding test, and the result demonstrates that compound I-9 is less likely to have bleeding risk than dabigatran etexilate. PMID:26537784

  15. Thrombin inhibits HMGB1-mediated proinflammatory signaling responses when endothelial protein C receptor is occupied by its natural ligand

    Directory of Open Access Journals (Sweden)

    Jong-Sup Bae

    2013-11-01

    Full Text Available High mobility group box 1 (HMGB1 is involved in thepathogenesis of vascular diseases. Unlike activated protein C(APC, the activation of PAR-1 by thrombin is known to elicitproinflammatory responses. To determine whether the occupancyof EPCR by the Gla-domain of APC is responsible for thePAR-1-dependent antiinflammatory activity of the protease, wepretreated HUVECs with the PC zymogen and then activatedPAR-1 with thrombin. It was found that thrombin downregulatesthe HMGB1-mediated induction of both TNF-? andIL-6 and inhibits the activation of both p38 MAPK and NF-?B inHUVECs pretreated with PC. Furthermore, thrombin inhibitedHMGB1-mediated hyperpermeability and leukocyte adhesion/migration by inhibiting the expression of cell adhesion moleculesin HUVECs if EPCR was occupied. Collectively, theseresults suggest the concept that thrombin can initiate proinflammatoryresponses in vascular endothelial cells through theactivation of PAR-1 may not hold true for normal vesselsexpressing EPCR under in vivo conditions. [BMB Reports 2013;46(11: 544-549

  16. 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.

  17. Sphingosine-1-phosphate induces thrombin receptor PAR-4 expression to enhance cell migration and COX-2 formation in human monocytes.

    Science.gov (United States)

    Mahajan-Thakur, Shailaja; Sostmann, Björn D; Fender, Anke C; Behrendt, Daniel; Felix, Stephan B; Schrör, Karsten; Rauch, Bernhard H

    2014-10-01

    Thrombin is not only a central factor in blood coagulation but also stimulates inflammatory processes, including monocyte responses, via activation of PARs. The signaling lipid S1P is a major determinant of monocyte function. Here, we established an interaction between S1P and human monocyte responses to thrombin. S1P induced PAR-1 and PAR-4 mRNA and total protein expression in human monocytes and U937 cells in a concentration (0.1-10 ?M)- and time (1-24 h)-dependent manner, respectively. However, only PAR-4 cell-surface expression was increased significantly by S1P, whereas PAR-1 remained unaffected. This response was associated with activation of the Akt, Erk, and p38 pathway and induction of COX-2 but not COX-1. PAR-4-mediated induction of COX-2 was prevented by the PI3K inhibitor LY (10 ?M). Preincubation of human monocytes with S1P (1 ?M; 16 h) resulted in an enhanced chemotaxis toward thrombin or to selective AP for PAR-4 but not PAR-1. Furthermore, down-regulation of PAR-4 transcription with siRNA attenuated the chemotactic response to thrombin and AP4. In conclusion, S1P enhances monocyte responses to thrombin via up-regulation of PAR-4 expression, which promotes cell migration and COX-2 abundance. This mechanism may facilitate monocyte recruitment to sites of vessel injury and inflammation. PMID:24990321

  18. Use of a Thrombin-gelatin Hemostatic Matrix (Surgiflo) in Spinal Surgery.

    Science.gov (United States)

    Gazzeri, Roberto; De Bonis, Costanzo; Galarza, Marcelo

    2014-11-01

    A variety of techniques have been used to stop venous bleeding from the spinal epidural space. These generally consist of packing with Surgicel®, fibrillar collagen or Gelfoam®. Bipolar coagulation may also be used to control bleeding from spinal venous plexus, but it may bear the risk of healthy nervous tissue injury: dissipation of heat from the tips of the bipolar forceps may induce thermal injury to adjacent neural structures. In the case of intraspinal bleeding, quick and safe hemostasis is mandatory to ensure adequate visualization and safe preparation so as to avoid damaging nerves and spinal medulla. In addition, quick and safe hemostasis reduces the duration of surgery. Efficient control of bleeding can thereby reduce perioperative morbidity. During 6 months, the authors performed more than 170 major spinal surgeries, and in 67 procedures they used injection of thrombin-gelatin hemostatic matrix (Surgiflo, Johnson & Johnson Wound Management, Somerville, NJ) into spinal epidural space to assist in hemostasis. When the venous bleeding continued from the epidural space after packing with hemostatic agents as Surgicel and fibrillar collagen, gelatin matrix was used to stop venous bleeding. In all cases, the results were judged to be excellent, with immediate stoppage of epidural bleeding, or good. No complications related to the thrombin-gelatin hemostatic matrix were encountered. The thrombin-gelatin matrix could represent a valuable tool when other hemostatic strategies are ineffective or suboptimal. It is safe and biocompatible when compared with hemostatic agents currently in use. This is the first study reporting the use of Surgiflo hemostatic matrix in spinal surgery. PMID:25419955

  19. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    International Nuclear Information System (INIS)

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated 125I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function

  20. 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.

  1. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

  2. Health Topic XML File Description

    Science.gov (United States)

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

  3. A new modified thrombin binding aptamer containing a 5?–5? inversion of polarity site

    OpenAIRE

    Martino, Luigi; Virno, Ada; Randazzo, Antonio; Virgilio, Antonella; Esposito, Veronica; Giancola, Concetta; Bucci, Mariarosaria; Cirino, Giuseppe; Mayol, Luciano

    2006-01-01

    The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5?–5? inversion of polarity site, namely d(3?GGT5?-5?TGGTGTGGTTGG3?), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(3?GGT5?-5?TGGTGTGGTTGG3?) is characterized by ...

  4. Use of dressing with human fibrin and thrombin during resection of a right atrial angiosarcoma

    Science.gov (United States)

    Bochenek, Maciej; Kapelak, Bogus?aw; Bartu?, Krzysztof; Urba?czyk, Ma?gorzata; Sadowski, Jerzy

    2015-01-01

    Primary malignant cardiac tumors are rare and are usually detected at an advanced stage of disease. Their location and infiltration often hinder surgical resection. Tissue sarcomas, especially angiosarcomas, are composed of irregular and delicate vascular tissue. The resection of such tumors from the heart is associated with a high risk of life-threatening bleeding that cannot be stopped with traditional surgical methods. We present a case report of the application of a dressing containing human fibrin and thrombin in order to prevent bleeding during the partial resection of advanced cardiac angiosarcoma in a 40-year-old patient. PMID:26336498

  5. Biophysical Investigation of GpIb? Binding to Thrombin Anion Binding Exosite II†

    OpenAIRE

    Sabo, T Michael; Maurer, Muriel C.

    2009-01-01

    Substrates and cofactors of the serine protease thrombin (IIa) employ two anion binding exosites (ABE-I and II) to aid in binding. On the surface of platelets resides the membrane bound receptor complex GpIb?/?-GpIX-GpV. IIa’s ABE-II is proposed to interact with an anionic portion of GpIb? which enhances IIa cleavage of PAR-1 and subsequent activation of platelets. In the current work, 1D and 2D NMR, analytical ultracentrifugation (AUC), and hydrogen deuterium exchange (HDX) coupled with MALD...

  6. 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 ... topical liquid (Pennsaid) is used to relieve osteoarthritis pain in the knees. Diclofenac is in a class ...

  7. Multiphoton Assisted Recombination

    International Nuclear Information System (INIS)

    We have observed multiphoton assisted recombination in the presence of a 38.8 GHz microwave field. Stimulated emission of up to ten microwave photons results in energy transfer from continuum electrons, enabling recombination. The maximum electron energy loss is far greater than the 2Up predicted by the standard 'simpleman's' model. The data are well reproduced by both an approximate analytic expression and numerical simulations in which the combined Coulomb and radiation fields are taken into account

  8. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

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

    International Nuclear Information System (INIS)

    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. 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. 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 < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-?TC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells. 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

  10. 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.…

  11. Pegasparaginase treatment alters thrombin generation by modulating the protein C and S system in acute lymphoblastic leukaemia/lymphoma.

    Science.gov (United States)

    Staddon, Jack H; Smock, Kristi J; Schiffman, Joshua D; Fluchel, Mark N; Engel, Michael E; Weyrich, Andrew S; Campbell, Robert A

    2015-10-01

    Paediatric patients with acute lymphoblastic leukaemia/lymphoma treated with pegasparaginase are at an increased risk of thrombosis. We evaluated changes in thrombin generation in the presence and absence of thrombomodulin using paired plasma samples collected from paediatric patients treated with pegasparaginase. Postpegasparaginase samples were significantly less sensitive to reductions in thrombin generation in the presence of thrombomodulin compared with prepegasparaginase, suggesting reduced protein C and S activity. This corresponded to a significant decrease in protein C and protein S antigen. Alterations in the protein C and S pathway may contribute to the increased risk of thrombosis in patients treated with pegasparaginase. PMID:26196196

  12. Comparison of antibody and aptamer receptors for the specific detection of thrombin with a nanometer gap-sized impedance biosensor.

    Science.gov (United States)

    Schlecht, U; Malavé, A; Gronewold, T; Tewes, M; Löhndorf, M

    2006-07-28

    Nanogap-impedance biosensors with electrode separations of 75 nm have been fabricated by means of standard optical lithography and a sacrificial layer technique. Due to a large surface-to-volume ratio and high sensitivity, these sensors are superior compared to open interdigitated electrode structures. As a model, the blood coagulation factor thrombin was detected. As specific receptors, either an antibody or a RNA-aptamer have been used. The microwave frequency impedance measurements showed that both ligands were equally suitable for the specific detection of thrombin. PMID:17723506

  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. Modulation of human uterine smooth muscle cell collagen contractility by thrombin, Y-27632, TNF alpha and indomethacin

    Directory of Open Access Journals (Sweden)

    Smith Terry J

    2009-01-01

    Full Text Available Abstract Background Preterm labour occurs in approximately 10% of pregnancies and is a major cause of infant morbidity and mortality. However, the pathways involved in regulating contractility in normal and preterm labour are not fully elucidated. Our aim was to utilise a human myometrial contractility model to investigate the effect of a number of uterine specific contractility agents in this system. Therefore, we investigated the contractile response of human primary uterine smooth muscle cells or immortalised myometrial smooth muscle cells cultured within collagen lattices, to known mediators of uterine contractility, which included thrombin, the ROCK-1 inhibitor Y-27632, tumour necrosis factor alpha (TNF alpha and the non-steroidal anti-inflammatory indomethacin. Methods Cell contractility was calculated over time, with the collagen gel contraction assay, utilising human primary uterine smooth muscle cells (hUtSMCs and immortalised myometrial smooth muscle cells (hTERT-HM: a decrease in collagen gel area equated to an increase in contractility. RNA was isolated from collagen embedded cells and gene expression changes were analysed by real time fluorescence reverse transcription polymerase chain reaction. Scanning electron and fluorescence microscopy were employed to observe cell morphology and cell collagen gel interactions. Statistical analysis was performed using ANOVA followed by Tukey's post hoc tests. Results TNF alpha increased collagen contractility in comparison to the un-stimulated collagen embedded hUtSMC cells, which was inhibited by indomethacin, while indomethacin alone significantly inhibited contraction. Thrombin augmented the contractility of uterine smooth muscle cell and hTERT-HM collagen gels, this effect was inhibited by the thrombin specific inhibitor, hirudin. Y-27632 decreased both basal and thrombin-induced collagen contractility in the hTERT-HM embedded gels. mRNA expression of the thrombin receptor, F2R was up-regulated in hUtSMCs isolated from collagen gel lattices, following thrombin-stimulated contractility. Conclusion TNF alpha and thrombin increased uterine smooth muscle cell collagen contractility while indomethacin had the opposite effect. Thrombin-induced collagen contractility resulted in F2R activation which may in part be mediated by the ROCK-1 pathway. This study established the in vitro human myometrial model as a viable method to assess the effects of a range of uterotonic or uterorelaxant agents on contractility, and also permits investigation of the complex regulatory pathways involved in mediating myometrial contractility at labour.

  15. Thrombin-induced IL-8/CXCL8 release is mediated by CK2, MSK1, and NF-?B pathways in human lung epithelial cells.

    Science.gov (United States)

    Lin, Chien-Huang; Shih, Chung-Hung; Chen, Bing-Chang

    2015-11-15

    Airway inflammation plays a major role in the pathophysiology of lung inflammatory diseases such as asthma. Thrombin, a serine protease, is known to mediate central functions in thrombosis and hemostasis and also plays a critical role in lung inflammation via producing chemokine release including interleukin (IL)-8/CXCL8. Our previous studies showed that c-Src- and Rac-dependent nuclear factor (NF)-?B signaling pathways participate in thrombin-induced IL-8/CXCL8 release in human lung epithelial cells. In this study, we further investigated the role of casein kinase 2 (CK2)/mitogen stress-activated protein kinase 1 (MSK1)-dependent p65 phosphorylation in thrombin-induced NF-?B activation and IL-8/CXCL8 release. Thrombin-induced IL-8/CXCL8 release was inhibited by CK2 inhibitors (apigenin and tetrabromobenzotriazole, TBB), small interfering RNA of CK2? (CK2? siRNA), and MSK1 siRNA. Treatment of cells with thrombin caused increases in CK2? phosphorylation at Ser209, which was inhibited by a protein kinase C ? (PKC?) inhibitor (Ro-32-0432). Thrombin-induced MSK1 phosphorylation at Ser581 and Akt phosphorylation at Ser473 were inhibited by apigenin. Moreover, the thrombin-induced increase in IL-8/CXCL8 release was attenuated by p65 siRNA. Stimulation of cells with thrombin resulted in an increase in p65 phosphorylation at Ser276, which was inhibited by apigenin and MSK1 siRNA. Thrombin-induced ?B-luciferase activity was also inhibited by apigenin and MSK1 siRNA. Taken together, these results show that thrombin activates the PKC?/CK2/MSK1 signaling pathways, which in turn initiates p65 phosphorylation and NF-?B activation, and ultimately induces IL-8/CXCL8 release in human lung epithelial cells. PMID:26463037

  16. Topical treatment of ichthyoses.

    Science.gov (United States)

    Fleckman, Philip; Newell, Brandon D; van Steensel, Maurice A; Yan, Albert C

    2013-01-01

    Management of ichthyoses is a complex and continuously dynamic process. Primary treatments of ichthyosis are by means of topical moisturizers and topical medications. Patients and families need to have reasonable and realistic expectations when it comes to topical therapy. Topical medications cannot cure the scaling, but can gradually reduce it and thus improve their condition. No one treatment regimen works for everyone, and the best topical therapy for each patient may be the result of months (or years) of painstaking effort on both the physician's and the patient's behalf. As patients get older and their activities and lifestyles change, so should their topical treatment regimen. Bear in mind that the more complex the skin care regimen and costly the topical treatments, the less likely a patient and their family will be compliant. PMID:23384017

  17. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Liu, Yan-Hong [Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin, Heilongjiang 150081 (China); Li, Yan; Wang, Jia-Ye [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Hattori, Toshio [Department of Emerging Infectious Diseases, Division of Internal Medicine, Graduate School of Medicine, Tohoku University, Sendai 9808574 (Japan); Ling, Hong, E-mail: lingh@ems.hrbmu.edu.cn [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Department of Parasitology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China); Zhang, Feng-Min, E-mail: fengminzhang@yahoo.com.cn [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China)

    2010-01-22

    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.

  19. Module-activity relationship of G-quadruplex based DNA aptamers for human thrombin.

    Science.gov (United States)

    Zavyalova, E; Golovin, A; Pavlova, G; Kopylov, A

    2013-01-01

    G-quadruplex based DNA aptamers for human thrombin are promising pharmaceuticals as anticoagulants. Initially discovered 15-mer DNA aptamer (15-TBA) has a minimal G-quadruplex structure which is able to inhibit thrombin. 15-TBA was modified and extended to improve aptamer activity and in vivo stability providing 31-TBA, NU172, RA-36, and some others as successful examples. In this paper an interplay between G-quadruplex (pharmacophore module) and additional modules has been studied. An original turbidimetric assay and conventional coagulation tests were applied to evaluate both inhibitory activity and type of inhibiting for aptamers constructed by exchanging the modules between 31- TBA and NU172. Additional modules strongly affect pharmacophore module inhibitory activity either enhancing or reducing it. RA-36 aptamer has two putative pharmacophore entities which also interplay being functionally non-equal. 5'- truncated RA-36 has half of the activity of RA-36, and the same as for 15-TBA. On the contrary 3'-truncated RA-36 has intermediate activity in between 15-TBA and RA-36. These results indicate fine regulation of G-quadruplex inhibitory activity by additional modules, as well as non-trivial behavior of joined pharmacophore modules. PMID:24083606

  20. Proteome changes in platelets activated by arachidonic acid, collagen, and thrombin

    Directory of Open Access Journals (Sweden)

    Suttnar Ji?í

    2010-11-01

    Full Text Available Abstract Background Platelets are small anucleated blood particles that play a key role in the control of bleeding. Platelets need to be activated to perform their functions and participate in hemostasis. The process of activation is accompanied by vast protein reorganization and posttranslational modifications. The goal of this study was to identify changes in proteins in platelets activated by different agonists. Platelets were activated by three different agonists - arachidonic acid, collagen, and thrombin. 2D SDS-PAGE (pI 4-7 was used to separate platelet proteins. Proteomes of activated and resting platelets were compared with each other by Progenesis SameSpots statistical software; and proteins were identified by nanoLC-MS/MS. Results 190 spots were found to be significantly different. Of these, 180 spots were successfully identified and correspond to 144 different proteins. Five proteins were found that had not previously been identified in platelets: protein CDV3 homolog, protein ETHE1, protein LZIC, FGFR1 oncogene partner 2, and guanine nucleotide-binding protein subunit beta-5. Using spot expression profile analysis, we found two proteins (WD repeat-containing protein 1 and mitochondrial glycerol-3-phosphate dehydrogenase that may be part of thrombin specific activation or signal transduction pathway(s. Conclusions Our results, characterizing the differences within proteins in both activated (by various agonists and resting platelets, can thus contribute to the basic knowledge of platelets and to the understanding of the function and development of new antiplatelet drugs.

  1. Computational study of some benzamidine-based inhibitors of thrombin-like snake venom proteinases

    Science.gov (United States)

    Henriques, Elsa S.; Nascimento, Marco A. C.; Ramos, Maria João

    Pit viper venoms contain a number of serine proteinases that, despite their observed coagulant thrombin-like action in vitro, exhibit a paradoxical benign defibrinogenating (anticoagulant) action in vivo, with clinical applications in preventing thrombi and improved blood circulation. Considering that several benzamidine-based inhibitors, some highly selective to thrombin, also inhibit the enzymatic activity of such venombins, the modeling of their enzyme-inhibitor interactions could provide valuable information on the topological factors that determine the divergences in activity. The first step, and the object of the present study, was to derive the necessary set of parameters, consistent with the CHARMM force field, and to perform molecular dynamics (MD) simulations on a few selected representatives of the inhibitors in question under physiological conditions. Bonding and van der Waals parameters were derived by analogy to similar ones in the existing force field. Net atomic charges were obtained with a restrained fitting to the molecular electrostatic potential generated at B3LYP/6-31G(d) level. The parameters were refined to reproduce the available experimental geometries and crystal data, and the MD simulations of the free inhibitors in aqueous solution at 298 K provided an insightful description of their available conformational space.

  2. 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.

  3. 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

  4. 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 * dendrimer s Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.328, year: 2008

  5. The Expression of the Thrombin Receptors PAR-3 and PAR-4 is Downregulated in Pancreatic Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Claudia Rudroff

    2015-05-01

    Full Text Available Background: Patients with pancreatic cancer frequently suffer from thrombosis as a consequence of excess thrombin generation. In addition to its role in the plasmatic coagulation cascade, thrombin induces numerous cellular effects by activating a unique group of G-protein-coupled receptors on the cell membrane, the proteinase-activated receptors (PARs. At present, PAR-1, PAR-3 and PAR-4 are known to be activated by thrombin. We previously demonstrated a putative role for PAR-1 in pancreatic cancer progression, but little is known about the physiological and pathophysiological roles of PAR-3 and PAR-4. In the present study, we examined the expression patterns of PAR-3 and PAR-4 in pancreatic tissue and pancreatic cancer cells. Methods: Tissue samples from three patients with pancreatic adenocarcinoma and six human pancreatic carcinoma cell lines were examined. Gene expression was analysed by RT-PCR and quantified by HPLC. Protein expression was determined by Western blot analysis. Data analysis was performed using ANOVA in SPSS. Results and Conclusion: In contrast to PAR-1, both PAR-3 and PAR-4 were expressed in healthy pancreases but downregulated in pancreatic cancer. The contrasting expression patterns of PAR-3 and PAR-4 compared with PAR-1 indicate that the mechanism that regulates the cellular effects of thrombin on tumor progression remains to be fully elucidated.

  6. 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.

  7. 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

  8. Syntacticized topics in Kurmuk

    DEFF Research Database (Denmark)

    Andersen, Torben

    2015-01-01

    This article argues that Kurmuk, a little-described Western Nilotic language, is characterized by a syntacticized topic whose grammatical relation is variable. In this language, declarative clauses have as topic an obligatory preverbal NP which is either a subject, an object or an adjunct. The grammatical relation of the topic is expressed by a voice-like inflection of the verb, here called orientation. While subject-orientation is morphologically unmarked, object-oriented and adjunct-oriented v...

  9. Thrombin Time

    Science.gov (United States)

    ... as part of an investigation of a possible bleeding disorder or inappropriate blood clot formation ( thrombotic episode ), particularly ... battery of tests typically required to evaluate a bleeding or thrombotic disorder. A significant percentage of people with decreased or ...

  10. Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

    OpenAIRE

    Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G; Court, Donald L.

    2009-01-01

    Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA sub...

