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Sample records for heparin binding activity

  1. Eosinophil cationic protein (ECP) can bind heparin and other glycosaminoglycans through its RNase active site.

    Science.gov (United States)

    Torrent, Marc; Nogués, M Victòria; Boix, Ester

    2011-01-01

    The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. During inflammation and eosinophilia disorders, ECP is secreted to the inflammation area, where it would contribute to the immune response. ECP secretion causes also severe damage to the host own tissues. ECP presents a high affinity for heparin and this property might be crucial for its immunomodulating properties, antipathogen action, and its toxicity against eukaryotic cells. ECP, also known as human RNase 3, belongs to the mammalian RNase A superfamily and its RNase activity is required for some of its biological properties. We have now proven that ECP heparin binding affinity depends on its RNase catalytic site, as the enzymatic activity is blocked by heparin. We have applied molecular modeling to analyze ECP binding to heparin representative probes, and identified protein residues at the catalytic and substrate binding sites that could contribute to the interaction. ECP affinity for heparin and other negatively charged glycosaminoglycans (GAGs) can explain not only its binding to the eukaryote cells glycocalix but also the reported high affinity for the specific carbohydrates at bacteria cell wall, promoting its antimicrobial action. 2010 John Wiley & Sons, Ltd.

  2. Structural and binding studies of SAP-1 protein with heparin.

    Science.gov (United States)

    Yadav, Vikash K; Mandal, Rahul S; Puniya, Bhanwar L; Kumar, Rahul; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2015-03-01

    SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity. © 2014 John Wiley & Sons A/S.

  3. Structure and function of a fungal adhesin that binds heparin and mimics thrombospondin-1 by blocking T cell activation and effector function.

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    T Tristan Brandhorst

    Full Text Available Blastomyces adhesin-1 (BAD-1 is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1 type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1.

  4. N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) inhibits the angiogenic activity of heparin-binding growth factors.

    Science.gov (United States)

    Nawaz, Imtiaz M; Chiodelli, Paola; Rezzola, Sara; Paganini, Giuseppe; Corsini, Michela; Lodola, Alessio; Di Ianni, Alessio; Mor, Marco; Presta, Marco

    2018-02-01

    The peptides N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) and BOC-Met-Leu-Phe (BOC1) are widely used antagonists of formyl peptide receptors (FPRs), BOC2 acting as an FPR1/FPR2 antagonist whereas BOC1 inhibits FPR1 only. Extensive investigations have been performed by using these FPR antagonists as a tool to assess the role of FPRs in physiological and pathological conditions. Based on previous observations from our laboratory, we assessed the possibility that BOC2 may exert also a direct inhibitory effect on the angiogenic activity of vascular endothelial growth factor-A (VEGF-A). Our data demonstrate that BOC2, but not BOC1, inhibits the angiogenic activity of heparin-binding VEGF-A 165 with no effect on the activity of the non-heparin-binding VEGF-A 121 isoform. Endothelial cell-based bioassays, surface plasmon resonance analysis, and computer modeling indicate that BOC2 may interact with the heparin-binding domain of VEGF-A 165 , thus competing for heparin interaction and preventing the binding of VEGF-A 165 to tyrosine kinase receptor VEGFR2, its phosphorylation and downstream signaling. In addition, BOC2 inhibits the interaction of a variety of heparin-binding angiogenic growth factors with heparin, including fibroblast growth factor 2 (FGF2) whose angiogenic activity is blocked by the compound. Accordingly, BOC2 suppresses the angiogenic potential of human tumor cell lines that co-express VEGF-A and FGF2. Thus, BOC2 appears to act as a novel multi-heparin-binding growth factor antagonist. These findings caution about the interpretation of FPR-focusing experimental data obtained with this compound and set the basis for the design of novel BOC2-derived, FPR independent multi-target angiogenesis inhibitors.

  5. Heparin (GAG-hed inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

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    López-Bayghen Esther

    2006-08-01

    Full Text Available Abstract Background High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR, plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs, such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Methods Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. Results We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Conclusion Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell

  6. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules

    OpenAIRE

    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Rehault-Godbert, Sophie

    2016-01-01

    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ova...

  7. Dual chain synthetic heparin-binding growth factor analogs

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    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

    2012-04-24

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  8. Dual chain synthetic heparin-binding growth factor analogs

    Science.gov (United States)

    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

    2009-10-06

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  9. A comparison of seven methods to analyze heparin in biomaterials: quantification, location, and anticoagulant activity

    NARCIS (Netherlands)

    Lammers, G.; Westerlo, E.M.A. van de; Versteeg, E.M.M.; Kuppevelt, A.H.M.S.M. van; Daamen, W.F.

    2011-01-01

    Glycosaminoglycans, like heparin, are frequently incorporated in biomaterials because of their capacity to bind and store growth factors and because of their hydrating properties. Heparin is also often used in biomaterials for its anticoagulant activity. Analysis of biomaterial-bound heparin is

  10. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)

    Heparin-binding proteins (HBPs) are essential constituents of human seminal fluid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affinity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser ...

  11. Mapping the heparin-binding site of the osteoinductive protein NELL1 by site-directed mutagenesis.

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    Takahashi, Kaneyoshi; Imai, Arisa; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D; Kuroda, Shun'ichi; Niimi, Tomoaki

    2015-12-21

    Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.

    Science.gov (United States)

    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Réhault-Godbert, Sophie

    2016-06-13

    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics.

  13. Crystallographic Analysis of Calcium-dependent Heparin Binding to Annexin A2

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    Shao,C.; Zhang, F.; Kemp, M.; Lindhardt, R.; Waisman, D.; Head, J.; Seaton, B.

    2006-01-01

    Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.

  14. Chemically modified, non-anticoagulant heparin derivatives are potent galectin-3 binding inhibitors and inhibit circulating galectin-3-promoted metastasis.

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    Duckworth, Carrie A; Guimond, Scott E; Sindrewicz, Paulina; Hughes, Ashley J; French, Neil S; Lian, Lu-Yun; Yates, Edwin A; Pritchard, D Mark; Rhodes, Jonathan M; Turnbull, Jeremy E; Yu, Lu-Gang

    2015-09-15

    Concentrations of circulating galectin-3, a metastasis promoter, are greatly increased in cancer patients. Here we show that 2- or 6-de-O-sulfated, N-acetylated heparin derivatives are galectin-3 binding inhibitors. These chemically modified heparin derivatives inhibited galectin-3-ligand binding and abolished galectin-3-mediated cancer cell-endothelial adhesion and angiogenesis. Unlike standard heparin, these modified heparin derivatives and their ultra-low molecular weight sub-fractions had neither anticoagulant activity nor effects on E-, L- or P-selectin binding to their ligands nor detectable cytotoxicity. Intravenous injection of such heparin derivatives (with cancer cells pre-treated with galectin-3 followed by 3 subcutaneous injections of the derivatives) abolished the circulating galectin-3-mediated increase in lung metastasis of human melanoma and colon cancer cells in nude mice. Structural analysis using nuclear magnetic resonance and synchrotron radiation circular dichroism spectroscopies showed that the modified heparin derivatives bind to the galectin-3 carbohydrate-recognition domain. Thus, these chemically modified, non-anticoagulant, low-sulfated heparin derivatives are potent galectin-3 binding inhibitors with substantial potential as anti-metastasis/cancer drugs.

  15. The role of VLA-4 binding for experimental melanoma metastasis and its inhibition by heparin.

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    Schlesinger, Martin; Roblek, Marko; Ortmann, Katrin; Naggi, Annamaria; Torri, Giangiacomo; Borsig, Lubor; Bendas, Gerd

    2014-05-01

    Heparin is known to efficiently attenuate metastasis in various tumour models by different mechanisms including inhibition of tumour cell contacts with soluble and cellular components such as inhibition of heparanase or P- and L-selectin. We recently showed that heparin efficiently binds to VLA-4 integrin in melanoma cells in vitro. Here we describe VLA-4 integrin as a mediator of melanoma metastasis that is inhibited by the low molecular weight heparin (LMWH) Tinzaparin. sh-RNA-mediated knock-down of VLA-4 integrin in B16F10 murine melanoma cells (B16F10-VLA-4kd) was performed and cell binding characteristics were investigated in vitro. Experimental metastasis of B16F10-VLA-4kd and B16F10 cells and interference by Tinzaparin were analysed in mice. VLA-4 knock-down of B16F10 cells resulted in loss of VCAM-1 binding, but preserved the capacity to bind platelets through P-selectin. The observed reduced metastasis of B16F10-VLA-4kd cells confirmed the role of VLA-4 in this process. However, loss of melanoma VLA-4 function hardly further affected reduction of metastasis in P-selectin deficient mice. Tinzaparin treatment of mice injected with B16F10 and B16F10-VLA-4kd cells significantly reduced metastasis suggesting its potential to block both P- and L-selectin and VLA-4 in vivo. The use of N-acetylated heparin, which has no VLA-4 binding activity but blocks P- and L-selectin was less efficient than Tinzaparin in mice injected with B16F10 cells and B16F10-VLA-4kd cells. These findings provide evidence that heparin inhibits experimental melanoma metastasis primarily by blocking VLA-4 and P-selectin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm

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    Marks, J.L.; Ax, R.L.

    1985-08-01

    The binding of the glycosaminoglycan (3H) heparin to bull spermatozoa was compared with nonreturn rates of dairy bulls. Semen samples from five bulls above and five below an average 71% nonreturn rate were used. Samples consisted of first and second ejaculates on a single day collected 1 d/wk for up to 5 consecutive wk. Saturation binding assays using (TH) heparin were performed to quantitate the binding characteristics of each sample. Scatchard plot analyses indicated a significant difference in the binding affinity for (TH) heparin between bulls of high and low fertility. Dissociation constants were 69.0 and 119.3 pmol for bulls of high and low fertility, respectively. In contrast, the number of binding sites for (TH) heparin did not differ significantly among bulls. Differences in binding affinity of (TH) heparin to bull sperm might be used to predict relative fertility of dairy bulls.

  17. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity.

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    Li, Binghan; Lu, Dan; Chen, Yuqing; Zhao, Minghui; Zuo, Li

    2016-04-22

    Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin's effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

  18. Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer

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    Giese Nathalia A

    2005-04-01

    Full Text Available Abstract Hsulf-1 is a newly identified enzyme, which has the ability to decrease the growth of hepatocellular, ovarian, and head and neck squamous cell carcinoma cells by interfering with heparin-binding growth factor signaling. Since pancreatic cancers over-express a number of heparin-binding growth factors and their receptors, the expression and function of this enzyme in pancreatic cancer was analyzed. Results Pancreatic cancer samples expressed significantly (22.5-fold increased Hsulf-1 mRNA levels compared to normal controls, and Hsulf-1 mRNA was localized in the cancer cells themselves as well as in peritumoral fibroblasts. 4 out of 8 examined pancreatic cancer cell lines expressed Hsulf-1, whereas its expression was below the level of detection in the other cell lines. Stable transfection of the Hsulf-1 negative Panc-1 pancreatic cancer cell line with a full length Hsulf-1 expression vector resulted in increased sulfatase activity and decreased cell-surface heparan-sulfate proteoglycan (HSPG sulfation. Hsulf-1 expression reduced both anchorage-dependent and -independent cell growth and decreased FGF-2 mediated cell growth and invasion in this cell line. Conclusion High expression of Hsulf-1 occurs in the stromal elements as well as in the tumor cells in pancreatic cancer and interferes with heparin-binding growth factor signaling.

  19. Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism.

    Science.gov (United States)

    Wei, Hudie; Cai, Haiyan; Wu, Jiawei; Wei, Zhenquan; Zhang, Fei; Huang, Xin; Ma, Lina; Feng, Lingling; Zhang, Ruoxi; Wang, Yunjie; Ragg, Hermann; Zheng, Ying; Zhou, Aiwu

    2016-11-25

    Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 Å resolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of ∼10 nm involving ∼8 monosaccharides and ∼6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 m(-1) s(-1), its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity

    Directory of Open Access Journals (Sweden)

    Binghan Li

    2016-04-01

    Full Text Available Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin’s effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4 and pre-osteoclasts (RAW264.7. In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand, M-CSF (macrophage colony-stimulating factor, and OPG (osteoprotegerin, which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG. However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

  1. Heparin interaction with protein-adsorbed surfaces

    NARCIS (Netherlands)

    Winterton, Lynn C.; Andrade, Joseph D.; Feijen, Jan; Kim, Sung Wan

    1986-01-01

    Albumin and fibrinogen show no binding affinity to varied molecular weights of heparin at physiological pH. Human plasma fibronectin was shown to bind heparins in both the solution and adsorbed states. Fibronectin was shown to have six active binding sites for heparins which may be sterically

  2. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Park, Yoon-Jeong, E-mail: parkyj@snu.ac.kr [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Dental Regenerative Biotechnology, Dental Research Institute and School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Chung, Chong-Pyoung, E-mail: ccpperio@snu.ac.kr [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Periodontology, College of Dentistry, Seoul National University, Seoul (Korea, Republic of)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer HBP sequence identified from HB-EGF has cell penetration activity. Black-Right-Pointing-Pointer HBP inhibits the NF-{kappa}B dependent inflammatory responses. Black-Right-Pointing-Pointer HBP directly blocks phosphorylation and degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer HBP inhibits nuclear translocation of NF-{kappa}B p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-{alpha} and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-{alpha} and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-{kappa}B-dependent inflammatory responses by directly blocking the phosphorylation and degradation of I{kappa}B{alpha} and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-{kappa}B. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

  3. A Polymer Therapeutic Having Universal Heparin Reversal Activity: Molecular Design and Functional Mechanism.

    Science.gov (United States)

    Kalathottukaren, Manu Thomas; Abbina, Srinivas; Yu, Kai; Shenoi, Rajesh A; Creagh, A Louise; Haynes, Charles; Kizhakkedathu, Jayachandran N

    2017-10-09

    Heparins are widely used to prevent blood clotting during surgeries and for the treatment of thrombosis. However, bleeding associated with heparin therapy is a concern. Protamine, the only approved antidote for unfractionated heparin (UFH) could cause adverse cardiovascular events. Here, we describe a unique molecular design used in the development of a synthetic dendritic polycation named as universal heparin reversal agent (UHRA), an antidote for all clinically used heparin anticoagulants. We elucidate the mechanistic basis for the selectivity of UHRA to heparins and its nontoxic nature. Isothermal titration calorimetry based binding studies of UHRAs having different methoxypolyethylene glycol (mPEG) brush structures with UFH as a function of solution conditions, including ionic strength, revealed that mPEG chains impose entropic penalty to the electrostatic binding. Binding studies confirm that, unlike protamine or N-UHRA (a truncated analogue of UHRA with no mPEG chains), the mPEG chains in UHRA avert nonspecific interactions with blood proteins and provide selectivity toward heparins through a combined steric repulsion and Donnan shielding effect (a balance of Fel and Fsteric). Clotting assays reveal that UHRA with mPEG chains did not adversely affect clotting, and neutralized UFH over a wide range of concentrations. Conversely, N-UHRA and protamine display intrinsic anticoagulant activity and showed a narrow concentration window for UFH neutralization. In addition, we found that mPEG chains regulate the size of antidote-UFH complexes, as revealed by atomic force microscopy and dynamic light scattering studies. UHRA molecules with mPEG chains formed smaller complexes with UFH, compared to N-UHRA and protamine. Finally, fluorescence and ELISA experiments show that UHRA disrupts antithrombin-UFH complexes to neutralize heparin's activity.

  4. Thrombin-inhibitory activity of whale heparin oligosaccharides.

    Science.gov (United States)

    Ototani, N; Kodama, C; Kikuchi, M; Yosizawa, Z

    1984-12-01

    Whale heparin was partially digested with a purified heparinase and the oligosaccharide fractions with 8-20 monosaccharide units were isolated from the digest by gel filtration on Sephadex G-50, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. A marked difference in the inhibitory activity for thrombin in the presence of antithrombin III was observed between the high-affinity fractions for antithrombin III of octasaccharide approximately hexadecasaccharide and those of octadecasaccharide approximately eicosasaccharide. The disaccharide compositions of these hexadeca-, octadeca-, and eicosasaccharides were analyzed by high-performance liquid chromatography after digestion with a mixture of purified heparitinases 1 and 2 and heparinase. The analytical data indicated that the proportions of trisulfated disaccharide (IdUA(2S)alpha 1----4GlcNS(6S)) and disulfated disaccharide (UA1----4GlcNS(6S)) increased with the manifestation of high thrombin-inhibitory activity, while that of monosulfated disaccharide (UA1----4GlcNS) decreased. The present observations, together with those so far reported, suggest that the presence of the former structural elements, specifically IdUA(2S)alpha 1----4GlcNS(6S), as well as the antithrombin III-binding pentasaccharide at the proper positions in the molecules of whale heparin oligosaccharides is essential for the manifestation of high inhibitory activity for thrombin in the presence of antithrombin III. The structural bases for the manifestation of the anticoagulant activity of whale and porcine heparins and their oligosaccharides are also discussed.

  5. Shape-persistent and adaptive multivalency: rigid transgeden (TGD) and flexible PAMAM dendrimers for heparin binding.

    Science.gov (United States)

    Bromfield, Stephen M; Posocco, Paola; Fermeglia, Maurizio; Tolosa, Juan; Herreros-López, Ana; Pricl, Sabrina; Rodríguez-López, Julián; Smith, David K

    2014-07-28

    This study investigates transgeden (TGD) dendrimers (polyamidoamine (PAMAM)-type dendrimers modified with rigid polyphenylenevinylene (PPV) cores) and compares their heparin-binding ability with commercially available PAMAM dendrimers. Although the peripheral ligands are near-identical between the two dendrimer families, their heparin binding is very different. At low generation (G1), TGD outperforms PAMAM, but at higher generation (G2 and G3), the PAMAMs are better. Heparin binding also depends strongly on the dendrimer/heparin ratio. We explain these effects using multiscale modelling. TGD dendrimers exhibit "shape-persistent multivalency"; the rigidity means that small clusters of surface amines are locally well optimised for target binding, but it prevents the overall nanoscale structure from rearranging to maximise its contacts with a single heparin chain. Conversely, PAMAM dendrimers exhibit "adaptive multivalency"; the flexibility means individual surface ligands are not so well optimised locally to bind heparin chains, but the nanostructure can adapt more easily and maximise its binding contacts. As such, this study exemplifies important new paradigms in multivalent biomolecular recognition. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors

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    Ha Na Kim

    2017-05-01

    Full Text Available Heparin and heparan sulfate are structurally-related carbohydrates with therapeutic applications in anticoagulation, drug delivery, and regenerative medicine. This study explored the effect of different bioreactor conditions on the production of heparin/heparan sulfate chains via the recombinant expression of serglycin in mammalian cells. Tissue culture flasks and continuously-stirred tank reactors promoted the production of serglycin decorated with heparin/heparan sulfate, as well as chondroitin sulfate, while the serglycin secreted by cells in the tissue culture flasks produced more highly-sulfated heparin/heparan sulfate chains. The serglycin produced in tissue culture flasks was effective in binding and signaling fibroblast growth factor 2, indicating the utility of this molecule in drug delivery and regenerative medicine applications in addition to its well-known anticoagulant activity.

  7. Interactions between nattokinase and heparin/GAGs.

    Science.gov (United States)

    Zhang, Fuming; Zhang, Jianhua; Linhardt, Robert J

    2015-12-01

    Nattokinase (NK) is a serine protease extracted from a traditional Japanese food called natto. Due to its strong fibrinolytic and thrombolytic activity, NK is regarded as a valuable dietary supplement or nutraceutical for the oral thrombolytic therapy. In addition, NK has been investigated for some other medical applications including treatment of hypertension, Alzheimer's disease, and vitreoretinal disorders. The most widely used clinical anticoagulants are heparin and low molecular weight heparins. The interactions between heparin and proteins modulate diverse patho-physiological processes and heparin modifies the activity of serine proteases. Indeed, heparin plays important roles in almost all of NK's potential therapeutically applications. The current report relies on surface plasmon resonance spectroscopy to examine NK interacting with heparin as well as other glycosaminoglycans (GAGs). These studies showed that NK is a heparin binding protein with an affinity of ~250 nM. Examination with differently sized heparin oligosaccharides indicated that the interaction between NK and heparin is chain-length dependent and the minimum size for heparin binding is a hexasaccharide. Studies using chemically modified heparin showed the 6-O-sulfo as well as the N-sulfo groups but not the 2-O-sulfo groups within heparin, are essential for heparin's interaction with NK. Other GAGs (including HS, DS, and CSE) displayed modest binding affinity to NK. NK also interfered with other heparin-protein interactions, including heparin's interaction with antithrombin and fibroblast growth factors.

  8. Impact of autoclave sterilization on the activity and structure of formulated heparin.

    Science.gov (United States)

    Beaudet, Julie M; Weyers, Amanda; Solakyildirim, Kemal; Yang, Bo; Takieddin, Majde; Mousa, Shaker; Zhang, Fuming; Linhardt, Robert J

    2011-08-01

    The stability of a formulated heparin was examined during its sterilization by autoclaving. A new method to follow loss in heparin binding to the serine protease inhibitor, antithrombin III, and the serine protease, thrombin, was developed using a surface plasmon resonance competitive binding assay. This loss in binding affinity correlated well with loss in antifactor IIa (thrombin) activity as well as antifactor Xa activity as measured using conventional amidolytic assays. Autoclaving also resulted in a modest breakdown of the heparin backbone as confirmed by a slight reduction in number-averaged and weight-averaged molecular weight and an increase in polydispersity. Although no clear changes were observed by nuclear magnetic resonance spectroscopy, disaccharide composition analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry suggested that loss of selected sulfo groups had taken place. It is this sulfo group loss that probably accounts for a decrease in the binding of autoclaved heparin to antithrombin III and thrombin as well as the observed decrease in its amidolytic activity. Copyright © 2011 Wiley-Liss, Inc.

  9. Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration.

    Science.gov (United States)

    Fu, Li; Zhang, Fuming; Li, Guoyun; Onishi, Akihiro; Bhaskar, Ujjwal; Sun, Peilong; Linhardt, Robert J

    2014-05-01

    The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin's core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  10. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)

    Prakash

    capacitation in the female reproductive tract is aided by. HBPs secreted by the male accessory sex glands (Miller et al. 1990). Seminal fluid HBPs are supposed to attach themselves to the sperm surface, especially lipids containing the phosphoryl-choline group, thus allowing heparin-like. GAGs in the female reproductive ...

  11. Structural Basis of Native CXCL7 Monomer Binding to CXCR2 Receptor N-Domain and Glycosaminoglycan Heparin

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    Aaron J. Brown

    2017-02-01

    Full Text Available CXCL7, a chemokine highly expressed in platelets, orchestrates neutrophil recruitment during thrombosis and related pathophysiological processes by interacting with CXCR2 receptor and sulfated glycosaminoglycans (GAG. CXCL7 exists as monomers and dimers, and dimerization (~50 μM and CXCR2 binding (~10 nM constants indicate that CXCL7 is a potent agonist as a monomer. Currently, nothing is known regarding the structural basis by which receptor and GAG interactions mediate CXCL7 function. Using solution nuclear magnetic resonance (NMR spectroscopy, we characterized the binding of CXCL7 monomer to the CXCR2 N-terminal domain (CXCR2Nd that constitutes a critical docking site and to GAG heparin. We found that CXCR2Nd binds a hydrophobic groove and that ionic interactions also play a role in mediating binding. Heparin binds a set of contiguous basic residues indicating a prominent role for ionic interactions. Modeling studies reveal that the binding interface is dynamic and that GAG adopts different binding geometries. Most importantly, several residues involved in GAG binding are also involved in receptor interactions, suggesting that GAG-bound monomer cannot activate the receptor. Further, this is the first study that describes the structural basis of receptor and GAG interactions of a native monomer of the neutrophil-activating chemokine family.

  12. Measurement of phenotype and absolute number of circulating heparin-binding hemagglutinin, ESAT-6 and CFP-10, and purified protein derivative antigen-specific CD4 T cells can discriminate active from latent tuberculosis infection.

    Science.gov (United States)

    Hutchinson, Paul; Barkham, Timothy M S; Tang, Wenying; Kemeny, David M; Chee, Cynthia Bin-Eng; Wang, Yee T

    2015-02-01

    The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154(+) TNF-α(+) cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Immobilization of NaIO4-treated heparin on PEG-modified 316L SS surface for high anti-thrombin-III binding.

    Science.gov (United States)

    Chuang, Tzu-Wen; Lin, Dong-Tsamn; Lin, Feng-Huei

    2008-09-01

    Poor compatibility between blood and metallic coronary artery stents is one reason for arterial restenosis; however, the immobilization of anticoagulant agents on the surface of the stent is a feasible method of improving stent compatibility. Heparin, a well-known anticoagulant, has been frequently used to coat the surfaces of certain biomaterials to attain blood compatibility. The compound 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide has often been utilized for the immobilization of heparin, but the critical carboxyl groups of heparin (with regards to heparin's anticoagulant activity) will be reduced by this method. This study examined possible methods of heparin immobilization without consuming these carboxyl groups. The 316L stainless steel surface was first activated with hexamethylene diisocyanate and then coupled with bis-amine-terminated poly (ethylene glycol) (BA-PEG) so as to create active amine groups. Sodium periodate (NaIO(4); SP) was then used to oxidize heparin to form aldehyde groups. The treated heparin could then be grafted onto the activated surface of the test material without losing its carboxyl groups. Effective surface modification of the hexamethylene diisocyanate-activated and BA-PEG-grafted 316L SS surface was confirmed using Fourier Transform Infrared Spectroscopy, electron spectroscopy for chemical analysis and a water contact angle test. After the heparin was immobilized on the BA-PEG-grafted 316L SS surface by SP, the surface showed an improvement in antithrombrin III (AT III) binding ability, its anticoagulant property, and hemocompatibility in comparison with heparin grafted by 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide.

  14. Sperm in poor quality semen from bulls during heat stress have a lower affinity for binding hydrogen-3 heparin

    Energy Technology Data Exchange (ETDEWEB)

    Ax, R.L.; Gilbert, G.R.; Shook, G.E.

    1987-01-01

    Binding assays with (/sup 3/H) heparin were performed using spermatozoa collected prior to, during, and following summer heat stress to dairy bulls. Ejaculates collected in August 1983 after a period of ambient temperatures exceeding 29.4/sup 0/C exhibited a high frequency of abnormal sperm, and motility was reduced in some samples. Sperm in samples collected during heat stress possessed dissociation constants for binding (/sup 3/H) heparin ranging from 134.5 to 163.2 nmol. In contrast, sperm in semen collected prior to and after heat stress had significantly lower dissociation constants (higher affinity) for (/sup 3/H)heparin, 12.9 to 56.4 nmol. The number of binding sites for (/sup 3/H) heparin on sperm did not change among collection periods. It was concluded that the binding affinity for (/sup 3/H) heparin may reflect membrane integrity of bull sperm.

  15. Specific binding of the glycosaminoglycan /sup 3/H-heparin to bull, monkey, and rabbit spermatozoa in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Handrow, R.R.; Boehm, S.K.; Lenz, R.W.; Robinson, J.A.; Ax, R.L.

    In Vitro binding and some binding parameters of the glycosaminoglycan heparin to viable epididymal or ejaculated bull spermatozoa, ejaculated rabbit spermatozoa, and frozen-thawed rhesus monkey spermatozoa were investigated. Nonspecific binding was affected only by the concentration of /sup 3/H-heparin, whereas specific binding was saturable, reversible, and dependent on the pH, temperature, and calcium concentration of the incubation medium. Magnesium concentration dependence was observed in the presence of calcium but could not be detected in the absence of calcium. Bound /sup 3/H-heparin was displaced by several orders of magnitude greater concentrations of chondroitin sulfate. Scatchard plot analysis suggested multiple binding affinities for /sup 3/H-heparin to spermatozoa. /sup 3/H-heparin was shown to bind to sperm heads and flagella. Fluorescein-labeled heparin bound to acrosomal, postacrosomal, and flagellar membranes. It was concluded that the specific binding of heparin involved a proteinaceous component on, or intercalated with, spermatozoal membranes. Thus, glycosaminoglycans present in the female reproductive tract may contribute to sperm capacitation and enhance the likelihood of successful fertilization in mammals.

  16. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities.

    Science.gov (United States)

    Long, D L; Ulici, V; Chubinskaya, S; Loeser, R F

    2015-09-01

    We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  17. Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

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    Wagner A S Judice

    Full Text Available Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold, and also decrease in k 3 (3.5-fold. The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25-S(-/(His(163-Im(+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

  18. Heparin-binding proteins of human seminal plasma: purification and characterization.

    Science.gov (United States)

    Kumar, Vijay; Hassan, Md Imtaiyaz; Kashav, Tara; Singh, Tej P; Yadav, Savita

    2008-12-01

    Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity-chromatography on Heparin-Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield. (c) 2008 Wiley-Liss, Inc.

  19. Identification of heparin-binding EGF-like growth factor (HB-EGF as a biomarker for lysophosphatidic acid receptor type 1 (LPA1 activation in human breast and prostate cancers.

    Directory of Open Access Journals (Sweden)

    Marion David

    Full Text Available Lysophosphatidic acid (LPA is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA₁₋₆. LPA receptor type 1 (LPA₁ signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA₁ is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. LPA₁ is known to induce IL-6 and IL-8 secretion, as also do LPA₂ and LPA₃. In this work, we first determined the LPA induced early-gene expression profile in three unrelated human cancer cell lines expressing different patterns of LPA receptors (PC3: LPA₁,₂,₆; MDA-MB-231: LPA1,2; MCF-7: LPA₂,₆. Among the set of genes upregulated by LPA only in LPA₁-expressing cells, we validated by QPCR and ELISA that upregulation of heparin-binding EGF-like growth factor (HB-EGF was inhibited by LPA₁-₃ antagonists (Ki16425, Debio0719. Upregulation and downregulation of HB-EGF mRNA was confirmed in vitro in human MDA-B02 breast cancer cells stably overexpressing LPA₁ (MDA-B02/LPA₁ and downregulated for LPA₁ (MDA-B02/shLPA1, respectively. At a clinical level, we quantified the expression of LPA₁ and HB-EGF by QPCR in primary tumors of a cohort of 234 breast cancer patients and found a significantly higher expression of HB-EGF in breast tumors expressing high levels of LPA₁. We also generated human xenograph prostate tumors in mice injected with PC3 cells and found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. All together our results demonstrate that HB-EGF is a new and relevant biomarker with potentially high value in

  20. Effectively enhancing cytotoxic and apoptotic effects of alpha-momorcharin by integrating a heparin-binding peptide.

    Science.gov (United States)

    Tan, Meng-Jie; Cao, Xue-Wei; Li, Peng-Fei; Zhai, Yi-Zhou; Zhou, Yu; Liu, Ye-Jun; Zhao, Jian; Wang, Fu-Jun

    2017-11-01

    Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein, has attracted a great deal of attention because of its antitumor activity. However, the cytotoxicity of α-MMC is limited due to insufficient cellular internalization in cancer cells. To enhance the cytotoxicity of α-MMC, a heparin-binding domain derived from heparin-binding epidermal growth factor (named heparin-binding peptide [HBP]) was used as a cell-penetrating peptide and fused to the C-terminus of α-MMC. This novel α-MMC-HBP fusion protein was expressed and purified with a Ni 2+ -resin. The N-glycosidase activity and DNase activity assay indicated that the introduction of HBP did not interfere with the intrinsic bioactivities of α-MMC. HBP was able to efficiently carry α-MMC into the tested cancer cells and significantly enhance the cytotoxic effects of α-MMC in a dose-dependent manner. This enhanced cytotoxic ability occurred due to the higher level of cell apoptosis induced by α-MMC-HBP, which was demonstrated in western blot analysis in which α-MMC-HBP triggered caspase 8, caspase 9, casapase 3, and PARP more intensely than α-MMC alone. α-MMC-HBP led to an upregulation of cleaved PARP and an increase in the Bax/Bcl-2 ratio. Our study provided a new practical way to greatly improve the antitumor activity of α-MMC, which could significantly expand the pharmaceutical applications of α-MMC. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  1. Heparin-binding protein (HBP): an early marker of respiratory failure after trauma?

    Science.gov (United States)

    Johansson, J; Brattström, O; Sjöberg, F; Lindbom, L; Herwald, H; Weitzberg, E; Oldner, A

    2013-05-01

    Trauma and its complications contribute to morbidity and mortality in the general population. Trauma victims are susceptible to acute respiratory distress syndrome (ARDS) and sepsis. Polymorphonuclear leucocytes (PMNs) are activated after trauma and there is substantial evidence of their involvement in the development of ARDS. Activated PMNs release heparin-binding protein (HBP), a granule protein previously shown to be involved in acute inflammatory reactions. We hypothesised that there is an increase in plasma HBP content after trauma and that the increased levels are related to the severity of the trauma or later development of severe sepsis and organ failure (ARDS). We investigated HBP in plasma samples within 36 h from trauma in 47 patients admitted to a level one trauma centre with a mean injury severity score (ISS) of 26 (21-34). ISS, admission sequential organ failure assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE) II scores were recorded at admission. ARDS and presence of severe sepsis were determined daily during intensive care. We found no correlation between individual maximal plasma HBP levels at admission and ISS, admission SOFA or APACHE II. We found, however, a correlation between HBP levels and development of ARDS (P = 0.026, n = 47), but not to severe sepsis. HBP is a potential biomarker candidate for early detection of ARDS development after trauma. Further research is required to confirm a casual relationship between plasma HBP and the development of ARDS. © 2013 The Acta Anaesthesiologica Scandinavica Foundation.

  2. Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II).

    Science.gov (United States)

    Rozenberg, G I; Espada, J; de Cidre, L L; Eiján, A M; Calvo, J C; Bertolesi, G E

    2001-01-01

    The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.

  3. Generation and characterization of conditional heparin-binding EGF-like growth factor knockout mice.

    Directory of Open Access Journals (Sweden)

    Atsushi Oyagi

    Full Text Available Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b altered dopamine and serotonin levels in the brain, (c decreases in spine density in neurons of the prefrontal cortex, (d reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA receptor and post-synaptic protein-95 (PSD-95, (e decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.

  4. hMPV Lineage Nomenclature and Heparin Binding

    Directory of Open Access Journals (Sweden)

    David Gordon

    2013-10-01

    Full Text Available Human metapneumovirus (hMPV, first described in 2001 [1], is responsible for causing serious respiratory illness in young children, the elderly and immunocompromised patients. Four distinct lineages of hMPV have been identified with the original nomenclature for these subgroups (A1, A2, B1 and B2, reported by van den Hoogen et al. [2], utilised by many. An alternate terminology (1A, 1B, 2A and 2B was also published by Ishiguro et al. in 2004 [3] which has been adopted by others. However, this has caused some confusion in the interpretation of publication results as the terminology is similar yet describes different subtypes. As a result, a number of investigators have made a submission to the International Committee on Taxonomy of Viruses (ICTV, ICTV taxonomic proposal 2012.012V for the official adoption of the original terminology as an approved nomenclature for hMPV [4]. We welcome this officially approved nomenclature which should provide clarification of these subtypes in future. Therefore to assist with the interpretation of our recently published research in the 2012 special issue of Viruses: Pneumoviruses and Metapneumoviruses entitled “Diversity in Glycosaminoglycan Binding Amongst hMPV G Protein Lineages” [5] we have updated the Figure 3 in this letter (see Figure 1, showing the proposed ICTV terminology compared to the Ishiguro classification (used in our publication. Note that in the original publication the alphanumeric order for the Ishiguro classification was transposed (e.g., 1A was referred to as A1.

  5. Thermodynamic parameters associated with the binding of adrenalin and norephedrine to heparin

    Energy Technology Data Exchange (ETDEWEB)

    Al-Ali, A.K.; Buchanan, J.D.; Power, D.M. (Salford Univ. (UK). Dept. of Biochemistry); Butler, J. (Christie Hospital and Holt Radium Inst., Manchester (UK). Paterson Labs.)

    1983-04-01

    Pulse radiolysis has been used to determine the thermodynamic parameters (..delta..G', ..delta..H' and ..delta..S') governing the binding of adrenalin and norephedrine to heparin. These complexes were completely dissociated by increasing concentrations of inorganic salts. Lower concentrations of divalent cations (e.g. Ca/sup 2 +/) than of monovalent cations (e.g. Na/sup +/) were necessary to effect dissociation of the complex. For each interaction an increase in drug binding occurred as the temperature was increased from ambient. However, a transition temperature was observed (48/sup 0/C) above which the drug was progressively released as the temperature was increased. These observations are discussed in terms of conformational changes induced in the polymer below and above its melting temperature.

  6. Heparin-binding correlates with increased efficiency of AAV1- and AAV6-mediated transduction of striated muscle, but negatively impacts CNS transduction.

    Science.gov (United States)

    Arnett, A L H; Beutler, L R; Quintana, A; Allen, J; Finn, E; Palmiter, R D; Chamberlain, J S

    2013-05-01

    Gene delivery vectors derived from adeno-associated virus (AAV) have great potential as therapeutic agents. rAAV1 and rAAV6, efficiently target striated muscle, but the mechanisms that determine their tropism remain unclear. It is known that AAV6, but not AAV1, interacts with heparin-sulfate proteoglycans (HSPG). HSPGs are not primary receptors for AAV6, but heparin interactions may affect tissue tropism and transduction. To investigate these possibilities, we generated rAAV1 and rAAV6 capsids that do or do not bind heparin. We evaluated the transduction profile of these vectors in vivo across multiple routes of administration, and found that heparin-binding capability influences tissue transduction in striated muscle and neuronal tissues. Heparin-binding capsids transduce striated muscle more efficiently than non-binding capsids, via both intramuscular and intravenous injection. However, rAAV6 achieved greater muscle transduction than the heparin-binding rAAV1 variant, suggesting that there are additional factors that influence differences in transduction efficiency between AAV1 and AAV6. Interestingly, the opposite trend was found when vectors were delivered via intracranial injection. Non-binding vectors achieved robust and widespread gene expression, whereas transduction via heparin-binding serotypes was substantially reduced. These data indicate that heparin-binding capability is an important determinant of transduction that should be considered in the design of rAAV-mediated gene therapies.

  7. Heparin-hyaluronic acid hydrogel in support of cellular activities of 3D encapsulated adipose derived stem cells.

    Science.gov (United States)

    Gwon, Kihak; Kim, Eunsol; Tae, Giyoong

    2017-02-01

    We have developed stem cell-responsive, heparin-hyaluronic acid (Hep-HA) hydrogel, crosslinked by thiolated heparin (Hep-SH) and methacrylated hyaluronic acid (HA-MA) via visible light mediated, thiol-ene reaction. Physical properties of the hydrogel (gelation time, storage modulus, and swelling ratio) were tunable by adjusting light intensity, initiator/polymer concentration, and precursor pH. Culture of human adipose derived mesenchymal stem cells (ADSCs) using this hydrogel was characterized and compared with the control hydrogels including Hep-PEG hydrogel, PEG-HA hydrogel. Sufficient initial adhesion and continuous proliferation of ADSCs in 2D were observed on both heparin-containing hydrogels (Hep-HA and Hep-PEG hydrogel) in contrast to no adhesion of ADSCs on PEG-HA hydrogel. On the other hand, in the case of 3D culture of encapsulated ADSCs, efficient cellular activities such as spreading, proliferation, migration, and differentiation of ADSCs were only observed in soft Hep-HA hydrogel compared to Hep-PEG or PEG-HA hydrogel with the similar modulus. The upregulated expressions of hyaluronidases in ADSCs encapsulated in Hep-HA hydrogel compared to the control hydrogels and effective degradation of the hydrogel by hyaluronidase imply that the degradation of hydrogel was necessary for 3D cellular activities. Thus, Hep-HA hydrogel, where heparin acts as a binding domain for ADSCs and HA acts as a degradation site by cell secreted enzymes, was efficient for 3D culture of human ADSCs without any additional modification using biological/chemical molecules. Stem cell-responsive hydrogel composed of heparin and hyaluronic acid was prepared by visible light-mediated thiol-ene reaction. Without additional modification using functional peptides for cell adhesion and matrix degradation, ADSCs encapsulated in this hydrogel showed efficient cellular activities such as spreading, proliferation, migration, and differentiation of ADSCs whereas control hydrogels missing

  8. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S. (Oregon HSU)

    2012-05-24

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  9. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

    2012-02-05

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  10. Characterizations of heparin-binding proteins in human urine by affinity purification-mass spectrometry and defining "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif.

    Science.gov (United States)

    Manissorn, Juthatip; Thongboonkerd, Visith

    2016-06-16

    Heparin-binding proteins (HBPs) are considered as potential modulators of kidney stone formation. However, HBPs had not been characterized in the urine previously. In this study, we applied affinity purification-mass spectrometry (AP-MS) using cellufine sulfate column chromatography and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-Q-TOF MS/MS) to identify HBPs in normal human urine. Using this approach, 83 HBPs were identified, including those involved in metabolic process, cellular process, immune system, developmental process, response to stimuli, cell communication, transport, cell adhesion and others. The AP-MS data were confirmed by Western blot analysis and chemico-protein interactions analysis using STITCH tool. In addition, 59, 55 and 51 identified HBPs had the known heparin-binding motifs "XBBXnBX", "XBXnBBX" and "XBBBnX", respectively. Moreover, a novel heparin-binding motif "L-x(2,3)-A-x(0,1)-L" was found in 58 identified HBPs using PRATT tool. The sensitivity and specificity of this novel motif were 85% and 100%, respectively, by validation using 20 known HBPs and 11 non-HBPs. We report herein for the first time a large number of HBPs in normal human urine and defined "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif. These findings will be useful to further understand the renal physiology and may also lead to identification of novel modulators of kidney stone formation. Heparin-binding proteins (HBPs) have several important roles in various biological processes, including kidney stone formation. However, HBPs had not been characterized in the urine. Our present work using affinity purification coupled to mass spectrometry (AP-MS) is the first large-scale study on HBPs in human urine. In addition to the three known heparin-binding motifs, "XBBXnBX", "XBXnBBX", and "XBBBnX", we successfully defined the amino acid pattern "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif. These findings will be useful to further

  11. Prophylactic heparin in patients with severe sepsis treated with drotrecogin alfa (activated)

    NARCIS (Netherlands)

    Levi, Marcel; Levy, Mitchell; Williams, Mark D.; Douglas, Ivor; Artigas, Antonio; Antonelli, Massimo; Wyncoll, Duncan; Janes, Jonathan; Booth, Frank V.; Wang, Dazhe; Sundin, David P.; Macias, William L.

    2007-01-01

    RATIONALE: Patients with severe sepsis frequently receive prophylactic heparin during drotrecogin alfa (activated) (DrotAA) treatment due to risk of venous thromboembolic events (VTEs). Biological plausibility exists for heparin to reduce DrotAA efficacy and/or increase bleeding. OBJECTIVES:

  12. Dimerisation and structural integrity of Heparin Binding Hemagglutinin A from Mycobacterium tuberculosis: implications for bacterial agglutination.

    Science.gov (United States)

    Esposito, Carla; Carullo, Paola; Pedone, Emilia; Graziano, Giuseppe; Del Vecchio, Pompea; Berisio, Rita

    2010-03-19

    Heparin Binding Hemagglutinin A (HBHA) is hitherto the sole virulence factor associated with tuberculosis dissemination from the lungs, the site of primary infection, to epithelial cells. We have previously reported the solution structure of HBHA, a dimeric and elongated molecule. Since oligomerisation of HBHA is associated with its ability to induce bacterial agglutination, we investigated this process using experimental and modelling techniques. We here identified a short segment of HBHA whose presence is mandatory for the stability of folded conformation, whose denaturation is a reversible two-state process. Our data suggest that agglutination-driven cell-cell interactions do not occur via association of HBHA monomers, nor via association of HBHA dimers and open the scenario to a possible trans-dimerisation process. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Epidermal transformation leads to increased perlecan synthesis with heparin-binding-growth-factor affinity

    DEFF Research Database (Denmark)

    Tapanadechopone, P; Tumova, S; Jiang, X

    2001-01-01

    cells was significantly increased in both mRNA and protein levels. JB6 perlecan was exclusively substituted with heparan sulphate, whereas that of RT101 contained some additional chondroitin sulphate. Detailed structural analysis of the heparan sulphate (HS) chains from perlecan of both cell types...... revealed that their overall sulphation and chain length were similar (approximately 60 kDa), but the HS chains of tumour-cell-derived perlecan were less sulphated. This resulted from reduced 2-O- and 6-O-sulphation, but not N-sulphation, and an increase in the proportion of unsulphated disaccharides....... Despite this, the heparan sulphate of RT101- and JB6-derived perlecan bound fibroblast growth factor-1, -2, -4 and -7 and heparin-binding epidermal growth factor with similar affinity. Therefore abundant tumour-derived perlecan may support the angiogenic responses seen in vivo and be a key player...

  14. Heparin-binding-hemagglutinin-induced IFN-gamma release as a diagnostic tool for latent tuberculosis.

    Directory of Open Access Journals (Sweden)

    Jean-Michel Hougardy

    Full Text Available BACKGROUND: The detection of latent tuberculosis infection (LTBI is a major component of tuberculosis (TB control strategies. In addition to the tuberculosis skin test (TST, novel blood tests, based on in vitro release of IFN-gamma in response to Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 (IGRAs, are used for TB diagnosis. However, neither IGRAs nor the TST can separate acute TB from LTBI, and there is concern that responses in IGRAs may decline with time after infection. We have therefore evaluated the potential of the novel antigen heparin-binding hemagglutinin (HBHA for in vitro detection of LTBI. METHODOLOGY AND PRINCIPAL FINDINGS: HBHA was compared to purified protein derivative (PPD and ESAT-6 in IGRAs on lymphocytes drawn from 205 individuals living in Belgium, a country with low TB prevalence, where BCG vaccination is not routinely used. Among these subjects, 89 had active TB, 65 had LTBI, based on well-standardized TST reactions and 51 were negative controls. HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI. PPD-based tests yielded 90.00% sensitivity and 70.00% specificity for the detection of LTBI, whereas the sensitivity and specificity for the ESAT-6-based tests were 40.74% and 90.91%, and those for the HBHA-based tests were 92.06% and 93.88%, respectively. The QuantiFERON-TB Gold In-Tube (QFT-IT test applied on 20 LTBI subjects yielded 50% sensitivity. The HBHA IGRA was not influenced by prior BCG vaccination, and, in contrast to the QFT-IT test, remote (>2 years infections were detected as well as recent (<2 years infections by the HBHA-specific test. CONCLUSIONS: The use of ESAT-6- and CFP-10-based IGRAs may underestimate the incidence of LTBI, whereas the use of HBHA may combine the operational advantages of IGRAs with high sensitivity and specificity for latent infection.

  15. Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

    Energy Technology Data Exchange (ETDEWEB)

    Han, Young-Hyun; Garron, Marie-Line; Kim, Hye-Yeon; Kim, Wan-Seok; Zhang, Zhenqing; Ryu, Kyeong-Seok; Shaya, David; Xiao, Zhongping; Cheong, Chaejoon; Kim, Yeong Shik; Linhardt, Robert J.; Jeon, Young Ho; Cygler, Miroslaw; (SNU); (Korea BSI); (McGill); (UST-Korea); (Rensselaer)

    2010-01-12

    Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a {beta}-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an ({alpha}/{alpha}){sub 6} fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.

  16. The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain

    DEFF Research Database (Denmark)

    Lorentsen, R H; Graversen, Jonas Heilskov; Caterer, N R

    2000-01-01

    Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded...... in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element....... Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6...

  17. Anti-angiogenic activity and antitumor efficacy of amphiphilic twin drug from ursolic acid and low molecular weight heparin

    Science.gov (United States)

    Cheng, Wenming; Zohra Dahmani, Fatima; Zhang, Juan; Xiong, Hui; Wu, Yuanyuan; Yin, Lifang; Zhou, Jianping; Yao, Jing

    2017-02-01

    Heparin, a potential blood anti-coagulant, is also known for its binding ability to several angiogenic factors through electrostatic interactions due to its polyanionic character. However, the clinical application of heparin for cancer treatment is limited by several drawbacks, such as unsatisfactory therapeutic effects and severe anticoagulant activity that could induce hemorrhaging. Herein, low molecular weight heparin (LMWH) was conjugated to ursolic acid (UA), which is also an angiogenesis inhibitor, by binding the amine group of aminoethyl-UA (UA-NH2) with the carboxylic groups of LMWH. The resulting LMWH-UA conjugate as an amphiphilic twin drug showed reduced anticoagulant activity and could also self-assemble into nanomicelles with a mean particle size ranging from 200-250 nm. An in vitro endothelial tubular formation assay and an in vivo Matrigel plug assay were performed to verify the anti-angiogenic potential of LMWH-UA. Meanwhile, the in vivo antitumor effect of LMWH-UA was also evaluated using a B16F10 mouse melanoma model. LMWH-UA nanomicelles were shown to inhibit angiogenesis both in vitro and in vivo. In addition, the i.v. administration of LMWH-UA to the B16F10 tumor-bearing mice resulted in a significant inhibition of tumor growth as compared to the free drug solutions. These findings demonstrate the therapeutic potential of LMWH-UA as a new therapeutic remedy for cancer therapy.

  18. An Antithrombin-Heparin Complex Increases the Anticoagulant Activity of Fibrin Clots

    Directory of Open Access Journals (Sweden)

    Lesley J. Smith

    2008-01-01

    Full Text Available Clotting blood contains fibrin-bound thrombin, which is a major source of procoagulant activity leading to clot extension and further activation of coagulation. When bound to fibrin, thrombin is protected from inhibition by antithrombin (AT + heparin but is neutralized when AT and heparin are covalently linked (ATH. Here, we report the surprising observation that, rather than yielding an inert complex, thrombin-ATH formation converts clots into anticoagulant surfaces that effectively catalyze inhibition of thrombin in the surrounding environment.

  19. Mycobacterial heparin-binding haemagglutinin adhesion-induced interferon & antibody for detection of tuberculosis.

    Science.gov (United States)

    Sun, Zhaogang; Nie, Lihui; Zhang, Xuxia; Li, Yan; Li, Chuanyou

    2011-04-01

    Mycobacterial heparin-binding haemagglutinin adhesin (HBHA) plays an important role in humoral and cellular immune response and is a potential diagnostic tool for tuberculosis (TB) serodiagnosis. This study was carried out to assess the usefulness of HBHA in TB clinics for differential diagnosis of pulmonary and extra-pulmonary TB (PTB, EPTB). In this study, 165 outpatients and 133 healthy volunteers were included to investigate the role of HBHA in TB diagnosis including the serodiagnostic tests and the interferon-γ release assays (IGRAs). The healthy volunteers were all without BCG vaccination including 73 subjects with purified protein derivative (PPD) (-) and 60 ones with PPD (+) (that is P-B- and P+B-). Of all the 165 outpatients 77 were PTB and 88 were EPTB. HBHA protein was used for serodiagnostic tests and IGRAs in peripheral blood mononuclear cells. HBHA-specific antibody levels in the serum of healthy subjects were significantly different from the patients with PTB or EPTB (PEPTB with limited sensitivity (77.08%; 95%CI, 62.69 to 87.97%) and specificity (87.50%; 95%CI, 74.75 to 95.27%). IFN-γ levels in the healthy (P+B- and P-B-) groups were significantly different (PEPTB subjects showed no difference in IFN-γ production. HBHA serodiagnostic test with IGRAs had the limited potential for use as auxiliary tools for the differential diagnosis of PTB and EPTB, since both methods showed low sensitivity and specificity.

  20. Comparative study of heparin-binding proteins profile of Murrah buffalo (Bubalus bubalis semen

    Directory of Open Access Journals (Sweden)

    S. S. Ramteke

    2014-09-01

    Full Text Available Aim: The experiment was conducted to study the total seminal plasma protein (TSPP and heparin-binding proteins (HBPs in relation to initial semen quality of buffalo bull. Materials and Methods: Semen from two Murrah buffalo bulls (bull no. 605 and 790 with mass motility of ≥3+ were used for the study and categorized into three groups (Group I- Mass motility 3+, Group II- Mass motility 4+ and Group III- Mass motility 5+. Seminal plasma from semen was separated by centrifugation. HBPs was isolated and purified from heparin-agarose affinity column by modified elution buffer. TSPP and isolated HBPs concentration was estimated by Lowry’s method. The purified HBPs were resolved on Sodium dodecyl sulfate polyacrylamide gel electrophoresis to check the protein profile of two bulls. Results: The mean values of TSPP concentrations in bull no. 605 and 790 in Group I, II and III were 30.64±0.12, 31.66±0.09, 32.53±0.19 and 28.51±0.09, 29.49±0.15, 30.45±0.17 mg/mL, respectively. The mean values of HBPs concentrations in bull no. 605 and 790 in Group I, II and III were 3.11±0.07, 3.32±0.06, 3.46±0.08 and 2.51±0.08, 2.91±0.05, 3.10±0.03 mg/mL, respectively. Both the values of TSPP and HBPs were significantly higher (p<0.01 in bull no. 605 when compared to 790 in all the three groups. 31 kDa HBP was more intensely present in bull no. 605, thus may indicate its superiority over bull no. 790 in relation to fertility potential. Conclusion: TSPP and HBPs shows variation in concentration with respect to initial semen quality. Furthermore, presence of fertility related 31 kDa HBPs in one of the bull may be an indication of high fertility of a bull. In future, in-vivo and in-vitro correlative study on larger basis is needed for the establishment of fertility-related HBPs in semen which might establish criteria for selection of buffalo bull with high fertility potential.

  1. Heparin inhibits Ca2+/calmodulin-dependent kinase II activation and c-fos induction in mesangial cells.

    OpenAIRE

    Miralem, T; Templeton, D M

    1998-01-01

    Like vascular smooth-muscle cells, rat mesangial cells (RMCs) display an anti-mitogenic response to heparin. In particular, heparin partially suppresses the ability of quiescent RMCs to enter the cell cycle and induce c-fos expression. When the mitogenic stimulus is serum, phorbol ester or platelet-derived growth factor, this response appears to result from the ability of heparin to suppress activation of the extracellular-signal-regulated kinase family of mitogen-activated protein kinases. H...

