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Sample records for heparin binding mechanism

  1. A mutation in the heparin-binding site of noggin as a novel mechanism of proximal symphalangism and conductive hearing loss.

    Science.gov (United States)

    Masuda, Sawako; Namba, Kazunori; Mutai, Hideki; Usui, Satoko; Miyanaga, Yuko; Kaneko, Hiroki; Matsunaga, Tatsuo

    2014-05-09

    The access of bone morphogenetic protein (BMP) to the BMP receptors on the cell surface is regulated by its antagonist noggin, which binds to heparan-sulfate proteoglycans on the cell surface. Noggin is encoded by NOG and mutations in the gene are associated with aberrant skeletal formation, such as in the autosomal dominant disorders proximal symphalangism (SYM1), multiple synostoses syndrome, Teunissen-Cremers syndrome, and tarsal-carpal coalition syndrome. NOG mutations affecting a specific function may produce a distinct phenotype. In this study, we investigated a Japanese pedigree with SYM1 and conductive hearing loss and found that it carried a novel heterozygous missense mutation of NOG (c.406C>T; p.R136C) affecting the heparin-binding site of noggin. As no mutations of the heparin-binding site of noggin have previously been reported, we investigated the crystal structure of wild-type noggin to investigate molecular mechanism of the p.R136C mutation. We found that the positively charged arginine at position 136 was predicted to be important for binding to the negatively charged heparan-sulfate proteoglycan (HSPG). An in silico docking analysis showed that one of the salt bridges between noggin and heparin disappeared following the replacement of the arginine with a non-charged cysteine. We propose that the decreased binding affinity of NOG with the p.R136C mutation to HSPG leads to an excess of BMP signaling and underlies the SYM1 and conductive hearing loss phenotype of carriers. Copyright © 2014 Elsevier Inc. All rights reserved.

  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. A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40

    Directory of Open Access Journals (Sweden)

    Nipaporn Ngernyuang

    2018-02-01

    Full Text Available The heparin-binding glycoprotein YKL-40 (CHI3L1 is intimately associated with microvascularization in multiple human diseases including cancer and inflammation. However, the heparin-binding domain(s pertinent to the angiogenic activity have yet been identified. YKL-40 harbors a consensus heparin-binding motif that consists of positively charged arginine (R and lysine (K (RRDK; residues 144–147; but they don't bind to heparin. Intriguingly, we identified a separate KR-rich domain (residues 334–345 that does display strong heparin binding affinity. A short synthetic peptide spanning this KR-rich domain successfully competed with YKL-40 and blocked its ability to bind heparin. Three individual point mutations, where alanine (A substituted for K or R (K337A, K342A, R344A, led to remarkable decreases in heparin-binding ability and angiogenic activity. In addition, a neutralizing anti-YKL-40 antibody that targets these residues and prevents heparin binding impeded angiogenesis in vitro. MDA-MB-231 breast cancer cells engineered to express ectopic K337A, K342A or R344A mutants displayed reduced tumor development and compromised tumor vessel formation in mice relative to control cells expressing wild-type YKL-40. These data reveal that the KR-rich heparin-binding motif is the functional heparin-binding domain of YKL-40. Our findings shed light on novel molecular mechanisms underlying endothelial cell angiogenesis promoted by YKL-40 in a variety of diseases.

  4. Effects of heparin on insulin binding and biological activity

    International Nuclear Information System (INIS)

    Kriauciunas, K.M.; Grigorescu, F.; Kahn, C.R.

    1987-01-01

    The effect of heparin, a polyanionic glycosaminoglycan known to alter the function of many proteins, on insulin binding and bioactivity was studied. Cultured human lymphocytes (IM-9) were incubated with varying concentrations of heparin, then extensively washed, and 125 I-labeled insulin binding was measured. Heparin at concentrations used clinically for anticoagulation (1-50 U/ml) inhibited binding in a dose-dependent manner; 50% inhibition of binding occurred with 5-10 U/ml. Scatchard analysis indicated that the decrease in binding was due to a decrease in both the affinity and the apparent number of available insulin receptors. The effect occurred within 10 min at 22 degrees C and persisted even after the cells were extensively washed. Inhibition of insulin binding also occurred when cells were preincubated with heparinized plasma or heparinized serum but not when cells were incubated with normal serum or plasma from blood anticoagulated with EDTA. By contrast, other polyanions and polycations, e.g., poly-L-glutamic acid, poly-L-lysine, succinylated poly-L-lysine, and histone, did not inhibit binding. Heparin also inhibited insulin binding in Epstein-Barr (EB) virus-transformed lymphocytes but had no effect on insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells. When isolated adipocytes were incubated with heparin, there was a dose-dependent inhibition of insulin-stimulated glucose oxidation and, to a lesser extent, of basal glucose oxidation. Although heparin has no effect on insulin binding to intact hepatoma cells, heparin inhibited both insulin binding and insulin-stimulated autophosphorylation in receptors solubilized from these cells

  5. A sensitive competitive binding assay for exogenous and endogenous heparins

    International Nuclear Information System (INIS)

    Dawes, J.; Pepper, D.S.

    1982-01-01

    A new type of assay for heparins has been devised, in which the test material competes with 125 I-labelled heparin for binding to protamine-Sepharose. The assay is very sensitive and will measure heparin concentrations down to 10 ng ml-1. It responds to both the degree of sulphation and the molecular weight of acidic polysaccharides, but is independent of their biological activities. It can be used to quantitate heparins in biological fluids after pretreatment of the samples with protease. In this way endogenous heparins were measured in normal human serum, plasma and urine. The assay is extremely versatile and has great potential for the investigation of endogenous and exogenous heparins

  6. Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

    Science.gov (United States)

    Lynch, D M; Howe, S E

    1985-11-01

    Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

  7. Improved assay for measuring heparin binding to bull sperm

    International Nuclear Information System (INIS)

    Miller, D.J.; Ax, R.L.

    1988-01-01

    The binding of heparin to sperm has been used to study capacitation and to rank relative fertility of bulls. Previous binding assays were laborious, used 10 7 sperm per assay point, and required large amounts of radiolabeled heparin. A modified heparin-binding assay is described that used only 5 x 10 4 cells per incubation well and required reduced amounts of [ 3 H] heparin. The assay was performed in 96-well Millititer plates, enabling easy incubation and filtering. Dissociation constants and concentrations of binding sites did not differ if analyzed by Scatchard plots, Woolf plots, or by log-logit transformed weighted nonlinear least squares regression, except in the case of outliers. In such cases, Scatchard analysis was more sensitive to outliers. Nonspecific binding was insignificant using nonlinear logistic fit regression and a proportion graph. The effects were tested of multiple free-thawing of sperm in either a commercial egg yolk extender, 40 mM Tris buffer with 8% glycerol, or 40 mM Tris buffer without glycerol. Freeze-thawing in extender did not affect the dissociation constant or the concentration of binding sites. However, freeze-thawing three times in 40 mM Tris reduced the concentration of binding sites and lowered the dissociation constant (raised the affinity). The inclusion of glycerol in the 40 mM Tris did not significantly affect the estimated dissociation constant or the concentration of binding sites as compared to 40 mM Tris without glycerol

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

    Indian Academy of Sciences (India)

    Prakash

    (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 ... Thus, the combined effects of seminal plasma components support the survival of ...... The BBXB motif of RANTES is the principal site for heparin binding and controls ...

  9. Detection of growth factor binding to gelatin and heparin using a photonic crystal optical biosensor

    International Nuclear Information System (INIS)

    Morgan, Abby W.; Chan, Leo L.; Sendemir-Urkmez, Aylin; Cunningham, Brian T.; Jamison, Russell D.

    2010-01-01

    Drug-carrier interactions are important to protein controlled release systems to protect the protein from denaturation and ensure properly timed release. A novel photonic crystal biosensor was used to investigate a gelatin-protein controlled release system to determine the amount of protein bound to the carrier at physiological conditions. The Biomolecular Interaction Detection (BIND) system reflects a narrow band of wavelengths when white light is shone incident to the grating. As mass is deposited onto the surface, the peak wavelength value is shifted due to changes in the optical density of the biosensor. The BIND system was used to detect the binding of growth factors onto acidic gelatin, basic gelatin, and heparin on the sensor surface. Through a series of experiments, including functionalizing the sensor, adjusting the ionic strength of the solution, adjusting the substrate concentration, and minimizing non-specific signal, the adsorption of the gelatins and heparin on the sensor was enhanced. The binding interaction of recombinant human transforming growth factor (rhTGF)-β1 and bone morphogenetic protein (rhBMP)-2 with the two types of gelatin and heparin were investigated. The strength of the interaction between rhTGF-β1 and the substrates is in the following order: heparin > acidic gelatin > basic gelatin. RhBMP-2 bound to the substrates but with less intensity than TGF-β1: heparin > basic gelatin > acidic gelatin. This work provides support for the controlled release mechanism through degradation of the gelatin carrier.

  10. Dansyl (5-dimethylaminonaphthalene-1-sulphonyl)-heparin binds antithrombin III and platelet factor 4 at separate sites

    Science.gov (United States)

    Piepkorn, Michael W.

    1981-01-01

    Antithrombin III binds to, and thereby augments the fluorescence of, dansyl-(5-dimethylaminonaphthalene-1-sulphonyl)-heparin; platelet factor 4 binding to the fluorescent heparin has little of this effect. Competition studies in which antithrombin III competes with platelet factor 4 for heparin binding demonstrate that heparin can simultaneously bind both proteins. PMID:7317004

  11. In vivo studies on the binding of heparin and its fractions with platelet factor 4

    International Nuclear Information System (INIS)

    Walz, D.A.; Hung, G.L.

    1985-01-01

    PF4 has a half-life in plasma of less than 3 minutes, and its rapid clearance appears to be a function of binding to the vascular endothelium. Once bound to the endothelium, PF4 can be released by heparin in a time-dependent manner; recovery is greater the sooner heparin is administered following PF4 infusion. This heparin-induced release of PF4 can be abolished if the heparin is first complexed with hexadimethrine bromide. Likewise, this heparin-induced release of PF4 is dependent upon the type of heparin used; low molecular weight heparin fractions and fragments do not cause the PF4 rebound seen with intact heparin. Thus, it would appear that low molecular weight forms of heparin are advantageous in that their in vivo administration would not be mediated by such platelet modulators as PF4

  12. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules

    International Nuclear Information System (INIS)

    Bashkin, P.; Doctrow, S.; Klagsbrun, M.; Svahn, C.M.; Folkman, J.; Vlodavsky, I.

    1989-01-01

    Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125 I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 x 10 12 binding sites/mm 2 ECM with an apparent k D of 610 nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 μg/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 μg/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descement's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response

  13. Removal of glycosaminoglycans from bovine granulosa cells contributes to increased binding of hydrogen-3 heparin

    Energy Technology Data Exchange (ETDEWEB)

    Ax, R.L.; Stodd, C.M.; Boehm, S.K.; Bellin, M.E.

    1986-02-01

    Granulosa cells from small or large bovine follicles were pretreated with enzymes that hydrolyze various glycosaminoglycans, and binding of (/sup 3/H)-heparin to the granulosa was measured. Binding of (/sup 3/H) heparin increased significantly after enzymatic pretreatments with chondroitinase ABC and fungal hyaluronidase, and similar results were obtained with granulosa from small and large follicles. No changes in binding of (/sup 3/H) heparin were detected after hydrolyses with chondroitinase AC and heparinase in either follicle size. Heparitinase, which hydrolyzes heparan sulfate, led to a significant 50% increase in binding of (/sup 3/H) heparin to granulosa from large follicles but was without effect in small follicles. These results suggest that the lower binding of (/sup 3/H) heparin, which has been reported with follicular enlargement, may be due to heparan sulfate occupying or obstructing binding sites for heparin on granulosa from large follicles.

  14. Layer-by-Layer Heparinization of the Cell Surface by Using Heparin-Binding Peptide Functionalized Human Serum Albumin.

    Science.gov (United States)

    Song, Guowei; Hu, Yaning; Liu, Yusheng; Jiang, Rui

    2018-05-20

    Layer-by-layer heparinization of therapeutic cells prior to transplantation is an effective way to inhibit the instant blood-mediated inflammatory reactions (IBMIRs), which are the major cause of early cell graft loss during post-transplantation. Here, a conjugate of heparin-binding peptide (HBP) and human serum albumin (HSA), HBP-HSA, was synthesized by using heterobifunctional crosslinker. After the first heparin layer was coated on human umbilical vein endothelial cells (HUVECs) by means of the HBP-polyethylene glycol-phospholipid conjugate, HBP-HSA and heparin were then applied to the cell surface sequentially to form multiple layers. The immobilization and retention of heparin were analyzed by confocal microscopy and flow cytometry, respectively, and the cytotoxity of HBP-HSA was further evaluated by cell viability assay. Results indicated that heparin was successfully introduced to the cell surface in a layer-by-layer way and retained for at least 24 h, while the cytotoxity of HBP-HSA was negligible at the working concentration. Accordingly, this conjugate provides a promising method for co-immobilization of heparin and HSA to the cell surface under physiological conditions with improved biocompatibility.

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

    International Nuclear Information System (INIS)

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

    1985-01-01

    The binding of the glycosaminoglycan [ 3 H] 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 [ 3 H] heparin were performed to quantitate the binding characteristics of each sample. Scatchard plot analyses indicated a significant difference in the binding affinity for [ 3 H] 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 [ 3 H] heparin did not differ significantly among bulls. Differences in binding affinity of [ 3 H] heparin to bull sperm might be used to predict relative fertility of dairy bulls

  16. Heparin-binding peptide amphiphile supramolecular architectures as platforms for angiogenesis and drug delivery

    Science.gov (United States)

    Chow, Lesleyann W.

    A fascinating phenomenon in nature is the self-assembly of molecules into a functional, hierarchical structure. In the past decade, the Stupp Laboratory has developed several classes of self-assembling biomaterials, one of which is the synthetic peptide amphiphile (PA). Self-assembling PAs are attractive and versatile biomolecules that can be customized for specific applications in regenerative medicine. In particular, a heparin-binding peptide amphiphile (HBPA) containing a specific heparin-binding peptide sequence was used here to induce angiogenesis and serve as a delivery vehicle for growth factors and small hydrophobic molecules. Throughout this dissertation, the HBPA/heparin system is used in different architectures for a variety of regenerative medicine applications. In one aspect of this work, hybrid scaffolds made from HBPA/heparin gelled on a poly(L-lactic acid) (PLLA) fiber mesh were used to promote angiogenesis to facilitate pancreatic islet transplantation for the treatment of type 1 diabetes. Delivery of growth factors with HBPA/PLLA scafflolds increased vessel density in vivo and correlated with improved transplant outcomes in a streptozotocin-induced diabetic mouse model. Soluble HBPA nanofiber architectures were also useful for islet transplantation applications. These nanofibers were used at concentrations below gelation to deliver growth factors into the dense islet cell aggregate, promoting cell survival and angiogenesis in vitro. The nanostructures infiltrated the islets and promoted the retention of heparin and growth factors within the islet. Another interesting growth factor release system discussed here is the HBPA membrane structure. HBPA was found to self-assemble with hyaluronic acid, a large biopolymer found in the body, into macroscopic, hierarchically-ordered membranes. Heparin was incorporated into these membranes and affected the membrane's mechanical properties and growth factor release. Human mesenchymal stem cells were also shown

  17. Endostatin competes with bFGF for binding to heparin-like glycosaminoglycans

    International Nuclear Information System (INIS)

    Reis, Renata C.M.; Schuppan, Detlef; Barreto, Aline C.; Bauer, Michael; Bork, Jens P.; Hassler, Gerda; Coelho-Sampaio, Tatiana

    2005-01-01

    Endostatin is a potent inhibitor of angiogenesis and tumor growth. Here, we used human endothelial cells from lung capillaries to investigate if endostatin competes with the proangiogenic growth factors, bFGF and VEGF, for binding to costimulatory heparan sulfate molecules. Endostatin inhibited 79% and 95% of the increase in proliferation induced by bFGF and VEGF 165 , respectively. The stimulatory effect of VEGF 165 was not affected by the presence of exogenous heparin, while that of bFGF was further enhanced in the presence of up to 0.1 μg/ml heparin. The heparin-binding protein protamine completely blocked bFGF-stimulated proliferation, while it did not affect the response to VEGF 165 . Simultaneous addition of endostatin and protamine led to additive effects both in inhibition of proliferation and induction of apoptosis. Although bFGF was found to bind more strongly to heparin-Sepharose than endostatin, the latter, but not the former, displaced protamine from heparin in solution, which supports the notion that endostatin can compete with bFGF for binding to heparan sulfate in vivo. Taken as a whole, our results demonstrate that there is a direct connection between the dependence of endostatin activity on heparin-like glycosaminoglycans and its ability to antagonize bFGF

  18. Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody

    International Nuclear Information System (INIS)

    Smith, J.W.; Dey, B.; Knauer, D.J.

    1990-01-01

    Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. The authors have previously proposed that the heparin binding site of ATIII residues within a region extending from amino acid residues 114-156. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation

  19. Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

    Science.gov (United States)

    Bolnick, Alan D; Bolnick, Jay M; Kohan-Ghadr, Hamid-Reza; Kilburn, Brian A; Pasalodos, Omar J; Singhal, Pankaj K; Dai, Jing; Diamond, Michael P; Armant, D Randall; Drewlo, Sascha

    2017-06-01

    Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB

  20. The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

    International Nuclear Information System (INIS)

    Sun Jian; Yu Jisheng; Yu Zhiwu; Zha Xiao; Wu Yuqing

    2012-01-01

    Graphial abstract: The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1. Highlights: ► Several driving forces contribute to the interaction between heparin and peptides. ► C-terminal of HPV L1 is a potential candidate for the attachment to host cells. ► The C-terminal peptides of HPV-16 and -18 L1 have different heparin-binding. ► The different heparin-binding provides an explanation for the distinct prevalences. - Abstract: The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.

  1. Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis.

    Science.gov (United States)

    Padmanabhan, Anand; Jones, Curtis G; Bougie, Daniel W; Curtis, Brian R; McFarland, Janice G; Wang, Demin; Aster, Richard H

    2015-01-01

    Antibodies specific for platelet factor 4 (PF4)/heparin complexes are the hallmark of heparin-induced thrombocytopenia and thrombosis (HIT), but many antibody-positive patients have normal platelet counts. The basis for this is not fully understood, but it is believed that antibodies testing positive in the serotonin release assay (SRA) are the most likely to cause disease. We addressed this issue by characterizing PF4-dependent binding of HIT antibodies to intact platelets and found that most antibodies testing positive in the SRA, but none of those testing negative, bind to and activate platelets when PF4 is present without any requirement for heparin (P HIT antibodies recognize PF4 in a complex with heparin, only a subset of these antibodies recognize more subtle epitopes induced in PF4 when it binds to CS, the major platelet glycosaminoglycan. Antibodies having this property could explain "delayed HIT" seen in some individuals after discontinuation of heparin and the high risk for thrombosis that persists for weeks in patients recovered from HIT. © 2015 by The American Society of Hematology.

  2. The N-terminal of a heparin-binding sperm membrane mitogen possess lectin-like sequence

    International Nuclear Information System (INIS)

    Mor, Visesato; Chatterjee, Tapati

    2007-01-01

    Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids- 1 A D T I V A V E L D T Y P N 14 -correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A

  3. 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....... There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D...... of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines...

  4. Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

    Science.gov (United States)

    Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

    2017-11-01

    Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  5. Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

    Science.gov (United States)

    Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-11-01

    Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

  6. Heparin binding sites on Ross River virus revealed by electron cryo-microscopy

    International Nuclear Information System (INIS)

    Zhang Wei; Heil, Marintha; Kuhn, Richard J.; Baker, Timothy S.

    2005-01-01

    Cell surface glycosaminoglycans play important roles in cell adhesion and viral entry. Laboratory strains of two alphaviruses, Sindbis and Semliki Forest virus, have been shown to utilize heparan sulfate as an attachment receptor, whereas Ross River virus (RRV) does not significantly interact with it. However, a single amino acid substitution at residue 218 in the RRV E2 glycoprotein adapts the virus to heparan sulfate binding and expands the host range of the virus into chicken embryo fibroblasts. Structures of the RRV mutant, E2 N218R, and its complex with heparin were determined through the use of electron cryo-microscopy and image reconstruction methods. Heparin was found to bind at the distal end of the RRV spikes, in a region of the E2 glycoprotein that has been previously implicated in cell-receptor recognition and antibody binding

  7. Thermodynamic parameters associated with the binding of adrenaline and norephedrine to heparin

    International Nuclear Information System (INIS)

    Ali-Ali, A.K.; Buchanan, J.D.; Power, D.M.; Butler, J.

    1983-01-01

    Pulse radiolysis was used to determine the thermodynamic parameters (ΔG', ΔH' and ΔS') governing the binding of adrenalin and norephedrine to heparin. The complexes were completely dissociated by increasing concentrations of inorganic salts. Lower concentrations of divalent cations (e.g. Ca 2+ ) were more necessary to affect dissociation than those of monovalent cations (e.g. Na + ). For each interaction, an increase in drug binding occurred as the temperature was increased from ambient. However, a transition temperature was observed (48 degC) above which the drug was progressively released as temperature was increased. These observations probably reflect conformational changes induced in the heparin below and above its melting temperature. (author)

  8. Aggregated forms of bull seminal plasma proteins and their heparin-binding activity

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Ryšlavá, H.; Liberda, J.; Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 69, - (2004), s. 616-630 ISSN 0010-0765 R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : bull seminal plasma proteins * heparin-binding proteins * aggregated forms of proteins Subject RIV: CE - Biochemistry Impact factor: 1.062, year: 2004

  9. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

    Science.gov (United States)

    Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

    2017-05-01

    High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

  10. Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding.

    Science.gov (United States)

    Shi, Shuiliang; Kelly, Brian J; Wang, Congrong; Klingler, Ken; Chan, Albert; Eckert, George J; Trippel, Stephen B

    2018-03-01

    Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Endothelial cell capture of heparin-binding growth factors under flow.

    Directory of Open Access Journals (Sweden)

    Bing Zhao

    2010-10-01

    Full Text Available Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2 was used for both the experiments and simulations. Our computational model was composed of three parts: (1 media flow equations, (2 mass transport equations and (3 cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that

  12. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

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

    Science.gov (United States)

    Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

    2000-04-01

    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 6-15) are shown to be of primary importance in heparin binding.

  14. Isolation of non-heprin-binding and heparin-binding proteins of boar prostate

    Czech Academy of Sciences Publication Activity Database

    Maňásková, Pavla; Liberda, J.; Tichá, M.; Jonáková, Věra

    2002-01-01

    Roč. 770, - (2002), s. 137-143 ISSN 1570-0232. [International Symposium /2./ - Separation in the BioSciences. Praha, 17.09.2001-20.09.2001] R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915 Keywords : isolation * prostatic proteins * heparin Subject RIV: CE - Biochemistry Impact factor: 1.913, year: 2002

  15. Mapping the heparin-binding site of the BMP antagonist gremlin by site-directed mutagenesis based on predictive modelling.

    Science.gov (United States)

    Tatsinkam, Arnold Junior; Mulloy, Barbara; Rider, Christopher C

    2015-08-15

    Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR-1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecular-mass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers. © 2015 Authors; published by Portland Press Limited.

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

    International Nuclear Information System (INIS)

    Al-Ali, A.K.; Buchanan, J.D.; Power, D.M.; Butler, J.

    1983-01-01

    Pulse radiolysis has been used to determine the thermodynamic parameters (ΔG', ΔH' and ΔS') 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 2+ ) than of monovalent cations (e.g. Na + ) 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 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. (author)

  17. Evidence for a saturable mechanism of disappearance of standard heparin in rabbits

    International Nuclear Information System (INIS)

    Boneu, B.; Caranobe, C.; Gabaig, A.M.; Dupouy, D.; Sie, P.; Buchanan, M.R.; Hirsh, J.