  11. Evidence supporting the use of recombinant activated factor VII in congenital bleeding disorders

    Directory of Open Access Journals (Sweden)

    Pär I Johansson

    2010-06-01

    Full Text Available Pär I Johansson, Sisse R OstrowskiCapital Region Blood Bank, Section for Transfusion Medicine, Rigshospitalet, University of Copenhagen, Copenhagen, DenmarkBackground: Recombinant activated factor VII (rFVIIa, NovoSeven® was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX.Objective: To review the evidence supporting the use of rFVIIa for the treatment of patients with congenital bleeding disorders.Patients and methods: English-language databases were searched in September 2009 for reports of randomized controlled trials (RCTs evaluating the ability of rFVIIa to restore hemostasis in patients with congenital bleeding disorders.Results: Eight RCTs involving 256 hemophilic patients with antibodies against coagulation factors, also known as inhibitors, were identified. The evidence supporting the use of rFVIIa in these patients was weak with regard to dose, clinical setting, mode of administration, efficacy, and adverse events, given the limited sample size of each RCT and the heterogeneity of the studies.Conclusion: The authors suggest that rFVIIa therapy in hemophilic patients with inhibitors should be based on the individual’s ability to generate thrombin and form a clot, and not on the patient’s weight alone. Therefore, assays for thrombin generation, such as whole-blood thromboelastography, have the potential to significantly improve the treatment of these patients.Keywords: hemophilia, inhibitors, coagulation factor VIII, coagulation factor IX, rFVIIa, NovoSeven, FEIBA, hemostasis, RCT

  12. 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.

  13. 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

  14. 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...

  15. Recombinant hormones in osteoporosis

    DEFF Research Database (Denmark)

    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.

  16. 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 ...

  17. The potential for changing prescribing patterns from warfarin to oral direct thrombin inhibitors: clinical scenarios.

    Science.gov (United States)

    Reiffel, James A

    2004-01-01

    Ximelagatran, a direct thrombin inhibitor, is currently being considered by the US Food and Drug Administration (FDA) for approval as an anticoagulant to manage thromboembolic disorders and prevent systemic embolism in patients with atrial fibrillation. If ximelagatran is approved, clinicians will have to decide which patients are candidates for this therapy, how to switch patients from warfarin to ximelagatran, and, if necessary, how to switch patients from ximelagatran to warfarin. In addition, clinicians will need to consider their approach to treating patients with new-onset atrial fibrillation as well as conditions that may require an adjustment in dosing. This article highlights some of these issues as well as current data that provide guidance on how to manage them; however, answers to other questions will not be available until after the FDA approves the package insert material and data from the SPORTIF trial become available. Therefore, clinicians should diligently follow the medical literature regarding the latest information on this agent. PMID:15619610

  18. Thromboelastography, thrombin generation test and thrombodynamics reveal hypercoagulability in patients with multiple myeloma.

    Science.gov (United States)

    Gracheva, Marina A; Urnova, Evdokiya S; Sinauridze, Elena I; Tarandovskiy, Ivan D; Orel, Elena B; Poletaev, Alexander V; Mendeleeva, Larisa P; Ataullakhanov, Fazoil I; Balandina, Anna N

    2015-12-01

    Patients with multiple myeloma (MM) are at increased risk of venous thromboembolism. Therefore, adequate laboratory control of hemostasis and subsequent adjustments of anticoagulant therapy are necessary. We studied hemostasis changes using thromboelastography (TEG), thrombin generation test (TGT) and thrombodynamics (TD) in primary MM patients (PMMpt, n = 25) and patients in remission (RMMpt, n = 34) during blood stem cell (BSC) mobilization. TD and TEG reveal hypercoagulability in PMMpt (*p < 0.05) in relation to healthy volunteers. There was no difference in any of the tests between PMMpt and RMMpt. We detected no heparin effect in 22% of patients one day after the onset of the prophylactic heparin treatment (500 IU/h) during BSC mobilization; tests shifted toward the hypercoagulability in 75% of patients one day after cyclophosphamide (4 g/m(2)) chemotherapy. Global hemostasis tests were in good agreement with each other, revealed hypercoagulability and heparin "resistance" in patients with MM and may be useful for therapy individualization. PMID:25907422

  19. Experiência inicial com o uso de adesivo tissular contendo trombina para tratamento do pseudo-aneurisma femoral Treatment of femoral pseudoaneurysm with thrombin tissue adhesive: initial experience

    Directory of Open Access Journals (Sweden)

    Daniel Mendes Pinto

    2006-03-01

    Full Text Available O pseudo-aneurisma (PSA após cateterização femoral tem sido diagnosticado com regularidade em serviços com grande movimento de intervenções percutâneas, com incidência variando de 0,05 a 6%. PSA femorais pequenos podem ser acompanhados até a resolução espontânea. As opções de tratamento são: compressão guiada por ultra-som, injeção de trombina para trombose do PSA e tratamento cirúrgico. A injeção percutânea de trombina tem a vantagem de ser um procedimento indolor e rápido. Podem ser utilizados trombina isolada ou preparados contendo trombina associada a fibrinogênio e fatores de coagulação. A experiência inicial dos autores de cinco casos tratados com injeção de adesivo tissular contendo trombina mostrou resultado satisfatório em quatro; um caso necessitou tratamento cirúrgico. Não houve sucesso com uso isolado de trombina humana, porém, ocorreu trombose imediata após injeção de preparado de trombina associada a fibrinogênio/fator XIII. Neste artigo, são discutidas as opções de tratamento dos PSA femorais e a técnica do uso de trombina percutânea.Pseudoaneurysms caused by femoral artery catheterization have been regularly diagnosed in medical units with a great number of percutaneous interventions, with a documented incidence between 0.05 and 6%. Small femoral pseudoaneurysms undergo spontaneous resolution. Treatment options are: ultrasound-guided compression, thrombin injection to induce pseudoaneurysm thrombosis and surgical treatment. Percutaneous thrombin injection has the advantage of being a fast and painless procedure. Both isolated thrombin and thrombin preparations with fibrinogen and coagulation factors can be used. The authors' initial experience with five cases treated with thrombin tissue adhesive showed successful results in four; one case required surgery. There was no success with isolated human thrombin, but immediate thrombosis was achieved after injection of thrombin associated to fibrinogen and factor XIII. In this article, the treatment options for femoral pseudoaneurysms and the technique of percutaneous thrombin are discussed.

  20. Experiência inicial com o uso de adesivo tissular contendo trombina para tratamento do pseudo-aneurisma femoral / Treatment of femoral pseudoaneurysm with thrombin tissue adhesive: initial experience

    Scientific Electronic Library Online (English)

    Daniel Mendes, Pinto; José Olimpio, Dias Júnior; Bernardo Lopes Cançado, Fonseca; Rodrigo Daniel, Moreialvar; Leonardo Ghizoni, Bez; Caetano de Sousa, Lopes.

    2006-03-01

    Full Text Available O pseudo-aneurisma (PSA) após cateterização femoral tem sido diagnosticado com regularidade em serviços com grande movimento de intervenções percutâneas, com incidência variando de 0,05 a 6%. PSA femorais pequenos podem ser acompanhados até a resolução espontânea. As opções de tratamento são: compre [...] ssão guiada por ultra-som, injeção de trombina para trombose do PSA e tratamento cirúrgico. A injeção percutânea de trombina tem a vantagem de ser um procedimento indolor e rápido. Podem ser utilizados trombina isolada ou preparados contendo trombina associada a fibrinogênio e fatores de coagulação. A experiência inicial dos autores de cinco casos tratados com injeção de adesivo tissular contendo trombina mostrou resultado satisfatório em quatro; um caso necessitou tratamento cirúrgico. Não houve sucesso com uso isolado de trombina humana, porém, ocorreu trombose imediata após injeção de preparado de trombina associada a fibrinogênio/fator XIII. Neste artigo, são discutidas as opções de tratamento dos PSA femorais e a técnica do uso de trombina percutânea. Abstract in english Pseudoaneurysms caused by femoral artery catheterization have been regularly diagnosed in medical units with a great number of percutaneous interventions, with a documented incidence between 0.05 and 6%. Small femoral pseudoaneurysms undergo spontaneous resolution. Treatment options are: ultrasound- [...] guided compression, thrombin injection to induce pseudoaneurysm thrombosis and surgical treatment. Percutaneous thrombin injection has the advantage of being a fast and painless procedure. Both isolated thrombin and thrombin preparations with fibrinogen and coagulation factors can be used. The authors' initial experience with five cases treated with thrombin tissue adhesive showed successful results in four; one case required surgery. There was no success with isolated human thrombin, but immediate thrombosis was achieved after injection of thrombin associated to fibrinogen and factor XIII. In this article, the treatment options for femoral pseudoaneurysms and the technique of percutaneous thrombin are discussed.

  1. 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; Rasmussen, Bjarne E.; Rasmussen, Lars M.; Andresen, Thomas Lars; Wengel, Jesper; Pasternak, Anna

    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 th...

  2. 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; Rasmussen, Leif Bjarne; Rasmussen, Lars M; Andresen, Thomas Lars; Wengel, Jesper; Pasternak, Anna

    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 th...

  3. Platelet protease-activated receptor (PAR)4, but not PAR1, associated with neutral sphingomyelinase responsible for thrombin-stimulated ceramide-NF-?B signaling in human platelets

    OpenAIRE

    Chen, Wei-Fan; Lee, Jie-Jen; Chang, Chao-Chien; Lin, Kuan-Hong; Wang, Shwu-Huey; Sheu, Joen-Rong

    2013-01-01

    Thrombin activates platelets mainly through protease-activated receptor (PAR)1 and PAR4. However, downstream platelet signaling between PAR1 and PAR4 is not yet well understood. This study investigated the relationship between nSMase/ceramide and the NF-?B signaling pathway in PARs-mediated human platelet activation. The LC-MS/MS, aggregometry, flow cytometry, immunoprecipitation, and mesenteric microvessels of mice were used in this study. Human platelets stimulated by thrombin, 3-OMS (a neu...

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

    International Nuclear Information System (INIS)

    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. (paper)

  5. PAR-1 activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand

    OpenAIRE

    Bae, Jong-Sup; REZAIE, ALIREZA R.

    2008-01-01

    We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by its natural ligand activated protein C (APC)/protein C switches the protease activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin from a disruptive to a protective effect in cultured human umbilical vein endothelial cells. Given the phenotypic differences between endothelial cells in venular and arterial beds, in this study we evaluated the signaling function of thrombin in human pulmon...

  6. Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.

    OpenAIRE

    Senger, D. R.; Perruzzi, C. A.; Papadopoulos-Sergiou, A; Van de Water, L

    1994-01-01

    Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleava...

  7. Radiative recombination rate coefficients

    International Nuclear Information System (INIS)

    Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation. (orig.)

  8. Radiative Recombination Rate Coefficients

    Science.gov (United States)

    Brinzanescu, O.; Brinzanescu, O.; Stöhlker, Th.; Stöhlker, Th.

    Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation.

  9. Radiative recombination rate coefficients

    Energy Technology Data Exchange (ETDEWEB)

    Brinzanescu, O. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); National Inst. for Laser, Plasma and Radiation Physics, Bucharest Magurele (Romania); Stoehlker, T. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); Frankfurt Univ. (Germany). Inst. fuer Kernphysik

    2001-07-01

    Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation. (orig.)

  10. Radiative recombination rate coefficients

    Energy Technology Data Exchange (ETDEWEB)

    Brinzanescu, O. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); National Inst. for Laser, Plasma and Radiation Physics, Bucharest (Romania); Stoehlker, T. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); Frankfurt Univ. (Germany). Inst. fuer Kernphysik

    2000-10-01

    Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation. (orig.)

  11. Intrachromosomal recombination in plants.

    OpenAIRE

    Peterhans, A; Schlüpmann, H; Basse, C; Paszkowski, J.

    1990-01-01

    Molecular evidence for intrachromosomal recombination between closely linked DNA repeats within the plant genome is presented. The non-overlapping complementary deletion derivatives of the selectable neomycin phosphotransferase gene (nptII), when intact conferring kanamycin resistance, were inserted into the genome of Nicotiana tabacum. The functional marker gene was restored with frequencies between 10(-4) and 10(-6) per proliferating cell clone. Prolonged tissue culture prior to kanamycin s...

  12. 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

  13. A Simplified Recombinant PSO

    OpenAIRE

    Bratton, Daniel; Blackwell, Tim M.

    2008-01-01

    Simplified forms of the particle swarm algorithm are very beneficial in contributing to understanding how a particle swarm optimization (PSO) swarm functions. One of these forms, PSO with discrete recombination, is extended and analyzed, demonstrating not just improvements in performance relative to a standard PSO algorithm, but also significantly different behavior, namely, a reduction in bursting patterns due to the removal of stochastic components from the update equations.

  14. Recombinant human milk proteins.

    Science.gov (United States)

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases. PMID:16902336

  15. ?-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 variants selected for autonomous growth

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

    Energy Technology Data Exchange (ETDEWEB)

    Park, Buem Jin; Sa, Young Seung; Kim, Yong Hwan; Kim, Young Hun [Kwangwoon University, Seoul (Korea, Republic of)

    2012-01-15

    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.

  17. Topical photodynamic therapy

    Directory of Open Access Journals (Sweden)

    Polja?ki Mirjana

    2006-01-01

    Full Text Available Topical photodynamic therapy is a therapeutic modality in development, thus arises grate interest among dermatologists worldwide. It is an effective therapy for actinic keratosis, superficial BCC and Bowenos disease. Treatment efficacy, good cosmetics, low risk of skin cancer, low invasiveness, low rate of adverse events, facility for treating multiple or large lesions, especially in poor healing sites and, for penile, digital and facial involvement, low general toxicity and possibility of repeating the treatments with the same efficiency, enable topical photodynamic therapy to become increasingly practiced treatment modality. Researching aimed topical photodynamic therapy to prove as a treatment modality for clinical use in other dermatoses, is in experimental phase. To answer the question when dermatologist should consider using topical photodynamic therapy treatment modatility, we are present available date.

  18. Sparse Topical Coding

    CERN Document Server

    Zhu, Jun

    2012-01-01

    We present sparse topical coding (STC), a non-probabilistic formulation of topic models for discovering latent representations of large collections of data. Unlike probabilistic topic models, STC relaxes the normalization constraint of admixture proportions and the constraint of defining a normalized likelihood function. Such relaxations make STC amenable to: 1) directly control the sparsity of inferred representations by using sparsity-inducing regularizers; 2) be seamlessly integrated with a convex error function (e.g., SVM hinge loss) for supervised learning; and 3) be efficiently learned with a simply structured coordinate descent algorithm. Our results demonstrate the advantages of STC and supervised MedSTC on identifying topical meanings of words and improving classification accuracy and time efficiency.

  19. Topics in Nuclear Astrophysics

    International Nuclear Information System (INIS)

    Some topics in nuclear astrophysics are discussed, e.g.: highly evolved stellar cores, stellar evolution (through the temperature analysis of stellar surface), nucleosynthesis and finally the solar neutrino problem. (L.C.)

  20. Diclofenac Topical (actinic keratosis)

    Science.gov (United States)

    Diclofenac topical gel (Solaraze; generic) is used to treat actinic keratosis (flat, scaly growths on the skin caused by too much sun exposure). Diclofenac is in a class of medications called nonsteroidal ...

  1. 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

  2. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    Science.gov (United States)

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528

  3. Flexibility of the thrombin-activatable fibrinolysis inhibitor pro-domain enables productive binding of protein substrates

    DEFF Research Database (Denmark)

    Valnickova, Zuzana; Sanglas, Laura; Arolas, Joan L; Petersen, Steen V; Schar, Christine; Otzen, Daniel; Aviles, Francesc X; Gomis-Ruth, F Xavier; Enghild, Jan J

    2010-01-01

    We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity towards large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared to other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active-site cleft. Here we further investigat...

  4. 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.

  5. Membrane Changes Associated with Platelet Activation: EXPOSURE OF ACTIN ON THE PLATELET SURFACE AFTER THROMBIN-INDUCED SECRETION

    OpenAIRE

    George, James N; Lyons, Roger M; Morgan, Rebecca K.

    1980-01-01

    The effect of aggregation and secretion on membrane proteins was studied in washed human platelets. Reversible aggregation without secretion was stimulated by ADP and secretion without aggregation was stimulated by thrombin in the presence of EDTA. No loss of platelet surface glycoproteins occurred during reversible ADP-induced platelet aggregation, as measured by quantitative polyacrylamide gel electrophoresis analysis of platelets that were labeled with 125I-diazotized diiodosulfanilic acid...

  6. 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...

  7. 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; Lambaa Altinok, Magda; Mumm, Hanne; Andersen, Marianne

    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...

  8. Thrombin induces fibronectin-specific migration of pulmonary microvascular endothelial cells: requirement of calcium/calmodulin-dependent protein kinase II

    OpenAIRE

    Meoli, David F.; White, R. James

    2009-01-01

    Pulmonary arterial hypertension (PAH) is a progressive disease of excess vasoconstriction and vascular cell proliferation that results in increased pulmonary vascular resistance and right heart failure. We have previously shown (66) that tissue factor expression is increased in the abnormal vessels of patients and rats with PAH. We hypothesized that tissue factor and its downstream mediator, thrombin, would promote migration of endothelial cells (EC) and the vascular pathology of PAH. Immunos...

  9. Epiregulin is a potent vascular smooth muscle cell-derived mitogen induced by angiotensin II, endothelin-1, and thrombin

    OpenAIRE

    Taylor, David S.; Cheng, Xinbo; Pawlowski, John E.; Wallace, Alison R.; Ferrer, Patricia; Molloy, Christopher J.

    1999-01-01

    Vasoactive GTP-binding protein-coupled receptor agonists such as angiotensin II (AII), endothelin-1 (ET-1), and ?-thrombin (?-Thr) have been reported to indirectly stimulate vascular smooth muscle cell (VSMC) proliferation by regulating the expression of one or more autocrine growth factors. Using ion-exchange, gel-filtration, and reverse-phase chromatographic purification methods, we isolated a major mitogenic protein present in AII-stimulated rat aortic smooth muscle...

  10. Thrombin and Its Receptor Enhance ST-Segment Elevation in Acute Myocardial Infarction by Activating the KATP Channel

    OpenAIRE

    Long, Ming; Yang, Lei; Huang, Genya; LIU, LIPING; Dong, Yugang; Du, Zhimin; Tang, Anli; Hu, Chenghen; Gu, Ruimin; Gao, Xiuren; Tang, Lilong

    2010-01-01

    ST-segment elevation is the major clinical criterion for committing patients with chest pain to have emergent coronary revascularizations; however, the mechanism responsible for ST-segment elevation is unknown. In a guinea pig model of ST-segment elevation acute myocardial infarction (AMI), local application of hirudin, a thrombin antagonist, significantly decreased AMI-induced ST-segment elevation in a dose-dependent manner. Hirudin-induced (5 antithrombin units [ATU]) decrease in ST elevati...

  11. Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation: Receptor Activation Mediates Extravillous Trophoblast Invasion in Vitro

    OpenAIRE

    Peter J O’Brien; Koi, Hideki; PARRY, Samuel; Brass, Lawrence F; Strauss, Jerome F; Wang, Li-Peng; John E. Tomaszewski; Christenson, Lane K.

    2003-01-01

    Proteolysis of the thrombin receptor, protease activated receptor-1 (PAR1), may enhance normal and pathological cellular invasion, and indirect evidence suggests that activation of PAR1 expressed by invasive extravillous trophoblasts (EVTs) influences human placentation. Here we describe PAR1, PAR2, and PAR3 protein distribution in the developing human placenta and implicate PAR1 and PAR2 activation in functions central to EVT invasion. PAR1, PAR2, and PAR3 are expressed in cultured 8- to 13-...

  12. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

    OpenAIRE

    Chen, Y; Grall, D; Salcini, A E; Pelicci, P.G. (Pier G.); Pouysségur, J; Van Obberghen-Schilling, E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we r...

  13. Effects of thrombin inhibition with melagatran on renal hemodynamics and function and liver integrity during early endotoxemia

    DEFF Research Database (Denmark)

    Nitescu, Nicoletta; Grimberg, Elisabeth; Ricksten, Sven-Erik; Marcussen, Niels; Nordlinder, Hans; Guron, Gregor

    2007-01-01

    Sepsis is associated with an activation of the coagulation system and multiorgan failure. The aim of the study was to examine the effects of selective thrombin inhibition with melagatran on renal hemodynamics and function, and liver integrity, during early endotoxemia. Endotoxemia was induced in thiobutabarbital-anesthetized rats by an intravenous bolus dose of lipopolysaccharide (LPS; 6 mg/kg). Sham-Saline, LPS-Saline, and LPS-Melagatran study groups received isotonic saline or melagatran immed...

  14. 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.

  15. Topical Acne Treatments and Pregnancy

    Science.gov (United States)

    Topical Acne Treatments and Pregnancy In every pregnancy, a woman starts out with a 3-5% chance of having ... from your health care provider. What are topical acne treatments? Topical acne treatments are medications applied directly ...

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

    Directory of Open Access Journals (Sweden)

    Dasnan Ismail

    2002-06-01

    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

  17. 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.

  18. 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.

  19. G-protein coupled and tyrosine kinase receptors: evidence that activation of the insulin-like growth factor I receptor is required for thrombin-induced mitogenesis of rat aortic smooth muscle cells.

    OpenAIRE

    Delafontaine, P; Anwar, A.; Lou, H.; Ku, L

    1996-01-01

    IGF I is an ubiquitous peptide that activates a membrane tyrosine kinase receptor and has autocrine/paracrine effects on vascular smooth muscle cells. Thrombin activates a G-protein coupled receptor and is also a mitogen for vascular smooth muscle cells. To assess the potential role of IGF I as a mediator of thrombin's effects, we characterized expression of IGF I and of its receptor on vascular smooth muscle cells exposed to thrombin. Thrombin dose-dependently decreased IGF I mRNA levels and...

  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. Characters and Topical Diversity

    DEFF Research Database (Denmark)

    Eriksson, Rune

    2014-01-01

    The purpose of this article is to contribute to our understanding of the difference between the bestseller and the non-bestseller in nonfiction. It is noticed that many bestsellers in nonfiction belongs to the sub-genre of creative nonfiction, but also that the topics in this kind of literature is largely ignored by the critics. Thus, the article tests how topics may work in creative nonfiction. Two Danish bestsellers belonging to the genre, Frank’s Mit smukke genom ( My Beautiful Genome), about...