  2. Manipulating the surface active and anticoagulant properties of heparin through amphiphilic molecular constructs

    Science.gov (United States)

    Mintz, Rosita Candida

    Cardiovascular devices implanted within the vasculature are subjected to non-specific adsorption of plasma proteins. This initiates the blood coagulation cascade and platelet adhesion and activation, leading to thrombus formation. In this thesis Heparin Alkyl Diblock (HAD) surfactants were developed to improve the blood compatibility of cardiovascular biomaterials. The material designs involved using heparin, a natural anticoagulant, to inhibit coagulation pathway enzymes and mimic the cell glycocalyx to provide a repulsive force field to inhibit non-specific protein adsorption. Type AB linear (HAD Cn, n = 6,10,12,18) and branched (HAD nx 18, n = 2,3,4) heparin surfactants were synthesized by end point coupling primary and secondary alkyl amines to heparin via reductive amination. Surfactant yields (83--4%) and anticoagulant activity (149.8 +/- 3.7--39.6 +/- 0.6 IU/mg) decreased with increased branching and hydrocarbon number. Surfactant adsorption, self assembly and molecular packing of HAD surfactants at the air/liquid and liquid/solid interface were a function of the number of hydrocarbons in the surfactant alkyl segment and the presence or absence of an ionic liquid phase. Increased molecular packing was observed at the air/PBS and PBS/graphite interface, relative to aqueous interfaces, resulting from buffer cations shielding heparin's negatively charged sulfate and carboxyl groups. At the PBS/graphite interface, the surfactant's apparent heparin head group cross section decreased in diameter (1.84 to 1.05 nm) and increased in tilt angle (75.7 to 81.9°) with increasing alkyl carbon number (n = 6 to 18). The heparin head group reached a minimum diameter, equivalent to the surfactant's diameter at the air/PBS interface (0.57 nm) just prior to 36 hydrocarbons in the surfactant. For surfactants with 36 to 78 hydrocarbons, the surfactant's heparin head group oriented normal to the graphite surface and alkyl overlap or interdigitation increased (0.02 to 0.59 nm

  3. Single amino acid changes can influence titer, heparin binding, and tissue tropism in different adeno-associated virus serotypes.

    Science.gov (United States)

    Wu, Zhijian; Asokan, Aravind; Grieger, Joshua C; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Samulski, R Jude

    2006-11-01

    Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors.

  4. Single Amino Acid Changes Can Influence Titer, Heparin Binding, and Tissue Tropism in Different Adeno-Associated Virus Serotypes▿

    OpenAIRE

    Wu, Zhijian; Asokan, Aravind; Joshua C Grieger; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Samulski, R. Jude

    2006-01-01

    Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 c...

  5. Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase

    DEFF Research Database (Denmark)

    Bowler, Russell P; Nicks, Mike; Olsen, Dorte Aa

    2002-01-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin...

  6. Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins.

    Science.gov (United States)

    Bjarnadóttir, Stefanía Guðrún; Flengsrud, Ragnar

    2014-01-01

    Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2mL, 10.7mg capacity) and piscine heparin (5mL, 2.7mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16cm×16cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1mg anti-human fibrinogen in 8.4mL resin) and serum albumin (0.42mg binding capacity in 14mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50cm×75μm)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Probing the impact of GFP tagging on Robo1-heparin interaction.

    Science.gov (United States)

    Zhang, Fuming; Moniz, Heather A; Walcott, Benjamin; Moremen, Kelley W; Wang, Lianchun; Linhardt, Robert J

    2014-05-01

    Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change.

  8. Antithrombin inactivation by neutrophil elastase requires heparin.

    Science.gov (United States)

    Jordan, R E; Nelson, R M; Kilpatrick, J; Newgren, J O; Esmon, P C; Fournel, M A

    1989-09-11

    In certain thrombotic states, large declines in the levels of functional circulating antithrombin occur, which may reflect the highly active nature of the endothelial surface in suppressing excessive amounts of activated coagulation enzymes. Alternatively, we have recently observed an unexpected and paradoxical in vitro functioning of heparin that could result in the inactivation of antithrombin in pathologic conditions. Specifically, antithrombin was rendered nonfunctional as an inhibitor of clotting enzymes as a result of a limited, heparin-dependent cleavage by neutrophil elastase. This inactivation occurred only in the presence of the active anticoagulant heparin fraction, which suggested that the heparin-antithrombin complex was the substrate for elastase attack. Interestingly, neutrophil elastase was found to bind tightly to heparin and heparin-like materials. Neutrophil elastase has been previously linked to nonspecific proteinolysis occurring in inflammatory thrombotic reactions. This affinity of both antithrombin and elastase for heparin suggests a novel mechanism of potential specificity. An important component of this hypothesis is the localization of the elastase/antithrombin reaction away from the high circulating levels of elastase inhibitors. The proposed inactivation of antithrombin on the vascular surface would likely occur only in pathologic states associated with neutrophil sequestration and activation. Nevertheless, this mechanism could lead to a localized reversal of the nonthrombogenic nature of the endothelium and potentially lead to significant reductions of functional antithrombin in certain disease states.

  9. Heparin Increases Food Intake through AgRP Neurons

    Directory of Open Access Journals (Sweden)

    Canjun Zhu

    2017-09-01

    Full Text Available Although the widely used anticoagulant drug heparin has been shown to have many other biological functions independent of its anticoagulant role, its effects on energy homeostasis are unknown. Here, we demonstrate that heparin level is negatively associated with nutritional states and that heparin treatment increases food intake and body weight gain. By using electrophysiological, pharmacological, molecular biological, and chemogenetic approaches, we provide evidence that heparin increases food intake by stimulating AgRP neurons and increasing AgRP release. Our results support a model whereby heparin competes with insulin for insulin receptor binding on AgRP neurons, and by doing so it inhibits FoxO1 activity to promote AgRP release and feeding. Heparin may be a potential drug target for food intake regulation and body weight control.

  10. Structural Characterization of the E2 Domain of APL-1, a C. Elegans Homolog of Human Amyloid Precursor Protein, and its Heparin Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Hoopes, J.; Liu, X; Xu, X; Demeler, B; Folta-Stogniew, E; Li, C; Ha, Y

    2010-01-01

    The amyloid {beta}-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.

  11. Evaluation of the activated partial thromboplastin time (APTT) sensitivity to heparin using five commercial reagents: implications for therapeutic monitoring.

    Science.gov (United States)

    Manzato, F; Mengoni, A; Grilenzoni, A; Lippi, G

    1998-12-01

    Heparin is an effective drug for prevention and treatment of thromboembolic conditions. Although several biological assays have been proposed for monitoring unfractionated heparin therapy, the measurement of the activated partial thromboplastin time (APTT) is the most widely employed test, and the overall risk of thromboembolic episodes was markedly reduced by maintaining APTT ratios above 1.5. However, the adjustment of the heparin therapy on the basis of APTT presents several questions which are still unresolved. Major discrepancies were found in APTTs performed using different reagents in both ex vivo and in vitro heparinized samples and occasionally with different lots of the same reagents; poor correlation was observed between APTT values and plasma heparin concentrations. In order to gain further insights into this phenomenon, we analysed the sensitivity to heparin of five commercial reagents for APTT measurement in 19 ex vivo heparinized samples. Differences were observed; correlation coefficients ranged from 0.820 to 0.985 and slopes of linear regressions from 0.26 to 1.14. Moreover, unsatisfactory correlations were obtained when APTT ratios were compared with heparin plasma concentrations in the same patients' samples. In the heparin therapeutic range of 0.35 - 0.70 U/ml, reagent-dependent differences were observed in the corresponding APTT values. These results point out a critical role of the assay methodology in monitoring heparin therapy by APTT. We suggest that reference materials and methods should be urgently identified, a universally agreed scale for reporting results should be established and reference ranges for the unfractionated heparin therapy should be reconsidered taking on account the reagent employed.

  12. Simultaneous immobilization of heparin and gentamicin on polypropylene textiles: a dual therapeutic activity.

    Science.gov (United States)

    Degoutin, Stéphanie; Jimenez, Maude; Chai, Feng; Pinalie, Thibaut; Bellayer, Severine; Vandenbossche, Marianne; Neut, Christel; Blanchemain, Nicolas; Martel, Bernard

    2014-11-01

    The aim of this work was to prepare a nonwoven polypropylene (PP) textile functionalized with bioactive molecules in order to improve simultaneously anticoagulation and antibacterial properties. The immobilization of either heparin (anticoagulation agent) or gentamicin (aminoglycoside class antibiotic) alone has already been proven to be effective on PP nonwoven textiles. In this work, we managed to go further, by immobilizing both heparin and gentamicin at the same time on one unique textile. A successive immersion in different heparin and gentamicin bathes successfully led to a dual drug coated textile, as confirmed by several characterization techniques (Fourier transform infrared-attenuated total reflectance, X-ray photoelectron spectroscopy, and scanning electron microscopy). The immobilization times were varied in order to determine the best compromise between cytocompatibility, anticoagulant effect, and antimicrobial activity. Short immersion times in gentamicin solutions confer very good antimicrobial activity to the textile and avoid cytotoxicity, whereas long immersion times in heparin solution were necessary to observe a significant anticoagulant effect. © 2013 Wiley Periodicals, Inc.

  13. Heparin-binding EGF-like growth factor mediates oxyhemoglobin-induced suppression of voltage-dependent potassium channels in rabbit cerebral artery myocytes.

    Science.gov (United States)

    Koide, Masayo; Penar, Paul L; Tranmer, Bruce I; Wellman, George C

    2007-09-01

    Oxyhemoglobin (OxyHb) can suppress voltage-dependent K(+) channel (K(V)) currents through protein tyrosine kinase activation, which may contribute to cerebral vasospasm following subarachnoid hemorrhage. Here we have tested the hypothesis that shedding of heparin-binding EGF-like growth factor (HB-EGF) and the resulting activation of the tyrosine kinase EGF receptor (EGFR) underlie OxyHb-induced K(V) channel suppression in the cerebral vasculature. With the use of the conventional whole cell patch-clamp technique, two EGFR ligands, EGF and HB-EGF, were found to mimic OxyHb-induced K(V) suppression in rabbit cerebral artery myocytes. K(V) current suppression by OxyHb or EGF ligands was eliminated by a specific EGFR inhibitor, AG-1478, but was unaffected by PKC inhibition. Compounds (heparin and CRM-197) that specifically interfere with HB-EGF signaling eliminated OxyHb-induced K(V) suppression, suggesting that HB-EGF is the EGFR ligand involved in this pathway. HB-EGF exists as a precursor protein that, when cleaved by matrix metalloproteases (MMPs), causes EGFR activation. MMP activation was detected in OxyHb-treated arteries by gelatin zymography. Furthermore, the MMP inhibitor (GM-6001) abolished OxyHb-induced K(V) current suppression. We also observed K(V) current suppression due to EGFR activation in human cerebral artery myocytes. In conclusion, these data demonstrate that OxyHb induces MMP activation, causing HB-EGF shedding and enhanced EGFR activity, ultimately leading to K(V) channel suppression. We propose that EGFR-mediated K(V) suppression contributes to vascular pathologies, such as cerebral vasospasm, and may play a more widespread role in the regulation of regional blood flow and peripheral resistance.

  14. Demonstration of specific binding of heparin to Plasmodium falciparum-infected vs. non-infected red blood cells by single-molecule force spectroscopy

    Science.gov (United States)

    Valle-Delgado, Juan José; Urbán, Patricia; Fernàndez-Busquets, Xavier

    2013-04-01

    Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level.Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force

  15. Novel Feature of Mycobacterium avium subsp. paratuberculosis, Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin

    Science.gov (United States)

    Lefrancois, Louise H.; Bodier, Christelle C.; Cochard, Thierry; Canepa, Sylvie; Raze, Dominique; Lanotte, Philippe; Sevilla, Iker A.; Stevenson, Karen; Behr, Marcel A.; Locht, Camille

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be

  16. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    Science.gov (United States)

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBA's effects on fused BSC cultures.

  17. Heparin binding chitosan derivatives for production of pro-angiogenic hydrogels for promoting tissue healing.

    Science.gov (United States)

    Yar, Muhammad; Shahzad, Sohail; Shahzadi, Lubna; Shahzad, Sohail Anjum; Mahmood, Nasir; Chaudhry, Aqif Anwar; Rehman, Ihtesham Ur; MacNeil, Sheila

    2017-05-01

    Our aim was to develop a biocompatible hydrogel that could be soaked in heparin and placed on wound beds to improve the vasculature of poorly vascularized wound beds. In the current study, a methodology was developed for the synthesis of a new chitosan derivative (CSD-1). Hydrogels were synthesized by blending CSD-1 for either 4 or 24h with polyvinyl alcohol (PVA). The physical/chemical interactions and the presence of specific functional groups were confirmed by Fourier transform infrared (FT-IR) spectroscopy and proton nuclear magnetic resonance (1H NMR). The porous nature of the hydrogels was confirmed by scanning electron microscopy (SEM). Thermal gravimetric analysis (TGA) showed that these hydrogels have good thermal stability which was slightly increased as the blending time was increased. Hydrogels produced with 24h of blending supported cell attachment more and could be loaded with heparin to induce new blood vessel formation in a chick chorionic allantoic membrane assay. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Photochemically synthesized heparin-based silver nanoparticles: an antimicrobial activity study

    Science.gov (United States)

    Rodriguez-Torres, Maria del Pilar; Acosta-Torres, Laura Susana; Díaz-Torres, Luis Armando

    2017-08-01

    The antimicrobial activity of silver nanoparticles has been extensively studied in the last years. Such nanoparticles constitute a potential and promising approach for the development of new antimicrobial systems especially due to the fact that several microorganisms are developing resistance to some already existing antimicrobial agents, therefore making antibacterial and antimicrobial studies on alternative materials necessary to overcome this issue. Silver nanoparticle concentration and size are determining factors on the antimicrobial activity of these nano systems. Heparin is a polysaccharide that belongs to the glycosaminoglycans (GAGs) family, molecules formed by a base disaccharide whose components are joined by a glycosidic linkage that is a repeating unit along their structure. It is highly sulfated making it a negatively charged material that is also widely used as an anticoagulant in Medicine because its biocompatibility besides it is also produced within the human body, specifically in the mast cells. Heparin alone possesses antimicrobial activity although it has not been studied very much in detail, it only has been demonstrated that it inhibits E. coli, P. aeruginosa, S. aureus and S. epidermidis, so taking this into account, this study is dedicated to assess UV photochemically-synthesized (λ=254 nm) heparin-based silver nanoparticles antimicrobial activity using the agar disk diffusion method complemented by the broth microdilution method to estimate de minimum inhibitory concentration (MIC), that is the lowest concentration at which an antimicrobial will inhibit visible growth of a microorganism. The strains used were the ones aforementioned to assess the antimicrobial activity degree these heparinbased nanoparticles exhibit.

  19. Effect of heparin and related glycosaminoglycan on PDGF-induced lung fibroblast proliferation, chemotactic response and matrix metalloproteinases activity

    Directory of Open Access Journals (Sweden)

    Masahiro Sasaki

    2000-01-01

    Full Text Available Fibroblast migration, proliferation, extacellular matrix protein synthesis and degradation are the key events in various biological and pathological processes in pulmonary fibrosis. In addition, biopsy specimens from the lungs of patients with plumomary fibrosis show increased numbers of mast cells which have metachromatic granules containing heparin, histamin and proteases. Little is known about how these products influence pulmonary fibrosis. In the present study, we investigated the effect of heparin and related glycosaminoglycans on PDGF-induced lung fibroblast proliferation and chemotactic response in vitro. In addition, we examined the effect of heparin on both the induction of matorix metalloproteinases (MMPs and MMPs activity in lung fibroblasts in vitro.

  20. Incomplete filling of lithium heparin tubes affects the activity of creatine kinase and gamma-glutamyltransferase.

    Science.gov (United States)

    Lippi, G; Avanzini, P; Cosmai, M; Aloe, R; Ernst, D

    2012-01-01

    This study aims to assess whether or not incomplete filling of primary lithium heparin tubes may influence the activity of creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT). Blood was drawn from 20 healthy volunteer using an identical sequence of tubes. First, a 6 mL, 13 x 100 mm 14 unit/mL lithium heparin Vacuette was filled and discharged. Then, three identical lithium heparin Vacuette tubes were filled, one to the nominal volume (i.e., full-draw tube), another with half of the nominal volume (half-draw tube) and the last with one-third of the nominal volume (low-draw tube). The plasma was separated and tested for CK (non-activated by N-acetylcysteine), AST and ALT on a Beckman Coulter Unicel DxC 800. Tests for CK were performed with a different reagent on a Beckman Coulter AU5800 (activated by N-acetylcysteine). Although the concentrations of ASL and ALT measured on the Unicel DxC and that of CK measured on the AU5800 did not change significantly across the different specimens, those of CK and GGT measured on the Unicel DxC 800 were significantly increased in the half-draw and low-draw tubes. The percentage bias of CK on the Unicel DxC 800 (using Bland Altman plots) was 3.3% and 7.9% for the half-draw and low-draw tubes, respectively, whereas that of GGT was 10.3% and 16.6% for the half-draw and low-draw tubes, respectively. These results suggest that short-draw lithium heparin tubes might be unsuitable for testing GGT and CK using specific combinations of reagents and instrumentation.

  1. Inactivation of human antithrombin by neutrophil elastase. Kinetics of the heparin-dependent reaction.

    Science.gov (United States)

    Jordan, R E; Nelson, R M; Kilpatrick, J; Newgren, J O; Esmon, P C; Fournel, M A

    1989-06-25

    Human neutrophil elastase catalyzes the inactivation of antithrombin by a specific and limited proteinolytic cleavage. This inactivation reaction is greatly accelerated by an active anticoagulant heparin subfraction with high binding affinity for antithrombin. A potentially complex reaction mechanism is suggested by the binding of both neutrophil elastase and antithrombin to heparin. The in vitro kinetic behavior of this system was examined under two different conditions: 1) at a constant antithrombin concentration in which the active anticoagulant heparin was varied from catalytic to saturating levels; and 2) at a fixed, saturating heparin concentration and variable antithrombin levels. Under conditions of excess heparin, the inactivation could be continuously monitored by a decrease in the ultraviolet fluorescence emission of the inhibitor. A Km of approximately 1 microM for the heparin-antithrombin complex and a turnover number of approximately 200/min was estimated from these analyses. Maximum acceleratory effects of heparin on the inactivation of antithrombin occur at heparin concentrations significantly lower than those required to saturate antithrombin. The divergence in acceleratory effect and antithrombin binding contrasts with the anticoagulant functioning of heparin in promoting the formation of covalent antithrombin-enzyme complexes and is likely to derive from the fact that neutrophil elastase is not consumed in the inactivation reaction. A size dependence was observed for the heparin effect since an anticoagulantly active octasaccharide fragment of heparin, with avid antithrombin binding activity, was without effect on the inactivation of antithrombin by neutrophil elastase. Despite the completely nonfunctional nature of elastase-cleaved antithrombin and the altered physical properties of the inhibitor as indicated by fluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the inactivated inhibitor exhibited a circulating half

  2. Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development.

    Science.gov (United States)

    Kim, Seonwook; Yang, Lihua; Kim, Seongu; Lee, Richard G; Graham, Mark J; Berliner, Judith A; Lusis, Aldons J; Cai, Lei; Temel, Ryan E; Rateri, Debra L; Lee, Sangderk

    2017-01-01

    The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

  3. A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; McCarthy, J B; Furcht, L T

    1993-01-01

    Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process......, as synthetic peptides coupled to ovalbumin, can support cell attachment. Only three of these sequences can promote focal adhesion formation when presented as multicopy complexes, and only one of these (WQPPRARI) retains this activity as free peptide. The major activity of this peptide resides in the sequence...... PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition...

  4. Heparin Oligosaccharides Inhibit Chemokine (CXC Motif) Ligand 12 (CXCL12) Cardioprotection by Binding Orthogonal to the Dimerization Interface, Promoting Oligomerization, and Competing with the Chemokine (CXC Motif) Receptor 4 (CXCR4) N Terminus*

    Science.gov (United States)

    Ziarek, Joshua J.; Veldkamp, Christopher T.; Zhang, Fuming; Murray, Nathan J.; Kartz, Gabriella A.; Liang, Xinle; Su, Jidong; Baker, John E.; Linhardt, Robert J.; Volkman, Brian F.

    2013-01-01

    The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics. PMID:23148226

  5. Low Anticoagulant Heparin Blocks Thrombin-Induced Endothelial Permeability in a PAR-Dependent Manner

    Science.gov (United States)

    Gonzales, Joyce N.; Kim, Kyung-mi; Zemskova, Marina A.; Rafikov, Ruslan; Heeke, Brenten; Varn, Matthew N.; Black, Stephen; Kennedy, Thomas P.; Verin, Alexander D.; Zemskov, Evgeny A.

    2014-01-01

    Acute lung injury and acute respiratory distress syndrome are accompanied by thrombin activation and fibrin deposition that enhances lung inflammation, activates endothelial cells and disrupts lung paracellular permeability. Heparin possesses anti-inflammatory properties but its clinical use is limited by hemorrhage and heparin induced thrombocytopenia. We studied the effects of heparin and low anticoagulant 2-O, 3-O desulfated heparin (ODSH) on thrombin-induced increases in paracellular permeability of cultured human pulmonary endothelial cells (EC). Pretreatment with heparin or ODSH blocked thrombin-induced decrease in the EC transendothelial electrical resistance (TER), attenuated thrombin-stimulated paracellular gap formation and actin cytoskeletal rearrangement. Our data demonstrated that heparin and ODSH had inhibitory effects on thrombin-induced RhoA activation and intracellular calcium elevation. Thrombin-stimulated phosphorylation of the cytoskeletal regulatory proteins, myosin light chain and ezrin/radixin/moesin, were also reduced. In these effects, low anticoagulant ODSH was more potent than heparin. Heparin or ODSH alone produced decreases in the EC TER that were abolished by siRNA-mediated depletion of the thrombin receptor, PAR-1. We also demonstrated that, in contrast to heparin, ODSH did not possess thrombin-binding activity. Results suggest that heparin and low anticoagulant ODSH, can interfere with thrombin-activated signaling. PMID:24469066

  6. Cell adhesion to fibrillin-1: identification of an Arg-Gly-Asp-dependent synergy region and a heparin-binding site that regulates focal adhesion formation

    DEFF Research Database (Denmark)

    Bax, Daniel V; Mahalingam, Yashithra; Cain, Stuart

    2007-01-01

    We have defined the molecular basis of cell adhesion to fibrillin-1, the major structural component of extracellular microfibrils that are associated with elastic fibres. Using human dermal fibroblasts, and recombinant domain swap fragments containing the Arg-Gly-Asp motif, we have demonstrated a......, and confirmed their role in focal adhesion formation. These integrin and syndecan adhesion motifs juxtaposed on fibrillin-1 are evolutionarily conserved and reminiscent of similar functional elements on fibronectin, highlighting their crucial functional importance....... a requirement for upstream domains for integrin-alpha(5)beta(1)-mediated cell adhesion and migration. An adjacent heparin-binding site, which supports focal adhesion formation, was mapped to the fibrillin-1 TB5 motif. Site-directed mutagenesis revealed two arginine residues that are crucial for heparin binding...

  7. New Insights in Thrombin Inhibition Structure-Activity Relationships by Characterization of Octadecasaccharides from Low Molecular Weight Heparin.

    Science.gov (United States)

    Mourier, Pierre A J; Guichard, Olivier Y; Herman, Fréderic; Sizun, Philippe; Viskov, Christian

    2017-03-08

    Low Molecular Weight Heparins (LMWH) are complex anticoagulant drugs that mainly inhibit the blood coagulation cascade through indirect interaction with antithrombin. While inhibition of the factor Xa is well described, little is known about the polysaccharide structure inhibiting thrombin. In fact, a minimal chain length of 18 saccharides units, including an antithrombin (AT) binding pentasaccharide, is mandatory to form the active ternary complex for LMWH obtained by alkaline β-elimination (e.g., enoxaparin). However, the relationship between structure of octadecasaccharides and their thrombin inhibition has not been yet assessed on natural compounds due to technical hurdles to isolate sufficiently pure material. We report the preparation of five octadecasaccharides by using orthogonal separation methods including size exclusion, AT affinity, ion pairing and strong anion exchange chromatography. Each of these octadecasaccharides possesses two AT binding pentasaccharide sequences located at various positions. After structural elucidation using enzymatic sequencing and NMR, in vitro aFXa and aFIIa were determined. The biological activities reveal the critical role of each pentasaccharide sequence position within the octadecasaccharides and structural requirements to inhibit thrombin. Significant differences in potency, such as the twenty-fold magnitude difference observed between two regioisomers, further highlights the importance of depolymerisation process conditions on LMWH biological activity.

  8. Heparin-Binding EGF-like Growth Factor (HB-EGF) Therapy for Intestinal Injury: Application and Future Prospects

    Science.gov (United States)

    Yang, Jixin; Su, Yanwei; Zhou, Yu; Besner, Gail E.

    2014-01-01

    Throughout the past 20 years, we have been investigating the potential therapeutic roles of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor family, in various models of intestinal injury including necrotizing enterocolitis (NEC), intestinal ischemia/reperfusion (I/R) injury, and hemorrhagic shock and resuscitation (HS/R). Our studies have demonstrated that HB-EGF acts as an effective mitogen, a restitution-inducing reagent, a cellular trophic factor, an anti-apoptotic protein and a vasodilator, via its effects on various cell types in the intestine. In the current paper, we have reviewed the application and therapeutic effects of HB-EGF in three classic animal models of intestinal injury, with particular emphasis on its protection of the intestines from NEC. Additionally, we have summarized the protective functions of HB-EGF on various target cells in the intestine. Lastly, we have provided a brief discussion focusing on the future development of HB-EGF clinical applications for the treatment of various forms of intestinal injury including NEC. PMID:24345808

  9. Heparin-binding EGF-like growth factor (HB-EGF) promotes cell migration and adhesion via focal adhesion kinase.

    Science.gov (United States)

    Su, Yanwei; Besner, Gail E

    2014-06-15

    Cell migration and adhesion are essential in intestinal epithelial wound healing and recovery from injury. Focal adhesion kinase (FAK) plays an important role in cell-extracellular matrix signal transduction. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) promotes intestinal epithelial cell (IEC) migration and adhesion in vitro. The present study was designed to determine whether FAK is involved in HB-EGF-induced IEC migration and adhesion. A scrape wound healing model of rat IECs was used to examine the effect of HB-EGF on FAK-dependent cell migration in vitro. Immunofluorescence and Western blot analyses were performed to evaluate the effect of HB-EGF on the expression of phosphorylated FAK (p-FAK). Cell adhesion assays were performed to determine the role of FAK in HB-EGF-induced cell adhesion on fibronectin (FN). HB-EGF significantly increased healing after scrape wounding, an effect that was reversed in the presence of an FAK inhibitor 14 (both with P HB-EGF increased p-FAK expression and induced p-FAK redistribution and actin reorganization in migrating rat IECs. Cell adhesion and spreading on FN were significantly increased by HB-EGF (P HB-EGF-induced cell adhesion and spreading on FN (both with P HB-EGF-mediated IEC migration and adhesion. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Mice with genetic deletion of the heparin-binding growth factor midkine exhibit early preclinical features of Parkinson's disease.

    Science.gov (United States)

    Prediger, Rui D S; Rojas-Mayorquin, Argelia E; Aguiar, Aderbal S; Chevarin, Caroline; Mongeau, Raymond; Hamon, Michel; Lanfumey, Laurence; Del Bel, Elaine; Muramatsu, Hisako; Courty, José; Raisman-Vozari, Rita

    2011-08-01

    There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson's disease (PD) begin many years before the appearance of the characteristic motor symptoms and that impairments in olfactory, cognitive and motor functions are associated with time-dependent disruption of dopaminergic neurotransmission in different brain areas. Midkine is a 13-kDa retinoic acid-induced heparin-binding growth factor involved in many biological processes in the central nervous system such as cell migration, neurogenesis and tissue repair. The abnormal midkine expression may be associated with neurochemical dysfunction in the dopaminergic system and cognitive impairments in rodents. Here, we employed adult midkine knockout mice (Mdk(-/-)) to further investigate the relevance of midkine in dopaminergic neurotransmission and in olfactory, cognitive and motor functions. Mdk(/-) mice displayed pronounced impairments in their olfactory discrimination ability and short-term social recognition memory with no gross motor alterations. Moreover, the genetic deletion of midkine decreased the expression of the enzyme tyrosine hydroxylase in the substantia nigra reducing partially the levels of dopamine and its metabolites in the olfactory bulb and striatum of mice. These findings indicate that the genetic deletion of midkine causes a partial loss of dopaminergic neurons and depletion of dopamine, resulting in olfactory and memory deficits with no major motor impairments. Therefore, Mdk(-/-) mice may represent a promising animal model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.

  11. A new chronometric assay to determine plasma antifactor Xa activity which is insensitive to the antithrombin activity of low molecular weight heparins.

    Science.gov (United States)

    Dignac, M; Gabaig, A M; Cambus, J P; Boneu, B

    1994-01-01

    Some commercially available chronometric assays are influenced by the residual antithrombin activity of low molecular weight heparins (LMWH) and they underestimate the ex vivo anti Xa activity. We have evaluated a new kit (Staclot-Heparin) highly specific for the anti Xa activity of LMWH. A comparison with the results given by a reference chromogenic method (Stachrom-Heparin) indicates a very good correlation between the 2 assays (r = 0.95, n = 59). This new assay is not influenced by vitamin K antagonist treatments. Clinical biologists now have the possibility of determining the anti Xa activity generated by LMWH easily and accurately, using a chronometric assay.

  12. Hemocompatibility improvement of perfusion-decellularized clinical-scale liver scaffold through heparin immobilization

    Science.gov (United States)

    Bao, Ji; Wu, Qiong; Sun, Jiu; Zhou, Yongjie; Wang, Yujia; Jiang, Xin; Li, Li; Shi, Yujun; Bu, Hong

    2015-01-01

    Whole-liver perfusion-decellularization is an attractive scaffold–preparation technique for producing clinical transplantable liver tissue. However, the scaffold’s poor hemocompatibility poses a major obstacle. This study was intended to improve the hemocompatibility of perfusion-decellularized porcine liver scaffold via immobilization of heparin. Heparin was immobilized on decellularized liver scaffolds (DLSs) by electrostatic binding using a layer-by-layer self-assembly technique (/h-LBL scaffold), covalent binding via multi-point attachment (/h-MPA scaffold), or end-point attachment (/h-EPA scaffold). The effect of heparinization on anticoagulant ability and cytocompatibility were investigated. The result of heparin content and release tests revealed EPA technique performed higher efficiency of heparin immobilization than other two methods. Then, systematic in vitro investigation of prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), platelet adhesion and human platelet factor 4 (PF4, indicates platelet activation) confirmed the heparinized scaffolds, especially the /h-EPA counterparts, exhibited ultralow blood component activations and excellent hemocompatibility. Furthermore, heparin treatments prevented thrombosis successfully in DLSs with blood perfusion after implanted in vivo. Meanwhile, after heparin processes, both primary hepatocyte and endothelial cell viability were also well-maintained, which indicated that heparin treatments with improved biocompatibility might extend to various hemoperfusable whole-organ scaffolds’ preparation. PMID:26030843

  13. Heparin-binding protein: an early indicator of critical illness and predictor of outcome in cardiac arrest.

    Science.gov (United States)

    Dankiewicz, Josef; Linder, Adam; Annborn, Martin; Rundgren, Malin; Friberg, Hans

    2013-07-01

    To investigate plasma levels of the neutrophil-borne heparin-binding protein (HBP) in patients with induced hypothermia after cardiac arrest (CA), and to study any association to severity of organ failure, incidence of infection and neurological outcome. This study included 84 patients with CA of mixed origin who were treated with hypothermia. Plasma samples from 7 time points during the first 72 h after return of spontaneous circulation (ROSC) were collected and analyzed for HBP with an ELISA. Outcomes were dichotomized: a cerebral performance category scale (CPC) of 1-2 at 6 months follow-up was considered a good outcome, a CPC of 3-5, a poor outcome. Patient data, including APACHE II and SOFA-scores were retrieved from the computerized system for quality assurance for intensive care. At 6 h and 12 h after CA, plasma levels of HBP were significantly higher among patients with a poor outcome. A receiver operated characteristics (ROC)-analysis yielded respective areas under curve (AUC) values of 0.68 and 0.70. This was similar to APACHE II and SOFA-score AUC values. There was a significant correlation between early elevated HBP-values and time to ROSC. HBP-levels were not higher in patients with infections at any time. Elevated HBP is an early indicator of organ failure and poor neurological outcome after CA, independent of microbial infection, and should be further evaluated in prospective trials. The temporal profile of HBP is suggestive of a role in the pathogenesis of critical illness after CA. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Increased Plasma Levels of Heparin-Binding Protein on Admission to Intensive Care Are Associated with Respiratory and Circulatory Failure.

    Directory of Open Access Journals (Sweden)

    Jonas Tydén

    Full Text Available Heparin-binding protein (HBP is released by granulocytes and has been shown to increase vascular permeability in experimental investigations. Increased vascular permeability in the lungs can lead to fluid accumulation in alveoli and respiratory failure. A generalized increase in vascular permeability leads to loss of circulating blood volume and circulatory failure. We hypothesized that plasma concentrations of HBP on admission to the intensive care unit (ICU would be associated with decreased oxygenation or circulatory failure.This is a prospective, observational study in a mixed 8-bed ICU. We investigated concentrations of HBP in plasma at admission to the ICU from 278 patients. Simplified acute physiology score (SAPS 3 was recorded on admission. Sequential organ failure assessment (SOFA scores were recorded daily for three days.Median SAPS 3 was 58.8 (48-70 and 30-day mortality 64/278 (23%. There was an association between high plasma concentrations of HBP on admission with decreased oxygenation (p<0.001 as well as with circulatory failure (p<0.001, after 48-72 hours in the ICU. There was an association between concentrations of HBP on admission and 30-day mortality (p = 0.002. ROC curves showed areas under the curve of 0,62 for decreased oxygenation, 0,65 for circulatory failure and 0,64 for mortality.A high concentration of HBP in plasma on admission to the ICU is associated with respiratory and circulatory failure later during the ICU care period. It is also associated with increased 30-day mortality. Despite being an interesting biomarker for the composite ICU population it's predictive value at the individual patient level is low.

  15. Heparin pharmacovigilance in Brazil.

    Science.gov (United States)

    Junqueira, Daniela Rezende Garcia; Viana, Thércia Guedes; Peixoto, Eliane R de M; Barros, Fabiana C R de; Carvalho, Maria das Graças; Perini, Edson

    2011-01-01

    To investigate the biological origin of injectable unfractioned heparin available in Brazilian market by discussing the impact of the profile of commercial products and the changes in heparin monograph on the drug safety. The Anvisa data base for the Registered Products of Pharmaceutical Companies and the Dictionary of Pharmaceutical Specialties (DEF 2008/2009) were searched. A survey with industries having an active permission for marketing the drug in Brazil was conducted. Five companies were granted a permission to market unfractioned heparin in Brazil. Three of them are porcine in origin and two of them are bovine in origin, with only one explicitly showing this information in the package insert. The effectiveness and safety of heparin studied in non-Brazilian populations may not represent the Brazilian reality, since most countries no longer produce bovine heparin. The currently marketed heparin has approximately 10% less anticoagulant activity than that previously produced and this change may have clinical implications. Evidence about the lack of dose interchangeability between bovine and porcine heparins and the unique safety profile of these drugs indicates the need to follow the treatment and the patients' response. Events threatening the patient's safety must be reported to the pharmacovigilance system in each particular country.

  16. Profiling Heparin-Chemokine Interactions Using Synthetic Tools

    Science.gov (United States)

    de Paz, Jose L.; Moseman, E. Ashley; Noti, Christian; Polito, Laura; von Andrian, Ulrich H.; Seeberger, Peter H.

    2009-01-01

    Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity. PMID:18030990

  17. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca2+ channels

    DEFF Research Database (Denmark)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin

    2015-01-01

    Our previous studies revealed that L-type voltage-dependent Ca2+ channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular...... domains of Cav1.2α1 to identify putative binding sites between the channel and hyaluronic acid or another class of polyanionic glycans, such as heparin/heparan sulfates. None of the tested peptides showed detectable interaction with hyaluronic acid, but two peptides derived from the first pore...

  18. The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

    Energy Technology Data Exchange (ETDEWEB)

    Prince, Robin N.; Schreiter, Eric R.; Zou, Peng; Wiley, H. S.; Ting, Alice Y.; Lee, Richard T.; Lauffenburger, Douglas A.

    2010-07-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGFlike domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane proform from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HBEGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

  19. Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Takayo; Yoshida, Yuichi [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Kiso, Shinichi, E-mail: kiso@gh.med.osaka-u.ac.jp [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Imai, Yasuharu [Department of Gastroenterology, Ikeda Municipal Hospital, Ikeda, Osaka (Japan); Higashiyama, Shigeki [Department of Biochemistry and Molecular Genetics, Ehime University, Graduate School of Medicine and Department of Cell Growth and Tumor Regulation, Proteo-Medicine Research Center (ProMRes), Ehime University, Shitsukawa, Toon, Ehime (Japan); Iwamoto, Ryo; Mekada, Eisuke [Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Takehara, Tetsuo [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan)

    2013-07-26

    Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

  20. Engineering of routes to heparin and related polysaccharides.

    Science.gov (United States)

    Bhaskar, Ujjwal; Sterner, Eric; Hickey, Anne Marie; Onishi, Akihiro; Zhang, Fuming; Dordick, Jonathan S; Linhardt, Robert J

    2012-01-01

    Anticoagulant heparin has been shown to possess important biological functions that vary according to its fine structure. Variability within heparin's structure occurs owing to its biosynthesis and animal tissue-based recovery and adds another dimension to its complex polymeric structure. The structural variations in chain length and sulfation patterns mediate its interaction with many heparin-binding proteins, thereby eliciting complex biological responses. The advent of novel chemical and enzymatic approaches for polysaccharide synthesis coupled with high throughput combinatorial approaches for drug discovery have facilitated an increased effort to understand heparin's structure-activity relationships. An improved understanding would offer potential for new therapeutic development through the engineering of polysaccharides. Such a bioengineering approach requires the amalgamation of several different disciplines, including carbohydrate synthesis, applied enzymology, metabolic engineering, and process biochemistry.

  1. Contact activation of plasmatic coagulation on polymeric membranes measured by the activity of kallikrein in heparinized plasma.

    Science.gov (United States)

    Groth, T; Synowitz, J; Malsch, G; Richau, K; Albrecht, W; Lange, K P; Paul, D

    1997-01-01

    Kallikrein is involved in the generation of bradykinin during extracorporal circulation, that is believed to play an important role in cases of anaphylactic shock during hemodialysis. Therefore, a method for the assessment of kallikrein generation was developed, based on the chromogenic substrate S-2302. Comparison of kallikrein-like activity on glass using citrate or heparinized plasma demonstrated enhanced activity in the presence of heparin. The applicability of the assay, and the time course of kallikrein generation was demonstrated with glass and cuprophan. Membranes based on pure polyacrylonitrile, or its copolymers differing in their content of acrylic acid, 2-hydroxyethyl acrylate, and allylsulphonate were investigated with respect to kallikrein-like activity, and physicochemical surface properties. It was found that high content in 2-hydroxyethyl acrylate, and acrylic acid caused a substantial activation of the contact system while low content in allylsulphonate (less than 2 mol%) did not result in enhanced kallikrein-like activity. The activating materials were characterized to be highly wettable, and had the most negative zeta potentials.

  2. Heparin-binding EGF-like growth factor (HB-EGF) stimulates the proliferation of Müller glia-derived progenitor cells in avian and murine retinas.

    Science.gov (United States)

    Todd, Levi; Volkov, Leo I; Zelinka, Chris; Squires, Natalie; Fischer, Andy J

    2015-11-01

    Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, heparin-binding EGF-like growth factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF not only stimulated MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Polymeric coatings that mimic the endothelium: combining nitric oxide release with surface-bound active thrombomodulin and heparin.

    Science.gov (United States)

    Wu, Biyun; Gerlitz, Bruce; Grinnell, Brian W; Meyerhoff, Mark E

    2007-10-01

    Multi-functional bilayer polymeric coatings are prepared with both controlled nitric oxide (NO) release and surface-bound active thrombomodulin (TM) alone or in combination with immobilized heparin. The outer-layer is made of CarboSil, a commercially available copolymer of silicone rubber (SR) and polyurethane (PU). The CarboSil is either carboxylated or aminated via an allophanate reaction with a diisocyanate compound followed by a urea-forming reaction between the generated isocyanate group of the polymer and the amine group of an amino acid (glycine), an oligopeptide (triglycine) or a diamine. The carboxylated CarboSil can then be used to immobilize TM through the formation of an amide bond between the surface carboxylic acid groups and the lysine residues of TM. Aminated CarboSil can also be employed to initially couple heparin to the surface, and then the carboxylic acid groups on heparin can be further used to anchor TM. Both surface-bound TM and heparin's activity are evaluated by chromogenic assays and found to be at clinically significant levels. The underlying NO release layer is made with another commercial SR-PU copolymer (PurSil) mixed with a lipophilic NO donor (N-diazeniumdiolated dibutylhexanediamine (DBHD/N(2)O(2))). The NO release rate can be tuned by changing the thickness of top coatings, and the duration of NO release at physiologically relevant levels can be as long as 2 weeks. The combination of controlled NO release as well as immobilized active TM and heparin from/on the same polymeric surface mimics the highly thromboresistant endothelium layer. Hence, such multifunctional polymer coatings should provide more blood-compatible surfaces for biomedical devices.

  4. Stepwise inhibition of T cell recruitment at post-capillary venules by orally active desulfated heparins in inflammatory arthritis.

    Science.gov (United States)

    Al Faruque, Hasan; Kang, Jin Hee; Hwang, Seung Rim; Sung, Shijin; Alam, Md Mahmudul; Sa, Keum Hee; Nam, Eon Jeong; Byun, Young Ro; Kang, Young Mo

    2017-01-01

    Identification of the structure-function relationship of heparin, particularly between 2-O-, 6-O-, and N-sulfation and its anticoagulant or anti-inflammatory activities, is critical in order to evaluate the biological effects of heparin, especially in conjunction with modifications for oral formulation. In this study, we demonstrated that removal of 2-O, 6-O, or N-desulfation and their hydrophobic modifications have differential effects on the blocking of interactions between sLeX and P-and L-selectins, with highest inhibition by 6-O desulfation, which was consistent with their in vivo therapeutic efficacies on CIA mice. The 6-O desulfation of lower molecular weight heparin (LMWH) retained the ability of LMWH to interfere with T cell adhesion via selectin-sLeX interactions. Furthermore, 6DSHbD coated on the apical surface of inflamed endothelium directly blocked the adhesive interactions of circulating T cells, which was confirmed in vivo by suppressing T cell adhesion at post-capillary venular endothelium. Thus, in series with our previous study demonstrating inhibition of transendothelial migration, oral delivery of low anticoagulant LMWH to venular endothelium of inflamed joint tissues ameliorated arthritis by the stepwise inhibition of T cell recruitment and provides a rationale for the development of modified oral heparins as innovative agents for the treatment of chronic inflammatory arthritis.

  5. Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

    Science.gov (United States)

    2012-01-01

    Background Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event. Methods Flagellar (Ff) and membrane (Mf) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis. Results The success of promastigotes subcellular fractionation led to the obtainment of Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 ± 2.0% of culture cells with adhered parasites): 30% (for HS 20μg/ml) and 16% (for HS 10μg/ml); HBP Mf (35.2% for 10μg/ml and 25.4% for 20μg/ml) and HBP Ff (10.0% for 10μg/ml and 31.4% for 20μg/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V

  6. Complex coacervation of lysozyme and heparin

    DEFF Research Database (Denmark)

    van de Weert, Marco; Andersen, Mia Bendix; Frokjaer, Sven

    2004-01-01

    To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability.......To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability....

  7. Incorporation of heparin into biomaterials.

    Science.gov (United States)

    Sakiyama-Elbert, Shelly E

    2014-04-01

    This review provides an overview of the incorporation of heparin into biomaterials with a focus on drug delivery and the use of heparin-based biomaterials for self-assembly of polymer networks. Heparin conjugation to biomaterials was originally explored to reduce the thrombogenicity of materials in contact with blood. Many of the conjugation strategies that were developed for these applications are still popular today for other applications. More recently heparin has been conjugated to biomaterials for drug delivery applications. Many of the delivery approaches have taken advantage of the ability of heparin to bind to a wide variety of growth factors, protecting them from degradation and potentiating interactions with cell surface receptors. More recently, the use of heparin as a base polymer for scaffold fabrication has also been explored, often utilizing non-covalent binding of heparin with peptides or proteins to promote self-assembly of hydrogel networks. This review will highlight recent advances in each of these areas. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  8. The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

    Science.gov (United States)

    Prince, Robin N.; Schreiter, Eric R.; Zou, Peng; Wiley, H. Steven; Ting, Alice Y.; Lee, Richard T.; Lauffenburger, Douglas A.

    2010-01-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGF-like domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane pro-form from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HB-EGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling. PMID:20530570

  9. Heparin Injection

    Science.gov (United States)

    ... moisture (not in the bathroom). Do not freeze heparin. It is important to keep all medication out of sight and reach of children as many containers (such as weekly pill minders and those for eye drops, creams, patches, and inhalers) are not child-resistant ...

  10. Involvement of three meningococcal surface-exposed proteins, the heparin-binding protein NhbA, the α-peptide of IgA protease and the autotransporter protease NalP, in initiation of biofilm formation

    KAUST Repository

    Arenas, Jesús

    2012-12-04

    Neisseria meningitidis is a common and usually harmless inhabitant of the mucosa of the human nasopharynx, which, in rare cases, can cross the epithelial barrier and cause meningitis and sepsis. Biofilm formation favours the colonization of the host and the subsequent carrier state. Two different strategies of biofilm formation, either dependent or independent on extracellular DNA (eDNA), have been described for meningococcal strains. Here, we demonstrate that the autotransporter protease NalP, the expression of which is phase variable, affects eDNA-dependent biofilm formation in N.meningitidis. The effect of NalP was found in biofilm formation under static and flow conditions and was dependent on its protease activity. Cleavage of the heparin-binding antigen NhbA and the α-peptide of IgA protease, resulting in the release of positively charged polypeptides from the cell surface, was responsible for the reduction in biofilm formation when NalP is expressed. Both NhbA and the α-peptide of IgA protease were shown to bind DNA. We conclude that NhbA and the α-peptide of IgA protease are implicated in biofilm formation by binding eDNA and that NalP is an important regulator of this process through the proteolysis of these surface-exposed proteins. © 2012 Blackwell Publishing Ltd.

  11. Electrostatic Forces as Dominant Interactions Between Proteins and Polyanions: an ESI MS Study of Fibroblast Growth Factor Binding to Heparin Oligomers

    Science.gov (United States)

    Minsky, Burcu Baykal; Dubin, Paul L.; Kaltashov, Igor A.

    2017-04-01

    The interactions between fibroblast growth factors (FGFs) and their receptors (FGFRs) are facilitated by heparan sulfate (HS) and heparin (Hp), highly sulfated biological polyelectrolytes. The molecular basis of FGF interactions with these polyelectrolytes is highly complex due to the structural heterogeneity of HS/Hp, and many details still remain elusive, especially the significance of charge density and minimal chain length of HS/Hp in growth factor recognition and multimerization. In this work, we use electrospray ionization mass spectrometry (ESI MS) to investigate the association of relatively homogeneous oligoheparins (octamer, dp8, and decamer, dp10) with acidic fibroblast growth factor (FGF-1). This growth factor forms 1:1, 2:1, and 3:1 protein/heparinoid complexes with both dp8 and dp10, and the fraction of bound protein is highly dependent on protein/heparinoid molar ratio. Multimeric complexes are preferentially formed on the highly sulfated Hp oligomers. Although a variety of oligomers appear to be binding-competent, there is a strong correlation between the affinity and the overall level of sulfation (the highest charge density polyanions binding FGF most strongly via multivalent interactions). These results show that the interactions between FGF-1 and Hp oligomers are primarily directed by electrostatics, and also demonstrate the power of ESI MS as a tool to study multiple binding equilibria between proteins and structurally heterogeneous polyanions.

  12. Heparin for assisted reproduction.

    Science.gov (United States)

    Akhtar, Muhammad A; Sur, Shyamaly; Raine-Fenning, Nick; Jayaprakasan, Kannamannadiar; Thornton, Jim G; Quenby, Siobhan

    2013-08-17

    Heparin as an adjunct in assisted reproduction (peri-implantation heparin) is given at or after egg collection or at embryo transfer during assisted reproduction. Heparin has been advocated to improve embryo implantation and clinical outcomes.  It has been proposed that heparin enhances the intra-uterine environment by improving decidualisation with an associated activation of growth factors and a cytokine expression profile in the endometrium that is favourable to pregnancy. To investigate whether the administration of heparin around the time of implantation (peri-implantation heparin) improves clinical outcomes in subfertile women undergoing assisted reproduction. A comprehensive and exhaustive search strategy was developed in consultation with the Trials Search Co-ordinator of the Cochrane Menstrual Disorders and Subfertility Group (MDSG). The strategy was used in an attempt to identify all relevant studies regardless of language or publication status (published, unpublished, in press, and in progress). Relevant trials were identified from both electronic databases and other resources (last search 6 May 2013). All randomised controlled trials (RCTs) were included where peri-implantation heparin was given during assisted reproduction. Peri-implantation low molecular weight heparin (LMWH) during IVF/ICSI was given at or after egg collection or at embryo transfer in the included studies. Live birth rate was the primary outcome. Two review authors independently assessed the eligibility and quality of trials and extracted relevant data. The quality of the evidence was evaluated using GRADE methods. Three RCTs (involving 386 women) were included in the review.Peri-implantation LMWH administration during assisted reproduction was associated with a significant improvement in live birth rate compared with placebo or no LMWH (odds ratio (OR) 1.77, 95% confidence interval (CI) 1.07 to 2.90, three studies, 386 women, I(2) = 51%, very low quality evidence with high

  13. Non-Anticoagulant Heparins Are Hepcidin Antagonists for the Treatment of Anemia

    Directory of Open Access Journals (Sweden)

    Maura Poli

    2017-04-01

    Full Text Available The peptide hormone hepcidin is a key controller of systemic iron homeostasis, and its expression in the liver is mainly regulated by bone morphogenetic proteins (BMPs, which are heparin binding proteins. In fact, heparins are strong suppressors of hepcidin expression in hepatic cell lines that act by inhibiting the phosphorylation of SMAD1/5/8 proteins elicited by the BMPs. The inhibitory effect of heparins has been demonstrated in cells and in mice, where subcutaneous injections of non-anticoagulant heparins inhibited liver hepcidin expression and increased iron bioavailability. The chemical characteristics for high anti-hepcidin activity in vitro and in vivo include the 2O-and 6O-sulfation and a molecular weight above 7 kDa. The most potent heparins have been found to be the super-sulfated ones, active in hepcidin suppression with a molecular weight as low as 4 kDa. Moreover, the alteration of endogenous heparan sulfates has been found to cause a reduction in hepcidin expression in vitro and in vivo, indicating that heparins act by interfering with the interaction between BMPs and components of the complex involved in the activation of the BMP/SMAD1/5/8 pathway. This review summarizes recent findings on the anti-hepcidin activity of heparins and their possible use for the treatment of anemia caused by hepcidin excess, including the anemia of chronic diseases.

  14. Applications of heparin and heparan sulfate microarrays.

    Science.gov (United States)

    Yin, Jian; Seeberger, Peter H

    2010-01-01

    Carbohydrate microarrays have become crucial tools for revealing the biological interactions and functions of glycans, primarily because the microarray format enables the investigation of large numbers of carbohydrates at a time. Heparan sulfate (HS) and heparin are the most structurally complex glycosaminoglycans (GAGs). In this chapter, we describe the preparation of a small library of HS/heparin oligosaccharides, and the fabrication of HS/heparin microarrays that have been used to establish HS/heparin-binding profiles. Fibroblast growth factors (FGFs), natural cytotoxicity receptors (NCRs), and chemokines were screened to illuminate the very important biological functions of these glycans. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  15. Performance of Activated Partial Thromboplastin Time (APTT): Determining Reagent Sensitivity to Factor Deficiencies, Heparin, and Lupus Anticoagulants.

    Science.gov (United States)

    Kershaw, Geoffrey

    2017-01-01

    The activated partial thromboplastin time (APTT) is a useful global assay for the assessment of the contact factor pathway of hemostasis and its inhibitors. The test is usually performed on fully automated analyzers using commercially prepared reagents. The three main clinical areas of interest are detection of factor deficiencies, detection of lupus anticoagulants and in the monitoring of therapy with unfractionated heparin. Methods are described here for assessing APTT reagents for their sensitivity to clotting time prolongation in each of these areas of interest.

  16. Advantages and pitfalls of combining intravenous antithrombin with nebulized heparin and tissue plasminogen activator in acute respiratory distress syndrome.

    Science.gov (United States)

    Rehberg, Sebastian; Yamamoto, Yusuke; Sousse, Linda E; Jonkam, Collette; Cox, Robert A; Prough, Donald S; Enkhbaatar, Perenlei

    2014-01-01

    Pulmonary coagulopathy has become an important therapeutic target in adult respiratory distress syndrome (ARDS). We hypothesized that combining intravenous recombinant human antithrombin (rhAT), nebulized heparin, and nebulized tissue plasminogen activator (TPA) more effectively improves pulmonary gas exchange compared with a single rhAT infusion, while maintaining the anti-inflammatory properties of rhAT in ARDS. Therefore, the present prospective, randomized experiment was conducted using an established ovine model. Following burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn, and 4 × 12 breaths of cold cotton smoke), 18 chronically instrumented sheep were randomly assigned to receive intravenous saline plus saline nebulization (control), intravenous rhAT (6 IU/kg/h) started 1 hour after injury plus saline nebulization (AT i.v.) or intravenous rhAT combined with nebulized heparin (10,000 IU every 4 hours, started 2 hours after injury), and nebulized TPA (2 mg every 4 hours, started 4 hours after injury) (triple therapy, n = 6 each). All animals were mechanically ventilated and fluid resuscitated according to standard protocols during the 48-hour study period. Both treatment approaches attenuated ARDS compared with control animals. Notably, triple therapy was associated with an improved PaO2/FiO2 ratio (p = 0.007), attenuated pulmonary obstruction (p = 0.02) and shunting (p = 0.025), as well as reduced ventilatory pressures (p heparin and nebulized TPA more effectively restores pulmonary gas exchange, but the anti-inflammatory effects of sole rhAT are abolished with the triple therapy. Interferences between the different anticoagulants may represent a potential explanation for these findings.

  17. Low-concentration heparin suppresses ionomycin-activated CAMK-II/EGF receptor- and ERK-mediated signaling in mesangial cells.

    Science.gov (United States)

    Song, Lifang; Xiao, Weiqun; Templeton, Douglas M

    2010-08-01

    Heparin and endogenous heparinoids inhibit the proliferation of smooth muscle cells, including renal mesangial cells; multiple effects on signaling pathways are well established, including effects on PKC, Erk, and CaMK-II. Many studies have used heparin at concentrations of 100 microg/ml or higher, whereas endogenous concentrations of heparinoids are much lower. Here we report the effects of low-concentration (1 microg/ml) heparin on activation of several kinases and subsequent induction of the c-fos gene in mesangial cells in response to the calcium ionophore, ionomycin, in the absence of serum factors. Ionomycin rapidly increases the phosphorylation of CaMK-II (by 30 s), and subsequently of the EGF receptor (EGFR), c-Src, and Erk 1/2. Low-dose heparin suppresses the ionomycin-dependent phosphorylation of EGFR, c-Src, and Erk 1/2, but not of CaMK-II, whereas inhibition of activated CaMK-II reduces phosphorylation of EGFR, c-Src, and Erk. Our data support a mechanism whereby heparin acts at the cell surface to suppress downstream targets of CaMK-II, including EGFR, leading in turn to a decrease in Erk- (but not c-Src-) dependent induction of c-fos.