    1987-01-01

    This work demonstrates that after bolus intravenous injection standard heparin (SH) disappearance results from the combination of a saturable and a non saturable mechanism. Pharmacokinetics and pharmacodynamics of SH were studied by measuring the disappearance of increasing doses (5 - 500 anti-factor Xa U/kg) of 125 I-heparin and of its biological effects. CPM curves allowed the determination of the half lives of heparin according to the dose injected. The half lives were clearly dose dependent and reached a plateau over 100 anti-factor Xa U/kg. The complex curve which describes the amount of heparin cleared per time unit after any given dose has been resolved into its two components reflecting a saturable and a non saturable mechanism of disappearance. For the doses less than 100 anti-factor Xa U/kg the saturable mechanism was preeminent and the anti-factor Xa activity disappearance followed an exponential pattern; for the doses less than 100 anti-factor Xa U/kg the contribution of the non saturable mechanism becomes more important and the anti-factor Xa activity disappearance followed a concave-convex pattern. Further experiments showed that the heparin half life shortened as the circulating anti-factor Xa activity decreased; this phenomenon may explain the concave-convex pattern of the curve of the anticoagulant effect observed after injection of large doses of SH

  18. Activation of M1 macrophages in sepsis-induced acute kidney injury in response to heparin-binding protein.

    Directory of Open Access Journals (Sweden)

    Li Xing

    Full Text Available In the early stage of sepsis, M1 macrophages result in the production of inflammatory mediators and AKI. Heparin-binding protein (HBP have been shown to play important roles in sepsis-induced AKI. In this study, we investigate the association of HBP with M1 macrophages in sepsis-induced AKI.Male C57BL6 mice were subjected to cecal ligation and puncture (CLP or sham surgery. Biochemical and histological renal damage was assessed. Macrophage infiltration was assessed by immunohistochemistry. RT-PCR was used to investigate the expression of heparin-binding protein (HBP, the inducible nitric oxide synthase (iNOS and arginase 1 (Arg-1 mRNAs. Western blots were performed to assay the tissue levels of HBP, tumor necrosis factor alpha (TNF-α and interleukin-6 (IL-6.High levels of HBP were obviously detected 24 h after sepsis-induced AKI. Heparin inhibited HBP expression during sepsis-induced AKI. The suppression of HBP expression by heparin injection after the establishment of sepsis-induced AKI resulted in a reduction in renal injury severity accompanied with a significant repression of M1 macrophage activation and expression of TNF-α and IL-6.HBP plays an important role in the initial inflammatory reaction associated with sepsis-induced AKI, presumably by activating M1 macrophages and suppressing TNF-α and IL-6 secretion.

  19. Amperometric Sensor for Heparin: Sensing Mechanism and Application in Human Blood Plasma Analysis

    Czech Academy of Sciences Publication Activity Database

    Langmaier, Jan; Olšák, J.; Samcová, E.; Samec, Zdeněk; Trojánek, Antonín

    2006-01-01

    Roč. 18, 13-14 (2006), s. 1329-1338 ISSN 1040-0397 R&D Projects: GA ČR GA203/04/0424 Institutional research plan: CEZ:AV0Z40400503 Keywords : heparin * amperometry * PVC membrane electrode * sensing mechanism * human blood plasma Subject RIV: CG - Electrochemistry Impact factor: 2.444, year: 2006

  20. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  1. Elevated urine levels of heparin-binding protein in children with urinary tract infection.

    Science.gov (United States)

    Kjölvmark, Charlott; Akesson, Per; Linder, Adam

    2012-08-01

    Urinary tract infection (UTI) is a common infection diagnosis in children, and efficient diagnosis and treatment are important to avoid serious complications. In this study we investigated whether urinary levels of neutrophil-derived heparin-binding protein (HBP) can be used as a marker of UTI in children. These results were compared to those of dipstick analysis, interleukin-6 (IL-6) analysis in urine, and bacterial culturing. Seventy-eight children aged 0-18 years with fever and/or symptoms indicating UTI were enrolled in a prospective consecutive study. Urine samples were cultured and analyzed with dipstick, and concentrations of HBP and IL-6 were measured. Fifteen patients were classified as having UTI, 30 patients had fever but were diagnosed with a non-urinary tract infection, and 33 patients had neither UTI nor fever. Using a urine HBP (U-HBP) cut-off level of 32 ng/mL, the sensitivity and specificity for detecting UTI were 93.3 and 90.3 %, respectively. Receiver operating characteristic curves demonstrated that U-HBP levels were a higher specificity indicator of UTI than urine white blood cell counts or urine IL-6 levels; they also showed a higher sensitivity than the results of the urine nitrite test. All patients with significant growth of clinically relevant bacteria had elevated U-HBP levels. The results indicate that rapid analysis of U-HBP can provide helpful guidance in the management of children with suspected UTI.

  2. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    Abstract Background The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infectio...

  3. 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.

  4. 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.

  5. Investigation of a Potential Protective Mechanism Against Heparin-Induced Thrombocytopenia in Patients on Chronic Intermittent Hemodialysis

    Science.gov (United States)

    Tanhehco, Yvette C.; Cuker, Adam; Rudnick, Michael; Sachais, Bruce S.

    2015-01-01

    BACKGROUND Heparin-induced thrombocytopenia (HIT) develops as a result of platelet (PLT) activation by anti-platelet factor 4 (PF4)/heparin complex antibodies. Despite repeated exposure to heparin, patients undergoing chronic intermittent hemodialysis (HD) rarely develop HIT. We investigated the possibility that HD decreases/removes PF4 from PLT surfaces and/or plasma, thereby disfavoring immune complex formation as a mechanism of protection against HIT. MATERIALS AND METHODS We enrolled 20 patients undergoing chronic HD at the Penn Presbyterian Medical Center. Blood samples were drawn before, during and after treatment in the presence and absence of heparin. PF4, PF4/heparin antibody, heparin, and P-selectin levels were measured. RESULTS No patients demonstrated clinical symptoms of HIT. PLT surface PF4 levels decreased and plasma PF4 levels increased concurrently with increase in plasma heparin concentration. In the absence of heparin, PLT surface and plasma PF4 levels were unchanged. Anti-PF4/heparin antibodies, which were non-functional by the serotonin release assay, were detectable in 8 patients. PLT surface P-selectin levels did not change during treatment. CONCLUSIONS Removal of PLT surface and/or plasma PF4 as a mechanism of protection against HIT in patients undergoing HD is not supported by the results of our study, although the transient decrease in PLT surface PF4 in the presence of large amounts of heparin remains a candidate mechanism. The small sample size, single type of dialyzer membrane, and early sampling time points may have led to the inability to detect changes in PF4 levels. Future studies should explore other potential protective mechanisms. PMID:23305841

  6. Degranulating mast cells in fibrotic regions of human tumors and evidence that mast cell heparin interferes with the growth of tumor cells through a mechanism involving fibroblasts

    International Nuclear Information System (INIS)

    Samoszuk, Michael; Kanakubo, Emi; Chan, John K

    2005-01-01

    The purpose of this study was to test the hypothesis that mast cells that are present in fibrotic regions of cancer can suppress the growth of tumor cells through an indirect mechanism involving peri-tumoral fibroblasts. We first immunostained a wide variety of human cancers for the presence of degranulated mast cells. In a subsequent series of controlled in vitro experiments, we then co-cultured UACC-812 human breast cancer cells with normal fibroblasts in the presence or absence of different combinations and doses of mast cell tryptase, mast cell heparin, a lysate of the human mast cell line HMC-1, and fibroblast growth factor-7 (FGF-7), a powerful, heparin-binding growth factor for breast epithelial cells. Degranulating mast cells were localized predominantly in the fibrous tissue of every case of breast cancer, head and neck cancer, lung cancer, ovarian cancer, non-Hodgkin's lymphoma, and Hodgkin's disease that we examined. Mast cell tryptase and HMC-1 lysate had no significant effect on the clonogenic growth of cancer cells co-cultured with fibroblasts. By contrast, mast cell heparin at multiple doses significantly reduced the size and number of colonies of tumor cells co-cultured with fibroblasts, especially in the presence of FGF-7. Neither heparin nor FGF-7, individually or in combination, produced any significant effect on the clonogenic growth of breast cancer cells cultured without fibroblasts. Degranulating mast cells are restricted to peri-tumoral fibrous tissue, and mast cell heparin is a powerful inhibitor of clonogenic growth of tumor cells co-cultured with fibroblasts. These results may help to explain the well-known ability of heparin to inhibit the growth of primary and metastatic tumors

  7. Degranulating mast cells in fibrotic regions of human tumors and evidence that mast cell heparin interferes with the growth of tumor cells through a mechanism involving fibroblasts

    Directory of Open Access Journals (Sweden)

    Kanakubo Emi

    2005-09-01

    Full Text Available Abstract Background The purpose of this study was to test the hypothesis that mast cells that are present in fibrotic regions of cancer can suppress the growth of tumor cells through an indirect mechanism involving peri-tumoral fibroblasts. Methods We first immunostained a wide variety of human cancers for the presence of degranulated mast cells. In a subsequent series of controlled in vitro experiments, we then co-cultured UACC-812 human breast cancer cells with normal fibroblasts in the presence or absence of different combinations and doses of mast cell tryptase, mast cell heparin, a lysate of the human mast cell line HMC-1, and fibroblast growth factor-7 (FGF-7, a powerful, heparin-binding growth factor for breast epithelial cells. Results Degranulating mast cells were localized predominantly in the fibrous tissue of every case of breast cancer, head and neck cancer, lung cancer, ovarian cancer, non-Hodgkin's lymphoma, and Hodgkin's disease that we examined. Mast cell tryptase and HMC-1 lysate had no significant effect on the clonogenic growth of cancer cells co-cultured with fibroblasts. By contrast, mast cell heparin at multiple doses significantly reduced the size and number of colonies of tumor cells co-cultured with fibroblasts, especially in the presence of FGF-7. Neither heparin nor FGF-7, individually or in combination, produced any significant effect on the clonogenic growth of breast cancer cells cultured without fibroblasts. Conclusion Degranulating mast cells are restricted to peri-tumoral fibrous tissue, and mast cell heparin is a powerful inhibitor of clonogenic growth of tumor cells co-cultured with fibroblasts. These results may help to explain the well-known ability of heparin to inhibit the growth of primary and metastatic tumors.

  8. Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate.

    Science.gov (United States)

    Vanpouille, Christophe; Denys, Agnès; Carpentier, Mathieu; Pakula, Rachel; Mazurier, Joël; Allain, Fabrice

    2004-09-01

    Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requirement of HS in pro-adhesive activity of CyPB. By using a strategy based on gel mobility shift assays with fluorophore-labelled oligosaccharides, we demonstrated that the minimal heparin unit required for efficient binding of CyPB is an octasaccharide. The mutants CyPB(KKK-) [where KKK- refers to the substitutions K3A(Lys3-->Ala)/K4A/K5A] and CyPB(DeltaYFD) (where Tyr14-Phe-Asp16 has been deleted) failed to interact with octasaccharides, confirming that the Y14FD16 and K3KK5 clusters are required for CyPB binding. Molecular modelling revealed that both clusters are spatially arranged so that they may act synergistically to form a binding site for the octasaccharide. We then demonstrated that heparin-derived octasaccharides and higher degree of polymerization oligosaccharides inhibited the interaction between CyPB and fluorophore-labelled HS chains purified from T-lymphocytes, and strongly reduced the HS-dependent pro-adhesive activity of CyPB. However, oligosaccharides or heparin were unable to restore adhesion of heparinase-treated T-lymphocytes, indicating that HS has to be present on the cell membrane to support the pro-adhesive activity of CyPB. Altogether, these results demonstrate that the octasaccharide is likely to be the minimal length unit required for efficient binding of CyPB to cell surface HS and consequent HS-dependent cell responses.

  9. A triad of lys12, lys41, arg78 spatial domain, a novel identified heparin binding site on tat protein, facilitates tat-driven cell adhesion.

    Directory of Open Access Journals (Sweden)

    Jing Ai

    Full Text Available Tat protein, released by HIV-infected cells, has a battery of important biological effects leading to distinct AIDS-associated pathologies. Cell surface heparan sulfate protoglycans (HSPGs have been accepted as endogenous Tat receptors, and the Tat basic domain has been identified as the heparin binding site. However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat-heparin interactions suggest that the basic domain is not the sole Tat heparin binding site. In the current study, an approach integrating computational modeling, mutagenesis, biophysical and cell-based assays was used to elucidate a novel, high affinity heparin-binding site: a Lys12, Lys41, Arg78 (KKR spatial domain. This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization. The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events.

  10. Pharmacological inhibition of heparin-binding EGF-like growth factor promotes peritoneal angiogenesis in a peritoneal dialysis rat model.

    Science.gov (United States)

    Li, Zhenyuan; Yan, Hao; Yuan, Jiangzi; Cao, Liou; Lin, Aiwu; Dai, Huili; Ni, Zhaohui; Qian, Jiaqi; Fang, Wei

    2018-04-01

    Molecular mechanisms of peritoneal dialysis (PD) ultrafiltration failure, peritoneal neo-angiogenesis, and fibrosis remain to be determined. We aimed to determine the role of heparin-binding EGF-like growth factor (HB-EGF) inhibition on angiogenesis of peritoneal membrane in a PD rat model. 32 male Wistar rats were assigned into (1) control group; (2) uremic non-PD group: subtotal nephrectomy-induced uremic rats without PD; (3) uremic rats subjected to PD: uremic rats that were dialyzed with Dianeal ® for 4 weeks; (4) CRM 197 group: dialyzed uremic rats were supplemented with CRM197, a specific HB-EGF inhibitor. Peritoneal transport function was examined by peritoneal equilibration test. Expression of HB-EGF and EGFR in peritoneal samples were examined by real-time PCR, immunohistochemical staining, and western blot. Progressive angiogenesis and fibrosis were observed in uremic PD rats, and there were associated with decreased net ultrafiltration (nUF), increased permeability of peritoneal membrane, and reduced expression of HB-EGF and EGFR protein and mRNA in uremic PD rats compared to uremic non-PD or control groups (both p CRM197 significantly induced peritoneal membrane permeability, decreased nUF, increased higher vessel density, and reduced pericyte count compared to that of uremic PD rats. The levels of HB-EGF and EGFR expression negatively correlated with vessel density in peritoneal membrane (both p < 0.001). PD therapy was associated with peritoneal angiogenesis, functional deterioration, and downregulation of HB-EGF/EGFR. Pharmacological inhibition of HB-EGF promoted PD-induced peritoneal angiogenesis and fibrosis and ultrafiltration decline, suggesting that HB-EGF downregulation contributes to peritoneal functional deterioration in the uremic PD rat model.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yar, Muhammad, E-mail: drmyar@ciitlahore.edu.pk [Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000 (Pakistan); Shahzad, Sohail [Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000 (Pakistan); Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur 63100 (Pakistan); Shahzadi, Lubna [Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000 (Pakistan); Shahzad, Sohail Anjum [Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060 (Pakistan); Mahmood, Nasir [Department of Allied Health Sciences and Chemical Pathology, University of Health Sciences, Lahore (Pakistan); Department of Human Genetics and Molecular Biology, University of Health Sciences, Lahore (Pakistan); Chaudhry, Aqif Anwar [Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000 (Pakistan); Rehman, Ihtesham ur [Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000 (Pakistan); Materials Science and Engineering, North Campus, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom); MacNeil, Sheila, E-mail: s.macneil@sheffield.ac.uk [Materials Science and Engineering, North Campus, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom)

    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 24 h 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 ({sup 1}H 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 24 h 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. - Highlights: • Chitosan based hydrogels were designed to stimulate angiogenesis. • Two new derivatives of chitosan were produced using a Mannich type reaction. • Blending a chitosan derivative with PVA gave a porous biocompatible hydrogel. • Heparin bound to the hydrogel on immersion changing its morphology. • Heparin loaded hydrogel stimulated blood vessel formation in a chick model.

  12. Role of the A+ helix in heparin binding to protein C inhibitor

    NARCIS (Netherlands)

    Elisen, M. G.; Maseland, M. H.; Church, F. C.; Bouma, B. N.; Meijers, J. C.

    1996-01-01

    Interactions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The

  13. N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

    International Nuclear Information System (INIS)

    Tang, Nan-Hong; Chen, Yan-Lin; Wang, Xiao-Qian; Li, Xiu-Jin; Wu, Yong; Zou, Qi-Lian; Chen, Yuan-Zhong

    2010-01-01

    Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved. The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively. Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity. Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents

  14. 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 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...

  15. 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

    Heparin-binding protein (HBP), an evolutionary ancient and biologically highly important molecule in inflammation, is an inactive serine protease due to mutations in the catalytic triad. The histidine (position 41) in the conserved sequence TAAHC is mutated to serine and this sequence (TAASC) pla...

  16. Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

    Czech Academy of Sciences Publication Activity Database

    Calvo, E.; Mizurini, D.M.; Sa-Nunes, A.; Ribeiro, J.M.C.; Andersen, J. F.; Mans, B.J.; Monteiro, R.Q.; Kotsyfakis, Michalis; Francischetti, I.M.B.

    2011-01-01

    Roč. 286, č. 32 (2011), 27998-28010 ISSN 0021-9258 Institutional research plan: CEZ:AV0Z60220518 Keywords : serpin * mosquito * Aedes albopictus * phospholipids * Factor Xa * heparin * binding affinity * coagulation * thrombus * bleeding Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 4.773, year: 2011

  17. Covalent Binding of Heparin to Functionalized PET Materials for Improved Haemocompatibility

    Directory of Open Access Journals (Sweden)

    Metod Kolar

    2015-03-01

    Full Text Available The hemocompatibility of vascular grafts made from poly(ethylene terephthalate (PET is insufficient due to the rapid adhesion and activation of blood platelets that occur upon incubation with whole blood. PET polymer was treated with NHx radicals created by passing ammonia through gaseous plasma formed by a microwave discharge, which allowed for functionalization with amino groups. X-ray photoelectron spectroscopy characterization using derivatization with 4-chlorobenzaldehyde indicated that approximately 4% of the –NH2 groups were associated with the PET surface after treatment with the gaseous radicals. The functionalized polymers were coated with an ultra-thin layer of heparin and incubated with fresh blood. The free-hemoglobin technique, which is based on the haemolysis of erythrocytes, indicated improved hemocompatibility, which was confirmed by imaging the samples using confocal optical microscopy. A significant decrease in number of adhered platelets was observed on such samples. Proliferation of both human umbilical vein endothelial cells and human microvascular endothelial cells was enhanced on treated polymers, especially after a few hours of cell seeding. Thus, the technique represents a promising substitute for wet-chemical modification of PET materials prior to coating with heparin.

  18. Heparin: The Silver Bullet of Aneurysmal Subarachnoid Hemorrhage?

    Directory of Open Access Journals (Sweden)

    Nicolas K. Khattar

    2018-03-01

    Full Text Available Various neurological diseases have recently been associated with neuroinflammation and worsening outcomes. Subarachnoid hemorrhage has been shown to generate a potent neuroinflammatory response. Heparin is a potential effective anti-inflammatory agent to prevent initial injury as well as delayed neurological decline. Different mechanisms of action for heparin have been proposed including, but not limited to the binding and neutralization of oxyhemoglobin, decreased transcription and signal transduction of endothelin-1, inhibition of binding to vessel wall selectins and vascular leakage into the subarachnoid space as well as direct binding and neutralization of inflammatory molecules. With a reasonably safe side-effect profile, heparin has shown significant promise in small series in human studies of aneurysmal subarachnoid hemorrhage in decreasing both initial and delayed neurological injury. Further studies are needed to validate various neuroprotective features of heparin in subarachnoid hemorrhage as well as other disease states.

  19. 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.

  20. 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.

  1. Enantiomeric and Diastereomeric Self-Assembled Multivalent (SAMul) Nanostructures - Understanding the Effects of Chirality on Binding to Polyanionic Heparin and DNA.

    Science.gov (United States)

    Thornalley, Kiri; Laurini, Erik; Pricl, Sabrina; Smith, David K

    2018-05-15

    A family of four self-assembling lipopeptides containing Ala-Lys peptides attached to a C16 aliphatic chain was synthesised. These compounds form two enantiomeric pairs that bear a diastereomeric relationship to one another (C16-L-Ala-L-Lys/C16-D-Ala-D-Lys) and (C16-D-Ala-L-Lys/C16-L-Ala-D-Lys). These diastereomeric pairs have very different critical micelle concentrations (CMCs), with LL/DD < DL/LD suggesting more effective assembly of the former. The self-assembled multivalent (SAMul) systems bind biological polyanions as result of the cationic lysine groups on their surfaces. Polyanion binding was investigated using dye displacement assays and isothermal calorimetry (ITC). On heparin binding, there was no significant enantioselectivity, but there was a binding preference for the diastereomeric assemblies with lower CMCs. Conversely, on binding DNA, there was a significant enantioselective preference for systems displaying D-lysine ligands, with a further slight preference for attachment to L-alanine, with the CMC being irrelevant. Binding to adaptive, ill-defined heparin has a large favourable entropic term, suggesting it depends primarily on the cationic SAMul nanostructure maximising surface contact with heparin, which can adapt, displacing solvent and other ions. Conversely, binding to well-defined, shape-persistent DNA has a larger favourable enthalpic term, and combined with the enantioselectivity, this allows us to suggest that its SAMul binding is based on optimised individual electrostatic interactions at the molecular level, with a preference for binding to D-lysine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

    DEFF Research Database (Denmark)

    Jeong, J; Han, I; Lim, Y

    2001-01-01

    of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C...

  3. Heparin kinetics

    International Nuclear Information System (INIS)

    Swart, C.A.M. de.

    1983-01-01

    The author has studied the kinetics of heparin and heparin fractions after intravenous administration in humans and in this thesis the results of this study are reported. Basic knowledge about the physico-chemical properties of heparin and its interactions with proteins resulting in anticoagulant and lipolytic effects are discussed in a review (chapter II), which also comprises some clinical aspects of heparin therapy. In chapter III the kinetics of the anticoagulant effect are described after intravenous administration of five commercial heparin preparations. A mathematical model is presented that fits best to these kinetics. The kinetics of the anticoagulant and lipolytic effects after intravenous injection of various 35 S-radiolabelled heparin fractions and their relationship with the disappearance of the radiolabel are described in chapter IV. Chapter V gives a description of the kinetics of two radiolabels after injection of in vitro formed complexes consisting of purified, 125 I-radiolabelled antithrombin III and various 35 S-radiolabelled heparin fractions. (Auth.)

  4. Overexpression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor Mediates Liver Fibrosis in Transgenic Mice.

    Science.gov (United States)

    Guo, Yongze; Ding, Qian; Chen, Lei; Ji, Chenguang; Hao, Huiyao; Wang, Jia; Qi, Wei; Xie, Xiaoli; Ma, Junji; Li, Aidi; Jiang, Xiaoyu; Li, Xiaotian; Jiang, Huiqing

    2017-08-01

    The role of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver fibrosis is not clear and is sometimes even contradictory. To clarify this role, a HB-EGF transgenic (Tg) mouse model was, for the first time, used to evaluate the functions of HB-EGF in liver fibrosis. For the in vivo study, carbon tetrachloride injection and bile duct ligation treatment were used to induce liver fibrosis in HB-EGF Tg mice and wild-type (WT) mice, respectively. Primary hepatic satellite cells (HSCs) were isolated from HB-EGF Tg and WT mice for the in vitro study. Compared with the WT mice, HB-EGF Tg mice were shown to develop more severe liver fibrosis when treated with carbon tetrachloride or bile duct ligation, with increased matrix metalloproteinases 13 activity and enhanced expression of fibrogenic genes including α-smooth muscle actin and collagen I. HB-EGF gene transfer led to an increase in proliferation and a decrease in apoptosis in primary HSCs. The ERK signaling pathway was more highly activated in primary HSCs from HB-EGF Tg mice than in those from WT mice. Our investigation confirmed the profibrotic effect of HB-EGF on the liver using a Tg mouse model. This result may contribute to the elucidation of HB-EGF as a therapeutic target in liver fibrosis. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

  5. 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

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

    International Nuclear Information System (INIS)

    Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Ángel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

    2006-01-01

    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. 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. 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 G 2 /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. 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 proliferation. Our data suggest that GAG-hed could have

  7. Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor.