  2. Affinity labeling of lysine-149 in the anion-binding exosite of human ?-thrombin with an N?-(dinitrofluorobenzyl)hirudin C-terminal peptide

    International Nuclear Information System (INIS)

    In order to define structural regions in thrombin that interact with hirudin, the N?-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin [GDFEEIPEEY(O35SO3)L (DNFB-[35S]Hir54-64)]. DNFB-[35S]Hir54-64 was reacted at a 10-fold molar excess with human ?-thrombin in phosphate-buffered saline at pH 7.4 and 23 degree C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single 35S-labeled band of Mr ?32,500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at Mr ?31,000. Incorporation of the 35S affinity reagent peptide was found markedly reduced when reaction with thrombin was performed in the presence of 5- and 20-fold molar excesses of unlabeled hirudin peptide, showing that a specific site was involved in complex formation. The human ?-thrombin-DNFB-Hir54-64 complex was reduced, S-carboxymethylated, and treated with pepsin. Peptic fragments were separated by reverse-phase HPLC revealing two major peaks containing absorbance at 310 nm. Automated Edman degradation of the peptide fragments allowed identification of Lys-149 of human thrombin as the major site of DNFB-Hir54-64 derivatization. These data suggest that the anionic C-terminal tail of hirudin interacts with an anion-binding exosite in human thrombin removed 18-20 angstrom from the catalytic apparatus

  3. 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.

    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 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.

  4. 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

  5. DIRECT SMEAR VS CELL BLOCK (PLASMA- THROMBIN CLOT METHOD: DIAGNOSTIC VALUE IN SEROSAL CAVITIES FLUIDS CYTOLOGY

    Directory of Open Access Journals (Sweden)

    P MAHZOUNI

    2000-03-01

    Full Text Available Introduction. To improve testing sensitivity, most laboratories use two or more preparation methods but in our laboratories only one method is used which is "direct smear". In this study we tried to evaluate the diagnostic value of cell block as adjunct to direct smear in the cytologic investigation of serosal cavities fluids. Methods. In a clinical trial study 62 specimens of serosal cavity fluids were investigated in AL-Zahrapathology laboratory (Get. 1998 to Get. 1999. Cytologic slides from each specimens were prepared in two methods: direct smear and cell block (plasma- thrombin clot method. Smears and cell blocks were studied separately by the same cytopathologist. The diagnosis were categorized as positive, negative, suspicious or unsatisfactory. Also, the time required for studing of each slides were noted. Findings. The findings indicated that there are discrepancy between direct smear and cell block methods in the number of "suspicious" cases. Also there is significant difference between the mean time needed for studing of direct smear and cell block. Conclusion. It is recommended that the remainer of each specimen should be kept in refrigerator in order to prepare cell blocks in suspicious cases of direct smear. This method facilitates making a more definite diagnosis and reducing the number of suspicious cases.

  6. Peptide affinity labels for thrombin and other trypsin-like proteases

    Science.gov (United States)

    Shaw, Elliott N. (Shoreham, NY); Kettner, Charles A. (Yaphank, NY)

    1982-03-09

    A peptide affinity label of the formula (I): ##STR1## wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C.sub.1 -C.sub.4 alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C.sub.1 -C.sub.6 acyl, and Q--(A)--.sub.n, wherein Q=hydrogen, aroyl, or C.sub.1 -C.sub.6 acyl, n=1-10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereof-containing, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH.sub.2 --, --CH.sub.2 --CH.sub.2 --,--CH.sub.2 --CH.sub.2 --CH.sub.2 --, --CH.dbd.CH-- and --CH(OH)--CH.sub.2. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent.

  7. Caspases and Thrombin Activity Regulation by Specific Serpin Inhibitors in Bovine Skeletal Muscle.

    Science.gov (United States)

    Gagaoua, Mohammed; Hafid, Kahina; Boudida, Yasmine; Becila, Samira; Ouali, Ahmed; Picard, Brigitte; Boudjellal, Abdelghani; Sentandreu, Miguel Angel

    2015-09-01

    In living cells, after activation, protein inhibitors constitute the last step of proteases activity regulation. This review intends to provide original information about a group of bovine muscle serine proteases inhibitors belonging to the Serpin superfamily and characterized at the gene and protein level. This report is the only one and the first to provide much information on this group of proteases inhibitors of the serpin type and their potential biological functions. Amongst the eight genes identified in bovine, three serpins were purified from the muscle tissue and characterized. These are two members of the bovSERPINA3 family, i.e., bovSERPINA3-1 and A3-3, and the last one is antithrombin III (AT-III or BovSERPINC1). BovSERPINA3 family comprises at least eight protein members encoded by different genes mapped on chromosome 7q23-q26 cluster. BovSERPINA3-1 and A3-3 were shown to locate within muscle cells and are cross-class inhibitors strongly active against trypsin as well as against human initiator and effector caspases 8 and 3. They constitute a key apoptosis control in mammals. They were thus expressed in proliferating and confluent myoblasts phases where cells must be alive but not in myotubes. Antithrombin III inhibits trypsin and, in a heparin dependent manner, thrombin. AT-III and its mRNA were expressed in muscle cells and in differentiating primary myoblasts in culture. PMID:26208691

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

    Directory of Open Access Journals (Sweden)

    Ornella Piazza

    2010-02-01

    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.

  9. Effect of ethanol on thrombin-induced platelet phospholipid breakdown and release of (TH)-5-hydroxytryptamine

    Energy Technology Data Exchange (ETDEWEB)

    Fenn, G.C.; Caberos, L.P.; Littleton, J.M.

    1985-01-01

    Ethanol has been reported previously to inhibit chemically-induced platelet aggregation and the release of platelet contents. In platelet suspensions the mechanical stimulus of stirring can induce slow aggregation and the loss of endogenous arachidonic acid from phospholipids by activation of platelet phospholipases. These changes are prevented by the presence of ethanol 20-100 mM, whereas, in unstirred suspensions, ethanol alone has no effect on platelet phospholipids. Under similar conditions of reduced platelet: platelet contact, chemical stimuli, such as thrombi, although unable to produce visible aggregation, still cause the release of (TH)-5-hydroxytryptamine from platelets and also initiate the breakdown of platelet phospholipids. Ethanol does not now inhibit the thrombin-induced release of platelet contents and has little effect on phosphatidylinositol breakdown, though it inhibits phosphatidylcholine breakdown. Ethanol may therefore inhibit platelet aggregation by reducing the effect of mechanical and chemical stimuli on the activation of phospholipase A2. In contrast ethanol has rather little effect on the receptor-mediated breakdown of phosphatidylinositol which is apparently sufficient to trigger the release of platelet contents.

  10. 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.

  11. Discovering Health Topics in Social Media Using Topic Models

    OpenAIRE

    Paul, Michael J.; Dredze, Mark

    2014-01-01

    By aggregating self-reported health statuses across millions of users, we seek to characterize the variety of health information discussed in Twitter. We describe a topic modeling framework for discovering health topics in Twitter, a social media website. This is an exploratory approach with the goal of understanding what health topics are commonly discussed in social media. This paper describes in detail a statistical topic model created for this purpose, the Ailment Topic Aspect Model (ATAM...

  12. Advanced Topics in Aerodynamics

    DEFF Research Database (Denmark)

    Filippone, Antonino

    1999-01-01

    "Advanced Topics in Aerodynamics" is a comprehensive electronic guide to aerodynamics,computational fluid dynamics, aeronautics, aerospace propulsion systems, design and relatedtechnology. We report data, tables, graphics, sketches,examples, results, photos, technical andscientific literature, for higher education, learning, reference, research and engineering services.

  13. Topical immunomodulators in dermatology

    Directory of Open Access Journals (Sweden)

    Khandpur Sujay

    2004-04-01

    Full Text Available Topical immunomodulators are agents that regulate the local immune response of the skin. They are now emerging as the therapy of choice for several immune-mediated dermatoses such as atopic dermatitis, contact allergic dermatitis, alopecia areata, psoriasis, vitiligo, connective tissue disorders such as morphea and lupus erythematosus, disorders of keratinization and several benign and malignant skin tumours, because of their comparable efficacy, ease of application and greater safety than their systemic counterparts. They can be used on a domiciliary basis for longer periods without aggressive monitoring. In this article, we have discussed the mechanism of action, common indications and side-effects of the commonly used topical immunomodulators, excluding topical steroids. Moreover, newer agents, which are still in the experimental stages, have also been described. A MEDLINE search was undertaken using the key words "topical immunomodulators, dermatology" and related articles were also searched. In addition, a manual search for many Indian articles, which are not indexed, was also carried out. Wherever possible, the full article was reviewed. If the full article could not be traced, the abstract was used.

  14. Selected topics in magnetism

    CERN Document Server

    Gupta, L C

    1993-01-01

    Part of the ""Frontiers in Solid State Sciences"" series, this volume presents essays on such topics as spin fluctuations in Heisenberg magnets, quenching of spin fluctuations by high magnetic fields, and kondo effect and heavy fermions in rare earths amongst others.

  15. Hadron Correlations and Parton Recombination

    OpenAIRE

    Fries, Rainer J.(Cyclotron Institute, Department of Physics & Astronomy, Texas A&M University, College Station, TX, 77843-3366, USA)

    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.

  16. Challenges of the management of severe hemophilia A with inhibitors: two case reports emphasizing the potential interest of a high-purity human Factor VIII/von Willebrand factor concentrate and individually tailored prophylaxis guided by thrombin-generation test.

    Science.gov (United States)

    Mathieu, Sophie; Crampe, Carine; Dargaud, Yesim; Lavigne-Lissalde, Géraldine; Escuriola-Ettingshausen, Carmen; Tardy, Brigitte; Meley, Roland; Thouvenin, Sandrine; Stephan, Jean L; Berger, Claire

    2015-12-01

    Severe hemophilia A is an X-linked bleeding disorder. Immune tolerance induction (ITI) is the best strategy of treatment when patients develop inhibitors. The objective is to illustrate the benefit of a high-purity human factor VIII/von Willebrand factor (VWF) concentrate (Octanate) in the management of ITI. We also wanted to raise the potential interest of laboratory assays such as thrombin-generation test (TGT) and epitope mapping. Two patients were treated during ITI, first with a recombinant FVIII and then with plasma-derived factor VIII without success, and, finally, with Octanate. Bypassing agents were used based on the results of TGT. Epitope mapping was performed during ITI therapy. These observations suggest the potential contribution of Octanate in the management of ITI in difficult cases. The use of bypassing agents can be necessary in prophylaxis or to treat bleedings, and may be guided by TGT results. Epitope mapping is used to describe the inhibitor. This article shows a decrease of the inhibitor directed against the C2 domain after initiation of Octanate. A high-purity human factor VIII/von Willebrand factor concentrate (Octanate) may be a valuable therapeutical option for ITI therapy. TGT and epitope mapping could be of help in the management of ITI. PMID:26517064

  17. 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 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)

  18. A 2D-DIGE-based proteomic analysis reveals differences in the platelet releasate composition when comparing thrombin and collagen stimulations

    Science.gov (United States)

    Vélez, Paula; Izquierdo, Irene; Rosa, Isaac; García, Ángel

    2015-01-01

    Upon stimulation, platelets release a high number of proteins (the releasate). There are clear indications that these proteins are involved in the pathogenesis of several diseases, such as atherosclerosis. In the present study we compared the platelet releasate following platelet activation with two major endogenous agonists: thrombin and collagen. Proteome analysis was based on 2D-DIGE and LC-MS/MS. Firstly, we showed the primary role of thrombin and collagen receptors in platelet secretion by these agonists; moreover, we demonstrated that GPVI is the primary responsible for collagen-induced platelet activation/aggregation. Proteomic analysis allowed the detection of 122 protein spots differentially regulated between both conditions. After excluding fibrinogen spots, down-regulated in the releasate of thrombin-activated platelets, 84 differences remained. From those, we successfully identified 42, corresponding to 37 open-reading frames. Many of the differences identified correspond to post-translational modifications, primarily, proteolysis induced by thrombin. Among others, we show vitamin K-dependent protein S, an anticoagulant plasma protein, is up-regulated in thrombin samples. Our results could have pathological implications given that platelets might be playing a differential role in various diseases and biological processes through the secretion of different subsets of granule proteins and microvesicles following a predominant activation of certain receptors. PMID:25645904

  19. The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.E.; Yan Zhu; O' Neill, S. (Harvard Medical School, Boston, MA (United States))

    1991-12-15

    Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolated and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.

  20. Topical photodynamic therapy

    OpenAIRE

    Polja?ki Mirjana; Jovanovi? Marina; Matovi? Ljubinka; Lugonja Branislava; Gaji? Branislava; Roš Tatjana

    2006-01-01

    Topical photodynamic therapy is a therapeutic modality in development, thus arises grate interest among dermatologists worldwide. It is an effective therapy for actinic keratosis, superficial BCC and Bowenos disease. Treatment efficacy, good cosmetics, low risk of skin cancer, low invasiveness, low rate of adverse events, facility for treating multiple or large lesions, especially in poor healing sites and, for penile, digital and facial involvement, low general toxicity and possibility of re...

  1. Topics on mathematical crystallography

    OpenAIRE

    Sunada, Toshikazu

    2014-01-01

    In July 2012 the General Assembly of the United Nations resolved that 2014 should be the International Year of Crystallography, 100 years since the award of the Nobel Prize for the discovery of X-ray diffraction by crystals. On this special occasion, we address several topics in mathematical crystallography. Especially motivated by the recent development in systematic design of crystal structures by both mathematicians and crystallographers, we discuss interesting relationsh...

  2. Topics in volatility models

    OpenAIRE

    Yi, Cong

    2010-01-01

    In this thesis I will present my PhD research work, focusing mainly on financial modelling of asset’s volatility and the pricing of contingent claims (financial derivatives), which consists of four topics: 1. Several changing volatility models are introduced and the pricing of European options is derived under these models; 2. A general local stochastic volatility model with stochastic interest rates (IR) is studied in the modelling of foreign exchange (FX) rates. The pricin...

  3. Topics in industrial mathematics

    International Nuclear Information System (INIS)

    Mathematical methods are widely used to solve practical problems arising in modern industry. This article outlines some of the topics relevant to AECL programmes. This covers the applications of transmission and neutron transport tomography to determine density distributions in rocks and two phase flow situations. Another example covered is the use of variational methods to solve the problems of aerosol migration and control theory. (author). 7 refs

  4. Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network.

    Science.gov (United States)

    Miszta, Adam; Pelkmans, Leonie; Lindhout, Theo; Krishnamoorthy, Ganeshram; de Groot, Philip G; Hemker, Coenraad H; Heemskerk, Johan W M; Kelchtermans, Hilde; de Laat, Bas

    2014-12-26

    Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate. PMID:25381443

  5. 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.

  6. A sensitive electrochemical aptasensor for thrombin detection based on exonuclease-catalyzed target recycling and enzyme-catalysis.

    Science.gov (United States)

    Yi, Huayu; Xu, Wenju; Yuan, Yali; Wu, Yongmei; Chai, Yaqin; Yuan, Ruo

    2013-09-15

    In the present study, a sensitive electrochemical aptasensor based on exonuclease-catalyzed target recycling and enzyme-catalysis was developed for thrombin (TB) detection. Firstly, the alcohol dehydrogenase (ADH) was abundantly embedded in the 3-(mercaptopropyl)trimethoxysilane (MPTS) sol with a 3-D network that exhibited tunable porosity and high thermal stability. ADH, as an alcohol oxidase, catalyzed the conversation of alcohol into acetaldehyde coupling with the production of NADH in the presence of NAD(+). Then the immobilized gold nanoparticles (AuNPs) could electrocatalyze the oxidation of NADH, finally promoting the redox reaction of the electroactive material methylene blue (MB) labeled on the hybrid double strand DNA (dsDNA). Furthermore, when the mixture of TB and RecJf exonuclease was introduced, TB combined with the thrombin aptamer II (TBA II) and the aptamer-TB complex was formed. And then, the RecJf exonuclease selectively degraded the TBA II from 5'?3', releasing the target TB into the solution. The free TB was reused to combine with other TBA II to accomplish the target recycling and realize the electrochemical signal amplification. In this way, excellent sensitivity of the aptasensor was obtained. The thrombin aptasensor achieved a detection limit of 1.7pM (defined as S/N=3) with a linear range from 5pM to 100nM. In addition, the proposed aptasensor had good stability and sensitivity, and would become a promising choice for the protein diagnostics in clinical analysis. PMID:23603135

  7. 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

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

    DEFF Research Database (Denmark)

    Skov Madsen, P; Hokland, P; Hokland, M

    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 ...

  9. In vitro assessment of Tc-99m labeled bovine thrombin and streptokinase-activated human plasmin: concise communication

    International Nuclear Information System (INIS)

    Bovine thrombin and streptokinase-activated human plasmin have been labeled with Tc-99m using stannous reduction of pertechnetate under physiological conditions (pH 7.4). The binding efficiency of radiotechnetium to these enzymes is greater than 94%, with less than 5% of reduced but unbound Tc-99m (Sn) complex as assayed by ascending paper radiochromatography using ITLC silica gel plate. Free or unbound pertechnetate is less than 1%. In vitro enzymatic analyses of the Tc-99m-labeled enzymes demonstrate no evidence of protein denaturation or significant loss of enzymatic activity after labeling. Both labeled enzymes are biochemically active in vitro with their respective substrates

  10. Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

    Science.gov (United States)

    Zang, Xiaonan; Zhang, Xuecheng; Mu, Xiaosheng; Liu, Bin

    2013-03-01

    This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mg L-1) than that from soluble protein (1.964 mg L-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

  11. Iodine chemistry in hydrogen recombiners

    International Nuclear Information System (INIS)

    Hydrogen recombiners, recently introduced in the French nuclear reactor buildings, display high temperature (up to about 900 deg. C) and several thousands square meters of a very reactive surface when operating during a severe accident scenario. Small scale analytical experiments show that cesium and cadmium iodides are unstable, and generate volatile iodine, when heated in an oven that reproduces most physico-chemical parameters of recombiner operation. Based on these results, and due to its potential with regard to the environmental source term of a severe accident, iodine chemistry in hydrogen recombiners deserves close and careful scrutiny. (authors)

  12. Recombining WMAP: constraints on ionizing and resonance radiation at recombination

    OpenAIRE

    Bean, Rachel; Melchiorri, Alessandro; Silk, Joe

    2003-01-01

    We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent CMB temperature and polarization spectra coming from 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...

  13. 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

  14. Topic and Topic-Comment Constructions in Mandarin Chinese.

    Science.gov (United States)

    Shi, Dingxu

    2000-01-01

    Attempts to provide a precise definition for topic and to derive most of the properties of topic from this definition. The main assumption is that the topic-comment construction is a syntactic device employed to fulfill certain discourse functions. (Author/VWL)

  15. Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines.

    Science.gov (United States)

    Burgey, Christopher S; Robinson, Kyle A; Lyle, Terry A; Sanderson, Philip E J; Lewis, S Dale; Lucas, Bobby J; Krueger, Julie A; Singh, Rominder; Miller-Stein, Cynthia; White, Rebecca B; Wong, Bradley; Lyle, Elizabeth A; Williams, Peter D; Coburn, Craig A; Dorsey, Bruce D; Barrow, James C; Stranieri, Maria T; Holahan, Marie A; Sitko, Gary R; Cook, Jacquelynn J; McMasters, Daniel R; McDonough, Colleen M; Sanders, William M; Wallace, Audrey A; Clayton, Franklin C; Bohn, Dennis; Leonard, Yvonne M; Detwiler, Theodore J; Lynch, Joseph J; Yan, Youwei; Chen, Zhongguo; Kuo, Lawrence; Gardell, Stephen J; Shafer, Jules A; Vacca, Joseph P

    2003-02-13

    Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives. PMID:12570369

  16. 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 tcu-PA/...

  17. Drugs and related topics.

    Science.gov (United States)

    Cereijo, Ines

    2006-01-01

    Drugs is a topic that was certainly an issue of discussion at this year's annual meeting. This Committee had the responsibility of organizing a half day symposium on "Pharmaceutical Authenticity and Safety" that took place September 12, 2005. This symposium aims at improving the critical points in the analytical pharmaceutical field related to traceability assessment, use of certified reference materials (CRMs), and proficiency testing implementation to get the highest quality of the obtained results. Recognized experts presented these topics. Also, other complementary subjects, such as the application of advanced analytical technologies to reach the authenticity and safety of the pharmaceutical drugs and drug products, their microbiological quality assessment, without disregarding an important topic such as sampling, was presented and discussed. The talks that were presented are the following: "Proficiency Testing as a Need in the Pharmaceutical Field," Arlene Fox (AOAC INTERNATIONAL, Gaithersburg, MD); "Implementation of Traceability in the Pharmaceutical Laboratory," Thomas Layloff, (Management Sciences for Health, Arlington, VA); "Harmonized Characteristics of Certified Reference Materials According to ISO Guides-Attractive also for Pharmaceutical Analysis," Hendrik Emons (Reference Materials, Unit Institute for Reference Materials and Measurements (IRMM), Joint Research Centre European Commission, (Geel, Belgium); "Importance of LC/MS/MS for the Fingerprinting of Pharmaceutical Drugs," Paul A. Steinberg, (Thermo Electron Corp., Woodstock, GA); "Process Analytical Technology (PAT) as a Way for Better Manufacturing and Quality Assurance," John F. Kauffman (Division of Pharmaceutical Analysis, U.S. Food and Drug Administration (St. Louis, MO); "Stability Testing for the Safety Assessment of Pharmaceuticals," Marta Vidal (Boeringher Ingelheim Argentina, Buenos Aires, Argentina); "Validation of Microbiological Methods for Sterile and Nonsterile Pharmaceutical Products," Michael Brodsky (Brodsky Consultants, Thornhill, ON, Canada); "The Relationship Between Pharmacopoeial Reference Standards and ISO REMCO Initiatives and Guides," Ronald G. Manning (United States Pharmacopoeia, Rockville, MD). PMID:16512255

  18. Topical Acne Treatments and Pregnancy

    Science.gov (United States)

    Topical Acne Treatments and Pregnancy In every pregnancy, a woman starts out with a 3-5% chance of having ... This sheet talks about whether exposure to topical acne treatments may increase the risk for birth defects ...