  18. Enhanced effect of BCG vaccine against pulmonary Mycobacterium tuberculosis infection in mice with lung Th17 response to mycobacterial heparin-binding hemagglutinin adhesin antigen.

    Science.gov (United States)

    Fukui, Masayuki; Shinjo, Kikuko; Umemura, Masayuki; Shigeno, Satoko; Harakuni, Tetsuya; Arakawa, Takeshi; Matsuzaki, Goro

    2015-12-01

    Although the BCG vaccine can prevent tuberculosis (TB) in infants, its ability to prevent adult pulmonary TB is reportedly limited. Therefore, development of a novel effective vaccine against pulmonary TB has become an international research priority. We have previously reported that intranasal vaccination of mice with a mycobacterial heparin-binding hemagglutinin adhesin (HBHA) plus mucosal adjuvant cholera toxin (CT) enhances production of IFN-γ and anti-HBHA antibody and suppresses extrapulmonary bacterial dissemination after intranasal infection with BCG. In the present study, the effects of intranasal HBHA + CT vaccine on murine pulmonary Mycobacterium tuberculosis (Mtb) infection were examined. Intranasal HBHA + CT vaccination alone failed to reduce the bacterial burden in the infected lung. However, a combination vaccine consisting of s.c. BCG priming and an intranasal HBHA + CT booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14 compared with BCG vaccine alone. Further, it was found that intranasal HBHA + CT vaccine enhanced not only IFN-γ but also IL-17A production by HBHA-specific T cells in the lung after pulmonary Mtb infection. Therefore, this combination vaccine may be a good candidate for a new vaccine strategy against pulmonary TB. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

  19. Women with homozygous AT deficiency type II heparin-binding site (HBS) are at high risk of pregnancy loss and pregnancy complications.

    Science.gov (United States)

    Kraft, Julia; Sunder-Plassmann, Raute; Mannhalter, Christine; Quehenberger, Peter; Tews, Gernot; Langer, Martin; Pabinger, Ingrid

    2017-06-01

    Data regarding outcome and therapy of pregnancies in patients with homozygous antithrombin (AT) deficiency are very rare. We conducted a retrospective, descriptive investigation with emphasis on the obstetric history of eight women with homozygous AT deficiency heparin-binding site (HBS), who had at least one pregnancy. The aim of the study was to get a better insight into the outcome and identify suitable management procedures of pregnancy in this rare disease. All patients suffered from homozygous AT deficiency caused by the mutation c.391C>T p.Leu131Phe in the AT gene (SERPINC1). The women reported in total 23 pregnancies; one pregnancy was excluded because of induced abortion. We found that only seven out of the 22 analyzed pregnancies ended with a live infant, all of them were born preterm. Among the 15 negative outcomes, seven were early pregnancy losses and eight were intrauterine fetal deaths. We found no clear association between treatment protocols and outcome. Eight pregnancies were not treated at all; all of them ended with pregnancy loss. We conclude that homozygous AT deficiency HBS, a form of severe thrombophilia, is associated with high risk of pregnancy loss and preterm delivery. Rigorous anticoagulation and/or replacement of AT during pregnancy may improve the outcome.

  20. Serum Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF) as a Biomarker for Primary Ovarian Cancer.

    Science.gov (United States)

    Miyata, Kohei; Yotsumoto, Fusanori; Fukagawa, Satoshi; Kiyoshima, Chihiro; Ouk, Nam Sung; Urushiyama, Daichi; Ito, Tomohiro; Katsuda, Takahiro; Kurakazu, Masamitsu; Araki, Ryota; Sanui, Ayako; Miyahara, Daisuke; Murata, Masaharu; Shirota, Kyoko; Yagi, Hiroshi; Takono, Tadao; Kato, Kiyoko; Yaegashi, Nobuo; Akazawa, Kohei; Kuroki, Masahide; Yasunaga, Shin'ichiro; Miyamoto, Shingo

    2017-07-01

    Ovarian cancer is the most lethal malignancy among gynaecological cancers. Although many anticancer agents have been developed for the treatment of ovarian cancer, it continues to have an extremely poor prognosis. Heparin-binding epidermal growth factor-like grown factor (HB-EGF) has been reported to be a rational therapeutic target for ovarian cancer. Here, we evaluated the clinical significance of serum HB-EGF by examining the association between prognosis and serum HB-EGF levels in patients with primary ovarian cancer. We found that high serum HB-EGF concentrations were significantly associated with poor prognosis in a combined cohort of patients with all stages of ovarian cancer, as well as in a subset of patients with advanced disease. In addition, serum HB-EGF levels increased as the cancer advanced. These data suggest that serum HB-EGF may be a target for the design of novel therapies for ovarian cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  1. Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice

    Directory of Open Access Journals (Sweden)

    Mitsuru Ando

    2014-01-01

    Full Text Available Sustained gene delivery of interferon (IFN γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity.

  2. Omeprazole and PGC-formulated heparin binding epidermal growth factor normalizes fasting blood glucose and suppresses insulitis in multiple low dose streptozotocin diabetes model

    Science.gov (United States)

    Castillo, Gerardo M.; Nishimoto-Ashfield, Akiko; Banerjee, Aryamitra A.; Landolfi, Jennifer A.; Lyubimov, Alexander V.; Bolotin, Elijah M.

    2013-01-01

    Purpose Our objective was to develop novel nanocarriers (protected graft copolymer, PGC) that improve the stability of heparin binding EGF (HBEGF) and gastrin and then to use PGC-formulated HBEGF (PGC-HBEGF) and Omeprazole (+/− PGC-gastrin) for normalizing fasting blood glucose (FBG) and improving islet function in diabetic mice. Method HBEGF, PGC-HBEGF, Omeprazole, Omeprazole+PGC-HBEGF, Omeprazole+PGC-gastrin+PGC-HBEGF and epidermal growth factor (EGF)+gastrin were tested in multiple low dose streptozotocin diabetic mice. Results Omeprazole+PGC-HBEGF normalized FBG and is better than EGF+gastrin at improving islet function and decreasing insulitis. Groups treated with Omeprazole, Omeprazole+PGC-HBEGF, or EGF+gastrin have significantly improved islet function versus saline control. All animals that received PGC-HBEGF had significantly reduced islet insulitis versus saline control. Non-FBG was lower for Omeprazole+PGC-gastrin+PGC-HBEGF but Omeprazole+PGC-HBEGF alone showed better FBG and glucose tolerance. Conclusions Omeprazole+PGC-HBEGF provides a sustained exposure to both EGFRA and gastrin, improves islet function, and decreases insulitis in multiple low dose streptozotocin diabetic mice. Although HBEGF or EGF elevates non-FBG, it facilitates a reduction of insulitis and, in the presence of Omeprazole, provides normalization of FBG at the end of treatment. The study demonstrates Omeprazole and PGC-HBEGF is a viable treatment for diabetes. PMID:23793991

  3. A flow cytometric assay of platelet activation marker P-selectin (CD62P) distinguishes heparin-induced thrombocytopenia (HIT) from HIT with thrombosis (HITT).

    Science.gov (United States)

    Jy, W; Mao, W W; Horstman, L L; Valant, P A; Ahn, Y S

    1999-10-01

    Heparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios approximately 2.5 vs. HIT ratios approximately 1.2; p HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.

  4. Expression of Heparin-Binding EGF-Like Growth Factor (HB-EGF) in Bovine Endometrium: Effects of HB-EGF and Interferon-τ on Prostaglandin Production.

    Science.gov (United States)

    Takatsu, K; Acosta, T J

    2015-06-01

    Heparin-binding EGF-like growth factor (HB-EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB-EGF, prostaglandins (PGs) and interferon-τ (IFN-τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB-EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB-EGF and IFN-τ on PGE2 and PGF2-α production by endometrial cells were investigated. RT-PCR analysis revealed that HB-EGF mRNA was greater at the mid-luteal stage than at the early and regressed luteal stages (p HB-EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p HB-EGF did not affect PGF2-α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2-α and PGE2 production by bovine endometrial stromal cells (p HB-EGF-stimulated PGF2-α (p  0.05) production by stromal cells. These results indicate that HB-EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN-τ increased their expression in cultured endometrial cells. HB-EGF and IFN-τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle. © 2015 Blackwell Verlag GmbH.

  5. Short arm region of laminin-5 gamma2 chain: structure, mechanism of processing and binding to heparin and proteins

    DEFF Research Database (Denmark)

    Sasaki, T; Göhring, W; Mann, K

    2001-01-01

    Laminin-5 is a typical component of several epithelial tissues and contains a unique gamma2 chain which can be proteolytically processed by BMP-1. This occurs in the N-terminal half of the gamma2 chain (606 residues), which consists of two rod-like tandem arrays of LE modules, LE1-3 and LE4...... in processed laminin-5 showed only a strong binding to fibulin-2. Immunological studies showed a similar partial processing in cell culture and tissues and the persistence of the released fragment in tissues. This indicated that both N-terminal regions of the gamma2 chain may have a function in vivo....

  6. Colorimetric assay of heparin in plasma based on the inhibition of oxidase-like activity of citrate-capped platinum nanoparticles.

    Science.gov (United States)

    You, Jyun-Guo; Liu, Yao-Wen; Lu, Chi-Yu; Tseng, Wei-Lung; Yu, Cheng-Ju

    2017-06-15

    We report citrate-capped platinum nanoparticles (Pt NPs) as oxidase mimetics for effectively catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), dopamine, and methylene blue in the presence of O2. To confirm oxidase-like activity of citrate-capped Pt NPs, their activity toward oxygen reduction reaction was studied using cyclic voltammetry and rotating ring-disk electrode method. The results obtained showed that Pt NP NPs can catalyze the oxidation of organic substrates to the colored product and the reduction of oxygen to water through a four-electron exchange process. Because the aggregation of Pt NPs can inhibit their oxidase-like activity and protamine can recognize heparin, we prepared the protamine-modified Pt NPs through direct adsorption on the surface of citrate-capped Pt NPs. The electrostatic attraction between heparin and protamine-stabilized Pt NPs induced nanoparticle aggregation, inhibiting their catalytic activity. Therefore, the lowest detectable heparin concentrations through UV-vis absorption and by the naked eye were estimated to be 0.3 and 60nM, respectively. Moreover, the proposed system enabled the determination of the therapeutic heparin concentration in a single drop of blood. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Gentamicin and bone morphogenic protein-2 (BMP-2)-delivering heparinized-titanium implant with enhanced antibacterial activity and osteointegration.

    Science.gov (United States)

    Lee, Deok-Won; Yun, Young-Pil; Park, Kyeongsoon; Kim, Sung Eun

    2012-04-01

    Insufficient bonding of implants to bone tissues and bacterial infections lead to the failure of titanium (Ti)-based orthopedic and dental implants. The aim of this study is to develop novel Ti implants that enhance osteoblast functions, while simultaneously decreasing bacterial infections. First, the surface of pristine Ti was functionalized with heparin-dopamine by mimicking a mussel adhesion mechanism. Gentamicin sulfate (GS) and/or bone morphogenic protein-2 (BMP-2) was then sequentially immobilized to the heparinized-Ti (Hep-Ti) surface. The compositions of pristine Ti and Hep-Ti with or without gentamicin and/or BMP-2 were characterized by X-ray photoelectron spectroscopy (XPS) and the growth of Staphylococcus aureus on the substrates was assayed. Osteoblast functions of all Ti substrates were investigated by cell proliferation assays, alkaline phosphatase (ALP) activity, and calcium deposition. The results showed that the growth of bacteria on GS/Hep-Ti and GS/BMP-2/Hep-Ti was significantly lower compared to that on the pristine Ti and BMP-2/Hep-Ti. In addition, BMP-2/Hep-Ti and GS/BMP-2/Hep-Ti significantly enhanced ALP activity and calcium mineral deposition of osteoblast cells. Taken together, GS/BMP-2/Hep-Ti could achieve the dual functions of excellent antibacterial activity and osteoblast function promotion. Therefore, dual drug (antibiotics and osteoinductive protein)-eluting Ti substrates such as GS/BMP-2/Hep-Ti are a promising material for the enhanced osteointegration and implant longevity in orthopedics and dentistry. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Latent transforming growth factor β-binding protein-3 and fibulin-1C interact with the extracellular domain of the heparin-binding EGF-like growth factor precursor

    Directory of Open Access Journals (Sweden)

    Eidels Leon

    2002-01-01

    Full Text Available Abstract Background The membrane-bound cell-surface precursor and soluble forms of heparin-binding epidermal growth factor-like growth factor (HB-EGF contribute to many cellular developmental processes. The widespread occurrence of HB-EGF in cell and tissue types has led to observations of its role in such cellular and tissue events as tumor formation, cell migration, extracellular matrix formation, wound healing, and cell adherence. Several studies have reported the involvement of such extracellular matrix proteins as latent transforming growth factor β-binding protein, TGF-β, and fibulin-1 in some of these processes. To determine whether HB-EGF interacts with extracellular matrix proteins we used the extracellular domain of proHB-EGF in a yeast two-hybrid system to screen a monkey kidney cDNA library. cDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated. Results From ≈ 3 × 106 screened monkey cDNA clones, cDNA clones were recovered that contained nucleotide sequences encoding domains of the monkey latent transforming growth factor-β binding protein-3 (MkLTBP-3 and fibulin-1C protein. The amino acid sequence derived from the MkLTBP-3 gene shared 98.6% identity with human LTBP-3 and 86.7% identity with mouse LTBP-3 amino acid sequences. The amino acid sequence derived from the monkey fibulin-1C gene shared 97.2% identity with human fibulin-1C. Yeast two-hybrid screens indicate that LTBP-3 and fibulin-1C interact with proHB-EGF through their calcium-binding EGF-like modules. Conclusions The interactions of the extracellular domain of proHB-EGF with LTBP-3 and fibulin-1C suggest novel functions for HB-EGF between cell and tissue surfaces.

  9. Heparin-binding epidermal growth factor expression in KATO-III cells after Helicobacter pylori stimulation under the influence of strychnos Nux vomica and Calendula officinalis.

    Science.gov (United States)

    Hofbauer, Roland; Pasching, Eva; Moser, Doris; Frass, Michael

    2010-07-01

    Previous studies have shown the stimulating effect of Helicobacter pylori on the gene expression of heparin-binding epidermal growth factor (HB-EGF) using the gastric epithelial cell line KATO-III. Strychnos Nux vomica (Nux vomica) and Calendula officinalis are used in highly diluted form in homeopathic medicine to treat patients suffering from gastritis and gastric ulcers. To investigate the influence of Nux vomica and Calendula officinalis on HB-EGF-like growth factor gene expression in KATO-III cells under the stimulation of H. pylori strain N6 using real-time PCR with and without addition of Nux vomica and Calendula officinalis as a 10c or 12c potency. Baseline expression and stimulation were similar to previous experiments, addition of Nux vomica 10c and Calendula officinalis 10c in a 43% ethanolic solution led to a significant reduction of H. pylori induced increase in gene expression of HB-EGF (reduced to 53.12+/-0.95% and 75.32+/-1.16% vs. control; p<0.05), respectively. Nux vomica 12c reduced HB-EGF gene expression even in dilutions beyond Avogadro's number (55.77+/-1.09%; p<0.05). Nux vomica 12c in a 21.5% ethanol showed a smaller effect (71.80+/-3.91%, p<0.05). This effect was only be observed when the drugs were primarily prepared in ethanol, not in aqueous solutions. The data suggest that both drugs prepared in ethanolic solution are potent inhibitors of H. pylori induced gene expression. 2010 Elsevier Ltd. All rights reserved.

  10. Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.

    Directory of Open Access Journals (Sweden)

    Maria Domina

    Full Text Available We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb epitopes. For this purpose, we used a novel mAb (designated 31E10/E7 directed against Neisserial Heparin-Binding Antigen (NHBA, a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128 in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.

  11. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5.

    Science.gov (United States)

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-08-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.

  12. Activated clotting time level with weight based heparin dosing during percutaneous coronary intervention and its determinant factors.

    Science.gov (United States)

    Soleimannejad, Majid; Aslanabadi, Naser; Sohrabi, Bahram; Shamshirgaran, Morteza; Separham, Ahmad; Kazemi, Babak; Khayati Shal, Ebrahim; Madadi, Reza; Shirzadi, Hamidreza; Azizi, Hoda; Ghaffari, Samad

    2014-01-01

    Percutaneous coronary intervention (PCI) may be associated with Thrombotic complications. Unfractionated heparin (UFH) is a potent and preferable antithrombotic agent during this procedure. Activated clotting time (ACT) is a good assay for accurate titration of UFH during PCI. The aim of this study was to evaluate ACT levels 10 minutes after administration of 100U/kg IV heparin and determining its associated factors. This study was performed in Madani hospital, Tabriz, Iran between January 2013 to January 2014. One hundred and two patients candidates for elective PCI were enrolled in the study. Data including demographic and risk factors were collected. The range of ACT was between 165 to 750 seconds (mean 319.8 seconds), 52 (51%) patients had ACT levels lower than 300sec and 12 (11.8%) patients had ACT levels between 300 to 350 seconds which is known optimal range and 38 (37.2%) cases had ACT levels above this value. Major risk factors had no effect on ACT value, but there was a trend to higher levels with increasing age (P=0.06). There was no difference in the rate of major or minor bleeding with respect to ACT levels (P=0.52). There was a trend to higher rate of minimal bleeding in those with ACT >350 sec (P=0.06). Weight based UFH injection may result in suboptimal anticoagulation during the procedure. Routine ACT measurement may be necessary to ascertain adequate anticoagulation. Major risk factors had no effect on ACT level and it was not associated with the rate of bleeding.

  13. Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

    Science.gov (United States)

    Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

    2015-01-01

    An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

  14. Sterilization of heparinized cuprophan hemodialysis membranes

    NARCIS (Netherlands)

    ten Hoopen, Hermina W.M.; Hinrichs, W.L.J.; Hinrichs, W.L.J.; Engbers, G.H.M.; Feijen, Jan

    1996-01-01

    The effects of sterilization of dry heparinized Cuprophan hemodialysis membranes by means of ethylene oxide (EtO) exposure, gamma irradiation, or steam on the anticoagulant activity and chemical characteristics of immobilized heparin and the permeability of the membrane were investigated.

  15. Heparin and heparin-induced thrombocytopenia

    African Journals Online (AJOL)

    2007-06-15

    Jun 15, 2007 ... pregnancy, renal failure and obesity.4 See Table I for a comparison of unfractionated heparin and LMWH. Fondaparinux (Arixtra) is a novel anticoagulant which takes the evolution of heparin even further. It is an indirect factor. Xa inhibitor which does not cross-react with HIT antibodies.5. Fondaparinux has ...

  16. Heparin-induced thrombocytopenia: towards standardization of platelet factor 4/heparin antigen tests.

    Science.gov (United States)

    Greinacher, A; Ittermann, T; Bagemühl, J; Althaus, K; Fürll, B; Selleng, S; Lubenow, N; Schellong, S; Sheppard, J I; Warkentin, T E

    2010-09-01

    Laboratory confirmation of heparin-induced thrombocytopenia (HIT) is based on detection of heparin-dependent platelet-activating antibodies. Platelet factor 4 (PF4)/heparin enzyme-immunoassays (EIA) are a widely available surrogate for platelet-activating antibodies. Defining the optical density (OD) reactivity profiles of a PF4/heparin EIA in reference subject and patient populations and the correlation of the EIA results (expressed in OD units) with the prevalence of platelet-activating antibodies. Using quantile regression we determined the 97.5th percentile of PF4/heparin-immunoglobulin G (IgG) EIA reactivities in non-heparin-treated individuals [blood donors (n = 935)] and patients before heparin therapy (n = 1207). In patients with suspected HIT, we compared the correlation of EIA-IgG reactivities (Greifswald laboratory; n = 2821) and the heparin-induced platelet activation assay (HIPA) with the correlation of reactivities of another EIA-IgG (McMaster laboratory; n = 1956) with the serotonin-release assay (SRA). PF4/heparin-IgG EIA OD reactivities had a lower OD 97.5th percentile in blood donors compared with patient groups before heparin treatment (P 0.9) when normalized OD ranges (maximum OD divided by 10) were used instead of absolute OD values. Results of PF4/heparin-IgG EIA should not be reported as only positive or negative as there is no single acceptable cut-off value. Instead, reporting PF4/heparin-IgG EIA OD results in ranges allows for risk-stratified prediction for presence of platelet-activating antibodies. Use of normalized OD ranges permits a standardized approach for inter-laboratory comparisons. © 2010 International Society on Thrombosis and Haemostasis.

  17. Heparin-binding correlates with increased efficiency of AAV1- and AAV6-mediated transduction of striated muscle, but negatively impacts CNS transduction

    OpenAIRE

    Arnett, Andrea L. H.; Lisa R Beutler; Quintana, Albert; Allen, James; Finn, Eric; Richard D Palmiter; Chamberlain, Jeffrey S.

    2012-01-01

    Gene delivery vectors derived from adeno-associated virus (AAV) have great potential as therapeutic agents. rAAV1 and rAAV6, efficiently target striated muscle, but the mechanisms that determine their tropism remain unclear. It is known that AAV6, but not AAV1, interacts with heparin-sulfate proteoglycans (HSPG). HSPGs are not primary receptors for AAV6, but heparin interactions may affect tissue tropism and transduction. To investigate these possibilities, we generated rAAV1 and rAAV6 capsid...

  18. Backbone dynamics of a biologically active human FGF-1 monomer, complexed to a hexasaccharide heparin-analogue, by {sup 15}N NMR relaxation methods

    Energy Technology Data Exchange (ETDEWEB)

    Canales-Mayordomo, Angeles; Fayos, Rosa [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain); Angulo, Jesus; Ojeda, Rafael [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Martin-Pastor, Manuel [Unidad de RM y Unidad de RMN de Biomoleculas Asociada al CSIC, Laboratorio de Estructura e Estructura de Biomoleculas Jose Carracido (Spain); Nieto, Pedro M.; Martin-Lomas, Manuel [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Lozano, Rosa; Gimenez-Gallego, Guillermo; Jimenez-Barbero, Jesus [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain)], E-mail: jjbarbero@cib.csic.es

    2006-08-15

    The binding site and backbone dynamics of a bioactive complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed heparin hexasaccharide has been investigated by HSQC and relaxation NMR methods. The comparison of the relaxation data for the free and bound states has allowed showing that the complex is monomeric, and still induces mutagenesis, and that the protein backbone presents reduced motion in different timescale in its bound state, except in certain points that are involved in the interaction with the fibroblast growth factor receptor (FGFR)

  19. Allergic anaphylaxis due to subcutaneously injected heparin

    Directory of Open Access Journals (Sweden)

    Anders Diana

    2013-01-01

    Full Text Available Abstract Heparins are one of the most used class of anticoagulants in daily clinical practice. Despite their widespread application immune-mediated hypersensitivity reactions to heparins are rare. Among these, the delayed-type reactions to s.c. injected heparins are well-known usually presenting as circumscribed eczematous plaques at the injection sites. In contrast, potentially life-threatening systemic immediate-type anaphylactic reactions to heparins are extremely rare. Recently, some cases of non-allergic anaphylaxis could be attributed to undesirable heparin contaminants. A 43-year-old patient developed severe anaphylaxis symptoms within 5–10 minutes after s.c. injection of enoxaparin. Titrated skin prick testing with wheal and flare responses up to an enoxaparin dilution of 1:10.000 indicated a probable allergic mechanism of the enoxaparin-induced anaphylaxis. The basophil activation test as an additional in-vitro test method was negative. Furthermore, skin prick testing showed rather broad cross-reactivity among different heparin preparations tested. In the presented case, history, symptoms, and results of skin testing strongly suggested an IgE-mediated allergic hypersensitivity against different heparins. Therefore, as safe alternative anticoagulants the patient could receive beneath coumarins the hirudins or direct thrombin inhibitors. Because these compounds have a completely different molecular structure compared with the heparin-polysaccharides.

  20. Plasminogen binding and activation by Mycoplasma fermentans.

    Science.gov (United States)

    Yavlovich, A; Higazi, A A; Rottem, S

    2001-04-01

    The binding of plasminogen to Mycoplasma fermentans was studied by an immunoblot analysis and by a binding assay using iodine-labeled plasminogen. The binding of 125I-labeled plasminogen was inhibited by unlabeled plasminogen, lysine, and lysine analog epsilon-aminocaproic acid. Partial inhibition was obtained by a plasminogen fragment containing kringles 1 to 3 whereas almost no inhibition was observed with a fragment containing kringle 4. Scatchard analysis revealed a dual-phase interaction, one with a dissociation constant (kd) of 0.5 microM and the second with a kd of 7.5 microM. The estimated numbers of plasminogen molecules bound were calculated to be 110 and 790 per cell, respectively. Autoradiograms of ligand blots containing M. fermentans membrane proteins incubated with 125I-labeled plasminogen identified two plasminogen-binding proteins of about 32 and 55 kDa. The binding of plasminogen to M. fermentans enhances the activation of plasminogen to plasmin by the urokinase-type plasminogen activator (uPA), as monitored by measuring the breakdown of chromogenic substrate S-2251. Enhancement was more pronounced with the low-molecular-weight and the single-chain uPA variants, known to have low plasminogen activator activities. The binding of plasminogen also promotes the invasion of HeLa cells by M. fermentans. Invasion was more pronounced in the presence of uPA, suggesting that the ability of the organism to invade host cells stems not only from its potential to bind plasminogen but also from the activation of plasminogen to plasmin.

  1. Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: inhibition of anticoagulant activity by low molecular weight heparin.

    Science.gov (United States)

    Dutta, Sumita; Gogoi, Debananda; Mukherjee, Ashis K

    2015-03-01

    In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA2-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 μmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA2-I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA2-I. The NnPLA2-I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA2-I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA2-I did not show cytotoxicity against human glioblastoma U87MG cells

  2. Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

    Directory of Open Access Journals (Sweden)

    S.A. Andrade

    2012-10-01

    Full Text Available Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH, male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv over 2 min, followed by iv administration of saline (SG or recombinant Lopap (rLopap at 1 µg/kg (LG1 or 10 µg/kg (LG10, 10 min after the injection of LMWH, in a blind manner. Control animals (CG were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT, prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

  3. A spectroscopic study of interaction of cationic dyes with heparin

    Directory of Open Access Journals (Sweden)

    R. Nandini

    2010-01-01

    Full Text Available The interaction of two cationic dyes namely, acridine orange and pinacyanol chloride with an anionic polyelectrolyte, heparin, has been investigated by spectrophotometric method.The polymer induced metachromasy in the dyes resulting in the shift of the absorption maxima of the dyes towards shorter wavelengths. The stability of the complexes formed between acridine orange and heparin was found to be lesser than that formed between pinacyanol chloride and heparin. This fact was further confirmed by reversal studies using alcohols, urea and surfactants. The interaction of acridine orange with heparin has also been investigated fluorimetrically.The interaction parameters revealed that binding between acridine orange and heparin arises due to electrostatic interaction while that between pinacyanol chloride and heparin is found to involve both electrostatic and hydrophobic forces. The effect of the structure of the dye in inducing metachromasy has also been discussed.

  4. Activated Hemostatic Biomarkers in Patients with Implanted Left Ventricle Assist Devices: Are Heparin and/or Clopidogrel Justified?

    Science.gov (United States)

    Pacholewicz, Jerzy; Kuliczkowski, Wiktor; Kaczmarski, Jacek; Zakliczyński, Michał; Garbacz, Marcin; Zembala, Marian; Serebruany, Victor

    2015-01-01

    Adequate anticoagulation represents a major problem for left ventricle assist device (LVAD) utilization in patients awaiting heart transplantation as well as for regeneration of the native heart. The proper management of hemostatic abnormalities during LVAD support may improve survival by reducing the incidence of hemorrhagic and/or thromboembolic complications. A 40-year-old man with implanted pulsatile LVAD due to dilated cardiomyopathy received aspirin and warfarin. The patient underwent serial weekly monitoring of hemostatic biomarkers including international normalization ratio, prothrombin time, prothrombin activity, activated partial thromboplastin time, fibrinogen, D-dimer, platelet aggregation induced by adenosine diphosphate and arachidonic acid, platelet count, and mean platelet volume. The external pump was exchanged three times - twice because of a clot formation in the blood chamber of the pump, and once according to the standard protocol. LVAD use was consistently associated with enhanced adenosine diphosphate-induced platelet aggregation independent from the timing of clot formation or external pump exchange. Among coagulation indices, increased D-dimer holds predictive value for clot formation. The fibrinogen level peaked before the first pump exchange and was twice as high than the average values. Gradual improvement in exercise capacity was observed 2 years after implantation, after which the patient underwent a controlled stress test in the stop mode of the LVAD and the device was successfully explanted. Serial assessment of hemostatic biomarkers may benefit and triage LVAD patients. Consistent platelet activation during long-term LVAD may justify the addition of clopidogrel, while high D-dimer and/or elevated fibrinogen may indicate adding heparin to the conventional antithrombotic regimen. Randomized evidence is needed to test such a hypothesis. © 2015 S. Karger AG, Basel.

  5. Hypersensitivity reactions to heparins.

    Science.gov (United States)

    Gonzalez-Delgado, Purificación; Fernandez, Javier

    2016-08-01

    This article provides an update on hypersensitivity reactions to heparins and novel oral anticoagulants, with special emphasis on diagnostic methods and management of patients. Although heparins are drugs widely used, hypersensitivity reactions are uncommon. Cutaneous delayed hypersensitivity reactions after subcutaneous administration affects up to 7.5% of patients. Heparin-induced thrombocytopenia is another unusual but severe condition in which early recognition is crucial. Immediate hypersensitivity reactions to heparins have been also reported, but with the novel oral anticoagulants are much more uncommon, although reports of exanthemas have been notified.Skin tests and subcutaneous provocation test are useful tools in the diagnosis of hypersensitivity reactions, except in heparin-induced thrombocytopenia in which biopsy of lesional skin and in-vitro tests are the modalities of choice to confirm the diagnosis.Management of hypersensitivity reactions includes finding an alternative depending on the type of reaction. Fondaparinux and novel oral anticoagulants may be safe alternatives. Delayed skin lesions after subcutaneous heparin are the most common type of hypersensitivity reactions, followed by life-threatening heparin-induced thrombocytopenia. Immediate reactions are uncommon. Allergologic studies may be useful to find an alternative option in patients with skin lesions in which heparin-induced thrombocytopenia has been previously excluded, as well as in heparin immediate reactions.

  6. [Heparin-induced thrombocytopenia: recent data].

    Science.gov (United States)

    Gruel, Y; Rollin, J; Leroux, D; Pouplard, C

    2014-03-01

    Despite less frequent, heparin-induced thrombocytopenia (HIT) remains a severe complication of treatment with heparin, and is important to diagnose and manage appropriately. HIT results from an atypical immune response to heparin, with the synthesis of IgG antibodies specific to heparin-modified platelet factor 4 (PF4) which activate platelets, leukocytes and the endothelium. This activation explains that low platelet count is associated with thrombotic events in 50% of patients. The diagnosis of HIT is sometimes evoked because of atypical manifestations (i.e. cutaneous necrosis, amnesia, hypotension or dyspnea following intravenous injection of heparin). Biological assays are always necessary to confirm HIT in case of clinical suspicion, and specific rapid tests are now available for detecting anti-PF4 antibodies. However, their specificity is poor and functional assays such as serotonin release assay or platelet aggregation test are often necessary. Argatroban that is a direct antithrombin drug can be used in patients with severe renal failure and will be preferred to danaparoid sodium in this situation. Fondaparinux is not licensed for treating confirmed HIT and can only be used in case of suspicion. The early detection of HIT is based on the monitoring of platelet count recommended in surgical patients receiving a low molecular weight heparin and in all patients treated with unfractionated heparin. Copyright © 2013. Published by Elsevier SAS.

  7. Stimulation of Fas agonistic antibody-mediated apoptosis by heparin-like agents suppresses Hsp27 but not Bcl-2 protective activity.

    Science.gov (United States)

    Manero, Florence; Ljubic-Thibal, Vesna; Moulin, Maryline; Goutagny, Nadège; Yvin, Jean-Claude; Arrigo, André-Patrick

    2004-01-01

    We report that in Jurkat T cells or freshly isolated T lymphocytes, physiological concentrations of high-molecular weight sulfated polysaccharides such as heparin, heparan sulfate, and dextran sulfate significantly increased the percentage of cell death induced by Fas IgM agonistic antibody. The phenomenon was caspase dependent and P53 independent and correlated with an increased accessibility of cell surface Fas receptors. We also observed that the Fas IgM agonistic antibody-dependent formation of sodium dodecyl sulfate (SDS)-resistant large structures containing Fas receptor was decreased in the presence of heparin-like agents. In contrast, the different agents had no effect when cell death was triggered by FasL, the natural ligand of Fas that does not generate SDS-resistant forms of Fas. Interestingly, the synergistic effect of heparin-like agents toward Fas IgM agonistic antibody-mediated cell death abolished Hsp27 antiapoptotic activity but did not alter much the protection generated by Bcl-2 expression.

  8. Stimulation of Fas agonistic antibody–mediated apoptosis by heparin-like agents suppresses Hsp27 but not Bcl-2 protective activity

    Science.gov (United States)

    Manero, Florence; Ljubic-Thibal, Vesna; Moulin, Maryline; Goutagny, Nadège; Yvin, Jean-Claude; Arrigo, André-Patrick

    2004-01-01

    We report that in Jurkat T cells or freshly isolated T lymphocytes, physiological concentrations of high– molecular weight sulfated polysaccharides such as heparin, heparan sulfate, and dextran sulfate significantly increased the percentage of cell death induced by Fas IgM agonistic antibody. The phenomenon was caspase dependent and P53 independent and correlated with an increased accessibility of cell surface Fas receptors. We also observed that the Fas IgM agonistic antibody–dependent formation of sodium dodecyl sulfate (SDS)–resistant large structures containing Fas receptor was decreased in the presence of heparin-like agents. In contrast, the different agents had no effect when cell death was triggered by FasL, the natural ligand of Fas that does not generate SDS-resistant forms of Fas. Interestingly, the synergistic effect of heparin-like agents toward Fas IgM agonistic antibody–mediated cell death abolished Hsp27 antiapoptotic activity but did not alter much the protection generated by Bcl-2 expression. PMID:15497502

  9. Neisseria meningitidis Opc invasin binds to the sulphated tyrosines of activated vitronectin to attach to and invade human brain endothelial cells.

    Directory of Open Access Journals (Sweden)

    Claudia Sa E Cunha

    2010-05-01

    Full Text Available The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR requiring sulphated tyrosines Y(56 and Y(59. Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified peptides spanning the vitronectin residues 43-68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain (45RGD(47 was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal

  10. Kinetic analysis of the clotting system in the presence of heparin and depolymerized heparin.

    Science.gov (United States)

    Heuck, C C; Baumann, P

    1991-01-01

    The kinetics of the activation of the plasmatic clotting system in the presence of heparin and depolymerized heparin (Kabi 2165), respectively, was compared with the kinetics of activation in plasma with isolated factor deficiency. Measurements were made with a chromogenic substrate method using Tos-Gly-Pro-Arg-p-nitroanilide acetate. The extinction curves were analyzed to determine the characteristics of a curve that was fitted to the experimental data to sufficiently describe the slope of the curves by constants. In the activated extrinsic clotting system, the action of heparin and depolymerized heparin results in a distribution pattern for the two relevant constants. K(1), defining the time of the point of inflection of the curve, and K(2), relating to the slope of the curve at the point of inflection, which is identical with the pattern observed in plasma with factor II deficiency. This distribution pattern can be explained by an inhibitory reaction on factor IIa, which is accelerated by the anticoagulant. In contrast, the pattern of K(2)/K(1) for the activated intrinsic system is identical with the pattern for plasma with factor X deficiency. Qualitative differences in the action of heparin and depolymerized heparin are not evident. The investigation confirms that the molecular action of heparin and depolymerized heparin as accelerators of the plasmatic clotting system is qualitatively the same. However, their action in the extrinsic and intrinsic system has different effects. Furthermore, the study reveals that constant K(2) is a more sensitive indicator to measure low heparin and depolymerized heparin activities than K(1) or its equivalent, the clotting time.

  11. Unfractionated and low-molecular-weight heparin and the phosphodiesterase inhibitors, IBMX and cilostazol, block ex vivo Equid Herpesvirus type-1-induced platelet activation

    Directory of Open Access Journals (Sweden)

    Tracy Stokol

    2016-11-01

    Full Text Available Equid herpes virus type-1 (EHV-1 is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins or platelet inhibitors that impede post-receptor thrombin signaling (phosphodiesterase [PDE] antagonists would inhibit EHV-1-induced platelet activation ex vivo. We exposed platelet-rich plasma collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque forming unit/cell in the presence or absence of unfractionated heparin (UFH, low-molecular-weight (LMWH heparin or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1 to 0.2 U/mL anti-Factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX or isoenzyme-3 selective (cilostazol PDE antagonists inhibit ex vivo EHV-1-induced platelet activation

  12. Quantitation of the mRNA expression of the epidermal growth factor system: selective induction of heparin-binding epidermal growth factor-like growth factor and amphiregulin expression by growth factor stimulation of prostate stromal cells.

    Science.gov (United States)

    Sørensen, B S; Tørring, N; Bor, M V; Nexo, E

    2000-09-01

    The epidermal growth factor (EGF) system is a rapidly expanding system of growth factors involved in many aspects of normal and cancerous growth. We have developed a method for the quantitation of mRNA coding for all six growth factors activating the human EGF receptor (HER-1) and for the quantitation of mRNA for the receptors HER-1 and its preferred dimerization partner, HER-2. The method is based on the generation of specific RNA standards, which are amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with the sample RNA and a set of calibrators. The resulting calibration curve is used to quantitate the unknown samples, which require only a single RT-PCR reaction. Our method has the advantage that quantitation is based on coamplification of an internal RNA standard, thereby controlling both the PCR and RT reactions. In addition, the RNA standards for all growth factors and receptors are combined in a single RT reaction, which minimizes variation and allows the quantitation of all eight mRNA species with only 0.1 microg RNA. This makes the method suitable for studies in which the supply of material is limited. The developed method has enabled us to demonstrate that prostate stromal cells in primary culture express EGF, heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, and epiregulin as well as the HER-1 and HER-2 receptors, whereas no transforming growth factor-alpha mRNA is found. Furthermore, activation of the EGF system in these cells by stimulation with HB-EGF or EGF in mitogenic doses causes a selective increase in the expression of amphiregulin and HB-EGF mRNA (more than 15-fold and 25-fold, respectively), whereas there is no increase in the expression of mRNA for the other growth factors or receptors. In accord with the increase in amphiregulin mRNA, the amount of amphiregulin peptide released from the cells is also increased. The selective induction of amphiregulin and HB-EGF by growth factor stimulation may represent a mechanism

  13. SUMO-1 possesses DNA binding activity

    Directory of Open Access Journals (Sweden)

    Wieruszeski Jean-Michel

    2010-05-01

    Full Text Available Abstract Background Conjugation of small ubiquitin-related modifiers (SUMOs is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs. Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood. Findings Using NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM. Conclusion This finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.

  14. Correlation between activated partial thromboplastin time and anti-Xa activity in patients who received low-molecular weight heparin as anticoagulation for haemodialysis.

    Science.gov (United States)

    Wong, Steve Siu-Man; Lau, Wai-Yan; Chan, Ping-Kwan; Wan, Ching-Kit; Cheng, Yuk-Lun

    2017-11-01

    Plasma anti-Xa activity, the recommended test to monitor low-molecular weight heparin (LMWH) therapy, is not readily available in many laboratories. In our clinical trials on the use of LMWH as anticoagulation for haemodialysis, a consistent prolongation of APTT in addition to the elevated anti-Xa activity was observed in the patients after LMWH administration. Hence, the paired anti-Xa activity and APTT data were re-analyzed. The APTT ratio, which was the proportional change in APTT from the baseline value after LMWH administration, was found to have a strong correlation with anti-Xa activity (coefficient of determination, R 2  = 0.72, P sensitivity was 88% and the specificity was 83.3% when an APTT ratio ≧1.4 was used as the cut point to predict the achievement of therapeutic anti-Xa activity. Our results illustrated that APTT is a potentially useful screening test to assess the degree of anticoagulation achieved by LMWH during haemodialysis, if the testing for plasma anti-Xa activity is not available. © 2017 Asian Pacific Society of Nephrology.

  15. Low Molecular Weight Heparin Inhibits Plasma Thrombin Generation via Direct Targeting of Factor IXa: Contribution of the Serpin-independent Mechanism

    Science.gov (United States)

    Buyue, Yang; Misenheimer, Tina M.; Sheehan, John P.

    2012-01-01

    Background While heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of factor IXa inhibition to heparin activity in human plasma remains incompletely defined. Objectives To determine the relevance of factor IXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity. Patient/Methods Thrombin generation was detected by fluorogenic substrate cleavage. Inhibitory potency (EC50) of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), Fondaparinux, and unfractionated heparin (UFH) was determined by plotting concentration versus relative velocity index (ratio +/− heparin). Inhibition was compared under factor IX-dependent and independent conditions (0.2 or 4 pM TF, respectively) in normal plasma, and in mock- or antithrombin/factor IX-depleted plasma supplemented with recombinant factor IX. Results UFH and Fondaparinux demonstrated similar potency under factor IX-dependent and independent conditions, whereas LMWH (2.9-fold) and ssLMWH (5.1-fold) demonstrated increased potency with limiting TF. UFH (62-fold) and Fondaparinux (42-fold) demonstrated markedly increased EC50 values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (2-fold) were less affected, with an EC50 within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing factor IX/antithrombin-depleted plasma with 90 nM recombinant factor IX possessing mutations in the heparin-binding exosite. Mutated factor IX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition. Conclusions Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin-independent interaction with the factor IXa heparin-binding exosite. PMID:22905983

  16. The use of heparin in preparing samples for blood-gas analysis.

    Science.gov (United States)

    Higgins, Chris

    2007-10-01

    Heparin is the only anticoagulant used to prepare samples for blood-gas analysis. There are two ways in which heparin can interfere with results. The first is high heparin concentration in blood, and the second is heparin dilution of blood if liquid rather than dried (lyophilized) heparin is used. Traditional blood-gas analytes (pH, pCO2, and pO2) are less affected than electrolytes (particularly ionized calcium), also measured on modern blood-gas analyzers. The sample requirements as far as heparin is concerned are thus less exacting if only pH, pCO2, and pO2 are to be measured. For these analytes, it is still essential that the heparin (either sodium or lithium) concentration is less than 200 IU/mL blood and that the blood is not diluted more than 5%. The inclusion of electrolytes in the test repertoire excludes the use of sodium heparin in favor of lithium heparin. The inclusion of ionized calcium in the test repertoire demands that the heparin should be lyophilized, and the concentration should not exceed 10 IU/mL blood, unless a specialized heparin that eliminates the effect of calcium binding by heparin is used. Whatever the heparin formulation, it is essential for accurate results that the correct volume of blood is sampled to achieve a correct heparin concentration (and dilution, if liquid heparin is used), and that blood and anticoagulant are well mixed immediately after sampling. One of the most common practical problems associated with blood-gas analysis is inadequate anticoagulation and the formation of small blood clots that can block the sample pathway of blood-gas analyzers and invalidate results. Inadequate mixing of specimen with heparin is usually the problem. Clearly, the lower the heparin concentration the greater is the risk that poor mixing technique will give rise to inadequate anticoagulation and the associated problems.

  17. Immune pathogenesis of heparin-induced thrombocytopenia.

    Science.gov (United States)

    Khandelwal, Sanjay; Arepally, Gowthami M

    2016-10-28

    The immune response to heparin is one of the most common drug-induced allergies, and yet, atypical for a drug hypersensitivity reaction. Whereas most drug-induced allergies are rare, idiosyncratic and life-long, the allergic response to heparin is common, predictable in certain clinical settings and transient. Advances in the last decade with regards to structural characterisation of the PF4/heparin antigenic complex, contributions of innate immunity and development of animal models have provided insights into the distinctive features of the HIT immune response. Recent descriptions of the crystal structure of the PF4/heparin complex, alongside other biophysical studies, have clarified the structural requirements for immunogenicity and heparin-dependency of antibody formation. Studies of interactions of PF4 with bacterial cell walls as well as epidemiologic associations of anti-PF4/heparin antibody formation and infection suggest a role for immune priming and explain the rapid evolution of an isotype-switched immune response in sensitised patients. Murine models have greatly facilitated investigations of cellular basis of the HIT response and identified a major role for T-cells and marginal zone B-cells, but key findings have yet to be validated in human disease. This chapter will summarise recent investigations of the HIT immune response in the context of major pathways of immune activation and identify areas of uncertainty.

  18. Multi-stage inhibition in breast cancer metastasis by orally active triple conjugate, LHTD4 (low molecular weight heparin-taurocholate-tetrameric deoxycholate).

    Science.gov (United States)

    Alam, Farzana; Al-Hilal, Taslim A; Park, Jooho; Choi, Jeong Uk; Mahmud, Foyez; Jeong, Jee-Heon; Kim, In-San; Kim, Sang Yoon; Hwang, Seung Rim; Byun, Youngro

    2016-04-01

    Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. To target human metastatic breast cancer as well as improving patient compliance, we developed an orally active low molecular weight heparin (LMWH)-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-β1 (TGF-β1) and CXCL12 exhibit enhanced metastatic activity during the initiation and progression stages of breast cancer, thus we direct the focus on investigating the antimetastatic effect of LHTD4/DCK complex by targeting TGF-β1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-β1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-β1 and CXCL12 (TGF-β1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-β1) and chemotaxis (mediated by CXCL12) were investigated. The in vivo anti-metastatic effect of LHTD4 was evaluated in an accelerated metastasis model and an orthotopic MDA-MB-231 breast cancer model. The obtained KD values of TGF-β1 and CXCL12 with LHTD4 were 0.85 and 0.019 μM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-β1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-β1R1 and CXCR4 showed that the effective inhibition of receptor phosphorylation was observed with the treatment of LHTD4. The expressions of epithelial to mesenchymal transition (EMT) marker proteins such as vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGF-β1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-β1 and CXCL12 on migration and

  19. Additive effect of heparin on the photoinactivation of Escherichia coli using tricationic P-porphyrins.

    Science.gov (United States)

    Matsumoto, Jin; Suzuki, Kou; Uezono, Hidekazu; Watanabe, Kaho; Yasuda, Masahide

    2017-12-01

    Polycationic porphyrins have received substantial attention in developing singlet oxygen-sensitizers for biological use such as in the photoinactivation of bacteria and photodynamic therapy (PDT) of tumor cells because they have strong binding affinities for DNA and proteins. However, these strong cellular interactions can retard elimination of the drug after PDT. Therefore, the studies on the interactions of porphyrins with other molecules present much interest, in order to modulate the sensitizers' activity or even remove them from the human body after PDT. Here, we studied the additive effect of heparin on the photoinactivation by polycationic porphyrins using Escherichia coli as a model cell. Tricationic P-porphyrin sensitizers substituted with an N-alkylpyridinium group (alkyl = pentyl (1a), hexyl (1b), and heptyl (1c)) or N-hexylammonium (1d) as the axial ligand were used. Additionally, dicationic Sb-porphyrin substituted with an N-hexylpyridinium group (1e) was prepared. We studied the additive effect of heparin on the photoinactivation of E. coli by 1a-1e. The bactericidal activities were evaluated using the half-life (T1/2 in min) of E. coli and the minimum effective concentrations ([P]) of the porphyrin sensitizers. In the absence of heparin, the [P] values were determined to be 0.4-0.5 μM for 1a-1c and 2.0 μM for 1d-1e. The bactericidal activity of 1a-1c was completely retarded by the addition of heparin (1.0 μM). However, the addition of heparin (1.0 μM) could not completely retard the bactericidal activity of 1d-1e whose [P] values were relatively large. It is suggested that tricationic 1a-1c adsorbed onto the anionic heparin through electrostatic interactions. The adsorption of 1 on heparin disturbs the uptake of 1 into E. coli cells. Thus, the addition of heparin was found to be a useful method for retarding photoinactivation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Glycosaminoglycan backbone is not required for the modulation of hemostasis: effect of different heparin derivatives and non-glycosaminoglycan analogs.

    Science.gov (United States)

    Bouças, Rodrigo I; Jarrouge-Bouças, Thais R; Lima, Marcelo A; Trindade, Edvaldo S; Moraes, Fabio A; Cavalheiro, Renan P; Tersariol, Ivarne L S; Hoppenstead, Debra; Fareed, Jawed; Nader, Helena B

    2012-06-01

    Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is

  1. Fibronectin binding modulates CXCL11 activity and facilitates wound healing.

    Directory of Open Access Journals (Sweden)

    Federico Tortelli

    Full Text Available Engineered biomatrices offer the potential to recapitulate the regenerative microenvironment, with important implications in tissue repair. In this context, investigation of the molecular interactions occurring between growth factors, cytokines and extracellular matrix (ECM has gained increasing interest. Here, we sought to investigate the possible interactions between the ECM proteins fibronectin (FN and fibrinogen (Fg with the CXCR3 ligands CXCL9, CXCL10 and CXCL11, which are expressed during wound healing. New binding interactions were observed and characterized. Heparin-binding domains within Fg (residues 15-66 of the β chain, Fg β15-66 and FN (FNI1-5, but not FNIII12-14 were involved in binding to CXCL10 and CXCL11 but not CXCL9. To investigate a possible influence of FN and Fg interactions with CXCL11 in mediating its role during re-epithelialization, we investigated human keratinocyte migration in vitro and wound healing in vivo in diabetic db/db mice. A synergistic effect on CXCL11-induced keratinocyte migration was observed when cells were treated with CXCL11 in combination with FN in a transmigration assay. Moreover, wound healing was enhanced in full thickness excisional wounds treated with fibrin matrices functionalized with FN and containing CXCL11. These findings highlight the importance of the interactions occurring between cytokines and ECM and point to design concepts to develop functional matrices for regenerative medicine.

  2. Strategy for the sequence analysis of heparin.

    Science.gov (United States)

    Liu, J; Desai, U R; Han, X J; Toida, T; Linhardt, R J

    1995-12-01

    The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been developed. This paper describes a strategy to sequence the GAG chains of heparin. Heparin, prepared from animal tissue, and processed by proteinases and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan heparin (containing small remnants of core protein). Raw porcine mucosal heparin was labelled on the amino termini of these core protein remnants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a mixture of heparin lyase I and III, resulted in a single NDBP-linkage region tetrasaccharide, which was characterized as deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp -(1-->O-Ser-NDBP (deltaUAp is 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid). Several NDBP-octasaccharides were isolated when NDBP-heparin was treated with only heparin lyase I. The structure of one of these NDBP-octasaccharides, deltaUAp2S(1-->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp(1-->3)-beta-D- Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser NDBP (S is sulphate, Ac is acetate), was determined by 1H-NMR and enzymatic methods. Enriched NDBP-heparin was treated with lithium hydroxide to release heparin, and the GAG chain was then labelled at xylose with 7-amino-1,3-naphthalene disulphonic acid (AGA). The resulting AGA-Xyl-heparin was sequenced on gradient PAGE using heparin lyase I and heparin lyase III. A predominant sequence in heparin at the protein core attachment site was deduced to be -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S (or60H) (1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNp2S6S( or 6OH)(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpAc (1

  3. Inhibitory Effect of Heparin on Gentamicin Concentrations in Blood

    Science.gov (United States)

    Regamey, Claude; Schaberg, Dennis; Kirby, William M. M.

    1972-01-01

    In monitoring gentamicin concentrations in the blood of patients with renal insufficiency, the assayed antibiotic concentration was found to be lower when the sample was drawn as heparinized plasma rather than as serum. This lowering of gentamicin concentrations by heparin was studied further by adding increasing doses of heparin and various amounts of gentamicin to human serum. With a range of 2 to 100 units of heparin per ml, gentamicin concentrations in the serum were lowered by 9 to 14%; with higher heparin concentrations, an even greater and increasing inhibition was noticed, reaching 56% for 1,000 units/ml. This inhibitory effect of heparin on gentamicin was reversible by dilution, indicating that it was not due to degradation or to formation of an inactive chemical complex. Underestimation by the laboratory of gentamicin concentrations in blood is likely to be greatest with capillary tubes, with which the concentration of heparin is especially high. With clinical heparinization, the amount of active heparin in the blood does not exceed 10 units/ml and is for the most part under 3 units/ml; thus, therapeutically significant inhibition of the antibiotic is unlikely in patients receiving anticoagulation. PMID:4670696

  4. Unfractionated heparin versus low molecular weight heparins for avoiding heparin-induced thrombocytopenia in postoperative patients.

    Science.gov (United States)

    Junqueira, Daniela R; Zorzela, Liliane M; Perini, Edson

    2017-04-21

    Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction presenting as a prothrombotic disorder related to antibody-mediated platelet activation. It is a paradoxical immune reaction resulting in thrombin generation in vivo, which leads to a hypercoagulable state and the potential to initiate venous or arterial thrombosis. A number of factors are thought to influence the incidence of HIT including the type and preparation of heparin (unfractionated heparin (UFH) or low molecular weight heparin (LMWH)) and the heparin-exposed patient population, with the postoperative patient population at higher risk.Although LMWH has largely replaced UFH as a front-line therapy, there is evidence supporting a lack of superiority of LMWH compared with UFH regarding prevention of deep vein thrombosis and pulmonary embolism following surgery, and similar frequencies of bleeding have been described with LMWH and UFH. The decision as to which of these two preparations of heparin to use may thus be influenced by harmful effects such as HIT. We therefore sought to determine the relative impact of UFH and LMWH on HIT in postoperative patients receiving thromboembolism prophylaxis. This is an update of a review first published in 2012. The objective of this review was to compare the incidence of heparin-induced thrombocytopenia (HIT) and HIT complicated by venous thromboembolism in postoperative patients exposed to unfractionated heparin (UFH) versus low molecular weight heparin (LMWH). For this update, the Cochrane Vascular Information Specialist searched the Specialised Register (May 2016), CENTRAL (2016, Issue 4) and trials registries. The authors searched Lilacs (June 2016) and additional trials were sought from reference lists of relevant publications. We included randomised controlled trials (RCTs) in which participants were postoperative patients allocated to receive prophylaxis with UFH or LMWH, in a blinded or unblinded fashion. Studies were excluded if they did not use

  5. Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets.