    Science.gov (United States)

    Armant, D Randall; Kilburn, Brian A; Petkova, Anelia; Edwin, Samuel S; Duniec-Dmuchowski, Zophia M; Edwards, Holly J; Romero, Roberto; Leach, Richard E

    2006-02-01

    Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is down regulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O(2) ( approximately 2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O(2) upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O(2), signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinase-mediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O(2) and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O(2) rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts.

  8. The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

    Science.gov (United States)

    Luo, Zhao; Cao, Xue-Wei; Li, Chen; Wu, Miao-Dan; Yang, Xu-Zhong; Zhao, Jian; Wang, Fu-Jun

    2016-11-01

    Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  9. Heparin Interaction with the Primed Polymorphonuclear Leukocyte CD11b Induces Apoptosis and Prevents Cell Activation

    Directory of Open Access Journals (Sweden)

    Meital Cohen-Mazor

    2015-01-01

    Full Text Available Heparin is known to have anti-inflammatory effects, yet the mechanisms are not completely understood. In this study, we tested the hypothesis that heparin has a direct effect on activated polymorphonuclear leukocytes (PMNLs, changing their activation state, and can explain its anti-inflammatory effect. To test our hypothesis, we designed both in vitro and ex vivo studies to elucidate the mechanism by which heparin modulates PMNL functions and therefore the inflammatory response. We specifically tested the hypothesis that priming of PMNLs renders them more susceptible to heparin. Amplified levels of CD11b and increased rate of superoxide release manifested PMNL priming. Increase in cell priming resulted in a dose-dependent increase in heparin binding to PMNLs followed by augmented apoptosis. Blocking antibodies to CD11b inhibited heparin binding and abolished the apoptotic response. Moreover, heparin caused a significant dose-dependent decrease in the rate of superoxide release from PMNLs, which was blunted by blocking antibodies to CD11b. Altogether, this study shows that the interaction of heparin with the PMNL CD11b results in cell apoptosis and explains heparin’s anti-inflammatory effects.

  10. Cellular adhesion responses to the heparin-binding (HepII) domain of fibronectin require heparan sulfate with specific properties

    DEFF Research Database (Denmark)

    Mahalingam, Yashithra; Gallagher, John T; Couchman, John R

    2006-01-01

    of fibronectin (HepII domain) through its HS chains. The fine structure of HS is critical to growth factor responses, and whether this extends to matrix ligands is unknown but is suggested from in vitro experiments. Cell attachment to HepII showed that heparin oligosaccharides of >or=14 sugar residues were...

  11. The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E.

    Science.gov (United States)

    Harris, Edward N; Weigel, Paul H

    2008-08-01

    The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341-17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. (125)I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.

  12. 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......M), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin–agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.......2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing...

  13. New Mechanisms of Mercury Binding to Peat

    Science.gov (United States)

    Nagy, K. L.; Manceau, A.; Gasper, J. D.; Ryan, J. N.; Aiken, G. R.

    2007-12-01

    Mercury can be immobilized in the aquatic environment by binding to peat, a solid form of natural organic matter. Binding mechanisms can vary in strength and reversibility, and therefore will control concentrations of bioreactive mercury, may explain rates of mercury methylation, and are important for designing approaches to improve water quality using natural wetlands or engineered phytoremediation schemes. In addition, strong binding between mercury and peat is likely to result in the fixation of mercury that ultimately resides in coal. The mechanisms by which aqueous mercury at low concentrations reacts with both dissolved and solid natural organic matter remain incompletely understood, despite recent efforts. We have identified three distinct binding mechanisms of divalent cationic mercury to solid peats from the Florida Everglades using EXAFS spectroscopic data (FAME beamline, European Synchrotron Radiation Facility (ESRF)) obtained on experimental samples as compared to relevant references including mercury-bearing solids and mercury bound to various organic molecules. The proportions of the three molecular configurations vary with Hg concentration, and two new configurations that involve sulfur ligands occur at Hg concentrations up to about 4000 ppm. The binding mechanism at the lowest experimental Hg concentration (60-80 ppm) elucidates published reports on the inhibition of metacinnabar formation in the presence of Hg-bearing solutions and dissolved natural organic matter, and also, the differences in extent of mercury methylation in distinct areas of the Florida Everglades.

  14. M/sub r/ 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor

    International Nuclear Information System (INIS)

    Moscatelli, D.; Joseph-Silverstein, J.; Manejias, R.; Rifkin, D.B.

    1987-01-01

    A M/sub r/ 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig grain along with the typical M/sub r/ 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The M/sub r/ 25,000 form, like the M/sub r/ 18,000 form was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The M/sub r/ 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentration of 0.1-10 ngml, the same range that was effective for guinea pig and human M/sub r/ 18,000 bFGFs. The binding of human 125 I-labeled bFGF to baby hamster kidney cells is inhibited equally by the M/sub r/ 25,000 guinea pig protein and the M/sub r/ 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the M/sub r/ 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the M/sub r/ 25,000 and M/sub r/ 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the M/sub r/ 25,000 guinea pig bFGF was converted to a M/sub r/ 18,000 protein. These results show that the two molecules are closely related and suggest that the M/sub r/ 25,000 protein shares substantial homology with the M/sub r/ 18,000 bFGF

  15. A Prevention of Pre-eclampsia with the Use of Acetylsalicylic Acid and Low-molecular Weight Heparin - Molecular Mechanisms.

    Science.gov (United States)

    Darmochwal-Kolarz, Dorota; Kolarz, Bogdan; Korzeniewski, Michal; Kimber-Trojnar, Zaneta; Patro-Malysza, Jolanta; Mierzynski, Radzisław; Przegalinska-Kałamucka, Monika; Oleszczuk, Jan

    Pre-eclampsia appears to be the main cause for the maternal and fetal morbidity and mortality. Pregnant women with pre-eclampsia are more likely to be threatened with conditions which potentially may be lethal, such as: disseminated intravascular coagulation, cerebral hemorrhage, liver and renal failure. Pregnancy complicated with pre-eclampsia is also associated with a greater risk for iatrogenic prematurity, intrauterine growth retardation, premature abruption of placenta, and even intrauterine fetal death. In the majority of cases the reasons for arterial hypertension among pregnant women remain obscure. For the past decades, there were many abortive attempts in the use of some microelements, vitamins or specific diets, such as polyunsaturated fatty acids, for the prophylaxis of pre-eclampsia. Recently, it has been shown that a prevention of pre-eclampsia with the use of a lowmolecular- weight heparins (LMWHs) and acetylsalicylic acid (ASA) could considerably reduce the frequency of preeclampsia. In this review, we present the studies concerning the applications of LMWHs and aspirin in the prophylaxis of pre-eclampsia and some important data about the mechanisms of anti-inflammatory actions of LMWHs and ASA.

  16. 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.

  17. 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.

  18. 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.

  19. Structural characterization of pharmaceutical heparins prepared from different animal tissues.

    Science.gov (United States)

    Fu, Li; Li, Guoyun; Yang, Bo; Onishi, Akihiro; Li, Lingyun; Sun, Peilong; Zhang, Fuming; Linhardt, Robert J

    2013-05-01

    Although most pharmaceutical heparin used today is obtained from porcine intestine, heparin has historically been prepared from bovine lung and ovine intestine. There is some regulatory concern about establishing the species origin of heparin. This concern began with the outbreak of mad cow disease in the 1990s and was exacerbated during the heparin shortage in the 2000s and the heparin contamination crisis of 2007-2008. Three heparins from porcine, ovine, and bovine were characterized through state-of-the-art carbohydrate analysis methods with a view profiling their physicochemical properties. Differences in molecular weight, monosaccharide and disaccharide composition, oligosaccharide sequence, and antithrombin III-binding affinity were observed. These data provide some insight into the variability of heparins obtained from these three species and suggest some analytical approaches that may be useful in confirming the species origin of a heparin active pharmaceutical ingredient. Copyright © 2013 Wiley Periodicals, Inc.

  20. Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds

    Science.gov (United States)

    Nguyen, Thi-Huong; Greinacher, Andreas; Delcea, Mihaela

    2015-05-01

    Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly understood. In this study, single molecule force spectroscopy (SMFS) is utilized to characterize the interaction of PF4 with heparins of defined length (5-, 6-, 8-, 12-, and 16-mers). Analysis of the force-distance curves shows that PF4/heparin binding strength rises with increasing heparin length. In addition, two binding pathways in the PF4/short heparins (=8-mers) are identified. We provide a model for the PF4/heparin complexes in which short heparins bind to one PF4 tetramer, while long heparins bind to two PF4 tetramers. We propose that the interaction between long heparins and PF4s is not only due to charge differences as generally assumed, but also due to hydrophobic interaction between two PF4s which are brought close to each other by long heparin. This complicated interaction induces PF4/heparin complexes more stable than other ligand-receptor interactions. Our results also reveal that the boundary between antigenic and non-antigenic heparins is between 8- and 12-mers. These observations are particularly important to understand processes in which PF4-heparin interactions are involved and to develop new heparin-derived drugs.Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly

  1. 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.

  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.

    2015-01-01

    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 stimulated not only 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. PMID:26500021

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

    International Nuclear Information System (INIS)

    Takemura, Takayo; Yoshida, Yuichi; Kiso, Shinichi; Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro; Imai, Yasuharu; Higashiyama, Shigeki; Iwamoto, Ryo; Mekada, Eisuke; Takehara, Tetsuo

    2013-01-01

    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

  4. Nuclear androgen receptors in human prostatic tissue. Extraction with heparin and estimation of the number of binding sites with different methods

    International Nuclear Information System (INIS)

    Foekens, J.A.; Bolt-de Vries, J.; Mulder, E.; Blankenstein, M.A.; Schroeder, F.H.; Molen, H.J. van der

    1981-01-01

    A procedure for the estimation of nuclear androgen receptors in benign prostatic hyperplastic tissue is described, which employs extraction of receptors from nuclei with buffers containing heparin. Extraction of a nuclear pellet with a heparin-containing (1 g/l) buffer appeared to have definite advantages over 0.4 mol/l KCl extraction. Heparin appeared to be twice as efficient in extracting androgen receptors. In addition aggregated receptor proteins, formed after storage at -80 0 C, were partly deaggregated by heparin. Specific isolation of the androgen receptor was performed using either agar gel electrophoresis, protamine sulphate precipitation or LH-20 gel filtration. A comparison was made between the amounts of estimated receptors with these different techniques. Protamine sulphate precipitation resulted in the highest estimates of receptor-bound 5α-[ 3 H]dihydrotestosterone ( 3 H-DHT). Treatment of the labelled nuclear extracts with a charcoal suspension prior to the receptor assay resulted in lower amounts of estimated androgen receptors. A method for routine evaluation of nuclear androgen receptors in prostatic tissue has been evaluated, which involves extraction of nuclear pellets with a heparin-containing (1 g/l) buffer, exchange labelling of the nuclear extracts for 20 h at 10 0 C and quantification of the receptors with protamine sulphate precipitation. (Auth.)

  5. 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.

  6. Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor.

    OpenAIRE

    Moscatelli, D; Joseph-Silverstein, J; Manejias, R; Rifkin, D B

    1987-01-01

    A Mr 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig brain along with the typical Mr 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The Mr 25,000 form, like the Mr 18,000 form, was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The Mr 25,000 guinea pig protein stimulated plasminogen activator pro...

  7. 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.

  8. M2 macrophages induce ovarian cancer cell proliferation via a heparin binding epidermal growth factor/matrix metalloproteinase 9 intercellular feedback loop.

    Science.gov (United States)

    Carroll, Molly J; Kapur, Arvinder; Felder, Mildred; Patankar, Manish S; Kreeger, Pamela K

    2016-12-27

    In ovarian cancer, a high ratio of anti-inflammatory M2 to pro-inflammatory M1 macrophages correlates with poor patient prognosis. The mechanisms driving poor tumor outcome as a result of the presence of M2 macrophages in the tumor microenvironment remain unclear and are challenging to study with current techniques. Therefore, in this study we utilized a micro-culture device previously developed by our lab to model concentrated paracrine signaling in order to address our hypothesis that interactions between M2 macrophages and ovarian cancer cells induce tumor cell proliferation. Using the micro-culture device, we determined that co-culture with M2-differentiated primary macrophages or THP-1 increased OVCA433 proliferation by 10-12%. This effect was eliminated with epidermal growth factor receptor (EGFR) or heparin-bound epidermal growth factor (HB-EGF) neutralizing antibodies and HBEGF expression in peripheral blood mononuclear cells from ovarian cancer patients was 9-fold higher than healthy individuals, suggesting a role for HB-EGF in tumor progression. However, addition of HB-EGF at levels secreted by macrophages or macrophage-conditioned media did not induce proliferation to the same extent, indicating a role for other factors in this process. Matrix metalloproteinase-9, MMP-9, which cleaves membrane-bound HB-EGF, was elevated in co-culture and its inhibition decreased proliferation. Utilizing inhibitors and siRNA against MMP9 in each population, we determined that macrophage-secreted MMP-9 released HB-EGF from macrophages, which increased MMP9 in OVCA433, resulting in a positive feedback loop to drive HB-EGF release and increase proliferation in co-culture. Identification of multi-cellular interactions such as this may provide insight into how to most effectively control ovarian cancer progression.

  9. Oscillatory mechanisms of process binding in memory.

    Science.gov (United States)

    Klimesch, Wolfgang; Freunberger, Roman; Sauseng, Paul

    2010-06-01

    A central topic in cognitive neuroscience is the question, which processes underlie large scale communication within and between different neural networks. The basic assumption is that oscillatory phase synchronization plays an important role for process binding--the transient linking of different cognitive processes--which may be considered a special type of large scale communication. We investigate this question for memory processes on the basis of different types of oscillatory synchronization mechanisms. The reviewed findings suggest that theta and alpha phase coupling (and phase reorganization) reflect control processes in two large memory systems, a working memory and a complex knowledge system that comprises semantic long-term memory. It is suggested that alpha phase synchronization may be interpreted in terms of processes that coordinate top-down control (a process guided by expectancy to focus on relevant search areas) and access to memory traces (a process leading to the activation of a memory trace). An analogous interpretation is suggested for theta oscillations and the controlled access to episodic memories. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  10. A sequential binding mechanism in a PDZ domain

    DEFF Research Database (Denmark)

    Chi, Celestine N; Bach, Anders; Engström, Åke

    2009-01-01

    that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants...

  11. 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

    International Nuclear Information System (INIS)

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung; Park, Yoon-Jeong; Chung, Chong-Pyoung

    2012-01-01

    Highlights: ► HBP sequence identified from HB-EGF has cell penetration activity. ► HBP inhibits the NF-κB dependent inflammatory responses. ► HBP directly blocks phosphorylation and degradation of IκBα. ► HBP inhibits nuclear translocation of NF-κB 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-α and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-α and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-κB-dependent inflammatory responses by directly blocking the phosphorylation and degradation of IκBα and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-κB. 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.

  12. 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.

  13. Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters.

    Science.gov (United States)

    Ziegler, André; Seelig, Joachim

    2004-01-01

    The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47-57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 +/- 2 kDa) has 6.3 +/- 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K0 = (6.0 +/- 0.6) x 10(5) M(-1) and a reaction enthalpy deltaHpep0 = -4.6 +/- 1.0 kcal/mol at 28 degrees C. The binding affinity, deltaGpep0, is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of deltaCp0 = +135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6-7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal

  14. Heparin and heparin-induced thrombocytopenia

    African Journals Online (AJOL)

    2007-06-15

    Jun 15, 2007 ... Heparin is one of the most widely used drugs. It is used routinely for treatment and thromboprophylaxis in a broad spectrum of conditions including venous thromboembolism, atrial fibrillation, acute coronary syndromes, peripheral vascular disease and to maintain the patency of indwelling catheters and ...

  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. Heparin-Induced Thrombocytopenia

    Science.gov (United States)

    ... HIT information card. Early identification of HIT and avoidance of inappropriate heparin therapy can help promote a ... Heart Association is a qualified 501(c)(3) tax-exempt organization. *Red Dress™ DHHS, Go Red™ AHA; ...

  17. 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, Jesú s; Nijland, Reindert; Rodriguez, Francisco J.; Bosma, Tom N. P.; Tommassen, Jan

    2012-01-01

    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.

  18. 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.

  19. Severe heparin osteoporosis in pregnancy.

    OpenAIRE

    Griffiths, H. T.; Liu, D. T.

    1984-01-01

    A case of severe osteoporosis following administration of low dose subcutaneous heparin in pregnancy is reported. Possible reasons for the condition are suggested which caution against the indiscriminate use of subcutaneous heparin in pregnancy.

  20. Binding Mechanisms in Selective Laser Sintering and Selective Laser Melting

    NARCIS (Netherlands)

    Kruth, J.P.; Mercelis, P.; Van Vaerenbergh, J.; van Vaerenbergh, J.; Froyen, L.; Rombouts, M.

    2005-01-01

    Purpose – This paper provides an overview of the different binding mechanisms in selective laser sintering (SLS) and selective laser melting (SLM), thus improving the understanding of these processes. Design/methodology/approach – A classification of SLS/SLM processes was developed, based on the

  1. 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

  2. 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

  3. N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Heparin Binding Ability

    Directory of Open Access Journals (Sweden)

    Chiung-Fang Chang

    2016-06-01

    Full Text Available R-spondin 1 (Rspo1 plays an essential role in stem cell biology by potentiating Wnt signaling activity. Despite the fact that Rspo1 holds therapeutic potential for a number of diseases, its biogenesis is not fully elucidated. All Rspo proteins feature two amino-terminal furin-like repeats, which are responsible for Wnt signal potentiation, and a thrombospondin type 1 (TSR1 domain that can provide affinity towards heparan sulfate proteoglycans. Using chemical inhibitors, deglycosylase and site-directed mutagenesis, we found that human Rspo1 and Rspo3 are both N-glycosylated at N137, a site near the C-terminus of the furin repeat 2 domain, and Rspo2 is N-glycosylated at N160, a position near the N-terminus of TSR1 domain. Elimination of N-glycosylation at these sites affects their accumulation in media but have no effect on the ability towards heparin. Introduction of the N-glycosylation site to Rspo2 mutant at the position homologous to N137 in Rspo1 restored full glycosylation and rescued the accumulation defect of nonglycosylated Rspo2 mutant in media. Similar effect can be observed in the N137 Rspo1 or Rspo3 mutant engineered with Rspo2 N-glycosylation site. The results highlight the importance of N-glycosylation at these two positions in efficient folding and secretion of Rspo family. Finally, we further showed that human Rspo1 is subjected to endoplasmic reticulum (ER quality control in N-glycan-dependent manner. While N-glycan of Rspo1 plays a role in its intracellular stability, it had little effect on secreted Rspo1. Our findings provide evidence for the critical role of N-glycosylation in the biogenesis of Rspo1.

  4. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  5. A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

    Directory of Open Access Journals (Sweden)

    Brian Krumm

    Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

  6. Antioxidant mechanism of milk mineral-high-affinity iron binding.

    Science.gov (United States)

    Allen, K; Cornforth, D

    2007-01-01

    Milk mineral (MM), a by-product of whey processing, is an effective antioxidant in meat systems, but the antioxidant mechanism has not been established. MM has been postulated to chelate iron and prevent iron-catalysis of lipid oxidation. The objective of this research was to examine this putative mechanism. MM was compared to sodium tripolyphosphate (STPP), calcium phosphate monobasic (CPM), and calcium pyrophosphate (CPP) to determine iron-binding capacity, sample solubility, and eluate soluble phosphorus after treating samples with a ferrous chloride standard. Scanning electron microscopy with energy-dispersive X-ray analysis was used to localize minerals on iron-treated MM particle surfaces. Histochemical staining for calcium was performed on raw and cooked ground beef samples with added MM. MM bound more iron per gram (P compounds, and was much less soluble (P iron across the MM particle surface, directly demonstrating iron binding to MM particles. Unlike other common chelating agents, such as STPP and citrate, histochemical staining demonstrated that MM remained insoluble in ground beef, even after cooking. The ability of MM to bind iron and remain insoluble may enhance its antioxidant effect by removing iron ions from solution. However, MM particles must be small and well distributed in order to adequately bind iron throughout the food system.

  7. Influence of heparin on radioimmunological assay of ACTH

    International Nuclear Information System (INIS)

    Dupouy, J.P.; Godaut, M.; Chatelain, A.

    1986-01-01

    1 - Heparin traps plasma ACTH, promoting the formation of aggregates with apparent high molecular weight as shown by chromatography on Sephadex G 50 fine columns. The percentage of 125 I-ACTH which appeared in the void volume of the column, increased linearly with the log. dose of heparin. 2 - Heparin at concentrations of up to 100 IU/ml does not impair ACTH adsorption on either silicic acid or Quso G 32 as well as further elution by acetic acid/acetone/water (I: 40: 59; V/V) or HCl O.I N. Silicic acid traps selectively ACTH but not heparin. 3 - Heparin interferes with direct RIA-ACTH in the plasma by decreasing 125 I-ACTH binding to the antibodies and modifying the slope of the standard curve. Unsuitable artefacts induced by heparin, as overestimation or underestimation of plasma ACTH levels by RIA, can be avoid by previous hormone extraction from heparinized plasmas. Such results emphasized the importance of the sample preparation in order to obtain consistent results [fr

  8. 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.

  9. 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.

  10. Heparin: Past, Present, and Future.

    Science.gov (United States)

    Oduah, Eziafa I; Linhardt, Robert J; Sharfstein, Susan T

    2016-07-04

    Heparin, the most widely used anticoagulant drug in the world today, remains an animal-derived product with the attendant risks of adulteration and contamination. A contamination crisis in 2007-2008 increased the impetus to provide non-animal-derived sources of heparin, produced under cGMP conditions. In addition, recent studies suggest that heparin may have significant antineoplastic activity, separate and distinct from its anticoagulant activity, while other studies indicate a role for heparin in treating inflammation, infertility, and infectious disease. A variety of strategies have been proposed to produce a bioengineered heparin. In this review, we discuss several of these strategies including microbial production, mammalian cell production, and chemoenzymatic modification. We also propose strategies for creating "designer" heparins and heparan-sulfates with various biochemical and physiological properties.

  11. Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues.

    Science.gov (United States)

    Jiang, Bin; Suen, Rachel; Wertheim, Jason A; Ameer, Guillermo A

    2016-12-12

    Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.

  12. Sterilization of heparinized cuprophan hemodialysis membranes

    OpenAIRE

    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. Sterilization did not result in a release of heparin or heparin fragments from heparinized Cuprophan. Sterilization of heparinized Cuprophan by means of EtO exposure and gamma irradiation induced a slight, i...

  13. Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles.

    Science.gov (United States)

    Tellier, Liane E; Miller, Tobias; McDevitt, Todd C; Temenoff, Johnna S

    2015-10-28

    Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.