  19. Topics in field theory

    CERN Document Server

    Karpilovsky, G

    1989-01-01

    This monograph gives a systematic account of certain important topics pertaining to field theory, including the central ideas, basic results and fundamental methods.Avoiding excessive technical detail, the book is intended for the student who has completed the equivalent of a standard first-year graduate algebra course. Thus it is assumed that the reader is familiar with basic ring-theoretic and group-theoretic concepts. A chapter on algebraic preliminaries is included, as well as a fairly large bibliography of works which are either directly relevant to the text or offer supplementary material of interest.

  20. Topical Treatment in Rhinology

    Directory of Open Access Journals (Sweden)

    Anamaria Gocea

    2014-02-01

    Full Text Available Far from being exhaustive, this paper aims to review the most illustrative topical therapies in rhinology, to show their patterns of action and their most suggestive characteristics proven by clinical trials and meta-analyses as tools of evidence based medecine. We describe several therapeutic clases: decongestants, antihistaminics, anticholinergics, antibiotics, disinfectants, antimicotics, fitotherapeutics, vitamins, immunotherapy and compounds. Furthermore, the nose is increasingly being used for the delivery of other drugs, ranging from hormone replacement therapy and growth hormone to insulin and anti-migraine medication (sumatriptan. The ability to directly reach the neuronal tissue in the olfactory niche and hence the brain makes this a very attractive challenge.

  1. Topics in Operator Theory

    CERN Document Server

    Ball, Joseph A; Helton, JWilliam; Rodman, Leiba; Spitkovsky, Iiya

    2010-01-01

    This is the first volume of a collection of original and review articles on recent advances and new directions in a multifaceted and interconnected area of mathematics and its applications. It encompasses many topics in theoretical developments in operator theory and its diverse applications in applied mathematics, physics, engineering, and other disciplines. The purpose is to bring in one volume many important original results of cutting edge research as well as authoritative review of recent achievements, challenges, and future directions in the area of operator theory and its applications.

  2. Topics in String Unification

    CERN Document Server

    Ibáñez, L E

    1991-01-01

    I discuss several aspects of strings as unified theories. After recalling the difficulties of the simplest supersymmetric grand unification schemes I emphasize the distinct features of string unification. An important role in constraining the effective low energy physics from strings is played by $duality$ symmetries. The discussed topics include the unification of coupling constants (computation of $\\sin ^2\\theta _W$ and $\\alpha _s$ at the weak scale), supersymmetry breaking through gaugino condensation, and properties of the induced SUSY-breaking soft terms. I remark that departures from universality in the soft terms are (in contrast to the minimal SUSY model) generically expected.

  3. Topics in circular statistics

    CERN Document Server

    Jammalamadaka, S Rao

    2001-01-01

    This research monograph on circular data analysis covers some recent advances in the field, besides providing a brief introduction to, and a review of, existing methods and models. The primary focus is on recent research into topics such as change-point problems, predictive distributions, circular correlation and regression, etc. An important feature of this work is the S-plus subroutines provided for analyzing actual data sets. Coupled with the discussion of new theoretical research, the book should benefit both the researcher and the practitioner. Contents: Circular Probability Distributions

  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. Topical Therapy Primer for Nondermatologists.

    Science.gov (United States)

    Jewell, Jolene R; Myers, Sarah A

    2015-11-01

    Topical medications are the foundation upon which dermatologic care is built. The proper use of topical therapeutics requires consideration of the active ingredient, potency, vehicle, and medication quantity. This article provides a concise but non-comprehensive list of topical medications used for acne, rosacea, psoriasis, actinic keratoses, and non-melanoma skin cancers. Common treatment regimens and pitfalls in prescribing topicals are discussed via clinical vignettes. PMID:26476246

  6. 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...

  7. Rapid Purification of Recombinant Histones

    OpenAIRE

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Peter B. Becker; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method “rapid histone purification” (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the mos...

  8. Construction of Gene Targeting Vectors by Recombineering

    OpenAIRE

    Lee, Song-Choon; Wang, Wei; Liu, Pentao

    2009-01-01

    Recombineering is a technology that utilizes the efficient homologous recombination functions encoded by ? phage to manipulate DNA in E.coli. Construction of knockout vectors has been greatly facilitated by recombineering as it allows one to choose any genomic region to manipulate. We describe here an efficient recombineering-based protocol for making mouse conditional knockout targeting vectors.

  9. 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 the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  10. Topics on continua

    CERN Document Server

    Macias, Sergio

    2005-01-01

    Specialized as it might be, continuum theory is one of the most intriguing areas in mathematics. However, despite being popular journal fare, few books have thoroughly explored this interesting aspect of topology. In Topics on Continua, Sergio Macías, one of the field's leading scholars, presents four of his favorite continuum topics: inverse limits, Jones's set function T, homogenous continua, and n-fold hyperspaces, and in doing so, presents the most complete set of theorems and proofs ever contained in a single topology volume. Many of the results presented have previously appeared only in research papers, and some appear here for the first time. After building the requisite background and exploring the inverse limits of continua, the discussions focus on Professor Jones''s set function T and continua for which T is continuous. An introduction to topological groups and group actions lead to a proof of Effros''s Theorem, followed by a presentation of two decomposition theorems. The author then offers an...

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

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Wenqing; Zhao, Wei [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Wang, Xiaoping [National Conservation of Snake Island and Laotieshan Mountain, Dalian, Liaoning, 116041 (China); Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    2007-08-01

    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 (V{sub M}) was calculated to be 2.13 Å{sup 3} Da{sup ?1} with one molecule in the asymmetric unit.

  12. 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

  13. 3D photonic crystal-based biosensor functionalized with quantum dot-based aptamer for thrombine detection

    Science.gov (United States)

    Lim, Chae Young; Choi, Eunpyo; Park, Youngkyu; Park, Jungyul

    2013-05-01

    In this paper, we propose a new technique for protein detection by using the enhancement of intensity in quantum dots (Qdot) whose emission is guided by 3D photonic crystal (PC) structures. For easy to use, we design the emitted light from the sensor can be recovered, when the chemical antibody (aptamer) conjugated with guard DNA (g-DNA) labeled with a quencher (Black FQ) hybridizes with the target proteins. In detail, we synthesis a Qdot-aptamer complex and then immobilize these complex on the PC surfaces. Next, we perform the hybridization of the Qdot-aptamer complex with g-DNA labeled with the quencher. It induces the quenching effect of fluoresce intensity in the Qdot-aptamer. In presence of target protein (thrombin), the Qdot-aptamer complex prefers to form the thrombin-aptamer complex: this results in the release of Black FQ-g-DNA and the quenched light intensity recovers into the original high intensity with Qdot. The intensity recovery varies quantitatively according to the level of the target protein concentration. This proposed sensor shows much higher detection sensitivity than the general fluorescent detection mechanism, which is functionalized on the flat surfaces because of the light guiding effect from 3D photonic crystal structures.

  14. Recombination of H and He in Yang-Mills Gravity

    CERN Document Server

    Katz, Daniel

    2014-01-01

    We investigate some aspects of the thermal history of the early universe according to Yang-Mills Gravity (YMG); a gauge theory of gravity set in flat spacetime. Specifically, equations for the ionization fractions of hydrogen and singly ionized helium during the recombination epoch are deduced analytically and then solved numerically. By considering several approximations we find that the presence of primordial helium and its interaction with Lyman series photons has a much stronger effect on the overall free electron density in YMG than it does in the standard, General Relativity (GR) based, model. Compared to the standard model recombination happens over a much larger range of temperatures, although there is still a very sharp temperature of last scattering around 2000 K. Since the ionization history of the universe is not directly observable we discuss how one may use it to predict the CMB power spectrum and thus test YMG. This topic will be explored in detail in an upcoming paper.

  15. 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

  16. Superconcentration and related topics

    CERN Document Server

    Chatterjee, Sourav

    2014-01-01

    A certain curious feature of random objects, introduced by the author as “super concentration,” and two related topics, “chaos” and “multiple valleys,” are highlighted in this book. Although super concentration has established itself as a recognized feature in a number of areas of probability theory in the last twenty years (under a variety of names), the author was the first to discover and explore its connections with chaos and multiple valleys. He achieves a substantial degree of simplification and clarity in the presentation of these findings by using the spectral approach. Understanding the fluctuations of random objects is one of the major goals of probability theory and a whole subfield of probability and analysis, called concentration of measure, is devoted to understanding these fluctuations. This subfield offers a range of tools for computing upper bounds on the orders of fluctuations of very complicated random variables. Usually, concentration of measure is useful when more direct prob...

  17. 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

  18. 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 * Salivary protein * Anti-thrombin * Anophelin * Hematophagy * Post-transcriptional regulation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2012

  19. Online transient isotachophoresis concentration by the pseudo-terminating electrolyte buffer for the separation of DNA-aptamer and its thrombin complex in poly(methyl methacrylate) microchip.

    Science.gov (United States)

    Wang, Jun; Zhang, Yong; Okamoto, Yukihiro; Kaji, Noritada; Tokeshi, Manabu; Baba, Yoshinobu

    2011-03-21

    Online automatic transient isotachophoresis concentration of DNA-aptamer and its thrombin complex by using one kind of pseudo-terminating electrolyte buffer in a cross-channel poly(methyl methacrylate) microchip is reported. Sample injection, transient concentration and separation were done continuously and controlled by a sequential voltage switching program, time-consuming steps and complicated chip design were not required. Peak resolution between DNA-aptamer and its thrombin complex was influenced by this novel pseudo-terminating electrolyte buffer, which was prepared by the addition of chemical component with slow mobility into the same buffer as leading electrolyte buffer. 1100-fold signal enhancement of thrombin complex was achieved by this transient isotachophoresis on a standard cross-form microchip. The concentration effect or standing time of transient isotachophoresis was proved to be influenced by the concentration of leading electrolyte ion and the concentration of pseudo-terminating electrolyte buffer ion (glycine). The transient concentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution for the size-based separation. The detection limit, taken as the lowest thrombin concentration at threefold S/N, was determined to be 0.5 amol in mass by this method. PMID:21270992

  20. Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides

    International Nuclear Information System (INIS)

    The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP

  1. 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...

  2. 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

  3. 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

  4. Topical Corticosteroid Addiction and Phobia

    OpenAIRE

    Ghosh, Aparajita; Sengupta, Sujata; Coondoo, Arijit; Jana, Amlan Kusum

    2014-01-01

    Corticosteroids, one of the most widely prescribed topical drugs, have been used for about six decades till date. However, rampant misuse and abuse down the years has given the drug a bad name. Topical steroid abuse may lead to two major problems which lie at the opposing ends of the psychosomatic spectrum. Topical steroid addiction, a phenomenon that came to be recognized about a decade after the introduction of the molecule is manifested as psychological distress and rebound phenomenon on s...

  5. 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

  6. Advanced verification topics

    CERN Document Server

    Bhattacharya, Bishnupriya; Hall, Gary; Heaton, Nick; Kashai, Yaron; Khan Neyaz; Kirshenbaum, Zeev; Shneydor, Efrat

    2011-01-01

    The Accellera Universal Verification Methodology (UVM) standard is architected to scale, but verification is growing and in more than just the digital design dimension. It is growing in the SoC dimension to include low-power and mixed-signal and the system integration dimension to include multi-language support and acceleration. These items and others all contribute to the quality of the SOC so the Metric-Driven Verification (MDV) methodology is needed to unify it all into a coherent verification plan. This book is for verification engineers and managers familiar with the UVM and the benefits it brings to digital verification but who also need to tackle specialized tasks. It is also written for the SoC project manager that is tasked with building an efficient worldwide team. While the task continues to become more complex, Advanced Verification Topics describes methodologies outside of the Accellera UVM standard, but that build on it, to provide a way for SoC teams to stay productive and profitable.

  7. Topics in statistical mechanics

    International Nuclear Information System (INIS)

    This thesis deals with four independent topics in statistical mechanics: (1) the dimer problem is solved exactly for a hexagonal lattice with general boundary using a known generating function from the theory of partitions. It is shown that the leading term in the entropy depends on the shape of the boundary; (2) continuum models of percolation and self-avoiding walks are introduced with the property that their series expansions are sums over linear graphs with intrinsic combinatorial weights and explicit dimension dependence; (3) a constrained SOS model is used to describe the edge of a simple cubic crystal. Low and high temperature results are derived as well as the detailed behavior near the crystal facet; (4) the microscopic model of the lambda-transition involving atomic permutation cycles is reexamined. In particular, a new derivation of the two-component field theory model of the critical behavior is presented. Results for a lattice model originally proposed by Kikuchi are extended with a high temperature series expansion and Monte Carlo simulation. 30 references

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

    International Nuclear Information System (INIS)

    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?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)

  9. 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)

  10. A multifunctional hemin@metal-organic framework and its application to construct an electrochemical aptasensor for thrombin detection

    Science.gov (United States)

    Xie, Shunbi; Ye, Jiawei; Yuan, Yali; Chai, Yaqin; Yuan, Ruo

    2015-10-01

    A new type of multifunctional metal-organic framework (MOF) has been synthesized by encapsulating hemin into the nano-sized Fe-MIL-88 MOFs (hemin@MOFs) and first applied in an electrochemical aptasensor to detect thrombin (TB) with the aid of an enzyme for signal amplification. The gold nanoparticle functionalized hemin@MOFs (Au/hemin@MOFs) have not only simultaneously served as redox mediators and solid electrocatalysts, but have also been utilized as an ideal loading platform to immobilize a large number of biomolecules. In this aptasensor, Au/hemin@MOFs conjugated with glucose oxidase (GOD) and thrombin binding aptamer (TBA II) were used as the secondary aptamer bioconjugates (Au/hemin@MOF-TBA II-GOD bioconjugates), and TB was sandwiched between Au/hemin@MOF-TBA II-GOD bioconjugates and the amino-terminated TBA I which was self-assembled on the gold nanoparticle (AuNP) modified electrode. The GOD could oxidize glucose into gluconic acid accompanied by the generation of H2O2. The generated H2O2 on the electrode surface was further electrocatalyzed by hemin@MOFs to amplify the electrochemical signal of hemin contained in hemin@MOFs. Therefore, the synthesized hemin@MOFs represented a new paradigm for multifunctional materials since it combined three different functions including serving as catalysts, redox mediators and loading platforms within a single material. With such an ingenious design, a wide linear range of 0.0001 nM to 30 nM was acquired with a relatively low detection limit of 0.068 pM for TB detection.A new type of multifunctional metal-organic framework (MOF) has been synthesized by encapsulating hemin into the nano-sized Fe-MIL-88 MOFs (hemin@MOFs) and first applied in an electrochemical aptasensor to detect thrombin (TB) with the aid of an enzyme for signal amplification. The gold nanoparticle functionalized hemin@MOFs (Au/hemin@MOFs) have not only simultaneously served as redox mediators and solid electrocatalysts, but have also been utilized as an ideal loading platform to immobilize a large number of biomolecules. In this aptasensor, Au/hemin@MOFs conjugated with glucose oxidase (GOD) and thrombin binding aptamer (TBA II) were used as the secondary aptamer bioconjugates (Au/hemin@MOF-TBA II-GOD bioconjugates), and TB was sandwiched between Au/hemin@MOF-TBA II-GOD bioconjugates and the amino-terminated TBA I which was self-assembled on the gold nanoparticle (AuNP) modified electrode. The GOD could oxidize glucose into gluconic acid accompanied by the generation of H2O2. The generated H2O2 on the electrode surface was further electrocatalyzed by hemin@MOFs to amplify the electrochemical signal of hemin contained in hemin@MOFs. Therefore, the synthesized hemin@MOFs represented a new paradigm for multifunctional materials since it combined three different functions including serving as catalysts, redox mediators and loading platforms within a single material. With such an ingenious design, a wide linear range of 0.0001 nM to 30 nM was acquired with a relatively low detection limit of 0.068 pM for TB detection. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04532k

  11. 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.

  12. KEGG PATHWAY / Homologous recombination [KEGG

    Lifescience Database Archive (English)

    Full Text Available PATHWAY: map03440 Entry map03440Pathway Name Homologous recombination Description Homologous rec ... drome mutated) are tumor suppressors that maintain genome ... integrity, at least in part, through HR. Class Gen ... 2MRX complex [PATH:map03440]M00295BRCA1-associated genome ... surveillance complex (BASC) [PATH:map03440] Diseas ...

  13. Improving recombinant protein purification yield

    Science.gov (United States)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  14. Recombination luminescence in rigid media

    International Nuclear Information System (INIS)

    When separated ion-pairs result from the ?-irradiation of pure or doped matrices or from solute photoionization, some of the photoejected electrons undergo spontaneous recombination, giving rise to the so-called isothermal luminescence (ITL). Cation-anion recombination takes place when molecular diffusion is possible; that is, it appears mostly in thermoluminescence (TL), upon warming irradiated samples. Electrons are photoextracted from matrix traps or from anions, and the neutralization luminescence is designated as stimulated luminescence (SL). ITL, TL, and SL all constitute very sensitive test for the presence of charged species. ITL decay kinetics throw some light on the recombination mechanism. In TL studies, the maxima of the glow curves correlate with phase transition temperatures (crystal ? crystal or glass ? crystal), providing insight into matrix molecular dynamics. From SL spectra, positive and negative charges can be discriminated, and various characteristics of the negative ions - electrons or anions - can be attained. The global SL irrespective of its spectrum composition, SL emission spectra, and SL excitation spectra or stimulation spectra are reviewed. The SL spectra, being specific of negative charged species, complement optical absorption spectroscopy when cations and anions have undistinguishable spectra. Even though radiative charge recombination constitutes the final step of ion-pair existence, it may serve to track back the successive stages of photoelectron life: electron ejection, matrix trapping or the attachments of electrons to molecules or radicals, and the release of trapped electrons. (Yamashita, S.)

  15. Recombination of Physical Unclonable Functions

    OpenAIRE

    Devadas, Srinivas; Yu, Meng-Day (Mandel)

    2010-01-01

    A new Physical Unclonable Function (PUF) construction is described, by treating silicon unique features extracted from PUF circuits as “genetic material” unique to each silicon, and recombining this chip-unique material in a way to obtain a combination of advantages not possible with the original PUF circuits, including altering PUF output statistics to better suit PUF-based key generation and authentication.

  16. The ?0 polarization and the recombination mechanism

    International Nuclear Information System (INIS)

    We use the recombination and the Thomas Precession Model to obtain a prediction for the ?0 polarization in the p+p ? ?0 + X reaction. We study the effect of the recombination function on the ?0 polarization. (author)

  17. Updated Topics in Healthcare Informatics

    OpenAIRE

    Takeda, Hiroshi

    2010-01-01

    This key note lecture introduces the role of IMIA, scope of healthcare informatics and some topics in healthcare informatics. Among updated topics, electronic patient record (EPR) and electronic health record (EHR) are featured. A new paradigm of clinical information systems, a document archiving and communication system (DACS) is also described and discussed.

  18. Linguistic Extensions of Topic Models

    Science.gov (United States)

    Boyd-Graber, Jordan

    2010-01-01

    Topic models like latent Dirichlet allocation (LDA) provide a framework for analyzing large datasets where observations are collected into groups. Although topic modeling has been fruitfully applied to problems social science, biology, and computer vision, it has been most widely used to model datasets where documents are modeled as exchangeable…

  19. 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 plasma conditions in which a population inversion between the energy levels of hydrogen can be generated. Population inversion is expected in plasmas for which three-body recombination makes a large contribution to the recombining processes and the effective recombination rate is larger than a critical value for a given electron density and temperature. Calculated results are presented in figures and tables. (author)

  20. Hot topic [editorial

    International Nuclear Information System (INIS)

    There is strong evidence for the human impact on climate change, but we should not ignore those who think otherwise. Unseasonably warm weather in many parts of Europe and North America last month will probably have added to the impression in many people's minds that climate change is a reality and that humans are guilty of warming our planet. The several hundred members of the United Nations' Intergovernmental Panel on Climate Change (IPCC) certainly think that the evidence for anthropogenic climate change is solid. Although Physics World was unable to obtain a copy of the IPCC's latest report on the science of climate change before its release date of 2 February - a clear sign of how sensitive its findings are - hints from those involved in writing the report suggest that the IPCC will have strengthened its conclusions, previously stated in 2001, that humans are heating up the Earth. While most scientists probably share this view, there are some who think otherwise. Many of those are either scientifically ill-informed or have dubious links with the energy industry. But some have genuine doubts. One bona fide sceptic is Richard Lindzen, a climate physicist from the Massachusetts Institute of Technology in the US, who was involved in preparing the IPCC's 2001 scientific report. While he does not dispute that the Earth is getting hotter, Lindzen thinks that, in all probability, the warming is largely the result of natural variations in the Earth's climate. Lindzen believes that climate models, although rooted in physics, contain far too many uncertainties to provide accurate forecasts. Indeed, mainstream climate physicists admit their computer models are far from perfect. Writing in their feature, for example, the chief scientist of the UK's Meteorological Office and colleagues describe how hard it is to incorporate the impact of clouds, which are much smaller than the resolution of the best models. They also warn that if clouds were modelled incorrectly, climate simulations 'would be seriously in error'. One may ask if this magazine should give space to Lindzen or those involved in geoengineering to air their views. Given the uncertainties still present within climate models and the potential costs of dealing with global warming, it would be wrong for Physics World to ignore those outside the mainstream. After all, as Richard Feynman once wrote: 'There is no harm in doubt and scepticism, for it is through these that new discoveries are made'. Physicists should never take anything at face value, not least a topic as important as climate change. (U.K.)

  1. 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)

  2. HOT TOPICS IN FOOD MICROBIOLOGY

    Science.gov (United States)

    Escherichia coli is a bacterium whose importance ranges from its role as a host for recombinant DNA manipulations to being one of the most well-recognized foodborne pathogens. Most E. coli strains are considered to be part of the normal microflora of the intestinal tract of humans and other warm-bl...