    Science.gov (United States)

    Bachelet, Laure; Bertholon, Isabelle; Lavigne, Damien; Vassy, Roger; Jandrot-Perrus, Martine; Chaubet, Frédéric; Letourneur, Didier

    2009-02-01

    P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate. Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators. The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC(50) of 20 nM as compared to 400 nM for heparin and affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the K(D) of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction. Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.

  6. Elucidation of IP6 and Heparin Interaction Sites and Conformational Changes in Arrestin-1 by Solution NMR†

    Science.gov (United States)

    Zhuang, Tiandi; Vishnivetskiy, Sergey A.; Gurevich, Vsevolod V.; Sanders, Charles R.

    2010-01-01

    Arrestins specifically bind activated and phosphorylated G protein-coupled receptors, and orchestrate both receptor trafficking, and channel signaling to G protein-independent pathways via direct interactions with numerous non-receptor partners. Here we report the first successful use of solution NMR to map the binding sites in arrestin-1 (visual arrestin) for two polyanionic compounds that mimic phosphorylated light-activated rhodopsin: inositol hexaphosphate (IP6) and heparin. This yielded a more complete identification of residues involved in the binding with these ligands than has previously been feasible. IP6 and heparin appear to bind to the same site on arrestin-1, centered on a positively charged region in the N-domain. We present the first direct evidence that both IP6 and heparin induced a complete release of the arrestin C-tail. These observations provide novel insight into the nature of arrestin transition from basal to active state and demonstrate the potential of NMR-based methods in the study of protein-protein interactions involving members of the arrestin family. PMID:21050017

  7. Identifying the functional part of heparin-binding protein (HBP) as a monocyte stimulator and the novel role of monocytes as HBP producers

    DEFF Research Database (Denmark)

    Schou, Morten; Djurup, René; Norris, Kjeld

    2011-01-01

    a crucial role when HBP binds to monocytes. We synthesized a 20-44 HBP peptide, cyclicized by a sulphur bridge, which encompasses this amino acid and functions as full-length HBP. Using a human monocyte cell line, we have shown that lipopolysaccharide (LPS)-triggered secretion of IL-6 is enhanced up to 10...

  8. Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction.

    Science.gov (United States)

    Craig, P A; Olson, S T; Shore, J D

    1989-04-05

    The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the "effective" antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the

  9. Influence of spacer length on heparin coupling efficiency and fibrinogen adsorption of modified titanium surfaces

    Directory of Open Access Journals (Sweden)

    Gbureck Uwe

    2007-07-01

    Full Text Available Abstract Background Chemical bonding of the drug onto surfaces by means of spacer molecules is accompanied with a reduction of the biological activity of the drug due to a constricted mobility since normally only short spacer molecule like aminopropyltrimethoxysilane (APMS are used for drug coupling. This work aimed to study covalent attachment of heparin to titanium(oxide surfaces by varying the length of the silane coupling agent, which should affect the biological potency of the drug due to a higher mobility with longer spacer chains. Methods Covalent attachment of heparin to titanium metal and TiO2 powder was carried out using the coupling agents 3-(Trimethoxysilyl-propylamine (APMS, N- [3-(Trimethoxysilylpropyl]ethylenediamine (Diamino-APMS and N1- [3-(Trimethoxy-silyl-propyl]diethylenetriamine (Triamino-APMS. The amount of bound coupling agent and heparin was quantified photometrically by the ninhydrin reaction and the tolidine-blue test. The biological potency of heparin was determined photometrically by the chromogenic substrate Chromozym TH and fibrinogen adsorption to the modified surfaces was researched using the QCM-D (Quartz Crystal Microbalance with Dissipation Monitoring technique. Results Zeta-potential measurements confirmed the successful coupling reaction; the potential of the unmodified anatase surface (approx. -26 mV shifted into the positive range (> + 40 mV after silanisation. Binding of heparin results in a strongly negatively charged surface with zeta-potentials of approx. -39 mV. The retaining biological activity of heparin was highest for the spacer molecule Triamino-APMS. QCM-D measurements showed a lower viscosity for adsorbed fibrinogen films on heparinised surfaces by means of Triamino-APMS. Conclusion The remaining activity of heparin was found to be highest for the covalent attachment with Triamino-APMS as coupling agent due to the long chain of this spacer molecule and therefore the highest mobility of the drug

  10. BB0347, from the lyme disease spirochete Borrelia burgdorferi, is surface exposed and interacts with the CS1 heparin-binding domain of human fibronectin.

    Directory of Open Access Journals (Sweden)

    Robert A Gaultney

    Full Text Available The causative agent of Lyme disease, Borrelia burgdorferi, codes for several known fibronectin-binding proteins. Fibronectin a common the target of diverse bacterial pathogens, and has been shown to be essential in allowing for the development of certain disease states. Another borrelial protein, BB0347, has sequence similarity with these other known fibronectin-binding proteins, and may be important in Lyme disease pathogenesis. Herein, we perform an initial characterization of BB0347 via the use of molecular and biochemical techniques. We found that BB0347 is expressed, produced, and presented on the outer surface of intact B. burgdorferi. We also demonstrate that BB0347 has the potential to be important in Lyme disease progression, and have begun to characterize the nature of the interaction between human fibronectin and this bacterial protein. Further work is needed to define the role of this protein in the borrelial infection process.

  11. Heparin conjugated quantum dots for in vitro imaging applications.

    Science.gov (United States)

    Maguire, Ciaran Manus; Mahfoud, Omar Kazem; Rakovich, Tatsiana; Gerard, Valerie Anne; Prina-Mello, Adriele; Gun'ko, Yurii; Volkov, Yuri

    2014-11-01

    In this work heparin-gelatine multi-layered cadmium telluride quantum dots (QDgel/hep) were synthesised using a novel 'one-pot' method. The QDs produced were characterised using various spectroscopic and physiochemical techniques. Suitable QDs were then selected and compared to thioglycolic acid stabilised quantum dots (QDTGA) and gelatine coated quantum dots (QDgel) for utilisation in in vitro imaging experiments on live and fixed permeabilised THP-1, A549 and Caco-2 cell lines. Exposure of live THP-1 cells to QDgel/hep resulted in localisation of the QDs to the nucleus of the cells. QDgel/hep show affinity for the nuclear compartment of fixed permeabilised THP-1 and A549 cells but remain confined to cytoplasm of fixed permeabilised Caco-2 cells. It is postulated that heparin binding to the CD11b receptor facilitates the internalisation of the QDs into the nucleus of THP-1 cells. In addition, the heparin layer may reduce the unfavourable thrombogenic nature of quantum dots observed in vivo. In this study, heparin conjugated quantum dots were found to have superior imaging properties compared to its native counterparts. The authors postulate that heparin binding to the CD11b receptor facilitates QD internalization to the nucleus, and the heparin layer may reduce the in vivo thrombogenic properties of quantum dots. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Unfractionated heparin versus low molecular weight heparin for avoiding heparin-induced thrombocytopenia in postoperative patients.

    Science.gov (United States)

    Junqueira, Daniela R G; Perini, Edson; Penholati, Raphael R M; Carvalho, Maria G

    2012-09-12

    Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction presenting as a prothrombotic disorder related to antibody-mediated platelet activation. It is a poorly understood paradoxical immune reaction resulting in thrombin generation in vivo, which leads to a hypercoagulable state and the potential to initiate venous or arterial thrombosis. A number of factors are thought to influence the incidence of HIT including the type and preparation of heparin (unfractionated heparin (UFH) or low molecular weight heparin (LMWH)) and the heparin-exposed patient population, with the postoperative patient population presenting a higher risk.Although LMWH has largely replaced UFH as a front-line therapy, there is evidence supporting a lack of superiority of LMWH compared with UFH regarding prevention of deep vein thrombosis and pulmonary embolism following surgery, and similar frequencies of bleeding have been described with LMWH and UFH. The decision as to which of these two preparations of heparin to use may thus be influenced by adverse reactions such as HIT. We therefore sought to determine the relative impact of UFH and LMWH specifically on HIT in postoperative patients receiving thromboembolism prophylaxis. The objective of this review was to compare the incidence of HIT and HIT complicated by thrombosis in patients exposed to UFH versus LMWH in randomised controlled trials (RCTs) of postoperative heparin therapy. The Cochrane Peripheral Vascular Diseases Group searched their Specialised Register (March 2012) and CENTRAL (2012, Issue 2). In addition, the authors searched LILACS (March 2012) and additional trials were sought from reference lists of relevant publications. We were interested in comparing the incidence of HIT occurring during exposure to UFH or LMWH after any surgical intervention. Therefore, we studied RCTs in which participants were postoperative patients allocated to receive UFH or LMWH, in a blinded or unblinded fashion. Eligible studies were

  13. Antiproliferative heparin (glycosaminoglycans) isolated from giant ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... source of these sulfated polysaccharides (Nader and. Dietrich, 1989) and it often corresponds up to 90% of the total GAG content of these organisms. Heparin and heap- rin-like substances have a wide range of important biolo- gical activities including inhibition of pulmonary artery smooth muscle cell ...

  14. A Simple Method for Discovering Druggable, Specific Glycosaminoglycan-Protein Systems. Elucidation of Key Principles from Heparin/Heparan Sulfate-Binding Proteins.

    Directory of Open Access Journals (Sweden)

    Aurijit Sarkar

    Full Text Available Glycosaminoglycans (GAGs affect human physiology and pathology by modulating more than 500 proteins. GAG-protein interactions are generally assumed to be ionic and nonspecific, but specific interactions do exist. Here, we present a simple method to identify the GAG-binding site (GBS on proteins that in turn helps predict high specific GAG-protein systems. Contrary to contemporary thinking, we found that the electrostatic potential at basic arginine and lysine residues neither identifies the GBS consistently, nor its specificity. GBSs are better identified by considering the potential at neutral hydrogen bond donors such as asparagine or glutamine sidechains. Our studies also reveal that an unusual constellation of ionic and non-ionic residues in the binding site leads to specificity. Nature engineers the local environment of Asn45 of antithrombin, Gln255 of 3-O-sulfotransferase 3, Gln163 and Asn167 of 3-O-sulfotransferase 1 and Asn27 of basic fibroblast growth factor in the respective GBSs to induce specificity. Such residues are distinct from other uncharged residues on the same protein structure in possessing a significantly higher electrostatic potential, resultant from the local topology. In contrast, uncharged residues on nonspecific GBSs such as thrombin and serum albumin possess a diffuse spread of electrostatic potential. Our findings also contradict the paradigm that GAG-binding sites are simply a collection of contiguous Arg/Lys residues. Our work demonstrates the basis for discovering specifically interacting and druggable GAG-protein systems based on the structure of protein alone, without requiring access to any structure-function relationship data.

  15. Heparin-associated thrombocytopenia: an update.

    Science.gov (United States)

    Fondu, P

    1995-01-01

    The use of heparin may be complicated by two types of thrombocytopenia (HAT): type I occurs early, is transient, and has no clinical relevance, while type II may lead to very severe manifestations (arterial or venous thromboses and more rarely bleedings), that are still underestimated by some clinicians. HAT-type II most frequently develops after use of therapeutic doses of unfractionated heparin (UH) but has also been described less frequently after use of very low doses of UH, of low molecular weight heparins (LMWH), and even of polysulfated glycosaminoglycosans devoid of anticoagulant action. The estimation of the incidence of HAT-type II and of related thromboses is a very difficult matter. Recent observations suggest that thromboses (notably venous) may be more frequent than previously estimated. HAT-type II pathophysiology includes the formation of immune complexes at the surface of platelets; the antigen has been shown to be most often platelet factor 4 bound to heparin while the antibody is recognized by platelet Fc gamma RII receptors. Thromboses result most probably from activation of both platelets (leading to the formation of microparticles) and endothelial cells. Several biological tests are presently available for diagnosing HAT-type II but none of them has been shown to be ideal. The prevention of HAT-type II requires history taking preference of LMWH to UH, early start of oral anticoagulation, and platelet monitoring from the fifth day of heparin therapy. The therapy of HAT-type II implies immediate discontinuation of heparin and avoidance of platelet transfusions, unless severe bleeding occurs. If further antithrombotic treatment is deemed necessary (probably in all cases), several options are possible but presently, the most recommended ones are Org 10172 or Ancrod; embolectomy or thrombolysis may also be required if a new thrombotic event has developed. A very difficult dilemma concerns patients previously sensitized to heparin and who present a

  16. Low molecular weight heparins.

    Science.gov (United States)

    Bergqvist, D

    1996-08-01

    Low molecular weight heparins (LMWHs) differ from unfractionated heparin (UFH) in a number of characteristics, which is probably due to differences in molecular weight distribution. From a clinical point of view the better subcutaneous bioavailability and longer biological half-life are important, making it sufficient to inject LMWHs once-daily only. For practical purposes it is also important that LMWHs be used without monitoring. They are effective as prophylaxis against postoperative venous thromboembolism after all types of surgery; in most studies, more effective than UFH. In most studies, this effect can be obtained safely and with less bleeding than with UFH. LMWHs compare favourably with UFH for starting treatment of deep vein thrombosis, as well as an anticoagulant during haemodialysis. Adverse effects such as thrombocytopenia and osteoporosis are more common with UFH than with LMWHs. Studies evaluating whether or not LMWHs can replace UFH in arterial diseases are still few with small sample sizes. Thus further systematic research is needed.

  17. Bovine and porcine heparins: different drugs with similar effects on human haemodialysis

    Science.gov (United States)

    2013-01-01

    Background Heparins from porcine and bovine intestinal mucosa differ in their structure and also in their effects on coagulation, thrombosis and bleeding. However, they are used as undistinguishable drugs. Methods We compared bovine and porcine intestinal heparin administered to patients undergoing a particular protocol of haemodialysis. We compared plasma concentrations of these two drugs and also evaluated how they affect patients and the dialyzer used. Results Compared with porcine heparin, bovine heparin achieved only 76% of the maximum plasma concentration as IU mL-1. This observation is consistent with the activities observed in the respective pharmaceutical preparations. When the plasma concentrations were expressed on weight basis, bovine heparin achieved a maximum concentration 1.5 fold higher than porcine heparin. The reduced anticoagulant activity and higher concentration, on weight basis, achieved in the plasma of patients under dialysis using bovine instead of porcine heparin did not affect significantly the patients or the dialyzer used. The heparin dose is still in a range, which confers security and safety to the patients. Discussion Despite no apparent difference between bovine and porcine intestinal heparins in the haemodialysis practice, these two types of heparins should be used as distinct drugs due to their differences in structure and biological effects. Conclusions The reduced anticoagulant activity achieved in the plasma of patients under dialysis using bovine instead of porcine heparin did not affect significantly the patients or the dialyzer. PMID:23763719

  18. Changes in heparin dose response slope during cardiac surgery: possible result in inaccuracy in predicting heparin bolus dose requirement to achieve target ACT.

    Science.gov (United States)

    Ichikawa, Junko; Mori, Tetsu; Kodaka, Mitsuharu; Nishiyama, Keiko; Ozaki, Makoto; Komori, Makiko

    2017-09-01

    The substantial interpatient variability in heparin requirement has led to the use of a heparin dose response (HDR) technique. The accuracy of Hepcon-based heparin administration in achieving a target activated clotting time (ACT) using an HDR slope remains controversial. We prospectively studied 86 adult patients scheduled for cardiac surgery requiring cardiopulmonary bypass. The total dose of calculated heparin required for patient and pump priming was administered simultaneously to achieve a target ACT of 450 s for HDR on the Hepcon HMS system. Blood samples were obtained after the induction of anesthesia, at 3 min after heparin administration and after the initiation of CPB to measure kaolin ACT, HDR slope, whole-blood heparin concentration based on the HDR slope and anti-Xa heparin concentration, antithrombin and complete blood count. The target ACT of 450 s was not achieved in 68.6% of patients. Compared with patients who achieved the target ACT, those who failed to achieve their target ACT had a significantly higher platelet count at baseline. Correlation between the HDR slope and heparin sensitivity was poor. Projected heparin concentration and anti-Xa heparin concentration are not interchangeable based on the Bland-Altman analysis. It can be hypothesized that the wide discrepancy in HDR slope versus heparin sensitivity may be explained by an inaccurate prediction of the plasma heparin level and/or the change in HDR of individual patients, depending on in vivo factors such as extravascular sequestration of heparin, decreased intrinsic antithrombin activity level and platelet count and/or activity.

  19. A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent.

    Science.gov (United States)

    Bett, Cyrus; Grgac, Ksenija; Long, Dianna; Karfunkle, Michael; Keire, David A; Asher, David M; Gregori, Luisa

    2017-05-01

    In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans. Thus, US heparin is currently sourced only from pig intestines. Availability of alternative sources of crude heparin, a life-saving drug, would benefit public health. Bovine heparin is an obvious option, but BSE clearance by the bovine heparin manufacturing process should be evaluated. To this end, using hamster 263K scrapie as a surrogate for BSE agent, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked scrapie agent. We removed aliquots from each step and analyzed them for residual abnormal prion protein (PrPTSE) using a sensitive in vitro method, real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity by 3.6 log10 and removed PrPTSE, measured as seeding activity, by 3.4 log10. NaOH treatment was the most effective removal step tested. We also investigated NaOH at different concentrations and pH: the results showed that as much as 5.2 log10 of PrPTSE seeding activity was removed at pH 12.5. Thus, changes to the concentration, treatment time, and temperature of alkaline extraction might further improve removal. Our results, using a basic heparin manufacturing process, inform efforts to reintroduce safe bovine heparin in the USA.

  20. Heparinization of a biomimetic bone matrix: integration of heparin during matrix synthesis versus adsorptive post surface modification.

    Science.gov (United States)

    König, Ulla; Lode, Anja; Welzel, Petra B; Ueda, Yuichiro; Knaack, Sven; Henß, Anja; Hauswald, Anke; Gelinsky, Michael

    2014-03-01

    This study intended to evaluate a contemporary concept of scaffolding in bone tissue engineering in order to mimic functions of the extracellular matrix. The investigated approach considered the effect of the glycosaminoglycan heparin on structural and biological properties of a synthetic biomimetic bone graft material consisting of mineralized collagen. Two strategies for heparin functionalization were explored in order to receive a three-component bone substitute material. Heparin was either incorporated during matrix synthesis by mixing with collagen prior to simultaneous fibril reassembly and mineralization (in situ) or added to the matrix after fabrication (a posteriori). Both methods resulted in an incorporation of comparable amounts of heparin, though its distribution in the matrix varied as indicated by TOF-SIMS analyses, and a similar modulation of their protein binding properties. Differential scanning calorimetry revealed that the thermal stability and thereby the degree of crosslinking of the heparinized matrices was increased. However, in contrast to the a posteriori modification, the in situ integration of heparin led to considerable changes of morphology and composition of the matrix: a more open network of collagen fibers yielding a more porous surface and a reduced mineral content were observed. Cell culture experiments with human mesenchymal stem cells (hMSC) revealed a strong influence of the mode of heparin functionalization on cellular processes, as demonstrated for proliferation and osteogenic differentiation of hMSC. Our results indicate that not only heparin per se but also the way of its incorporation into a collagenous matrix determines the cell response. In conclusion, the a posteriori modification was beneficial to support adhesion, proliferation and differentiation of hMSC.

  1. Vinculin activation is necessary for complete talin binding.

    Science.gov (United States)

    Golji, Javad; Lam, Johnny; Mofrad, Mohammad R K

    2011-01-19

    Focal adhesions are critical to a number of cellular processes that involve mechanotransduction and mechanical interaction with the cellular environment. The growth and strengthening of these focal adhesions is dependent on the interaction between talin and vinculin. This study investigates said interaction and how vinculin activation influences it. Using molecular dynamics, the interaction between talin's vinculin binding site (VBS) and vinculin's domain 1 (D1) is simulated both before and after vinculin activation. The simulations of VBS binding to vinculin before activation suggest the proximity of the vinculin tail to D1 prevents helical movement in D1 and thus prevents binding of VBS. In contrast, interaction of VBS with activated vinculin shows the possibility of complete VBS insertion into D1. In the simulations of both activated and autoinhibited vinculin where VBS fails to fully bind, VBS demonstrates significant hydrophobic interaction with surface residues in D1. These interactions link VBS to D1 even without its proper insertion into the hydrophobic core. Together these simulations suggest VBS binds to vinculin with the following mechanism: 1), VBS links to D1 via surface hydrophobic interactions; 2), vinculin undergoes activation and D1 is moved away from the vinculin tail; 3), helices in D1 undergo conformational change to allow VBS binding; and 4), VBS inserts itself into the hydrophobic core of D1. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Simulated Thrombin Generation in the Presence of Surface-Bound Heparin and Circulating Tissue Factor.

    Science.gov (United States)

    Dydek, E Victoria; Chaikof, Elliot L

    2016-04-01

    An expanded computational model of surface induced thrombin generation was developed that includes hemodynamic effects, 22 biochemical reactions and 44 distinct chemical species. Surface binding of factors V, VIII, IX, and X was included in order to more accurately simulate the formation of the surface complexes tenase and prothrombinase. In order to model these reactions, the non-activated, activated and inactivated forms were all considered. This model was used to investigate the impact of surface bound heparin on thrombin generation with and without the additive effects of thrombomodulin (TM). In total, 104 heparin/TM pairings were evaluated (52 under venous conditions, 52 under arterial conditions), the results demonstrating the synergistic ability of heparin and TM to reduce thrombin generation. Additionally, the role of circulating tissue factor (TF(p)) was investigated and compared to that of surface-bound tissue factor (TF(s)). The numerical results suggest that circulating TF has the power to amplify thrombin generation once the coagulation cascade is already initiated by surface-bound TF. TF(p) concentrations as low as 0.01 nM were found to have a significant impact on total thrombin generation.

  3. Intratracheal heparin improves plastic bronchitis due to sulfur mustard analog.

    Science.gov (United States)

    Houin, Paul R; Veress, Livia A; Rancourt, Raymond C; Hendry-Hofer, Tara B; Loader, Joan E; Rioux, Jacqueline S; Garlick, Rhonda B; White, Carl W

    2015-02-01

    Inhalation of sulfur mustard (SM) and SM analog, 2-chloroethyl ethyl sulfide (CEES), cause fibrinous cast formation that occludes the conducting airways, similar to children with Fontan physiology-induced plastic bronchitis. These airway casts cause significant mortality and morbidity, including hypoxemia and respiratory distress. Our hypothesis was that intratracheal heparin, a highly cost effective and easily preserved rescue therapy, could reverse morbidity and mortality induced by bronchial cast formation. Sprague-Dawley rats were exposed to 7.5% CEES via nose-only aerosol inhalation to produce extensive cast formation and mortality. The rats were distributed into three groups: non-treated, phosphate-buffered saline (PBS)-treated, and heparin-treated groups. Morbidity was assessed with oxygen saturations and clinical distress. Blood and bronchoalveolar lavage fluid (BALF) were obtained for analysis, and lungs were fixed for airway microdissection to quantify the extent of airway cast formation. Heparin, given intratracheally, improved survival (100%) when compared to non-treated (75%) and PBS-treated (90%) controls. Heparin-treated rats also had improved oxygen saturations, clinical distress and airway cast scores. Heparin-treated rats had increased thrombin clotting times, factor Xa inhibition and activated partial thromboplastin times, indicating systemic absorption of heparin. There were also increased red blood cells (RBCs) in the BALF in 2/6 heparin-treated rats compared to PBS-treated control rats. Intratracheal heparin 1 hr after CEES inhalation improved survival, oxygenation, airway obstruction, and clinical distress. There was systemic absorption of heparin in rats treated intratracheally. Some rats had increased RBCs in BALF, suggesting a potential for intrapulmonary bleeding if used chronically after SM inhalation. © 2014 Wiley Periodicals, Inc.

  4. Construction of mussel-inspired coating via the direct reaction of catechol and polyethyleneimine for efficient heparin immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yujie [School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); The Institute of Biomaterials and Surface Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Luo, Rifang, E-mail: lrifang@126.com [School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); The Institute of Biomaterials and Surface Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Shen, Fangyu; Tang, Linlin [School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); The Institute of Biomaterials and Surface Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Wang, Jin, E-mail: jinxxwang@263.net [School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); The Institute of Biomaterials and Surface Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Huang, Nan [School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); The Institute of Biomaterials and Surface Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2015-02-15

    Highlights: • Catechol (CA) and PEI copolymerization was a mimetic and dopamine-like coating method. • CA/PEI film provided amine groups and was effective in heparin immobilization. • CA/PEI coating could inhibit smooth muscle cell proliferation. • CA/PEI coating did not show any significant cytotoxicity to endothelial cell. - Abstract: Dopamine could self-polymerize to form the coating on various substrates and the co-existence of catechols and amines was crucial in performing such polymerization process. In this work, a mimetic approach of coating formation was carried out based on the co-polymerization of catechol (CA) and polyethyleneimine (PEI). Mussel-inspired CA/PEI coating was deposited on 316L stainless steel (SS). Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectroscopy (XPS) demonstrated the successful coating formation. QCM measurement showed good affinity of heparin immobilization on CA/PEI coating surface ascribed to the amine groups. Herein, vascular cell-material interactions like endothelial cells (ECs) and smooth muscle cells (SMCs) were also investigated. Interestingly, CA/PEI and heparin modified coatings presented no cytotoxicity to ECs, however to a certain extent, decreased SMCs proliferation. Moreover, heparin-binding surface presented significant anti-platelet adhesion and activation properties. These results effectively suggested that the mussel-inspired CA/PEI coating might be promising when served as a platform for biomolecule immobilization.

  5. Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy.

    Science.gov (United States)

    Chammas, Oliver; Bonass, William A; Thomson, Neil H

    2017-05-01

    The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP>Heparin or HepS>DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Binding and Activation of Human Plasminogen by Mycobacterium tuberculosis

    Science.gov (United States)

    Monroy, Verónica; Amador, Angelica; Ruiz, Blanca; Espinoza-Cueto, Patricia; Xolalpa, Wendy; Mancilla, Raul; Espitia, Clara

    2000-01-01

    The first evidence of the interaction of Mycobacterium tuberculosis with the plasminogen system is herein reported. By FACScan analysis and affinity blotting, lysine-dependent binding of plasminogen to M. tuberculosis was demonstrated. The binding molecules were 30-, 60-, and 66-kDa proteins present in cell wall and soluble protein extracts. The activation of plasminogen, which occurred only in presence of fibrin and was not inhibited by the host serpin, α2-antiplasmin, was also demonstrated. PMID:10858253

  7. Collection of heparinized plasma by plasmapheresis

    NARCIS (Netherlands)

    van der Meer, P. F.; Vrielink, H.; Pietersz, R. N.; Dekker, W. J.; Reesink, H. W.

    1999-01-01

    BACKGROUND AND OBJECTIVES: Heparinized plasma can be used for exchange transfusions in neonates and is usually collected by drawing whole blood using heparin as anticoagulant. The heparinized red blood cells and buffy coat cannot be used and are therefore discarded. To collect heparinized plasma

  8. Enzyme activation through the utilization of intrinsic dianion binding energy.

    Science.gov (United States)

    Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

    2017-03-01

    We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed.

  9. Transmyocardial drilling revascularization combined with heparinized bFGF-incorporating stent activates resident cardiac stem cells via SDF-1/CXCR4 axis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Guang-Wei [Department of Cardiac Surgery and Neurology, The First Hospital of China Medical University, Shenyang 110001 (China); Wen, Ti [College of Life Science, Nankai University, Tianjin 300036 (China); Gu, Tian-Xiang, E-mail: cmugtx@sina.com [Department of Cardiac Surgery and Neurology, The First Hospital of China Medical University, Shenyang 110001 (China); Li-Ling, Jesse [Department of Medical Genetics, China Medical University, Shenyang 110001 (China); Institute of Medical Genetics, School of Life Science and Key Laboratory for Bio-resources and Eco-environment of the Ministry of Education, Sichuan University, Chengdu 610064 (China); Wang, Chun; Zhao, Ye; Liu, Jing; Wang, Ying [Department of Cardiac Surgery and Neurology, The First Hospital of China Medical University, Shenyang 110001 (China); Liu, Tian-Jun; Lue, Feng [Institute of Biomedical Engineering, Peking Union Medical College, Beijing 100730 (China)

    2012-02-15

    Objective: To investigate whether transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor (bFGF)-incorporating degradable stent implantation (TMDRSI) can promote myocardial regeneration after acute myocardial infarction (AMI). Methods: A model of AMI was generated by ligating the mid-third of left anterior descending artery (LAD) of miniswine. After 6 h, the animals were divided into none-treatment (control) group (n = 6) and TMDRSI group (n = 6). For TMDRSI group, two channels with 3.5 mm in diameter were established by a self-made drill in the AMI region, into which a stent was implanted. Expression of stromal cell-derived factor-1{sub {alpha}} (SDF-1{sub {alpha}}) and CXC chemokine receptor 4 (CXCR4), cardiac stem cell (CSC)-mediated myocardial regeneration, myocardial apoptosis, myocardial viability, and cardiac function were assessed at various time-points. Results: Six weeks after the operation, CSCs were found to have differentiated into cardiomyocytes to repair the infarcted myocardium, and all above indices showed much improvement in the TMDRSI group compared with the control group (P < 0.001). Conclusions: The new method has shown to be capable of promoting CSCs proliferation and differentiation into cardiomyocytes through activating the SDF-1/CXCR4 axis, while inhibiting myocardial apoptosis, thereby enhancing myocardial regeneration following AMI and improving cardiac function. This may provide a new strategy for myocardial regeneration following AMI. -- Highlights: Black-Right-Pointing-Pointer The effects of TMDR and bFGF-stent on myocardial regeneration were studied in a pig model of AMI. Black-Right-Pointing-Pointer TMDR and bFGF-stent implantation activated CSCs via the SDF-1/CXCR4 axis. Black-Right-Pointing-Pointer CSC-mediated myocardial regeneration improved cardiac function. Black-Right-Pointing-Pointer It may be a new therapeutic strategy for AMI.

  10. Low-molecular weight heparin increases circulating sFlt-1 levels and enhances urinary elimination.

    Directory of Open Access Journals (Sweden)

    Henning Hagmann

    Full Text Available RATIONALE: Preeclampsia is a devastating medical complication of pregnancy which leads to maternal and fetal morbidity and mortality. While the etiology of preeclampsia is unclear, human and animal studies suggest that excessive circulating levels of soluble fms-like tyrosine-kinase-1 (sFlt-1, an alternatively spliced variant of VEGF-receptor1, contribute to the signs and symptoms of preeclampsia. Since sFlt-1 binds to heparin and heparan sulfate proteoglycans, we hypothesized that the anticoagulant heparin, which is often used in pregnancy, may interfere with the levels, distribution and elimination of sFlt-1 in vivo. OBJECTIVE: We systematically determined serum and urine levels of angiogenic factors in preeclamptic women before and after administration of low molecular weight heparin and further characterized the interaction with heparin in biochemical studies. METHODS AND RESULTS: Serum and urine samples were used to measure sFlt-1 levels before and after heparin administration. Serum levels of sFlt-1 increased by 25% after heparin administration in pregnant women. The magnitude of the increase in circulating sFlt-1 correlated with initial sFlt-1 serum levels. Urinary sFlt-1 levels were also elevated following heparin administration and levels of elimination were dependent on the underlying integrity of the glomerular filtration barrier. Biochemical binding studies employing cation exchange chromatography revealed that heparin bound sFlt-1 had decreased affinity to negatively charged surfaces when compared to sFlt-1 alone. CONCLUSION: Low molecular weight heparin administration increased circulating sFlt1 levels and enhanced renal elimination. We provide evidence that both effects may be due to heparin binding to sFlt1 and masking the positive charges on sFlt1 protein.

  11. Mannan-binding lectin activates C3 and the

    DEFF Research Database (Denmark)

    Selander, B.; Martensson, U.; Weintraub, A.

    2006-01-01

    Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL) or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera...

  12. In vitro effects of heparin and tissue factor pathway inhibitor on factor VII assays. possible implications for measurements in vivo after heparin therapy

    DEFF Research Database (Denmark)

    Bladbjerg, E-M; Larsen, L F; Ostergaard, P

    2000-01-01

    The coagulant activity of blood coagulation factor VII (FVII:C) can be lowered by changes in lifestyle and by therapeutic intervention, e.g. heparin infusion. The question is, however, whether FVII:C determined ex vivo is a valid measure of the FVII activity in vivo. We measured plasma FVII......:C, activated FVII (FVIIa), FVII protein (FVII:Ag), tissue factor pathway inhibitor (TFPI), triglycerides, and free fatty acids (FFA) before and 15 min after infusion of a bolus of unfractionated heparin (50 IU/kg body weight) in 12 healthy subjects. Additionally, we conducted in vitro experiments...... activity by means of FVII clotting assays. These assays should therefore not be used to measure the coagulation status of patients in heparin therapy, unless extraordinary precautions are taken to eliminate TFPI and heparin effects ex vivo. The observed effect of heparin on FVII:Ag should be investigated...

  13. HDAC Inhibitors without an Active Site Zn2+-Binding Group

    DEFF Research Database (Denmark)

    Vickers, Chris J.; Olsen, Christian Adam; Leman, Luke J.

    2012-01-01

    Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn2+ ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off......-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure−activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn2+-binding group. The lead compounds (e.g., 15 and 26) display good...... potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/ off competitive inhibitor of HDACs 1−3 with Ki values of 49, 33, and 37 nM, respectively. Our proof...

  14. Synergistic action of heparin and serum on basic fibroblast growth factor-modulated DNA synthesis and mitochondrial activity of cultured bovine corneal endothelial cells

    NARCIS (Netherlands)

    Hoppenreijs, V. P.; Pels, E.; Felten, P. C.; Ruijter, J. M.; Vrensen, G. F.; Treffers, W. F.

    1996-01-01

    Basic fibroblast growth factor (bFGF) is a major mitogen and chemoattractant for many cell types. The synergistic role of fetal bovine serum (FBS) and heparin on the modulation of tissue-cultured bovine corneal endothelial cells by bFGF was studied. Cell modulation was assessed by DNA synthesis

  15. Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα.

    Science.gov (United States)

    Fang, Changming; Filipp, Fabian V; Smith, Jeffrey W

    2012-04-01

    Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.

  16. Polyguluronate sulfate and its oligosaccharides but not heparin promotes FGF19/FGFR1c signaling

    Science.gov (United States)

    Lan, Ying; Zeng, Xuan; Guo, Zhihua; Zeng, Pengjiao; Hao, Cui; Zhao, Xia; Yu, Guangli; Zhang, Lijuan

    2017-06-01

    Fibroblast growth factor 19(FGF19) functions as a hormone by affecting glucose metabolism. FGF19 improves glucose tolerance when overexpressed in mice with impaired glucose tolerance or diabetes. A functional cellular FGF19 receptor consists of FGF receptor (FGFR) and glycosaminoglycan complexed with either α Klotho or β Klotho. Interestingly, in mice with diet-induced diabetes, a single injection of FGF1 is enough to restore blood sugar levels to a healthy range. FGF1 binds heparin with high affinity whereas FGF19 does not, indicating that polysaccharides other than heparin might enhance FGF19/FGFR signaling. Using a FGFs/FGFR1c signaling-dependent BaF3 cell proliferation assay, we discovered that polyguluronate sulfate (PGS) and its oligosaccharides, PGS12 and PGS25, but not polyguluronate (PG), a natural marine polysaccharide, enhanced FGF19/FGFR1c signaling better than that of heparin based on 3H-thymidine incorporation. Interestingly, PGS6, PGS8, PGS10, PGS12, PGS25, and PGS, but not PG, had comparable FGF1/FGFR1c signal-stimulating activity compared to that of heparin. These results indicated that PGS and its oligosaccharides were excellent FGF1/FGFR1c and FGF19/FGFR1c signaling enhancers at cellular level. Since the inexpensive PGS and PGS oligosaccharides can be absorbed through oral route, these seaweed-derived compounds merit further investigation as novel agents for the treatment of type 2 diabetes through enhancing FGF1/FGFR1c and FGF19/FGFR1c signaling in future.

  17. Effects of surface-bound and intravenously administered heparin on cell-surface interactions: inflammation and coagulation.

    Science.gov (United States)

    Johnson, G; Curry, B; Cahalan, L; Prater, R; Biggerstaff, J; Hussain, A; Gartner, M; Cahalan, P

    2013-05-01

    Intravenous administration of heparin and heparin-bonded extracorporeal circuits are frequently used to mitigate the deleterious effects of blood contact with synthetic materials. The work described here utilized human blood in a micro-perfusion circuit to experimentally examine the effects of intravenous and surface-bound heparin on cellular activation. Activation markers of coagulation and of the inflammatory response were examined using flow cytometry; specifically, markers of platelet, monocyte, polymorphonuclear leukocyte (PMN), and lymphocyte activation were quantified. The results indicate that surface-bound heparin reduces the inflammatory response whereas systemically administered heparin does not. This finding has important implications for blood-contacting devices, particularly within the context of recently elucidated connections between inflammation pathways and coagulation disorders. Data presented indicate that surface-bound heparin and intravenously administered heparin play distinct, but vital roles in rendering biomaterial surfaces compatible with blood.

  18. Heparin release from thermosensitive hydrogels

    NARCIS (Netherlands)

    Gutowska, Anna; Bae, You Han; Feijen, Jan; Kim, Sung Wan

    1992-01-01

    Thermosensitive hydrogels (TSH) were synthesized and investigated as heparin releasing polymers for the prevention of surface induced thrombosis. TSH were synthesized with N-isopropyl acrylamide (NiPAAm) copolymerized with butyl methacrylate (BMA) (hydrophobic) or acrylic acid (AAc) (hydrophilic)

  19. Determination of activated plasma fibronectin using radioactive labelled collagen I

    DEFF Research Database (Denmark)

    Fenger, M

    1984-01-01

    The plasma concentration of biological active fibronectin was assayed by a protein binding assay using 125I-collagen I as ligand and heparin as activator. The standard curve is linear for a fibronectin range of 1.1-11 pmol (0.5-5.0 micrograms) and the coefficient of variation was less than 10......%. The active or activable fibronectin was compared to the immunoreactive fibronectin in plasma from patients with various bacterial diseases. Similar concentrations were detected by the two assays suggesting that all the circulating fibronectin was functionally active. The assay was also applied to determine...... the structure-function relationship of heparin and heparansulphate in activation of fibronectin. Low-sulphated heparansulphate from umbilical cords and heparin-activated fibronectin but the effect was uncorrelated to anticoagulation activity. Only a small fraction of the heparin was actually capable...

  20. The suicide substrate reaction between plasminogen activator inhibitor 1 acid thrombin is regulated by the cofactors vitronectin and heparin

    NARCIS (Netherlands)

    van Meijer, M.; Smilde, A.; Tans, G.; Nesheim, M. E.; Pannekoek, H.; Horrevoets, A. J.

    1997-01-01

    The interaction of thrombin with plasminogen activator inhibitor 1 (PAI-1) is shown to result in the simultaneous formation of both cleaved PAI-1 and a sodium dodecyl sulfate-stable thrombin-PAI-1 complex. The kinetics of this reaction can be described by a ''suicide substrate'' mechanism that

  1. The Use of Heparin during Endovascular Peripheral Arterial Interventions: A Synopsis

    Directory of Open Access Journals (Sweden)

    Arno M. Wiersema

    2016-01-01

    Full Text Available A large variety exists for many aspects of the use of heparin as periprocedural prophylactic antithrombotics (PPAT during peripheral arterial interventions (PAI. This variation is present, not only within countries, but also between them. Due to a lack of (robust data, no systematic review on the use of heparin during PAI could be justified. A synopsis of all available literature on heparin during PAI describes that heparin is used on technical equipment to reduce the thrombogenicity and in the flushing solution with saline. Heparin could have a cumulative anticoagulant effect when used in combination with ionic contrast medium. No level-1 evidence exists on the use of heparin. A measurement of actual anticoagulation status by means of an activated clotting time should be mandatory.

  2. Low-dose versus high-dose heparinization during arteriovenous carbon dioxide removal.

    Science.gov (United States)

    Murphy, J A; Savage, C M; Alpard, S K; Deyo, D J; Jayroe, J B; Zwischenberger, J B

    2001-11-01

    The purpose of this study was to compare low-dose (LD) and high-dose (HD) systemic heparinization in a prospective randomized study of arteriovenous carbon dioxide removal (AVCO2R) during acute respiratory distress syndrome, using a commercially available heparin-coated oxygenator. Adult sheep (n = 13) received an LD50 smoke inhalation and 40% TBSA third degree cutaneous flame burn injury. At 40-48 h post-injury, animals underwent cannulation of the carotid artery and jugular vein and were then randomized to HD heparin (activated clotting time, ACT > 300s, n = 6) and LD heparin (ACT heparin (ACT heparin-coated oxygenator does not increase thrombogenicity during AVCO2R for smoke/burn-induced severe lung injury in sheep.

  3. Single-step synthesis of heparin-doped polypyrrole nanoparticles for delivery of angiogenic factor.

    Science.gov (United States)

    Xiong, Gordon M; Yap, Yi Zhen; Choong, Cleo

    2016-04-01

    To perform one-pot synthesis of heparin-immobilized polypyrrole (PPy) nanoparticles and evaluate the use of these nanoparticles for the delivery of VEGF. Heparin-stabilized synthesis of PPy nanoparticles was performed via oxidative polymerization. VEGF-bound PPy-heparin nanoparticles were delivered to endothelial cells and bioactivity of VEGF was assessed by Matrigel tube formation. Size-controllable synthesis of heparin-doped PPy nanoparticles was achieved, and heparin promoted the conjugation of VEGF. Angiogenic activity of the VEGF-conjugated PPy nanoparticles was verified. Heparin-doped PPy nanoparticles can be synthesized using one-pot reaction and provide a delivery platform by which VEGF can be conjugated onto.

  4. Preparation of Low Molecular Weight Heparin by Microwave Discharge Electrodeless Lamp/TiO2 Photo-Catalytic Reaction.

    Science.gov (United States)

    Lee, Do-Jin; Kim, Byung Hoon; Kim, Sun-Jae; Kim, Jung-Sik; Lee, Heon; Jung, Sang-Chul

    2015-01-01

    An MDEL/TiO2 photo-catalyst hybrid system was applied, for the first time, for the production of low molecular weight heparin. The molecular weight of produed heparin decreased with increasing microwave intensity and treatment time. The abscission of the chemical bonds between the constituents of heparin by photo-catalytic reaction did not alter the characteristics of heparin. Formation of by-products due to side reaction was not observed. It is suggested that heparin was depolymerized by active oxygen radicals produced during the MDEL/TiO2 photo-chemical reaction.

  5. Heparin and insulin in the management of hypertriglyceridemia-associated pancreatitis: case series and literature review.

    Science.gov (United States)

    Kuchay, Mohammad Shafi; Farooqui, Khalid J; Bano, Tarannum; Khandelwal, Manoj; Gill, Harmandeep; Mithal, Ambrish

    2017-01-01

    Severe hypertriglyceridemia accounts for up to 7% of all cases of acute pancreatitis. Heparin and insulin activate lipoprotein lipase (LPL), thereby reducing plasma triglyceride levels. However, the safety and efficacy of heparin and insulin in the treatment of hypertriglyceridemia-associated acute pancreatitis have not been well established yet. We successfully used heparin and insulin as first-line therapy in four consecutive patients with acute pancreatitis secondary to hypertriglyceridemia. In a literature search, we revised almost all reports published to date of patients managed successfully with this combination. Heparin and insulin appear to be a safe, effective, and inexpensive first-line therapy for hypertriglyceridemia-associated acute pancreatitis.

  6. Increased accuracy in heparin and protamine administration decreases bleeding: a pilot study

    DEFF Research Database (Denmark)

    Runge, Marx; Møller, Christian H; Steinbrüchel, Daniel A

    2009-01-01

    of the study was to evaluate whether a heparin-protamine titration system, Hemochron RxDx, could reduce postoperative bleeding and blood transfusion. Fifty-three patients were included prospectively over a 6-month period. The test group (RxDx group; 28 patients) received heparin and protamine doses calculated...... using the Hemochron RxDx system, which performs a baseline activated clotting time (ACT) value together with a heparin response test. An accurate heparin dose was calculated based on the Bull dose/response curve. Protamine doses were calculated by the same method. In the control group (25 patients...

  7. Activated macrophage survival is coordinated by TAK1 binding proteins.

    Directory of Open Access Journals (Sweden)

    September R Mihaly

    Full Text Available Macrophages play diverse roles in tissue homeostasis and immunity, and canonically activated macrophages are critically associated with acute inflammatory responses. It is known that activated macrophages undergo cell death after transient activation in some settings, and the viability of macrophages impacts on inflammatory status. Here we report that TGFβ- activated kinase (TAK1 activators, TAK1-binding protein 1 (TAB1 and TAK1-binding protein 2 (TAB2, are critical molecules in the regulation of activated macrophage survival. While deletion of Tak1 induced cell death in bone marrow derived macrophages even without activation, Tab1 or Tab2 deletion alone did not profoundly affect survival of naïve macrophages. However, in lipopolysaccharide (LPS-activated macrophages, even single deletion of Tab1 or Tab2 resulted in macrophage death with both necrotic and apoptotic features. We show that TAB1 and TAB2 were redundantly involved in LPS-induced TAK1 activation in macrophages. These results demonstrate that TAK1 activity is the key to activated macrophage survival. Finally, in an in vivo setting, Tab1 deficiency impaired increase of peritoneal macrophages upon LPS challenge, suggesting that TAK1 complex regulation of macrophages may participate in in vivo macrophage homeostasis. Our results demonstrate that TAB1 and TAB2 are required for activated macrophages, making TAB1 and TAB2 effective targets to control inflammation by modulating macrophage survival.

  8. Optimized Treatment of Heparinized Blood Fractions to Make Them Suitable for Analysis.

    Science.gov (United States)

    Sánchez-Fito, María Teresa; Oltra, Elisa

    2015-08-01

    It has been known for decades that many cytokines, such as IL-2, IL-6, and IL-12, bind to heparin. Even though some enzyme-linked immunosorbent assays (ELISA) use antibody-recognizing epitopes not affected by this binding, ELISA manufacturers often warn that heparinized plasma or serum fractions containing more than 3 IU (international units)/mL of heparin should not be used in assays so as to prevent heparin interference in the reaction. In addition, enzyme-based nucleic acid amplifications from heparinized samples have been shown defective by several research groups. The aim of this study was to determine optimal degradation and/or removal of heparin from heparinized blood samples to best turn them into fractions for appropriate ELISA and RT-PCR analysis. A colorimetric reporter assay based on the metachromatic effect of the binding of heparin to toluidine blue was shown to be a low-cost effective method to discriminate assay compatible blood fractions with heparin levels below 3 IU/mL. Heparin removal from human blood fractions was best achieved by treatment with either Bacteroides Heparinase II or the less expensive Heparinase I at a final concentration of 0.1 U/μL and incubations at 30°C for a period between 30 min and 4 h, or by adsorption to Ecteola slurries at a concentration of 20 mg/mL for 20 min at room temperature (RT). The fact that both enzymatic and resin-based optimized treatments allowed for replication of the readings obtained with heparin-free equivalent fractions in both ELISA and RT-PCR assays indicates they should be appropriate for quantitative studies such as expression profiling at both the protein and nucleic acid level. The cost-effective protocols developed in this study could make heparinized, otherwise unusable, blood-derived collections suitable for analysis by ELISA and RT-PCR amplifications, among other analyses, enhancing the possibilities for studying valuable bio-banked heparinized human samples.

  9. A pyrene-based fluorescent sensor for ratiometric detection of heparin and its complex with heparin for reversed ratiometric detection of protamine in aqueous solution

    Science.gov (United States)

    Gong, Weiwei; Wang, Shihuai; Wei, Yuting; Ding, Liping; Fang, Yu

    2017-01-01

    An imidazolium-modified pyrene derivative, IPy, was used for ratiometric detection of heparin, and its complex with heparin was used for reversed ratiometric detection of protamine in both aqueous solution and serum samples. The cationic fluorescent probe could interact with anionic heparin via electrostatic interaction to bring about blue-to-green fluorescence changes as monomer emission significantly decreases and excimer increases. The binary combination of IPy and heparin could be further used for green-to-blue detection of protamine since heparin prefers to bind to protamine instead of the probe due to its stronger affinity with protamine. The cationic probe shows high sensitivity to heparin with a low detection limit of 8.5 nM (153 ng/mL) and its combination with heparin displays high sensitivity to protamine with a detection limit as low as 15.4 nM (107.8 ng/mL) according to the 3σ IUPAC criteria. Moreover, both sensing processes are fast and can be performed in serum solutions, indicating possibility for practical applications.

  10. Anti-tumor activity of a novel HS-mimetic-vascular endothelial growth factor binding small molecule.

    Directory of Open Access Journals (Sweden)

    Kazuyuki Sugahara

    Full Text Available The angiogenic process is controlled by variety of factors of which the vascular endothelial growth factor (VEGF pathway plays a major role. A series of heparan sulfate mimetic small molecules targeting VEGF/VEGFR pathway has been synthesized. Among them, compound 8 (2-butyl-5-chloro-3-(4-nitro-benzyl-3H-imidazole-4-carbaldehyde was identified as a significant binding molecule for the heparin-binding domain of VEGF, determined by high-throughput-surface plasmon resonance assay. The data predicted strong binding of compound 8 with VEGF which may prevent the binding of VEGF to its receptor. We compared the structure of compound 8 with heparan sulfate (HS, which have in common the functional ionic groups such as sulfate, nitro and carbaldehyde that can be located in similar positions of the disaccharide structure of HS. Molecular docking studies predicted that compound 8 binds at the heparin binding domain of VEGF through strong hydrogen bonding with Lys-30 and Gln-20 amino acid residues, and consistent with the prediction, compound 8 inhibited binding of VEGF to immobilized heparin. In vitro studies showed that compound 8 inhibits the VEGF-induced proliferation migration and tube formation of mouse vascular endothelial cells, and finally the invasion of a murine osteosarcoma cell line (LM8G7 which secrets high levels of VEGF. In vivo, these effects produce significant decrease of tumor burden in an experimental model of liver metastasis. Collectively, these data indicate that compound 8 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of endothelial cell migration and angiogenesis mediated by VEGF. In conclusion, compound 8 may normalize the tumor vasculature and microenvironment in tumors probably by inhibiting the binding of VEGF to its receptor.

  11. Stimulation of Fas agonistic antibody–mediated apoptosis by heparin-like agents suppresses Hsp27 but not Bcl-2 protective activity

    OpenAIRE

    Manero, Florence; Ljubic-Thibal, Vesna; Moulin, Maryline; Goutagny, Nadège; Yvin, Jean-Claude; Arrigo, André-Patrick

    2004-01-01

    We report that in Jurkat T cells or freshly isolated T lymphocytes, physiological concentrations of high– molecular weight sulfated polysaccharides such as heparin, heparan sulfate, and dextran sulfate significantly increased the percentage of cell death induced by Fas IgM agonistic antibody. The phenomenon was caspase dependent and P53 independent and correlated with an increased accessibility of cell surface Fas receptors. We also observed that the Fas IgM agonistic antibody–dependent forma...

  12. RNA Aptamer Binds Heparin-Binding Epidermal Growth Factor-Like Growth Factor with High Affinity and Specificity and Neutralizes Its Activity

    Directory of Open Access Journals (Sweden)

    Masaki Yamato

    2017-09-01

    Conclusion: We identified a novel RNA aptamer that bound with high affinity and specificity to rhHB-EGF and potently inhibited the rhHB-EGF-mediated phosphorylation of EGFR. The anti-HB-EGF aptamer may be a promising therapeutic agent for specifically neutralizing HB-EGF signaling.

  13. Thrombomodulin binding selects the catalytically active form of thrombin

    Science.gov (United States)

    Handley, Lindsey D.; Treuheit, Nicholas A.; Venkatesh, Varun J.; Komives, Elizabeth A.

    2015-01-01

    Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the 4th, 5th, and most of the 6th EGF-like domain (TM456m) has been prepared that has much improved solubility, thrombin-binding capacity, and anticoagulant activity over previous TM456 constructs. In the present work we compare backbone amide exchange of human α-thrombin in three states: apo, PPACK-bound, and TM456m-bound. Beyond causing decreased amide exchange at their binding sites, TM and PPACK both cause decreased amide exchange in regions more distant from the active site, within the γ-loop and the adjacent N-terminus of the heavy chain. The decreased amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, HDXMS results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation. PMID:26468766

  14. [Therapeutic indications of low molecular weight heparins].

    Science.gov (United States)

    Samama, M M; Michaut-Paterno, F

    1991-11-01

    The depolymerisation of the various chains of unfractionated heparin (UFH) by chemical or enzymatic reactions provides so-called low molecular weight heparin (LMWH), with an average molecular weight of approximately 5000 daltons. The specific biological and pharmacokinetic properties of LMWH with greater inhibition of factor Xa than of thrombin activity, less interaction with platelets, better bioavailability and a longer half life of anti-Xa activity, suggest possible new therapeutic applications. The hypothesis of reducing the risk of haemorrhage related to the antithrombin activity and the incidence of heparin-induced thrombocytopenia whilst preserving effective antithrombotic action has stimulated clinical and biological research. Clinical trials of prophylaxis of venous thrombo-embolism have been undertaken mainly in surgical patients. The results have shown identical if not better efficacy of LMWH compared to UFH in general surgical and above all orthopedic patients in whom subcutaneous heparin is only effective with a strict protocol which is difficult to adhere to in routine practice (adaptation of dosage to activated partial thromboplastin time). The risk of bleeding was not significantly lower using LMWH at the specified dosage, which in the latter indication, is twice that used in general surgery. There are many indications of prophylaxis of thromboembolism in the medical specialties but, paradoxically, LMWH has not been widely studied because of the difficulties in performing the therapeutic trials. Except in rare cases (extreme body weights, renal failure, haemorrhagic disease, thrombotic or haemorrhagic complications) the evaluation of amidolytic anti-Xa activity does not seem to be necessary. More recently, LMWH has been studied in a small number of trials for the treatment of deep venous thrombosis (DVT). The therapeutic efficacy is identical if not better than that of UFH without increasing the risk of bleeding. Biological monitoring seems to be

  15. Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin.

    Science.gov (United States)

    Handley, Lindsey D; Treuheit, Nicholas A; Venkatesh, Varun J; Komives, Elizabeth A

    2015-11-03

    Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the fourth, fifth, and most of the sixth EGF-like domain (TM456m) that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity versus those of previous TM456 constructs. In this work, we compare backbone amide exchange of human α-thrombin in three states: apo, D-Phe-Pro-Arg-chloromethylketone (PPACK)-bound, and TM456m-bound. Beyond causing a decreased level of amide exchange at their binding sites, TM and PPACK both cause a decreased level of amide exchange in other regions including the γ-loop and the adjacent N-terminus of the heavy chain. The decreased level of amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, hydrogen-deuterium exchange mass spectrometry results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation.

  16. Lipoprotein lipase deficiency due to long-term heparinization presenting as severe hypertriglyceridaemia in pregnancy.