  14. Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme

    International Nuclear Information System (INIS)

    Zhang Yun-Xin

    2014-01-01

    Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical mechanics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provide a new way to understand biophysical processe by statistical mechanics analysis. (interdisciplinary physics and related areas of science and technology)

  15. Increased expression of heparin binding EGF (HB-EGF), amphiregulin, TGF alpha and epiregulin in androgen-independent prostate cancer cell lines.

    DEFF Research Database (Denmark)

    Tørring, Niels; Sørensen, Boe Sandahl; Nexø, Ebba

    2000-01-01

    BACKGROUND: The proliferation of androgen-independent prostate cancer cell lines has previously been shown to be influenced by an autocrine loop of the epidermal growth factor (EGF) system. This observation has alerted us to study the expression of ligands and receptors from the EGF......-system in prostate cell lines. METHODS: The expression of the EGF system was determined by quantitative RT-PCR and ELISA in the normal prostate epithelial cell line (PNT1A), in the androgen sensitive-(LNCaP), and the androgen-independent (DU145 and PC3) prostate cancer cell lines. RESULTS: The expression of m...... which exhibit low expression of HER1. Similar results were obtained by ELISA. CONCLUSIONS: The data indicates a selective up-regulation of a subclass of ligands of the EGF-system in androgen-independent prostate cancer cell lines. We suggest this could be a mechanism to escape androgen dependence...

  16. Evaluation of binding energies by using quantum mechanical methods

    International Nuclear Information System (INIS)

    Postolache, Cristian; Matei, Lidia; Postolache, Carmen

    2002-01-01

    Evaluation of binding energies (BE) in molecular structure is needed for modelling chemical and radiochemical processes by quantum-chemical methods. An important field of application is evaluation of radiolysis and autoradiolysis stability of organic and inorganic compounds as well as macromolecular structures. The current methods of calculation do not allow direct determination of BE but only of total binding energies (TBE) and enthalpies. BEs were evaluated indirectly by determining the homolytic dissociation energies. The molecular structures were built and geometrically optimized by the molecular mechanics methods MM+ and AMBER. The energy minimizations were refined by semi-empirical methods. Depending on the chosen molecular structure, the CNDO, INDO, PM3 and AM1 methods were used. To reach a high confidence level the minimizations were done for gradients lower than 10 -3 RMS. The energy values obtained by the difference of the fragment TBLs, of the transition states and initial molecular structures, respectively, were associated to the hemolytic fragmentation energy and BE, respectively. In order to evaluate the method's accuracy and to establish the application fields of the evaluation methods, the obtained values of BEs were compared with the experimental data taken from literature. To this goal there were built, geometrically optimized by semi-empirical methods and evaluated the BEs for 74 organic and inorganic compounds (alkanes, alkene, alkynes, halogenated derivatives, alcohols, aldehydes, ketones, carboxylic acids, nitrogen and sulfur compounds, water, hydrogen peroxide, ammonia, hydrazine, etc. (authors)

  17. 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

  18. 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.

  19. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  20. Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

    Energy Technology Data Exchange (ETDEWEB)

    Dahms, Sven O., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Mayer, Magnus C. [Freie Universität Berlin, Thielallee 63, 14195 Berlin (Germany); Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow (Germany); Roeser, Dirk [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Multhaup, Gerd [McGill University Montreal, Montreal, Quebec H3G 1Y6 (Canada); Than, Manuel E., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)

    2015-03-01

    Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

  1. Selective interaction of heparin with the variable region 3 within surface glycoprotein of laboratory-adapted feline immunodeficiency virus.

    Directory of Open Access Journals (Sweden)

    Qiong-Ying Hu

    Full Text Available Heparan sulfate proteoglycans (HSPG can act as binding receptors for certain laboratory-adapted (TCA strains of feline immunodeficiency virus (FIV and human immunodeficiency virus (HIV. Heparin, a soluble heparin sulfate (HS, can inhibit TCA HIV and FIV entry mediated by HSPG interaction in vitro. In the present study, we further determined the selective interaction of heparin with the V3 loop of TCA of FIV. Our current results indicate that heparin selectively inhibits infection by TCA strains, but not for field isolates (FS. Heparin also specifically interferes with TCA surface glycoprotein (SU binding to CXCR4, by interactions with HSPG binding sites on the V3 loop of the FIV envelope protein. Peptides representing either the N- or C-terminal side of the V3 loop and containing HSPG binding sites were able to compete away the heparin block of TCA SU binding to CXCR4. Heparin does not interfere with the interaction of SU with anti-V3 antibodies that target the CXCR4 binding region or with the interaction between FS FIV and anti-V3 antibodies since FS SU has no HSPG binding sites within the HSPG binding region. Our data show that heparin blocks TCA FIV infection or entry not only through its competition of HSPG on the cell surface interaction with SU, but also by its interference with CXCR4 binding to SU. These studies aid in the design and development of heparin derivatives or analogues that can inhibit steps in virus infection and are informative regarding the HSPG/SU interaction.

  2. Raman spectroscopy in investigations of mechanism of binding of human serum albumin to molecular probe fluorescein

    International Nuclear Information System (INIS)

    Vlasova, I M; Saletsky, A M

    2008-01-01

    The mechanism of binding of molecular probe fluorescein to molecules of human serum albumin was studied by the Raman spectroscopy method. The position of binding Center on human serum albumin molecule for fluorescein is determined. The amino acid residues of albumin molecule, participating in binding of fluorescein at different pH values of solution, are established. The conformation rearrangements of globules of human serum albumin, taking place at binding of fluorescein at different pH values of solution, are registered

  3. N-acetyl-heparin attenuates acute lung injury caused by acid aspiration mainly by antagonizing histones in mice.

    Science.gov (United States)

    Zhang, Yanlin; Zhao, Zanmei; Guan, Li; Mao, Lijun; Li, Shuqiang; Guan, Xiaoxu; Chen, Ming; Guo, Lixia; Ding, Lihua; Cong, Cuicui; Wen, Tao; Zhao, Jinyuan

    2014-01-01

    Acute lung injury (ALI) is the leading cause of death in intensive care units. Extracellular histones have recently been recognized to be pivotal inflammatory mediators. Heparin and its derivatives can bind histones through electrostatic interaction. The purpose of this study was to investigate 1) the role of extracellular histones in the pathogenesis of ALI caused by acid aspiration and 2) whether N-acetyl-heparin (NAH) provides more protection than heparin against histones at the high dose. ALI was induced in mice via intratracheal instillation of hydrochloric acid (HCl). Lethality rate, blood gas, myeloperoxidase (MPO) activity, lung edema and pathological changes were used to evaluate the degree of ALI. Heparin/NAH was administered intraperitoneally, twice a day, for 3 days or until death. Acid aspiration caused an obvious increase in extracellular histones. A significant correlation existed between the concentration of HCl aspirated and the circulating histones. Heparin/NAH (10 mg/kg) improved the lethality rate, blood gas, MPO activity, lung edema and pathological score. At a dose of 20 mg/kg, NAH still provided protection, however heparin tended to aggravate the injury due to hemorrhagic complications. The specific interaction between heparin and histones was verified by the binding assay. In summary, high levels of extracellular histones can be pathogenic in ALI caused by acid aspiration. By neutralizing extracellular histones, heparin/NAH can offer similar protection at the moderate doses. At the high dose, NAH provides better protection than heparin.

  4. How to give a heparin shot

    Science.gov (United States)

    ... you put the injection. Storing Your Heparin and Supplies Ask your pharmacist how to store your heparin ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  5. DNA-cisplatin binding mechanism peculiarities studied with single molecule stretching experiments

    Science.gov (United States)

    Crisafuli, F. A. P.; Cesconetto, E. C.; Ramos, E. B.; Rocha, M. S.

    2012-02-01

    We propose a method to determine the DNA-cisplatin binding mechanism peculiarities by monitoring the mechanical properties of these complexes. To accomplish this task, we have performed single molecule stretching experiments by using optical tweezers, from which the persistence and contour lengths of the complexes can be promptly measured. The persistence length of the complexes as a function of the drug total concentration in the sample was used to deduce the binding data, from which we show that cisplatin binds cooperatively to the DNA molecule, a point which so far has not been stressed in binding equilibrium studies of this ligand.

  6. Eldecalcitol (ED-71), an analog of 1α,25(OH)2D3, inhibits the growth of squamous cell carcinoma (SCC) cells in vitro and in vivo by down-regulating expression of heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) and FGF-2.

    Science.gov (United States)

    Shintani, T; Takatsu, F; Rosli, S N Z; Usui, E; Hamada, A; Sumi, K; Hayashido, Y; Toratani, S; Okamoto, Tetsuji

    2017-10-01

    Heparin-binding protein 17 (HBp17)/fibroblast growth factor-binding protein-1 (FGFBP-1) was first purified from medium conditioned by A431 cells for its capacity to bind to fibroblast growth factors 1 and 2 (FGF-1 and -2). Among FGF family members, FGF-2 is a potent mitogen for various cell types, including vascular endothelial cells, fibroblasts, and cancer cells such as oral squamous cell carcinoma (OSCC) cells. Besides being well known in bone metabolism, the active form of vitamin D 3 , i.e., 1α,25(OH) 2 D 3 (1,25D 3 ), was reported to have protective effects for heart disease and cancer. Previously, we reported that 1,25D 3 inhibited HBp17/FGFBP-1 expression in OSCC cell lines through NF-κB inhibition (IκBα activation) and resulted in the inactivation of FGF-2. In this study, we examined the potential anti-tumor effect of ED-71, an analog of 1α,25(OH) 2 D 3 , for squamous cell carcinoma cells in vitro and in vivo. The cell lines used were OSCC cell lines (NA-HO-1-n-1 and UE-HO-1-u-1), established from oral cancer patients in our laboratory, and an epidermoid carcinoma/SCC cell line (A431). The growth assay in serum-free culture revealed that ED-71 inhibited the growth of the cancer cell lines in a dose-dependent manner. In addition, ED-71 suppressed HBp17/FGFBP-1 expression by inhibiting the NF-κB pathway as did 1,25D 3 . Furthermore, a luciferase reporter assay revealed that the promoter activity of HBp17/FGFBP-1 (region between -217 and +61) was down-regulated by ED-71. Oral administration of ED-71 significantly inhibited the growth of A431-derived tumors in athymic nude mice. Immunohistochemical analysis revealed that the expression of HBp17/FGFBP-1, FGF-2, CD31, and Ki-67 in the tumors of ED71-treated group was down-regulated in comparison to control. These results suggest that ED-71 possesses potential anti-tumor activity for SCCs both in vitro and in vivo. This compound may act directly on the tumor cells or on endothelial cells by modulating the

  7. Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2

    Directory of Open Access Journals (Sweden)

    Yan Li

    2015-04-01

    Full Text Available Cyclin-dependent kinase 2 (CDK2 is a crucial regulator of the eukaryotic cell cycle. However it is well established that monomeric CDK2 lacks regulatory activity, which needs to be aroused by its positive regulators, cyclins E and A, or be phosphorylated on the catalytic segment. Interestingly, these activation steps bring some dynamic changes on the 3D-structure of the kinase, especially the activation segment. Until now, in the monomeric CDK2 structure, three binding sites have been reported, including the adenosine triphosphate (ATP binding site (Site I and two non-competitive binding sites (Site II and III. In addition, when the kinase is subjected to the cyclin binding process, the resulting structural changes give rise to a variation of the ATP binding site, thus generating an allosteric binding site (Site IV. All the four sites are demonstrated as being targeted by corresponding inhibitors, as is illustrated by the allosteric binding one which is targeted by inhibitor ANS (fluorophore 8-anilino-1-naphthalene sulfonate. In the present work, the binding mechanisms and their fluctuations during the activation process attract our attention. Therefore, we carry out corresponding studies on the structural characterization of CDK2, which are expected to facilitate the understanding of the molecular mechanisms of kinase proteins. Besides, the binding mechanisms of CDK2 with its relevant inhibitors, as well as the changes of binding mechanisms following conformational variations of CDK2, are summarized and compared. The summary of the conformational characteristics and ligand binding mechanisms of CDK2 in the present work will improve our understanding of the molecular mechanisms regulating the bioactivities of CDK2.

  8. Disappearance of a low molecular weight heparin fraction (CY 216) differs from standard heparin in rabbits

    International Nuclear Information System (INIS)

    Boneu, B.; Buchanan, M.R.; Caranobe, C.; Gabaig, A.M.; Dupouy, D.; Sie, P.; Hirsh, J.

    1987-01-01

    In previous studies, we have reported that standard heparin (SH) was cleared by two mechanisms, a saturable mechanism which predominated at low doses (less than 100 anti-factor Xa U/kg) and a non-saturable mechanism which predominated at higher doses, when the first mechanism became saturated. In this study, we examined the importance of these two mechanisms in the disappearance of a low molecular weight heparin fraction (LMWH) (CY 216), by comparing the pharmacokinetics and the pharmacodynamics of a wide range of doses of SH and CY 216 (1.5 to 500 anti-factor Xa U/kg). Pharmacokinetics was measured as the disappearance of 125 I-radiolabelled SH or CY 216. Pharmacodynamics was measured as the disappearance of the anti-factor Xa activity of SH and CY 216. We found that the saturable mechanism contributed little to the disappearance of CY 216 and that it was cleared predominantly by the non-saturable mechanism at all doses tested. Thus, at low doses (less than 100 anti-factor Xa U/kg), SH was cleared more rapidly than CY 216, whereas at higher doses, CY 216 was cleared more rapidly than SH. We conclude that the mechanism of disappearance of LMWH's differ significantly from those of SH, and that this difference may explain the apparent prolonged anticoagulant activity of LMWH's within the therapeutic range doses

  9. Interference of heparin in carcinoembryonic antigen radioimmunoassays

    International Nuclear Information System (INIS)

    Wu, J.T.

    1983-01-01

    A false Roche carcinoembryonic antigen (CEA) activity could be detected in all commercial and noncommercial heparin preparations examined. The possibility of 'due to contamination' has been ruled out. Using the Roche procedure, heparin solutions, in the absence of CEA, gave positive CEA activity; on the other hand, no CEA activity was detected in solutions containing only heparin when the Abbott Kit was used. When heparin was present in specimens containing CEA, the Abbott Kit underestimated the CEA activity, whereas the Roche Kit gave false elevated values. However, the negative effect of heparin could be reduced by heat treatment in the presence of plasma proteins. (Auth.)

  10. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

    DEFF Research Database (Denmark)

    List, K; Høyer-Hansen, G; Rønne, E

    1999-01-01

    Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interfer......Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance......) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former...

  11. Analysis and characterization of heparin impurities.

    Science.gov (United States)

    Beni, Szabolcs; Limtiaco, John F K; Larive, Cynthia K

    2011-01-01

    This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations.

  12. Mechanisms of Staphylococcus epidermidis adhesion to model biomaterial surfaces: Establising a link between thrombosis and infection

    Science.gov (United States)

    Higashi, Julie Miyo

    Infections involving Staphylococcus epidermidis remain a life threatening complication associated with the use of polymer based cardiovascular devices. One of the critical steps in infection pathogenesis is the adhesion of the bacteria to the device surface. Currently, mechanisms of S. epidermidis adhesion are incompletely understood, but are thought to involve interactions between bacteria, device surface, and host blood elements in the form of adsorbed plasma proteins and surface adherent platelets. Our central hypothesis is that elements participating in thrombosis also promote S. epidermidis adhesion by specifically binding to the bacterial surface. The adhesion kinetics of S. epidermidis RP62A to host modified model biomaterial surface octadecyltrichlorosilane (OTS) under hydrodynamic shear conditions were characterized. Steady state adhesion to adsorbed proteins and surface adherent platelets was achieved at 90-120 minutes and 60-90 minutes, respectively. A dose response curve of S. epidermidis adhesion in the concentration range of 10sp7{-}10sp9 bac/mL resembled a multilayer adsorption isotherm. Increasing shear stress was found to LTA, and other LTA blocking agents significantly decreased S. epidermidis adhesion to the fibrin-platelet clots, suggesting that this interaction between S. epidermidis and fibrin-platelet clots is specific. Studies evaluated the adhesion of S. epidermidis to polymer immobilized heparin report conflicting results. Paulsson et al., showed that coagulase negative staphylococci adhered in comparable numbers to both immobilized heparin and nonheparinized surfaces, while exhibiting significantly greater adhesion to both surfaces than S. aureus. Preadsorption of the surfaces with specific heparin binding plasma proteins vitronectin, fibronectin, laminin, and collagen significantly increased adhesion. It was postulated that immobilized heparin contained binding sites for the plasma proteins, exposing bacteria binding domains of the

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.

    Science.gov (United States)

    Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A; Esko, Jeffrey D; Linhardt, Robert J; Sharfstein, Susan T

    2012-03-01

    Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Convective Leakage Makes Heparin Locking of Central Venous Catheters Ineffective Within Seconds: Experimental Measurements in a Model Superior Vena Cava.

    Science.gov (United States)

    Barbour, Michael C; McGah, Patrick M; Ng, Chin H; Clark, Alicia M; Gow, Kenneth W; Aliseda, Alberto

    2015-01-01

    Central venous catheters (CVCs), placed in the superior vena cava (SVC) for hemodialysis or chemotherapy, are routinely filled while not in use with heparin, an anticoagulant, to maintain patency and prevent thrombus formation at the catheter tip. The heparin-locking procedure, however, places the patient at risk for systemic bleeding, as heparin is known to leak from the catheter into the blood stream. We provide evidence from detailed in vitro experiments that shows the driving mechanism behind heparin leakage to be convective-diffusive transport due to the pulsatile flow surrounding the catheter. This novel mechanism is supported by experimental planar laser-induced fluorescence (PLIF) and particle image velocimetry (PIV) measurements of flow velocity and heparin transport from a CVC placed inside a model SVC inside a pulsatile flow loop. The results predict an initial, fast (<10 s), convection-dominated phase that rapidly depletes the concentration of heparin in the near-tip region, the region of the catheter with side holes. This is followed by a slow, diffusion-limited phase inside the catheter lumen, where the concentration is still high, that is insufficient at replenishing the lost heparin concentration in the near-tip region. The results presented here, which are consistent with previous in vivo estimates of 24 hour leakage rates, predict that the concentration of heparin in the near-tip region is essentially zero for the majority of the interdialytic phase, rendering the heparin locking procedure ineffective.

  16. Heparin and glutathione II: correlation between decondensation of bull sperm cells and its nucleons.

    Science.gov (United States)

    Delgado, N M; Flores-Alonso, J C; Rodríguez-Hernández, H M; Merchant-Larios, H; Reyes, R

    2001-01-01

    The correlation between the kinetics of bull sperm nuclear and nucleon decondensation induced by the action of physiological concentrations of heparin/GSH was studied. Sperm and nucleon suspensions were incubated at 37 degrees C in salt medium, at a constant concentration of either heparin or GSH and increasing concentrations of the other reagent. Even though nucleons are pretreated with DTT/CTAB, when they are incubated alone with GSH for 96 h, they remain intact, no matter which concentration is employed, and it was impossible to observe the slightest sign of nuclei decondensation. Therefore, rupture of disulfide bridges is not the main mechanism to induce nuclei decondensation and perhaps the GSH role resides in potentate the heparin effect by increasing its negative charge. Nevertheless, nucleons reach 95% of chromatin decondensation in the presence of heparin plus GSH or heparin alone. The fact that the correlation between heparin and GSH concentrations needed to induce sperm nuclei decondensation was 3- to 4-fold greater that in nucleons might be due to the complete lack of nucleon membranes. Heparin/GSH seem to induce nuclei decondensation by an ionic chromatin charge neutralization mechanism.

  17. Lanthanide shift reagents, binding, shift mechanisms and exchange

    International Nuclear Information System (INIS)

    Boer, J.W.M. de

    1977-01-01

    Paramagnetic lanthanide shift reagents, when added to a solution of a substrate, induce shifts in the nuclear magnetic resonance (NMR) spectrum of the substrate molecules. The induced shifts contain information about the structure of the shift reagent substrate complex. The structural information, however, may be difficult to extract because of the following effects: (1) different complexes between shift reagent and substrate may be present in solution, e.g. 1:1 and 1:2 complexes, and the shift observed is a weighed average of the shifts of the substrate nuclei in the different complexes; (2) the Fermi contact interaction, arising from the spin density at the nucleus, contributes to the induced shift; (3) chemical exchange effects may complicate the NMR spectrum. In this thesis, the results of an investigation into the influence of these effects on the NMR spectra of solutions containing a substrate and LSR are presented. The equations describing the pseudo contact and the Fermi contact shift are derived. In addition, it is shown how the modified Bloch equations describing the effect of the chemical exchange processes occurring in the systems studied can be reduced to the familiar equations for a two-site exchange case. The binding of mono- and bifunctional ethers to the shift reagent are reported. An analysis of the induced shifts is given. Finally, the results of the experiments performed to study the exchange behavior of dimethoxyethane and heptafluorodimethyloctanedionato ligands are presented

  18. Proteolytic activation transforms heparin cofactor II into a host defense molecule.

    Science.gov (United States)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

    2013-06-15

    The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.

  19. The Effect of Low Molecular Weight Heparins on Fracture Healing.

    Science.gov (United States)

    Kapetanakis, Stylianos; Nastoulis, Evangelos; Demesticha, Theano; Demetriou, Thespis

    2015-01-01

    Venous Thromboembolism is a serious complication in the trauma patient. The most commonly studied and used anticoagulant treatment in prophylaxis of thrombosis is heparin. The prolonged use of unfractionated heparin has been connected with increased incidence of osteoporotic fractures. Low molecular-weight-heparins (LMWHs) have been the golden rule in antithrombotic therapy during the previous two decades as a way to overcome the major drawbacks of unfractioned heparin. However there are few studies reporting the effects of LMWHs on bone repair after fractures. This review presents the studies about the effects of LMWHs on bone biology (bone cells and bone metabolism) and underlying the mechanisms by which LMWHs may impair fracture healing process. The authors' research based on literature concluded that there are no facts and statistics for the role of LMWHs on fracture healing process in humans and the main body of evidence of their role comes from in vitro and animal studies. Further large clinical studies designed to compare different types of LMWHs, in different dosages and in different patient or animal models are needed for exploring the effects of LMWHs on fracture healing process.

  20. 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

  1. 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

  2. Fragment-based quantum mechanical calculation of protein-protein binding affinities.

    Science.gov (United States)

    Wang, Yaqian; Liu, Jinfeng; Li, Jinjin; He, Xiao

    2018-04-29

    The electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method has been successfully utilized for efficient linear-scaling quantum mechanical (QM) calculation of protein energies. In this work, we applied the EE-GMFCC method for calculation of binding affinity of Endonuclease colicin-immunity protein complex. The binding free energy changes between the wild-type and mutants of the complex calculated by EE-GMFCC are in good agreement with experimental results. The correlation coefficient (R) between the predicted binding energy changes and experimental values is 0.906 at the B3LYP/6-31G*-D level, based on the snapshot whose binding affinity is closest to the average result from the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation. The inclusion of the QM effects is important for accurate prediction of protein-protein binding affinities. Moreover, the self-consistent calculation of PB solvation energy is required for accurate calculations of protein-protein binding free energies. This study demonstrates that the EE-GMFCC method is capable of providing reliable prediction of relative binding affinities for protein-protein complexes. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

  3. Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins.

    Science.gov (United States)

    Hanas, J S; Rodgers, J S; Bantle, J A; Cheng, Y G

    1999-11-01

    The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys(2)His(2) zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 microM lead ions and completely inhibited by 10 to 20 microM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.