  3. Topic Model for Graph Mining.

    Science.gov (United States)

    Xuan, Junyu; Lu, Jie; Zhang, Guangquan; Luo, Xiangfeng

    2015-12-01

    Graph mining has been a popular research area because of its numerous application scenarios. Many unstructured and structured data can be represented as graphs, such as, documents, chemical molecular structures, and images. However, an issue in relation to current research on graphs is that they cannot adequately discover the topics hidden in graph-structured data which can be beneficial for both the unsupervised learning and supervised learning of the graphs. Although topic models have proved to be very successful in discovering latent topics, the standard topic models cannot be directly applied to graph-structured data due to the "bag-of-word" assumption. In this paper, an innovative graph topic model (GTM) is proposed to address this issue, which uses Bernoulli distributions to model the edges between nodes in a graph. It can, therefore, make the edges in a graph contribute to latent topic discovery and further improve the accuracy of the supervised and unsupervised learning of graphs. The experimental results on two different types of graph datasets show that the proposed GTM outperforms the latent Dirichlet allocation on classification by using the unveiled topics of these two models to represent graphs. PMID:25616091

  4. Topics in lightwave transmission systems

    CERN Document Server

    Li, Tingye

    1991-01-01

    Topics in Lightwave Transmission Systems is a second volume of a treatise on optical fiber communications that is devoted to the science, engineering, and application of information transmission via optical fibers. The first volume, published in 1985, dealt exclusively with fiber fabrication. The present volume contains topics that pertain to subsystems and systems. The book contains five chapters and begins with discussions of transmitters and receivers, which are basic to systems now operating in the field. Subsequent chapters cover topics relating to coherent systems: frequency and phase m

  5. Nondisjunction of chromosome 15: Origin and recombination

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. (Baylor College of Medicine, Houston, TX (United States)); Langlois, S. (Univ. of Britisch Columbia, Vancouver (Canada)); Morris, M.A.; Malcolm, S.

    1993-09-01

    Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N=27) and Angelman syndrome patients (N-5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, more paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. 33 refs., 1 fig., 7 tabs.

  6. Mechanisms of sister chromatid recombination

    International Nuclear Information System (INIS)

    Studies using T948 as a model system have been carried out aimed at elucidating the mechanism of sister chromatid recombination (SCR). Characterization of U.V. light- and x-ray-induced SCR, the relationiship between SCR induction and DNA repair using rad mutations, and the relationship between SCR induction and the time of cell division using cdc mutations are presented. It has been supposed that SCR is induced at the phase of S-G2 following DNA replication, that postreplication break of DNA strands is strongly involved in the induction of SCR, and that induction type of SCR, i.e., conversion type or recombination type, is dependent upon the type of molecular damage of DNA. (Namekawa, K.)

  7. Inhibited Recombination of Charged Magnetoexcitons

    OpenAIRE

    Okamura, H; Heiman, D; 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 exc...

  8. Recombinant bacteriophage lysins as antibacterials

    OpenAIRE

    Fenton, Mark; Ross, Paul; McAuliffe, Olivia; O'Mahony, Jim; Coffey, Aidan

    2010-01-01

    With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these e...

  9. Clindamycin and Benzoyl Peroxide Topical

    Science.gov (United States)

    The combination of clindamycin and benzoyl peroxide is used to treat acne. Clindamycin and benzoyl peroxide are in a class of medications called topical antibiotics. The combination of clindamycin and benzoyl peroxide works by killing the bacteria ...

  10. Two topics in quantum chromodynamics

    International Nuclear Information System (INIS)

    The two topics are (1) estimates of perturbation theory coefficients for R(e+e- ? hadrons), and (2) the virtual-photon structure function, with emphasis on the analytic behavior in its squared mass. 20 refs., 4 figs., 2 tabs

  11. Present topics of nuclear energy

    International Nuclear Information System (INIS)

    The report is discussing the topics: Reprocessing of spent fuel elements; Final storage of radioactive wastes; Effects of thermal power plants upon the climate; Safeguarding of nuclear facilities and fissionable materials; Properties and possibilities of plutonium. (orig./HP)

  12. Topics on the FORM software

    International Nuclear Information System (INIS)

    These notes studies the compilation with FORM software as applied to high energy physics, covering the following topics: Command structures, statistics and numbers, Dirac matrices, optimization control, Gamma matrices, errors and polynomial substitution

  13. 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...

  14. p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation.

    OpenAIRE

    Papkoff, J; Chen, R. H.; Blenis, J; Forsman, J.

    1994-01-01

    Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mito...

  15. 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

  16. Salmon and Human Thrombin Differentially Regulate Radicular Pain, Glial-Induced Inflammation and Spinal Neuronal Excitability through Protease-Activated Receptor-1

    OpenAIRE

    Smith, Jenell R.; Syre, Peter P.; Oake, Shaina A.; Nicholson, Kristen J.; Weisshaar, Christine L.; Cruz, Katrina; Bucki, Robert; Baumann, Bethany C.; Janmey, Paul A.; Winkelstein, Beth A.

    2013-01-01

    Chronic neck pain is a major problem with common causes including disc herniation and spondylosis that compress the spinal nerve roots. Cervical nerve root compression in the rat produces sustained behavioral hypersensitivity, due in part to the early upregulation of pro-inflammatory cytokines, the sustained hyperexcitability of neurons in the spinal cord and degeneration in the injured nerve root. Through its activation of the protease-activated receptor-1 (PAR1), mammalian thrombin can enha...

  17. Topics of Bioengineering in Wikipedia

    Directory of Open Access Journals (Sweden)

    Vassia Atanassova

    2009-10-01

    Full Text Available The present report aims to give a snapshot of how topics from the field of bioengineering (bioinformatics, bioprocess systems, biomedical engineering, biotechnology, etc. are currently covered in the free electronic encyclopedia Wikipedia. It also offers insights and information about what Wikipedia is, how it functions, how and when to cite Wikipedian articles, if necessary. Several external wikis, devoted to topics of bioengineering, are also listed and reviewed.

  18. Psoriasis: consensus on topical therapies

    DEFF Research Database (Denmark)

    van de Kerkhof, P C M; Barker, J; Griffiths, C E M; Kragballe, K; Mason, J; Menter, A; Papp, K

    2008-01-01

    Objective A consensus conference was convened to evaluate the topical treatment of psoriasis. Participants Members of the International Psoriasis Council (IPC) with broad clinical experience in the treatment of psoriasis and a specialist in meta- and pharmacoeconomic analyses were invited to participate on the consensus panel. Those accepting the invitation convened in Saariselkä, Finland. Evidence An advisory group on topical treatments was nominated by the organizing panel members. All partici...

  19. Key Topics in Sports Medicine

    OpenAIRE

    Amir Ali Narvani; Panagiotis Thomas; Burce Lynn

    2006-01-01

    Key Topics in Sports Medicine is a single quick reference source for sports and exercise medicine. It presents the essential information from across relevant topic areas, and includes both the core and emerging issues in this rapidly developing field. It covers: 1) Sports injuries, rehabilitation and injury prevention, 2) Exercise physiology, fitness testing and training, 3) Drugs in sport, 4) Exercise and health promotion, 5) Sport and exercise for special and clinical populations, 6) The ps...

  20. Pharmacogenetics of ophthalmic topical ?-blockers

    OpenAIRE

    Sidjanin, Duska J.; McCarty, Catherine A.; Patchett, Richard; Smith, Edward; Wilke, Russell A

    2008-01-01

    Glaucoma is the second leading cause of blindness worldwide. The primary glaucoma risk factor is elevated intraocular pressure. Topical ?-blockers are affordable and widely used to lower intraocular pressure. Genetic variability has been postulated to contribute to interpersonal differences in efficacy and safety of topical ?-blockers. This review summarizes clinically significant polymorphisms that have been identified in the ?-adrenergic receptors (ADRB1, ADRB2 and ADRB3). The implications ...

  1. Recombinant factor XIII and congenital factor XIII deficiency: an update from human and animal studies

    Directory of Open Access Journals (Sweden)

    Inbal A

    2013-10-01

    Full Text Available Aida InbalThrombosis and Hemostasis Unit, Hematology Institute, Beilinson Hospital, Rabin Medical Center, Petach Tikva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, IsraelAbstract: Factor XIII (FXIII is a protransglutaminase composed of two catalytic A subunits and two carrier B subunits. An intracellular form of FXIII is present in monocytes/macrophages and platelets as a homodimer of two A subunits. Following activation by thrombin, FXIII becomes plasma transglutaminase, which crosslinks ?-glutamyl-?-lysine residues of fibrin chains and thereby stabilizes the fibrin clot. FXIII deficiency results in a moderate to severe hemorrhagic disorder, abnormal wound healing in about 30% of patients, and recurrent abortion in homozygous females. More than 800 cases of FXIII deficiency have been reported, most of them due to mutation in the FXIII-A gene, resulting in FXIII-A deficiency. Among mutations causing FXIII-A deficiency, 50% are missense mutations. Only 16 mutations in the FXIII-B gene have been published. Routine laboratory tests are normal in patients with FXIII deficiency, and the diagnosis is established by demonstration of decreased FXIII activity and antigen. Plasma-derived, virus-inactivated factor XIII concentrate is the treatment of choice. The low plasma levels of FXIII (about 5% required to control bleeding and its long half-life make monthly prophylactic therapy feasible. Recently, recombinant FXIII concentrate with a half-life similar to that of native FXIII has been developed and tested in a multinational clinical study. This new product appears to be safe and appropriate for lifelong prophylactic treatment of patients with FXIII-A deficiency.Keywords: recombinant FXIII concentrate, FXIII deficiency

  2. Thrombin receptor activation elicits rapid protein tyrosine phosphorylation and stimulation of the raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligate autocrine mechanism promoting cell proliferation induced by G-protein-coupled receptor agonist.

    OpenAIRE

    Molloy, C J; Pawlowski, J E; Taylor, D S; Turner, C E; Weber, H; Peluso, M.

    1996-01-01

    Treatment of quiescent rat aortic smooth muscle cells with either alpha-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) resulted in pronounced increases in [3H]thymidine incorporation that were concentration dependent and reached a maximum of approximately 15-fold above serum-starved controls. However, in contrast to FBS, PDGF-BB, or basic fibroblast growth factor (bFGF), that initiated DNA synthesis promptly after 16-19 h, thymidine incorporation in response to thrombin was...

  3. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, ?, and N15, the integrase from the Streptomyces phage, ?C31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

  4. Effects of nuclear mutations for recombination and repair functions and of caffeine on mitochondrial recombination

    International Nuclear Information System (INIS)

    Studies of both prokaryotic and eukaryotic organisms indicate that pathways governing repair of damage to nuclear DNA caused by x-ray or ultraviolet irradiation overlap with those controlling recombination. Fourteen nuclear mutants of Saccharomyces cerevisiae were tested in order to determine whether these mutant genes affected mitochondrial recombination. None of the mutations studied significantly affected mitochondrial recombination. The nuclear recombination and repair pathways studied do not overlap with the nuclear pathway which controls recombination of mitochondrial DNA. A second set of experiments was designed to test the effect of caffeine on both nuclear and mitochondrial recombination in Saccharomyces cerevisiae. (U.S.)

  5. 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.

  6. mRNA expression of genes involved in inflammation and haemostasis in equine fibroblast-like synoviocytes following exposure 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.

  7. Topical corticosteroids: Abuse and Misuse

    Directory of Open Access Journals (Sweden)

    Yugandar Inakanti

    2015-04-01

    Full Text Available Background: The Topical Corticosteroids are among the most commonly prescribed medication in an out-patient dermatology setting since they were first introduced in early 1950s. Probably no other group of drugs has had such a profound impact on the specialty as Topical Corticosteroid. They provide rapid symptomatic relief in almost all inflammatory dermatoses, especially in the short term. Multiple pathways including rebound vasodilatation and proinflammatory cytokine release have been proposed as the mechanism for such reactions. Aim: To study various adverse effects of topical corticosteroids misuse over face. Materials and Methods: 130 patients with a history of topical corticosteroid use on face for minimum 1 month duration were included in this study. Results: Majority of patients were between age group of 21 to 30 (65.4%. Female sex preponderance over male sex with 67.8%. Majority of patients were House wives (49% followed by Employees (23%. Duration of application of TC was 3-6 months (77% in majority of cases. Most commonly abused TC was Betamethasone Valerate (79.2%. Conclusion: Topical Corticosteroid should not be used on the face unless it is under strict dermatological supervision.

  8. 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.

  9. Effect of ethanol on thrombin-induced platelet phospholipid breakdown and release of [3H]-5-hydroxytryptamine

    International Nuclear Information System (INIS)

    Ethanol has been reported previously to inhibit chemically-induced platelet aggregation and the release of platelet contents. In platelet suspensions the mechanical stimulus of stirring can induce slow aggregation and the loss of endogenous arachidonic acid from phospholipids by activation of platelet phospholipases. These changes are prevented by the presence of ethanol 20-100 mM, whereas, in unstirred suspensions, ethanol alone has no effect on platelet phospholipids. Under similar conditions of reduced platelet: platelet contact, chemical stimuli, such as thrombi, although unable to produce visible aggregation, still cause the release of [3H]-5-hydroxytryptamine from platelets and also initiate the breakdown of platelet phospholipids. Ethanol does not now inhibit the thrombin-induced release of platelet contents and has little effect on phosphatidylinositol breakdown, though it inhibits phosphatidylcholine breakdown. Ethanol may therefore inhibit platelet aggregation by reducing the effect of mechanical and chemical stimuli on the activation of phospholipase A2. In contrast ethanol has rather little effect on the receptor-mediated breakdown of phosphatidylinositol which is apparently sufficient to trigger the release of platelet contents

  10. Recombinant DNA production of spider silk proteins.

    Science.gov (United States)

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    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 recombinant spider silks. PMID:24119078

  11. Recombination activity enhancement by stress in silicon

    OpenAIRE

    Gundel, P.; Schubert, M.C.; Heinz, F.D.; Warta, W.

    2010-01-01

    The recombination activity of grain boundaries and precipitate colonies is analyzed with submicron spatial resolution and compared to the surrounding stress field. This analysis reveals a positive correlation between tensile stress and recombination activity and a negative correlation between compressive stress and recombination activity. This correlation can be explained by the stress induced mobility enhancement due to the strong piezoresistance of silicon. This observation could lead to a ...

  12. Topical corticosteroid addiction and phobia

    Directory of Open Access Journals (Sweden)

    Aparajita Ghosh

    2014-01-01

    Full Text Available Corticosteroids, one of the most widely prescribed topical drugs, have been used for about six decades till date. However, rampant misuse and abuse down the years has given the drug a bad name. Topical steroid abuse may lead to two major problems which lie at the opposing ends of the psychosomatic spectrum. Topical steroid addiction, a phenomenon that came to be recognized about a decade after the introduction of the molecule is manifested as psychological distress and rebound phenomenon on stoppage of the drug. The rebound phenomenon, which can affect various parts of the body particularly the face and the genitalia has been reported by various names in the literature. TC phobia which lies at the opposite end of the psychiatric spectrum of steroid abuse has been reported particularly among parents of atopic children. Management of both conditions is difficult and frustrating. Psychological counseling and support can be of immense help in both the conditions.

  13. Quantum mechanics II advanced topics

    CERN Document Server

    Rajasekar, S

    2015-01-01

    Quantum Mechanics II: Advanced Topics uses more than a decade of research and the authors’ own teaching experience to expound on some of the more advanced topics and current research in quantum mechanics. A follow-up to the authors introductory book Quantum Mechanics I: The Fundamentals, this book begins with a chapter on quantum field theory, and goes on to present basic principles, key features, and applications. It outlines recent quantum technologies and phenomena, and introduces growing topics of interest in quantum mechanics. The authors describe promising applications that include ghost imaging, detection of weak amplitude objects, entangled two-photon microscopy, detection of small displacements, lithography, metrology, and teleportation of optical images. They also present worked-out examples and provide numerous problems at the end of each chapter.

  14. Consequences of recombination on traditional phylogenetic analysis.

    DEFF Research Database (Denmark)

    Schierup, M H; Hein, J

    2000-01-01

    We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mtDNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination. With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may not be immediately detectable in a data set. The phylogenies when recombination is present superficially resemble phylogenies for sequences from an exponentially growing population. However, exponential growth has a different effect on statistics such as Tajima's D. Furthermore, ignoring recombination leads to a large overestimation of the substitution rate heterogeneity and the loss of the molecular clock. These results are discussed in relation to viral and mtDNA data sets. Udgivelsesdato: 2000-Oct

  15. Human Insulin from Recombinant DNA Technology

    Science.gov (United States)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  16. Selected topics in nuclear structure

    International Nuclear Information System (INIS)

    The Fourth International Conference on selected topics in nuclear structure was held at Dubna in July 1994 on recent experimental and theoretical investigations in nuclear structure. Topics discussed were the following: nuclear structure at low-energy excitations (collective quasiparticle phenomena, proton-neutron interactions, microscopic and phenomenological theories of nuclear structure; nuclear structure studies with charged particles. heavy ions, neutrons and photons; nuclei at high angular momenta and superdeformation, structure and decay properties of giant resonances, charge-exchange resonances and ?-decay; semiclassical approach of large amplitude collective motion and structure of hot nuclei

  17. Topics in millimeter wave technology

    CERN Document Server

    Button, Kenneth

    1988-01-01

    Topics in Millimeter Wave Technology, Volume 1 presents topics related to millimeter wave technology, including fin-lines and passive components realized in fin-lines, suspended striplines, suspended substrate microstrips, and modal power exchange in multimode fibers. A miniaturized monopulse assembly constructed in planar waveguide with multimode scalar horn feeds is also described. This volume is comprised of five chapters; the first of which deals with the analysis and synthesis techniques for fin-lines as well as the various passive components realized in fin-line. Tapers, discontinuities,

  18. Topics in M-theory

    International Nuclear Information System (INIS)

    We give a brief history of the passage from strings to branes and we review some aspects of the following topics in M-theory: (a) an extended brane scan, (b) superembedding approach to the dynamics of superbranes and (c) supermembranes in anti de Sitter space, singletons and massless higher spin field theories. (author)

  19. Selected topics in nuclear structure

    International Nuclear Information System (INIS)

    19. winter school in Zakopane was devoted to selected topics in nuclear structure such as: production of spin resonances, heavy ions reactions and their applications to the investigation of high spin states, octupole deformations, excited states and production of new elements etc. The experimental data are ofen compared with theoretical predictions. Report contains 28 papers. (M.F.W.)

  20. Two topics in quantum chromodynamics

    Energy Technology Data Exchange (ETDEWEB)

    Bjorken, J.D.

    1989-12-01

    The two topics are (1) estimates of perturbation theory coefficients for R(e{sup +}e{sup -} {yields} hadrons), and (2) the virtual-photon structure function, with emphasis on the analytic behavior in its squared mass. 20 refs., 4 figs., 2 tabs.

  1. Topics in optics and music

    Science.gov (United States)

    Sparks, Andrew W.

    2012-10-01

    While the use of optics in the playback of music has been a tremendously successful technology and laser light shows are a common occurrence, other intersections of optics and music tend to be less well known. Topics such as optics-based instruments, performance tools and effects, instrument characterization and manufacturing, recording, playback, and signal processing are explored.

  2. Topic Map for Authentic Travel

    OpenAIRE

    Wandsvik,Atle; Zare, Mehdi

    2007-01-01

    E-business is a new trend in Internet use. Authentic travel is an approach to travel and travel business which helps the traveler experience what is authentic in the travel destination. But how can the traveler find those small authentic spots and organize them together to compose a vacation? E-business techniques, combined withTopic Maps, can help.

  3. The pharmacology of topical analgesics.

    Science.gov (United States)

    Barkin, Robert L

    2013-07-01

    Pain management of patients continues to pose challenges to clinicians. Given the multiple dimensions of pain--whether acute or chronic, mild, moderate, or severe, nociceptive or neuropathic--a multimodal approach may be needed. Fortunately, clinicians have an array of nonpharmacologic and pharmacologic treatment choices; however, each modality must be chosen carefully, because some often used oral agents are associated with safety and tolerability issues that restrict their use in certain patients. In particular, orally administered nonsteroidal antiinflammatory drugs, opioids, antidepressants, and anticonvulsants are known to cause systemic adverse effects in some patients. To address this problem, a number of topical therapies in various therapeutic classes have been developed to reduce systemic exposure and minimize the risks of patients developing adverse events. For example, topical nonsteroidal anti-inflammatory drug formulations produce a site-specific effect (ie, cyclo-oxygenase inhibition) while decreasing the systemic exposure that may lead to undesired effects in patients. Similarly, derivatives of acetylsalicylic acid (ie, salicylates) are used in topical analgesic formulations that do not significantly enter the patient's systemic circulation. Salicylates, along with capsaicin, menthol, and camphor, compose the counterirritant class of topical analgesics, which produce analgesia by activating and then desensitizing epidermal nociceptors. Additionally, patches and creams that contain the local anesthetic lidocaine, alone or co-formulated with other local anesthetics, are also used to manage patients with select acute and chronic pain states. Perhaps the most common topical analgesic modality is the cautious application of cutaneous cold and heat. Such treatments may decrease pain not by reaching the target tissue through systemic distribution, but by acting more directly on the affected tissue. Despite the tolerability benefits associated with avoiding systemic circulation, topically applied analgesics are associated with application-site reactions in patients, such as dryness, erythema, burning, and discoloration. Furthermore, some adverse events that have been observed in patients may be suggestive of some degree of systemic exposure. This article reviews the mechanisms of action, pharmacokinetics, and tolerability of topical treatments for the management of patient pain. PMID:24547599

  4. 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.

  5. Recombinant Swinepox Virus for Veterinary Vaccine Development.

    Science.gov (United States)

    Fan, Hong-Jie; Lin, Hui-Xing

    2016-01-01

    Poxvirus-vectors have been widely used in vaccine development for several important human and animal diseases; some of these vaccines have been licensed and used extensively. Swinepox virus (SPV) is well suited to develop recombinant vaccines because of its large packaging capacity for recombinant DNA, its host range specificity, and its ability to induce appropriate immune responses. PMID:26458836

  6. Titania Photocatalysis beyond Recombination: A Critical Review

    OpenAIRE

    Bunsho Ohtani

    2013-01-01

    This short review paper shows the significance of recombination of a photoexcited electron and a hole in conduction and valence bands, respectively, of a titania photocatalyst, since recombination has not yet been fully understood and has not been evaluated adequately during the past several decades of research on heterogeneous photocatalysis.