    OpenAIRE

    Watts, G F; Cameron, J; Henderson, A; Richmond, W.

    1991-01-01

    A case of severe hypertriglyceridaemia presenting in the third trimester of pregnancy in a woman on long-term heparin prophylaxis is described. The hypertriglyceridaemia was attributed to impaired clearance of triglyceride-rich lipoprotein particles secondary to heparin-induced reduction in the activity of the lipolytic enzyme, lipoprotein lipase.

  17. Vinculin Activation Is Necessary for Complete Talin Binding

    OpenAIRE

    Golji, Javad; Lam, Johnny; Mofrad, Mohammad R.K.

    2011-01-01

    Focal adhesions are critical to a number of cellular processes that involve mechanotransduction and mechanical interaction with the cellular environment. The growth and strengthening of these focal adhesions is dependent on the interaction between talin and vinculin. This study investigates said interaction and how vinculin activation influences it. Using molecular dynamics, the interaction between talin's vinculin binding site (VBS) and vinculin's domain 1 (D1) is simulated both before and a...

  18. Mechanical prophylaxis is a heparin-independent risk for anti–platelet factor 4/heparin antibody formation after orthopedic surgery

    Science.gov (United States)

    Bito, Seiji; Migita, Kiyoshi; Nakamura, Mashio; Shinohara, Kazuhito; Sato, Tomotaro; Tonai, Takeharu; Shimizu, Motoyuki; Shibata, Yasuhiro; Kishi, Kazuhiko; Kubota, Chikara; Nakahara, Shinnosuke; Mori, Toshihito; Ikeda, Kazuo; Ota, Shusuke; Minamizaki, Takeshi; Yamada, Shigeru; Shiota, Naofumi; Kamei, Masataka; Motokawa, Satoru

    2016-01-01

    Platelet-activating antibodies, which recognize platelet factor 4 (PF4)/heparin complexes, induce spontaneous heparin-induced thrombocytopenia (HIT) syndrome or fondaparinux-associated HIT without exposure to unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). This condition mostly occurs after major orthopedic surgery, implying that surgery itself could trigger this immune response, although the mechanism is unclear. To investigate how surgery may do so, we performed a multicenter, prospective study of 2069 patients who underwent total knee arthroplasty (TKA) or hip arthroplasty. Approximately half of the patients received postoperative thromboprophylaxis with UFH, LMWH, or fondaparinux. The other half received only mechanical thromboprophylaxis, including dynamic (intermittent plantar or pneumatic compression device), static (graduated compression stockings [GCSs]), or both. We measured anti-PF4/heparin immunoglobulins G, A, and M before and 10 days after surgery using an immunoassay. Multivariate analysis revealed that dynamic mechanical thromboprophylaxis (DMT) was an independent risk factor for seroconversion (odds ratio [OR], 2.01; 95% confidence interval [CI], 1.34-3.02; P = .001), which was confirmed with propensity-score matching (OR, 1.99; 95% CI, 1.17-3.37; P = .018). For TKA, the seroconversion rates in patients treated with DMT but no anticoagulation and in patients treated with UFH or LMWH without DMT were similar, but significantly higher than in patients treated with only GCSs. The proportion of patients with ≥1.4 optical density units appeared to be higher among those treated with any anticoagulant plus DMT than among those not treated with DMT. Our study suggests that DMT increases risk of an anti-PF4/heparin immune response, even without heparin exposure. This trial was registered to www.umin.ac.jp/ctr as #UMIN000001366. PMID:26659923

  19. The uses of heparin to treat burn injury.

    Science.gov (United States)

    Oremus, Mark; Hanson, Mark; Whitlock, Richard; Young, Ed; Gupta, Alok; Dal Cin, Arianna; Archer, Carolyn; Raina, Parminder

    2006-12-01

    effectiveness of other treatments, or whether treatment effectiveness varied according to (a) the method of applying heparin to (b) burn etiology. Four studies mentioned contraindications to using heparin to treat burns. These contraindications were bleeding diathesis, bleeding history, active bleeding or associated trauma with potential bleeding, active intestinal ulcer, thrombocytopenia, liver disease, renal disorders, or allergy to heparin. There is no strong evidence in the 19 abstracted articles to suggest that heparin should be used in the treatment of burn injury on account of its non-anticoagulant properties.

  20. Covalent co-immobilization of heparin/laminin complex that with different concentration ratio on titanium surface for selectively direction of platelets and vascular cells behavior

    Science.gov (United States)

    Wang, Jian; Chen, Yuan; Liu, Tao; Wang, Xue; Liu, Yang; Wang, Yuan; Chen, Junying; Huang, Nan

    2014-10-01

    Surface biofunctional modification of coronary artery stent to improve the hemocompatibility and selectively accelerate endothelium regeneration but prevent restenosis have been become a new hotspot. For this, a novel method was developed in this work by co-immobilization of Ln and heparin complex on poly-L-lysine modified Ti surface. Take the advantage of the specific interaction between Ln and heparin, Ln and heparin complexes with different concentration ratios were set up for creating different exposure density of these two types of biomolecules. According to biocompatibility evaluation results, the Hep/Ln complexes modified surface displayed less platelet adhesion and activation. Especially, on L(150)H and L(200)H surface, the AT III binding quantity, APTT value and anti-coagulation property of modified surface were significantly promoted. Furthermore, the adherent density and proliferation activity of ECs and EPCs were positively correlated with Ln concentration. Notably, the proliferation of both ECs and EPCs on L(100)H, L(150)H and L(200)H surface were greatly promoted. Another hand, the proliferation activity of SMCs was significantly inhibited on Hep/Ln modified surfaces, which was considered mainly due to the inhibitory effect of heparin to SMCs. According to the existing results, this study demonstrated that in a certain range of heparin and laminin concentration ratio, the biological behavior of platelets, ECs, EPCs and SMCs could be selectively directed. We suggested that this article provided a potential method to construct an adequate platform on a stent surface for accelerate endothelialization with low side effects.

  1. Therapeutic monitoring of unfractionated heparin - trials and tribulations.

    Science.gov (United States)

    Baluwala, Israfil; Favaloro, Emmanuel J; Pasalic, Leonardo

    2017-07-01

    Heparin is one of the oldest biological medicines with an established role in prevention and treatment of arterial and venous thromboembolism. Published therapeutic ranges for unfractionated heparin (UFH) mostly precede the large increase in the number of activated partial thromboplastin time (APTT) reagent/instrument combinations that now show wide variability. Areas covered: This paper explores the use of UFH, the development of heparin therapeutic ranges (HTRs), and the strengths and limitations of the methods used to monitor heparin's anticoagulant effect. Expert commentary: Despite longstanding use of UFH for management of thromboembolic conditions, the optimal test for monitoring UFH remains undetermined. Although used extensively for monitoring UFH, routine APTT-derived HTRs are based on limited science that may have little relevance to current laboratory practice. Anti-FXa levels may provide better and more reliable HTRs; however, even these levels show considerable inter-laboratory variation, and there are insufficient clinical studies proving improved clinical efficacy. Alternative tests for monitoring UFH reported over time have not been proven effective nor feasible, secondary to technical or cost issues, or lack of general adoption. Thus, despite limited evidence of clinical utility, an uncomfortable marriage of convenience represented by heparin laboratory monitoring is unlikely to be terminated in the immediate future.

  2. Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.

    Science.gov (United States)

    Baik, Jong Youn; Dahodwala, Hussain; Oduah, Eziafa; Talman, Lee; Gemmill, Trent R; Gasimli, Leyla; Datta, Payel; Yang, Bo; Li, Guoyun; Zhang, Fuming; Li, Lingyun; Linhardt, Robert J; Campbell, Andrew M; Gorfien, Stephen F; Sharfstein, Susan T

    2015-07-01

    Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 μg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. IVIg for Treatment of Severe Refractory Heparin-Induced Thrombocytopenia.

    Science.gov (United States)

    Padmanabhan, Anand; Jones, Curtis G; Pechauer, Shannon M; Curtis, Brian R; Bougie, Daniel W; Irani, Mehraboon S; Bryant, Barbara J; Alperin, Jack B; Deloughery, Thomas G; Mulvey, Kevin P; Dhakal, Binod; Wen, Renren; Wang, Demin; Aster, Richard H

    2017-09-01

    Heparin-induced thrombocytopenia (HIT) complicated by severe thrombocytopenia and thrombosis can pose significant treatment challenges. Use of alternative anticoagulants in this setting may increase bleeding risks, especially in patients who have a protracted disease course. Additional therapies are lacking in this severely affected patient population. We describe three patients with HIT who had severe thromboembolism and prolonged thrombocytopenia refractory to standard treatment but who achieved an immediate and sustained response to IVIg therapy. The mechanism of action of IVIg was evaluated in these patients and in five additional patients with severe HIT. The impact of a common polymorphism (H/R 131) in the platelet IgG receptor FcγRIIa on IVIg-mediated inhibition of platelet activation was also examined. At levels attained in vivo, IVIg inhibits HIT antibody-mediated platelet activation. The constant domain of IgG (Fc) but not the antigen-binding portion (Fab) is required for this effect. Consistent with this finding, IVIg had no effect on HIT antibody binding in a solid-phase HIT immunoassay (platelet factor 4 enzyme-linked immunoassay). The H/R131 polymorphism in FcγRIIa influences the susceptibility of platelets to IVIg treatment, with the HH131 genotype being most susceptible to IVIg-mediated inhibition of antibody-induced activation. However, at high doses of IVIg, activation of platelets of all FcγRIIa genotypes was significantly inhibited. All three patients did well on long-term anticoagulation therapy with direct oral anticoagulants. These studies suggest that IVIg treatment should be considered in patients with HIT who have severe disease that is refractory to standard therapies. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  4. Genetic influences on mannan-binding lectin (MBL) and mannan-binding lectin associated serine protease-2 (MASP-2) activity

    DEFF Research Database (Denmark)

    Sørensen, GL; Petersen, I; Thiel, Steffen

    2007-01-01

    The lectin pathway of the complement system is activated when Mannan-binding lectin (MBL) in complex with MASP-2 binds microorganisms. Polymorphisms in both genes are responsible for low serum levels, which associate with increased risk of infection and autoimmune disease. The present study...

  5. Genetic influences on Mannan-binding lectin (MBL) and Mannan-binding lectin associated serine protease-2 (MASP-2) activity

    DEFF Research Database (Denmark)

    Sorensen, Grith L; Petersen, Inge; Thiel, Steffen

    2007-01-01

    The lectin pathway of the complement system is activated when Mannan-binding lectin (MBL) in complex with MASP-2 binds microorganisms. Polymorphisms in both genes are responsible for low serum levels, which associate with increased risk of infection and autoimmune disease. The present study...

  6. Quantitative compositional analysis of heparin using exhaustive heparinase digestion and strong anion exchange chromatography

    Directory of Open Access Journals (Sweden)

    Pierre Mourier

    2015-03-01

    Full Text Available Heparin is a linear sulfated polysaccharide widely used therapeutically as an anticoagulant. It is also the starting material for manufacturing low-molecular-weight heparins (LMWH. Quality control of heparin and LMWH is critical to ensure the safety and therapeutic activity of the final product. However due to their complex and heterogeneous structure, orthogonal analytical techniques are needed to characterize the building blocks of heparin. One of the state-of-the-art methods for heparin analysis is based on complete enzymatic digestion using a mixture of heparinases I, II, and III, followed by the separation of the resulting oligosaccharides by liquid chromatography. The European Pharmacopoeia strong anion-exchange chromatographic method, used to quantify 1,6-anhydro derivatives in enoxaparin, is here applied to the analysis of the heparin building blocks. Their quantification, namely the determination of their average w/w percentage in the heparin chain, is obtained after identification of all components including glycoserine derivatives and 3-O sulfated di- and tetrasaccharides. This work therefore provides a comprehensive overview of the building blocks of unfractionated heparin, including those chemically modified by the manufacturing process, either within the polysaccharide chain or at its reducing end.

  7. Facile immobilization of heparin on bioabsorbable iron via mussel adhesive protein (MAPs

    Directory of Open Access Journals (Sweden)

    Xuchen Xu

    2014-10-01

    Full Text Available Motivated by adhesive proteins in mussels, strategies using dopamine to modified surface have become particularly attractive. In the present work, we developed a novel and convenient method to modify the biodegradable Fe plates with heparin. Iron was first treated by a facile one-step pH-induced polymerization of dopamine, and then a high density heparin was successfully grafted onto the surface via coupling with polydopamine (PDA active layer. Heparin immobilization contributed much longer blood clotting coagulation time than the pure Fe sample, and hence reduced the risk of thrombosis. Cell viability tests suggested that the heparin modified Fe plates were more favorable to the proliferation of ECV304 cells. In summary, the heparin modified Fe plates with good anti-thrombus properties and inhibiting the proliferation of VSMC cells provide great prospects for biodegradable iron.

  8. Two different corticosteroid-binding globulin variants that lack cortisol-binding activity in a greek woman.

    Science.gov (United States)

    Hill, L A; Vassiliadi, D A; Simard, M; Pavlaki, A; Perogamvros, I; Hadjidakis, D; Hammond, G L

    2012-11-01

    Corticosteroid-binding globulin (CBG), encoded by SERPINA6, is the principal plasma binding protein for cortisol. Most nonsynonymous single-nucleotide polymorphisms that alter the production or function of CBG occur rarely, and their clinical significance remains obscure. Serum and DNA were obtained from a Greek woman with low morning cortisol levels and from family members. SERPINA6 exons were sequenced, and serum CBG was measured by ELISA and cortisol-binding capacity assay. Recombinant CBG variants were produced for detailed functional studies. A novel heterozygous c.1282G>C transversion in exon 5 of SERPINA6, resulting in a p.Trp393Ser (W371S) substitution, was identified in the proband, who was also heterozygous for single-nucleotide polymorphisms encoding the CBG Lyon (D367N) and CBG A224S variants. The proband had no measurable plasma cortisol-binding activity despite a CBG level of 273 nm by ELISA. She inherited CBG W371S from her mother whose plasma cortisol-binding capacity was approximately 50% lower than the CBG measurements by ELISA (314 nm). The proband's father and four children were heterozygous for CBG D367N; their CBG levels by ELISA were normal, but corresponding cortisol-binding capacity measurements were 50% lower. Pedigree analysis revealed that W371S segregates with A224 and that D367N and W371S segregate separately. Recombinant CBG D367N and CBG W371S had no measureable cortisol-binding activity. A new CBG Athens (W371S) variant that lacks cortisol-binding activity has been identified in a carrier of the cortisol-binding deficient CBG Lyon (D367N) variant. Analyses of CBG levels in this pedigree illustrate how immunoassays fail to accurately reflect cortisol-binding activity.

  9. Heparin improves oxygenation and minimizes barotrauma after severe smoke inhalation in an ovine model.

    Science.gov (United States)

    Cox, C S; Zwischenberger, J B; Traber, D L; Traber, L D; Haque, A K; Herndon, D N

    1993-04-01

    Inhalation injury is one of the main causes of mortality in burn victims. The tracheobronchial epithelium sloughs and combines with a protein rich exudate to form casts of the airways that can lead to obstruction. We studied the effects of a continuous infusion of heparin on the acute pulmonary injury that occurs after smoke inhalation injury in sheep. Twelve ewes with vascular catheters received a standardized smoke inhalation injury and mechanical ventilation according to protocol for 72 hours. The heparin group (n = 6) received a 400 unit per kilogram bolus of heparin followed by a continuous infusion to maintain the activated clotting time between 250 to 300 seconds. The control group (n = 6) received a saline solution vehicle. Hemodynamics, blood gases and plasma samples for conjugated dienes were taken every six hours. At necropsy, pulmonary tissue was collected for histologic findings, polymorphonuclear neutrophil leukosequestration, wet-to-dry weight ratios and conjugated dienes. PaO2 to FIO2 ratios were improved in the heparin group compared with the control group at 12 to 72 hours after injury, and peak airway pressures were higher in the control group compared with the heparin group. Positive end expiratory pressure requirements were higher in the control group compared with the heparin group. There were significantly fewer airway tracheobronchial casts as determined by our tracheobronchial casts scoring system (2.4 +/- 0.4 versus 0.67 +/- 0.21) and confirmed by histologic examination. Pulmonary blood-free wet-to-dry weight ratios were higher in the control group compared with the heparin group (6.4 +/- 0.5 versus 5.2 +/- 0.1; p heparin. Heparin decreases tracheobronchial cast formation, improves oxygenation, minimizes barotrauma and reduces pulmonary edema in an ovine model of severe smoke inhalation injury. Heparin does not reduce oxygen free radical activity after smoke inhalation injury.

  10. Stability of Ceftazidime and Heparin in Four Different Types of Peritoneal Dialysis Solutions.

    Science.gov (United States)

    Kandel, Surendra; Zaidi, Syed Tabish R; Wanandy, S Troy; Ming, Long C; Castelino, Ronald L; Sud, Kamal; Patel, Rahul P

    2018-01-01

    Intraperitoneal (IP) administration of ceftazidime is recommended for the treatment of peritoneal dialysis-associated peritonitis (PDAP) from Pseudomonas. Patients with PDAP may also need IP heparin to overcome problems with drainage of turbid peritoneal dialysis (PD) fluids and blockage of catheters with fibrin. Physico-chemical stability of ceftazidime and heparin, and biological stability of heparin in many types of PD solutions is unknown. Therefore, we investigated the stability of ceftazidime and heparin in 4 types of PD solutions. A total of 12 PD bags (3 for each type of solution) containing ceftazidime and heparin were prepared and stored at 4°C for 120 hours, and then at 25°C for 6 hours, and finally at 37°C for 12 hours. An aliquot was withdrawn after predefined time points and analyzed for the concentration of ceftazidime and heparin using high-performance liquid-chromatography (HPLC). Samples were assessed for pH, color changes, particle content, and anticoagulant activity of heparin. Ceftazidime and heparin retained more than 91% of their initial concentration when stored at 4°C for 120 hours followed by storage at 25°C for 6 hours and then at 37°C for 12 hours. Heparin retained more than 95% of its initial activity throughout the study period. Particle formation was not detected at any time under the storage conditions. The pH and color remained essentially unchanged throughout the study. Ceftazidime-heparin admixture retains its stability over long periods of storage at different temperatures, allowing its potential use for PDAP treatment in outpatient and remote settings. Copyright © 2018 International Society for Peritoneal Dialysis.

  11. Microbes bind complement inhibitor factor H via a common site.

    Directory of Open Access Journals (Sweden)

    T Meri

    Full Text Available To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH. FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20. We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii. We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site."

  12. Serum Albumin Binding and Esterase Activity: Mechanistic Interactions with Organophosphates

    Directory of Open Access Journals (Sweden)

    Nikolay V. Goncharov

    2017-07-01

    Full Text Available The albumin molecule, in contrast to many other plasma proteins, is not covered with a carbohydrate moiety and can bind and transport various molecules of endogenous and exogenous origin. The enzymatic activity of albumin, the existence of which many scientists perceive skeptically, is much less studied. In toxicology, understanding the mechanistic interactions of organophosphates with albumin is a special problem, and its solution could help in the development of new types of antidotes. In the present work, the history of the issue is briefly examined, then our in silico data on the interaction of human serum albumin with soman, as well as comparative in silico data of human and bovine serum albumin activities in relation to paraoxon, are presented. Information is given on the substrate specificity of albumin and we consider the possibility of its affiliation to certain classes in the nomenclature of enzymes.

  13. Heparin removal in three intraoperative blood savers in cardiac surgery.

    Science.gov (United States)

    Rougé, P; Fourquet, D; Depoix-Joseph, J P; Nguyen, F; Barthélémy, R

    1993-01-01

    The aim of the study was to compare the residual heparin in the composition of autologous blood retransfusion units harvested during cardiac surgery under extra-corporeal circulation with three different intraoperative autologous blood savers. In this institutionally approved study, thirty patients undergoing CABG were randomly assigned to three groups according to the intraoperative blood saver used during the procedure: {HAEMONETICS Cell Saver IV (n=10)--DIDECO/SHILEY STAT (n=11)--BRAT 250 (n=9)}. Anaesthesia and conduct of bypass were identical for all patients. The initial heparin dose was 300IU-kg -1 and was supplemented to maintain an activated coagulation time over 480s. The harvested blood was processed according to the procedure defined by each equipment manufacturer. The biological study was performed on the first blood sediments sampled before administering protamine to the patient. Blood cell count, residual heparinemia assessed by its anti-Xa activity using an amidolytic method {STACHROM HEPARIN--DIAGNOSTICA STAGO}, and weight of the blood sediment proteins were determined. Demographic data did not differ between groups. Despite a slight but significant difference between groups, the three devices provided virtual elimination of heparin. The total protein content was significantly higher in the BRAT 250 group. There was a highly significant positive correlation between the anti-Xa activity and total protein content. Haematologic data were within clinically acceptable ranges.

  14. Heparin and Carboxymethylchitosan Metal Nanoparticles: An Evaluation of Their Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Adriana Bava

    2013-01-01

    Full Text Available In the search for noninvasive diagnostic techniques and new therapies, “nanosystems”, which are capable of binding and targeting bioactive molecules, are becoming increasingly important. In this context, biocompatible coatings are gaining interest, not only for their biological effects but also because they are considered capable to mask nanoparticle toxicity. In this work, we have compared the toxicity of nanoparticles coated with heparin and carboxymethylchitosan in the SKOV-3 cell line. Our results indicate that heparin and carboxymethylchitosan coatings do not guarantee the decrease of nanoparticle intrinsic toxicity which is often envisaged. Nonetheless, these coatings provide the opportunity for further functionalization with a variety of biomolecules for their use in theranostics.

  15. How much heparin do we really need to go on pump? A rethink of current practices.

    LENUS (Irish Health Repository)

    Shuhaibar, M N

    2012-02-03

    OBJECTIVES: Patients undergoing myocardial revascularisation using extracorporeal circulation require heparin anticoagulation. We aimed to evaluate the effect of reducing heparin dosage on target activated clotting time (ACT) and postoperative blood loss. METHODS: In a prospective randomised trial, 195 patients undergoing isolated primary CABG were randomised into four groups A, B, C, and D receiving an initial heparin dosage of 100, 200, 250 and 300 iu\\/kg, respectively. Extra incremental heparin (50 iu\\/kg) was added if required to achieve a target ACT of 480 s before initiating cardiopulmonary bypass. Postoperative blood loss was measured from the time of heparin reversal to drain removal 24h later. RESULTS: Target ACT was achieved in 0, 63, 68.3 and 82.4% of patients in groups A, B, C and D, respectively, after the initial dose of heparin. In group B, of those not achieving target act a single increment of heparin was sufficient to achieve target ACT in further 18.6%. The mean ACT after the initial dose in groups B, C and D was 482.9, 519 and 588 s, respectively (P<0.05). Postoperative blood loss in millilitre per kilogram was directly proportional to preoperative heparin dose. CONCLUSIONS: Patients receiving lower dose of heparin has lower postoperative blood loss. Of those achieving the target ACT, group B was significantly the closest to the target ACT. A starting dose of 200 iu\\/kg of heparin and if necessary one 50 iu\\/kg increment achieved target ACT in 81.5% of patients. The added benefit of significant drop in postoperative blood loss is evident.

  16. Postoperative Bleeding After Change in Heparin Supplier: A Cardiothoracic Center Experience.

    Science.gov (United States)

    Bojan, Mirela; Fischer, Andreas; Narayanasamy, Ashok; Yea, Paul; Dunnett, Eleanor; Kelleher, Andrea

    2017-10-01

    Unfractionated heparin is a mixture of glycosaminoglycans with different pharmacologic and pharmacokinetic properties. The literature suggests that blood loss after cardiac surgery is related to both elevated postoperative heparin concentrations and the potency of different heparin brands. An audit of the observed increase in the incidence of cardiac surgery-related bleeding after change in heparin supplier. Patient characteristics were compared between groups before and after a change in heparin brands. Tertiary cardiothoracic center. All patients undergoing cardiac surgery between August 1, 2011, and April 30, 2012. None. Two hundred eighty patients underwent surgery before a change in heparin brands and 216 after a change. Their preoperative and intraoperative characteristics were similar. Postoperative chest tube drainages and blood transfusions were significantly greater after the change in heparin brands (postoperative chest drainage 476.8 ± 393.1 v 344.8 ± 323.2 mL/6 h and 1,062.2 ± 738.8 v 841.8 ± 567.4 mL/24 h, respectively; both p brand. The likelihood ratio chi-square test for nested models identified an added predictive value of the heparin brand when included as a predictor of bleeding (chest drainage >800 mL/6 h) in a model comprising recirculation, assessed using either an elevated anti-factor X activity or ratio between nonheparinase R and heparinase-modified R. It is likely that the observed increase in postoperative bleeding was related to the pharmacologic properties of the new heparin brand rather than a higher incidence of heparin recirculation. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Genetic influences on mannan-binding lectin (MBL) and mannan-binding lectin associated serine protease-2 (MASP-2) activity

    DEFF Research Database (Denmark)

    Sørensen, Grith Lykke; Petersen, Inge; Thiel, Steffen

    2007-01-01

    The lectin pathway of the complement system is activated when Mannan-binding lectin (MBL) in complex with MASP-2 binds microorganisms. Polymorphisms in both genes are responsible for low serum levels, which associate with increased risk of infection and autoimmune disease. The present study...... includes 1215 MBL measurements and 1214 MASP-2 activity measurements in healthy Danish adult twins. Total MASP-2 activity was estimated by C4 cleaving activity of samples diluted in an excess of MBL. Twin-twin correlations were higher in monozygotic (MZ) than in dizygotic (DZ) twins for both traits...

  18. Heparin-induced thrombocytopenia and thrombosis.

    Science.gov (United States)

    Patel, Vipul P; Bong, Matthew; Di Cesare, Paul E

    2007-05-01

    Heparin-induced thrombocytopenia (HIT) and heparin induced thrombocytopenia with thrombosis (HITT) ar rare complications associated with use of unfractionate heparin (UFH) or low-molecular-weight heparin (LMWH) HIT is a benign clinical condition characterized by a mil drop in platelet count with no clinical significance. HIT is an immune-mediated reaction associated with a wide spread "hypercoagulable" state resulting in arterial an venous thrombosis. There is a higher incidence of HIT with UFH use than with LMWH use. Orthopedic surger patients are at higher risk for developing HITT than are patients who receive prophylactic heparin for cardiovascular surgery or medical reasons. Therapy for patients suspected of having HITT should begin with immedi ate discontinuation of heparin in any form followed by pharmacologic inhibition with thrombin (e.g., recombinant hirudin [lepirudin], argatroban, danaparoid sodium).

  19. Characterization of Helicobacter pylori sigma54 promoter-binding activity.

    Science.gov (United States)

    Pereira, Lara E; Brahmachary, Priyanka; Hoover, Timothy R

    2006-06-01

    Several Helicobacter pylori flagellar genes require sigma(54) for their transcription. Predicted H. pylori sigma(54)-dependent promoters display a preference for A at position -23 instead of C or T as occurs in promoters from most other bacteria. Substitution of the A at position -23 of the H. pylori flaB promoter with a C did not effect expression of a flaB'-'xylE reporter gene in H. pylori, whereas T or G substitutions at this position drastically reduced expression. Results of gel mobility shift assays that used DNA probes corresponding to core promoter sequences and a H. pylori sigma(54) protein fused to the Escherichia coli maltose-binding protein suggested that H. pylori sigma(54) has a higher affinity for promoters with an A at the -23 position. The failure to observe an effect on expression for the flaB mutant promoter with the A to C substitution at the -23 position indicates that sequences flanking the core promoter region may assist binding of H. pylori sigma(54) to the mutant flaB promoter. Alternatively, H. pylori RNA polymerase or the sigma(54)-dependent activator FlgR may compensate for the reduced affinity of sigma(54) for the mutant flaB promoter.

  20. An assay for the mannan-binding lectin pathway of complement activation

    DEFF Research Database (Denmark)

    Petersen, Steen Vang; Thiel, S; Jensen, L

    2001-01-01

    The mannan-binding lectin (MBL) pathway of complement activation has been established as the third pathway of complement activation. MBL is a carbohydrate-binding serum protein, which circulates in complex with serine proteases known as mannan-binding lectin associated serine proteases (MASPs...

  1. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...

  2. Neutralisation of the anti-coagulant effects of heparin by histones in blood plasma and purified systems.

    Science.gov (United States)

    Longstaff, Colin; Hogwood, John; Gray, Elaine; Komorowicz, Erzsebet; Varjú, Imre; Varga, Zoltán; Kolev, Krasimir

    2016-03-01

    Neutrophil extracellular traps (NETs) composed primarily of DNA and histones are a link between infection, inflammation and coagulation. NETs promote coagulation and approaches to destabilise NETs have been explored to reduce thrombosis and treat sepsis. Heparinoids bind histones and we report quantitative studies in plasma and purified systems to better understand physiological consequences. Unfractionated heparin (UFH) was investigated by activated partial thromboplastin time (APTT) and alongside low-molecular-weight heparins (LMWH) in purified systems with thrombin or factor Xa (FXa) and antithrombin (AT) to measure the sensitivity of UFH or LMWH to histones. A method was developed to assess the effectiveness of DNA and non-anticoagulant heparinoids as anti-histones. Histones effectively neutralised UFH, the IC50 value for neutralisation of 0.2 IU/ml UFH was 1.8 µg/ml histones in APTT and 4.6 µg/ml against 0.6 IU/ml UFH in a purified system. Histones also inhibited the activities of LMWHs with thrombin (IC50 6.1 and 11.0 µg/ml histones, for different LMWHs) or FXa (IC50 7.8 and 7.0 µg/ml histones). Direct interactions of UFH and LMWH with DNA and histones were explored by surface plasmon resonance, while rheology studies showed complex effects of histones, UFH and LMWH on clot resilience. A conclusion from these studies is that anticoagulation by UFH and LMWH will be compromised by high affinity binding to circulating histones even in the presence of DNA. A complete understanding of the effects of histones, DNA and heparins on the haemostatic system must include an appreciation of direct effects on fibrin and clot structure.

  3. Low molecular weight heparin versus unfractionated heparin in the initial treatment of venous thromboembolism

    NARCIS (Netherlands)

    Hettiarachchi, R. J.; Prins, M. H.; Lensing, A. W.; Buller, H. R.

    1998-01-01

    In this review, we analyze data from randomized trials in which low molecular weight heparin was compared with unfractionated heparin, both to estimate the treatment effect of low molecular weight heparin in the initial treatment of venous thromboembolism and to evaluate the effect of the varied

  4. A photoacoustic tool for therapeutic drug monitoring of heparin (Conference Presentation)

    Science.gov (United States)

    Wang, Junxin; Hartanto, James; Jokerst, Jesse V.

    2017-03-01

    Heparin is used broadly in cardiac, pulmonary, surgical, and vascular medicine to treat thrombotic disorders with over 500 million doses per year globally. Despite this widespread use, it has a narrow therapeutic window and is one of the top three medication errors. The active partial thromboplastin time (PTT) monitors heparin, but this blood test suffers from long turnaround times, a variable reference range, and limited utility with low molecular weight heparin. Here, we describe an imaging technique that can monitor heparin concentration and activity in real time using photoacoustic spectroscopy via methylene blue as a simple and Federal Drug Agency-approved contrast agent. We found a strong correlation between heparin concentration and photoacoustic signal measured in phosphate buffered saline (PBS) and blood (R2>0.90). Clinically relevant concentrations were detected in blood with a heparin detection limit of 0.28 U/mL and a low molecular weight heparin (enoxaparin) detection limit of 72 μg/mL. We validated this imaging approach by correlation to the PTT (Pearson's r = 0.86; p<0.05) as well as with protamine sulfate treatment. To the best of our knowledge, this is the first report to use imaging data to monitor anticoagulation.

  5. Heparin-induced thrombocytopenia: a review of concepts regarding a dangerous adverse drug reaction.

    Science.gov (United States)

    Junqueira, Daniela Rezende Garcia; Carvalho, Maria das Graças; Perini, Edson

    2013-01-01

    Heparin is a natural agent with antithrombotic action, commercially available for therapeutic use as unfractionated heparin and low molecular weight heparin. Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin that promotes antibody-mediated platelet activation. HIT is defined as a relative reduction in platelet count of 50% (even when the platelet count at its lowest level is above>150 x 10(9)/L) occurring within five to 14 days after initiation of the therapy. Thrombocytopenia is the main feature that directs the clinical suspicion of the reaction and the increased risk of thromboembolic complications is the most important and paradoxical consequence. The diagnosis is a delicate issue, and requires a combination of clinical probability and laboratory tests for the detection of platelet activation induced by HIT antibodies. The absolute risk of HIT has been estimated between 1% and 5% under treatment with unfractionated heparin, and less than 1% with low molecular weight heparin. However, high-quality evidence about the risk of HIT from randomized clinical trials is scarce. In addition, information on the frequency of HIT in developing countries is not widely available. This review aims to provide a better understanding of the key features of this reaction and updated information on its frequency to health professionals and other interested parties. Knowledge, familiarity, and access to therapeutic options for the treatment of this adverse reaction are mandatory to minimize the associated risks, improving patient safety. Copyright © 2013 Elsevier Editora Ltda. All rights reserved.

  6. Development and evaluation of a fluorescence microplate assay for quantification of heparins and other sulfated carbohydrates.

    Science.gov (United States)

    Lühn, Susanne; Schrader, Thomas; Sun, Wei; Alban, Susanne

    2010-05-01

    Due to their complex composition, quantification of heparins is difficult. On the one hand there are many biological tests, which only indirectly detect effects of the antithrombin-binding material. On the other hand direct quantitative methods are available but they are often insensitive, challenging, time-consuming or expensive. The aim of this study was to develop a sensitive, rapid, simple as well as inexpensive direct quantification assay suitable for routine analysis. Based on Polymer-H, a novel heparin complexing, fluorescent labeled synthetic polymer (lambda((ex)) 320nm, lambda((em)) 510nm), a microplate assay was developed and optimized. The specificity of the assay was evaluated by structure-assay response relationships studies using structurally defined glucan sulfates, heparins, and other natural and synthetic sulfated carbohydrates. The fluorescence intensity of Polymer-H (7.5microg/ml) showed to be concentration-dependently amplified by heparins as well as by other sulfated carbohydrates. The best sensitivity, accuracy and linearity were observed in a range from 0.63 to 5.0microg/ml heparins. No differences in the fluorescence between various heparins were observed, so that only one calibration curve is needed. In addition, all types of carbohydrates with a degree of sulfation (DS)> approximately 1.2 and a M(r)>3000 can be quantified as well. By own calibration curves also other sulfated carbohydrates like fondaparinux or other glycosaminoglycans (DS>0.4) can be determined. Copyright 2009 Elsevier B.V. All rights reserved.

  7. Retinoblastoma-binding Protein 1 Has an Interdigitated Double Tudor Domain with DNA Binding Activity*

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-01-01

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10–100 μm; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1. PMID:24379399

  8. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  9. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Directory of Open Access Journals (Sweden)

    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  10. Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xinyue, E-mail: liux22@rpi.edu [National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong, 250100 (China); Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); St Ange, Kalib, E-mail: stangk2@rpi.edu [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); Wang, Xiaohua, E-mail: wangx35@rpi.edu [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); School of Computer and Information, Hefei University of Technology, Hefei (China); Lin, Lei, E-mail: Linl5@rpi.edu [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); Zhang, Fuming, E-mail: zhangf2@rpi.edu [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); and others

    2017-04-08

    Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical β-eliminative cleavage of porcine intestinal heparin. Lovenox{sup ®}, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox{sup ®}, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin. - Highlights: • Low molecular weight heparins prepared from different heparin parents were analyzed. • An integrated analytical

  11. Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

    Science.gov (United States)

    Du, Ping; Sun, Shiqi; Dong, Jinjie; Zhi, Xiaoying; Chang, Yanyan; Teng, Zhidong; Guo, Huichen; Liu, Zaixin

    2017-04-01

    The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification. Copyright © 2016. Published by Elsevier B.V.

  12. A model ternary heparin conjugate by direct covalent bond strategy applied to drug delivery system.

    Science.gov (United States)

    Wang, Ying; Xin, Dingcheng; Hu, Jiawen; Liu, Kaijian; Pan, Jiangao; Xiang, Jiannan

    2009-01-01

    A model ternary heparin conjugate by direct covalent bond strategy has been developed, in which modified heparin using active mix anhydride as intermediate conjugates with model drug molecule and model specific ligand, respectively. Designed ester bonds between model drug and heparin facilitate hydrolysis kinetics research. The strategy can be extended to design and synthesize a targeted drug delivery system. The key point is to use mixed anhydride groups as activating intermediates to mediate the synthesis of the ternary heparin conjugate. Formation of mixed anhydride is detected by the conductimetry experiment. The ternary heparin conjugate is characterized by (13)C NMR, FT-IR and GPC, respectively. The decreased trend on degree of substitution (DS) is consistent with that of introduced anticancer drug and specific ligand in drug delivery system. Moreover, their anticoagulant activity is evaluated by measuring activated partial thromboplastin time (APTT) and anti-factor Xa activity. The results show that model ternary heparin conjugate with reduced anticoagulant activity may avoid the risk of severe hemorrhagic complication during the administration and is potential to develop a safe and effective drug delivery system on anticancer research.

  13. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    Energy Technology Data Exchange (ETDEWEB)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-05-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. /sup 14/C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell.

  14. Heparin and Heparin-Derivatives in Post-Subarachnoid Hemorrhage Brain Injury: A Multimodal Therapy for a Multimodal Disease

    Directory of Open Access Journals (Sweden)

    Erik G. Hayman

    2017-05-01

    Full Text Available Pharmacologic efforts to improve outcomes following aneurysmal subarachnoid hemorrhage (aSAH remain disappointing, likely owing to the complex nature of post-hemorrhage brain injury. Previous work suggests that heparin, due to the multimodal nature of its actions, reduces the incidence of clinical vasospasm and delayed cerebral ischemia that accompany the disease. This narrative review examines how heparin may mitigate the non-vasospastic pathological aspects of aSAH, particularly those related to neuroinflammation. Following a brief review of early brain injury in aSAH and heparin’s general pharmacology, we discuss potential mechanistic roles of heparin therapy in treating post-aSAH inflammatory injury. These roles include reducing ischemia-reperfusion injury, preventing leukocyte extravasation, modulating phagocyte activation, countering oxidative stress, and correcting blood-brain barrier dysfunction. Following a discussion of evidence to support these mechanistic roles, we provide a brief discussion of potential complications of heparin usage in aSAH. Our review suggests that heparin’s use in aSAH is not only safe, but effectively addresses a number of pathologies initiated by aSAH.

  15. DNA binding specificity and cleavage activity of Pacmmar transposase.

    Science.gov (United States)

    Delaurière, Laurence; Chénais, Benoît; Pradier, Elisabeth; Hardivillier, Yann; Renault, Sylvaine; Casse, Nathalie

    2009-08-04

    Mariner-like elements (MLEs) are members of the Tc1/mariner superfamily of transposable elements which transpose by a "cut and paste" mechanism. Most of the MLEs characterized to date are transpositionally inactive due to the accumulation of mutations in their transposase gene. Here, we report the biochemical study of two copies of the Pacmmar element (Pacmmar1.1 and Pacmmar1.2), isolated from the coastal crab Pachygrapsus marmoratus. These two copies present an open reading frame encoding a putative active transposase. Using an in vitro transposition assay, we show that Pacmmar transposases are unable to perform by themselves the transposition reaction. However, we demonstrate by an electrophoretic mobility shift assay that both transposases bind specifically to the inverted terminal repeat of the Pacmmar element. Moreover, an in vitro cleavage assay showed that both transposases have the capacity to cleave the transposon. The in vitro cleavage activity of Pacmmar transposases appears imprecise, suggesting the requirement of specific host factors or the presence of mutations which have modified the cleavage specificity of the enzyme.

  16. Voltammetric determination of heparin based on its interaction with malachite green

    Directory of Open Access Journals (Sweden)

    Xueliang Niu

    2008-08-01

    Full Text Available In this paper malachite green (MG was used as a bioprobe to determine heparin concentration by linear sweep voltammetry on the dropping mercury working electrode (DME. In Britton-Robinson (B-R buffer solution of pH 1.5, MG had a well-defined second order derivative linear sweep voltammetric reductive peak at –0.618 V (vs. SCE. After the addition of heparin into the MG solution, the reductive peak current decreased apparently without the movement of peak potential. Based on the difference of the peak current, a new voltammetric method for the determination of heparin was established. The conditions for the binding reaction and the electrochemical detection were optimized. Under the selected experimental conditions the difference of peak current was directly proportional to the concentration of heparin in the range from 0.3 to 10.0 mg/L with the linear regression equation as ∆ip″ (nA = 360.19 C (mg/L + 178.88 (n = 15, γ = 0.998 and the detection limit as 0.28 mg/L (3σ. The effects of coexisting substances such as metal ions, amino acids on the determination of heparin were investigated and the results showed that this method had good selectivity. This method was further applied to determine the heparin content in heparin sodium injection samples with satisfactory results and good recovery. The stoichiometry of the biocomplex was calculated by the electrochemical method and the binding mechanism was further discussed.

  17. A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin

    National Research Council Canada - National Science Library

    Jeske, Walter P; Hoppensteadt, Debra; Gray, Angel; Walenga, Jeanine M; Cunanan, Josephine; Myers, Lauren; Fareed, Jawed; Bayol, Alain; Rigal, Hélène; Viskov, Christian

    2011-01-01

    ... units within a certain chain length that impart the various and heterogeneous biological activities of heparin. The most notable illustration of the heterogeneous sulfation pattern is the presence of a particular 3-O sulfate group required for specific interaction of heparin with antithrombin (AT) [2] . This 3-O sulfate group is present on approximately o...

  18. The use of fondaparinux for the treatment of venous thromboembolism in a patient with heparin-induced thombocytopenia and thrombosis caused by heparin flushes

    Directory of Open Access Journals (Sweden)

    Alex C Spyropoulos

    2008-06-01

    Full Text Available Alex C Spyropoulos1, Sharyl Magnuson1, Sei Keng Koh21Clinical Thrombosis Center, Lovelace Medical Center, Albuquerque, NM, USA; 2Department of Pharmacy, Singapore General Hospital, SingaporeAbstract: Heparin-induced thrombocytopenia (HIT is an immunologic drug reaction characterized by paradoxical association with venous and arterial thrombosis. The syndrome is caused by IgG antibodies that are reactive against complexes of platelet factor 4 and heparin. Fondparinux does not bind to platelet factor 4, is structurally too short to induce an antibody response, and could in theory be a useful agent to treat HIT. A 69-year-old white female presented with a lower extremity extensive iliofemoral deep vein thrombosis after a right total knee arthroplasty and was subsequently found to have a pulmonary embolism. The patient was noted to have heparin flushes during her operation. Her platelet drop decreased >50% from baseline during initiation of antithrombotic therapy. She was started on subcutaneous fondaparinux 7.5 mg once daily injection. Her serotonin release assay and enzyme-linked immunosorbent assay for heparin antibodies were positive for HIT. Her platelet count nadir was 60 × 103/mm3 on day 5 and the platelet count rebounded after 8 days of fondaparinux therapy. No recurrent thrombotic or bleeding events were noted throughout her therapy. Anecdotal reports have shown that fondaparinux can be a useful agent to treat HIT with or without thrombosis.Keywords: fondaparinux, heparin-induced thrombocytopenia with thrombosis (HITT

  19. Determination of activated plasma fibronectin using radioactive labelled collagen I

    DEFF Research Database (Denmark)

    Fenger, M

    1984-01-01

    The plasma concentration of biological active fibronectin was assayed by a protein binding assay using 125I-collagen I as ligand and heparin as activator. The standard curve is linear for a fibronectin range of 1.1-11 pmol (0.5-5.0 micrograms) and the coefficient of variation was less than 10...

  20. Covalent co-immobilization of heparin/laminin complex that with different concentration ratio on titanium surface for selectively direction of platelets and vascular cells behavior

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jian; Chen, Yuan; Liu, Tao; Wang, Xue; Liu, Yang; Wang, Yuan; Chen, Junying, E-mail: chenjy@263.net; Huang, Nan

    2014-10-30

    Highlights: • Extracellular matrix inspired surface modification with fibronectin, heparin and VEGF to construct a favorable microenvironment for selectively anticoagulant and promote endothelialization. • Take the advantage of specific intermolecular interaction, the bioactivity of above biomolecules was more efficiently maintained in compared with the common used covalent immobilization method. • Poly-l-lysine was used as a novel interlayer for surface amination, and in comparison, PLL coating was more feasible and the degradation product had no harm to human body. - Abstract: Surface biofunctional modification of coronary artery stent to improve the hemocompatibility and selectively accelerate endothelium regeneration but prevent restenosis have been become a new hotspot. For this, a novel method was developed in this work by co-immobilization of Ln and heparin complex on poly-L-lysine modified Ti surface. Take the advantage of the specific interaction between Ln and heparin, Ln and heparin complexes with different concentration ratios were set up for creating different exposure density of these two types of biomolecules. According to biocompatibility evaluation results, the Hep/Ln complexes modified surface displayed less platelet adhesion and activation. Especially, on L(150)H and L(200)H surface, the AT III binding quantity, APTT value and anti-coagulation property of modified surface were significantly promoted. Furthermore, the adherent density and proliferation activity of ECs and EPCs were positively correlated with Ln concentration. Notably, the proliferation of both ECs and EPCs on L(100)H, L(150)H and L(200)H surface were greatly promoted. Another hand, the proliferation activity of SMCs was significantly inhibited on Hep/Ln modified surfaces, which was considered mainly due to the inhibitory effect of heparin to SMCs. According to the existing results, this study demonstrated that in a certain range of heparin and laminin concentration ratio

  1. Antiproliferative heparin (glycosaminoglycans) isolated from giant ...

    African Journals Online (AJOL)

    Heparin was isolated from two bivalve mollusks, Tridacna maxima (giant clam) and Perna viridis (green mussel). The isolated heparin was quantified in crude as well as purified samples and they were estimated as 2.72 and 2.2 g/kg (in crude) and 260 and 248 mg/g (in purified samples) in T. maxima and P. viridis, ...

  2. HEPARIN-INDUCED THROMBOCYTOPAENIA/THROMBOSIS: A ...

    African Journals Online (AJOL)

    2009-12-02

    Dec 2, 2009 ... bleeding, allergic reactions, skin necrosis, osteoporosis ... This study found a significantly higher incidence of heparin induced antibody formation in the UFH group than in the. LMWH heparin group (20.7% vs 7.5% p<0.001). The patients on short term .... Tietge et al (30) presented a case report of a 76 year ...

  3. Extracellular matrix inspired surface functionalization with heparin, fibronectin and VEGF provides an anticoagulant and endothelialization supporting microenvironment

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xue [Key Laboratory of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Liu, Tao [Key Laboratory of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai’an (China); Chen, Yuan [Key Laboratory of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Zhang, Kun [Key Laboratory of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); School of Life Science, Zhengzhou University, Zhengzhou (China); Maitz, Manfred F. [Key Laboratory of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Hohe Str. 06, 01069 Dresden (Germany); Pan, Changjiang [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai’an (China); Chen, Junying, E-mail: chenjy@263.net [Key Laboratory of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Huang, Nan [Key Laboratory of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China)

    2014-11-30

    Highlights: • Surface modification with fibronectin, heparin and VEGF could selectively anticoagulant and promote endothelialization. • The bioactivity of biomolecules was more efficiently maintained via specific intermolecular interaction. • Poly-l-lysine interlayer was more feasible and the degradation product had no harm to human body. - Abstract: The biocompatibility of currently used coronary artery stent is still far from perfect, which closely related to insufficient endothelialization and thrombus formation. In this study, heparin, fibronectin and VEGF were immobilized on Ti surface to construct a multifunctional microenvironment with favorable properties to inhibit thrombosis formation and promote endothelialization simultaneously. The microenvironment on Ti surface was characterized in detail and demonstrated that the Hep/Fn/VEGF biofunctional coating was constructed successfully on Ti surface. The influence of surface properties such as chemical composition, roughness, hydrophilicity, and binding density of biomolecules on the performances of hemocompatibility and cytocompatibility was evaluated and discussed. Modified surface significantly enhanced the AT III binding density and prolonged the clotting time. In vitro platelet adhesion and activation assays further proved that the modified surface presented favorable anti-coagulant property. In addition, the proliferation of endothelial progenitor cells (EPCs) and endothelial cells (ECs) on the Hep/Fn/VEGF biofunctional coating was significantly promoted. In conclusion, the Hep/Fn/VEGF biofunctional coating was successfully constructed with desirable anticoagulant and endothelialization supporting properties. This work may provide a promising approach for biofunctional surface modification of coronary artery stent to acquire a desired multifunctional microenvironment.

  4. Transthyretin-binding activity of contaminants in blood from polar bear (Ursus maritimus) cubs.

    Science.gov (United States)

    Bytingsvik, Jenny; Simon, Eszter; Leonards, Pim E G; Lamoree, Marja; Lie, Elisabeth; Aars, Jon; Derocher, Andrew E; Wiig, Oystein; Jenssen, Bjørn M; Hamers, Timo

    2013-05-07

    We determined the transthyretin (TTR)-binding activity of blood-accumulating contaminants in blood plasma samples of approximately 4-months-old polar bear (Ursus maritimus) cubs from Svalbard sampled in 1998 and 2008. The TTR-binding activity was measured as thyroxine (T4)-like equivalents (T4-EQMeas). Our findings show that the TTR-binding activity related to contaminant levels was significantly lower (45%) in 2008 than in 1998 (mean ± standard error of mean: 1998, 2265 ± 231 nM; 2008, 1258 ± 170 nM). Although we cannot exclude a potential influence of between-year differences in capture location and cub body mass, our findings most likely reflect reductions of TTR-binding contaminants or their precursors in the arctic environment (e.g., polychlorinated biphenyls [PCBs]). The measured TTR-binding activity correlated positively with the cubs' plasma levels of hydroxylated PCBs (OH-PCBs). No such association was found between TTR-binding activity and the plasma levels of perfluoroalkyl substances (PFASs). The OH-PCBs explained 60 ± 7% and 54 ± 4% of the TTR-binding activity in 1998 and 2008, respectively, and PFASs explained ≤1.2% both years. Still, almost half the TTR-binding activity could not be explained by the contaminants we examined. The considerable levels of TTR-binding contaminants warrant further effect directed analysis (EDA) to identify the contaminants responsible for the unexplained part of the observed TTR-binding activity.

  5. Basic Amino Acid Residues of Human Eosinophil Derived Neurotoxin Essential for Glycosaminoglycan Binding

    Science.gov (United States)

    Hung, Ta-Jen; Chang, Wei-Tang; Tomiya, Noboru; Lee, Yuan-Chuan; Chang, Hao-Teng; Chen, Chien-Jung; Kuo, Ping-Hsueh; Fan, Tan-chi; Chang, Margaret Dah-Tsyr

    2013-01-01

    Human eosinophil derived neurotoxin (EDN), a granule protein secreted by activated eosinophils, is a biomarker for asthma in children. EDN belongs to the human RNase A superfamily possessing both ribonucleolytic and antiviral activities. EDN interacts with heparin oligosaccharides and heparin sulfate proteoglycans on bronchial epithelial Beas-2B cells. In this study, we demonstrate that the binding of EDN to cells requires cell surface glycosaminoglycans (GAGs), and the binding strength between EDN and GAGs depends on the sulfation levels of GAGs. Furthermore, in silico computer modeling and in vitro binding assays suggest critical roles for the following basic amino acids located within heparin binding regions (HBRs) of EDN 34QRRCKN39 (HBR1), 65NKTRKN70 (HBR2), and 113NRDQRRD119 (HBR3) and in particular Arg35, Arg36, and Arg38 within HBR1, and Arg114 and Arg117 within HBR3. Our data suggest that sulfated GAGs play a major role in EDN binding, which in turn may be related to the cellular effects of EDN. PMID:24065103

  6. Immobilization of heparin on the surface of polypropylene non-woven fabric for improvement of the hydrophilicity and blood compatibility.

    Science.gov (United States)

    Li, Rong; Wang, Hengdong; Wang, Wenfeng; Ye, Yin

    2013-01-01

    A polypropylene non-woven fabric (PPNWF) was exposed to oxygen plasma to produce peroxides on its surface. These peroxides were used to initiate graft polymerization of acrylic acid (AA) on the surface of PPNWF. Direct heparinization was accomplished via a reaction between heparin and PP-PAA (AA grafted PPNWF) which was activated by EDC (N-ethyl-N'-[3-(dimethylamino)propyl] carbodiimide). Indirect heparinized PPNWF was prepared by grafting poly(ethylene oxide) (PEO) on a PP-PAA surface to form PP-PAA-PEO, followed by reaction with heparin which was activated by EDC before use. The surface modified PPNWFs were characterized by attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy, electron spectroscopy for chemical analysis (ESCA) and contact angle goniometry. It was found that hydrophilicity was greatly improved, as indicated by the decrease of the water contact angle from 142 to 33°. In vitro blood compatibility evaluation of modified PPNWFs, including hemolysis rate, platelet adhesion, plasma protein adsorption and activated partial thromboplastin time (APTT) was investigated. The results suggested that both heparinized PPNWFs showed lower hemolysis rates and better platelet anti-adhesion than non-heparinized controls. Furthermore, PPNWF obtained via indirect immobilization of heparin showed better hydrophilicity and blood compatibility than direct heparinization of PPNWF.

  7. DNA binding and cleavage activity of a structurally characterized Ni ...

    Indian Academy of Sciences (India)

    The binding constant (Kb) and the linear Stern-Volmer quenching constant (Ksv) of the complex have been determined as 9.23 × 10 4 M−1 and 2.0 × 10 4 M−1, respectively. Spectroscopic and hydrodynamic investigations revealed groove or electrostatic nature of binding of 1 with DNA. 1 is also found to induce oxidative ...

  8. Controllable production of low molecular weight heparins by combinations of heparinase I/II/III.

    Science.gov (United States)

    Wu, Jingjun; Zhang, Chong; Mei, Xiang; Li, Ye; Xing, Xin-Hui

    2014-01-30

    Enzymatic depolymerization of heparin by heparinases is promising for production of low molecular weight heparins (LMWHs) as anticoagulants, due to its mild reaction conditions and high selectivity. Here, different heparinase combinations were used to depolymerize heparin. Heparinase I and heparinase II can depolymerize heparin more efficiently than heparinase III, respectively, but heparinase III was the best able to protect the anticoagulant activities of LMWHs. Heparinase III and heparinase I/II combinations were able to efficiently depolymerize heparin to LMWHs with higher anticoagulant activity than the LMWHs produced by the respective heparinase I and heparinase II. HepIII and HepI is the best combination for maintaining high anti-IIa activity (75.7 ± 4.21 IU/mg) at the same Mw value. Furthermore, considering both the changes in molecular weight and anticoagulant activity, the action patterns of heparinase I and heparinase II were found not to follow the exolytic and processive depolymerizing mechanism from the reducing end of heparin. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Cutaneous reactions to heparin therapy: when are they caused by heparin allergy?