  4. Cancer Cell Adhesion and Metastasis: Selectins, Integrins, and the Inhibitory Potential of Heparins

    Directory of Open Access Journals (Sweden)

    Gerd Bendas

    2012-01-01

    Full Text Available Cell adhesion molecules play a significant role in cancer progression and metastasis. Cell-cell interactions of cancer cells with endothelium determine the metastatic spread. In addition, direct tumor cell interactions with platelets, leukocytes, and soluble components significantly contribute to cancer cell adhesion, extravasation, and the establishment of metastatic lesions. Clinical evidence indicates that heparin, commonly used for treatment of thromboembolic events in cancer patients, is beneficial for their survival. Preclinical studies confirm that heparin possesses antimetastatic activities that lead to attenuation of metastasis in various animal models. Heparin contains several biological activities that may affect several steps in metastatic cascade. Here we focus on the role of cellular adhesion receptors in the metastatic cascade and discuss evidence for heparin as an inhibitor of cell adhesion. While P- and L-selectin facilitation of cellular contacts during hematogenous metastasis is being accepted as a potential target of heparin, here we propose that heparin may also interfere with integrin activity and thereby affect cancer progression. This review summarizes recent findings about potential mechanisms of tumor cell interactions in the vasculature and antimetastatic activities of heparin.

  5. Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

    Science.gov (United States)

    Wang, Congzhou; Jin, Yingzi; Desai, Umesh R; Yadavalli, Vamsi K

    2015-06-01

    The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale. Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Binding mechanism and dynamic conformational change of C subunit of PKA with different pathways.

    Science.gov (United States)

    Chu, Wen-Ting; Chu, Xiakun; Wang, Jin

    2017-09-19

    The catalytic subunit of PKA (PKAc) exhibits three major conformational states (open, intermediate, and closed) during the biocatalysis process. Both ATP and substrate/inhibitor can effectively induce the conformational changes of PKAc from open to closed states. Aiming to explore the mechanism of this allosteric regulation, we developed a coarse-grained model and analyzed the dynamics of conformational changes of PKAc during binding by performing molecular dynamics simulations for apo PKAc, binary PKAc (PKAc with ATP, PKAc with PKI), and ternary PKAc (PKAc with ATP and PKI). Our results suggest a mixed binding mechanism of induced fit and conformational selection, with the induced fit dominant. The ligands can drive the movements of Gly-rich loop as well as some regions distal to the active site in PKAc and stabilize them at complex state. In addition, there are two parallel pathways (pathway with PKAc-ATP as an intermediate and pathway PKAc-PKI as an intermediate) during the transition from open to closed states. By molecular dynamics simulations and rate constant analyses, we find that the pathway through PKAc-ATP intermediate is the main binding route from open to closed state because of the fact that the bound PKI will hamper ATP from successful binding and significantly increase the barrier for the second binding subprocess. These findings will provide fundamental insights of the mechanisms of PKAc conformational change upon binding.

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

    International Nuclear Information System (INIS)

    Liu, Xinyue; St Ange, Kalib; Wang, Xiaohua; Lin, Lei; Zhang, Fuming

    2017-01-01

    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"®, 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"®, 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 approach relied

  8. 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

  9. Heparin-based hydrogels with tunable sulfation & degradation for anti-inflammatory small molecule delivery.

    Science.gov (United States)

    Peng, Yifeng; Tellier, Liane E; Temenoff, Johnna S

    2016-08-16

    Sustained release of anti-inflammatory agents remains challenging for small molecule drugs due to their low molecular weight and hydrophobicity. Therefore, the goal of this study was to control the release of a small molecule anti-inflammatory agent, crystal violet (CV), from hydrogels fabricated with heparin, a highly sulfated glycosaminoglycan capable of binding positively-charged molecules such as CV. In this system, both electrostatic interactions between heparin and CV and hydrogel degradation were tuned simultaneously by varying the level of heparin sulfation and varying the amount of dithiothreitol within hydrogels, respectively. It was found that heparin sulfation significantly affected CV release, whereby more sulfated heparin hydrogels (Hep and Hep(-N)) released CV with near zero-order release kinetics (R-squared values between 0.96-0.99). Furthermore, CV was released more quickly from fast-degrading hydrogels than slow-degrading hydrogels, providing a method to tune total CV release between 5-15 days while maintaining linear release kinetics. In particular, N-desulfated heparin hydrogels exhibited efficient CV loading (∼90% of originally included CV), near zero-order CV release kinetics, and maintenance of CV bioactivity after release, making this hydrogel formulation a promising CV delivery vehicle for a wide range of inflammatory diseases.

  10. Analysis of electric moments of RNA-binding proteins: implications for mechanism and prediction

    Directory of Open Access Journals (Sweden)

    Sarai Akinori

    2011-02-01

    Full Text Available Abstract Background Protein-RNA interactions play important role in many biological processes such as gene regulation, replication, protein synthesis and virus assembly. Although many structures of various types of protein-RNA complexes have been determined, the mechanism of protein-RNA recognition remains elusive. We have earlier shown that the simplest electrostatic properties viz. charge, dipole and quadrupole moments, calculated from backbone atomic coordinates of proteins are biased relative to other proteins, and these quantities can be used to identify DNA-binding proteins. Closely related, RNA-binding proteins are investigated in this study. In particular, discrimination between various types of RNA-binding proteins, evolutionary conservation of these bulk electrostatic features and effect of conformational changes by complex formation are investigated. Basic binding mechanism of a putative RNA-binding protein (HI1333 from Haemophilus influenza is suggested as a potential application of this study. Results We found that similar to DNA-binding proteins (DBPs, RNA-binding proteins (RBPs also show significantly higher values of electric moments. However, higher moments in RBPs are found to strongly depend on their functional class: proteins binding to ribosomal RNA (rRNA constitute the only class with all three of the properties (charge, dipole and quadrupole moments being higher than control proteins. Neural networks were trained using leave-one-out cross-validation to predict RBPs from control data as well as pair-wise classification capacity between proteins binding to various RNA types. RBPs and control proteins reached up to 78% accuracy measured by the area under the ROC curve. Proteins binding to rRNA are found to be best distinguished (AUC = 79%. Changes in dipole and quadrupole moments between unbound and bound structures were small and these properties are found to be robust under complex formation. Conclusions Bulk electric

  11. Thermodynamics and binding mechanism of polyphenon-60 with human lysozyme elucidated by calorimetric and spectroscopic techniques

    International Nuclear Information System (INIS)

    Yasmeen, Shama; Riyazuddeen

    2017-01-01

    Highlights: • Thermodynamics of the binding of Lys with polypenone-60 were studied. • The binding was found to be exothermic. • Polyphenon-60 quenches the fluorescence of Lys through static quenching. • Polyphenon-60 binds to Lys through hydrogen binding. • Conformational changes of Lys were studied using circular dichorism. - Abstract: Protein-drug interaction offer information of the structural features that determine the therapeutic effectiveness of drug and have become an attractive research field in life science, chemistry, and clinical medicine. Interaction of pharmacologically important antioxidant drug polyphenon-60 with human lysozyme (Lys) at physiological pH 7.4 has been studied by using calorimetric and various spectroscopic techniques. UV–visible spectroscopy results indicate the complex formation between Lys and polyphenon-60. The binding constant, quenching mechanism and the number of binding sites were determined by the fluorescence quenching spectra of Lys in presence of polyphenon-60. Fluorescence data indicate that the polyphenon-60 interact with Lys through static quenching mechanism with binding affinity of 2.9 × 10 4 M −1 . The average binding distance between drug and Lys was found to be 2.89 nm on the basis of the theory of Förster's energy transfer. Isothermal titration calorimetry (ITC) data reveals the thermodynamic investigations which suggest that the interaction of Lys and polyphenon-60 through exothermic process and enthalpy driven and also explore that the polyphenon-60 binds in both sites of Lys with high and low affinity. Hydrogen bonding (high affinity) and hydrophobic interactions (low affinity) are the major forces in stabilizing the drug protein complex. Far-UV CD and FTIR results deciphere the conformational alterations in the secondary structure of Lys.

  12. 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.

  13. Tannic acid and chromic chloride-induced binding of protein to red cells: a preliminary study of possible binding sites and reaction mechanisms.

    Science.gov (United States)

    Hunt, A F; Reed, M I

    1990-07-01

    The binding mechanisms and binding sites involved in the tannic acid and chromic chloride-induced binding of protein to red cells were investigated using the binding of IgA paraprotein to red cells as model systems. Inhibition studies of these model systems using amino acid homopolymers and compounds (common as red cell membrane constituents) suggest that the mechanisms involved are similar to those proposed for the conversion of hide or skin collagen to leather, as in commercial tanning. These studies also suggest that tannic acid-induced binding of IgA paraprotein to red cells involves the amino acid residues of L-arginine, L-lysine, L-histidine, and L-proline analogous to tanning with phenolic plant extracts. The amino acid residues of L-aspartate, L-glutamate and L-asparagine are involved in a similar manner in chronic chloride-induced binding of protein to red cells.

  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. 21 CFR 864.7525 - Heparin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

  16. 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.

  17. Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

    International Nuclear Information System (INIS)

    Knott, Michael; Best, Robert B.

    2014-01-01

    Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an “induced fit” or “conformational selection” mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD

  18. Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

    Energy Technology Data Exchange (ETDEWEB)

    Knott, Michael [Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Best, Robert B., E-mail: robertbe@helix.nih.gov [Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520 (United States)

    2014-05-07

    Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an “induced fit” or “conformational selection” mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD.

  19. Insight into the binding mechanism of imipenem to human serum albumin by spectroscopic and computational approaches.

    Science.gov (United States)

    Rehman, Md Tabish; Shamsi, Hira; Khan, Asad U

    2014-06-02

    The mechanism of interaction between imipenem and HSA was investigated by various techniques like fluorescence, UV.vis absorbance, FRET, circular dichroism, urea denaturation, enzyme kinetics, ITC, and molecular docking. We found that imipenem binds to HSA at a high affinity site located in subdomain IIIA (Sudlow's site I) and a low affinity site located in subdomain IIA.IIB. Electrostatic interactions played a vital role along with hydrogen bonding and hydrophobic interactions in stabilizing the imipenem.HSA complex at subdomain IIIA, while only electrostatic and hydrophobic interactions were present at subdomain IIA.IIB. The binding and thermodynamic parameters obtained by ITC showed that the binding of imipenem to HSA was a spontaneous process (ΔGD⁰(D)= -32.31 kJ mol(-1) for high affinity site and ΔGD⁰(D) = -23.02 kJ mol(-1) for low affinity site) with binding constants in the range of 10(4)-10(5) M(-1). Spectroscopic investigation revealed only one binding site of imipenem on HSA (Ka∼10(4) M(-1)). FRET analysis showed that the binding distance between imipenem and HSA (Trp-214) was optimal (r = 4.32 nm) for quenching to occur. Decrease in esterase-like activity of HSA in the presence of imipenem showed that Arg-410 and Tyr-411 of subdomain IIIA (Sudlow's site II) were directly involved in the binding process. CD spectral analysis showed altered conformation of HSA upon imipenem binding. Moreover, the binding of imipenem to subdomain IIIA (Sudlow's site II) of HSA also affected its folding pathway as clear from urea-induced denaturation studies.

  20. Statistical-mechanical lattice models for protein-DNA binding in chromatin

    International Nuclear Information System (INIS)

    Teif, Vladimir B; Rippe, Karsten

    2010-01-01

    Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.

  1. Longer peptide can be accommodated in the MHC class I binding site by a protrusion mechanism

    DEFF Research Database (Denmark)

    Stryhn, A; Pedersen, L O; Holm, A

    2000-01-01

    and C termini of a bound peptide interact through hydrogen bonding networks to conserved residues at either end of the class I binding site. Accordingly, it is thought that the termini are fixed and that only minor variations in peptide size are possible through a central bulging mechanism. We find...

  2. 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.

  3. How Robust Is the Mechanism of Folding-Upon-Binding for an Intrinsically Disordered Protein?

    Science.gov (United States)

    Bonetti, Daniela; Troilo, Francesca; Brunori, Maurizio; Longhi, Sonia; Gianni, Stefano

    2018-04-24

    The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (N TAIL ) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of N TAIL upon binding to XD by measuring the effect on both the folding and binding steps of N TAIL when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of N TAIL of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Preparation and characterization of microspheres of albumin-heparin conjugates

    NARCIS (Netherlands)

    Kwon, Glen S.; Bae, You Han; Kim, Sung Wan; Cremers, Harry; Cremers, H.F.M.; Feijen, Jan

    1991-01-01

    Albumin-heparin microspheres have been prepared as a new drug carrier. A soluble albumin-heparin conjugate was synthesized by forming amide bonds between human serum albumin and heparin. After purification the albumin-heparin conjugate was crosslinked in a water-in-oil emulsion to form

  5. Click-coated, heparinized, decellularized vascular grafts.

    Science.gov (United States)

    Dimitrievska, Sashka; Cai, Chao; Weyers, Amanda; Balestrini, Jenna L; Lin, Tylee; Sundaram, Sumati; Hatachi, Go; Spiegel, David A; Kyriakides, Themis R; Miao, Jianjun; Li, Guoyun; Niklason, Laura E; Linhardt, Robert J

    2015-02-01

    A novel method enabling the engineering of a dense and appropriately oriented heparin-containing layer on decellularized aortas has been developed. Amino groups of decellularized aortas were first modified to azido groups using 3-azidobenzoic acid. Azide-clickable dendrons were attached onto the azido groups through "alkyne-azide" click chemistry, affording a tenfold amplification of adhesions sites. Dendron end groups were finally decorated with end-on modified heparin chains. Heparin chains were oriented like heparan sulfate groups on native endothelial cells surface. X-ray photoelectron spectroscopy, nuclear magnetic resonance imaging, mass spectrometry and Fourier transform infrared FTIR spectroscopy were used to characterize the synthesis steps, building the final heparin layered coatings. The continuity of the heparin coating was verified using fluorescent microscopy and histological analysis. The efficacy of heparin linkage was demonstrated with factor Xa anti-thrombogenic assay and platelet adhesion studies. The results suggest that oriented heparin immobilization to decellularized aortas may improve the in vivo blood compatibility of decellularized aortas and vessels. Copyright © 2014 Acta Materialia Inc. All rights reserved.

  6. RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

    Science.gov (United States)

    Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

    2014-10-17

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Functionalization of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds via surface heparinization for bone tissue engineering.

    Science.gov (United States)

    Jiang, Tao; Khan, Yusuf; Nair, Lakshmi S; Abdel-Fattah, Wafa I; Laurencin, Cato T

    2010-06-01

    Scaffolds exhibiting biological recognition and specificity play an important role in tissue engineering and regenerative medicine. The bioactivity of scaffolds in turn influences, directs, or manipulates cellular responses. In this study, chitosan/poly(lactic acid-co-glycolic acid) (chitosan/PLAGA) sintered microsphere scaffolds were functionalized via heparin immobilization. Heparin was successfully immobilized on chitosan/PLAGA scaffolds with controllable loading efficiency. Mechanical testing showed that heparinization of chitosan/PLAGA scaffolds did not significantly alter the mechanical properties and porous structures. In addition, the heparinized chitosan/PLAGA scaffolds possessed a compressive modulus of 403.98 +/- 19.53 MPa and a compressive strength of 9.83 +/- 0.94 MPa, which are in the range of human trabecular bone. Furthermore, the heparinized chitosan/PLAGA scaffolds had an interconnected porous structure with a total pore volume of 30.93 +/- 0.90% and a median pore size of 172.33 +/- 5.89 mum. The effect of immobilized heparin on osteoblast-like MC3T3-E1 cell growth was investigated. MC3T3-E1 cells proliferated three dimensionally throughout the porous structure of the scaffolds. Heparinized chitosan/PLAGA scaffolds with low heparin loading (1.7 microg/scaffold) were shown to be capable of stimulating MC3T3-E1 cell proliferation by MTS assay and cell differentiation as evidenced by elevated osteocalcin expression when compared with nonheparinized chitosan/PLAGA scaffold and chitosan/PLAGA scaffold with high heparin loading (14.1 microg/scaffold). This study demonstrated the potential of functionalizing chitosan/PLAGA scaffolds via heparinization with improved cell functions for bone tissue engineering applications.

  8. Oral heparin results in the appearance of heparin fragments in the plasma of rats

    International Nuclear Information System (INIS)

    Larsen, A.K.; Lund, D.P.; Langer, R.; Folkman, J.

    1986-01-01

    We have previously shown that angiogenesis inhibition and tumor regression can be accomplished by combinations of heparin or heparin fragments with cortisone. Oral heparin was also effective in combination with cortisone. We now show that a single oral dose of [ 35 S]heparin or [ 3 H]heparin (15,000 units/kg) results in continuous release of radioactive material into the bloodstream for at least 12 hr. This is associated with the presence of anti-factor Xa activity at a level of approximately equal to 0.1 unit/ml. The radioactive material is identified as oligo-, di-, and monosaccharides by its behavior in chromatographic systems, its possession of anti-factor Xa activity, and the effect of treatment with bacterial heparinase. The heparin fragments are extensively metabolized to fragments without anti-factor Xa activity that are readily subject to urinary excretion

  9. Two Differential Binding Mechanisms of FG-Nucleoporins and Nuclear Transport Receptors

    Directory of Open Access Journals (Sweden)

    Piau Siong Tan

    2018-03-01

    Full Text Available Summary: Phenylalanine-glycine-rich nucleoporins (FG-Nups are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC. Previous studies showed that nuclear transport receptors (NTRs were found to interact with FG-Nups by forming an “archetypal-fuzzy” complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1. Association and dissociation rate constants are more than an order of magnitude lower than in the archetypal-fuzzy complex between FG-Nup153 and NTRs. Unexpectedly, this behavior appears not to be encoded selectively into CRM1 but rather into the FG-Nup214 sequence. The same distinct binding mechanisms are unperturbed in O-linked β-N-acetylglucosamine-modified FG-Nups. Our results have implications for differential roles of distinctly spatially distributed FG-Nup⋅NTR interactions in the cell. : Archetypal-fuzzy complexes found in most FG-Nucleoporin⋅nuclear transport receptor complexes allow fast yet specific nuclear transport. Tan et al. show that FG-Nup214, located at the periphery of the nuclear pore complex, binds to CRM1⋅RanGTP via a coupled reconfiguration-binding mechanism, which can enable different functionalities e.g., cargo release. Keywords: intrinsically disordered protein, glycosylation, FG-Nup, nuclear transport receptors, binding mechanism, single-molecule FRET, molecular dynamics simulations

  10. 77 FR 7584 - Draft Guidance for Industry on Heparin for Drug and Medical Device Use; Monitoring Crude Heparin...

    Science.gov (United States)

    2012-02-13

    ... strategies to ensure that the heparin supply chain is not contaminated with OSCS or any non- porcine origin... device manufacturers of finished products, and others to the potential risk of crude heparin...) among patients injected with heparin sodium in 2008, FDA identified the contaminant OSCS in heparin API...

  11. 78 FR 38058 - Guidance for Industry on Heparin for Drug and Medical Device Use: Monitoring Crude Heparin for...

    Science.gov (United States)

    2013-06-25

    ... public health. FDA developed this guidance to alert manufacturers to the risks of crude heparin contaminants and to recommend strategies to ensure that the heparin supply chain is not contaminated with OSCS... heparin sodium in 2008, FDA identified the contaminant OSCS in crude heparin sourced from China. FDA is...

  12. Distinct mechanisms of a phosphotyrosyl peptide binding to two SH2 domains.

    Science.gov (United States)

    Pang, Xiaodong; Zhou, Huan-Xiang

    2014-05-01

    Protein phosphorylation is very common post-translational modification, catalyzed by kinases, for signaling and regulation. Phosphotyrosines frequently target SH2 domains. The spleen tyrosine kinase (Syk) is critical for tyrosine phosphorylation of multiple proteins and for regulation of important pathways. Phosphorylation of both Y342 and Y346 in Syk linker B is required for optimal signaling. The SH2 domains of Vav1 and PLC-γ both bind this doubly phosphorylated motif. Here we used a recently developed method to calculate the effects of Y342 and Y346 phosphorylation on the rate constants of a peptide from Syk linker B binding to the SH2 domains of Vav1 and PLC-γ. The predicted effects agree well with experimental observations. Moreover, we found that the same doubly phosphorylated peptide binds the two SH2 domains via distinct mechanisms, with apparent rigid docking for Vav1 SH2 and dock-and-coalesce for PLC-γ SH2.

  13. Heparin- induced thrombocytopenia (HIT: a case report of CABG patient

    Directory of Open Access Journals (Sweden)

    Alireza Jahangirifard

    2016-08-01

    Full Text Available Heparin- induced thrombocytopenia (HIT is an antibody mediated adverse effect of heparin therapy which is classified into two subtypes, HITI which is non-immune, spontaneously reversible thrombocytopenia and; HITII which is an autoimmune-mediated adverse effect of heparin therapy. In this case report, we described a 65-year old male patient with HITII after coronary artery bypass grafting.Key words: Heparin- induced thrombocytopenia, Heparin- induced thrombosis, coronary artery bypass grafting.

  14. Biomimetic synthesis and biocompatibility evaluation of carbonated apatites template-mediated by heparin

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Yi [Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies, School and Hospital of Stomatology, Peking University, Beijing 100081 (China); Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Sun, Yuhua [Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies, School and Hospital of Stomatology, Peking University, Beijing 100081 (China); Chen, Xiaofang [Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Zhu, Peizhi, E-mail: pzzhu@umich.edu [Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055 (United States); Wei, Shicheng, E-mail: sc-wei@pku.edu.cn [Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies, School and Hospital of Stomatology, Peking University, Beijing 100081 (China); Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China)

    2013-07-01

    Biomimetic synthesis of carbonated apatites with good biocompatibility is a promising strategy for the broadening application of apatites for bone tissue engineering. Most researchers were interested in collagen or gelatin-based templates for synthesis of apatite minerals. Inspired by recent findings about the important role of polysaccharides in bone biomineralization, here we reported that heparin, a mucopolysaccharide, was used to synthesize carbonated apatites in vitro. The results indicated that the Ca/P ratio, carbon content, crystallinity and morphology of the apatites varied depending on the heparin concentration and the initial pH value. The morphology of apatite changed from flake-shaped to needle-shaped, and the degree of crystallinity decreased with the increasing of heparin concentration. Biocompatibility of the apatites was tested by proliferation and alkaline phosphatase activity of MC3T3-E1 cells. The results suggested that carbonated apatites synthesized in the presence of heparin were more favorable to the proliferation and differentiation of MC3T3-E1 cells compared with traditional method. In summary, the heparin concentration and the initial pH value play a key role in the chemical constitution and morphology, as well as biological properties of apatites. These biocompatible nano-apatite crystals hold great potential to be applied as bioactive materials for bone tissue engineering. - Highlights: • Heparin was used as a template to synthesize needle-shaped nano-apatite. • Changing the pH value and concentration led to different properties of apatite. • Apatite prepared by heparin was more favorable to the osteogenic differentiation. • Possible synthesis mechanism of apatite templated by heparin was described.

  15. Interactions of poly(amidoamine) dendrimers with human serum albumin: binding constants and mechanisms.

    Science.gov (United States)

    Giri, Jyotsnendu; Diallo, Mamadou S; Simpson, André J; Liu, Yi; Goddard, William A; Kumar, Rajeev; Woods, Gwen C

    2011-05-24

    The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K(b)) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To gain insight into the mechanisms of HSA binding to PAMAM dendrimers, we combined (1)H NMR, saturation transfer difference (STD) NMR, and NMR diffusion ordered spectroscopy (DOSY) of dendrimer-HSA complexes with atomistic molecular dynamics (MD) simulations of dendrimer conformation in aqueous solutions. The binding measurements show that the HSA binding constants (K(b)) of PAMAM dendrimers depend on dendrimer size and terminal group chemistry. The NMR (1)H and DOSY experiments indicate that the interactions between HSA and PAMAM dendrimers are relatively weak. The (1)H NMR STD experiments and MD simulations suggest that the inner shell protons of the dendrimers groups interact more strongly with HSA proteins. These interactions, which are consistently observed for different dendrimer generations (G0-NH(2)vs G4-NH(2)) and terminal groups (G4-NH(2)vs G4-OH with amidoethanol groups), suggest that PAMAM dendrimers adopt backfolded configurations as they form weak complexes with HSA proteins in aqueous solutions at physiological pH (7.4).