  7. Titania Photocatalysis beyond Recombination: A Critical Review

    Directory of Open Access Journals (Sweden)

    Bunsho Ohtani

    2013-11-01

    Full Text Available This short review paper shows the significance of recombination of a photoexcited electron and a hole in conduction and valence bands, respectively, of a titania photocatalyst, since recombination has not yet been fully understood and has not been evaluated adequately during the past several decades of research on heterogeneous photocatalysis.

  8. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; 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.

  9. 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.

  10. Recombinant organisms for production of industrial products

    OpenAIRE

    Adrio, Jose-Luis; Arnold L. Demain

    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...

  11. Constraining topic maps : a TMCL declarative implementation

    OpenAIRE

    Ramalho, José Carlos; Librelotto, Giovani Rubert; Henriques, Pedro Rangel

    2005-01-01

    This paper describes the design of an XML language to formally specify constraints over Topic Maps (XTche). This language allows to express contextual conditions on classes of Topic Maps that are further processed by a XSLT based processor. With XTche, a topic map designer defines a set of restrictions that guarantee that a particular topic map is semantically valid. Topic Maps tend to grow quite fast. Most times the designer has some restrictions in mind like: what kind of ...

  12. Glycosylation of recombinant antibody therapeutics.

    Science.gov (United States)

    Jefferis, Royston

    2005-01-01

    The adaptive immune system has the capacity to produce antibodies with a virtually infinite repertoire of specificities. Recombinant antibodies specific for human targets are established in the clinic as therapeutics and represent a major new class of drug. Therapeutic efficacy depends on the formation of complexes with target molecules and subsequent activation of downstream biologic effector mechanisms that result in elimination of the target. The activation of effector mechanisms is dependent on structural characteristics of the antibody molecule that result from posttranslational modifications, in particular, glycosylation. The production of therapeutic antibody with a consistent human glycoform profile has been and remains a considerable challenge to the biopharmaceutical industry. Recent research has shown that individual glycoforms of antibody may provide optimal efficacy for selected outcomes. Thus a further challenge will be the production of a second generation of antibody therapeutics customized for their clinical indication. PMID:15903235

  13. Recombination at silicon dangling bonds

    International Nuclear Information System (INIS)

    In the past, pulsed electrically detected magnetic resonance experiments (pEDMR) with silicon dangling bonds (db) in hydrogenated microcrystalline silicon (?c-Si:H) showed that at low temperatures, two db recombination mechanisms exist where electrons are captured (i) by dbs directly (db-dc) or (ii) via band-tail states (tail-db). Here, similar experiments on hydrogenated amorphous silicon (a-Si:H) and crystalline silicon/silicondioxide interfaces (c-Si/SiO2) are presented. They show that at low temperatures, only the db-dc is detectable at dbs in the c-Si/SiO2 interface (Pb centers) while in a-Si:H, only tail-db processes are observed

  14. Staphylocidal action of thrombin-induced platelet microbicidal protein is influenced by microenvironment and target cell growth phase.

    Science.gov (United States)

    Koo, S P; Yeaman, M R; Bayer, A S

    1996-09-01

    Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic peptide released from rabbit platelets following exposure to thrombin in vitro. This peptide exerts potent in vitro microbicidal activity against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. It is known that the microbicidal actions of other cationic antimicrobial peptides (e.g., neutrophil defensins) are influenced by environmental factors and target cell growth phase. However, whether these parameters affect tPMP microbicidal activity has not been studied. Thus, we assessed the in vitro bactericidal activity of tPMP against two tPMP-susceptible strains, Bacillus subtilis ATCC 6633 and S. aureus 502A, in various target cell growth phases or under various microenvironmental conditions. The conditions studied included differing bacterial growth phase (logarithmic versus stationary), temperature (range, 4 to 42 degrees C), pH (range, 4.5 to 8.5), cationicity (range, 0.1 mM to 2 M), anionicity (range, 0.08 to 5 microM), and neutral carbohydrates ranging in molecular weight (MW) from 180 to 37,700 (range, 50 to 500 mM) as well as rabbit platelet-free plasma and serum. tPMP staphylocidal activity was greater against logarithmic- than stationary-phase cells. tPMP bactericidal activity against both B. subtilis and S. aureus was directly correlated with temperature and pH, with microbicidal activity exhibited near the physiological range (37 to 42 degrees C and pH 7.2 to 8.5, respectively). The presence of cations (Na+, K+, Ca2+, and Mg2+) decreased tPMP bactericidal activity in a time- and concentration-dependent manner, with complete inhibition at monovalent or divalent cation concentrations of > or = 250 or > or = 10 mM, respectively. Staphylocidal activity of tPMP was also inhibited by the polyanions polyanetholsulfonic acid and polyaspartic acid, at 0.1 and 0.4 microM, respectively. Coincident exposure with low-MW carbohydrates (glucose, sucrose, and melezitose) did not affect tPMP staphylocidal activity. However, higher-MW carbohydrates (raffinose and dextrans) decreased tPMP activity in a manner directly proportional to their concentration and MW. Solute-mediated inhibition of tPMP bactericidal activity was independent of solute osmolality but directly related to the duration of tPMP-solute coexposure. tPMP enhanced the staphylocidal activities of platelet-free plasma and heat-inactivated serum, while the activity of normal serum was not affected. These collective observations suggest that tPMP retains antimicrobial activities under physiological conditions which are likely to be relevant to host defense in vivo. PMID:8751926

  15. 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.

  16. 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)

  17. Amplified thrombin aptasensor based on alkaline phosphatase and hemin/G-quadruplex-catalyzed oxidation of 1-naphthol.

    Science.gov (United States)

    Yang, Zhe-Han; Zhuo, Ying; Yuan, Ruo; Chai, Ya-Qin

    2015-05-20

    An alkaline phosphatase (ALP)-based biosensor can in situ generate an electroactive product by enzymatic hydrolysis of inactive substrates. To obtain a higher signal-to-background ratio, a chemical redox cycling signal-amplified strategy based on the addition of a strong reducing agent has often be applied in the construction of ALP-based biosensors. However, the strong reducing agent not only affects the activity of ALP but also readily reacts with dissolved oxygen, leading to inaccurate results. In this work, a new signal-amplified strategy for a thrombin (TB) aptasensor based on the catalytic oxidation of ALP-generated products, 1-naphthol (NP), using hemin/G-quadruplex DNAzymes was reported. We implemented gold-nanoparticle-decorated zinc oxide nanoflowers (Au-ZnO) as the matrix for immobilizing ALP and TB aptamer (TBA) and then labeled it with hemin to form hemin/G-quadruplex/ALP/Au-ZnO bioconjugates (TBA II bioconjugates). Through a "sandwich" reaction, TBA II bioconjugates were captured on the electrode surface. The amplified signal was carried out in two steps: (i) an ALP-catalyzed inactive substrate, 1-naphthyl phosphate (NPP), in situ produces NP on the surface of the electrode; (ii) on the one hand, NP as a new reactant could be directly electrooxidized and generated an electrochemical signal, but, on the other hand, NP could be oxidized by hemin/G-quadruplex in the presence of H2O2, resulting in amplification of the electrochemical signal. The proposed TB aptasensor achieved a linear range of 1 pM to 30 nM with a detection limit of 0.37 pM (defined as S/N = 3). PMID:25907268

  18. 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.

  19. Is there any role of thrombin activatable fibrinolysis inhibitor in the development of a hypercoagulable state in gastric cancer

    Directory of Open Access Journals (Sweden)

    Eser Mehmet

    2012-08-01

    Full Text Available Abstract Background The purpose of this study was to investigate plasma levels of thrombin activatable fibrinolysis inhibitor (TAFI and TAFI’s relationship with coagulation markers (prothrombin fragment 1?+?2 in gastric cancer patients. Methods Thirty-three patients with gastric adenocarcinoma and 29 healthy control subjects were prospectively enrolled in the study. Patients who had a history of secondary malignancy, thrombosis related disease, oral contraceptive use, diabetes mellitus, chronic renal failure or similar chronic metabolic disease were excluded from the study. A fasting blood sample was drawn from patients to determine the plasma levels of TAFI and Prothrombin Fragment 1?+?2 (F 1?+?2. In addition, data on patient age, sex, body mass index (BMI and stage of disease were recorded. The same parameters, except stage of disease, were also recorded for the control group. Subsequently, we assessed the difference in the levels of TAFI and F 1?+?2 between the patient and control groups. Moreover, we investigated the relation of TAFI and F 1?+?2 levels with age, sex, BMI and stage of disease in the gastric cancer group. Results There were no statistical differences in any demographic variables (age, gender and BMI between the groups (Table 1. The mean plasma TAFI levels of the gastric cancer group (69.4?±?33.1 and control group (73.3?±?27.5 were statistically similar (P?=?0.62. The mean plasma F 1?+?2 level in the gastric cancer group was significantly higher than for those in the control group (549.7?±?325.3 vs 151.9?±?67.1, respectively; P? Conclusion In our study, TAFI levels of gastric cancer patients were similar to healthy subjects. The results of our study suggest that TAFI does not play a role in pathogenesis of the hypercoagulable state in gastric cancer patients.

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

    Directory of Open Access Journals (Sweden)

    Manal S. Fawzy

    2015-06-01

    Full Text Available 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.

  1. Thrombin-activatable fibrinolysis inhibitor activity and global fibrinolytic capacity in Type 1 diabetes: evidence for normal fibrinolytic state.

    Science.gov (United States)

    Harmanci, Ayla; Kandemir, Nurgun; Dagdelen, Selcuk; Gonc, Nazli; Buyukasik, Yahya; Alikasifoglu, Ayfer; Kirazli, Serafettin; Ozon, Alev; Gurlek, Alper

    2006-01-01

    Hypofibrinolysis is a state that is commonly observed in type 2 diabetic patients, a finding also possibly related to obesity and insulin resistance. There is little information, however, regarding the status of fibrinolytic system in Type 1 diabetes, in particular as reflected by thrombin-activatable fibrinolysis inhibitor (TAFI) activity and global fibrinolytic capacity (GFC). To provide information in this respect, 30 Type 1 diabetic patients (median age=16) and 28 healthy controls (median age=14) were enrolled in this study. The median duration of diabetes was 7 years, and median HbA(1c) was 8.85% (range: 5.5-11.9%) in the diabetic group. None of the patients had macrovascular complications. Microvascular complications were present in a total of eight patients (nephropathy: n=5; retinopathy: n=3). A comparison of the TAFI activity between the patient (median 84.9, range: 71.5-103.3%) and the control groups (median=83.3, range: 63.7-97.4%) yielded no statistically significant difference (P=.950). Similarly, GFC was comparable between the two groups (median=8.22, range: 0.72-22.38 microg/ml, and median=13.32, range: 3.0-23.22 microg/ml, respectively, in the diabetic and control groups, P=.086). TAFI activity did not significantly correlate with age, albumin excretion, fasting plasma glucose, HbA(1c), D-dimer, and fibrinogen by Spearman rank correlation test. There was as a significant inverse correlation between GFC and TAFI activity (r=-.414, P=.006). Contrary to the previous observations in Type 2 diabetes, our data suggest that fibrinolytic activity is not adversely affected by Type 1 diabetes, and it has no relationship with the degree of metabolic control. PMID:16389166

  2. Effective inhibition of thrombin by Rhipicephalus microplus serpin-15 (RmS-15) obtained in the yeast Pichia pastoris.

    Science.gov (United States)

    Xu, Tao; Lew-Tabor, Ala; Rodriguez-Valle, Manuel

    2016-02-01

    The cattle tick (Rhipicephalus microplus) affects cattle industries in tropical and subtropical countries because it is the vector of babesiosis and anaplasmosis which constitutes a threat to the health of cattle. During blooding feeding, ticks secrete saliva containing a complex of bioactive molecules into the injured site to evade host's defensive responses. Serine protease inhibitors (serpins) are important anti-haemostatic molecules present in tick saliva that are necessary for a successful blood feeding. Several serpin sequences have been reported in R. microplus but there is a gap of information about their functions during host-parasite interactions. In this study, the RmS-15 expressed in the yeast Pichia pastoris was characterised using kinetic assays and in vitro analysis. The inhibitory enzymatic assays conducted showed that RmS-15 is a physiological inhibitor of thrombin with a stoichiometric inhibition (SI) of 1.5 and high inhibition affinity with ka=9.3±0.5×104M(-1)s(-1). RmS-15 delayed the clotting of plasma in a dose-dependent manner as determined in a recalcification time assay. Significant elevated ELISA titres were observed in tick resistant and susceptible cattle on day 28 after the tick infestation (p<0.001). This data suggests direct contact of RmS-15 with the immune system of the host at the tick-feeding site. The present study contributed to the understanding of the biological functions of R. microplus serpins during host-parasite interactions which contributes to the design of future innovative methods for tick control. PMID:26530984

  3. Containment air circulation for optimal hydrogen recombination

    International Nuclear Information System (INIS)

    An accepted first-line defense for hydrogen mitigation is to design for the hydrogen to be rapidly mixed with the containment atmosphere and diluted to below flammability concentrations. Then, as hydrogen continues to be produced in the longer term, recombiners can be used to remove hydrogen: recombiners can be located in forced-air ducts or passive recombiners can be distributed within containment and the heat of recombination used to promote local air circulation. However, this principle does not eliminate the possibility of high hydrogen concentrations at locations removed from the recombiners. An improvement on this strategy is to arrange for a specific, buoyancy-driven, overall circulation of the containment atmosphere such that the recombiners can be located within the recirculation flow, immediately downstream of the hydrogen source. This would make the mixing process more predictable and solve the mass-transfer problem associated with distributed recombiners. Ideally, the recombiners would be located just above the hydrogen source so that the heat of recombination would assist the overall circulation. In this way, the hydrogen would be removed as close as possible to the source, thereby minimizing the amount of hydrogen immediately downstream of the source and reducing the hydrogen concentration to acceptable levels at other locations. Such a strategy requires the containment volume to be divided into an upflow path, past the hydrogen source and the recombiner, and a downflow path to complete the circuit. The flow could be generated actively using fans or passively using buoyancy forces arising from the difference in density of gases in the upfiow and downflow paths; the gases in the downflow path being cooled at an elevated heat sink. (author)

  4. Roles of thrombin and platelet membrane glycoprotein IIb/IIIa in platelet-subendothelial deposition after angioplasty in an ex vivo whole artery model

    International Nuclear Information System (INIS)

    Platelet deposition at the site of injury caused by balloon angioplasty is associated with acute closure and restenosis. In a new ex vivo whole artery angioplasty model, the authors examined the roles of thrombin inhibition with D-Phe-Pro-ArgCH2Cl (PPACK) and inhibition of the platelet membrane fibrinogen receptor glycoprotein IIb/IIIa (GPIIb/IIIa) with monoclonal antibody 7E3 on platelet deposition at the site of balloon injury. Fresh rabbit aortas were mounted in a perfusion chamber. One half of the mounted arterial segment was dilated with a standard angioplasty balloon catheter and the uninjured half served as the control segment. The vessels were perfused with human blood at physiological pressure and shear rates of 180-250 second-1 for 30 minutes. Platelet deposition was measured using 111In-labeled platelets and scanning electron microscopy. With heparin (2 units/ml) anticoagulation, 8.2 ± 2.2 x 10(6) platelets/cm2 were deposited at the site of balloon injury compared with 0.7 ± 0.2 x 10(6) platelets/cm2 on uninjured segments (p less than 0.02, n = 7). PPACK was tested at a concentration (10 microM) that totally inhibited platelet aggregation in response to thrombin. 7E3 was tested at a concentration (10 micrograms/ml) that totally inhibited platelet aggregation. Platelet deposition at the site of balloon injury was reduced 47% by PPACK and 70% by 7E3 compared with heparin. At shear rates seen in nonstenotic coronary arteries, PPACK and 7E3 are more effective than heparin in reducing platelet deposition at the site of balloon injury. The significant inhibition of platelet deposition by PPACK demonstrates the importance of heparin-resistant thrombin in platelet thrombus formation

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

    International Nuclear Information System (INIS)

    Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: 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

  6. 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

  7. 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.

  8. Topic Graph Generation for Query Navigation Use of Frequency Classes for Topic Extraction

    CERN Document Server

    Niwa, Y; Iwayama, M; Takano, A; Nitta, Y; Niwa, Yoshiki; Nishioka, Shingo; Iwayama, Makoto; Takano, Akihiko; Nitta, Yoshihiko

    1997-01-01

    To make an interactive guidance mechanism for document retrieval systems, we developed a user-interface which presents users the visualized map of topics at each stage of retrieval process. Topic words are automatically extracted by frequency analysis and the strength of the relationships between topic words is measured by their co-occurrence. A major factor affecting a user's impression of a given topic word graph is the balance between common topic words and specific topic words. By using frequency classes for topic word extraction, we made it possible to select well-balanced set of topic words, and to adjust the balance of common and specific topic words.

  9. Retapamulin: A newer topical antibiotic

    Directory of Open Access Journals (Sweden)

    D Dhingra

    2013-01-01

    Full Text Available Impetigo is a common childhood skin infection. There are reports of increasing drug resistance to the currently used topical antibiotics including fusidic acid and mupirocin. Retapamulin is a newer topical agent of pleuromutilin class approved by the Food and Drug Administration for treatment of impetigo in children and has been recently made available in the Indian market. It has been demonstrated to have low potential for the development of antibacterial resistance and a high degree of potency against poly drug resistant Gram-positive bacteria found in skin infections including Staphylococcus aureus strains. The drug is safe owing to low systemic absorption and has only minimal side-effect of local irritation at the site of application.

  10. Do scientists trace hot topics?

    CERN Document Server

    Wei, Tian; Wu, Chensheng; Yan, XiaoYong; Fan, Ying; Di, Zengru; Wu, Jinshan

    2013-01-01

    Do scientists follow hot topics in their scientific investigations? In this paper, by performing analysis to papers published in the American Physical Society (APS) Physical Review journals, it is found that papers are more likely to be attracted by hot fields, where the hotness of a field is measured by the number of papers belonging to the field. This indicates that scientists generally do follow hot topics. However, there are qualitative differences among scientists from various countries, among research works regarding different number of authors, different number of affiliations and different number of references. These observations could be valuable for policy makers when deciding research funding and also for individual researchers when searching for scientific projects.

  11. 76 FR 81806 - Ophthalmic and Topical Dosage Form New Animal Drugs; Ivermectin Topical Solution

    Science.gov (United States)

    2011-12-29

    ...FDA-2011-N-0003] Ophthalmic and Topical Dosage Form...Ivermectin Topical Solution AGENCY: Food and Drug...treated with a topical solution of ivermectin. DATES...Pour-On, a topical solution used on cattle to control...follows: PART 524--OPHTHALMIC AND TOPICAL DOSAGE...

  12. Probabilistic analysis and related topics

    CERN Document Server

    Bharucha-Reid, A T

    1983-01-01

    Probabilistic Analysis and Related Topics, Volume 3 focuses on the continuity, integrability, and differentiability of random functions, including operator theory, measure theory, and functional and numerical analysis. The selection first offers information on the qualitative theory of stochastic systems and Langevin equations with multiplicative noise. Discussions focus on phase-space evolution via direct integration, phase-space evolution, linear and nonlinear systems, linearization, and generalizations. The text then ponders on the stability theory of stochastic difference systems and Marko

  13. Topics in clinical oncology. 15

    International Nuclear Information System (INIS)

    The monograph comprising primarily papers on topical subjects of oncology and cancer research, contains also a selection of papers presented at the 2. Congress of the Czechoslovak Society of Nuclear Medicine and Radiation Hygiene. Seven papers were selected on behalf of their subject related to clinical oncology. All of them were iputted in INIS; five of them deal with the scintiscanning of the skeleton of cancer patients, one with radioimmunodetection of tumors, and one with radionuclide lymphography. (A.K.)

  14. Topics in combinatorial pattern matching

    DEFF Research Database (Denmark)

    Vildhøj, Hjalte Wedel

    2015-01-01

    This dissertation studies problems in the general theme of combinatorial pattern matching. More specifically, we study the following topics: Longest Common Extensions. We revisit the longest common extension (LCE) problem, that is, preprocess a string T into a compact data structure that supports fast LCE queries. An LCE query takes a pair (i, j) of indices in T and returns the length of the longest common prefix of the suffixes of T starting at positions i and j. Such queries are also commonly ...

  15. Probabilistic analysis and related topics

    CERN Document Server

    Bharucha-Reid, A T

    1979-01-01

    Probabilistic Analysis and Related Topics, Volume 2 focuses on the integrability, continuity, and differentiability of random functions, as well as functional analysis, measure theory, operator theory, and numerical analysis.The selection first offers information on the optimal control of stochastic systems and Gleason measures. Discussions focus on convergence of Gleason measures, random Gleason measures, orthogonally scattered Gleason measures, existence of optimal controls without feedback, random necessary conditions, and Gleason measures in tensor products. The text then elaborates on an

  16. Hot topics from the Tevatron

    Energy Technology Data Exchange (ETDEWEB)

    Glenzinski, D.; /Fermilab

    2008-01-01

    The Tevatron Run-II began in March 2001. To date, both the CDF and D0 experiments have collected 1 fb{sup -1} of data each. The results obtained from this data set were summarized at this conference in 39 parallel session presentations covering a wide range of topics. The author summarizes the most important of those results here and comments on some of the prospects for the future.

  17. Topical Ocular Delivery of NSAIDs

    OpenAIRE

    Ahuja, Munish; Dhake, Avinash S.; Sharma, Surendra K; Dipak K. Majumdar

    2008-01-01

    In ocular tissue, arachidonic acid is metabolized by cyclooxygenase to prostaglandins which are the most important lipid derived mediators of inflammation. Presently nonsteroidal anti-inflammatory drugs (NSAIDs) which are cyclooxygenase (COX) inhibitors are being used for the treatment of inflammatory disorders. NSAIDs used in ophthalmology, topically, are salicylic-, indole acetic-, aryl acetic-, aryl propionic- and enolic acid derivatives. NSAIDs are weak acids with pKa mostly between 3.5 a...

  18. A Synthetic Congener Modeled on a Microbicidal Domain of Thrombin- Induced Platelet Microbicidal Protein 1 Recapitulates Staphylocidal Mechanisms of the Native Molecule?