    Directory of Open Access Journals (Sweden)

    Giuliana Zisa

    2013-03-01

    Full Text Available Introduction: Little is known about the incidence and causes of heparin-induced skin lesions. The most commonly reported causes are delayed-type hypersensitivity reactions. We describe 3 patients who were referred to our staff between March and October 2009 for suspected heparin allergies. All were scheduled to undergo major surgery (cardiovascular or orthopedic. Materials and methods: All 3 patients reported the development of itchy, erythematous rashes a few days after the subcutaneous administration of heparin (nadroparin calcium in cases 1 and 2, unspecified in case 3. Each of them underwent a diagnostic work-up for heparin allergy, which included prick and intradermal tests with commonly used heparins and patch testing with undiluted heparins and disinfectants. Results: Patch tests with disinfectants were negative in all 3 cases. In case 2, all allergological tests were negative. In cases 1 and 3, delayed positivity emerged for nadroparin calcium and at least one other heparin tested. Intravenous and/or subcutaneous provocation testing was done with an alternative heparin which produced negative results in skin tests (heparin sodium in case 1, pentasaccharide fondaparinux in case 3. In both cases the alternative drug was tolerated. After our evaluation, all 3 patients underwent surgery with no heparin-related complications. Discussion: The presenting clinical features in these 3 cases provided no information on which reactions were likely to be allergic: all 3 patients presented with similar local delayed reaction. The allergic reactions were identified only after cutaneous testing.

  10. Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.

    Science.gov (United States)

    Quax, P H; Grimbergen, J M; Lansink, M; Bakker, A H; Blatter, M C; Belin, D; van Hinsbergh, V W; Verheijen, J H

    1998-05-01

    Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no

  11. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

    2014-09-15

    Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

  12. Data for chitin binding activity of Moringa seed resistant protein (MSRP

    Directory of Open Access Journals (Sweden)

    Anudeep Sandanamudi

    2016-12-01

    Full Text Available Chitin binding activity of moringa seed resistant protein (MSRP isolated from defatted moringa seed flour was investigated in the present study “Characterization of soluble dietary fiber from Moringa oleifera seeds and its immunomodulatory effects” (S. Anudeep, V.K. Prasanna, S.M. Adya, C. Radha, 2016 [1]. The assay reaction mixture contained 0.4 mg/ml of MSRP and different amounts (20–100 mg of chitin. MSRP exhibited binding activity over wide range of chitin concentration. Maximum binding activity was observed at 80 mg of chitin. The property of MSRP to bind chitin can be exploited for its purification.

  13. Data for chitin binding activity of Moringa seed resistant protein (MSRP).

    Science.gov (United States)

    Sandanamudi, Anudeep; Bharadwaj, Kishan R; Cheruppanpullil, Radha

    2016-12-01

    Chitin binding activity of moringa seed resistant protein (MSRP) isolated from defatted moringa seed flour was investigated in the present study "Characterization of soluble dietary fiber from Moringa oleifera seeds and its immunomodulatory effects" (S. Anudeep, V.K. Prasanna, S.M. Adya, C. Radha, 2016) [1]. The assay reaction mixture contained 0.4 mg/ml of MSRP and different amounts (20-100 mg) of chitin. MSRP exhibited binding activity over wide range of chitin concentration. Maximum binding activity was observed at 80 mg of chitin. The property of MSRP to bind chitin can be exploited for its purification.

  14. Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα[S

    Science.gov (United States)

    Fang, Changming; Filipp, Fabian V.; Smith, Jeffrey W.

    2012-01-01

    Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA. PMID:22223860

  15. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A

    Directory of Open Access Journals (Sweden)

    Manuela Maurer

    2016-04-01

    Full Text Available The periplasmic oligopeptide binding protein A (OppA represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK, but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

  16. Increased activity of the mannan-binding lectin complement activation pathway in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Ytting, H; Jensenius, Jens Christian; Christensen, I J

    2004-01-01

    BACKGROUND: Postoperative bacterial infectious complications are frequent in patients with colorectal cancer (CRC), with subsequent increased recurrence rates and poor prognosis. Deficiency of the mannan-binding lectin (MBL) complement activation pathway may cause increased risk of infection......: Serum MBL concentrations and MBL/MASP activity were determined using immunofluorometric assays. The levels are presented as the median, inter-quartile range and range. RESULTS: Serum MBL levels were significantly (P cancer (1384 (400-2188) ng/mL) (median......, inter-quartile range) compared with levels in healthy blood donors (924 (230-1476) ng/mL). Similarly, the MBL/MASP activity was significantly (P

  17. Safety and potential anticoagulant effects of nebulised heparin in burns patients with inhalational injury at Singapore General Hospital Burns Centre.

    Science.gov (United States)

    Yip, Lian Yee; Lim, Yen Fang; Chan, Hong Ngee

    2011-11-01

    Nebulised heparin, N-acetylcysteine (NAC) and salbutamol were shown to decrease reintubation rates, incidence of atelectasis and mortality in paediatric patients and reduce lung injury scores in adult burns patients with inhalational lung injury (ILI). Nebulised heparin, NAC and salbutamol treatment protocol was introduced in Singapore General Hospital (SGH) Burns Centre in 2006. However, safety data on the use of nebulised heparin and NAC for burns patients with ILI is not well established. In this study, we investigated the safety and potential anticoagulant effects of nebulised heparin in burns patients with ILI. A retrospective study with historical control was conducted. The treatment group consisted of 52 mechanically ventilated adult patients, with a diagnosis of ILI as confirmed by bronchoscopy, admitted to burn intensive care unit (BICU) from the year 2006 to 2009. The group was treated with nebulised heparin, NAC and salbutamol. The control group consists of 11 mechanically ventilated BICU ILI patients treated from year 2001 to 2005 before protocol initiation. Blood coagulation indices (prothrombin time (PT), activated partial thromboplastin time (APTT) and platelet count) were monitored and bleeding incidences were assessed. Blood coagulation indices did not suggest an increase risk of bleeding with nebulised heparin. The APTT, PT and platelet count followed a similar trend for both groups over 7 days. No clinically significant increase in bleeding risk was found to be associated with nebulised heparin. Nebulised heparin was not found to potentiate the risk of bleeding in burns patients with ILI. Copyright © 2011 Elsevier Ltd and ISBI. All rights reserved.

  18. Transthyretin-Binding Activity of Contaminants in Blood from Polar Bear (Ursus maritimus) Cubs

    NARCIS (Netherlands)

    Bytingsvik, J.; Simon, E.; Leonards, P.E.G.; Lamoree, M.H.; Lie, E.; Aars, J.; Derocher, A. E.; Wiig, O.; Jenssen, B.M.; Hamers, T.

    2013-01-01

    We determined the transthyretin (TTR)-binding activity of blood-accumulating contaminants in blood plasma samples of approximately 4-months-old polar bear (Ursus maritimus) cubs from Svalbard sampled in 1998 and 2008. The TTR-binding activity was measured as thyroxine (T4)-like equivalents (T4-EQ

  19. Revisiting the Response Mechanism of Polymeric Membrane Based Heparin Electrodes

    OpenAIRE

    Bell, Andrea K.; Höfler, Lajos; Meyerhoff, Mark E.

    2011-01-01

    Potentiometric membrane electrodes that respond to heparin and other polyanions were introduced in the early 1990s. Herein, the mechanism of polymer membrane electrode type heparin sensors is revisited. The extraction/diffusion of heparin is studied via both potentiometric and impedance spectroscopic techniques using a pre-fractionated heparin preparation that contains polyanionic species > 10000 Daltons. The reversal in EMF response using this heparin preparation indicates diffusion of highe...

  20. Influence of low molecular weight heparin on cancer patients’ survival

    Directory of Open Access Journals (Sweden)

    V. V. Ptushkin

    2013-01-01

    Full Text Available There is an evidence of interaction between the hemostasis system and tumor progression factors. It is known that in addition to the fibrin formation and platelets activation, thrombin can influence many cells function interacting with protease-activating receptors including tumor cells. These receptors are involved in the malignant cell phenotype formation (adhesion, proliferation, proteolysis. Thrombin can also affect angiogenesis by stimulating endothelial cells penetration through basal membrane and its migration with new vessels formation. Furthermore, it can cause the release of main neoangiogenesis promoter – vascular endothelial growth factor. All of the above and many other linkages of coagulation and tumor create a theoretical background of possible affecting tumor by regulation of the coagulation activity. Thepromise of this approach is controversial, but there is some clinical and experimental evidence of their effectiveness. The most used group ofdrugs for this purpose was heparins. Several retrospective studies have shown a benefit of low molecular weight heparins (LMWH over unfractionated heparin in cancer patient survival. The appearance of a new heparins group – ultra LMWH are of interest from this point ofview and their possible use in cancer patients. To date bemiparin and semuloparin are used in clinic. Both (bemiparin about 3600 kDa,semuloparin 3000 kDa have substancially reduced molecular weight as compared with the smallest of LMWH – enoxaparin (4600 kDa.Use of bemiparin in patients with small cell lung cancer receiving chemotherapy resulted in increased of 2-year survival rate compared to the control group (68.6 % vs. 29.4 %, p = 0.0042.

  1. Influence of low molecular weight heparin on cancer patients’ survival

    Directory of Open Access Journals (Sweden)

    V. V. Ptushkin

    2014-07-01

    Full Text Available There is an evidence of interaction between the hemostasis system and tumor progression factors. It is known that in addition to the fibrin formation and platelets activation, thrombin can influence many cells function interacting with protease-activating receptors including tumor cells. These receptors are involved in the malignant cell phenotype formation (adhesion, proliferation, proteolysis. Thrombin can also affect angiogenesis by stimulating endothelial cells penetration through basal membrane and its migration with new vessels formation. Furthermore, it can cause the release of main neoangiogenesis promoter – vascular endothelial growth factor. All of the above and many other linkages of coagulation and tumor create a theoretical background of possible affecting tumor by regulation of the coagulation activity. Thepromise of this approach is controversial, but there is some clinical and experimental evidence of their effectiveness. The most used group ofdrugs for this purpose was heparins. Several retrospective studies have shown a benefit of low molecular weight heparins (LMWH over unfractionated heparin in cancer patient survival. The appearance of a new heparins group – ultra LMWH are of interest from this point ofview and their possible use in cancer patients. To date bemiparin and semuloparin are used in clinic. Both (bemiparin about 3600 kDa,semuloparin 3000 kDa have substancially reduced molecular weight as compared with the smallest of LMWH – enoxaparin (4600 kDa.Use of bemiparin in patients with small cell lung cancer receiving chemotherapy resulted in increased of 2-year survival rate compared to the control group (68.6 % vs. 29.4 %, p = 0.0042.

  2. Fondaparinux (ARIXTRA) as an alternative anti-thrombotic prophylaxis when there is hypersensitivity to low molecular weight and unfractionated heparins.

    Science.gov (United States)

    Parody, Rocio; Oliver, Arturo; Souto, Juan Carlos; Fontcuberta, Jordi

    2003-11-01

    During the last decade, new anticoagulant drugs with anti-factor-Xa properties have been described (1, 2). Among them is fondaparinux that has been licensed recently. It is a pentasaccharide mimicking the site where heparin binds to antithrombin III (1). This new drug has produced very promising clinical results in the prophylaxis of venous thrombosis after orthopedic surgery (3). Here we report two different clinical situations in which fondaparinux has yielded a successful outcome: first, a patient with repeated cutaneus reaction to several different low molecular weight heparins (LMWH), and second, a patient with severe heparin-induced thrombocytopenia (HIT). We decided to use fondaparinux in both cases since it is commercially available in Spain and mostly because the absence of in vitro cross-reaction with heparins, as discussed later.

  3. Characterization of microtubule-binding and dimerization activity of Giardia lamblia end-binding 1 protein.

    Science.gov (United States)

    Kim, Juri; Nagami, Sara; Lee, Kyu-Ho; Park, Soon-Jung

    2014-01-01

    End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1102-238, but not rGlEB11-184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB11-238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.

  4. Characterization of Microtubule-Binding and Dimerization Activity of Giardia lamblia End-Binding 1 Protein

    Science.gov (United States)

    Kim, Juri; Nagami, Sara; Lee, Kyu-Ho; Park, Soon-Jung

    2014-01-01

    End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1102–238, but not rGlEB11–184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB11–238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia. PMID:24828878

  5. Diacylglycerol kinase theta and zeta isoforms : regulation of activity, protein binding partners and physiological functions

    NARCIS (Netherlands)

    Los, Alrik Pieter

    2007-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the

  6. Heparin-binding EGF-like growth factor, which acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9, which up-regulates functional receptors and diphtheria toxin sensitivity.

    OpenAIRE

    Iwamoto, R; Higashiyama, S.; Mitamura, T; Taniguchi, N.; Klagsbrun, M; Mekada, E

    1994-01-01

    DRAP27, the monkey homolog of human CD9 antigen (DRAP27/CD9) and diphtheria toxin receptor (DTR) were expressed in mouse L cells. L cells transfected transiently with both DRAP27/CD9 and DTR cDNA bound approximately 10 times more diphtheria toxin (DT) than cells transfected with DTR alone. Stable L cell transfectants expressing both DTR and DRAP27/CD9 (LCH-1 cells) had 15 times more cell surface DT-binding sites and were 20 times more sensitive to DT than were stable L cell transfectants expr...

  7. Anticoagulation and endothelial cell behaviors of heparin-loaded graphene oxide coating on titanium surface

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Chang-Jiang, E-mail: panchangjiang@hyit.edu.cn [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an 223003 (China); Pang, Li-Qun [Department of General Surgery, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an 223300 (China); Gao, Fei [Zhejiang Zylox Medical Devices Co., Ltd., Hangzhou 310000 (China); Wang, Ya-Nan; Liu, Tao; Ye, Wei; Hou, Yan-Hua [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an 223003 (China)

    2016-06-01

    Owing to its unique physical and chemical properties, graphene oxide (GO) has attracted tremendous interest in many fields including biomaterials and biomedicine. The purpose of the present study is to investigate the endothelial cell behaviors and anticoagulation of heparin-loaded GO coating on the titanium surface. To this end, the titanium surface was firstly covered by the polydopamine coating followed by the deposition of the GO coating. Heparin was finally loaded on the GO coating to improve the blood compatibility. The results of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) indicated that the heparin-loaded GO coating was successfully created on the titanium surface. The scanning electron microscopy (SEM) images indicated that a relative uniform GO coating consisting of multilayer GO sheets was formed on the substrate. The hydrophilicity of the titanium surface was enhanced after the deposition of GO and further improved significantly by the loading heparin. The GO coating can enhance the endothelial cell adhesion and proliferation as compared with polydopamine coating and the blank titanium. Loading heparin on the GO coating can significantly reduce the platelet adhesion and prolong the activated partial thromboplastin time (APTT) while not influence the endothelial cell adhesion and proliferation. Therefore, the heparin-loaded GO coating can simultaneously enhance the cytocompatibility to endothelial cells and blood compatibility of biomaterials. Because the polydopamine coating can be easily prepared on most of biomaterials including polymer, ceramics and metal, thus the approach of the present study may open up a new window of promising an effective and efficient way to promote endothelialization and improve the blood compatibility of blood-contact biomedical devices such as intravascular stents. - Highlights: • Heparin-loaded graphene oxide coating was

  8. Low-dose intravenous heparin infusion in patients with aneurysmal subarachnoid hemorrhage: a preliminary assessment

    Science.gov (United States)

    Simard, J. Marc; Aldrich, E. Francois; Schreibman, David; James, Robert F.; Polifka, Adam; Beaty, Narlin

    2015-01-01

    Object Aneurysmal subarachnoid hemorrhage (aSAH) predisposes to delayed neurological deficits, including stroke and cognitive and neuropsychological abnormalities. Heparin is a pleiotropic drug that antagonizes many of the pathophysiological mechanisms implicated in secondary brain injury after aSAH. Methods The authors performed a retrospective analysis in 86 consecutive patients with Fisher Grade 3 aSAH due to rupture of a supratentorial aneurysm who presented within 36 hours and were treated by surgical clipping within 48 hours of their ictus. Forty-three patients were managed postoperatively with a low-dose intravenous heparin infusion (Maryland low-dose intravenous heparin infusion protocol: 8 U/kg/hr progressing over 36 hours to 10 U/kg/hr) beginning 12 hours after surgery and continuing until Day 14 after the ictus. Forty-three control patients received conventional subcutaneous heparin twice daily as deep vein thrombosis prophylaxis. Results Patients in the 2 groups were balanced in terms of baseline characteristics. In the heparin group, activated partial thromboplastin times were normal to mildly elevated; no clinically significant hemorrhages or instances of heparin-induced thrombocytopenia or deep vein thrombosis were encountered. In the control group, the incidence of clinical vasospasm requiring rescue therapy (induced hypertension, selective intraarterial verapamil, and angioplasty) was 20 (47%) of 43 patients, and 9 (21%) of 43 patients experienced a delayed infarct on CT scanning. In the heparin group, the incidence of clinical vasospasm requiring rescue therapy was 9% (4 of 43, p = 0.0002), and no patient suffered a delayed infarct (p = 0.003). Conclusions In patients with Fisher Grade 3 aSAH whose aneurysm is secured, postprocedure use of a low-dose intravenous heparin infusion may be safe and beneficial. PMID:24032706

  9. How to give a heparin shot

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/patientinstructions/000661.htm How to give a heparin shot To use the sharing ... nurse or other health professional will teach you how to prepare the medicine and give the shot. The ...

  10. Increased unfractionated heparin requirements with decreasing body mass index in pregnancy.

    Science.gov (United States)

    Patil, Avinash S; Clapp, Tracy; Gaston, Piyamas K; Kuhl, David; Rinehart, Eliza; Meyer, Norman L

    2016-12-01

    Pregnant women receiving low-molecular-weight heparin for therapeutic anticoagulation are often converted to unfractionated heparin in anticipation of labor. We aim to characterize the impact of maternal body mass index on attainment of target anticoagulation during the conversion process. We conducted a five-year retrospective study of a pregnancy cohort converted from low-molecular-weight heparin to unfractionated heparin in the third trimester. Patient demographics, anticoagulation regimens, and clinical outcomes were extracted from the medical record. Nonparametric statistical methods were used for analysis by body mass index (35). Thirty-one subjects were evenly distributed by body mass index (p = 0.97). Linear regression revealed an inverse correlation between patient body mass index and unfractionated heparin dose needed to achieve therapeutic anticoagulation (p = 0.04). Subjects with body mass index > 35 attained therapeutic activated partial thromboplastin time levels at 18 U (Units)/kg/h, while subjects with body mass index body mass index < 30 during pregnancy. This paradoxical relationship may be explained by physiologic characteristics that increase unfractionated heparin elimination, including diminished adiposity and increased renal clearance.

  11. Structure-activity relations in binding of perfluoroalkyl compounds to human thyroid hormone T3 receptor.

    Science.gov (United States)

    Ren, Xiao-Min; Zhang, Yin-Feng; Guo, Liang-Hong; Qin, Zhan-Fen; Lv, Qi-Yan; Zhang, Lian-Ying

    2015-02-01

    Perfluoroalkyl compounds (PFCs) have been shown to disrupt thyroid functions through thyroid hormone receptor (TR)-mediated pathways, but direct binding of PFCs with TR has not been demonstrated. We investigated the binding interactions of 16 structurally diverse PFCs with human TR, their activities on TR in cells, and the activity of perfluorooctane sulfonate (PFOS) in vivo. In fluorescence competitive binding assays, most of the 16 PFCs were found to bind to TR with relative binding potency in the range of 0.0003-0.05 compared with triiodothyronine (T3). A structure-binding relationship for PFCs was observed, where fluorinated alkyl chain length longer than ten, and an acid end group were optimal for TR binding. In thyroid hormone (TH)-responsive cell proliferation assays, PFOS, perfluorohexadecanoic acid, and perfluorooctadecanoic acid exhibited agonistic activity by promoting cell growth. Furthermore, similar to T3, PFOS exposure promoted expression of three TH upregulated genes and inhibited three TH downregulated genes in amphibians. Molecular docking analysis revealed that most of the tested PFCs efficiently fit into the T3-binding pocket in TR and formed a hydrogen bond with arginine 228 in a manner similar to T3. The combined in vitro, in vivo, and computational data strongly suggest that some PFCs disrupt the normal activity of TR pathways by directly binding to TR.

  12. Aminoglycosylation can enhance the G-quadruplex binding activity of epigallocatechin.

    Directory of Open Access Journals (Sweden)

    Li-Ping Bai

    Full Text Available With the aim of enhancing G-quadruplex binding activity, two new glucosaminosides (16, 18 of penta-methylated epigallocatechin were synthesized by chemical glycosylation. Subsequent ESI-TOF-MS analysis demonstrated that these two glucosaminoside derivatives exhibit much stronger binding activity to human telomeric DNA and RNA G-quadruplexes than their parent structure (i.e., methylated EGC (14 as well as natural epigallocatechin (EGC, 6. The DNA G-quadruplex binding activity of 16 and 18 is even more potent than strong G-quadruplex binder quercetin, which has a more planar structure. These two synthetic compounds also showed a higher binding strength to human telomeric RNA G-quadruplex than its DNA counterpart. Analysis of the structure-activity relationship revealed that the more basic compound, 16, has a higher binding capacity with DNA and RNA G-quadruplexes than its N-acetyl derivative, 18, suggesting the importance of the basicity of the aminoglycoside for G-quadruplex binding activity. Molecular docking simulation predicted that the aromatic ring of 16 π-stacks with the aromatic ring of guanine nucleotides, with the glucosamine moiety residing in the groove of G-quadruplex. This research indicates that glycosylation of natural products with aminosugar can significantly enhance their G-quadruplex binding activities, thus is an effective way to generate small molecules targeting G-quadruplexes in nucleic acids. In addition, this is the first report that green tea catechin can bind to nucleic acid G-quadruplex structures.

  13. Genome-wide regulation of TATA-binding protein activity

    NARCIS (Netherlands)

    van Werven, F.J.

    2009-01-01

    Transcription, the synthesis of RNA from a DNA template, is a well-controlled process. TATA binding protein (TBP) recruitment to promoters is essential for transcription by all three RNA polymerases, and often is the rate-limiting step of transcription initiation. TBP is incorporated into different

  14. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

    Science.gov (United States)

    Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

    2011-05-31

    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

  15. Platelet Factor 4 Binds to Vascular Proteoglycans and Controls Both Growth Factor Activities and Platelet Activation.

    Science.gov (United States)

    Lord, Megan S; Cheng, Bill; Farrugia, Brooke L; McCarthy, Simon; Whitelock, John M

    2017-03-10

    Platelet factor 4 (PF4) is produced by platelets with roles in both inflammation and wound healing. PF4 is stored in platelet α-granules bound to the glycosaminoglycan (GAG) chains of serglycin. This study revealed that platelet serglycin is decorated with chondroitin/dermatan sulfate and that PF4 binds to these GAG chains. Additionally, PF4 had a higher affinity for endothelial-derived perlecan heparan sulfate chains than serglycin GAG chains. The binding of PF4 to perlecan was found to inhibit both FGF2 signaling and platelet activation. This study revealed additional insight into the ways in which PF4 interacts with components of the vasculature to modulate cellular events. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jingsong; Campobasso, Nino; Biju, Mangatt P.; Fisher, Kelly; Pan, Xiao-Qing; Cottom, Josh; Galbraith, Sarah; Ho, Thau; Zhang, Hong; Hong, Xuan; Ward, Paris; Hofmann, Glenn; Siegfried, Brett; Zappacosta, Francesca; Washio, Yoshiaki; Cao, Ping; Qu, Junya; Bertrand, Sophie; Wang, Da-Yuan; Head, Martha S.; Li, Hu; Moores, Sheri; Lai, Zhihong; Johanson, Kyung; Burton, George; Erickson-Miller, Connie; Simpson, Graham; Tummino, Peter; Copeland, Robert A.; Oliff, Allen (GSKPA)

    2014-10-02

    c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the {alpha}I helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the {alpha}I helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

  17. Modulation of the homophilic interaction between the first and second Ig modules of neural cell adhesion molecule by heparin

    DEFF Research Database (Denmark)

    Kulahin, Nikolaj; Rudenko, Olga; Kiselyov, V.

    2005-01-01

    The second Ig module (IgII) of the neural cell adhesion molecule (NCAM) is known to bind to the first Ig module (IgI) of NCAM (so-called homophilic binding) and to interact with heparan sulfate and chondroitin sulfate glycoconjugates. We here show by NMR that the heparin and chondroitin sulfate......II. Accordingly, we show that treatment of cerebellar granule neurons (CGNs) with heparin inhibits NCAM-mediated outgrowth. In contrast, treatment with heparinase III or chondroitinase ABC abrogates NCAM-mediated neurite outgrowth in CGNs emphasizing the importance of the presence of heparan/chondroitin sulfates...

  18. In situ formation of poly(vinyl alcohol–heparin hydrogels for mild encapsulation and prolonged release of basic fibroblast growth factor and vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Justine J Roberts

    2016-11-01

    Full Text Available Heparin-based hydrogels are attractive for controlled growth factor delivery, due to the native ability of heparin to bind and stabilize growth factors. Basic fibroblast growth factor and vascular endothelial growth factor are heparin-binding growth factors that synergistically enhance angiogenesis. Mild, in situ encapsulation of both basic fibroblast growth factor and vascular endothelial growth factor and subsequent bioactive dual release has not been demonstrated from heparin-crosslinked hydrogels, and the combined long-term delivery of both growth factors from biomaterials is still a major challenge. Both basic fibroblast growth factor and vascular endothelial growth factor were encapsulated in poly(vinyl alcohol-heparin hydrogels and demonstrated controlled release. A model cell line, BaF32, was used to show bioactivity of heparin and basic fibroblast growth factor released from the gels over multiple days. Released basic fibroblast growth factor promoted higher human umbilical vein endothelial cell outgrowth over 24 h and proliferation for 3 days than the poly(vinyl alcohol-heparin hydrogels alone. The release of vascular endothelial growth factor from poly(vinyl alcohol-heparin hydrogels promoted human umbilical vein endothelial cell outgrowth but not significant proliferation. Dual-growth factor release of basic fibroblast growth factor and vascular endothelial growth factor from poly(vinyl alcohol-heparin hydrogels resulted in a synergistic effect with significantly higher human umbilical vein endothelial cell outgrowth compared to basic fibroblast growth factor or vascular endothelial growth factor alone. Poly(vinyl alcohol-heparin hydrogels allowed bioactive growth factor encapsulation and provided controlled release of multiple growth factors which is beneficial toward tissue regeneration applications.

  19. Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin.

    Science.gov (United States)

    Rostagno, A A; Schwarzbauer, J E; Gold, L I

    1999-03-01

    Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to

  20. Inhaled unfractionated heparin improves abnormalities of alveolar coagulation, fibrinolysis and inflammation in endotoxemia-induced lung injury rats.

    Science.gov (United States)

    Wang, Zong-Yu; Wu, Sheng-Nan; Zhu, Zhao-Zhong; Yang, Ba-Xian; Zhu, Xi

    2013-01-01

    Acute lung injury/acute respiratory distress syndrome presents with not only local inflammation, but also pulmonary coagulopathy which is characterized by an alveolar procoagulant response, anticoagulant inhibition, fibrinolytic supression and fibrin deposition. We thus had hypothesized that if aerosolized unfractionated heparin was inhaled into alveolar spaces, it could block the procoagulant tendency, lessen depletion of coagulation factors, and even influence the inflammatory response. We also assessed the effects of different administration regimens of heparin. Male Wistar rats were given inhaled heparin starting 30 minutes before (prophylactic heparin) or 2 hours after (therapeutic heparin) intravenous lipopolysaccharide (LPS) was administered at 6-hour intervals; control groups received inhaled normal saline with or without being exposed to LPS. Thrombin-antithrombin complexes, activated protein C, tissue type and urokinase type plasminogen activators (t-PA/u-PA), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-α, interleukin-6 in bronchoalveolar lavage, and lung tissue myeloperoxidase activity, and histology score were measured at three time-points. PAI-1/(t-PA + u-PA) was calculated based on the before-mentioned parameters. Statistical analysis was made using one-way analysis of variance (ANOVA) with post hoc test or Student's t test in the case of heterogeneity of variance. An alveolar procoagulant reaction, depressed fibrinolysis, and inflammatory response occurred in endotoxemia-induced lung injury. Local prophylactic application of heparin attenuated coagulation and early inflammation, promoted fibrinolysis, and reduced the histology score. Therapeutic application of heparin had similar, but weaker effects. Intrapulmonary application of unfractionated heparin by inhalation might inhibit alveolar procoagulant reaction and the early inflammatory response, promote fibrinolysis, and alleviate pulmonary pathology in endotoxemia-induced lung

  1. Complementary three-dimensional quantitative structure-activity relationship modeling of binding affinity and functional potency

    DEFF Research Database (Denmark)

    Tosco, Paolo; Ahring, Philip K; Dyhring, Tino

    2009-01-01

    Complementary 3D-QSAR modeling of binding affinity and functional potency is proposed as a tool to pinpoint the molecular features of the ligands, and the corresponding amino acids in the receptor, responsible for high affinity binding vs those driving agonist behavior and receptor activation...

  2. Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding.

    Science.gov (United States)

    Watanabe, Yuta; Ishimori, Koichiro; Uchida, Takeshi

    2017-02-12

    HBP23, a 23-kDa heme-binding protein identified in rats, is a member of the peroxiredoxin (Prx) family, the primary peroxidases involved in hydrogen peroxide catabolism. Although HBP23 has a characteristic Cys-Pro heme-binding motif, the significance of heme binding to Prx family proteins remains to be elucidated. Here, we examined the effect of heme binding to human peroxiredoxin-1 (PRX1), which has 97% amino acid identity to HBP23. PRX1 was expressed in Escherichia coli and purified to homogeneity. Spectroscopic titration demonstrated that PRX1 binds heme with a 1:1 stoichiometry and a dissociation constant of 0.17 μM. UV-vis spectra of heme-PRX1 suggested that Cys52 is the axial ligand of ferric heme. PRX1 peroxidase activity was lost upon heme binding, reflecting the fact that Cys52 is not only the heme-binding site but also the active center of peroxidase activity. Interestingly, heme binding to PRX1 caused a decrease in the toxicity and degradation of heme, significantly suppressing H2O2-dependent heme peroxidase activity and degradation of PRX1-bound heme compared with that of free hemin. By virtue of its cytosolic abundance (∼20 μM), PRX1 thus functions as a scavenger of cytosolic hemin (dual role; Cys-dependent peroxidase activity and cytosolic heme scavenger. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Zinc-regulated DNA binding of the yeast Zap1 zinc-responsive activator.

    Directory of Open Access Journals (Sweden)

    Avery G Frey

    Full Text Available The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1.

  4. Zinc-Regulated DNA Binding of the Yeast Zap1 Zinc-Responsive Activator

    Science.gov (United States)

    Frey, Avery G.; Bird, Amanda J.; Evans-Galea, Marguerite V.; Blankman, Elizabeth; Winge, Dennis R.; Eide, David J.

    2011-01-01

    The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1. PMID:21799889

  5. [Methods of heparin therapy in surgical patients with thrombohemorrhagic syndrome].

    Science.gov (United States)

    Gasanov, F D

    2012-01-01

    The results of comparative study of different heparin medications efficacy in patients with thrombohemorrhagic syndrome (THS) are presented. The study was conducted in 286 patients with THS as a result of peritonitis of various etiology (174 patients), massive hemorrhage, shock, microcirculation disorders (112 patients). Heparin therapy carried out in 249 patients (87.1%), 37 patients (12.9%) had no heparin therapy./ In "heparin" group 193 patients (77.5%) received low molecular weight heparin (LMWH), 56 patients (22.5%) took unfractionated heparin (UFH). LWMH demonstrated high efficiency with fewer hemorrhagic complications in comparison with UFH/.

  6. Increased activity of the mannan-binding lectin complement activation pathway in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Ytting, H; Jensenius, J C; Christensen, Ib Jarle

    2004-01-01

    BACKGROUND: Postoperative bacterial infectious complications are frequent in patients with colorectal cancer (CRC), with subsequent increased recurrence rates and poor prognosis. Deficiency of the mannan-binding lectin (MBL) complement activation pathway may cause increased risk of infection......: Serum MBL concentrations and MBL/MASP activity were determined using immunofluorometric assays. The levels are presented as the median, inter-quartile range and range. RESULTS: Serum MBL levels were significantly (P cancer (1384 (400-2188) ng/mL) (median...... in the colon or rectum, and disease stages according to Dukes' classification. No statistical difference (P=0.20) in frequency of MBL deficiency was found between the patients (20%) and the donors (27%). CONCLUSIONS: Overall, the MBL complement activation pathway is significantly increased in patients...

  7. Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation.

    Directory of Open Access Journals (Sweden)

    Ran Sun

    2015-12-01

    Full Text Available Trichinella spiralis expresses paramyosin (Ts-Pmy as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host's immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated.The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration.Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9.

  8. SPATHOLOBUS SUBERECTUS STEM EXTRACT IMPROVES THE PROTECTIVE EFFECT OF HEPARIN ON CERULEIN-INDUCED PANCREATITIS.

    Science.gov (United States)

    Shao, Zhengyi

    2017-01-01

    The present study evaluates the effect of Spatholobus suberectus stem extract (SS) in the management of pancreatitis alone and in combination with heparin. Pancreatitis was induced pancreatitis by cerulean (50μg/kg, i.p.) five times at an interval of 1 h without any pretreatment of drug. Rats were treated with SS (100 and 200 mg/kg, p. o.) and heparin (150 U/kg, i.p.) alone and in combination for the duration of a week. Later pancreatic weight and blood flow was estimated and different biochemical parameters like concentration of D-dimer and Interleukin 1β (IL-Ιβ) and activity of amylase and lipase were determined in blood of pancreatitis rats. Moreover effect of drug treatment on DNA synthesis and histopathology was also estimated on cerulean induced pancreatitis rats. Results of this study suggest that treatment with SS alone and in combination with heparin significantly increase in prothrombin time and pancreatic blood flow than negative control group. There was significant decrease in concentration of IL-Ιβ and D-dimer and activity of amylase and lipase in SS and heparin treated group than negative control group. Pancreatic DNA synthesis was also found to be reduced in SS and heparin alone and in combination treated group. Histopathology study also reveals that treatment with SS and heparin alone and in combination reduces edema, hemorrhages, leukocyte infiltration in the TS of pancreatic tissues. Present study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway and potentiates the effect of heparin in the management of pancreatitis.

  9. Alzheimer's Disease: Another Target for Heparin Therapy

    Directory of Open Access Journals (Sweden)

    Luigi Bergamaschini

    2009-01-01

    Full Text Available Alzheimer's disease (AD is the leading cause of dementia and cognitive decline in the elderly. Brain tissue changes indicate that the two main proteins involved in AD are amyloid-β(A-β, which is associated with the formation of senile amyloid plaques, and tau, which is associated with the formation of neurofibrillary tangles. Although a central role for A-β in the pathogenesis of AD is indisputable, considerable evidence indicates that A-β production is not the sole culprit in AD pathology. AD is also accompanied by an inflammatory response that contributes to irreversible changes in neuronal viability and brain function, and accumulating evidence supports the pivotal role of complement and contact systems in its pathogenesis and progression. The complexity of AD pathology provides numerous potential targets for therapeutic interventions. Compounds that interact directly with A-β protein or interfere with its production and/or aggregation can reduce the inflammatory and neurotoxic effects of A-β, and heparin, a glycosaminoglycan mixture currently used in the prophylaxis and treatment of thrombosis, might be a candidate, as recent research has been extended to consider its nonanticoagulant properties, including its modulation of various proteases and anti-inflammatory activity.

  10. Plasmodium vivax MSP-1 peptides have high specific binding activity to human reticulocytes.

    Science.gov (United States)

    Rodríguez, Luis Eduardo; Urquiza, Mauricio; Ocampo, Marisol; Curtidor, Hernando; Suárez, Jorge; García, Javier; Vera, Ricardo; Puentes, Alvaro; López, Ramses; Pinto, Martha; Rivera, Zuly; Patarroyo, Manuel Elkin

    2002-01-31

    Plasmodium vivax merozoites have high preferential ability to interact with and invade reticulocytes, although these cells correspond to only 2% of the red blood cells (RBC) population. P. vivax merozoite surface protein-1 (Pv-MSP-1) is believed to have an important role in attachment and invasion process. Using 88 non-overlapping 20-mer peptides, covering the entire Pv-MSP-1 Belem strain sequence, RBC and reticulocyte binding assays were performed. Fourteen sequences were identified with high specific binding activity to reticulocytes, but only three had high specific binding activity to mature erythrocytes. These peptides showed affinity constant values between 20 and 150nM, indicating a strong interaction between these sequences and reticulocyte receptors. Critical residues in binding to reticulocytes for these peptides were determined by competition binding assays with glycine scanning analogues. All high binding peptides bind to reticulocyte surface proteins having a molecular mass of around 18-20kDa which are not present in mature RBC. Interestingly, some high activity binding peptides (HABPs) are located close to the hypothesised 42 and 19kDa fragment cleavage sites for this protein, suggesting that these sequences have an important role in target cell attachment and invasion process by Pv-MSP-1.HABPs may be clustered in two regions, with region I being located between amino acids 280-719, and region II between amino acids 1060-1599 with higher than 25% identity level. A P. falciparum MSP-1 antigenic domain binds to RBCs and inhibits parasite invasion. Peptides 1721 and 1724 bind with high activity to reticulocytes in homologous Pv-MSP-1, suggesting similar functions for these two sequences.

  11. Labelling of pneumococcal penicillin-binding proteins with (/sup 3/H)propionyl-ampicillin. A rapid method for monitoring penicillin-binding activity

    Energy Technology Data Exchange (ETDEWEB)

    Hakenbeck, R. (Max-Planck-Institut fuer Molekulare Genetik, Berlin (Germany, F.R.)); Kohiyama, M. (Paris-7 Univ., 75 (France). Inst. de Biologie Moleculaire)

    1982-08-01

    Penicillin-binding proteins (PBPs) are membrane components ubiquitous to all bacteria examined so far. Some of them are present in only a few copies per cell. The conventional method of visualizing these proteins consists in binding of radioactive penicillin to the fractions containing PBPs followed by SDS-PAGE and finally fluorography. Although this procedure is laborious, it is necessary for the determination of the identity as well as for the quantification of each PBP. On the other hand, when penicillin-binding conditions are to be examined or binding activity has to be followed through fractionation and purification of PBPs, no fast monitoring device for these proteins has been available. The authors developed a rapid and easy assay for penicillin-binding activity with a filter-binding technique using (/sup 3/H)propionyl ampicillin (/sup 3/H-PA) of high specific activity. As little 2..mu..g of crude membranes obtained from the highly penicillin-sensitive, ..beta..-lactamase-negative organism Streptococcus pneumoniae, are sufficient to detect binding activity. In this paper they describe optimum conditions for the assay of PBPs and show that this binding activity correlates with the presence of native penicillin-binding proteins.

  12. The effect of heparin on pregnancy associated plasma protein-A concentration in healthy, non-pregnant individuals.

    Science.gov (United States)

    Jespersen, Camilla H B; Vestergaard, Kirstine R; Schou, Morten; Teisner, Børge; Iversen, Kasper

    2015-08-01

    The objective of this study was to determine the differences in pregnancy associated plasma protein-A (PAPP-A) concentrations in heparin naive and heparin treated healthy men and non-pregnant women, to find a possible difference in different age groups, and to determine the response in PAPP-A concentration to repeated injections of unfractionated heparin. Twenty-five healthy, non-pregnant volunteers divided into five groups (determined by gender and age) received 5000 IU unfractionated heparin intravenously. Five young men received an additional 5000 IU after 90 and 180 min. Blood samples to determine PAPP-A concentration and APTT were drawn at different time points. Injection of heparin elicited increase in and rapid normalization of PAPP-A concentrations in all subjects. The group of 20-30-year-old never-pregnant women had lower responses than the individuals of the four other groups. The difference was not significant (p > 0.05). Repeated injections of heparin caused additional peaks in PAPP-A concentration of about the same sizes as the first peak. We observed an increase in time to normalization of PAPP-A concentration (from 75-90 min to 90-150 min) and APTT levels with repeated injections. We observed a rapid normalization of PAPP-A. Our result has a great similarity to the half-life of unfractionated heparin. This result combined with the finding of equally sized peaks in PAPP-A concentration, and that all of this was found in healthy, non-pregnant individuals, suggests that heparin might compete for a binding-site on PAPP-A or with PAPP-A itself for a common receptor in healthy arterial vessels. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  13. Investigation of in vitro Opioid Receptor Binding Activities of Some Turkish Salvia species

    Directory of Open Access Journals (Sweden)

    Özge Gündüz Çınar

    2011-01-01

    Full Text Available Kappa Opioid Peptide Receptor (KOPr activation produces analgesic, psychotomimetic, diuretic and antipruritic effects. KOPr ligands are investigated for their potential roles in the treatment of addiction, depression, feeding behavior, psychosis and schizophrenia. In this study the methanolic extracts of a number of Salvia species which are native to Turkey (S. tomentosa, S. tchihatcheffii , S. rosifolia, S. dichroantha and S. sclarea were tested for their potential binding to opioid receptors in rat brain membranes and Chinese Hamster Ovary Cells expressing human KOPr (CHO-KOPh. [ 3H]Diprenorphine, an unselective opioid antagonist, was utilized in the radioligand receptor binding assays. All extracts (0.11 mg/ml inhibited the [ 3H]Diprenorphine binding with ranging KOPr binding affinities. More than 50% inhibition of diprenorphine binding was shown only with Salvia dichroantha and Salvia sclarea both in rat brain membranes and CHO-KOPh membranes.Among them Salvia sclarea deserves further investigation for its active component(s and its pharmacological characterization. This study clearly demonstrates the potential opioid receptor binding activities of several Turkish Salvia species. This work constitutes the first study on in vitro opioid receptor binding activities of Salvia species from the Turkish flora.

  14. Binding of the urokinase-type plasminogen activator to its cell surface receptor is inhibited by low doses of suramin

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Danø, K

    1993-01-01

    .e. trypan blue and Evans blue) did prove inhibitory. The inhibition found with suramin showed a concentration dependence consistent with a mixed competitive and noncompetitive mechanism. The off-rate of prebound ligand was accelerated by the drug. It is speculated that the present effect may contribute...... micrograms/ml when using U937 cells and a ligand concentration of 0.3 nM. This concentration of the drug is well below the serum levels found in suramin-treated patients. Inhibition of binding was also demonstrated at the molecular level, using chemical cross-linking or an enzyme-linked immunosorbent assay......-type technique based on the ligand interaction. The inhibition was not caused by a mere polyanion effect since polysulfates such as heparin, heparan sulfate, and pentosan polysulfate were non-inhibitory or showed only a very weak inhibition. However, polysulfonated compounds with structures resembling suramin (i...

  15. short communication binding of nickel and zinc ions with activated ...

    African Journals Online (AJOL)

    a

    products in the production of activated carbon for environmental pollution monitoring and control, we report on the use of activated carbon obtained from sugar cane fibre for the removal of nickel and zinc ions from aqueous solution. EXPERIMENTAL.

  16. Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding site

    Energy Technology Data Exchange (ETDEWEB)

    McVittie, L.D.; Sibley, D.R.

    1989-01-01

    A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either (/sup 3/H)-N-(1-(2-thienyl)cyclohexyl)piperidine ((/sup 3/H)TCP) or (/sup 3/H)MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4/degrees/C. In the presence of detergent, (/sup 3/H)TCP binding exhibits a K/sub d/ of 250 nM, a B/sub max/ of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor (/sup 3/H)TCP binding: K/sub d/ = 48 nM, M/sub max/ = 1.13 pmol/mg protein.

  17. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  18. Platelet-activating factor (PAF) receptor binding activity of the roots of Enicosanthellum pulchrum.

    Science.gov (United States)

    Nordin, Noraziah; Jalil, Juriyati; Jantan, Ibrahim; Murad, Shahnaz

    2012-03-01

    Enicosanthellum pulchrum (King) Heusden (Annonaceae) is a coniferous tree that is confined to mountain forests. The chemical constituents of this species have been studied previously; however, its biological activity has never been investigated before and is reported here for the first time. The extracts, fractions and compounds from the roots of E. pulchrum were investigated for their inhibitory effects on platelet-activating factor (PAF) receptor binding to rabbit platelets using (3)H-PAF as a ligand. The PAF receptor binding inhibitory effect using rabbit platelets was determined in vitro by measuring the difference between total amount of bound (3)H-PAF in the presence and the absence of excess unlabelled PAF. The compounds were isolated by bioassay-guided fractionation and their structures were elucidated by spectroscopic techniques. Among the extracts tested, the ethyl acetate extract was the most active with 85.6% inhibition, while hexane and methanol extracts showed 40.2 and 42.5% inhibition, respectively. Fractionation of the ethyl acetate extract using vacuum liquid chromatography (VLC) yielded six fractions AEA(I--VI). Chromatography fraction AEA(VI) yielded a new compound, 1-(2',3',4'-trimethoxyphenyl)hexan-1-ol, while fraction AEA(III) afforded three compounds, namely liriodenine, cleistopholine and dehydroanonaine. 1-(2',3',4'-Trimethoxyphenyl)hexan-1-ol, cleistopholine and dehydroanonaine showed relatively strong inhibition with IC(50) values of 26.6, 50.2 and 45.4 µM, respectively. The results suggest that these compounds could be responsible for the PAF antagonistic activity of the ethyl acetate extract of this plant.

  19. Grades of 43 fish species in Japan based on IgE-binding activity.

    Science.gov (United States)

    Koyama, Harumi; Kakami, Michiko; Kawamura, Makiko; Tokuda, Reiko; Kondo, Yasuto; Tsuge, Ikuya; Yamada, Kazue; Yasuda, Toshitaka; Urisu, Atsuo

    2006-09-01

    Hypersensitivity reactions to fish are a common food allergy, but IgE-binding activity to fish species have not been fully elucidated. The aim of this study was to identify fish with high binding activity to IgE in sera from Japanese fish-hypersensitive individuals. 38 children with a history of at least one episode of hypersensitivity after ingestion of fish were enrolled and 34 children with no history of reactions and negative IgE results for at least five kinds of fish antigen were included as controls. Using a radioallergosorbent test, we examined IgE-binding to each fish species using sera from fish-hypersensitive subjects. Fish were then graded according to IgE-binding activity. Many fish species, including red salmon, silver salmon, yellowfin tuna, big eyed tuna, Atlantic tuna, saurel, skipper, yellowtail, Japanese sardine, bonita and mackerel had high IgE-binding activity. All of these fish are abundantly consumed in Japan. The hypersensitivity reactions experienced by many subjects occurred after ingestion of species with high IgE-binding activity. Only halibut (Osteichthyes) and sharks (Chondrichthyes) had low IgE-binding activity. A correlation was observed between IgE levels and expression of symptoms after fish ingestion. High consumption of salmon, tuna, scad (including saurel), skipper, yellowtail, sardine, bonita and mackerel in Japan might be the cause of the high IgE-binding activity of these species. The grades of fish species consumed widely in Japan are likely to be useful for nutritional instruction of fish-allergic patients.

  20. Cholesterol Crystals Activate the Lectin Complement Pathway via Ficolin-2 and Mannose-Binding Lectin

    DEFF Research Database (Denmark)

    Pilely, Katrine; Rosbjerg, Anne; Genster, Ninette

    2016-01-01

    Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind...... CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using...... recombinant proteins, specific inhibitors, as well as deficient and normal sera. Additionally, we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2...

  1. Recurrent intradialytic heparin induced anaphylaxis: workup and management

    OpenAIRE

    Santosa, Amelia; Tan, Seng Hoe; Cheng, Yew Kuang

    2013-01-01

    Heparin has been widely used for intradialytic anticoagulation since the 1940s. Heparin induced anaphylaxis can be life threatening, mandating early recognition and intervention. However, due to its relative rarity many physicians remain unaware. We report the case of a 70-year-old woman requiring dialysis, who developed recurrent anaphylaxis to intradialytic heparin. We describe a systematic approach to confirm the suspected heparin allergy, which must include an evaluation of predisposing f...

  2. Self-assembled FUS binds active chromatin and regulates gene transcription

    Science.gov (United States)

    Yang, Liuqing; Gal, Jozsef; Chen, Jing; Zhu, Haining

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Fused in sarcoma (FUS) is a DNA/RNA binding protein and mutations in FUS cause a subset of familial ALS. Most ALS mutations are clustered in the C-terminal nuclear localization sequence of FUS and consequently lead to the accumulation of protein inclusions in the cytoplasm. It remains debatable whether loss of FUS normal function in the nucleus or gain of toxic function in the cytoplasm plays a more critical role in the ALS etiology. Moreover, the physiological function of FUS in the nucleus remains to be fully understood. In this study, we found that a significant portion of nuclear FUS was bound to active chromatin and that the ALS mutations dramatically decreased FUS chromatin binding ability. Functionally, the chromatin binding is required for FUS transcription activation, but not for alternative splicing regulation. The N-terminal QGSY (glutamine-glycine-serine-tyrosine)-rich region (amino acids 1–164) mediates FUS self-assembly in the nucleus of mammalian cells and the self-assembly is essential for its chromatin binding and transcription activation. In addition, RNA binding is also required for FUS self-assembly and chromatin binding. Together, our results suggest a functional assembly of FUS in the nucleus under physiological conditions, which is different from the cytoplasmic inclusions. The ALS mutations can cause loss of function in the nucleus by disrupting this assembly and chromatin binding. PMID:25453086

  3. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity ...

    Indian Academy of Sciences (India)

    activity, anti-cancer and anti-microbial activities.9 12. The design of small complexes that bind and react with DNA is one of the interesting activities of bioinor- ganic chemist. Hence it is important to investigate more efficient drugs that target DNA. In recent years,13 15 there is some interest towards synthesis, DNA interac-.

  4. A novel application of multi-wavelength TIRF spectroscopy for real time monitoring of antithrombin interactions with immobilized heparin.

    Science.gov (United States)

    Klinth, J E; Larsson, R; Andersson, P O; Ekdahl, K Nilsson

    2006-04-15

    Real time interactions of antithrombin (AT) with Corline Heparin Surfaces (CHS) with one and two layers of heparin conjugate have been examined using a multi-wavelength TIRF spectroscopy technique with continuous flow. Fluorescently labeled AT, adsorbed from citrated human blood plasma, showed significantly higher signals on CHS compared to the cationic surface used to attach the heparin conjugate. The AT binding to CHS was very stable, also after exposure to soluble heparin at a concentration of 1.5 IU/mL. Only a few percent of the bound AT were displaced from the surfaces by AT present in plasma after long-term exposure to plasma. In contrast, larger amounts of the freshly added AT had adsorbed to the surfaces, especially to the surface with two layers of heparin conjugate, indicating the presence of unsaturated AT binding sites. The amount of AT bound to the different surfaces was quantified after elution using an enzyme immunoassay (EIA). Characteristic emission spectra of proteins and fluorophores of labeled proteins, obtained at the surfaces after a long-term exposure to plasma, confirmed their presence at the surfaces. The multi-wavelength TIRF technique proved to be a useful tool when combined with other techniques to study the time course of interactions of fluorescently labeled proteins with biomaterials, even in a complex environment such as plasma.

  5. Heparin-coated cardiopulmonary bypass circuits selectively deplete the pattern recognition molecule ficolin-2 of the lectin complement pathway in vivo

    DEFF Research Database (Denmark)

    Hein, Estrid; Munthe-Fog, L; Thiara, A S

    2015-01-01

    of infections. Thus, we investigated the biocompatibility of the recognition molecules of the lectin pathway in two different types of cardiopulmonary bypass circuits. Bloods were drawn at five time-points before, during and postoperatively from 30 patients undergoing elective cardiac surgery. Patients were......The complement system can be activated via the lectin pathway by the recognition molecules mannose-binding lectin (MBL) and the ficolins. Ficolin-2 exhibits binding against a broad range of ligands, including biomaterials in vitro, and low ficolin-2 levels are associated with increased risk...... randomized into two groups using different coatings of cardiopulmonary bypass circuits, Phisio® (phosphorylcholine polymer coating) and Bioline® (albumin-heparin coating). Concentrations of MBL, ficolin-1, -2 and -3 and soluble C3a and terminal complement complex (TCC) in plasma samples were measured...

  6. Zinc ions bind to and inhibit activated protein C

    DEFF Research Database (Denmark)

    Zhu, Tianqing; Ubhayasekera, Wimal; Nickolaus, Noëlle

    2010-01-01

    Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was ...

  7. short communication binding of nickel and zinc ions with activated ...

    African Journals Online (AJOL)

    a

    ABSTRACT. Activated carbon was prepared from sugar cane fibre by carbonizing at 500 oC for 30 minutes. ... Equilibrium sorption of nickel and zinc ions by the activated carbon was studied using a range of metal ion .... which means that limited sorption cycles will be required in the treatment of waste water containing the ...

  8. Economic evaluation of low-molecularweight heparin in the ...

    African Journals Online (AJOL)

    Objective. To undertake an economic evaluation of the administration and monitoring costs of the two different forms of heparin in patients with unstable coronary artery disease (DCAD). Study design. Equivalent efficacy was found for lowmolecular- weight heparin (LMWH) and for unfractionated heparin (UFH) in the ...