  16. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo

    2010-03-28

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

  17. Formation Mechanism and Binding Energy for Body-Centred Regular Icosahedral Structure of Li13 Cluster

    International Nuclear Information System (INIS)

    Liu Weina; Li Ping; Gou Qingquan; Zhao Yanping

    2008-01-01

    The formation mechanism for the body-centred regular icosahedral structure of Li 13 cluster is proposed. The curve of the total energy versus the separation R between the nucleus at the centre and nuclei at the apexes for this structure of Li 13 has been calculated by using the method of Gou's modified arrangement channel quantum mechanics (MACQM). The result shows that the curve has a minimal energy of -96.951 39 a.u. at R = 5.46a 0 . When R approaches to infinity, the total energy of thirteen lithium atoms has the value of -96.564 38 a.u. So the binding energy of Li 13 with respect to thirteen lithium atoms is 0.387 01 a.u. Therefore the binding energy per atom for Li 13 is 0.029 77 a.u. or 0.810 eV, which is greater than the binding energy per atom of 0.453 eV for Li 2 , 0.494 eV for Li 3 , 0.7878 eV for Li 4 , 0.632 eV for Li 5 , and 0.674 eV for Li 7 calculated by us previously. This means that the Li 13 cluster may be formed stably in a body-centred regular icosahedral structure with a greater binding energy

  18. Cationization of heparin for film applications

    Czech Academy of Sciences Publication Activity Database

    Šimkovic, I.; Mendichi, R.; Kelnar, Ivan; Filip, J.; Hricovíni, M.

    2015-01-01

    Roč. 115, 22 January (2015), s. 551-558 ISSN 0144-8617 Institutional support: RVO:61389013 Keywords : heparin * cationization * NMR Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.219, year: 2015

  19. In vitro binding of 67Ga to Ehrlich ascites tumor cells

    International Nuclear Information System (INIS)

    Kojima, S.; Kubodera, A.

    1984-01-01

    The binding of 67 Ga to Ehrlich ascites tumor cells (ETC) was studied in vitro. Acid mucopolysaccharide (AMPS) present at the cell surface of ETC was identified as heparan sulfate (HS). The extent of 67 Ga binding to ETC reached a plateau (ca. 10% of the added dose) at 1-2 h after the start of incubation. The binding was higher under neutral or alkaline conditions than under acidic conditions. Heparin and heparitinase treatment both significantly decreased the extent of 67 Ga binding to ETC. Mild treatment with protease, including trypsin or papain, also decreased the binding. On the contrary, the treatment with trypsin under severe conditions markedly increased the extent of 67 Ga binding to ETC. These results support the hypothesis that HS plays an important role as a 67 Ga receptor in the mechanism of gallium binding to ETC. (orig.)

  20. Quantitative ligand and receptor binding studies reveal the mechanism of interleukin-36 (IL-36) pathway activation.

    Science.gov (United States)

    Zhou, Li; Todorovic, Viktor; Kakavas, Steve; Sielaff, Bernhard; Medina, Limary; Wang, Leyu; Sadhukhan, Ramkrishna; Stockmann, Henning; Richardson, Paul L; DiGiammarino, Enrico; Sun, Chaohong; Scott, Victoria

    2018-01-12

    IL-36 cytokines signal through the IL-36 receptor (IL-36R) and a shared subunit, IL-1RAcP (IL-1 receptor accessory protein). The activation mechanism for the IL-36 pathway is proposed to be similar to that of IL-1 in that an IL-36R agonist (IL-36α, IL-36β, or IL-36γ) forms a binary complex with IL-36R, which then recruits IL-1RAcP. Recent studies have shown that IL-36R interacts with IL-1RAcP even in the absence of an agonist. To elucidate the IL-36 activation mechanism, we considered all possible binding events for IL-36 ligands/receptors and examined these events in direct binding assays. Our results indicated that the agonists bind the IL-36R extracellular domain with micromolar affinity but do not detectably bind IL-1RAcP. Using surface plasmon resonance (SPR), we found that IL-1RAcP also does not bind IL-36R when no agonist is present. In the presence of IL-36α, however, IL-1RAcP bound IL-36R strongly. These results suggested that the main pathway to the IL-36R·IL-36α·IL-1RAcP ternary complex is through the IL-36R·IL-36α binary complex, which recruits IL-1RAcP. We could not measure the binding affinity of IL-36R to IL-1RAcP directly, so we engineered a fragment crystallizable-linked construct to induce IL-36R·IL-1RAcP heterodimerization and predicted the binding affinity during a complete thermodynamic cycle to be 74 μm The SPR analysis also indicated that the IL-36R antagonist IL-36Ra binds IL-36R with higher affinity and a much slower off rate than the IL-36R agonists, shedding light on IL-36 pathway inhibition. Our results reveal the landscape of IL-36 ligand and receptor interactions, improving our understanding of IL-36 pathway activation and inhibition. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. A coupled channel study on a binding mechanism of positronic alkali atoms

    International Nuclear Information System (INIS)

    Kubota, Yoshihiro; Kino, Yasushi

    2008-01-01

    In order to investigate the binding mechanism of weakly bound states of positronic alkali atoms, we calculate the energies and wavefunctions using the Gaussian expansion method (GEM) where a positronium (Ps)-alkali ion channel and a positron-alkali atom channel are explicitly introduced. The energies of the bound states are updated using a model potential that reproduces well the observed energy levels of alkali atoms. The binding mechanism of the positronic alkali atom is analyzed by the wavefunctions obtained. The structure of the positronic alkali atom has been regarded as a Ps cluster orbiting the alkali ion, which is described by the Ps-alkali ion channel. We point out that the fraction having the positron-alkali atom configuration is small but plays an indispensable role for the weakly bound system

  3. Behavioural evidence for separate mechanisms of audiovisual temporal binding as a function of leading sensory modality.

    Science.gov (United States)

    Cecere, Roberto; Gross, Joachim; Thut, Gregor

    2016-06-01

    The ability to integrate auditory and visual information is critical for effective perception and interaction with the environment, and is thought to be abnormal in some clinical populations. Several studies have investigated the time window over which audiovisual events are integrated, also called the temporal binding window, and revealed asymmetries depending on the order of audiovisual input (i.e. the leading sense). When judging audiovisual simultaneity, the binding window appears narrower and non-malleable for auditory-leading stimulus pairs and wider and trainable for visual-leading pairs. Here we specifically examined the level of independence of binding mechanisms when auditory-before-visual vs. visual-before-auditory input is bound. Three groups of healthy participants practiced audiovisual simultaneity detection with feedback, selectively training on auditory-leading stimulus pairs (group 1), visual-leading stimulus pairs (group 2) or both (group 3). Subsequently, we tested for learning transfer (crossover) from trained stimulus pairs to non-trained pairs with opposite audiovisual input. Our data confirmed the known asymmetry in size and trainability for auditory-visual vs. visual-auditory binding windows. More importantly, practicing one type of audiovisual integration (e.g. auditory-visual) did not affect the other type (e.g. visual-auditory), even if trainable by within-condition practice. Together, these results provide crucial evidence that audiovisual temporal binding for auditory-leading vs. visual-leading stimulus pairs are independent, possibly tapping into different circuits for audiovisual integration due to engagement of different multisensory sampling mechanisms depending on leading sense. Our results have implications for informing the study of multisensory interactions in healthy participants and clinical populations with dysfunctional multisensory integration. © 2016 The Authors. European Journal of Neuroscience published by Federation

  4. Sliding mechanics of coated composite wires and the development of an engineering model for binding.

    Science.gov (United States)

    Zufall, S W; Kusy, R P

    2000-02-01

    A tribological (friction and wear) study, which was designed to simulate clinical sliding mechanics, was conducted as part of an effort to determine the suitability of poly(chloro-p-xylylene) coatings for composite orthodontic archwires. Prototype composite wires, having stiffnesses similar to those of current initial and intermediate alignment wires, were tested against stainless steel and ceramic brackets in the passive and active configurations (with and without angulation). Kinetic coefficient of friction values, which were determined to quantify sliding resistances as functions of the normal forces of ligation, had a mean that was 72% greater than uncoated wire couples at 0.43. To improve analysis of the active configuration, a mathematical model was developed that related bracket angulation, bracket width, interbracket distance, wire geometry, and wire elastic modulus to sliding resistance. From this model, kinetic coefficients of binding were determined to quantify sliding resistances as functions of the normal forces of binding. The mean binding coefficient was the same as that of uncoated wire couples at 0.42. Although penetrations through the coating were observed on many specimens, the glass-fiber reinforcement within the composite wires was undamaged for all conditions tested. This finding implies that the risk of glass fiber release during clinical use would be eliminated by the coating. In addition, the frictional and binding coefficients were still within the limits outlined by conventional orthodontic wire-bracket couples. Consequently, the coatings were regarded as an improvement to the clinical acceptability of composite orthodontic archwires.

  5. Carboxamide SIRT1 inhibitors block DBC1 binding via an acetylation-independent mechanism

    Science.gov (United States)

    Hubbard, Basil P; Loh, Christine; Gomes, Ana P; Li, Jun; Lu, Quinn; Doyle, Taylor LG; Disch, Jeremy S; Armour, Sean M; Ellis, James L; Vlasuk, George P; Sinclair, David A

    2013-01-01

    SIRT1 is an NAD+-dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1. PMID:23892437

  6. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Nuemket, Nipawan [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu [Creative Research Institution ' Sousei,' Hokkaido University, Sapporo 001-0021 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tsukamoto, Kentaro; Tsuji, Takao [Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192 (Japan); Nakamura, Keiji; Kozaki, Shunji [Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531 (Japan); Yao, Min [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao, E-mail: tanaka@castor.sci.hokudai.ac.jp [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan)

    2011-07-29

    Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

  7. Heparin-Functionalized chitosan/ κ-carrageenan complexes as potential scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Dofeliz, Joni L.; Rojas, Nina Rosario L.

    2015-01-01

    Cell-based approaches to tissue regeneration are playing an increasingly important role in bone and cartilage repair. This is made possible through the use of growth factors, which are signaling molecules that induce a number of effects such as cell proliferation, migration and differentiation. But problems arise as these growth factors tend to be expensive, short-lived and slow moving through the extracellular matrix, making them very inefficient in their current form of introduction. One such growth factor is basic fibroblast growth factor (bFGF). The general objective of this study is to construct heparin-functionalized chitosan/κ-carrageenan scaffolds, which when bound to basic fibroblast growth factor (bFGF), may be used in the culture of mesenchymal stem cells for differentiation into cartilage. In this study, gels of semi-interpenetrating networks (semi-IPN) of chitosan and κ-carrageenan (2:4:1 blend ration) were prepared using calcium chloride as a cross-linker. The gels were then functionalized with heparin, which is known for its growth factor binding ability, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as cross-linker. The functionalized gels were then reshaped into scaffold on the wells of 48-well plates through freeze-dry method. The scaffolds were found to be realatively porous with pore sizes larger than 100 μm which satisfies the requirements for cell culture. Subsequent thermogravimetric analysis shows that the scaffolds were relatively stable with a degradation temperature of 277°C. Additionally, there was no observable separation of the individual degradation temperatures of chitosan and κ-carrageenan, confirming that a single miscible phase in the form of a semi-IPN structure was created. Results of scanning electron microscopy also showed that the scaffolds were stable under 2 hours of UV sterilization. The FTIR spectra of the heparinized PEC showed characteristic absorption bands (S=O asymetric stretch) in the area of 1160

  8. ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism

    Science.gov (United States)

    Liao, Jielou

    2004-03-01

    Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

  9. Mechanisms of membrane binding of small GTPase K-Ras4B farnesylated hypervariable region.

    Science.gov (United States)

    Jang, Hyunbum; Abraham, Sherwin J; Chavan, Tanmay S; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I; Nussinov, Ruth; Gaponenko, Vadim

    2015-04-10

    K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

    Science.gov (United States)

    Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

  11. A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains.

    Directory of Open Access Journals (Sweden)

    Alexandr P Kornev

    2008-04-01

    Full Text Available Cyclic nucleotides (cAMP and cGMP regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1 the phosphate binding cassette (PBC, which binds the cAMP ribose-phosphate, 2 the "hinge," a flexible helix, which contacts the PBC, 3 the beta(2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4 a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif. The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the beta(2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.

  12. Preparation and characterization of chitosan-heparin composite matrices for blood contacting tissue engineering

    International Nuclear Information System (INIS)

    He Qing; Gong Kai; Gong Yandao; Zhang Xiufang; Ao Qiang; Zhang Lihai; Hu Min

    2010-01-01

    Chitosan has been widely used for biomaterial scaffolds in tissue engineering because of its good mechanical properties and cytocompatibility. However, the poor blood compatibility of chitosan has greatly limited its biomedical utilization, especially for blood contacting tissue engineering. In this study, we exploited a polymer blending procedure to heparinize the chitosan material under simple and mild conditions to improve its antithrombogenic property. By an optimized procedure, a macroscopically homogeneous chitosan-heparin (Chi-Hep) blended suspension was obtained, with which Chi-Hep composite films and porous scaffolds were fabricated. X-ray photoelectron spectroscopy and sulfur elemental analysis confirmed the successful immobilization of heparin in the composite matrices (i.e. films and porous scaffolds). Toluidine blue staining indicated that heparin was distributed homogeneously in the composite matrices. Only a small amount of heparin was released from the matrices during incubation in normal saline for 10 days. The composite matrices showed improved blood compatibility, as well as good mechanical properties and endothelial cell compatibility. These results suggest that the Chi-Hep composite matrices are promising candidates for blood contacting tissue engineering.

  13. From simple receptors to complex multimodal percepts: a first global picture on the mechanisms involved in perceptual binding.

    Science.gov (United States)

    Velik, Rosemarie

    2012-01-01

    The binding problem in perception is concerned with answering the question how information from millions of sensory receptors, processed by millions of neurons working in parallel, can be merged into a unified percept. Binding in perception reaches from the lowest levels of feature binding up to the levels of multimodal binding of information coming from the different sensor modalities and also from other functional systems. The last 40 years of research have shown that the binding problem cannot be solved easily. Today, it is considered as one of the key questions to brain understanding. To date, various solutions have been suggested to the binding problem including: (1) combination coding, (2) binding by synchrony, (3) population coding, (4) binding by attention, (5) binding by knowledge, expectation, and memory, (6) hardwired vs. on-demand binding, (7) bundling and binding of features, (8) the feature-integration theory of attention, and (9) synchronization through top-down processes. Each of those hypotheses addresses important aspects of binding. However, each of them also suffers from certain weak points and can never give a complete explanation. This article gives a brief overview of the so far suggested solutions of perceptual binding and then shows that those are actually not mutually exclusive but can complement each other. A computationally verified model is presented which shows that, most likely, the different described mechanisms of binding act (1) at different hierarchical levels and (2) in different stages of "perceptual knowledge acquisition." The model furthermore considers and explains a number of inhibitory "filter mechanisms" that suppress the activation of inappropriate or currently irrelevant information.

  14. From simple receptors to complex multimodal percepts: A first global picture on the mechanisms involved in perceptual binding

    Directory of Open Access Journals (Sweden)

    Rosemarie eVelik

    2012-07-01

    Full Text Available The binding problem in perception is concerned with answering the question how information from millions of sensory receptors, processed by millions of neurons working in parallel, can be merged into a unified percept. Binding in perception reaches from the lowest levels of feature binding up to the levels of multimodal binding of information coming from the different sensor modalities and also from other functional systems. The last 40 years of research have shown that the binding problem cannot be solved easily. Today, it is considered as one of the key questions to brain understanding. To date, various solutions have been suggested to the binding problem including: (1 combination coding, (2 binding by synchrony, (3 population coding, (4 binding by attention, (5 binding by knowledge, expectation, and memory, (6 hardwired versus on-demand binding, (7 bundling and binding of features, (8 the feature-integration theory of attention, (9 synchronization through top-down processes. Each of those hypotheses addresses important aspects of binding. However, each of them also suffers from certain weak points and can never give a complete explanation. This article gives a brief overview of the so far suggested solutions of perceptual binding and then shows that those are actually not mutually exclusive but can complement each other. A computationally verified model is presented which shows that, most likely, the different described mechanisms of binding act (1 at different hierarchical levels and (2 in different stages of perceptual knowledge acquisition. The model furthermore considers and explains a number of inhibitory filter mechanisms that suppress the activation of inappropriate or currently irrelevant information.

  15. Heparin octasaccharide decoy liposomes inhibit replication of multiple viruses

    Science.gov (United States)

    Hendricks, Gabriel L.; Velazquez, Lourdes; Pham, Serena; Qaisar, Natasha; Delaney, James C.; Viswanathan, Karthik; Albers, Leila; Comolli, James C.; Shriver, Zachary; Knipe, David M.; Kurt-Jones, Evelyn A.; Fygenson, Deborah K.; Trevejo, Jose M.

    2016-01-01

    Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as ‘molecular sinks’ and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform. PMID:25637710

  16. Binding energy and mechanical stability of single- and multi-walled carbon nanotube serpentines

    International Nuclear Information System (INIS)

    Zhao, Junhua; Lu, Lixin; Rabczuk, Timon

    2014-01-01

    Recently, Geblinger et al. [Nat. Nanotechnol. 3, 195 (2008)] and Machado et al. [Phys. Rev. Lett. 110, 105502 (2013)] reported the experimental and molecular dynamics realization of S-like shaped single-walled carbon nanotubes (CNTs), the so-called CNT serpentines. We reported here results from continuum modeling of the binding energy γ between different single- and multi-walled CNT serpentines and substrates as well as the mechanical stability of the CNT serpentine formation. The critical length for the mechanical stability and adhesion of different CNT serpentines are determined in dependence of E i I i , d, and γ, where E i I i and d are the CNT bending stiffness and distance of the CNT translation period. Our continuum model is validated by comparing its solution to full-atom molecular dynamics calculations. The derived analytical solutions are of great importance for understanding the interaction mechanism between different single- and multi-walled CNT serpentines and substrates

  17. Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

    Science.gov (United States)

    Neupane, Durga P; Avalos, Dante; Fullam, Stephanie; Roychowdhury, Hridindu; Yukl, Erik T

    2017-10-20

    Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  19. Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

    Science.gov (United States)

    Rice, Austin J; Harrison, Alistair; Alvarez, Frances J D; Davidson, Amy L; Pinkett, Heather W

    2014-05-23

    Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. DEGRADATION AND INTRAHEPATIC COMPATIBILITY OF ALBUMIN-HEPARIN CONJUGATE MICROSPHERES

    NARCIS (Netherlands)

    CREMERS, HFM; WOLF, RFE; BLAAUW, EH; SCHAKENRAAD, JM; LAM, KH; NIEUWENHUIS, P; VERRIJK, R; KWON, G; BAE, YH; KIM, SW; FEIJEN, J

    The in vitro degradation properties of glutaraldehyde cross-linked albumin and albumin-heparin conjugate microspheres (AMS and AHCMS respectively) were evaluated using light microscopy, turbidity measurements and heparin release determinations, showing that the microspheres are degraded by

  1. The Panitumumab EGFR Complex Reveals a Binding Mechanism That Overcomes Cetuximab Induced Resistance.

    Directory of Open Access Journals (Sweden)

    E Allen Sickmier

    Full Text Available Panitumumab and cetuximab target the epidermal growth factor receptor for the treatment of metastatic colorectal cancer. These therapies provide a significant survival benefit to patients with metastatic colorectal cancer with wild-type RAS. A single point mutation in the ectodomain of EGFR (S468R confers acquired or secondary resistance in cetuximab treated patients, which is not observed in panitumumab-treated patients. Structural and biophysical studies presented here show this mutation directly blocks cetuximab binding to EGFR domain III and describes a unique mechanism by which panitumumab uses a central cavity to accommodate this mutation.

  2. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A., E-mail: alashkov83@gmail.com [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2012-05-15

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  3. Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation

    Directory of Open Access Journals (Sweden)

    Olgun eGuvench

    2015-06-01

    Full Text Available The extracellular N-terminal hyaluronan binding domain (HABD of CD44 is a small globular domain that confers hyaluronan (HA binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA binding site from a low affinity to a high affinity state; in the partially-disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently-attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD.

  4. 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.

  5. Endogenous heparin levels in the controlled asthmatic patient ...

    African Journals Online (AJOL)

    Background. Since heparin possesses anti-inflammatory properties, it is hypothesised that asthmatic patients have decreased levels of circulating heparin compared with healthy individuals. Design. We compared endogenous heparin levels in controlled asthmatic patients (53 adults) from the Asthma Clinic at ...

  6. Heparin increases food intake through AgRP neurons

    Science.gov (United States)

    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 t...

  7. Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

    International Nuclear Information System (INIS)

    Ahluwalia, Unnati; Deep, Shashank

    2012-01-01

    Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

  8. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  9. Safety and Efficacy of Argatroban in the Management of Heparin-Induced Thrombocytopenia

    Directory of Open Access Journals (Sweden)

    Bernd Saugel

    2011-01-01

    Full Text Available Heparin-induced thrombocytopenia (HIT is a life-threatening adverse reaction to heparin therapy that is characterized by thrombocytopenia and an increased risk of venous and arterial thrombosis. According to guidelines, in patients with strongly suspected or confirmed HIT all sources of heparin have to be discontinued and an alternative, nonheparin anticoagulant for HIT treatment must immediately be started. For both the prophylaxis of thrombembolic events in HIT and the treatment of HIT with thrombosis the direct thrombin inhibitor argatroban is approved in the United States. The objective of this review is to describe the mechanism of action and the pharmacokinetic profile of argatroban, to characterize argatroban regarding its safety and therapeutic efficacy and to discuss its place in therapy in HIT.

  10. Structural and quantum mechanical computations to elucidate the altered binding mechanism of metal and drug with pyrazinamidase from Mycobacterium tuberculosis due to mutagenicity.

    Science.gov (United States)

    Rasool, Nouman; Iftikhar, Saima; Amir, Anam; Hussain, Waqar

    2018-03-01

    Pyrazinamide is known to be the most effective treatment against tuberculosis disease and is known to have bacteriostatic action. By targeting the bacterial spores, this drug reduces the chances for the progression of the infection in organisms. In recent years, increased instances of the drug resistance of bacterial strains are reported. Pyrazinamidase, activator for pyrazinamide, leads to resistance against the drug due to mutagenicity across the world. The present study aimed at the quantum mechanistic analysis of mutations in pyrazinamidase to gain insights into the mechanism of this enzyme. Quantum mechanical calculations were performed to analyse the effect of mutations at the metal coordination site using ORCA software program. Moreover, conformational changes in PZase binding cavity has also been analysed due to mutations of binding pocket residues using CASTp server. In order to elucidate the behaviour of the mutant pyrazinamidase, docking of PZA in the binding pocket of PZase was performed using AutoDock Vina. Analysis of results revealed that iron showed weak binding with the metal coordination site of the mutant proteins due to alteration in electron transfer mechanism. The binding cavity of the mutant PZase has undergone major conformational changes as the volume of pocket increased due to bulky R-chains of mutated amino acids. These conformational changes lead to weak binding of the drug at binding cavity of PZase and reduce the drug activation mechanism leading to increased drug resistance in the bacterial strains. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Sugar and pH dual-responsive mesoporous silica nanocontainers based on competitive binding mechanisms

    Science.gov (United States)

    Yilmaz, M. Deniz; Xue, Min; Ambrogio, Michael W.; Buyukcakir, Onur; Wu, Yilei; Frasconi, Marco; Chen, Xinqi; Nassar, Majed S.; Stoddart, J. Fraser; Zink, Jeffrey I.