    OpenAIRE

    Xiong, Yan Q; Bayer, Arnold S; Elazegui, Lisa; Yeaman, Michael R

    2006-01-01

    Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a staphylocidal peptide released by activated platelets. This peptide initiates its microbicidal activity by membrane permeabilization, with ensuing inhibition of intracellular macromolecular synthesis. RP-1 is a synthetic congener modeled on the C-terminal microbicidal ?-helix of tPMP-1. This study compared the staphylocidal mechanisms of RP-1 with those of tPMP-1, focusing on isogenic tPMP-1-susceptible (ISP479C) and -resistant (I...

  19. 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.

  20. Percutaneous thrombin injection treatment of a gluteal pseudoaneurysm following radiofrequency ablation of a hip osteoid osteoma in a 6-year-old boy.

    Science.gov (United States)

    Kumar, Abhishek; Ramchand, Tekchand; Contractor, Sohail

    2014-12-01

    Osteoid osteomas are benign bone lesions that present with bone pain in children and young adults. Over the last 2 decades, radiofrequency ablation has become the mainstay of treatment and is now preferred over surgical resection. Major complications of the procedure are very rare, consisting mostly of local skin burns. We present a case of a child presenting with a gluteal pseudoaneursym following CT-guided radiofrequency ablation of an acetabular osteoid osteoma, which was then treated successfully with percutaneous thrombin injection. PMID:25015326

  1. Electron-ion recombination rates for merged-beams experiments

    International Nuclear Information System (INIS)

    Energy dependence of the electron-ion recombination rates are studied for different recombination processes (radiative recombination, three-body recombination, dissociative recombination) for Maxwellian relative velocity distribution of arbitrary asymmetry. The results are discussed in context of the electron-ion merged beams experiments in cooling ion storage rings. The question of indication of a possible contribution of the three-body recombination to the measured recombination rates versus relative energy is particularly addressed. Its influence on the electron beam temperature derived from the energy dependence of recombination rate is discussed

  2. V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element.

    OpenAIRE

    Roch, F A; Hobi, R; Berchtold, M W; Kuenzle, C C

    1997-01-01

    The immunoglobulin heavy chain intron enhancer (Emu) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Emu, possibly affecting V(D)J recombination. Our work shows that ...

  3. 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.

  4. On-line Hot Topic Recommendation Using Tolerance Rough Set Based Topic Clustering

    Directory of Open Access Journals (Sweden)

    Yonghui Wu

    2010-04-01

    Full Text Available In this paper we present our research of online hot topic detection and label extraction method for our hot topic recommendation system. Using a new topical feature selection method, the feature space is compressed suitable for an online system. The tolerance rough set model is used to enriching the small set of topical feature words to a topical approximation space. According to the distance defined on the topical approximation space, the web pages are clustered into groups which will be merged with document overlap. The topic labels are extracted based on the approximation topical space enriched with the useful but high frequency topical words dropped by the clustering process. The experiments show that our method could generate more information abundant classes and more topical class labels, alleviate the topical drift caused by the non-topical and noise words.

  5. Recombinant vaccine for canine parvovirus in dogs.

    OpenAIRE

    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-as...

  6. Recombination in narrow-gapped semiconductors

    International Nuclear Information System (INIS)

    In narrow-gapped semiconductors of the type Hgsub(1-x)Cdsub(x)Te as well as in lead chalcogenides and their mixed crystals with energy gaps of some tenths of eV, the band-band recombination processes dominate if the samples are sufficiently perfect in their crystal lattices. The relative importance of the radiative or Auger recombination depends on the width of the energy gap and the charge carrier concentration. In the extreme case of very narrow energy gaps plasmon and one-electron recombination occurs additionally

  7. On-line Hot Topic Recommendation Using Tolerance Rough Set Based Topic Clustering

    OpenAIRE

    Yonghui Wu; Yuxin Ding; Xiaolong Wang; Jun Xu

    2010-01-01

    In this paper we present our research of online hot topic detection and label extraction method for our hot topic recommendation system. Using a new topical feature selection method, the feature space is compressed suitable for an online system. The tolerance rough set model is used to enriching the small set of topical feature words to a topical approximation space. According to the distance defined on the topical approximation space, the web pages are clustered into groups which will be mer...

  8. Dielectronic recombination of boronlike argon

    International Nuclear Information System (INIS)

    We present measurements and calculations of ?n=0 dielectronic recombination resonances of boronlike argon between 0.2 and 6 eV. A storage ring equipped with an electron cooler was used for the measurements. Methods employed to reduce the electron energy distribution and improve the accuracy of resonance energy measurements have yielded an energy resolution of 30 meV full width at half maximum at low energies, and an energy uncertainty better than 30 meV. The high energy resolution results from the use of an adiabatic expansion technique to reduce the transverse electron energy distribution. The improved accuracy in energy determinations is achieved through the inclusion of variations in the ion velocity, which occur during scans of the electron velocity, in the relative velocity transformations. Calculations of the resonance strengths and energies were made using two different methods, multiconfiguration Dirac-Fock and multiconfiguration Breit-Pauli approximations. A comparison of the experimental data to the calculations shows fair agreement in both the spectral features and integrated intensities above 3 eV. However, poor agreement is found below 3 eV. copyright 1996 The American Physical Society

  9. Dabigatran and its reversal with recombinant factor VIIa and prothrombin complex concentrate : a Sonoclot in vitro study

    DEFF Research Database (Denmark)

    SØlbeck, Sacha; Nilsson, Caroline U

    2014-01-01

    OBJECTIVE: Dabigatran is a new oral direct thrombin inhibitor. No specific antidote exists in the event of hemorrhage, but prothrombin complex concentrate (PCC) and recombinant activated factor VII (rFVIIa) are suggested therapies. Sonoclot is a bedside viscoelastic instrument for monitoring the coagulation process in whole blood. The aim of this study was to investigate the effect of dabigatran and reversal with PCC and rFVIIa, as monitored by the Sonoclot. METHODS: Citrated whole blood was drawn and mixed in vitro with dabigatran, dabigatran + PCC or dabigatran + rFVIIa and analyzed with three different Sonoclot cuvettes: Glassbead, kaolin and tissue factor (diluted) activated. RESULTS: The Sonoclot detected in vitro-induced anticoagulation due to dabigatran with the glassbead- and kaolin-activated cuvettes. There was no reversing effect of PCC, probably due to the presence of heparin in the PCC we used. There was no certain reversing effect of rFVIIa. CONCLUSIONS: The Sonoclot can detect the anticoagulant effect of dabigatran. Our results do not support efficient reversal of dabigatran with PCC and rFVIIa, or alternatively do not support the ability of Sonoclot to detect a reversing effect of the PCC and rFVIIa in our study. Clinical studies of dabigatran-treated patients with severe bleeding are called for, as well as the continued development of specific antidotes and monitoring techniques.

  10. Topical Pain Relievers May Cause Burns

    Science.gov (United States)

    ... Articulos en Espanol Topical Pain Relievers May Cause Burns Get Consumer Updates by E-mail Consumer Updates ... rare, have ranged from mild to severe chemical burns with use of such brand-name topical muscle ...

  11. Environmental Health Topics from A to Z

    Science.gov (United States)

    ... Education Home Page Brochures & Fact Sheets Environmental Health Topics Science Education Kids' Pages Research Home Page At ... Science Resources for Scientists National Toxicology Program Research Topics All Scientists All Research Groups Funding Opportunities Home ...

  12. Potential theory—selected topics

    CERN Document Server

    Aikawwa, Hiroaki

    1996-01-01

    The first part of these lecture notes is an introduction to potential theory to prepare the reader for later parts, which can be used as the basis for a series of advanced lectures/seminars on potential theory/harmonic analysis. Topics covered in the book include minimal thinness, quasiadditivity of capacity, applications of singular integrals to potential theory, L(p)-capacity theory, fine limits of the Nagel-Stein boundary limit theorem and integrability of superharmonic functions. The notes are written for an audience familiar with the theory of integration, distributions and basic functional analysis.

  13. Topics in atomic collision theory

    CERN Document Server

    Geltman, Sydney; Brueckner, Keith A

    1969-01-01

    Topics in Atomic Collision Theory originated in a course of graduate lectures given at the University of Colorado and at University College in London. It is recommended for students in physics and related fields who are interested in the application of quantum scattering theory to low-energy atomic collision phenomena. No attention is given to the electromagnetic, nuclear, or elementary particle domains. The book is organized into three parts: static field scattering, electron-atom collisions, and atom-atom collisions. These are in the order of increasing physical complexity and hence necessar

  14. Topics in commutative ring theory

    CERN Document Server

    Watkins, John J

    2009-01-01

    Topics in Commutative Ring Theory is a textbook for advanced undergraduate students as well as graduate students and mathematicians seeking an accessible introduction to this fascinating area of abstract algebra. Commutative ring theory arose more than a century ago to address questions in geometry and number theory. A commutative ring is a set-such as the integers, complex numbers, or polynomials with real coefficients--with two operations, addition and multiplication. Starting from this simple definition, John Watkins guides readers from basic concepts to Noetherian rings-one of

  15. Topical timolol and serum lipoproteins.

    OpenAIRE

    WEST, J; Longstaff, S

    1990-01-01

    Oral timolol taken for the treatment of systemic hypertension has been shown to affect adversely serum lipoprotein levels. In a 15-week study on 19 patients topical timolol therapy for raised intraocular pressure was found to have no significant adverse effect on serum lipoprotein levels. This is reassuring in view of the large number of patients on this form of long term therapy, and selection of the type of beta blocker to use should not be influenced by lipid changes associated with the or...

  16. A highly sensitive electrochemical aptasensor for thrombin detection using functionalized mesoporous silica@multiwalled carbon nanotubes as signal tags and DNAzyme signal amplification.

    Science.gov (United States)

    Zhang, Juan; Chai, Yaqin; Yuan, Ruo; Yuan, Yali; Bai, Lijuan; Xie, Shunbi

    2013-11-21

    In this work, we demonstrated a novel sensitive sandwich-type pseudobienzyme aptasensor for thrombin detection. Greatly amplified sensitivity was based on mesoporous silica-multiwalled carbon nanotube (mSiO2@MWCNT) nanocomposites as enhanced materials and a pseudobienzyme electrocatalytic system. Firstly, the mSiO2@MWCNT nanocomposites not only have good biocompatibility and a suitable microenvironment for stabilizing the aptamer assembly, but also can load large amounts of electron mediator thionine (Thi), platinum nanoparticles (PtNPs) and hemin/G-quadruplex bioelectrocatalytic complex. Moreover, in the presence of H2O2 in an electrolytic cell, the synergistic reaction of PtNPs and hemin/G-quadruplex bioelectrocatalyzed the reduction of H2O2, dramatically amplifying the response signals of electron mediator Thi and improving the sensitivity. Secondly, dendrimer functionalized reduced graphene oxide (PAMAM-rGO) as the biosensor platform enhanced the surface area for the immobilization of abundant primary aptamers as well as facilitated electron transfer from Thi to the electrode, thus amplifying the detection response. Using the above multiple effects, the approach showed a high sensitivity and a wider linearity for the detection of thrombin in the range between 0.0001 nM and 80 nM with a detection limit of 50 fM. This new design avoided the fussy labeling process and the spatial distribution of each sequentially acting enzyme, which provided an ideal candidate for the development of a sensitive and simple bioanalytical platform. PMID:24081001

  17. 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.

  18. Topical tacrolimus as treatment of atopic dermatitis

    OpenAIRE

    Masutaka Furue; Satoshi Takeuchi

    2009-01-01

    Masutaka Furue, Satoshi TakeuchiDepartment of Dermatology, Faculty of Medical Sciences, Kyushu University, Fukuoka, JapanAbstract: Atopic dermatitis (AD) is a common, chronic, relapsing, severely pruritic, eczematous skin disease. The mainstays of treatment for AD are topical tacrolimus and topical steroids. Tacrolimus, a calcineurin inhibitor, not only complements existing treatment options but also overcomes some of the drawbacks of topical steroid therapy when given topically and thus meet...

  19. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed.

  20. PRODUCTION OF RECOMBINANT PROTEINS IN INSECT CELLS

    Directory of Open Access Journals (Sweden)

    Christian Kollewe

    2013-01-01

    Full Text Available Among the wide range of methods and expression hosts available for the heterologous production of recombinant proteins, insect cells are ideal for the production of complex proteins requiring extensive post-translational modification. This review article provides an overview of the available insect-cell expression systems and their properties, focusing on the widely-used Baculovirus Expression Vector System (BEVS. We discuss the different strategies used to generate recombinant baculovirus vectors and show how advanced techniques for virus titer determination can accelerate the production of recombinant proteins. The stable transfection of insect cells is an alternative to BEVS which has recently been augmented with recombinase-mediated cassette exchange for site-specific gene integration. We consider the advantages and limitations of these techniques for the production of recombinant proteins in insect cells and compare them to other expression platforms.

  1. Dielectronic recombination of hydrogen-like ions

    International Nuclear Information System (INIS)

    Decay dynamics of dielectronic recombination (DR) processes of H-like titanium ions was investigated with an electron beam ion trap. In the DR of H-like ions a K-shell vacancy is available even after the decay of the doubly excited state produced by the recombination. Therefore secondary X-ray emission is possible. An observed X-ray spectrum of DR obtained in the present experiment was well reproduced theoretically by taking into account the secondary X-rays

  2. 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 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.

  3. 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...

  4. Breaking the sound barrier in recombination fronts

    OpenAIRE

    Williams, R. J. R.; J. E. Dyson

    1995-01-01

    We exploit a generic instability in the integration of steady, sonic, near-isothermal flows to find the complete transition diagram for recombination fronts (for a model system of equations). The instability requires the integration of the flow equations for speeds between the isothermal and adiabatic sound speeds to be performed with particular care. As a result of this, the previous work of Newman & Axford on the structure of recombination fronts neglected an important cla...

  5. Recombination energy in double white dwarf formation

    OpenAIRE

    Nandez, Jose L. A.; Ivanova, Natalia; Lombardi Jr., James C.

    2015-01-01

    In this Letter we investigate the role of recombination energy during a common envelope event. We confirm that taking this energy into account helps to avoid the formation of the circumbinary envelope commonly found in previous studies. For the first time, we can model a complete common envelope event, with a clean compact double white dwarf binary system formed at the end. The resulting binary orbit is almost perfectly circular. In addition to considering recombination ener...

  6. Recombinant DNA production of spider silk proteins

    OpenAIRE

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio 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...

  7. Co-factor activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  8. Persistence and Loss of Meiotic Recombination Hotspots

    OpenAIRE

    Pineda-Krch, Mario; Redfield, Rosemary J

    2005-01-01

    The contradiction between the long-term persistence of the chromosomal hotspots that initiate meiotic recombination and the self-destructive mechanism by which they act strongly suggests that our understanding of recombination is incomplete. This “hotspot paradox” has been reinforced by the finding that biased gene conversion also removes active hotspots from human sperm. To investigate the requirements for hotspot persistence, we developed a detailed computer simulation model of their activi...

  9. Topical report review status: Volume 10

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-03-01

    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 by the NRC. This report is published annually.

  10. Current topics in nail surgery

    Science.gov (United States)

    Alam; Scher

    1999-10-01

    BACKGROUND: Nail surgery can be performed in an office-based dermatology practice with a limited amount of specialized equipment and training. Several excellent reviews have been published in recent years that detail the techniques of nail surgery for both the novice and the experienced practitioner. OBJECTIVE: In this article recent developments in nail surgery are discussed. Topics that are treated include the general principles of nail surgery, including epidemiologic issues, studies of nail anatomy, instrumentation, and anesthesia. The reconstruction of injuries and congenital defects involving the nail is explained, and the role of the hand surgeon clarified. Appropriate removal of tumours and cysts is considered, with special attention to the management of malignant lesions. The controversy regarding more or less conservative management of melanonychia striata is addressed, and the need for early diagnosis of subungual melanoma is emphasized. Other topics are surgical management of ingrown nails and onychomycosis. Newer areas of nail surgery, such as laser surgery of the nail, psychodermatology of the nail, and the role of primary care physicians in simple nail surgery are also examined. PMID:10575165

  11. Determination of recombination in Mycoplasma hominis

    DEFF Research Database (Denmark)

    Jacobsen, Iben SØgaard; Boesen, Thomas

    2002-01-01

    Mycoplasma hominis has been previously described as a heterogeneous species, and in the present study intraspecies diversity of 20 M. hominis isolates from different individuals was analyzed using parts of the unlinked gyrase B (gyrB), elongation factor Tu (tuf), SRalpha homolog (ftsY), hitB-hitL, excinuclease ABC subunit A (uvrA) and glyceraldehyde-3-phosphate dehydrogenase (gap) genes. The level of variability of these M. hominis genes was low compared with the housekeeping genes from Helicobacter pylori and Neisseria meningitidis, but only few M. hominis isolates had identical sequences in all genes indicating the presence of recombination. In order to test for intergenic recombination, phylogenetic trees were reconstructed for each of the genes but no well-supported bifurcating phylogenetic trees could be obtained. The genes were tested for intragenic recombination using the correlation between linkage disequilibrium and distance between the segregating sites, by the homoplasy ratio (H ratio), and by compatibility matrices. The gap gene showed well-supported evidence for high levels of recombination, whereas recombination was less frequent and not significant within the other genes. The analysis revealed intergenic and intragenic recombination in M. hominis and this may explain the high intraspecies variability. The results obtained in the present study may be of importance for future population studies of Mycoplasma species.

  12. Electron - ion recombination processes - an overview

    International Nuclear Information System (INIS)

    Extensive theoretical and experimental studies have been carried out for the past 20 years on electron - ion recombination processes, as they are applied to the analysis of astrophysical and laboratory plasmas. We review the basic understanding gained through these efforts, with emphasis on some of the more recent progress made in recombination theory as the recombining system is affected by time-dependent electric fields and plasma particles at low temperature. Together with collisional ionization and excitation processes, recombination is important in determining ionization balance and excited-state population in non-equilibrium plasmas. The radiation emitted by plasmas is usually the principal medium with which to study the plasma condition, as it is produced mainly during the recombination and decay of excited states of ions inside the plasma. This is especially true when the plasma under study is not readily accessible by direct probes, as in astrophysical plasmas. Moreover, external probes may sometimes cause undesirable disturbances of the plasma. Electron-ion recombination proceeds in several different modes. The direct modes include three-body recombination (TBR) and one-step radiative recombination (RR), all to the ground- and singly-excited states of the target ions. By contrast, the indirect resonant mode is a two-step dielectronic recombination (DR), which proceeds first with the formation of doubly-excited states by radiationless excitation/capture. The resonant states thus formed may relax by autoionization and/or radiative cascades. For more exotic modes of recombination, we consider off-shell dielectronic recombination (radiative DR = RDR), in which an electron capture is accompanied by simultaneous radiative emission and excitation of the target ion. Some discussion on attachment of electrons to neutral atoms, resulting in the formation of negative ions, is also given. When resonance states involve one or more electrons in high Rydberg states, presence of an external or intrinsic electric field in the vicinity of the target ions can seriously affect the ionic states involved and the resulting reaction rates. Such perturbative fields can be intrinsic, as in the case of the plasma ion field, or externally imposed. A proper theoretical treatment of this difficult problem is crucial in understanding the recombination process which takes place in a field contaminated environment. The simple off-shell dressing procedure of high Rydberg states by a time-dependent field is reviewed, and the possibility of an anomalously large enhancement in the rates, due to the momentum coherence effect (MCE), is discussed. The presently available data on recombination rates are summarized, and several important deficiencies and future directions for further research are pointed out. Based on the detailed calculations for a number of cases, several empirical rate formulae for RR and DR processes have been generated to summarize the data for ready applications. As the collection of atoms is cooled to very low temperatures, T 8 Ryd, and the bound electrons are ionized by laser irradiation to states of very precisely controlled energies, the prospect for accurate experimental measurements of very-low-energy recombination rates is considered, where the electron temperature can be very low. Therefore, it is of interest to reconsider theoretically some new phenomena which may occur at such cold environments, in which the electron de Broglie wavelength can be very large, and both the density and coherent effects, as well as possible field effects, must be properly taken into account. Finally, a broader understanding of the various recombination processes may be achieved by studying their relationships to other reactions initiated by electron, ion and photon impact. (author)

  13. Multilingual Topic Models for Unaligned Text

    CERN Document Server

    Boyd-Graber, Jordan

    2012-01-01

    We develop the multilingual topic model for unaligned text (MuTo), a probabilistic model of text that is designed to analyze corpora composed of documents in two languages. From these documents, MuTo uses stochastic EM to simultaneously discover both a matching between the languages and multilingual latent topics. We demonstrate that MuTo is able to find shared topics on real-world multilingual corpora, successfully pairing related documents across languages. MuTo provides a new framework for creating multilingual topic models without needing carefully curated parallel corpora and allows applications built using the topic model formalism to be applied to a much wider class of corpora.

  14. Distortion of the CMB Spectrum by Primeval Hydrogen Recombination

    OpenAIRE

    Boschan, Peter; Biltzinger, Peter

    1996-01-01

    We solve the recombination equation by taking into account the induced recombinations and a physical cut off in the hydrogen spectrum. The effective recombination coefficient is parametrized as a function of temperature and free electron density and is about a factor of four larger than without the induced recombination. This accelerates the last stage of the recombination processes and diminishes the residual ionization by a factor of about 2.6. The number and energy distri...

  15. 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)

  16. Topics in Number Theory Conference

    CERN Document Server

    Andrews, George; Ono, Ken

    1999-01-01

    From July 31 through August 3,1997, the Pennsylvania State University hosted the Topics in Number Theory Conference. The conference was organized by Ken Ono and myself. By writing the preface, I am afforded the opportunity to express my gratitude to Ken for beng the inspiring and driving force behind the whole conference. Without his energy, enthusiasm and skill the entire event would never have occurred. We are extremely grateful to the sponsors of the conference: The National Sci­ ence Foundation, The Penn State Conference Center and the Penn State Depart­ ment of Mathematics. The object in this conference was to provide a variety of presentations giving a current picture of recent, significant work in number theory. There were eight plenary lectures: H. Darmon (McGill University), "Non-vanishing of L-functions and their derivatives modulo p. " A. Granville (University of Georgia), "Mean values of multiplicative functions. " C. Pomerance (University of Georgia), "Recent results in primality testing. " C. ...