  9. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    Science.gov (United States)

    de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2015-01-01

    In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

  10. ATP binding by NLRP7 is required for inflammasome activation in response to bacterial lipopeptides.

    Science.gov (United States)

    Radian, Alexander D; Khare, Sonal; Chu, Lan H; Dorfleutner, Andrea; Stehlik, Christian

    2015-10-01

    Nucleotide-binding oligimerization domain (NOD)-like receptors (NLRs) are pattern recognition receptors (PRRs) involved in innate immune responses. NLRs encode a central nucleotide-binding domain (NBD) consisting of the NAIP, CIITA, HET-E and TP1 (NACHT) domain and the NACHT associated domain (NAD), which facilitates receptor oligomerization and downstream inflammasome signaling. The NBD contains highly conserved regions, known as Walker motifs, that are required for nucleotide binding and hydrolysis. The NLR containing a PYRIN domain (PYD) 7 (NLRP7) has been recently shown to assemble an ASC and caspase-1-containing high molecular weight inflammasome complex in response to microbial acylated lipopeptides and Staphylococcus aureus infection. However, the molecular mechanism responsible for NLRP7 inflammasome activation is still elusive. Here we demonstrate that the NBD of NLRP7 is an ATP binding domain and has ATPase activity. We further show that an intact nucleotide-binding Walker A motif is required for NBD-mediated nucleotide binding and hydrolysis, oligomerization, and NLRP7 inflammasome formation and activity. Accordingly, THP-1 cells expressing a mutated Walker A motif display defective NLRP7 inflammasome activation, interleukin (IL)-1β release and pyroptosis in response to acylated lipopeptides and S. aureus infection. Taken together, our results provide novel insights into the mechanism of NLRP7 inflammasome assembly. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    Directory of Open Access Journals (Sweden)

    Sinara Mônica Vitalino de Almeida

    2015-06-01

    Full Text Available In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide derivatives (3a–h were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z-2-(acridin-9-ylmethylene-N- (4-chlorophenyl hydrazinecarbothioamide (3f, while the most active compound in antiproliferative assay was (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide (3a. There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

  12. Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfateβ-1→3GalNAc(4-Sulfateβ-1→] motifs in dermatan sulfate on heparin cofactor II activity

    Directory of Open Access Journals (Sweden)

    Sugahara Kazuyuki

    2011-05-01

    Full Text Available Abstract Background Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra and Stolidobranchia (Halocynthia pyriformis and Styela plicata. Despite the identical disaccharide backbone, consisting of [→4IdoA(2Sβ-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi and Phlebobranchia (Ciona intestinalis, aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. Results Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfateβ-1→3GalNAcβ-1

  13. Tetranectin Binds to the Kringle 1-4 Form of Angiostatin and Modifies Its Functional Activity

    DEFF Research Database (Denmark)

    Mogues, Tirsit; Etzerodt, Michael; Hall, Crystal

    2004-01-01

    Tetranectin is a plasminogen kringle 4 domain-binding protein present in plasma and various tissue locations. Decreased plasma tetranectin or increased tetranectin in stroma of cancers correlates with cancer progression and adverse prognosis. A possible mechanism through which tetranectin could...... influence cancer progression is by altering activities of plasminogen or the plasminogen fragment, angiostatin. Tetranectin was found to bind to the kringle 1-4 form of angiostatin (AST $;{\\text{K1-4}}$ ). In addition, tetranectin inhibited binding of plasminogen or AST $;{\\text{K1-4}}$ to extracellular...

  14. Structure-activity studies on the potentiation of benzodiazepine receptor binding by ethylenediamine analogues and derivatives.

    OpenAIRE

    Morgan, P. F.; Stone, T. W.

    1983-01-01

    The effect of ethylenediamine analogues on in vitro binding of [3H]-diazepam to crude cerebral cortical synaptosomal membranes in the rat was studied. Ethylenediamine significantly increased [3H]-diazepam binding to a maximum potentiation of 154% control (EC50 = 1.8 X 10(-4) M) and was the most active compound studied in terms of both potency and the maximum potentiation observed. Potentiation of [3H]-diazepam binding by ethylenediamine analogues is dependent on carbon-chain length, appears t...

  15. The role of heparin in sepsis: much more than just an anticoagulant.

    Science.gov (United States)

    Li, Xu; Ma, Xiaochun

    2017-11-01

    Despite progress in antibiotic treatment, mechanical ventilation, fluid resuscitation and blood glucose maintenance, sepsis remains a cause of high mortality in the intensive care unit to date, there are no proven treatment strategies for the routine management of septic patients. The extensive interaction between inflammation and coagulation contributes to the basic pathophysiology of sepsis. Thus, the agents that attenuate the activation of both inflammation and coagulation may improve the outcome in sepsis. Apart from the well-known anticoagulant effects of heparin, it also possesses various immunomodulatory properties and protects glycocalyx from shedding. Hence, heparin seems to be such an agent. Immunothrombosis plays an important role in early host defence against bacterial dissemination, thus the proper timing for anticoagulant therapy should be determined. We review the available experimental and clinical data supporting the use of heparin in sepsis. At this time the use of heparin in the treatment of sepsis is conflicting. Future trials of heparin therapy for sepsis should concentrate on the very severely ill patients, in whom benefit is most likely to be demonstrated. © 2017 John Wiley & Sons Ltd.

  16. Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

    The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

  17. Trehalose diester glycolipids are superior to the monoesters in binding to Mincle, activation of macrophages invitro and adjuvant activity invivo

    DEFF Research Database (Denmark)

    Huber, Alexandra; Kallerup, Rie S.; Korsholm, Karen S.

    2016-01-01

    augmented by all TDXs tested, in a wide concentration range. In contrast, the TMXs triggered macrophage activation only at high concentrations. Macrophage activation by all TDXs required Mincle, but was independent of MyD88. The superior capacity of TDXs for activating macrophages was paralleled by direct...... binding of TDXs, but not of TMXs, to a Mincle-Fc fusion protein. Insertion of a short polyethylene glycol between the sugar and acyl chain in TDS reduced Mincle-binding and macrophage activation. Immunization of mice with cationic liposomes containing the analogues demonstrated the superior adjuvant...

  18. Serum Protein Binding and the Antimicrobial Activities of Garcinia ...

    African Journals Online (AJOL)

    The kinetics of bactericidal activity of the methanolic extracts of powdered seeds of Garcinia kola (Heckel), Kola acuminata and Kola nitida obtained by soxhlet extraction, were studied using two bacterial sensitive strains of Staphylococcus aureus and Pseudomonas aeruginosa. The study showed that the three extracts at ...

  19. From DNA binding to transcriptional activation: Is the TALE complete?

    Science.gov (United States)

    Bobola, Nicoletta

    2017-09-04

    How transcription factors (TFs) control enhancer and promoter functions to effect changes in gene expression is an important question. In this issue, Hau et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201701154) show that the TALE TF MEIS recruits the histone modifier PARP1/ARTD1 at promoters to decompact chromatin and activate transcription. © 2017 Bobola.

  20. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    Science.gov (United States)

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease.

  1. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    Energy Technology Data Exchange (ETDEWEB)

    Shlomai, Amir, E-mail: amirsh@tasmc.health.gov.il [Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel); Institute for Gastroenterology and Liver disease, Tel-Aviv Sourasky Medical Center, 6 Weizmann street, Tel-Aviv (Israel); Shaul, Yosef [Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel)

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  2. Type III effector activation via nucleotide binding, phosphorylation, and host target interaction.

    Directory of Open Access Journals (Sweden)

    Darrell Desveaux

    2007-03-01

    Full Text Available The Pseudomonas syringae type III effector protein avirulence protein B (AvrB is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]-interacting protein. RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 A resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s. We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.

  3. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.; Tamás, Markus J.

    2015-12-28

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

  4. Thermodynamic analysis on the binding of heavy metals onto extracellular polymeric substances (EPS) of activated sludge.

    Science.gov (United States)

    Sheng, Guo-Ping; Xu, Juan; Luo, Hong-Wei; Li, Wen-Wei; Li, Wei-Hua; Yu, Han-Qing; Xie, Zhi; Wei, Shi-Qiang; Hu, Feng-Chun

    2013-02-01

    Metal binding to microbial extracellular polymeric substances (EPS) greatly influences the distribution of heavy metals in microbial aggregates, soil and aquatic systems in nature. In this work, the thermodynamic characteristics of the binding between aqueous metals (with copper ion as an example) and EPS of activated sludge were investigated. Isothermal titration calorimetry was employed to estimate the thermodynamic parameters for the binding of Cu²⁺ onto EPS, while three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy with parallel factor analysis was used for quantifying the complexation of Cu²⁺ with the EPS. The binding mechanisms were further explored by X-ray absorption fine structure (XAFS) and Fourier transform infrared (FTIR) spectroscopy analysis. The results show that the proteins and humic substances in EPS were both strong ligands for Cu²⁺. The binding capacity N, binding constant K, binding enthalpy ΔH were calculated as 5.74 × 10⁻² mmol/g, 2.18 × 10⁵ L/mol, and -11.30 kJ/mol, respectively, implying that such a binding process was exothermic and thermodynamically favorable. The binding process was found to be driven mainly by the entropy change of the reaction. A further investigation shows that Cu²⁺ bound with the oxygen atom in the carboxyl groups in the EPS molecules of activated sludge. This study facilitates a better understanding about the roles of EPS in protecting microbes against heavy metals. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Miz-1 activates gene expression via a novel consensus DNA binding motif.

    Directory of Open Access Journals (Sweden)

    Bonnie L Barrilleaux

    Full Text Available The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences--ATCGGTAATC and ATCGAT (Mizm1 and Mizm2--bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the in vivo relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed in vivo to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate.

  6. Heparin-Induced Thrombocytopenia Antibody Test

    Science.gov (United States)

    ... High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV ... Sources Used in Previous Reviews (© 1995-2011). Unit Code 81904: Heparin-PF4 Antibody (HIT), Serum. Mayo Clinic ...

  7. Cargo binding activates myosin VIIA motor function in cells.

    Science.gov (United States)

    Sakai, Tsuyoshi; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

    2011-04-26

    Myosin VIIA, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating myosin VIIA as a cargo-transporting motor. Human myosin VIIA was present throughout cytosol, but it moved to the tip of filopodia upon the formation of dimer induced by dimer-inducing reagent. The forced dimer of myosin VIIA translocated its cargo molecule, MyRip, to the tip of filopodia, whereas myosin VIIA without the forced dimer-forming module does not translocate to the filopodial tips. These results suggest that dimer formation of myosin VIIA is important for its cargo-transporting activity. On the other hand, myosin VIIA without the forced dimerization module became translocated to the filopodial tips in the presence of cargo complex, i.e., MyRip/Rab27a, and transported its cargo complex to the tip. Coexpression of MyRip promoted the association of myosin VIIA to vesicles and the dimer formation. These results suggest that association of myosin VIIA monomers with membrane via the MyRip/Rab27a complex facilitates the cargo-transporting activity of myosin VIIA, which is achieved by cluster formation on the membrane, where it possibly forms a dimer. Present findings support that MyRip, a cargo molecule, functions as an activator of myosin VIIA transporter function.

  8. Calcium-binding properties of a calcium-dependent protein kinase from Plasmodium falciparum and the significance of individual calcium-binding sites for kinase activation.

    Science.gov (United States)

    Zhao, Y; Pokutta, S; Maurer, P; Lindt, M; Franklin, R M; Kappes, B

    1994-03-29

    Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates with 45Ca2+. Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca(2+)-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca(2+)-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca(2+)-induced conformational change and the Ca(2+)-dependent activation. Mutations at sites III and IV have minor effects.

  9. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    Carbohydrate active enzymes, particularly those that are active on polysaccharides, are often found associated with carbohydrate binding modules (CBMs), which can play several roles in supporting enzyme function, such as localizing the enzyme to the substrate. However, the presence of CBMs is not...

  10. Binding among Select Episodic Elements Is Altered via Active Short-Term Retrieval

    Science.gov (United States)

    Bridge, Donna J.; Voss, Joel L.

    2015-01-01

    Of the many elements that comprise an episode, are any disproportionately bound to the others? We tested whether active short-term retrieval selectively increases binding. Individual objects from multiobject displays were retrieved after brief delays. Memory was later tested for the other objects. Cueing with actively retrieved objects facilitated…

  11. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    Science.gov (United States)

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms. © 2014 International Society for Neurochemistry.

  12. Correction: Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfateβ-1→3GalNAc(4-Sulfateβ-1→] motifs in dermatan sulfate on heparin cofactor II activity

    Directory of Open Access Journals (Sweden)

    Sugahara Kazuyuki

    2011-07-01

    Full Text Available Abstract After the publication of the work entitled "Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfateβ-1→3GalNAc(4-Sulfateβ-1→] motifs in dermatan sulfate on heparin cofactor II activity", by Kozlowski et al., BMC Biochemistry 2011, 12:29, we found that the legends to Figures 2 to 5 contain serious mistakes that compromise the comprehension of the work. This correction article contains the correct text of the legends to Figures 2 to 5.

  13. Characterization of a benzyl-phenoxy-ethanamine binding protein in Trypanosoma equiperdum and the possible relation between binding affinity and trypanocidal activity.

    Science.gov (United States)

    Betbeder, D; Perie, J J; Baltz, T; Poirot, M; Faye, J C

    1993-04-01

    A new family of benzyl-phenoxy-ethanamine derivatives has been assayed for trypanocidal activity. Using tritiated morpholino-benzyl-phenoxy-ethanamine as a probe, it is shown that this ligand is able to bind specifically to a protein contained in extracts of Trypanosoma equiperdum. The binding is saturable and of high affinity (KD = 4 nM: Bmax = 200 fmol (mg protein)-1). The in vitro activities of the investigated compounds against this parasite correlate with their affinities to the putative binding site. Moreover, using an azido functionalized morpholino-benzyl-phenoxyethanamine as photoprobe a major M(r) = 40,000 protein was specifically revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This molecular weight corresponds with the previously observed value determined for the antioestrogen binding site protein of rat liver which has been shown to specifically bind antioestrogens of the triphenylethylene family and phenoxyethanamine derivatives.

  14. Revisiting the Response Mechanism of Polymeric Membrane Based Heparin Electrodes.

    Science.gov (United States)

    Bell, Andrea K; Höfler, Lajos; Meyerhoff, Mark E

    2012-01-01

    Potentiometric membrane electrodes that respond to heparin and other polyanions were introduced in the early 1990s. Herein, the mechanism of polymer membrane electrode type heparin sensors is revisited. The extraction/diffusion of heparin is studied via both potentiometric and impedance spectroscopic techniques using a pre-fractionated heparin preparation that contains polyanionic species > 10000 Daltons. The reversal in EMF response using this heparin preparation indicates diffusion of higher MW heparin fragments to the backside of the membrane. Diffusion coefficients are calculated using a novel formula derived from the phase boundary potential model and Fick's second law of diffusion. Impedance spectroscopy is also employed to show that high MW heparin species are extracted and diffuse across the PVC membranes.

  15. Cargo binding activates myosin VIIA motor function in cells

    OpenAIRE

    Sakai, Tsuyoshi; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

    2011-01-01

    Myosin VIIA, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating myosin VIIA as a cargo-transporting motor. Human myosin VIIA was present ...

  16. Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II.

    Science.gov (United States)

    Zhu-Salzman, K; Shade, R E; Koiwa, H; Salzman, R A; Narasimhan, M; Bressan, R A; Hasegawa, P M; Murdock, L L

    1998-12-08

    Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

  17. Sense oligonucleotide competition for gene promoter binding and activation.

    Science.gov (United States)

    Cutroneo, Kenneth R; Chiu, Jen-Fu

    2003-01-01

    Considerable evidence has ensued on the importance of growth factors during regeneration both for cell replication and for stimulation of reparative cells to synthesize and secrete extracellular matrix components. During the healing process if the growth factor concentration is too high because of over-expression, abnormal wound healing and tissue fibrosis will occur. The growth factor concentration at the wound site may be controlled by gene therapy and the titration of gene dosage. However, if there is a narrow window between the beneficial effects and adverse effects of gene therapy, oligonucleotide approaches may be used concurrently with gene therapy to control growth factor concentration(s) at the wound site. Antisense oligos offer a method to control the concentration of growth factors at the level of translation. A novel method using sense oligos to the proalpha1 (I) collagen gene to inhibit gene transcription and collagen synthesis has recently been reported. The exogenous modified oligodeoxynucleotide competes with the cis-element (i.e. the transforming growth factor-beta (TGF-beta) element) in the distal 5'-flanking region of the proalpha1 (I) collagen gene for the trans-acting factor (i.e. the TGF-beta activator protein complex), thereby down regulating promoter activity of the proalpha1 (I) collagen gene and inhibiting type I collagen synthesis. The oligonucleotide approaches, both antisense and sense therapies, may be used to regulate over-expression of growth factors and thereby either eliminate or lessen the potential adverse effects of gene therapy.

  18. Structural insights into human peroxisome proliferator activated receptor delta (PPAR-delta selective ligand binding.

    Directory of Open Access Journals (Sweden)

    Fernanda A H Batista

    Full Text Available Peroxisome proliferator activated receptors (PPARs δ, α and γ are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328 in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.

  19. Chicken mannose-binding lectin function in relation to antibacterial activity towards Salmonella enterica

    DEFF Research Database (Denmark)

    Ulrich-Lynge, Sofie Louise; Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann

    2015-01-01

    Mannose-binding lectin (MBL) is a C-type serum lectin of importance in innate immunity. Low serum concentrations of MBL have been associated with greater susceptibility to infections. In this study, binding of purified chicken MBL (cMBL) to Salmonella enterica subsp. enterica (S. enterica......) serotypes B, C1 and D was investigated by flow cytometry, and Staphylococcus aureus (S. aureus) was used for comparison. For S. enterica the C1 serotypes were the only group to exhibit binding to cMBL. Furthermore, functional studies of the role of cMBL in phagocytosis and complement activation were...... performed. Spiking with cMBL had a dose-dependent effect on the HD11 phagocytic activity of S. enterica subsp. enterica serovar Montevideo, and a more pronounced effect in a carbohydrate competitive assay. This cMBL dose dependency of opsonophagocytic activity by HD11 cells was not observed for S. aureus...

  20. Calmodulin-binding transcription activators and perspectives for applications in biotechnology.

    Science.gov (United States)

    Shen, Chenjia; Yang, Yanjun; Du, Liqun; Wang, Huizhong

    2015-12-01

    In recent years, a novel family of calmodulin-binding transcription activators (CAMTAs) has been reported in various species. The CAMTAs share a conserved domain organization, with a CG-1 DNA-binding domain, a transcription factor immunoglobulin domain, several ankyrin repeats, a calmodulin-binding domain, and a varying number of IQ motifs. CAMTAs participate in transcriptional regulation by recognizing and binding to a specific cis-element: (G/A/C)CGCG(C/G/T). Plants suffer from the environmental challenges, including abiotic and biotic stresses. Investigations in various plant species indicate a broad range of CAMTA functions involved in developmental regulation, environmental stress response, and hormone cross talk. In this review, we focus on the expression patterns and biological functions of CAMTAs to explore their probable applications in biotechnology. Furthermore, the identification and phylogenetic analysis of CAMTAs in crops could open new perspectives for enhancing stress tolerance, which could lead to improved crop production.

  1. In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata

    Directory of Open Access Journals (Sweden)

    L. Cardilo-Reis

    2006-11-01

    Full Text Available In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10% of mammalian heparin and about 5% as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5% (mean ± SD without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4%, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13% without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.

  2. Viscoelastic blood coagulation measurement with Sonoclot predicts postoperative bleeding in cardiac surgery after heparin reversal.

    Science.gov (United States)

    Bischof, Dominique B; Ganter, Michael T; Shore-Lesserson, Linda; Hartnack, Sonja; Klaghofer, Richard; Graves, Kirk; Genoni, Michele; Hofer, Christoph K

    2015-01-01

    The aim of the study was to determine if Sonoclot with its sensitive glass bead-activated, viscoelastic test can predict postoperative bleeding in patients undergoing cardiac surgery at predefined time points. A prospective, observational clinical study. A teaching hospital, single center. Consecutive patients undergoing cardiac surgery (N = 300). Besides routine laboratory coagulation studies and heparin management with standard (kaolin) activated clotting time, additional native blood samples were analyzed on a Sonoclot using glass bead-activated tests. Glass bead-activated clotting time, clot rate, and platelet function were recorded immediately before anesthesia induction and at the end of surgery after heparin reversal but before chest closure. Primary outcome was postoperative blood loss (chest tube drainage at 4, 8, and 12 hours postoperatively). Secondary outcome parameters were transfusion requirements, need for surgical re-exploration, time of mechanical ventilation, length of intensive care unit and hospital stay, and hospital morbidity and mortality. Patients were categorized into "bleeders" and "nonbleeders." Patient characteristics, operations, preoperative standard laboratory parameters, and procedural times were comparable between bleeders and nonbleeders except for sex and age. Bleeders had higher rates of transfusions, surgical re-explorations, and complications. Only glass bead measurements by Sonoclot after heparin reversal before chest closure but not preoperatively were predictive for increased postoperative bleeding. Sonoclot with its glass bead-activated tests may predict the risk for postoperative bleeding in patients undergoing cardiac surgery at the end of surgery after heparin reversal but before chest closure. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Data for chitin binding activity of Moringa seed resistant protein (MSRP)

    OpenAIRE

    Sandanamudi, Anudeep; Bharadwaj, Kishan R.; Cheruppanpullil, Radha

    2016-01-01

    Chitin binding activity of moringa seed resistant protein (MSRP) isolated from defatted moringa seed flour was investigated in the present study “Characterization of soluble dietary fiber from Moringa oleifera seeds and its immunomodulatory effects” (S. Anudeep, V.K. Prasanna, S.M. Adya, C. Radha, 2016) [1]. The assay reaction mixture contained 0.4 mg/ml of MSRP and different amounts (20–100 mg) of chitin. MSRP exhibited binding activity over wide range of chitin concentration. Maximum bindin...

  4. The antibacterial activity of peptides derived from human beta-2 glycoprotein I is inhibited by protein H and M1 protein from Streptococcus pyogenes

    NARCIS (Netherlands)

    Nilsson, Maria; Wasylik, Sylwia; Mörgelin, Matthias; Olin, Anders I.; Meijers, Joost C. M.; Derksen, Ronald H. W. M.; de Groot, Philip G.; Herwald, Heiko

    2008-01-01

    During the last years, the importance of antibacterial peptides has attracted considerable attention. We report here that peptides derived from the fifth domain of beta-2 glycoprotein I (beta(2)GPI), a human heparin binding plasma protein, have antibacterial activities against Gram-positive and

  5. Penicillin binding proteins as danger signals: meningococcal penicillin binding protein 2 activates dendritic cells through Toll-like receptor 4.

    Directory of Open Access Journals (Sweden)

    Marcelo Hill

    Full Text Available Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml ± 0.1, CD80 (LOGEC50 = 4.88 µg/ml ± 0.15 and CD86 (LOGEC50 = 5.36 µg/ml ± 0.1. This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7 ± 5.1% cells versus 12 ± 2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed thatPBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.

  6. Penicillin binding proteins as danger signals: meningococcal penicillin binding protein 2 activates dendritic cells through Toll-like receptor 4.

    Science.gov (United States)

    Hill, Marcelo; Deghmane, Ala-Eddine; Segovia, Mercedes; Zarantonelli, Maria Leticia; Tilly, Gaëlle; Blancou, Philippe; Bériou, Gaëlle; Josien, Régis; Anegon, Ignacio; Hong, Eva; Ruckly, Corinne; Antignac, Aude; El Ghachi, Meriem; Boneca, Ivo Gomperts; Taha, Muhamed-Kheir; Cuturi, Maria Cristina

    2011-01-01

    Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml ± 0.1), CD80 (LOGEC50 = 4.88 µg/ml ± 0.15) and CD86 (LOGEC50 = 5.36 µg/ml ± 0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7 ± 5.1% cells versus 12 ± 2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed thatPBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.

  7. General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

    Science.gov (United States)

    Chang, Tianjun; Qi, Cui; Meng, Jie; Zhang, Nan; Bing, Tao; Yang, Xianda; Cao, Zehui; Shangguan, Dihua

    2013-01-01

    G-quadruplexes (G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs) are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes) have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

  8. General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

    Directory of Open Access Journals (Sweden)

    Tianjun Chang

    Full Text Available G-quadruplexes (G4s are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

  9. Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

    DEFF Research Database (Denmark)

    Helledie, T; Antonius, M; Sorensen, R V

    2000-01-01

    Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty a...

  10. Myelin basic protein stimulates plasminogen activation via tissue plasminogen activator following binding to independent l-lysine-containing domains.

    Science.gov (United States)

    Gonzalez-Gronow, Mario; Fiedler, Jenny L; Farias Gomez, Cristian; Wang, Fang; Ray, Rupa; Ferrell, Paul D; Pizzo, Salvatore V

    2017-08-26

    Myelin basic protein (MBP) is a key component of myelin, the specialized lipid membrane that encases the axons of all neurons. Both plasminogen (Pg) and tissue-type plasminogen activator (t-PA) bind to MBP with high affinity. We investigated the kinetics and mechanisms involved in this process using immobilized MBP and found that Pg activation by t-PA is significantly stimulated by MBP. This mechanism involves the binding of t-PA via a lysine-dependent mechanism to the Lys91 residue of the MBP NH2-terminal region Asp82 -Pro99, and the binding of Pg via a lysine-dependent mechanism to the Lys122 residue of the MBP COOH-terminal region Leu109-Gly126. In this context, MBP mimics fibrin and because MBP is a plasmin substrate, our results suggest direct participation of the Pg activation system on MBP physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Post-transplant IVC occlusion and thrombosis treated with tPA, heparin, and sharp recanalization.

    Science.gov (United States)

    Mindikoglu, Ayse L; Miller, Jonathan S; Borge, Marc A; Van Thiel, David H

    2005-03-01

    Complete inferior vena cava (IVC) thrombosis can be a lethal complication in a liver transplant recipient. The case of a 52-year-old liver transplant recipient, who developed complete IVC as well as left iliofemoral thrombosis, is reported. After treatment with combined tissue plasminogen activator (tPA) and heparin, the IVC was successfully recanalized with sharp dissection, balloon dilatation, and stent placement.

  12. Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations.

    Science.gov (United States)

    Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek W; Mai, Sabine; Cremer, Christoph

    2017-05-05

    Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Alderete, J.F.

    1984-08-01

    Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites.

  14. Plasmodium falciparum acid basic repeat antigen (ABRA) peptides: erythrocyte binding and biological activity.

    Science.gov (United States)

    Curtidor, H; Urquiza, M; Suarez, J E; Rodriguez, L E; Ocampo, M; Puentes, A; Garcia, J E; Vera, R; Lopéz, R; Ramirez, L E; Pinzon, M; Patarroyo, M E

    2001-08-14

    Non overlapping 20-mer peptides, covering the complete sequence of acid basic repeat antigen (ABRA) of Plasmodium falciparum, were synthesised and tested in binding assays to erythrocytes. Five peptides localised in the N-terminal region coded 2148 (121LQSHKKLIKALKKNIESYQN(140)), 2149 (141KKHLIYKNKSYNPLLLSCVK(160)), 2150 (161KMNMLKENVDYIQKNQNLFK(180)), 2152 (201YKSQGHKKETSQNQNENNDN(220)) and 2153 (221QKYQEVNDEDDVNDEEDTND(240)) specifically bind to erythrocytes. These peptides bind independently of the peptide and erythrocyte charge, with high affinity (Kd between 70 and 180 nM) and the hydrophobic interaction is important for this binding ( approximately 30% hydrophobic critical residues). These results allow us define a specific erythrocyte binding region (residues 121-240), which may bound to at least three different binding sites on erythrocytes. Peptide 2153 shares the underlined sequence 221QKYQEVNDEDDVNDEEDTND(240) with an earlier 18-mer peptide recognised by human exposed sera. Peptides number 2148 and 2149 in vitro inhibit erythrocyte invasion by merozoites. We found that 2149 peptide and some of its glycine analogues show specific haemolytic and/or antimicrobial activity. We discuss a possible role of ABRA or its regions in the merozoite invasion of erythrocyte.

  15. Ligand binding reduces SUMOylation of the peroxisome proliferator-activated receptor γ (PPARγ activation function 1 (AF1 domain.

    Directory of Open Access Journals (Sweden)

    Rolf Diezko

    Full Text Available Peroxisome proliferator-activated receptor gamma (PPARγ is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1 domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.

  16. The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus

    Directory of Open Access Journals (Sweden)

    Enjuanes Luis

    2011-09-01

    Full Text Available Abstract Background Transmissible gastroenteritis virus (TGEV has a sialic acid binding activity that is believed to be important for enteropathogenicity, but that has so far appeared to be dispensable for infection of cultured cells. The aims of this study were to determine the effect of sialic acid binding for the infection of cultured cells under unfavorable conditions, and comparison of TGEV strains and mutants, as well as the avian coronavirus IBV concerning their dependence on the sialic acid binding activity. Methods The infectivity of different viruses was analyzed by a plaque assay after adsorption times of 5, 20, and 60 min. Prior to infection, cultured cells were either treated with neuraminidase to deplete sialic acids from the cell surface, or mock-treated. In a second approach, pre-treatment of the virus with porcine intestinal mucin was performed, followed by the plaque assay after a 5 min adsorption time. A student's t-test was used to verify the significance of the results. Results Desialylation of cells only had a minor effect on the infection by TGEV strain Purdue 46 when an adsorption period of 60 min was allowed for initiation of infection. However, when the adsorption time was reduced to 5 min the infectivity on desialylated cells decreased by more than 60%. A TGEV PUR46 mutant (HAD3 deficient in sialic acid binding showed a 77% lower titer than the parental virus after a 5 min adsorption time. After an adsorption time of 60 min the titer of HAD3 was 58% lower than that of TGEV PUR46. Another TGEV strain, TGEV Miller, and IBV Beaudette showed a reduction in infectivity after neuraminidase treatment of the cultured cells irrespective of the virion adsorption time. Conclusions Our results suggest that the sialic acid binding activity facilitates the infection by TGEV under unfavorable environmental conditions. The dependence on the sialic acid binding activity for an efficient infection differs in the analyzed TGEV strains.

  17. A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

    Science.gov (United States)

    Hoggard, Timothy; Shor, Erika; Müller, Carolin A.; Nieduszynski, Conrad A.; Fox, Catherine A.

    2013-01-01

    Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time. PMID:24068963

  18. Angiomotin binding-induced activation of Merlin/NF2 in the Hippo pathway.

    Science.gov (United States)

    Li, Youjun; Zhou, Hao; Li, Fengzhi; Chan, Siew Wee; Lin, Zhijie; Wei, Zhiyi; Yang, Zhou; Guo, Fusheng; Lim, Chun Jye; Xing, Wancai; Shen, Yuequan; Hong, Wanjin; Long, Jiafu; Zhang, Mingjie

    2015-07-01

    The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4(DCAF1). Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin's auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin's binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin's auto-inhibitory tail does not obviously alter Merlin's conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.

  19. Gestalt-binding of tropomyosin on actin during thin filament activation.

    Science.gov (United States)

    Lehman, William; Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Raunser, Stefan

    2013-08-01

    Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin-tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.

  20. Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity.

    Science.gov (United States)

    Lee, Hojoon X; Mendes, Fabio A; Plouhinec, Jean-Louis; De Robertis, Edward M

    2009-11-01

    In Xenopus embryos, a dorsal-ventral patterning gradient is generated by diffusing Chordin/bone morphogenetic protein (BMP) complexes cleaved by BMP1/Tolloid metalloproteinases in the ventral side. We developed a new BMP1/Tolloid assay using a fluorogenic Chordin peptide substrate and identified an unexpected negative feedback loop for BMP4, in which BMP4 inhibits Tolloid enzyme activity noncompetitively. BMP4 binds directly to the CUB (Complement 1r/s, Uegf [a sea urchin embryonic protein] and BMP1) domains of BMP1 and Drosophila Tolloid with high affinity. Binding to CUB domains inhibits BMP4 signaling. These findings provide a molecular explanation for a long-standing genetical puzzle in which antimorphic Drosophila tolloid mutant alleles displayed anti-BMP effects. The extensive Drosophila genetics available supports the relevance of the interaction described here at endogenous physiological levels. Many extracellular proteins contain CUB domains; the binding of CUB domains to BMP4 suggests a possible general function in binding transforming growth factor-beta (TGF-beta) superfamily members. Mathematical modeling indicates that feedback inhibition by BMP ligands acts on the ventral side, while on the dorsal side the main regulator of BMP1/Tolloid enzymatic activity is the binding to its substrate, Chordin.

  1. D-fructose-binding proteins in bull seminal plasma: Isolation and characterization

    Czech Academy of Sciences Publication Activity Database

    Liberda, J.; Kraus, Marek; Ryšlavá, H.; Vlasáková, M.; Jonáková, Věra; Tichá, M.

    2001-01-01

    Roč. 47, č. 4 (2001), s. 113-119 ISSN 0015-5500 R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915 Keywords : bull seminal plasma * non-heparin-binding and heparin-binding proteins * D-fructose-binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

  2. DNA binding and cleavage activity by a mononuclear iron (II) Schiff ...

    Indian Academy of Sciences (India)

    DNA binding and cleavage activity by a mononuclear iron(II)Schiff base complex: Synthesis and structural characterization. Abhijit Pal Bhaskar Biswas Merry Mitra Subramaniyam Rajalakshmi Chandra Shekhar Purohit Soumitra Hazra Gopinatha Suresh Kumar Balachandran Unni Nair Rajarshi Ghosh. Volume 125 Issue 5 ...

  3. Autolytic Activity and Plasma Binding Study of Aap, a Novel Minor Autolysin of Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Ramina Mahboobi

    2016-04-01

    Full Text Available Pneumococcal autolysins are enzymes involved in cell wall turnover and cellular division physiologically. They have been found to be involved in the pneumococcus pathogenesis. The aim of this study was to identify the autolytic activity of Spr1754 as a novel protein of Streptococcus pneumoniae. Moreover, the binding of the recombinant protein to plasma proteins was also determined. The spr1754 gene was amplified by PCR and cloned into the pET21a(+ prokaryotic expression vector. The constructed pET21a(+/spr1754 recombinant plasmid was transformed into E. coli Origami (DE3 and induced using IPTG. The recombinant protein of Spr1754 was purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis using anti-His tag monoclonal antibody. Autolytic activity and the ability of the recombinant protein in binding to plasma proteins were performed using zymogram analysis and western blot, respectively. The spr1754 with expected size was cloned and overexpressed in Escherichia coli Origami (DE3, successfully. After purification of the Spr1754 recombinant protein, the autolytic activity was observed by zymography. Of the four plasma proteins used in this study, binding of lactoferrin to Spr1754 recombinant protein was shown. The Spr1754 recombinant protein has a bifunctional activity, i.e., as being autolysin and lactoferrin binding and designated as Aap (autolytic/ adhesion/ pneumococcus. Nevertheless, characterization of the Aap needs to be followed using gene inactivation and cell wall localization.

  4. DNA binding and cleavage activity of a structurally characterized Ni(II)

    Indian Academy of Sciences (India)

    1375–1381. c Indian Academy of Sciences. DOI 10.1007/s12039-015-0900-4. DNA binding and cleavage activity of a structurally characterized Ni(II). Schiff base complex. SARAT CHANDRA KUMARa, ABHIJIT PALa, MERRY MITRAa,. V M MANIKANDAMATHAVANb, CHIA -HER LINc, BALACHANDRAN UNNI NAIRb,∗.

  5. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity ...

    Indian Academy of Sciences (India)

    ... lanthanum complex and 10-coordinated in the cerium complex. The coordination polyhedra around the lanthanum and cerium were found to have distorted icosahedron and distorted bicapped square antiprism respectively. DNA binding and nuclease activity of these complexes were also investigated in the present work.

  6. Bioinspired Heparin Nanosponge Prepared by Photo-crosslinking for Controlled Release of Growth Factors

    DEFF Research Database (Denmark)

    Choi, Won Il; Sahu, Abhishek; Vilos, Cristian

    2017-01-01

    Growth factors have great therapeutic potential for various disease therapy and tissue engineering applications. However, their clinical efficacy is hampered by low bioavailability, rapid degradation in vivo and non-specific biodistribution. Nanoparticle based delivery systems are being evaluated...... factor binding ability. Four different growth factors, bFGF, VEGF, BMP-2, and HGF were successfully encapsulated into Hep-NS. In vitro studies showed sustained release of all the growth factors for almost 60 days and the rate of release was directly dependent on the amount of heparin in Hep......-NS. The released growth factors retained their bioactivity as assessed by a cell proliferation assay. This heparin nanosponge is therefore a promising nanocarrier for the loading and controlled release of growth factors....

  7. Multifunctional silk-heparin biomaterials for vascular tissue engineering applications.

    Science.gov (United States)

    Seib, F Philipp; Herklotz, Manuela; Burke, Kelly A; Maitz, Manfred F; Werner, Carsten; Kaplan, David L

    2014-01-01

    Over the past 30 years, silk has been proposed for numerous biomedical applications that go beyond its traditional use as a suture material. Silk sutures are well tolerated in humans, but the use of silk for vascular engineering applications still requires extensive biocompatibility testing. Some studies have indicated a need to modify silk to yield a hemocompatible surface. This study examined the potential of low molecular weight heparin as a material for refining silk properties by acting as a carrier for vascular endothelial growth factor (VEGF) and improving silk hemocompatibility. Heparinized silk showed a controlled VEGF release over 6 days; the released VEGF was bioactive and supported the growth of human endothelial cells. Silk samples were then assessed using a humanized hemocompatibility system that employs whole blood and endothelial cells. The overall thrombogenic response for silk was very low and similar to the clinical reference material polytetrafluoroethylene. Despite an initial inflammatory response to silk, apparent as complement and leukocyte activation, the endothelium was maintained in a resting, anticoagulant state. The low thrombogenic response and the ability to control VEGF release support the further development of silk for vascular applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Effects of nadroparin, enoxaparin, and unfractionated heparin on endogenous factor Xa and IIa formation and on thrombelastometry profiles.

    Science.gov (United States)

    Cvirn, Gerhard; Wagner, Thomas; Juergens, Guenther; Koestenberger, Martin

    2009-01-01

    Measurements of anti-FXa or anti-FIIa (thrombin) activities are conventional tests for biological monitoring of low molecular weight heparin (LMWH) or unfractionated heparin treatment. It was the aim of our study to assess the anticoagulant efficacy of the LMWHs nadroparin and enoxaparin and that of unfractionated heparin not by the above-mentioned isolated measurements but in the physiological environment of clotting plasma or whole blood. The effects of increasing amounts of nadroparin, enoxaparin, or unfractionated heparin on the time-course of FXa or FIIa formation were investigated in tissue factor activated platelet-poor plasma using a subsampling technique and chromogenic substrates. Moreover, the anticoagulant efficacy of these drugs was also investigated in whole blood triggered by the physiological relevant activator collagen/endogenous thrombin using thrombelastometry. Nadroparin is as efficient as enoxaparin concerning suppression of endogenous FXa or FIIa formation. The two LMWHs are capable of suppressing the formation of FIIa as efficient as that of FXa. Compared with equivalent anti-FXa activity, unfractionated heparin is markedly more efficient in suppressing the formation of FXa and FIIa than the LMWHs. Corresponding results were obtained in whole blood. The anticoagulant efficacy of nadroparin was comparable with that of enoxaparin and the influence of unfractionated heparin on thrombelastometry parameters was markedly stronger than that of the two LMWHs. We conclude that LMWHs are efficient inhibitors not only of endogenous FXa formation but also of endogenous FIIa formation. Under our experimental conditions, the anticoagulant efficacy of nadroparin was comparable with that of enoxaparin but markedly lower than that of unfractionated heparin.

  9. Heparin-conjugated polyethylenimine for gene delivery.

    Science.gov (United States)

    Jeon, Oju; Yang, Hee Seok; Lee, Tae-Jin; Kim, Byung-Soo

    2008-12-18

    A major problem when using cationic polymers for gene delivery is that transfection is strongly inhibited by the presence of serum. This shortcoming limits the application of cationic polymers for systematic gene delivery in vivo. Due to the shielding effect of heparin, heparin conjugation to cationic polymers may improve the in vivo gene transfection efficiency. In this study, the transfection efficiency of heparin-conjugated polyethylenimine (HCPEI) with a low molecular weight of 1800 Da was compared to the transfection efficiencies of polyethylenimine with a low molecular weight of 1800 Da (PEI1800), polyethylenimine with a high molecular weight of 25,000 Da (PEI25k), and Lipofectamine. The size of the HCPEI/plasmid DNA (pDNA) complex is approximately 250 nm. HCPEI has a proton-buffering effect and HCPEI/pDNA has higher blood compatibility and a lower cytotoxicity than PEI25k/pDNA and Lipofectamine/pDNA. For in vitro transfection of rabbit smooth muscle cells in serum-free medium, the transfection efficiency of HCPEI/pDNA was not significantly different from those of PEI25k/pDNA and Lipofectamine/pDNA. Importantly, in serum-containing medium, the transfection efficiency of HCPEI/pDNA was significantly higher than those of PEI25k/pDNA and Lipofectamine/pDNA. For vascular endothelial growth factor (VEGF) gene transfection to mouse ischemic limbs, HCPEI/pDNA exhibited significantly higher VEGF expression and more extensive neovascularization than PEI/pDNA and Lipofectamine/pDNA. Taken together, heparin conjugation to PEI improves the in vivo gene transfection efficiency of PEI.

  10. Activation of the ATR kinase by the RPA-binding protein ETAA1

    DEFF Research Database (Denmark)

    Haahr, Peter; Hoffmann, Saskia; Tollenaere, Maxim A X

    2016-01-01

    Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway...... in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically......, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate...

  11. Insulin induces a transcriptional activation of epiregulin, HB-EGF and amphiregulin, by a PI3K-dependent mechanism: identification of a specific insulin-responsive promoter element

    DEFF Research Database (Denmark)

    Ornskov, Dorthe; Nexo, Ebba; Sørensen, Boe Sandahl

    2007-01-01

    Previously we have shown that insulin-stimulation of RT4 bladder cancer cells leads to increased proliferation, which require HER1 activation, and is accompanied by increased mRNA expression of the EGF-ligands heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), and epiregulin (EPI...

  12. Electrophoresis for the analysis of heparin purity and quality

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J.

    2012-01-01

    The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007–2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. PMID:22736353

  13. Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli

    NARCIS (Netherlands)

    Snijder, H.J.; Kingma, R.L.; Kalk, K.H.; Dekker, N.; Egmond, M.R.|info:eu-repo/dai/nl/112946593; Dijkstra, B.W.

    2010-01-01

    Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of phospholipids. Enzymatic activity is regulated by reversible dimerisation and calcium-binding. We have investigated the role of calcium by X-ray crystallography. In monomeric OMPLA, one calcium ion

  14. POT1-independent single-strand telomeric DNA binding activities in Brassicaceae.

    Science.gov (United States)

    Shakirov, Eugene V; McKnight, Thomas D; Shippen, Dorothy E

    2009-06-01

    Telomeres define the ends of linear eukaryotic chromosomes and are required for genome maintenance and continued cell proliferation. The extreme ends of telomeres terminate in a single-strand protrusion, termed the G-overhang, which, in vertebrates and fission yeast, is bound by evolutionarily conserved members of the POT1 (protection of telomeres) protein family. Unlike most other model organisms, the flowering plant Arabidopsis thaliana encodes two divergent POT1-like proteins. Here we show that the single-strand telomeric DNA binding activity present in A. thaliana nuclear extracts is not dependent on POT1a or POT1b proteins. Furthermore, in contrast to POT1 proteins from yeast and vertebrates, recombinant POT1a and POT1b proteins from A. thaliana, and from two additional Brassicaceae species, Arabidopsis lyrata and Brassica oleracea (cauliflower), fail to bind single-strand telomeric DNA in vitro under the conditions tested. Finally, although we detected four single-strand telomeric DNA binding activities in nuclear extracts from B. oleracea, partial purification and DNA cross-linking analysis of these complexes identified proteins that are smaller than the predicted sizes of BoPOT1a or BoPOT1b. Taken together, these data suggest that POT1 proteins are not the major single-strand telomeric DNA binding activities in A. thaliana and its close relatives, underscoring the remarkable functional divergence of POT1 proteins from plants and other eukaryotes.

  15. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

    2015-01-01

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

  16. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Heparin monitoring: From blood tube to microfluidic device

    OpenAIRE

    Harris, L. F.; Killard, A.

    2012-01-01

    Heparin anticoagulant therapy has been pivotal in both the treatment and prophylaxis of thrombotic disease for many decades. It remains standard practice to monitor unfractionated heparin (UFH) therapy due to its unpredictable pharmacokinetics. The advent of low molecular weight heparins (LMWHs) reduced the need for continuous laboratory monitoring due to the improved dose-response relationships and pharmacokinetics of these drugs. However, special patient cohorts exist where monitoring becom...

  18. In Vitro Analyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

    Science.gov (United States)

    Miceli, Marisa H.; Bernardo, Stella M.; Ku, T. S. Neil; Walraven, Carla

    2012-01-01

    Infections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin on Candida albicans biofilms and planktonic cells have not been previously studied. Therefore, we sought to determine the in vitro effect of a heparin sodium preparation (HP) on biofilms and planktonic cells of C. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformed C. albicans biofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P < 0.0001). Pure-H, MP, and PP each inhibited C. albicans biofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H have in vitro antifungal activity against C. albicans mature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention of C. albicans biofilms is warranted. PMID:21986822

  19. Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

    Directory of Open Access Journals (Sweden)

    Previato Jose O

    2008-05-01

    Full Text Available Abstract Background The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs. In the present study, nuclear magnetic resonance (NMR was used to map the binding site(s of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. Results The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr. Conclusion Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.

  20. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    Energy Technology Data Exchange (ETDEWEB)

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  1. Factor XI anion-binding sites are required for productive interactions with polyphosphate.

    Science.gov (United States)

    Geng, Y; Verhamme, I M; Smith, S A; Cheng, Q; Sun, M; Sheehan, J P; Morrissey, J H; Gailani, D

    2013-11-01

    Conversion of factor XI (FXI) to FXIa is enhanced by polymers of inorganic phosphate (polyP). This process requires FXI to bind to polyP. Each FXIa subunit contains anion-binding sites (ABSs) on the apple 3 (A3) and catalytic domains that are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin. To determine the importance of FXI ABSs to polyP enhancement of FXI activation. Recombinant FXI variants lacking one or both ABSs were tested in polyP-dependent purified protein systems, plasma clotting assays, and a murine thrombosis model. In the presence of polyP, activation rates for FXI lacking either ABS were reduced compared with wild-type FXI, and FXI lacking both sites had an even greater defect. In contrast to heparin, polyP binding to FXIa did not enhance inhibition by antithrombin and did not interfere with FXIa activation of FIX. FXI lacking one or both ABSs does not reconstitute FXI-deficient plasma as well as wild-type FXI when polyP was used to initiate coagulation. In FXI-deficient mice, FXI lacking one or more ABSs was inferior to wild-type FXI in supporting arterial thrombus formation. The ABSs on FXIa that are required for expression of heparin's cofactor activity during protease inhibition by antithrombin are also required for expression of polyP cofactor activity during FXI activation. These sites may contribute to FXI-dependent thrombotic processes. © 2013 International Society on Thrombosis and Haemostasis.

  2. Does ′heparin-induced thrombocytopenia′ hit our minds?

    Directory of Open Access Journals (Sweden)

    Arun R Thangavel

    2016-01-01

    Full Text Available Unfractionated heparin is a widely used drug to prevent deep vein thrombosis and pulmonary emboli in patients at risk. With the advent of newer anticoagulants having lesser side effects, its use has diminished but not out of service. Here, we report a case of deep venous thrombosis, in a patient on prophylactic dose of heparin, which was later found to be a manifestation of heparin-induced thrombocytopenia (HIT. Thrombosis in the presence of heparin prophylaxis should be considered as HIT rather than a failure of anticoagulation.

  3. Heparin in the treatment of burns: a review.

    Science.gov (United States)

    Saliba, M J

    2001-06-01

    Burns are difficult to treat, wounds with complex local and systemic pathology and high mortality, that often heal slowly with scars and contractures. Glycosaminoglycans (GAGs) have been used in parenteral and topical application studies. These studies have uncovered anticoagulative, antiinflammatory and neoangiogenic properties, which may stimulate tissue repair and reepithelializing effects. The endogenous GAGs utilized in treating burns are heparin, dermatan sulfate, heparan sulfate, keratin sulfate, chondroitin-4- and chondroitin-6-sulfate, and hyaluronic acid. Heparin, the most sulfated and acidic GAG, has been used parenterally, topically, by inhalation, in pellet, and in bioengineered membranes. Heparin relieved pain, inhibited clotting and inflammation, restored blood flow, and enhanced healing. Heparin effects that improved and reduced burn care were time, dose, pH, site, source and duration related in studies. Potential adverse effects with heparin use are bleeding, thrombocytopenia and allergy. Heparin preserved lung and improved function. Heparin preserved intestinal integrity and reduced bacterial translocation. Collagen restoration was enhanced. The healed skin was smooth. Heparin reduced needs for pain medicine, topical antibiotics, resuscitation fluids, blood, water baths, debridement, surgery and grafts. Cost of treatments were reduced. Although not as yet fully substantiated, topical heparin therapy of burns may be a useful addition to the range of available treatments for burn wounds.

  4. Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

    DEFF Research Database (Denmark)

    Henriksen, Jens; Roepstorff, Peter; Ringborg, Lene H.

    2006-01-01

    Heparin and heparin-derived components are widely applied anticoagulant drugs used for amongst other applications medical treatment of deep vein thrombosis and pulmonary embolism. Depolymerisation of native heparin results in complex mixtures of sulfated linear oligosaccharides that are usually...

  5. Direct binding and activation of protein kinase C isoforms by steroid hormones.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2008-10-01

    The non-genomic action of steroid hormones regulates a wide variety of cellular responses including regulation of ion transport, cell proliferation, migration, death and differentiation. In order to achieve such plethora of effects steroid hormones utilize nearly all known signal transduction pathways. One of the key signalling molecules regulating the non-genomic action of steroid hormones is protein kinase C (PKC). It is thought that rapid action of steroids hormones results from the activation of plasma membrane receptors; however, their molecular identity remains elusive. In recent years, an increasing number of studies have pointed at the selective binding and activation of specific PKC isoforms by steroid hormones. This has led to the hypothesis that PKC could act as a receptor as well as a transducer of the non-genomic effects of these hormones. In this review we summarize the current knowledge of the direct binding and activation of PKC by steroid hormones.

  6. Designing a new chitinase with more chitin binding and antifungal activity.

    Science.gov (United States)

    Matroodi, Soheila; Motallebi, Mostafa; Zamani, Mohammadreza; Moradyar, Mehdi

    2013-08-01

    Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin binding domain (ChBD). We have produced a chimeric chitinase with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Serratia marcescens Chitinase B. The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the enzyme activity of the chimeric chitinase when compared with the native Chit42. The chimeric chitinase showed higher antifungal activity toward phytopathogenic fungi.

  7. Anandamide capacitates bull spermatozoa through CB1 and TRPV1 activation.

    Directory of Open Access Journals (Sweden)

    María Gracia Gervasi

    2011-02-01

    Full Text Available Anandamide (AEA, a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1 and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate

  8. Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).

    Science.gov (United States)

    Wang, Tao; Chen, Xiangjun; Han, Jun; Ma, Sichun; Wang, Jianmei; Li, Xufeng; Zhang, Hui; Liu, Zhibin; Yang, Yi

    2016-07-01

    SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Exploring the DNA binding mode of transition metal based biologically active compounds

    Science.gov (United States)

    Raman, N.; Sobha, S.

    2012-01-01

    Few novel 4-aminoantipyrine derived Schiff bases and their metal complexes were synthesized and characterized. Their structural features and other properties were deduced from the elemental analysis, magnetic susceptibility and molar conductivity as well as from mass, IR, UV-vis, 1H NMR and EPR spectral studies. The binding of the complexes with CT-DNA was analyzed by electronic absorption spectroscopy, viscosity measurement, and cyclic voltammetry. The interaction of the metal complexes with DNA was also studied by molecular modeling with special reference to docking. The experimental and docking results revealed that the complexes have the ability of interaction with DNA of minor groove binding mode. The intrinsic binding constants ( Kb) of the complexes with CT-DNA were found out which show that they are minor groove binders. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the pUC19 DNA in the presence of AH 2 (ascorbic acid). Moreover, the oxidative cleavage studies using distamycin revealed the minor groove binding for the newly synthesized 4-aminoantipyrine derived Schiff bases and their metal complexes. Evaluation of antibacterial activity of the complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Klebsiella pneumoniae exhibited that the complexes have potent biocidal activity than the free ligands.

  10. High-dose heparin fails to improve acute lung injury following smoke inhalation in sheep.

    Science.gov (United States)

    Murakami, Kazunori; Enkhbaatar, Perenlei; Shimoda, Katsumi; Mizutani, Akio; Cox, Robert A; Schmalstieg, Frank C; Jodoin, Jeffrey M; Hawkins, Hal K; Traber, Lillian D; Traber, Daniel L

    2003-04-01

    Thrombin is involved in various inflammatory responses. In sepsis, coagulation abnormalities are major complications. Acute lung injury is one of the most life-threatening problems that can result from sepsis. We hypothesized that high-dose heparin might be effective in attenuating acute lung injury in our sepsis model. Female sheep ( n =16) were surgically prepared for the study. After a tracheotomy, 48 breaths of cotton smoke (heparin infusion group ( n =6), a Ringer's lactate infusion group ( n =6), and a sham-injury group ( n =4; surgically prepared in the same fashion but receiving no inhalation injury or bacteria). The treatment was started 1 h after the insult, and was continued thereafter for 24 h. The dose of heparin was adjusted by monitoring to target an activated clotting time of between 300 and 400 s (baseline=approx. 150 s). Sheep exposed to lung injury presented with typical hyperdynamic cardiovascular changes, including an increased cardiac output and a fall in systemic vascular resistance. There was a decrease in the arterial partial pressure of O(2). In conclusion, high-dose heparin did not prevent lung dysfunction in this model, in which acute lung injury was induced by combined smoke and septic challenge.

  11. Functional importance of the DNA binding activity of Candida albicans Czf1p.

    Directory of Open Access Journals (Sweden)

    Ivana Petrovska

    Full Text Available The human opportunistic pathogen Candida albicans undergoes a reversible morphological transition between the yeast and hyphal states in response to a variety of signals. One such environmental trigger is growth within a semisolid matrix such as agar medium. This growth condition is of interest because it may mimic the growth of C. albicans in contact with host tissue during infection. During growth within a semisolid matrix, hyphal growth is positively regulated by the transcriptional regulator Czf1p and negatively by a second key transcriptional regulator, Efg1p. Genetic studies indicate that Czf1p, a member of the zinc-cluster family of transcriptional regulators, exerts its function by opposing the inhibitory influence of Efg1p on matrix-induced filamentous growth. We examined the importance of the two known activities of Czf1p, DNA-binding and interaction with Efg1p. We found that the two activities were separable by mutation allowing us to demonstrate that the DNA-binding activity of Czf1p was essential for its role as a positive regulator of morphogenesis. Surprisingly, however, interactions with Efg1p appeared to be largely dispensable. Our studies provide the first evidence of a key role for the DNA-binding activity of Czf1p in the morphological yeast-to-hyphal transition triggered by matrix-embedded growth.

  12. Quantitative structure-activity relationships for estrogen receptor binding affinity of phenolic chemicals.

    Science.gov (United States)

    Hu, Jian-Ying; Aizawa, Takako

    2003-03-01

    The estrogen receptor (ER) binding affinities of 25 compounds including 15 industrial phenolic chemicals, two phytoestrogens, three natural steroids and one man-made steroid were detected by a binding competition assay. The 17 industrial phenolic chemicals were selected as objective compounds because they are possibly released from epoxy and polyester-styrene resins used in lacquer coatings of concrete tank and lining of steel pipe in water supply system. A quantitative structure-activity relationship (QSAR) for structurally diverse phenols, nine alkylphenols with only one alkyl group, four hydroxyl biphenyls, bisphenol A and four natural and man-made estrogens was established by applying a quantum chemical modeling method. Logarithm of octanol-water coefficient (logPow), molecular volume (V(m)), and energies of the highest occupied molecular orbital ( epsilon (HOMO)) and lowest unoccupied molecular orbital ( epsilon (LUMO)) were selected as hydrophobic, steric (V(m)), and electronic chemical descriptors, respectively. Chemicals capable of ER binding had large V(m) and high epsilon (HOMO), while the effects of logPow and epsilon (LUMO) on the binding affinity could not be identified. The QSAR made successful predictions for the three phytoestrogens. Also, the successful prediction of ER-binding affinity for biochanin A, another phytoestrogen, two indicators of pH (phenolphthalin and phenolphthalein) and one alkylphenolic chemical with three alkyl groups (4-methyl-2,6-di-butyl-phenol), by amending the V(m) in the above-mentioned QSAR according to the electron-density distribution (or HOMO density) is an additional step in the elucidation of chemical steric and electronic parameters for predicting the binding affinities of phenolic compounds.