    2014-12-01

    A sugar and pH dual-responsive controlled release system, which is highly specific towards molecular stimuli, has been developed based on the binding between catechol and boronic acid on a platform of mesoporous silica nanoparticles (MSNs). By grafting phenylboronic acid stalks onto the silica surface, catechol-containing β-cyclodextrins can be attached to the orifices of the MSNs' nanopores through formation of boronate esters which block access to the nanopores. These esters are stable enough to prevent cargo molecules from escaping. The boronate esters disassociate in the presence of sugars, enabling the molecule-specific controlled-release feature of this hybrid system. The rate of release has been found to be tunable by varying both the structures and the concentrations of sugars, as a result of the competitive binding nature associated with the mechanism of its operation. Acidification also induces the release of cargo molecules. Further investigations show that the presence of both a low pH and sugar molecules provides cooperative effects which together control the rate of release.A sugar and pH dual-responsive controlled release system, which is highly specific towards molecular stimuli, has been developed based on the binding between catechol and boronic acid on a platform of mesoporous silica nanoparticles (MSNs). By grafting phenylboronic acid stalks onto the silica surface, catechol-containing β-cyclodextrins can be attached to the orifices of the MSNs' nanopores through formation of boronate esters which block access to the nanopores. These esters are stable enough to prevent cargo molecules from escaping. The boronate esters disassociate in the presence of sugars, enabling the molecule-specific controlled-release feature of this hybrid system. The rate of release has been found to be tunable by varying both the structures and the concentrations of sugars, as a result of the competitive binding nature associated with the mechanism of its operation

  12. Screening of Lactobacillus strains for their ability to bind benzo(a)pyrene and the mechanism of the process.

    Science.gov (United States)

    Zhao, Hongfei; Zhou, Fang; Qi, Yeqiong; Dziugan, Piotr; Bai, Fengling; Walczak, Piotr; Zhang, Bolin

    2013-09-01

    In order to investigate the binding ability of Lactobacillus strains to Benzo(a)pyrene (BaP), 15 strains were analysed. L. plantarum CICC 22135 and L. pentosus CICC 23163 exhibited high efficiency in removing BaP from aqueous medium; the binding rates were 66.76% and 64.31%, respectively. This process was affected by temperature, incubation time and pH, and cell viability was not necessary for the binding ability. Additionally, both strains, especially strain CICC 23163 showed high specificity in binding BaP. The cell-BaP complexes were stable in aqueous medium. The mechanism of binding was investigated by examining the binding ability of different components of the microorganism cells. The results revealed that peptidoglycans played an important role in binding BaP and its structural integrity was required. Consequently, we proposed that the mechanism of this process was a physisorption and peptidoglycan was the main binding site. These two strains may be used for dietary detoxification in human diet and animal feed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Blood interaction with a Bioline heparin coated HIA-VAD : A study on calves

    NARCIS (Netherlands)

    vanderKamp, KWHJ; Magielse, CPE; Elstrodt, JM; vanderMeer, J; vanOeveren, W; Rakhorst, G

    The blood compatibility of ventricular assist devices developed by the Helmholtz institute Aachen (HA-VAD's) was tested on calves. Seven calves received a non-coated HIA-VAD (control) and three a Bioline heparin coated device. The circulatory support of these HIA-VAD's lasted one week. Mechanical

  14. Characterization of heparin aerosols generated in jet and ultrasonic nebulizers

    DEFF Research Database (Denmark)

    Bendstrup, K.E.; Newhouse, M.T.; Pedersen, Ole Finn

    1999-01-01

    Inhaled heparin has been used for asthma treatment, but results have been inconsistent, probably due to highly varying lung doses. We determined the output per unit time and the particle size distributions of sodium heparin, calcium heparin, and low molecular weight (LMW) heparin formulations in ...... on the exhalation filter, and 15,000 IU was captured on the inhalation filter (inhaled mass). This corresponds to a respirable mass of 10,000 IU of heparin with a high probability of reaching the lower respiratory tract in normal healthy adults....

  15. 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 ...

  16. Biological distribution of 51Cr-heparin

    International Nuclear Information System (INIS)

    Almeida, M.A.T.M. de.

    1979-01-01

    The kinetics of heparin in normal Wistar rats using the radioactive tracer 51 Cr, has been studied. The labeled and purified 51 Cr-heparin was injected into rats intravenously and by intraperitoneal injection. In measuring the radioactivity of organs it was possible to conclude that the tissues rich in mast cells, liver and spleen, were found to take up the greater amounts of heparin. The curve that represents the logarithm of the concentration of heparin versus time is biexponential. The half-lives of the two exponential were determined. The volume of distribution, the rate constant and the renal clearance were determined by the values of the plasma levels and urinary excretions. The biological half-time, the turnover rate and the turnover time were determined by measuring the residual radioactivity of the total body and urinary excretions. With the data obtained from the mentioned experiments a compartmental model was performed in which the plasma is the central compartment for the distribution of the drug, exchanging with another extraplasmatic compartment and finally the drug being stored in reticulo endothelial system cells. (Author) [pt

  17. The microtubule associated protein END BINDING 1 represses root responses to mechanical cues.

    Science.gov (United States)

    Gleeson, Laura; Squires, Shannon; Bisgrove, Sherryl R

    2012-05-01

    The ability of roots to navigate around rocks and other debris as they grow through the soil requires a mechanism for detecting and responding to input from both touch and gravity sensing systems. The microtubule associated protein END BINDING 1b (EB1b) is involved in this process as mutants have defects responding to combinations of touch and gravity cues. This study investigates the role of EB1b in root responses to mechanical cues. We find that eb1b-1 mutant roots exhibit an increase over wild type in their response to touch and that the expression of EB1b genes in transgenic mutants restores the response to wild type levels, indicating that EB1b is an inhibitor of the response. Mutant roots are also hypersensitive to increased levels of mechanical stimulation, revealing the presence of another process that activates the response. These findings are supported by analyses of double mutants between eb1b-1 and seedlings carrying mutations in PHOSPHOGLUCOMUTASE (PGM), ALTERED RESPONSE TO GRAVITY1 (ARG1), or TOUCH3 (TCH3), genes that encode proteins involved in gravity sensing, signaling, or touch responses, respectively. A model is proposed in which root responses to mechanical cues are modulated by at least two competing regulatory processes, one that promotes touch-mediated growth and another, regulated by EB1b, which dampens root responses to touch and enhances gravitropism. © 2012. Published by Elsevier Ireland Ltd. All rights reserved.

  18. Binding and segmentation via a neural mass model trained with Hebbian and anti-Hebbian mechanisms.

    Science.gov (United States)

    Cona, Filippo; Zavaglia, Melissa; Ursino, Mauro

    2012-04-01

    Synchronization of neural activity in the gamma band, modulated by a slower theta rhythm, is assumed to play a significant role in binding and segmentation of multiple objects. In the present work, a recent neural mass model of a single cortical column is used to analyze the synaptic mechanisms which can warrant synchronization and desynchronization of cortical columns, during an autoassociation memory task. The model considers two distinct layers communicating via feedforward connections. The first layer receives the external input and works as an autoassociative network in the theta band, to recover a previously memorized object from incomplete information. The second realizes segmentation of different objects in the gamma band. To this end, units within both layers are connected with synapses trained on the basis of previous experience to store objects. The main model assumptions are: (i) recovery of incomplete objects is realized by excitatory synapses from pyramidal to pyramidal neurons in the same object; (ii) binding in the gamma range is realized by excitatory synapses from pyramidal neurons to fast inhibitory interneurons in the same object. These synapses (both at points i and ii) have a few ms dynamics and are trained with a Hebbian mechanism. (iii) Segmentation is realized with faster AMPA synapses, with rise times smaller than 1 ms, trained with an anti-Hebbian mechanism. Results show that the model, with the previous assumptions, can correctly reconstruct and segment three simultaneous objects, starting from incomplete knowledge. Segmentation of more objects is possible but requires an increased ratio between the theta and gamma periods.

  19. Human Adenosine A2A Receptor: Molecular Mechanism of Ligand Binding and Activation

    Directory of Open Access Journals (Sweden)

    Byron Carpenter

    2017-12-01

    Full Text Available Adenosine receptors (ARs comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs. ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR, making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes.

  20. Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

    Science.gov (United States)

    Chen, Qiang; Kinde, Monica N; Arjunan, Palaniappa; Wells, Marta M; Cohen, Aina E; Xu, Yan; Tang, Pei

    2015-09-08

    Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

  1. Leveraging non-binding instruments for global health governance: reflections from the Global AIDS Reporting Mechanism for WHO reform.

    Science.gov (United States)

    Taylor, A L; Alfven, T; Hougendobler, D; Tanaka, S; Buse, K

    2014-02-01

    As countries contend with an increasingly complex global environment with direct implications for population health, the international community is seeking novel mechanisms to incentivize coordinated national and international action towards shared health goals. Binding legal instruments have garnered increasing attention since the World Health Organization adopted its first convention in 2003. This paper seeks to expand the discourse on future global health lawmaking by exploring the potential value of non-binding instruments in global health governance, drawing on the case of the 2001 United Nations General Assembly Special Session Declaration of Commitment on HIV/AIDS. In other realms of international concern ranging from the environment to human rights to arms control, non-binding instruments are increasingly used as effective instruments of international cooperation. The experience of the Global AIDS Reporting Mechanism, established pursuant to the Declaration, evidences that, at times, non-binding legal instruments can offer benefits over slower, more rigid binding legal approaches to governance. The global AIDS response has demonstrated that the use of a non-binding instrument can be remarkably effective in galvanizing increasingly deep commitments, action, reporting compliance and ultimately accountability for results. Based on this case, the authors argued that non-binding instruments deserve serious consideration by the international community for the future of global health governance, including in the context of WHO reform. Copyright © 2013 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

  2. Surface biomimetic modification with laminin-loaded heparin/poly-L-lysine nanoparticles for improving the biocompatibility

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Tao, E-mail: 11140021@hyit.edu.cn [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an (China); Hu, Youdong [Department of Geriatrics, The Affiliated Huai' an Hospital of Xuzhou Medical College, Huai' an (China); Tan, Jianying [Key Lab. of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Liu, Shihui [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an (China); Chen, Junying [Key Lab. of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Guo, Xin; Pan, Changjiang [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an (China); Li, Xia, E-mail: xial_li@qq.com [Department of Geriatrics, The Affiliated Huai' an Hospital of Xuzhou Medical College, Huai' an (China)

    2017-02-01

    Late thrombus and restenosis caused by delayed endothelialization and insufficient biocompatibility of polymer coating continue to be the greatest limitations of drug-eluting stents. In this study, based on the specific structure of vascular basement membrane, a novel biomimetic nano-coating was constructed by incorporating laminin into electrostatic-assembled heparin/poly-L-lysine nanoparticles. Alteration of heparin and poly-L-lysine concentration ratio in a certain range has no significantly influence nanoparticle size, uniformity and stability, but may affect the chemical property and subsequently the binding efficiency to dopamine-coated titanium surface. By use of this feature, four different nanoparticles were synthesized and immobilized on titanium surface for creating gradient nanoparticle binding density. According to in vitro biocompatibility evaluation, the nanoparticle modified surfaces were found to effectively block coagulation pathway and reduce thrombosis formation. Moreover, NP10L and NP15L modified surface with relatively low heparin exposing density (4.9 to 7.1 μg/cm2) showed beneficial effect in selective promoting EPCs and ECs proliferation, as well as stimulating cell migration and NO synthesis. - Highlights: • A novel laminin-loaded anticoagulant nanoparticle was prepared and used for titanium surface modification. • The nanoparticle binding density was adjustable by alteration the concentration ratio of heparin and poly-L-lysine. • In a certain range of NPs density, the surface was found to selectively direct platelet and vascular cells behavior.

  3. Surface biomimetic modification with laminin-loaded heparin/poly-L-lysine nanoparticles for improving the biocompatibility

    International Nuclear Information System (INIS)

    Liu, Tao; Hu, Youdong; Tan, Jianying; Liu, Shihui; Chen, Junying; Guo, Xin; Pan, Changjiang; Li, Xia

    2017-01-01

    Late thrombus and restenosis caused by delayed endothelialization and insufficient biocompatibility of polymer coating continue to be the greatest limitations of drug-eluting stents. In this study, based on the specific structure of vascular basement membrane, a novel biomimetic nano-coating was constructed by incorporating laminin into electrostatic-assembled heparin/poly-L-lysine nanoparticles. Alteration of heparin and poly-L-lysine concentration ratio in a certain range has no significantly influence nanoparticle size, uniformity and stability, but may affect the chemical property and subsequently the binding efficiency to dopamine-coated titanium surface. By use of this feature, four different nanoparticles were synthesized and immobilized on titanium surface for creating gradient nanoparticle binding density. According to in vitro biocompatibility evaluation, the nanoparticle modified surfaces were found to effectively block coagulation pathway and reduce thrombosis formation. Moreover, NP10L and NP15L modified surface with relatively low heparin exposing density (4.9 to 7.1 μg/cm2) showed beneficial effect in selective promoting EPCs and ECs proliferation, as well as stimulating cell migration and NO synthesis. - Highlights: • A novel laminin-loaded anticoagulant nanoparticle was prepared and used for titanium surface modification. • The nanoparticle binding density was adjustable by alteration the concentration ratio of heparin and poly-L-lysine. • In a certain range of NPs density, the surface was found to selectively direct platelet and vascular cells behavior.

  4. Quantum mechanics/molecular mechanics modeling of photoelectron spectra: the carbon 1s core-electron binding energies of ethanol-water solutions.

    Science.gov (United States)

    Löytynoja, T; Niskanen, J; Jänkälä, K; Vahtras, O; Rinkevicius, Z; Ågren, H

    2014-11-20

    Using ethanol-water solutions as illustration, we demonstrate the capability of the hybrid quantum mechanics/molecular mechanics (QM/MM) paradigm to simulate core photoelectron spectroscopy: the binding energies and the chemical shifts. An integrated approach with QM/MM binding energy calculations coupled to preceding molecular dynamics sampling is adopted to generate binding energies averaged over the solute-solvent configurations available at a particular temperature and pressure and thus allowing for a statistical assessment with confidence levels for the final binding energies. The results are analyzed in terms of the contributions in the molecular mechanics model-electrostatic, polarization, and van der Waals-with atom or bond granulation of the corresponding MM charge and polarizability force-fields. The role of extramolecular charge transfer screening of the core-hole and explicit hydrogen bonding is studied by extending the QM core to cover the first solvation shell. The results are compared to those obtained from pure electrostatic and polarizable continuum models. Particularly, the dependence of the carbon 1s binding energies with respect to the ethanol concentration is studied. Our results indicate that QM/MM can be used as an all-encompassing model to study photoelectron binding energies and chemical shifts in solvent environments.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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

  6. Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yezhong [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China); College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China); Zhou Bo [College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China); Zhang Xiaoping; Huang Ping [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China); Li Chaohong [Education Ministry Key Laboratory for Oral Biomedical Engineering, School of Stomatology, Wuhan University, Wuhan 430072 (China); Liu Yi [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023 (China) and College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China)], E-mail: prof.liuyi@263.net

    2009-04-30

    The interaction between malachite green (MG) and bovine serum albumin (BSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by MG was the result of the formation of the MG-BSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (K{sub a}) between MG and BSA at four different temperatures were obtained to be 3.734 x 10{sup 4}, 3.264 x 10{sup 4}, 2.718 x 10{sup 4}, and 2.164 x 10{sup 4} L mol{sup -1}, respectively. The enthalpy change ({delta}H) and entropy change ({delta}S) were calculated to be -27.25 kJ mol{sup -1} and -11.23 J mol{sup -1} K{sup -1}, indicating that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. Site marker competitive experiments indicated that the binding of MG to BSA primarily took place in sub-domain IIA. The binding distance (r) between MG and the tryptophan residue of BSA was obtained to be 4.79 nm according to Foerster theory of non-radioactive energy transfer. The conformational investigation showed that the presence of MG decreased the {alpha}-helical content of BSA (from 62.6% to 55.6%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules.

  7. Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods

    International Nuclear Information System (INIS)

    Zhang Yezhong; Zhou Bo; Zhang Xiaoping; Huang Ping; Li Chaohong; Liu Yi

    2009-01-01

    The interaction between malachite green (MG) and bovine serum albumin (BSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by MG was the result of the formation of the MG-BSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (K a ) between MG and BSA at four different temperatures were obtained to be 3.734 x 10 4 , 3.264 x 10 4 , 2.718 x 10 4 , and 2.164 x 10 4 L mol -1 , respectively. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -27.25 kJ mol -1 and -11.23 J mol -1 K -1 , indicating that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. Site marker competitive experiments indicated that the binding of MG to BSA primarily took place in sub-domain IIA. The binding distance (r) between MG and the tryptophan residue of BSA was obtained to be 4.79 nm according to Foerster theory of non-radioactive energy transfer. The conformational investigation showed that the presence of MG decreased the α-helical content of BSA (from 62.6% to 55.6%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules

  8. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Directory of Open Access Journals (Sweden)

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  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. Heparin molecularly imprinted polymer thin flm on gold electrode by plasma-induced graft polymerization for label-free biosensor.

    Science.gov (United States)

    Orihara, Kouhei; Hikichi, Atsushi; Arita, Tomohiko; Muguruma, Hitoshi; Yoshimi, Yasuo

    2018-03-20

    Heparin, a highly sulfated glycosaminoglycan, is an important biomaterial having biological and therapeutic functionalities such as anticoagulation, regeneration, and protein stabilization. This study addresses a label-free quartz crystal microbalance (QCM) biosensor for heparin detection based on a macromolecularly imprinted polymer (MIP) as an artificial recognition element. We demonstrate the novel strategy for MIP in the form of thin film on a gold (Au) electrode with the plasma-induced graft polymerization (PIP) technique. The procedure of PIP is as follows: (i) Hexamethyldisiloxane plasma-polymerized thin film (PPF) as a pre-coating scaffold of active species for PIP (post-polymerization) is deposited on an Au electrode. (ii) The PPF/Au electrode is soaked in an water solution containing heparin (template), (2-(methacryloxy)-ethyl)trimethylammonium chloride acrylamide (functional monomer), acrylamide, and N,N-methylenebisacrylamide (crosslinker). Double bonds of monomer and crosslinker attacked by residually active species in pre-coating PPF cause radical chain reaction. Consequently, a growing polymer network of 20 nm thickness of PIP-MIP thin film is formed and grafted on the PPF/Au surface. (iii) The PIP-MIP/PPF/Au is washed by sodium chloride solution so as to remove the template. Non-imprinted polymer (NIP) is carried out like the same procedure without a template. The AFM, XPS, and QCM measurements show that the PIP process facilitates macromolecularly surface imprinting of template heparin where the template is easily removed and is rapidly rebound to PIP-MIP without a diffusional barrier. The heparin-PIP-MIP specifically binds to heparin compared with heparin analog chondroitin sulfate C (selective factor: 4.0) and a detectable range of heparin in the presence of CS (0.1 wt%) was 0.001-0.1 wt%. The PIP-NIP does not show selectivity between them. The evaluated binding kinetics are association (k a  = 350 ± 100 M -1  s -1

  11. Binding of Divalent Cations to Polygalacturonate: A Mechanism Driven by the Hydration Water.

    Science.gov (United States)

    Huynh, Uyen T D; Lerbret, Adrien; Neiers, Fabrice; Chambin, Odile; Assifaoui, Ali

    2016-02-11

    We have investigated the interactions between polygalacturonate (polyGal) and four divalent cations (M(2+) = Ba(2+), Ca(2+), Mg(2+), Zn(2+)) that differ in size and affinity for water. Our results evidence that M(2+)-polyGal interactions are intimately linked to the affinity of M(2+) for water. Mg(2+) interacts so strongly with water that it remains weakly bound to polyGal (polycondensation) by sharing water molecules from its first coordination shell with the carboxylate groups of polyGal. In contrast, the other cations form transient ionic pairs with polyGal by releasing preferentially one water molecule (for Zn(2+)) or two (for Ca(2+) and Ba(2+)), which corresponds to monodentate and bidentate binding modes with carboxylates, respectively. The mechanism for the binding of these three divalent cations to polyGal can be described by two steps: (i) monocomplexation and formation of point-like cross-links between polyGal chains (at low M(2+)/Gal molar ratios, R) and (ii) dimerization (at higher R). The threshold molar ratio, R*, between these two steps depends on the nature of divalent cations and is lower for calcium ions (R* 0.3). This difference may be explained by the intermediate affinity of Ca(2+) for water with respect to those of Zn(2+) and Ba(2+), which may induce the formation of cross-links of intermediate flexibility. By comparison, the lower and higher flexibilities of the cross-links formed by Zn(2+) and Ba(2+), respectively, may shift the formation of dimers to higher molar ratios (R*).

  12. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Juan Du

    Full Text Available Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin or polymeric form (F-actin. Members of the actin-depolymerizing factor (ADF/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1 in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin.

  13. Sensitive detection of oversulfated chondroitin sulfate in heparin sodium or crude heparin with a colorimetric microplate based assay.

    Science.gov (United States)

    Sommers, Cynthia D; Mans, Daniel J; Mecker, Laura C; Keire, David A

    2011-05-01

    In this work we describe a 96-well microplate assay for oversulfated chondroitin sulfate A (OSCS) in heparin, based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N'-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. The assay takes advantage of several unique properties of heparin, OSCS, and LPTP, including OSCS inhibition of heparinase I and II activity, the molecular weight dependence of heparin-LPTP spectral shifts, and the distinct association of heparin fragments and OSCS to LPTP. These factors combine to enable detection of the presence of 0.003% w/w spiked OSCS in 10 μg of heparin sodium active pharmaceutical ingredient (API) using a plate reader and with visual detection to 0.1% levels. The same detection limit for OSCS was observed in the presence of 10% levels of dermatan sulfate (DS) or chondroitin sulfate A (CSA) impurities. In addition, we surveyed a selection of crude heparin samples received by the agency in 2008 and 2009 to determine average and extreme DS, CSA, and galactosamine weight percent levels. In the presence of these impurities and the variable heparin content in the crude heparin samples, spiked OSCS was reliably detected to the 0.1% w/w level using a plate reader. Finally, authentically OSCS contaminated heparin sodium API and crude samples were distinguished visually by color from control samples using the LPTP/heparinase test.

  14. Mechanism of selective VEGF-A binding by neuropilin-1 reveals a basis for specific ligand inhibition.

    Directory of Open Access Journals (Sweden)

    Matthew W Parker

    Full Text Available Neuropilin (Nrp receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF family of proangiogenic cytokines and the semaphorin 3 (Sema3 family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5 isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.