  17. Synergetics introduction and advanced topics

    CERN Document Server

    Haken, Hermann

    2004-01-01

    This book is an often-requested reprint of two classic texts by H. Haken: "Synergetics. An Introduction" and "Advanced Synergetics". Synergetics, an interdisciplinary research program initiated by H. Haken in 1969, deals with the systematic and methodological approach to the rapidly growing field of complexity. Going well beyond qualitative analogies between complex systems in fields as diverse as physics, chemistry, biology, sociology and economics, Synergetics uses tools from theoretical physics and mathematics to construct an unifying framework within which quantitative descriptions of complex, self-organizing systems can be made. This may well explain the timelessness of H. Haken's original texts on this topic, which are now recognized as landmarks in the field of complex systems. They provide both the beginning graduate student and the seasoned researcher with solid knowledge of the basic concepts and mathematical tools. Moreover, they admirably convey the spirit of the pioneering work by the founder of ...

  18. KEY TOPICS IN SPORTS MEDICINE

    Directory of Open Access Journals (Sweden)

    Amir Ali Narvani

    2006-12-01

    Full Text Available Key Topics in Sports Medicine is a single quick reference source for sports and exercise medicine. It presents the essential information from across relevant topic areas, and includes both the core and emerging issues in this rapidly developing field. It covers: 1 Sports injuries, rehabilitation and injury prevention, 2 Exercise physiology, fitness testing and training, 3 Drugs in sport, 4 Exercise and health promotion, 5 Sport and exercise for special and clinical populations, 6 The psychology of performance and injury. PURPOSE The Key Topics format provides extensive, concise information in an accessible, easy-to-follow manner. AUDIENCE The book is targeted the students and specialists in sports medicine and rehabilitation, athletic training, physiotherapy and orthopaedic surgery. The editors are authorities in their respective fields and this handbook depends on their extensive experience and knowledge accumulated over the years. FEATURES The book contains the information for clinical guidance, rapid access to concise details and facts. It is composed of 99 topics which present the information in an order that is considered logical and progressive as in most texts. Chapter headings are: 1. Functional Anatomy, 2. Training Principles / Development of Strength and Power, 3. Biomechanical Principles, 4. Biomechanical Analysis, 5. Physiology of Training, 6. Monitoring of Training Progress, 7. Nutrition, 8. Hot and Cold Climates, 9. Altitude, 10. Sport and Travelling, 11. Principles of Sport Injury Diagnosis, 12. Principles of Sport and Soft Tissue Management, 13. Principles of Physical Therapy and Rehabilitation, 14. Principles of Sport Injury Prevention, 15. Sports Psychology, 16. Team Sports, 17. Psychological Aspects of Injury in Sport, 18. Injury Repair Process, 19. Basic Biomechanics of Tissue Injury, 20. Plain Film Radiography in Sport, 21. Nuclear Medicine, 22. Diagnostic Ultrasound, 23. MRI Scan, 24. Other Imaging, 5. Head Injury, 26. Eye Injury, 27. Injury to the Face, Nose, Ear, 28. Dental Problems, 29. Protective Headwear and Facial Protection in Sport, 30. Spinal Injury: Functional Anatomy and General Biomechanics, 31. Cervical Spine Injuries, 32. Thoracolumbar Spine Injuries, 33. Spine Related Syndromes, 34. Chest Injuries, 35. Abdominal Injuries and Abdominal Wall Injuries, 36. Urinary Tract Injuries, 37. The Shoulder Disorders, 38. Acromioclavicular Joint Disorders, 39. The Elbow Joint, 40. The Forearm and Wrist, 41. Hand Injuries, 42. Neurological Injury Affecting the Upper Limb, 43. Buttock Pain, 44. Groin Pain, 45. Thigh Injuries. ASSESSMENT Based on graduate programme teaching practice and with an international team of contributors, this is a valuable and practical resource for all those interested in sports and exercise medicine, including sports clinicians, general practitioners, team doctors, orthopaedic surgeons, accident and emergency doctors and physiotherapists. It is concise and well organized in its presentation, creating an effective textbook. I believe, therefore, the book will serve as a first-rate teaching tool and reference for students and specialists in sports medicine and rehabilitation, athletic training, physiotherapy. Students will enjoy the format of this book

  19. Conclusion from the fifth topic

    International Nuclear Information System (INIS)

    The topic ''mechanics and alteration kinetics of glasses'' is a crucial point for the understanding of the long-term behaviour of nuclear glasses. Kinetic models used in simulation are based on the works made by Grambow who imputes the control of the alteration kinetics of borosilicate glasses to the desorption of the ortho-silica acid produced at the reactive interface. The ensuing kinetics law requires the existence of an equilibrium of the silica at the interface glass/gel and the existence of a linear concentration gradient dissolved in the interstitial gel solution. The role of the gel needs further studies to be well understood. The difficulty lies in the fact that the composition and the structure of the gel varies with time, space (anisotropy) and with the conditions of alteration (temperature, pH, flowrate...). (A.C.)

  20. Neutron transport simulation (selected topics)

    International Nuclear Information System (INIS)

    Neutron transport simulation is usually performed for criticality, power distribution, activation, scattering, dosimetry and shielding problems, among others. During the last fifteen years, innovative technological applications have been proposed (Accelerator Driven Systems, Energy Amplifiers, Spallation Neutron Sources, etc.), involving the utilization of intermediate energies (hundreds of MeV) and high-intensity (tens of mA) proton accelerators impinging in targets of high Z elements. Additionally, the use of protons, neutrons and light ions for medical applications (hadrontherapy) impose requirements on neutron dosimetry-related quantities (such as kerma factors) for biologically relevant materials, in the energy range starting at several tens of MeV. Shielding and activation related problems associated to the operation of high-energy proton accelerators, emerging space-related applications and aircrew dosimetry-related topics are also fields of intense activity requiring as accurate as possible medium- and high-energy neutron (and other hadrons) transport simulation. These applications impose specific requirements on cross-section data for structural materials, targets, actinides and biologically relevant materials. Emerging nuclear energy systems and next generation nuclear reactors also impose requirements on accurate neutron transport calculations and on cross-section data needs for structural materials, coolants and nuclear fuel materials, aiming at improved safety and detailed thermal-hydraulics and radiation damage studies. In this review paper, the state-of-the-art in the computational tools and methodologies available to perform neutron transport simulation is presented. Proton- and neutron-induced cross-section data needs and requirements are discussed. Hot topics are pinpointed, prospective views are provided and future trends identified.

  1. Neutron transport simulation (selected topics)

    Science.gov (United States)

    Vaz, P.

    2009-10-01

    Neutron transport simulation is usually performed for criticality, power distribution, activation, scattering, dosimetry and shielding problems, among others. During the last fifteen years, innovative technological applications have been proposed (Accelerator Driven Systems, Energy Amplifiers, Spallation Neutron Sources, etc.), involving the utilization of intermediate energies (hundreds of MeV) and high-intensity (tens of mA) proton accelerators impinging in targets of high Z elements. Additionally, the use of protons, neutrons and light ions for medical applications (hadrontherapy) impose requirements on neutron dosimetry-related quantities (such as kerma factors) for biologically relevant materials, in the energy range starting at several tens of MeV. Shielding and activation related problems associated to the operation of high-energy proton accelerators, emerging space-related applications and aircrew dosimetry-related topics are also fields of intense activity requiring as accurate as possible medium- and high-energy neutron (and other hadrons) transport simulation. These applications impose specific requirements on cross-section data for structural materials, targets, actinides and biologically relevant materials. Emerging nuclear energy systems and next generation nuclear reactors also impose requirements on accurate neutron transport calculations and on cross-section data needs for structural materials, coolants and nuclear fuel materials, aiming at improved safety and detailed thermal-hydraulics and radiation damage studies. In this review paper, the state-of-the-art in the computational tools and methodologies available to perform neutron transport simulation is presented. Proton- and neutron-induced cross-section data needs and requirements are discussed. Hot topics are pinpointed, prospective views are provided and future trends identified.

  2. Performance testing of passive autocatalytic recombiners (PARs)

    Energy Technology Data Exchange (ETDEWEB)

    Blanchat, T. [Sandia National Laboratories, Albuquerque, NM (United States); Malliakos, A. [U.S. Nuclear Regulatory Commission, Washington, DC (United States)

    1997-03-01

    Passive autocatalytic recombiners (PARs) have been under consideration in the U.S. as a combustible gas control system in advanced light water reactor (ALWR) containments for design basis and severe accidents. PARs do not require a source of power. Instead they use palladium or platinum as a catalyst to recombine hydrogen and oxygen gases into water vapor upon contact with the catalyst. Energy from the recombination of hydrogen with oxygen is released at a relatively slow but continuous rate into the containment which prevents the pressure from becoming too high. The heat produced creates strong buoyancy effects which increases the influx of the surrounding gases to the recombiner. These natural convective flow currents promote mixing of combustible gases in the containment. PARs are self-starting and self-feeding under a very wide range of conditions. The recombination rate of the PAR system needs to be great enough to keep the concentration of hydrogen (or oxygen) below acceptable limits. There are several catalytic recombiner concepts under development worldwide. The USNRC is evaluating a specific design of a PAR which is in an advanced stage of engineering development and has been proposed for ALWR designs. Sandia National laboratories (SNL), under the sponsorship and the direction of the USNRC, is conducting an experimental program to evaluate the performance of PARs. The PAR will be tested at the SURTSEY facility at SNL. The test plan currently includes the following experiments: experiments will be conducted to define the startup characteristics of PARs (i.e., to define what is the lowest hydrogen concentration that the PAR starts recombining the hydrogen with oxygen); experiments will be used to define the hydrogen depletion rate of PARs as a function of hydrogen concentration; and experiments will be used to define the PAR performance in the presence of high concentrations of steam. (author)

  3. Performance testing of passive autocatalytic recombiners (PARs)

    International Nuclear Information System (INIS)

    Passive autocatalytic recombiners (PARs) have been under consideration in the U.S. as a combustible gas control system in advanced light water reactor (ALWR) containments for design basis and severe accidents. PARs do not require a source of power. Instead they use palladium or platinum as a catalyst to recombine hydrogen and oxygen gases into water vapor upon contact with the catalyst. Energy from the recombination of hydrogen with oxygen is released at a relatively slow but continuous rate into the containment which prevents the pressure from becoming too high. The heat produced creates strong buoyancy effects which increases the influx of the surrounding gases to the recombiner. These natural convective flow currents promote mixing of combustible gases in the containment. PARs are self-starting and self-feeding under a very wide range of conditions. The recombination rate of the PAR system needs to be great enough to keep the concentration of hydrogen (or oxygen) below acceptable limits. There are several catalytic recombiner concepts under development worldwide. The USNRC is evaluating a specific design of a PAR which is in an advanced stage of engineering development and has been proposed for ALWR designs. Sandia National laboratories (SNL), under the sponsorship and the direction of the USNRC, is conducting an experimental program to evaluate the performance of PARs. The PAR will be tested at the SURTSEY facility at SNL. The test plan currently includes the following experiments: experiments will be conducted to define the startup characteristics of PARs (i.e., to define what is the lowest hydrogen concentration that the PAR starts recombining the hydrogen with oxygen); experiments will be used to define the hydrogen depletion rate of PARs as a function of hydrogen concentration; and experiments will be used to define the PAR performance in the presence of high concentrations of steam. (author)

  4. Recent Advances In Topical Therapy In Dermatology

    Directory of Open Access Journals (Sweden)

    Mohan Thappa Devinder

    2003-01-01

    Full Text Available With changing times various newer topical agents are introduced in the field of dermatology. Tacrolimus and pimecrolimus are immunisuppressants, which are effective topically and are tried in the management of atopic dermatitis as well as other disorders including allergic contact dermatitis, atrophic lichen planus, pyoderma gangrenosum. Imiquimod, an immune response modifier, is presently in use for genital warts but has potentials as anti- tumour agent and in various other dermatological conditions when used topically. Tazarotene is a newer addition to the list of topical reginoids, which is effective in psoriasis and has better effect in combination with calcipotriene, phototherapy and topical costicosteroids. Tazarotene and adapelene are also effective in inflammatory acne. Calcipotriol, a vitamin D analogue has been introduced as a topical agent in the treatment of psoriasis. Steroid components are also developed recently which will be devoid of the side effects but having adequate anti-inflammatory effect. Topical photodynamic therapy has also a wide range of use in dermatology. Newer topical agents including cidofovir, capsaicin, topical sensitizers, topical antifungal agents for onychomycosis are also of use in clinical practice. Other promising developments include skin substitutes and growth factors for wound care.

  5. Microarrayed recombinant allergens for diagnosis of allergy.

    Science.gov (United States)

    Harwanegg, C; Laffer, S; Hiller, R; Mueller, M W; Kraft, D; Spitzauer, S; Valenta, R

    2003-01-01

    We suggest that the coapplication of recombinant allergens and microarray technology can lead to the development of new forms of multi-allergen tests which allow the determining and monitoring of complex sensitization profiles of allergic patients in single assays. The allergen extracts which have so far been used for diagnosis only allowed the determining of whether an allergic patient is sensitized against a particular allergen source, but the disease-eliciting allergens could not be identified. Through the application of recombinant DNA technology a rapidly growing panel of recombinant allergen molecules has become available which meanwhile comprises the epitope spectrum of most of the important allergen sources. We demonstrate that microarray technology can be used to establish multi-allergen tests consisting of microarrayed recombinant allergen molecules. Microarrayed recombinant allergens can be used to determine and monitor the profile of disease-eliciting allergens using single tests that require minute amounts of serum from allergic patients. The wealth of diagnostic information gained through microarray-based allergy testing will likely improve diagnosis, prevention and treatment of allergy. PMID:12534543

  6. 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.

  7. Human recombinant lysosomal enzymes produced in microorganisms.

    Science.gov (United States)

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  8. 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.

  9. Generation of Modified Pestiviruses by Targeted Recombination

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Friis, Martin Barfred

    Infectious cDNA clones are a prerequisite for directed genetic manipulation of pestivirus RNA genomes. We have developed a novel strategy to facilitate manipulation and rescue of modified pestiviruses from infectious cDNA clones based on bacterial artificial chromosomes (BACs). The strategy involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E.coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome and hence is not limited to the use of internal restriction sites. Rescue of modified pestiviruses can be obtained by electroporation of cell cultures with full-length RNA transcripts in vitro transcribed from the recombined BAC clones. We have used this approach to generate a series of new pestivirus BACs modified within different genomic regions and infectious pestiviruses have been rescued from several of these new constructs,demonstrating that recombination-mediated mutagenesis of pestivirus BACs provides a useful tool for expediting the construction of recombinant pestiviruses.

  10. Towards universal systems for recombinant gene expression

    Directory of Open Access Journals (Sweden)

    Sørensen Hans

    2010-04-01

    Full Text Available Abstract Recombinant gene expression is among the most important techniques used both in molecular and medical research and in industrial settings. Today, two recombinant expression systems are particularly well represented in the literature reporting on recombinant expression of specific genes. According to searches in the PubMed citation database, during the last 15 years 80% of all recombinant genes reported on in the literature were expressed in either the enterobacterium Escherichia coli or the methylotropic yeast Pichia pastoris. Nevertheless, some eukaryotic proteins are misfolded or inadequately posttranslationally modified in these expression systems. This situation demands identification of other recombinant expression systems that enable the proper expression of the remaining eukaryotic genes. As of now, a single universal system allowing expression of all target genes is still a distant goal. In this light, thorough experimental screening for systems that can yield satisfying quantity and quality of target protein is required. In recent years, a number of new expression systems have been described and used for protein production. Two systems, namely Drosophila melanogaster S2 insect cells and human embryonic kidney 293 (HEK293 cells stably expressing the EBNA-1 gene, show exceptional promise. The time has come to identify a few well-performing systems that will allow us to express, purify, and characterize entire eukaryotic genomes.

  11. Human alpha-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester: a kinetic, thermodynamic and X-ray crystallographic study

    OpenAIRE

    MILLA, Paola; BALLIANO, Gianni

    1997-01-01

    Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0°C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarb...

  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. Tamoxifen-Containing Eye Drops Successfully Trigger Cre-Mediated Recombination in the Entire Eye.

    Science.gov (United States)

    Schlecht, Anja; Leimbeck, Sarah V; Tamm, Ernst R; Braunger, Barbara M

    2016-01-01

    Embryonic lethality in mice with targeted gene deletion is a major issue that can be circumvented by using Cre-loxP-based animal models. Various inducible Cre systems are available, e.g. such that are activated following tamoxifen treatment, and allow deletion of a specific target gene at any desired time point during the life span of the animal. In this study, we describe the efficiency of topical tamoxifen administration by eye drops using a Cre- reporter mouse strain (R26R). We report that tamoxifen-responsive CAGGCre-ER (TM) mice show a robust Cre- mediated recombination throughout the entire eye. PMID:26427451

  14. Recombinant human erythropoietin in sports: a review

    Directory of Open Access Journals (Sweden)

    Bento Rafael Maia de Almeida

    2003-01-01

    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.

  15. Jet Fragmentation via Recombination of Parton Showers

    CERN Document Server

    Han, Kyong Chol; Ko, Che Ming

    2016-01-01

    We propose to model hadronization of parton showers in QCD jets through a hybrid approach involving quark recombination and string fragmentation. This is achieved by allowing gluons at the end of the perturbative shower evolution to undergo a non-perturbative splitting into quark and antiquark pairs, then applying a Monte-Carlo version of instantaneous quark recombination, and finally subjecting remnant quarks (those which have not found a recombination partner) to Lund string fragmentation. When applied to parton showers from the PYTHIA Monte Carlo event generator, the final hadron spectra from our calculation compare quite well to PYTHIA jets that have been hadronized with the default Lund string fragmentation. Our new approach opens up the possibility to generalize hadronization to jets embedded in a quark gluon plasma.

  16. Tracking topic birth and death in LDA.

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Andrew T.; Robinson, David Gerald

    2011-09-01

    Most topic modeling algorithms that address the evolution of documents over time use the same number of topics at all times. This obscures the common occurrence in the data where new subjects arise and old ones diminish or disappear entirely. We propose an algorithm to model the birth and death of topics within an LDA-like framework. The user selects an initial number of topics, after which new topics are created and retired without further supervision. Our approach also accommodates many of the acceleration and parallelization schemes developed in recent years for standard LDA. In recent years, topic modeling algorithms such as latent semantic analysis (LSA)[17], latent Dirichlet allocation (LDA)[10] and their descendants have offered a powerful way to explore and interrogate corpora far too large for any human to grasp without assistance. Using such algorithms we are able to search for similar documents, model and track the volume of topics over time, search for correlated topics or model them with a hierarchy. Most of these algorithms are intended for use with static corpora where the number of documents and the size of the vocabulary are known in advance. Moreover, almost all current topic modeling algorithms fix the number of topics as one of the input parameters and keep it fixed across the entire corpus. While this is appropriate for static corpora, it becomes a serious handicap when analyzing time-varying data sets where topics come and go as a matter of course. This is doubly true for online algorithms that may not have the option of revising earlier results in light of new data. To be sure, these algorithms will account for changing data one way or another, but without the ability to adapt to structural changes such as entirely new topics they may do so in counterintuitive ways.

  17. Topical therapies in inflammatory bowel disease

    OpenAIRE

    Frei, Pascal; Biedermann, Luc; Manser, Christine N.; Wilk, Maike; Manz, Michael; Vavricka, Stephan R; Rogler, Gerhard

    2012-01-01

    Due to misunderstandings about their effectiveness and feasibility, topical (or rectal) therapies with aminosalicylates (5-aminosalicylic acid, 5-ASA) and steroids are often underused in patients with ulcerative colitis (UC). However, many of these patients could be treated solely with rectal/topical therapies, or could benefit from them in combination with oral therapies. We review the evidence for topical therapies containing 5-ASA and budesonide in UC and discuss how these therapies can be...

  18. A Topic Modeling Toolbox Using Belief Propagation

    OpenAIRE

    Zeng, Jia

    2012-01-01

    Latent Dirichlet allocation (LDA) is an important hierarchical Bayesian model for probabilistic topic modeling, which attracts worldwide interests and touches on many important applications in text mining, computer vision and computational biology. This paper introduces a topic modeling toolbox (TMBP) based on the belief propagation (BP) algorithms. TMBP toolbox is implemented by MEX C++/Matlab/Octave for either Windows 7 or Linux. Compared with existing topic modeling packa...

  19. Online Belief Propagation for Topic Modeling

    OpenAIRE

    Zeng, Jia; Liu, Zhi-Qiang; Cao, Xiao-Qin

    2012-01-01

    The batch latent Dirichlet allocation (LDA) algorithms play important roles in probabilistic topic modeling, but they are not suitable for processing big data streams due to high time and space compleixty. Online LDA algorithms can not only extract topics from big data streams with constant memory requirements, but also detect topic shifts as the data stream flows. In this paper, we present a novel and easy-to-implement online belief propagation (OBP) algorithm that infers t...

  20. The Virtual Robotics Laboratory; TOPICAL

    International Nuclear Information System (INIS)

    The growth of the Internet has provided a unique opportunity to expand research collaborations between industry, universities, and the national laboratories. The Virtual Robotics Laboratory (VRL) is an innovative program at Oak Ridge National Laboratory (ORNL) that is focusing on the issues related to collaborative research through controlled access of laboratory equipment using the World Wide Web. The VRL will provide different levels of access to selected ORNL laboratory secondary education programs. In the past, the ORNL Robotics and Process Systems Division has developed state-of-the-art robotic systems for the Army, NASA, Department of Energy, Department of Defense, as well as many other clients. After proof of concept, many of these systems sit dormant in the laboratories. This is not out of completion of all possible research topics. but from completion of contracts and generation of new programs. In the past, a number of visiting professors have used this equipment for their own research. However, this requires that the professor, and possibly his/her students, spend extended periods at the laboratory facility. In addition, only a very exclusive group of faculty can gain access to the laboratory and hardware. The VRL is a tool that enables extended collaborative efforts without regard to geographic limitations