  13. BRCA1 DNA-binding activity is stimulated by BARD1.

    Science.gov (United States)

    Simons, Amanda M; Horwitz, Andrew A; Starita, Lea M; Griffin, Karen; Williams, R Scott; Glover, J N Mark; Parvin, Jeffrey D

    2006-02-15

    The breast- and ovarian-specific tumor suppressor BRCA1 has been implicated in numerous cellular processes, including transcription, ubiquitination, and DNA repair. Its tumor suppression activity is tightly linked to that of BARD1, a protein that heterodimerizes with BRCA1. It has been previously shown that BRCA1 binds to DNA, an interesting functional observation in light of the genetic data linking BRCA1 to DNA repair pathways. In this work, we reexamine the DNA-binding properties of BRCA1, comparing them with the DNA-binding properties of the BRCA1/BARD1 heterodimer. Because nuclear BRCA1 exists as a heterodimer with BARD1, it is likely that in vitro studies of the heterodimer will provide a more accurate model of physiologic conditions. Our results indicate that whereas BARD1 cannot directly bind DNA, it does enhance DNA binding by BRCA1. This is a surprising observation as both DNA-binding domains are distal to the BARD1-interacting RING domain of BRCA1. Further analysis of the dimerization reveals that the BRCA1/BARD1 interaction is not limited to the amino-terminal RING domains of each protein. The carboxyl terminus of BRCA1 contributes significantly to the stability of the heterodimer. We also show that the presence of BARD1 has a secondary effect, as autoubiquitination of BRCA1/BARD1 heterodimers additionally enhances the affinity of BRCA1 for DNA. Together, these data suggest that BRCA1 and BARD1 heterodimerization is stabilized via domains not previously thought to interact and that BARD1 acts in both ubiquitination-dependent and ubiquitination-independent ways to influence the role of BRCA1 in DNA repair.

  14. Activated charcoal forms non-IgE binding complexes with peanut proteins.

    Science.gov (United States)

    Vadas, Peter; Perelman, Boris

    2003-07-01

    Conventional management of peanut-induced anaphylaxis is composed of administration of epinephrine, antihistamine, and steroid and stabilization of airway, ventilatory, and circulatory function. Therapies directed toward slowing or preventing further absorption of peanut protein from the gastrointestinal tract after accidental ingestion have not been a routine part of management. The purpose of this study was to determine the ability of activated charcoal to complex with peanut protein, thereby preventing its binding to either peanut-specific IgE or peanut-specific IgG. Peanut protein was coincubated with micronized activated charcoal suspension at pH 3.5 or 7.4. Peanut protein complexed with charcoal was removed by centrifugation. Binding of residual peanut protein to peanut-specific IgG was measured by a sandwich ELISA assay. Also, ability of uncomplexed peanut protein to bind to peanut-specific IgE was determined by Western blot and by skin prick testing in subjects with peanut allergy. Activated charcoal (AC) formed complexes with peanut protein, effectively competing for binding with peanut-specific IgG in a sandwich ELISA assay. AC complexed efficiently with peanut protein at both neutral and acidic pH in as little as 60 seconds. AC was also able to remove IgE-binding peanut allergens from solution as determined by Western blot and by skin prick testing in subjects with peanut allergy. A ratio of 200 mg of AC to 1 mg peanut protein was required for complete removal of peanut protein from solution. AC was able to complex with peanut protein within food matrices such as ice cream and chocolate. The data presented herein show that AC removes both IgE-binding and IgG-binding peanut proteins from solution rapidly at both neutral and acidic pH. These data suggest that administration of AC may be useful as an adjunct to slow or to prevent further absorption of peanut protein from the gastrointestinal tract after accidental ingestion by individuals with peanut

  15. Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase

    OpenAIRE

    Lin, Xiukun; Liu, Jun; Maley, Frank; Chu, Edward

    2003-01-01

    The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA bin...

  16. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ming V. [Program of Cardiovascular Sciences, Houston, TX 77030 (United States); Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Chen, Weiqin [Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Harmancey, Romain N. [Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip [Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Taegtmeyer, Heinrich [Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Chan, Lawrence, E-mail: lchan@bcm.tmc.edu [Program of Cardiovascular Sciences, Houston, TX 77030 (United States); Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); St. Luke' s Episcopal Hospital, Houston, TX 77030 (United States)

    2010-05-07

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

  17. Inhibition activity of wild berry juice fractions against Streptococcus pneumoniae binding to human bronchial cells.

    Science.gov (United States)

    Huttunen, Sanna; Toivanen, Marko; Arkko, Satu; Ruponen, Marika; Tikkanen-Kaukanen, Carina

    2011-01-01

    Bacterial adhesion to the cell surface is a crucial step before infection can take place. Inhibition of bacterial binding offers a novel preventive approach against infections. Cranberry (Vaccinium macrocarpon Ait.) juice has been found to have antiadhesive activity against different bacteria. Streptococcus pneumoniae is an important pathogen and the most common cause for pneumonia, meningitis, and otitis media. In this study the inhibitory activity of cranberry (Vaccinium oxycoccos L.), bilberry (Vaccinium myrtillus L.) and crowberry (Empetrum nigrum and Empetrum hermaphroditum L.) juice fractions against pneumococcal binding was tested using human bronchial cells (Calu-3) as an adhesion model. In addition, the antimicrobial activity of the berry juice fractions was tested. It was found that the studied berry juice fractions had antiadhesion activity and cranberry juice was the most active. The adhesion inhibition activity of cranberry juice was nearly 90% at a concentration of 8.7 mg/g of soluble solids. The antimicrobial activity of the studied berry juice fractions was found to be remarkable; pneumococcal growth was inhibited totally at a concentration of ∼86 mg/g. Both antiadhesion and antimicrobial activities were reduced after solid-phase extraction of the berry juices, which may suggest molecular synergistic effects of the berry juice molecules against S. pneumoniae. The findings indicate that cranberry, bilberry and crowberry juices have potential against pneumococcal infections. Copyright © 2010 John Wiley & Sons, Ltd.

  18. The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

    Science.gov (United States)

    Lim, Khai Lone; Amir, Amirah; Lau, Yee Ling; Fong, Mun Yik

    2017-08-11

    The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated. The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes. Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

  19. Radioreceptor assay to study the affinity of benzodiazepines and their receptor binding activity in human plasma including their active metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Dorow, R.G.; Seidler, J.; Schneider, H.H. (Schering A.G., Berlin (Germany, F.R.))

    1982-04-01

    A radioreceptor assay has been established to measure the receptor affinities of numerous benzodiazepines in clinical use. The time course of receptor binding activity was studied by this method in the plasma of eight healthy subjects randomly treated with 1mg lormetazepam (Noctamid(R)), 2mg flunitrazepam (Rohypnol(R)), and 10mg diazepam (Valium(R)), and placebo on a cross-over basis. Blood samples were collected up to 154h after treatment. Receptor affinities of numerous benzodiazepines in vitro show good correlation with therapeutic human doses (r=0.96) and may be predictive of drug potency in man. Mean peak plasma levels of lormetazepam binding equivalents were 4.8+-1 ng/ml at 2h after lormetazepam, 7.2+-1.8 ng/ml at 8h after flunitrazepam, and 17.9+-2.7 ng/ml at 15h after diazepam. Plasma elimination halflives of benzodiazepine binding equivalents were 9.3, 23 and 63h, respectively. Slow elimination of benzodiazepine binding equivalents following flunitrazepam and diazepam may be due to persistent active metabolites.

  20. Spectroscopic analysis on the binding interaction of biologically active pyrimidine derivative with bovine serum albumin

    Directory of Open Access Journals (Sweden)

    Vishwas D. Suryawanshi

    2016-02-01

    Full Text Available A biologically active antibacterial reagent, 2–amino-6-hydroxy–4–(4-N, N-dimethylaminophenyl-pyrimidine-5-carbonitrile (AHDMAPPC, was synthesized. It was employed to investigate the binding interaction with the bovine serum albumin (BSA in detail using different spectroscopic methods. It exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus which are common food poisoning bacteria. The experimental results showed that the fluorescence quenching of model carrier protein BSA by AHDMAPPC was due to static quenching. The site binding constants and number of binding sites (n≈1 were determined at three different temperatures based on fluorescence quenching results. The thermodynamic parameters, enthalpy change (ΔH, free energy (ΔG and entropy change (ΔS for the reaction were calculated to be 15.15 kJ/mol, –36.11 kJ/mol and 51.26 J/mol K according to van't Hoff equation, respectively. The results indicated that the reaction was an endothermic and spontaneous process, and hydrophobic interactions played a major role in the binding between drug and BSA. The distance between donor and acceptor is 2.79 nm according to Förster's theory. The alterations of the BSA secondary structure in the presence of AHDMAPPC were confirmed by UV–visible, synchronous fluorescence, circular dichroism (CD and three-dimensional fluorescence spectra. All these results indicated that AHDMAPPC can bind to BSA and be effectively transported and eliminated in the body. It can be a useful guideline for further drug design.

  1. Non-coding single nucleotide variants affecting estrogen receptor binding and activity.

    Science.gov (United States)

    Bahreini, Amir; Levine, Kevin; Santana-Santos, Lucas; Benos, Panayiotis V; Wang, Peilu; Andersen, Courtney; Oesterreich, Steffi; Lee, Adrian V

    2016-12-13

    Estrogen receptor (ER) activity is critical for the development and progression of the majority of breast cancers. It is known that ER is differentially bound to DNA leading to transcriptomic and phenotypic changes in different breast cancer models. We investigated whether single nucleotide variants (SNVs) in ER binding sites (regSNVs) contribute to ER action through changes in the ER cistrome, thereby affecting disease progression. Here we developed a computational pipeline to identify SNVs in ER binding sites using chromatin immunoprecipitation sequencing (ChIP-seq) data from ER+ breast cancer models. ER ChIP-seq data were downloaded from the Gene Expression Omnibus (GEO). GATK pipeline was used to identify SNVs and the MACS algorithm was employed to call DNA-binding sites. Determination of the potential effect of a given SNV in a binding site was inferred using reimplementation of the is-rSNP algorithm. The Cancer Genome Atlas (TCGA) data were integrated to correlate the regSNVs and gene expression in breast tumors. ChIP and luciferase assays were used to assess the allele-specific binding. Analysis of ER ChIP-seq data from MCF7 cells identified an intronic SNV in the IGF1R gene, rs62022087, predicted to increase ER binding. Functional studies confirmed that ER binds preferentially to rs62022087 versus the wild-type allele. By integrating 43 ER ChIP-seq datasets, multi-omics, and clinical data, we identified 17 regSNVs associated with altered expression of adjacent genes in ER+ disease. Of these, the top candidate was in the promoter of the GSTM1 gene and was associated with higher expression of GSTM1 in breast tumors. Survival analysis of patients with ER+ tumors revealed that higher expression of GSTM1, responsible for detoxifying carcinogens, was correlated with better outcome. In conclusion, we have developed a computational approach that is capable of identifying putative regSNVs in ER ChIP-binding sites. These non-coding variants could potentially regulate

  2. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination

    DEFF Research Database (Denmark)

    Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan

    2014-01-01

    Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening...... conditions. Furthermore a marked improvement in the correlation was found by using kinase constructs containing the catalytic domain in presence of additional domains or subunits....

  3. Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Kožíšek, Milan

    2014-01-01

    to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn(2+) binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence......-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein....

  4. Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

    Science.gov (United States)

    Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

    2016-09-01

    In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

  5. Grades of 43 Fish Species in Japan Based on IgE-binding Activity

    Directory of Open Access Journals (Sweden)

    Harumi Koyama

    2006-01-01

    Conclusions: A correlation was observed between IgE levels and expression of symptoms after fish ingestion. High consumption of salmon, tuna, scad (including saurel, skipper, yellowtail, sardine, bonita and mackerel in Japan might be the cause of the high IgE-binding activity of these species. The grades of fish species consumed widely in Japan are likely to be useful for nutritional instruction of fish-allergic patients.

  6. Low molecular weight heparin (CY-216) versus unfractionated heparin in chronic hemodialysis.

    Science.gov (United States)

    Grau, E; Sigüenza, F; Maduell, F; Linares, M; Olaso, M A; Martinez, R; Caridad, A

    1992-01-01

    In 14 patients undergoing chronic hemodialysis, we investigated the safety and efficacy of the low molecular fragment (CY-216) in comparison to unfractionated heparin (UFH) in the prevention of clotting in the extracorporeal circuit (ECC). In this study, 168 hemodialysis sessions were undertaken with UFH in 2 bolus doses (5,437 +/- 1,477 SD IU) and 231 with CY-216 in a single bolus dose [initial dose 150 anti-Xa U Institut Choay (IC)/kg]. There were no clots in the bubble trap in any UFH sessions, and 14.8% had coagulated fibers in the dialyzer. Clotting in the bubble trap was observed in 2 CY-216 sessions (0.8%) and coagulated fibers in 22.6% of the sessions. At the end of the study, the mean dose of CY-216 was 250 anti-Xa UIC/kg but a dose of 350 anti-Xa UIC/kg was needed in the 2 patients treated by recombinant human erythropoietin. Anti-Xa levels at the end of the runs were higher (0.47 +/- 0.1 U/ml) in the CY-216 group than in the UFH group (0.28 +/- 0.1 U/ml). There was a correlation between anti-Xa levels and efficacy in the CY-216 group. An anti-Xa activity above 0.4 U/ml was needed in order to minimize thrombus formation. Antithrombin III-protease complexes (ATM) and D dimer fibrin derivatives (D dimer) were used as thrombotic markers but they were of little value for the detection of fibrin formation in the ECC. Our findings suggest that CY-216 administered as a single bolus dose seems to be of similar effectiveness to UFH.

  7. Randomized comparison of a novel anticoagulant, vasoflux, and heparin as adjunctive therapy to streptokinase for acute myocardial infarction: results of the VITAL study (Vasoflux International Trial for Acute Myocardial Infarction Lysis)

    NARCIS (Netherlands)

    Peters, R. J.; Spickler, W.; Théroux, P.; White, H.; Gibson, M.; Molhoek, P. G.; Anderson, H. V.; Weitz, J. I.; Hirsh, J.; Weaver, W. D.

    2001-01-01

    Vasoflux is a low-molecular-weight heparin derivative that inhibits factor IXa activation of factor X and catalyzes fibrin-bound thrombin inactivation by heparin cofactor II. We studied whether vasoflux improves the results of thrombolysis with streptokinase for acute myocardial infarction. We

  8. Metal Ions Activate Vascular Endothelial Cells and Increase Lymphocyte Chemotaxis and Binding

    Science.gov (United States)

    Ninomiya, James T.; Kuzma, Scott A.; Schnettler, Timothy J.; Krolikowski, John G.; Struve, Janine A.; Weihrauch, Dorothee

    2014-01-01

    Metal on metal articulations in hip arthroplasty offer advantages, including lower volumetric wear compared to conventional metalonpolyethylene bearings, and increased resistance to dislocation. Reports described early failures, with histologic features similar to a Type IV immune response. Mechanisms by which metal wear products cause this reaction are not completely understood. We hypothesized a mechanism through direct activation of endothelial cells (ECs) by metal ions, resulting in both vasculitis and accumulation of lymphocytes without prior immune sensitization. Effects of metal ions were evaluated using human ECs in culture. Alterations in chemotactic proteins IL8 and MCP1 were assessed, as was upregulation of the adhesion molecule ICAM-1 and lymphocyte binding to ECs. Cobalt increased secretion of IL8 and MCP1 significantly, and upregulated the expression of ICAM-1 in ECs compared to stimulation by chromium and controls. Binding of lymphocytes to ECs and transEC migration were both significantly increased by cobalt but not chromium. These findings suggest that cobalt contributes more to the activation of ECs and lymphocyte binding than chromium without an allergic response. Some of the adverse tissue reactions to implants with components made of cobalt–chromium–molybdenium alloys may be due in part to activation of the endothelium by metal ions. PMID:23629852

  9. Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation.

    Directory of Open Access Journals (Sweden)

    Matti Myllykoski

    Full Text Available The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.

  10. Synthesis, DNA Binding and Topoisomerase I Inhibition Activity of Thiazacridine and Imidazacridine Derivatives

    Directory of Open Access Journals (Sweden)

    Elizabeth Almeida Lafayette

    2013-12-01

    Full Text Available Thiazacridine and imidazacridine derivatives have shown promising results as tumors suppressors in some cancer cell lines. For a better understanding of the mechanism of action of these compounds, binding studies of 5-acridin-9-ylmethylidene-3-amino-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-imidazolidin-4-one and 3-acridin-9-ylmethyl-thiazolidin-2,4-dione with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopy and circular dichroism spectroscopy were performed. The binding constants ranged from 1.46 × 104 to 6.01 × 104 M−1. UV-Vis, fluorescence and circular dichroism measurements indicated that the compounds interact effectively with ctDNA, both by intercalation or external binding. They demonstrated inhibitory activities to human topoisomerase I, except for 5-acridin-9-ylmethylidene-2-thioxo-1,3-thiazolidin-4-one. These results provide insight into the DNA binding mechanism of imidazacridines and thiazacridines.

  11. Activator Protein-1: redox switch controlling structure and DNA-binding

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Zhou; Machius, Mischa; Nestler, Eric J.; Rudenko, Gabby

    2017-09-07

    The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a ‘redox switch’ centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the ‘OFF’ state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins.

  12. Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

    Energy Technology Data Exchange (ETDEWEB)

    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike (Vanderbilt)

    2010-11-16

    14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} for fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.

  13. [Oxygenator thrombosis without heparin resistance in polycythemia vera].

    Science.gov (United States)

    Lehot, J-J; Was, B; Dendeleu, L; Jegaden, O

    2012-05-01

    A 55-year-old male with a history of positive HIV serology and polycythemia vera underwent coronary artery bypass graft surgery with normothermic extracorporeal circulation. Following heparin administration the activated clotting time (ACT) was 633 seconds (Hemocron with kaolin). Lower than expected arterial and venous oxygen partial pressures together with high pressure (350 mmHg) in the arterial line upstream of the oxygenator were observed. Because of these signs the oxygenator was changed during the procedure. The outcome was uneventful. Electronic microscopic examination of the oxygenator membrane and thermic exchanger revealed fibrin and platelet deposits. Similar cases are described in the literature during polycythemia vera. Therefore the prevention might be a preoperative treatment with antiplatelet therapy in polycytemia vera. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  14. Heparin Leakage in Central Venous Catheters by Hemodynamic Transport

    Science.gov (United States)

    Barbour, Michael; McGah, Patrick; Gow, Kenneth; Aliseda, Alberto

    2014-11-01

    Central venous catheters (CVCs), placed in the superior vena cava for hemodialysis, are routinely filled with heparin, an anticoagulant, while not in use to maintain patency and prevent thrombus formation at the catheter tip. However, the heparin-lock procedure places the patient at risk for systemic bleeding incidences, as heparin is known to leak into the blood stream. We propose that the driving mechanism behind heparin leakage is advective-diffusive transport due to the pulsatile blood flow surrounding the catheter tip. This novel hypothesis is based on Planar Laser Induced Fluorescence (PLIF) measurements of heparin transport from a CVC placed inside an in vitro pulsatile flow loop and validated with CFD simulations. The results show an initial, fast (catheter lumen, where concentration is still high, that is insufficient at replenishing the lost heparin at the tip. These results, which estimate leakage rates consistent with published in vivo data, predict that the concentration of heparin at the catheter tip is effectively zero for the majority of the interdialytic phase, rendering the heparin lock ineffective.

  15. Anticoagulant effect of low molecular weight heparin on central ...

    African Journals Online (AJOL)

    Purpose: To analyse the effect of low molecular weight heparin on venous catheters in haemodialysis patients. Methods: This study included 140 eligible patients who were randomly and evenly divided into two groups, viz, a study group that received low molecular weight heparin and a control group that received ...

  16. 99m Tc-labeled heparin test in orthopaedic surgery

    Energy Technology Data Exchange (ETDEWEB)

    Bouvier, J.F.; Lafon, J.C.; Colin, M.; Chatelut, J.; Beaubatie, F. (Hopital Universitaire Dupuytren, Limoges (France))

    1983-06-30

    99m Tc-labeled heparin test was performed for early detection of phlebitis or pulmonary embolism after orthopaedic prothesis. Heparinic treatment and surgery per se were demonstrated to have no effect on the results. If this test demonstrates a statistical difference for pathologic patients, it is of greater value to consider ratio between rates before and after intervention.

  17. Citrate anticoagulation for CRRT in children: comparison with heparin.

    Science.gov (United States)

    Fernández, Sara Nicole; Santiago, Maria José; López-Herce, Jesús; García, Miriam; Del Castillo, Jimena; Alcaraz, Andrés José; Bellón, Jose María

    2014-01-01

    Regional anticoagulation with citrate is an alternative to heparin in continuous renal replacement therapies, which may prolong circuit lifetime and decrease hemorrhagic complications. A retrospective comparative cohort study based on a prospective observational registry was conducted including critically ill children undergoing CRRT. Efficacy, measured as circuit survival, and secondary effects of heparin and citrate were compared. 12 patients on CRRT with citrate anticoagulation and 24 patients with heparin anticoagulation were analyzed. Median citrate dose was 2.6 mmol/L. Median calcium dose was 0.16 mEq/kg/h. Median heparin dose was 15 UI/kg/h. Median circuit survival was 48 hours with citrate and 31 hours with heparin (P = 0.028). 66.6% of patients treated with citrate developed mild metabolic alkalosis, which was directly related to citrate dose. There were no cases of citrate intoxication: median total calcium/ionic calcium index (CaT/I) of 2.16 and a maximum CaT/I of 2.33, without metabolic acidosis. In the citrate group, 45.5% of patients developed hypochloremia and 27.3% hypomagnesemia. In the heparin group, 27.8% developed hypophosphatemia. Three patients were moved from heparin to citrate to control postoperatory bleeding. In conclusion citrate is a safe and effective anticoagulation method for CRRT in children and it achieves longer circuit survival than heparin.

  18. Citrate Anticoagulation for CRRT in Children: Comparison with Heparin

    Directory of Open Access Journals (Sweden)

    Sara Nicole Fernández

    2014-01-01

    Full Text Available Regional anticoagulation with citrate is an alternative to heparin in continuous renal replacement therapies, which may prolong circuit lifetime and decrease hemorrhagic complications. A retrospective comparative cohort study based on a prospective observational registry was conducted including critically ill children undergoing CRRT. Efficacy, measured as circuit survival, and secondary effects of heparin and citrate were compared. 12 patients on CRRT with citrate anticoagulation and 24 patients with heparin anticoagulation were analyzed. Median citrate dose was 2.6 mmol/L. Median calcium dose was 0.16 mEq/kg/h. Median heparin dose was 15 UI/kg/h. Median circuit survival was 48 hours with citrate and 31 hours with heparin (P=0.028. 66.6% of patients treated with citrate developed mild metabolic alkalosis, which was directly related to citrate dose. There were no cases of citrate intoxication: median total calcium/ionic calcium index (CaT/I of 2.16 and a maximum CaT/I of 2.33, without metabolic acidosis. In the citrate group, 45.5% of patients developed hypochloremia and 27.3% hypomagnesemia. In the heparin group, 27.8% developed hypophosphatemia. Three patients were moved from heparin to citrate to control postoperatory bleeding. In conclusion citrate is a safe and effective anticoagulation method for CRRT in children and it achieves longer circuit survival than heparin.

  19. Increased accuracy in heparin and protamine administration decreases bleeding

    DEFF Research Database (Denmark)

    Runge, Marx; Møller, Christian H; Steinbrüchel, Daniel A

    2009-01-01

    Three to 5 percent of the patients undergoing cardiac surgery are reoperated because of bleeding. When a surgical cause can be excluded, heparin/protamine mismatch may be considered. Insufficient reversal of heparin and overdosing of protamine may cause postoperative bleeding. The purpose of the ...

  20. Heparin-containing block copolymers; Part I: Surface characterization

    NARCIS (Netherlands)

    Vulić, I.; Pijpers, A.P.; Okano, T.; Kim, S.W.; Feijen, Jan

    1993-01-01

    Newly synthesized heparin-containing block copolymers, consisting of a hydrophobic block of polystyrene (PS), a hydrophilic spacer-block of poly(ethylene oxide) (PEO) and covalently bound heparin (Hep) as bioactive block, were coated on aluminium, glass, polydimethylsiloxane (PDMS), PS or Biomer

  1. ORIGINAL ARTICLES Endogenous heparin levels in the controlled ...

    African Journals Online (AJOL)

    the hypothesis that asthmatic patients have lower levels of circulating endogenous heparin than healthy ... of stimuli in guinea pig and rat models of allergic asthma, as well as in asthmatic patients.3 .... 2008; 585: 375-384. 4. Ceyhan B, Celikel T. Effect of inhaled heparin on methacholine-induced bronchial hyperreactivity.

  2. Release of macromolecules from albumin-heparin microspheres

    NARCIS (Netherlands)

    Kwon, Glen S.; Bae, You Han; Cremers, H.F.M.; Cremers, Harry; Feijen, Jan; Kim, Sung Wan

    1992-01-01

    Hydrophilic microspheres based on albumin-heparin conjugates have been prepared as a macromolecular delivery system. The soluble albumin-heparin conjugate was synthesized and crosslinked in a water-in-oil emulsion with glutaraldehyde to form microspheres in the same manner as for albumin microsphere

  3. Anticoagulant effect of low molecular weight heparin on central ...

    African Journals Online (AJOL)

    Thus, low molecular weight heparin seems to be more suitable as an anticoagulant solution in patients with venous catheters. Keywords: Low molecular weight heparin, Haemodialysis, Central venous catheter, Vascular access. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), ...

  4. Voltammetric determination of heparin based on its interaction with

    African Journals Online (AJOL)

    a

    For example, Jiao et al. investigated the interaction between some cationic dyes such as azure A, methylene blue and azure B with heparin by absorption spectrophotometry [13]. Sun et al. applied brilliant cresyl blue and neutral red for spectrophotometric determination of heparin [14,. 15]. Liu et al. established a resonance ...

  5. A systematic review of heparin to treat burn injury.

    Science.gov (United States)

    Oremus, Mark; Hanson, Mark D; Whitlock, Richard; Young, Edward; Archer, Carolyn; Dal Cin, Arianna; Gupta, Alok; Raina, Parminder

    2007-01-01

    This systematic review was conducted to assess the evidence for using heparin to treat burn injury. The following databases were searched for relevant studies: MEDLINE, EMBASE, CINAHL, The Cochrane Central Database of Controlled Trials, Web of Science, and BIOSIS. Additional searches involved the reference lists of included studies, the "grey " literature (eg, government reports), and consultations with experts to obtain unpublished manuscripts. Included studies were summarized descriptively and in tabular form, and assessed for methodological quality. A metaanalysis was conducted to obtain a summary estimate for the association between heparin use and postburn mortality. Nine studies were abstracted and included in the review. Five studies contained adult and pediatric patients, one contained adults only, and three contained pediatric patients only. Burn etiologies included flame, scald, thermal, or smoke inhalation. Heparin administration was done topically, subcutaneously, intravenously, or via aerosol. Heparin was reported to have a beneficial impact on mortality, graft and wound healing, and pain control. For mortality, the overall estimate (relative risk) of heparin's effect was 0.32 (95% confidence interval = 0.18-0.57). Heparin's reported benefits may be severely biased because the abstracted studies were beset by poor methodological quality (eg, inadequate definitions of treatment and outcome, no control of confounding). Given poor study quality, there is no strong evidence to indicate that heparin can improve clinical outcomes in the treatment of burn injury. Further research is needed to assess the clinical utility of using heparin in the treatment of burn injury.

  6. Prophylaxis of postoperative thromboembolism with low molecular weight heparins

    DEFF Research Database (Denmark)

    Jørgensen, L N; Wille-Jørgensen, P; Hauch, O

    1993-01-01

    to placebo or dextran and at least as efficient as unfractionated heparin in the prevention of deep vein thrombosis (DVT). Compared with unfractionated heparin, one of the LMWH preparations significantly reduced the total incidence of DVT. The rate of non-fatal pulmonary embolism was 0.49 per cent...

  7. [Delayed-type hypersensitivity to heparin: diagnosis and therapeutic management].

    Science.gov (United States)

    Nosbaum, A; Pralong, P; Rozieres, A; Dargaud, Y; Nicolas, J-F; Bérard, F

    2012-05-01

    Heparin is widely used as an anticoagulant and is indicated in the prevention and treatment of thromboembolic disorders. Heparin-induced delayed-type hypersensitivity presents as eczematous lesions, either at the injection site or generally, and affects 7.5% of patients on heparin. This poses diagnostic and therapeutic issues, since an alternative anticoagulant treatment is essential and the risk of cross-reactivity may be as high as 80%, depending on the type of heparin used. If delayed-type hypersensitivity is suspected, heparin-induced thrombocytopenia must first be ruled out, and heparin should be stopped. Fondaparinux is currently the first-line alternative, with a risk of cross-reactivity estimated at only 10%. The switch from a low-molecular-weight heparin (LMWH) to another LMWH is no longer recommended. The use of unfractionated heparin, danaparoid or hirudin may be warranted in the event of recurrence with fondaparinux, and an immuno-allergological work-up is needed to specify the exact profile of cross-allergies. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  8. Enhancement of sperm-zona pellucida (ZP) binding capacity by activation of protein kinase A and C pathways in certain infertile men with defective sperm-ZP binding.

    Science.gov (United States)

    Liu, D Y; Liu, M L; Baker, H W G

    2009-01-01

    Defective sperm-zona pellucida (ZP) binding (DSZPB) is a common cause of failure of fertilization in vitro. This study was to determine if DSZPB is caused by defective pathways upstream of protein kinase A (PKA) and C (PKC), or reduced protein tyrosine phosphorylation (TP). Infertile men with DSZPB and either normal sperm morphology (NSM) > or = 14% (n = 15) or ZP binding test was performed by incubation of motile sperm with oocytes for 2 h with or without dibutyryl cyclic AMP (dbcAMP, PKA activator) or phorbol myristate acetate (PMA, PKC activator). TP of capacitated sperm in medium was assessed by immunofluorescence with an anti-phosphotyrosine monoclonal antibody. For normal sperm with normal sperm-ZP binding, both PMA and dbcAMP significantly enhanced sperm-ZP binding in a dose-response manner. Only dbcAMP, but not PMA, significantly increased TP of capacitated sperm. In DSZPB men with severe teratozoospermia (NSM ZP binding, despite dbcAMP significantly increasing the TP of capacitated sperm for all samples. In contrast, for DSZPB with NSM > or = 14%, PMA caused significantly increased sperm binding up to normal levels (> or =40 sperm bound/ZP) in five men, and dbcAMP had a similar result in two men. Again TP was significantly enhanced only by dbcAMP, but not by PMA. There is defective signalling in pathways upstream of PKC and PKA in some men with DSZPB and normal semen analysis. Stimulation of TP by dbcAMP does not enhance sperm-ZP binding capacity in DSZPB men with low TP, regardless of sperm morphology.

  9. Heparin-Related Thrombocytopenia Triggered by Severe Status of Systemic Lupus Erythematosus and Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Satoshi Suzuki

    2016-01-01

    Full Text Available A patient with severe lupus nephritis developed thrombocytopenia during treatment with high-dose steroids. In addition to viral- or disease-induced cytopenia, the pathology was believed to arise from diverse contributing factors, such as thrombotic microangiopathy and heparin-related thrombocytopenia (HIT. By combining plasma exchange therapy and intravenous cyclophosphamide, we successfully controlled the SLE activity and improved the thrombocytopenia. An antecedent bacterial infection or SLE activity is believed to have contributed to the concurrent HIT.

  10. Acidosis induced by carbon dioxide insufflation decreases heparin potency: a risk factor for thrombus formation.

    Science.gov (United States)

    Gorter, Karin A M; Stehouwer, Marco C; Van Putte, Bart P; Vlot, Eline A; Urbanus, Rolf T

    2017-04-01

    Since the introduction of CO2 insufflation during open heart surgery in our hospital, we incidentally observed thrombus formation in the dissected heart, in the pericardium and in the cardiotomy reservoir of the cardiopulmonary bypass system. Furthermore, we measured very high levels of pCO2, causing severe acidosis, in stagnant blood in the pericardium and cardiotomy reservoir. In this in vitro study, we assessed the influence of acidosis and hypothermia on heparin potency and thrombin formation. We assessed heparin potency in function of pH (pH 5.0-7.4) and temperature (24-37°C) by comparing the activated partial thromboplastin time in platelet-poor plasma between samples with and without unfractionated heparin. We measured thrombin formation in platelet-poor plasma by means of fluorescent, calibrated, automated thrombography in function of pH (pH 5.0-7.4) and temperature (24-37°C). The parameters of interest were the endogenous thrombin potential and the peak amount of thrombin generation. The major finding of this study is the significant decrease in the efficiency of unfractionated heparin in delaying thrombus formation at acidotic (pH 5.0-7.0) conditions (p=0.034-0.05). Furthermore, we found that thrombin formation is significantly increased at hypothermic (24-34°C) conditions (p=acidosis may lead to a decreased heparin potency. Acidosis, as induced by CO2 insufflation, may predispose patients to incidental thrombus formation in stagnant blood in the open thorax and in the cardiotomy reservoir. Hypothermia might further increase this risk. Therefore, we recommend reconsidering the potential advantages and disadvantages of using CO2 insufflation during cardiopulmonary bypass.

  11. The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.

    Science.gov (United States)

    Jongruja, Nujarin; You, Dong-Ju; Kanaya, Eiko; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2010-11-01

    Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg(2+)-dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg(2+)-dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg(2+) ion are arranged ideally. © 2010 The Authors Journal compilation © 2010 FEBS.

  12. Graft patency after off-pump coronary artery bypass surgery is inferior even with identical heparinization protocols

    DEFF Research Database (Denmark)

    Houlind, Kim Christian; Fenger-Grøn, Morten; Juel Holme, Susanne

    2014-01-01

    OBJECTIVE: To determine whether graft patency after on-pump and off-pump coronary artery bypass surgery is similar when performed using the same heparinization protocol. METHODS: In a randomized, controlled, multicenter trial, 900 patients more than 70 years of age received either on-pump or off......-pump coronary artery bypass surgery. Heparin was given to achieve an activated clotting time of 400 seconds before arteriotomy in both groups. After the procedure, protamine sulfate was given to revert the activated clotting time to less than 120 seconds. Coronary angiography was performed 6 months after...

  13. A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity.

    Science.gov (United States)

    Das, Mrinal Kumar; Sharma, Radhey Shyam; Mishra, Vandana

    2011-12-01

    Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC(50) against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Stress-related changes of benzodiazepine binding inhibitory activity (B.B.I.A.) in humans.

    Science.gov (United States)

    Marazziti, D; Michelini, S; Giannaccini, G; Martini, C; Castrogiovanni, P; Cassano, G B; Lucacchini, A

    1990-01-01

    The presence of benzodiazepine binding inhibitory activity (B.B.I.A.) in sera from 44 psychiatric patients and from 14 healthy volunteers, prompted us to investigate whether or not this activity underwent changes in stressful situations. We measured the inhibitory units (IU) of deproteinized sera of 12 subjects, immediately before and 2 weeks after sitting for a difficult university exam. Our results showed significantly higher IU values (i.e., higher B.B.I.A. concentrations) in the samples taken just before the exam. This preliminary finding clearly suggests the involvement of B.B.I.A. in anxiety mechanisms.

  15. Silver nanoparticles-loaded activated carbon fibers using chitosan as binding agent: Preparation, mechanism, and their antibacterial activity

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Chengli, E-mail: tcl-lily@mail.zjxu.edu.cn [College of Mechanical and Electrical Engineering, Jiaxing University, Jiaxing 314001 (China); Hu, Dongmei [College of Mechanical Science and Engineering, Jilin University, Changchun 130022 (China); Cao, Qianqian [College of Mechanical and Electrical Engineering, Jiaxing University, Jiaxing 314001 (China); Yan, Wei [Department of Environmental Science and Engineering, Xi’an Jiaotong University, Xi’an 710049 (China); Xing, Bo [College of Mechanical and Electrical Engineering, Jiaxing University, Jiaxing 314001 (China)

    2017-02-01

    Highlights: • Chitosan was firstly introduced as binding agent for AgNPs loading on ACF surface. • Molecular dynamics simulation was used to explore the AgNPs loading mechanism. • Loading mechanism was proposed based on the experimental and simulation results. • Antibacterial AgNPs-loaded ACF showed use potential for water disinfection. - Abstract: The effective and strong adherence of silver nanoparticles (AgNPs) to the substrate surface is pivotal to the practical application of those AgNPs-modified materials. In this work, AgNPs were synthesized through a green and facile hydrothermal method. Chitosan was introduced as the binding agent for the effective loading of AgNPs on activated carbon fibers (ACF) surface to fabricate the antibacterial material. Apart from conventional instrumental characterizations, i. e., scanning electron microscope (SEM), X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), zeta potential and Brunauer-Emmett-Teller (BET) surface area measurement, molecular dynamics simulation method was also applied to explore the loading mechanism of AgNPs on the ACF surface. The AgNPs-loaded ACF material showed outstanding antibacterial activity for S. aureus and E. coli. The combination of experimental and theoretical calculation results proved chitosan to be a promising binding agent for the fabrication of AgNPs-loaded ACF material with excellent antibacterial activity.

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

    DEFF Research Database (Denmark)

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan

    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...

  17. Inhibition of allergic airway responses by heparin derived oligosaccharides: identification of a tetrasaccharide sequence

    Directory of Open Access Journals (Sweden)

    Ahmed Tahir

    2012-01-01

    Full Text Available Abstract Background Previous studies showed that heparin's anti-allergic activity is molecular weight dependent and resides in oligosaccharide fractions of Objective To investigate the structural sequence of heparin's anti-allergic domain, we used nitrous acid depolymerization of porcine heparin to prepare an oligosaccharide, and then fractionated it into disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide fractions. The anti-allergic activity of each oligosaccharide fraction was tested in allergic sheep. Methods Allergic sheep without (acute responder and with late airway responses (LAR; dual responder were challenged with Ascaris suum antigen with and without inhaled oligosaccharide pretreatment and the effects on specific lung resistance and airway hyperresponsiveness (AHR to carbachol determined. Additional inflammatory cell recruitment studies were performed in immunized ovalbumin-challenged BALB/C mice with and without treatment. Results The inhaled tetrasaccharide fraction was the minimal effective chain length to show anti-allergic activity. This fraction showed activity in both groups of sheep; it was also effective in inhibiting LAR and AHR, when administered after the antigen challenge. Tetrasaccharide failed to modify the bronchoconstrictor responses to airway smooth muscle agonists (histamine, carbachol and LTD4, and had no effect on antigen-induced histamine release in bronchoalveolar lavage fluid in sheep. In mice, inhaled tetrasaccharide also attenuated the ovalbumin-induced peribronchial inflammatory response and eosinophil influx in the bronchoalveolar lavage fluid. Chemical analysis identified the active structure to be a pentasulfated tetrasaccharide ([IdoU2S (1→4GlcNS6S (1→4 IdoU2S (1→4 AMan-6S] which lacked anti-coagulant activity. Conclusions These results demonstrate that heparin tetrasaccharide possesses potent anti-allergic and anti-inflammatory properties, and that the domains responsible for anti

  18. The Rickettsia Surface Cell Antigen 4 Applies Mimicry to Bind to and Activate Vinculin*

    Science.gov (United States)

    Park, HaJeung; Lee, Jun Hyuck; Gouin, Edith; Cossart, Pascale; Izard, Tina

    2011-01-01

    Pathogenic Rickettsia species cause high morbidity and mortality, especially R. prowazekii, the causative agent of typhus. Like many intracellular pathogens, Rickettsia exploit the cytoskeleton to enter and spread within the host cell. Here we report that the cell surface antigen sca4 of Rickettsia co-localizes with vinculin in cells at sites of focal adhesions in sca4-transfected cells and that sca4 binds to and activates vinculin through two vinculin binding sites (VBSs) that are conserved across all Rickettsia. Remarkably, this occurs through molecular mimicry of the vinculin-talin interaction that is also seen with the IpaA invasin of the intracellular pathogen Shigella, where binding of these VBSs to the vinculin seven-helix bundle head domain (Vh1) displaces intramolecular interactions with the vinculin tail domain that normally clamp vinculin in an inactive state. Finally, the vinculin·sca4-VBS crystal structures reveal that vinculin adopts a new conformation when bound to the C-terminal VBS of sca4. Collectively, our data define the mechanism by which sca4 activates vinculin and interacts with the actin cytoskeleton, and they suggest important roles for vinculin in Rickettsia pathogenesis. PMID:21841197

  19. Binding affinity of triphenyl acrylonitriles to estrogen receptors: quantitative structure-activity relationships.

    Science.gov (United States)

    Bolboacă, Sorana D; Marta, Monica M; Jäntschi, Lorentz

    2010-01-01

    The quantitative structure-activity relationship approach was applied to understand the relative binding affinity of triphenyl acrylonitriles to estrogen receptors. A sample of previously studied triphenyl acrylonitriles was divided into training (18 compounds) and test sets (7 compounds) using a stratified random approach. The molecular descriptor family on vertices cutting (MDFV) approach was used in order to translate the structural information into descriptors. The relationship between binding activity and structural descriptors was identified using the multiple linear regression procedure. An optimal three-parameter equation with a determination coefficient of 0.9580 and a cross-validation leave-one-out parameter of 0.9408 was identified. The optimal model was assessed on a test set and a determination coefficient of 0.9004 was obtained. The MDFV model proved not to be significantly different from the previously reported model in terms of goodness-of-fit. In terms of information criteria (Akaike's, Bayesian, Amemiya, and Hannan-Quinn) and Kubinyi function, the MDFV model proved to perform better than the previously reported model. The optimal MDFV model was able to explain approximately 96% of the total variance in the estrogenic binding relative affinity of triphenyl acrylonitriles and to have estimation and prediction abilities. Although there were no significant differences in terms of goodness-of-fit, the MDFV model proved to exhibit better information parameters compared to the previously reported model using the same number of molecular descriptors.

  20. Change of Ferritin-binding Activity in the Serum of Foal after Birth.

    Science.gov (United States)

    Ohya, Takushi; Kondo, Takashi; Yoshikawa, Yasunaga; Watanabe, Kiyotaka; Orino, Koichi

    2011-01-01

    In mammal circulation, various ferritin-binding proteins (FBPs) are thought to be involved in the clearance of circulating ferritin after complex formation with it. However, horse FBPs are known to cause inhibitory effects on ferritin immunoassay due to the concealment of the ferritin molecule to anti-ferritin antibodies used in the ferritin immunoassay. These inhibitory effects are eliminated by heat treatment of horse serum at 75°C for 15 min. The inhibitory effects on ferritin immunoassay in the sera of ten foal sera (5 females and 5 males) from 1 to 18 months were detected during all periods, and ferritin concentrations of the foal sera increased 20-100% as compared with those of untreated sera by same heat treatment. Ferritin concentrations of heat-treated foal sera increased after birth, reaching to ferritin levels of adult horse at 9 months of age. Thereafter, although serum ferritin concentrations fell down at 12 months of age, these concentrations increased to adult levels at 15 months of age again. The ratio of ferritin concentration of heat-treated serum to that of the untreated serum was regarded as an apparent ferritin-binding activity. Ferritin-binding activities in the sera of foals showed peak at 2 and 4 months of age in females and males, respectively. These results suggested that horse FBPs were heat unstable, and FBPs may play an important role in iron metabolism at early developmental stage.

  1. Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme

    Directory of Open Access Journals (Sweden)

    Caitlin Siobhan Byrt

    2012-11-01

    Full Text Available A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM to a synthetic glycosyl hydrolase (GH improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the carbohydrate-binding module (CBM of the tomato (Solanum lycopersicum SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using CMC, MUC and native crystalline cellulose assays. The presence of the CBM substantially improved the endo-glucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.

  2. Recrystallization inhibition in ice due to ice binding protein activity detected by nuclear magnetic resonance

    Directory of Open Access Journals (Sweden)

    Jennifer R. Brown

    2014-09-01

    Full Text Available Liquid water present in polycrystalline ice at the interstices between ice crystals results in a network of liquid-filled veins and nodes within a solid ice matrix, making ice a low porosity porous media. Here we used nuclear magnetic resonance (NMR relaxation and time dependent self-diffusion measurements developed for porous media applications to monitor three dimensional changes to the vein network in ices with and without a bacterial ice binding protein (IBP. Shorter effective diffusion distances were detected as a function of increased irreversible ice binding activity, indicating inhibition of ice recrystallization and persistent small crystal structure. The modification of ice structure by the IBP demonstrates a potential mechanism for the microorganism to enhance survivability in ice. These results highlight the potential of NMR techniques in evaluation of the impact of IBPs on vein network structure and recrystallization processes; information useful for continued development of ice-interacting proteins for biotechnology applications.

  3. Cerium Binding Activity of Pectins Isolated from the Seagrasses Zostera marina and Phyllospadix iwatensis

    Directory of Open Access Journals (Sweden)

    Valeri Kovalev

    2012-04-01

    Full Text Available Cerium binding activity of three different water soluble pectin compounds of different origin was studied in a batch sorption system. The Langmuir, Freundlich and BET sorption models were adopted to describe the binding reactions between metal ions and pectin molecules. The Langmuir model provided the best fit. Within the pH range from 4.0 to 6.0, the largest amount of the cerium ions was bound by pectin isolated from the seagrass Phylospadix iwatensis in comparison to pectin extracted from the seagrass Zostera marina and pectin obtained from citrus peel (commercial grade. The Langmuir constants were also highest for the pectin samples isolated from the seagrass P. iwatensis. The results obtained from this study suggest that pectin is a prospective source for the development of radioisotope-removing pharmaceuticals.

  4. Cerium binding activity of pectins isolated from the seagrasses Zostera marina and Phyllospadix iwatensis.

    Science.gov (United States)

    Khotimchenko, Yuri; Khozhaenko, Elena; Kovalev, Valeri; Khotimchenko, Maxim

    2012-04-01

    Cerium binding activity of three different water soluble pectin compounds of different origin was studied in a batch sorption system. The Langmuir, Freundlich and BET sorption models were adopted to describe the binding reactions between metal ions and pectin molecules. The Langmuir model provided the best fit. Within the pH range from 4.0 to 6.0, the largest amount of the cerium ions was bound by pectin isolated from the seagrass Phylospadix iwatensis in comparison to pectin extracted from the seagrass Zostera marina and pectin obtained from citrus peel (commercial grade). The Langmuir constants were also highest for the pectin samples isolated from the seagrass P. iwatensis. The results obtained from this study suggest that pectin is a prospective source for the development of radioisotope-removing pharmaceuticals.

  5. Pentadecapeptide BPC 157 reduces bleeding time and thrombocytopenia after amputation in rats treated with heparin, warfarin or aspirin.

    Science.gov (United States)

    Stupnisek, Mirjana; Franjic, Sandra; Drmic, Domagoj; Hrelec, Masa; Kolenc, Danijela; Radic, Bozo; Bojic, Davor; Vcev, Aleksandar; Seiwerth, Sven; Sikiric, Predrag

    2012-05-01

    Recently, in rat abdominal aorta terminoterminal-anastomosis the stable gastric pentadecapeptide BPC 157 prevents obstructive thrombus formation and rapidly destroys already formed obstructive thrombus. Also, BPC 157 wound healing may signify the clot as conductive matrix or "scaffold" to speed up wound healing process, and decrease bleeding. Here, in rats, BPC 157 (10 μg/kg, 10 ng/kg) improved always reduced bleeding time and amount of bleeding after (tail) amputation only, heparin (250 mg/kg, 25mg/kg, 10mg/kg i.v.), warfarin (1.5mg/kg i.g. once daily for 3 consecutive days), aspirin (0.1g/kg i.g. (once daily/3 consecutive days) or 1.0 g/kg i.p. once), and amputation associated with those agents application. BPC 157 counteracting regimens (i.v., i.p., i.g. (immediately after any challenge)) correspondingly follow the route of bleeding-agents application. All heparin-, warfarin-, and aspirin-rats and normal-rats that received BPC 157 exhibited lesser fall in platelets count. BPC 157 attenuated over-increased APTT-, TT-values in 10mg/kg heparin-rats, but did not influence heparin activity (anti-Xa test). Indicatively, unless counteracted in BPC 157 rats, excessive bleeding-acute thrombocytopenia (BPC 157 markedly prolongs the survival time (heparin-rats, 25mg/kg, right foot amputation). Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Three different chromogenic methods do not give equivalent anti-Xa levels for patients on therapeutic low molecular weight heparin (dalteparin) or unfractionated heparin.

    Science.gov (United States)

    Kovacs, M J; Keeney, M; MacKinnon, K; Boyle, E

    1999-02-01

    In this study we compare three chromogenic methods (IL-Heparin, Stachrom Heparin and Heparin Sigma) on two different instruments (ACL300+ and AMAX CS190) for patients on dalteparin (n = 41), a low molecular weight heparin or unfractionated heparin (n = 50). For dalteparin the mean anti-Xa levels for IL-Heparin, Stachrom Heparin and Heparin Sigma were 0.27, 0.30 and 0.21 U/ml, respectively, while for heparin they were 0.52, 0.55 and 0.41 U/ml, respectively. To test for instrument specific effects, IL-Heparin and Stachrom Heparin were repeated on both instruments on 42 patients receiving unfractionated heparin. For IL-Heparin the mean anti-Xa levels on the AMAX CS190 and ACL300+ were 0.51 and 0.59 U/ml, respectively, while for Stachrom Heparin they were 0.55 and 0.67 anti-Xa U/ml. We conclude that different chromogenic anti-Xa methods do not give equivalent anti-Xa levels for the same samples. Moreover, the differences are clinically significant. This is not explained entirely by instrumentation effects. Recommended therapeutic ranges may need to be method and instrument specific.

  7. Anadenanthera colubrina (Vell.) Brenan produces steroidal substances that are active against Alternaria alternata (Fr.) Keissler and that may bind to oxysterol-binding proteins.

    Science.gov (United States)

    Campos, Viviane Ac; Perina, Fabiano J; Alves, Eduardo; Sartorelli, Jaqueline; Moura, Amanda M; Oliveira, Denilson F

    2014-12-01

    In previous studies, the extract from Anadenanthera colubrina was active against Alternaria alternata in vitro and reduced the disease caused by this fungus on Murcott tangor fruits to levels that have been obtained using commercial fungicides. Therefore, the goal of the present work was to isolate and identify the active substances in this extract and identify in silico their protein target in the fungus. The bioguided fractionation of the methanol extract from the fruits of A. colubrina resulted in the isolation of β-sitosterol and β-sitosteryl linoleate, which had minimal inhibitory concentrations (MICs) of 250 and 500 µg mL(-1) , respectively, against A. alternata. Under the same conditions, the MICs for two commercial fungicides were 1250 and 19 µg mL(-1) . In silico studies showed that these steroidal substances bind well to oxysterol-binding proteins from Saccharomyces cerevisiae. β-Sitosterol and β-sitosteryl linoleate, produced by A. colubrina, are active against A. alternata. In silico studies suggest that these substances may act by binding to oxysterol-binding proteins. Therefore, both substances and these proteins have potential use in the development of new steroidal structures and analogues to control the disease caused by A. alternata. © 2014 Society of Chemical Industry.

  8. Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity

    DEFF Research Database (Denmark)

    Bindesbøll, Christian; Fan, Qiong; Nørgaard, Rikke C

    2015-01-01

    Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription...

  9. Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Ruzanski, Christian

    2014-01-01

    Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half ...

  10. Spectrofluorimetric determination of heparin using doxycycline-europium probe

    Energy Technology Data Exchange (ETDEWEB)

    Li Jing [Department of Chemistry, Shandong Normal University, Jinan 250014 (China); Liu Jinkai [Department of Chemistry, Shandong Normal University, Jinan 250014 (China); Zhu Xiaojing [Department of Chemistry, Shandong Normal University, Jinan 250014 (China); Peng Qian [Department of Chemistry, Shandong Normal University, Jinan 250014 (China); Jiang Chongqiu [Department of Chemistry, Shandong Normal University, Jinan 250014 (China)]. E-mail: jiangchongqiu@sdnu.edu.cn

    2005-06-15

    A new spectrofluorimetric method was developed for the determination of the trace amount of heparin (Hep). Using doxycycline (DC)-europium ion (Eu{sup 3+}) as a fluorescent probe, in the buffer solution of pH=8.9, Hep can remarkably enhance the fluorescence intensity of the DC-Eu{sup 3+} complex at {lambda}=612 nm and the enhanced fluorescence intensity of Eu{sup 3+} ion is in proportion to the concentration of Hep. Optimum conditions for the determination of Hep were also investigated. The linear range and detection limit for the determination of Hep are 0.04-0.8 {mu}g/mL and 19.7 ng/mL, respectively. This method is simple, practical, and relatively free of interference from coexisting substances and can be successfully applied to assess Hep in biological samples. By the Rosenthal graphic method, the association constant and binding numbers of Hep with the probe are 6.60x10{sup 4} L/mol and 33.9. Moreover, the enhancement mechanism of the fluorescence intensity in the DC-Eu{sup 3+} system and the DC-Eu{sup 3+}-Hep-CTMAB system have been also discussed.

  11. Requirement for an A-tract structure at the binding site of phage phi 29 transcriptional activator.

    Science.gov (United States)

    Nuez, B; Rojo, F; Salas, M

    1994-03-25

    The Bacillus subtilis phage phi 29 transcriptional activator, protein p4, binds to the 5'-AACT-TTTT-15 base-pair spacer-AAAATGTT-3' inverted repeat. In this communication, we study the influence in protein p4 binding of the DNA helical structure within the protein p4 recognition sequences, 5'-AAAATAG-3'. Protein p4 could efficiently bind to a modified target in which the A-tracts had been changed into T-tracts (a different sequence with a similar structure). Binding was lost when the structure of the binding site was modified by an interrupting C residue. The results suggest that the DNA helical structure of the A-tracts is critical for p4 binding. Two models are described that would explain how protein p4 recognized its target sequences on the DNA.

  12. Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters.

    Science.gov (United States)

    Thangasamy, Samina