  15. Influence of binding material of PZT coating on microresonator's electrical and mechanical properties

    Science.gov (United States)

    Janusas, Giedrius; Guobiene, Asta; Palevicius, Arvydas; Brunius, Alfredas; Cekas, Elingas; Baltrusaitis, Valentinas; Sakalys, Rokas

    2017-06-01

    Microresonators are fundamental components integrated in hosts of MEMS applications: covering the automotive sector, the telecommunication industry, electronic equipment for surface/material characterization and motion sensing, and etc. The aim of this paper is to investigate the mechanical and electrical properties of PZT film fabricated with three binding materials: polyvinyl butyral (PVB), polymethyl methacrylate (PMMA) and polystyrene (PS) and to evaluate applicability in control of microresonators Q factor. Micro particles of PZT powder were mixed with 20% solution of PVB, PMMA and PS in benzyl alcohol. For investigation of mechanical and electrical properties multilayer cantilevers were made. Obtained PZT and polymer paste was screen printed on copper (thickness 40 μm) using polyester monofilament screen meshes (layer thickness 50 μm) and dried for 30 min at 100°C. Electric dipoles of the PZT particles in composite material were aligned using high voltage generator (5 kV) and a custom-made holder. Electric field was held for 30 min. Surfaces of the applied films were investigated by Atomic Force Microscope NanoWizard(R)3 NanoScience. Dynamic and electrical characteristics of the multilayer were investigated using laser triangular displacement sensor LK-G3000. The measured vibration amplitude and generated electrical potential was collected with USB oscilloscope PicoScope 3424. As the results showed, these cantilevers were able to transform mechanical strain energy into electric potential and, v.v. However, roughness of PZT coatings with PMMA and PS were higher, what could be the reason of the worse quality of the top electrode. However, the main advantage of the created composite piezoelectric material is the possibility to apply it on any uniform or non-uniform vibrating surface and to transform low frequency vibrations into electricity.

  16. Increased synthesis of heparin affin regulatory peptide in the perforant path lesioned mouse hippocampal formation

    DEFF Research Database (Denmark)

    Poulsen, F R; Lagord, C; Courty, J

    2000-01-01

    Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is a developmentally regulated extracellular matrix protein that induces cell proliferation and promotes neurite outgrowth in vitro as well as pre- and postsynaptic developmental...... differentiation in vivo. Here we have investigated the expression of HARP mRNA and protein in the perforant path lesioned C57B1/6 mouse hippocampal formation from 1 to 35 days after surgery. This type of lesion induces a dense anterograde and terminal axonal degeneration, activation of glial cells, and reactive...... axonal sprouting within the perforant path zones of the fascia dentata and hippocampus as well as axotomy-induced retrograde neuronal degeneration in the entorhinal cortex. Analysis of sham- and unoperated control mice showed that HARP mRNA is expressed in neurons and white and gray matter glial cells...

  17. Electrophoresis for the analysis of heparin purity and quality.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J

    2012-06-01

    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. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

    Science.gov (United States)

    Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

    2015-01-01

    Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

  19. 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.

  20. Immunomodulating effects of heparin on human B cell proliferation

    International Nuclear Information System (INIS)

    Wasik, Maria; Stepien-Sopniewska, Barbara; Gorski, Andrzej

    1993-01-01

    Recent data indicate that heparin may act as an immunomodulator. In this paper we have analyzed the effect of this agent on human B cell proliferation ''in vitro'' induced by ''S. aureus'' Cowan. The action of heparin is complex, but there was a trend for inhibition of B cell responses obtained from defibrinated but not heparinized blood samples. This suggest that heparin interacts with platelet products (growth factors, cytokines) and the results of such interactions determine the final effect. (author). 6 refs, 4 figs

  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. Effect of buffer at nanoscale molecular recognition interfaces - electrostatic binding of biological polyanions.

    Science.gov (United States)

    Rodrigo, Ana C; Laurini, Erik; Vieira, Vânia M P; Pricl, Sabrina; Smith, David K

    2017-10-19

    We investigate the impact of an over-looked component on molecular recognition in water-buffer. The binding of a cationic dye to biological polyanion heparin is shown by isothermal calorimetry to depend on buffer (Tris-HCl > HEPES > PBS). The heparin binding of self-assembled multivalent (SAMul) cationic micelles is even more buffer dependent. Multivalent electrostatic molecular recognition is buffer dependent as a result of competitive interactions between the cationic binding interface and anions present in the buffer.

  3. Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model.

    Science.gov (United States)

    Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong

    2016-04-05

    Thrombin-binding aptamer (TBA) with the sequence 5'GGTTGGTGTGGTTGG3' could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.

  4. Two-stage DNA compaction induced by silver ions suggests a cooperative binding mechanism

    Science.gov (United States)

    Jiang, Wen-Yan; Ran, Shi-Yong

    2018-05-01

    The interaction between silver ions and DNA plays an important role in the therapeutic use of silver ions and in related technologies such as DNA sensors. However, the underlying mechanism has not been fully understood. In this study, the dynamics of Ag+-DNA interaction at a single-molecule level was studied using magnetic tweezers. AgNO3 solutions with concentrations ranging from 1 μM to 20 μM led to a 1.4-1.8 μm decrease in length of a single λ-DNA molecule, indicating that Ag+ has a strong binding with DNA, causing the DNA conformational change. The compaction process comprises one linear declining stage and another sigmoid-shaped stage, which can be attributed to the interaction mechanism. Considering the cooperative effect, the sigmoid trend was well explained using a phenomenological model. By contrast, addition of silver nanoparticle solution induced no detectable transition of DNA. The dependence of the interaction on ionic strength and DNA concentration was examined via morphology characterization and particle size distribution measurement. The size of the Ag+-DNA complex decreased with an increase in Ag+ ionic strength ranging from 1 μM to 1 mM. Morphology characterization confirmed that silver ions induced DNA to adopt a compacted globular conformation. At a fixed [AgNO3]:[DNA base pairs] ratio, increasing DNA concentration led to increased sizes of the complexes. Intermolecular interaction is believed to affect the Ag+-DNA complex formation to a large extent.

  5. Differential Mechanisms for SHP2 Binding and Activation Are Exploited by Geographically Distinct Helicobacter pylori CagA Oncoproteins

    Directory of Open Access Journals (Sweden)

    Takeru Hayashi

    2017-09-01

    Full Text Available Helicobacter pylori East Asian CagA is more closely associated with gastric cancer than Western CagA. Here we show that, upon tyrosine phosphorylation, the East Asian CagA-specific EPIYA-D segment binds to the N-SH2 domain of pro-oncogenic SHP2 phosphatase two orders of magnitude greater than Western CagA-specific EPIYA-C. This high-affinity binding is achieved via cryptic interaction between Phe at the +5 position from phosphotyrosine in EPIYA-D and a hollow on the N-SH2 phosphopeptide-binding floor. Also, duplication of EPIYA-C in Western CagA, which increases gastric cancer risk, enables divalent high-affinity binding with SHP2 via N-SH2 and C-SH2. These strong CagA bindings enforce enzymatic activation of SHP2, which endows cells with neoplastic traits. Mechanistically, N-SH2 in SHP2 is in an equilibrium between stimulatory “relaxed” and inhibitory “squeezed” states, which is fixed upon high-affinity CagA binding to the “relaxed” state that stimulates SHP2. Accordingly, East Asian CagA and Western CagA exploit distinct mechanisms for SHP2 deregulation.

  6. Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

    Science.gov (United States)

    Poda, Suresh B; Kobayashi, Masakazu; Nachane, Ruta; Menon, Veena; Gandhi, Adarsh S; Budac, David P; Li, Guiying; Campbell, Brian M; Tagmose, Lena

    2015-10-01

    Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-β-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

  7. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

    Science.gov (United States)

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

    2010-01-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.—Kovacs, E., Harmat, V., Tóth, J., Vértessy, B. G., Módos, K., Kardos, J., Liliom, K. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. PMID:20522785

  8. DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

    Science.gov (United States)

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N1-methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ-and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained. PMID:24339041

  9. Structural and functional characterization of the interaction between cyclophilin B and a heparin-derived oligosaccharide.

    Science.gov (United States)

    Hanoulle, Xavier; Melchior, Aurélie; Sibille, Nathalie; Parent, Benjamin; Denys, Agnès; Wieruszeski, Jean-Michel; Horvath, Dragos; Allain, Fabrice; Lippens, Guy; Landrieu, Isabelle

    2007-11-23

    The chemotaxis and integrin-mediated adhesion of T lymphocytes triggered by secreted cyclophilin B (CypB) depend on interactions with both cell surface heparan sulfate proteoglycans (HSPG) and the extracellular domain of the CD147 membrane receptor. Here, we use NMR spectroscopy to characterize the interaction of CypB with heparin-derived oligosaccharides. Chemical shift perturbation experiments allowed the precise definition of the heparan sulfate (HS) binding site of CypB. The N-terminal extremity of CypB, which contains a consensus sequence for heparin-binding proteins was modeled on the basis of our experimental NMR data. Because the HS binding site extends toward the CypB catalytic pocket, we measured its peptidyl-prolyl cis-trans isomerase (PPIase) activity in the absence or presence of a HS oligosaccharide toward a CD147-derived peptide. We report the first direct evidence that CypB is enzymatically active on CD147, as it is able to accelerate the cis/trans isomerization of the Asp(179)-Pro(180) bond in a CD147-derived peptide. However, HS binding has no significant influence on this PPIase activity. We thus conclude that the glycanic moiety of HSPG serves as anchor for CypB at the cell surface, and that the signal could be transduced by CypB via its PPIase activity toward CD147.

  10. Naked eye detection of infertility based on sperm protamine-induced aggregation of heparin gold nanoparticles.

    Science.gov (United States)

    Vidya, Raj; Saji, Alex

    2018-05-01

    The development of an easy to use, one-pot, environmentally friendly, non-invasive and label-free colorimetric probe for the determination of semen protamines, the biochemical marker of male fertility, using heparin gold nanoparticles (HAuNPs) is presented. The affinity of HAuNPs for protamines was due to the electrostatic interactions between polycationic protamine and polyanionic heparin. The binding of HAuNPs to protamine was characterized by variation in the plasmon absorption spectra followed by a visibly observable colour change of the solution from red to blue. We observed a red shift in the plasmon peak and the method exhibited linearity in the range of 10-70 ng/mL with a detection limit of 5 ng/mL, which is much lower than that reported for colorimetric sensors of protamine. The colour change and the variation in the absorbance of HAuNPs were highly specific for protamines in the presence of different interfering compounds and the method was successfully applied for determining protamine in real samples of semen and serum. Rather than a quantitative estimation, it seems that the method provides a quick screening between a large array of positive and negative samples and, moreover, it maintains the privacy of the user. The method appears to be simple and would be very useful in third-world countries where high-tech diagnostic aids are inaccessible to the majority of the population. Graphical Abstract Heparin gold nanoparticles aided visual detection of infertility.

  11. Insights into the mechanism of ligand binding to octopine dehydrogenase from Pecten maximus by NMR and crystallography.

    Directory of Open Access Journals (Sweden)

    Sander H J Smits

    Full Text Available Octopine dehydrogenase (OcDH from the adductor muscle of the great scallop, Pecten maximus, catalyzes the NADH dependent, reductive condensation of L-arginine and pyruvate to octopine, NAD(+, and water during escape swimming and/or subsequent recovery. The structure of OcDH was recently solved and a reaction mechanism was proposed which implied an ordered binding of NADH, L-arginine and finally pyruvate. Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures. Furthermore, the crystal structure of the OcDH/NADH/agmatine complex was determined which suggests a key role of the side chain of L-arginine in protein cataylsis. Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail.

  12. SPECIFIC ASPECTS OF INTERACTION OF PLATELETS WITH THE HEPARINIZED MATERIALS

    Directory of Open Access Journals (Sweden)

    E.A. Nemets

    2012-01-01

    Full Text Available Comparative analysis of anticoagulant nature on medical materials testing was done. It was found that change of citrate by heparin is accompanied by significant changes in platelet adhesion and activation. This results allowed us to arrive at a conclusion about reasonability of heparin usage as anticoagulant in in vitro testing. 

  13. 99m Tc-labeled heparin test in orthopaedic surgery

    International Nuclear Information System (INIS)

    Bouvier, J.F.; Lafon, J.C.; Colin, M.; Chatelut, J.; Beaubatie, F.

    1983-01-01

    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 [fr

  14. 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.

  15. Heparin release from thermosensitive polymer coatings: in vivo studies

    NARCIS (Netherlands)

    Gutowska, Anna; Bae, You Han; Jacobs, Harvey; Mohammad, Fazal; Mix, Donald; Feijen, Jan; Kim, Sung Wan

    1995-01-01

    Biomer/poly(N-isopropylacrylamide)/[poly(NiPAAm)] thermosensitive polymer blends were prepared and their application as heparin-releasing polymer coatings for the prevention of surface-induced thrombosis was examined. The advantage of using poly(NiPAAm)-based coatings as heparin-releasing polymers

  16. Heparin-induced thrombocytopenia: real-world issues.

    Science.gov (United States)

    Linkins, Lori-Ann; Warkentin, Theodore E

    2011-09-01

    Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating antibodies. HIT sera often activate platelets without needing heparin-such heparin-"independent" platelet activation can be associated with HIT beginning or worsening despite stopping heparin ("delayed-onset HIT"). We address important issues in HIT diagnosis and therapy, using a recent cohort of HIT patients to illustrate influences of heparin type; triggers for HIT investigation; serological features of heparin-independent platelet activation; and treatment. In our cohort of recent HIT cases ( N = 13), low-molecular-weight heparin (dalteparin) was a common causative agent ( N = 8, 62%); most patients were diagnosed after HIT-thrombosis had occurred; and danaparoid was the most frequently selected treatment. Heparin-independent platelet activation was common (7/13 [54%]) and predicted slower platelet count recovery (>1 week) among evaluable patients (5/5 vs 1/6; P = 0.015). In our experience with argatroban-treated patients, HIT-associated consumptive coagulopathy confounds anticoagulant monitoring. Our observations provide guidance on practical aspects of HIT diagnosis and management. Thieme Medical Publishers.

  17. 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 ...

  18. Molecular modeling study on the allosteric inhibition mechanism of HIV-1 integrase by LEDGF/p75 binding site inhibitors.

    Directory of Open Access Journals (Sweden)

    Weiwei Xue

    Full Text Available HIV-1 integrase (IN is essential for the integration of viral DNA into the host genome and an attractive therapeutic target for developing antiretroviral inhibitors. LEDGINs are a class of allosteric inhibitors targeting LEDGF/p75 binding site of HIV-1 IN. Yet, the detailed binding mode and allosteric inhibition mechanism of LEDGINs to HIV-1 IN is only partially understood, which hinders the structure-based design of more potent anti-HIV agents. A molecular modeling study combining molecular docking, molecular dynamics simulation, and binding free energy calculation were performed to investigate the interaction details of HIV-1 IN catalytic core domain (CCD with two recently discovered LEDGINs BI-1001 and CX14442, as well as the LEDGF/p75 protein. Simulation results demonstrated the hydrophobic domain of BI-1001 and CX14442 engages one subunit of HIV-1 IN CCD dimer through hydrophobic interactions, and the hydrophilic group forms hydrogen bonds with HIV-1 IN CCD residues from other subunit. CX14442 has a larger tert-butyl group than the methyl of BI-1001, and forms better interactions with the highly hydrophobic binding pocket of HIV-1 IN CCD dimer interface, which can explain the stronger affinity of CX14442 than BI-1001. Analysis of the binding mode of LEDGF/p75 with HIV-1 IN CCD reveals that the LEDGF/p75 integrase binding domain residues Ile365, Asp366, Phe406 and Val408 have significant contributions to the binding of the LEDGF/p75 to HIV1-IN. Remarkably, we found that binding of BI-1001 and CX14442 to HIV-1 IN CCD induced the structural rearrangements of the 140 s loop and oration displacements of the side chains of the three conserved catalytic residues Asp64, Asp116, and Glu152 located at the active site. These results we obtained will be valuable not only for understanding the allosteric inhibition mechanism of LEDGINs but also for the rational design of allosteric inhibitors of HIV-1 IN targeting LEDGF/p75 binding site.

  19. Mechanism of DNA–binding loss upon single-point mutation in p53

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    loss in protein−DNA binding affinity and specificity upon single point ..... we computed the root–mean–square–deviations (RMSDs) of each residue's ...... Petsko G and Ringe D 1984 Fluctuations in protein structure from. X-ray diffraction; Annu.

  20. Role of Ca2+ in the binding mechanism of EHDP-Tc complex to bone

    International Nuclear Information System (INIS)

    Langevelde, A. van; Huisman, C.M.; Driessen, O.M.J.; Pauwels, E.K.J.

    1979-01-01

    Experiments are described testing the hypothesis that 99m-Tc-EHDP complex travels to bone as a unit and dissociates at the bone binding site because of the high affinity of EHDP for hydroxyapatite after which technetium binds separately. The results indicate that technetium is not dissociated from EHDP in binding to hydroxyapatite, but the EHDP-Tc-ligand stays intact. It is postulated that calcium plays an important role in bone-labelling with EHDP-Tc complex and that in fact the EHDP-Ca-Tc complex is the binding agent. Only by assuming the presence of this agent could the action of magnesium-ions or of excess calcium-ions be explained. (Auth./C.F.)

  1. Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

    OpenAIRE

    Davidson, Edgar; Bryan, Christopher; Fong, Rachel H.; Barnes, Trevor; Pfaff, Jennifer M.; Mabila, Manu; Rucker, Joseph B.; Doranz, Benjamin J.

    2015-01-01

    Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epito...

  2. Molecular modeling reveals the novel inhibition mechanism and binding mode of three natural compounds to staphylococcal α-hemolysin.

    Directory of Open Access Journals (Sweden)

    Jiazhang Qiu

    Full Text Available α-Hemolysin (α-HL is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG, Oroxin A (ORA, and Oroxin B (ORB, when inhibiting the hemolytic activity of α-HL, could bind to the "stem" region of α-HL. This was completed using conventional Molecular Dynamics (MD simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA indicated that because of the inhibitors that bind to the "stem" region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.

  3. The asymmetric binding of PGC-1α to the ERRα and ERRγ nuclear receptor homodimers involves a similar recognition mechanism.

    Directory of Open Access Journals (Sweden)

    Maria Takacs

    Full Text Available PGC-1α is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts together with the estrogen-related receptors (ERRα and ERRγ in the regulation of mitochondrial and metabolic gene networks. Dimerization of the ERRs is a pre-requisite for interactions with PGC-1α and other coactivators, eventually leading to transactivation. It was suggested recently (Devarakonda et al that PGC-1α binds in a strikingly different manner to ERRγ ligand-binding domains (LBDs compared to its mode of binding to ERRα and other nuclear receptors (NRs, where it interacts directly with the two ERRγ homodimer subunits.Here, we show that PGC-1α receptor interacting domain (RID binds in an almost identical manner to ERRα and ERRγ homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1α RID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions. NMR studies further defined the limits of PGC-1α RID that interacts with ERRs. Consistent with these findings, the solution structures of PGC-1α/ERRα LBDs and PGC-1α/ERRγ LBDs complexes share an identical architecture with an asymmetric binding of PGC-1α to homodimeric ERR.These studies provide the molecular determinants for the specificity of interactions between PGC-1α and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Our work indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators to homodimers.

  4. Use of heparin in the investigation of obscure gastrointestinal bleeding

    International Nuclear Information System (INIS)

    Mernagh, J.R.; O'Donovan, N.; Somers, S.; Gill, G.; Sridhar, S.

    2001-01-01

    To determine if the administration of heparin improves the predictive value of angiography in the investigation of obscure gastrointestinal (GI) bleeding. 18 patients with a history of chronic GI bleeding were investigated with angiography. For 6 patients, the cause of GI bleeding was established with angiography; the 12 patients who had negative results were given heparin for 24 h and were reassessed with angiography. After heparin administration, the source of GI bleeding was determined with angiography for 6 of the remaining 12 patients. Thus, heparinization increased diagnostic yield from 33% (6 of 18) to 67% (12 of 18). No significant complications, such as uncontrolled GI bleeding, occurred. Heparinization improves the diagnostic yield of angiography when obscure GI bleeding is being investigated. (author)

  5. Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

    Directory of Open Access Journals (Sweden)

    Hyock Joo Kwon

    Full Text Available Ledipasvir, a direct acting antiviral agent (DAA targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants.

  6. 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.

  7. Printed microfluidic filter for heparinized blood.

    Science.gov (United States)

    Bilatto, Stanley E R; Adly, Nouran Y; Correa, Daniel S; Wolfrum, Bernhard; Offenhäusser, Andreas; Yakushenko, Alexey

    2017-05-01

    A simple lab-on-a-chip method for blood plasma separation was developed by combining stereolithographic 3D printing with inkjet printing, creating a completely sealed microfluidic device. In some approaches, one dilutes the blood sample before separation, reducing the concentration of a target analyte and increasing a contamination risk. In this work, a single drop (8  μ l) of heparinized whole blood could be efficiently filtered using a capillary effect without any external driving forces and without dilution. The blood storage in heparin tubes during 24 h at 4 °C initiated the formation of small crystals that formed auto-filtration structures in the sample upon entering the 3D-printed device, with pores smaller than the red blood cells, separating plasma from the cellular content. The total filtration process took less than 10 s. The presented printed plasma filtration microfluidics fabricated with a rapid prototyping approach is a miniaturized, fast and easy-to-operate device that can be integrated into healthcare/portable systems for point-of-care diagnostics.

  8. Combined quantum mechanics/molecular mechanics (QM/MM) simulations for protein-ligand complexes: free energies of binding of water molecules in influenza neuraminidase.

    Science.gov (United States)

    Woods, Christopher J; Shaw, Katherine E; Mulholland, Adrian J

    2015-01-22

    The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

  9. 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.

  10. 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.

  11. Trans-Binding Mechanism of Ubiquitin-like Protein Activation Revealed by a UBA5-UFM1 Complex

    Directory of Open Access Journals (Sweden)

    Walaa Oweis

    2016-09-01

    Full Text Available Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s. Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation.

  12. Elucidation of the binding mechanism of coumarin derivatives with human serum albumin.

    Directory of Open Access Journals (Sweden)

    Archit Garg

    Full Text Available Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×10(5 M(-1, -7.175 Kcal M(-1 for coumarin derivative (CD enamide; 0.837±0.01×10(5 M(-1, -6.685 Kcal M(-1 for coumarin derivative (CD enoate, and 0.606±0.01×10(5 M(-1, -6.49 Kcal M(-1 for coumarin derivative methylprop (CDM enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases.

  13. Interactions of Poly(amidoamine) Dendrimers with Human Serum Albumin: Binding Constants and Mechanisms

    OpenAIRE

    Giri, Jyotsnendu; Diallo, Mamadou S.; Simpson, André J.; Liu, Yi; Goddard, William A., III; Kumar, Rajeev; Woods, Gwen C.

    2011-01-01

    The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K_b) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To g...

  14. 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

    to overcome these limitations. Herein, we have developed a thermosensitive heparin nanosponge (Hep-NS) by a one step photopolymerization reaction between diacrylated pluronic and thiolated heparin molecules. The amount of heparin in Hep-NS was precisely controlled by varying the heparin amount in the reaction...

  15. Differences in Binding and Monitoring Mechanisms Contribute to Lifespan Age Differences in False Memory

    Science.gov (United States)

    Fandakova, Yana; Shing, Yee Lee; Lindenberger, Ulman

    2013-01-01

    Based on a 2-component framework of episodic memory development across the lifespan (Shing & Lindenberger, 2011), we examined the contribution of memory-related binding and monitoring processes to false memory susceptibility in childhood and old age. We administered a repeated continuous recognition task to children (N = 20, 10-12 years),…

  16. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    DEFF Research Database (Denmark)

    Wong, Mei Mei Jaslyn Elizabeth; Midtgaard, Søren Roi; Gysel, Kira

    2015-01-01

    of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering...

  17. Ligand binding and activation mechanism og the glucagon-like peptide-1 receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye

    GLP-1R interacts with receptor agonists. The thesis includes four studies, which investigate different aspects of these interactions. The first study elucidates GLP-1 binding to the extracellular domain of GLP-1R (ECD) (Study I), whereas the second study identifies receptor domains important for small...

  18. Current Trends in Heparin Use During Arterial Vascular Interventional Radiology

    International Nuclear Information System (INIS)

    Durran, Alexandra C.; Watts, Christopher

    2012-01-01

    Purpose: This study was designed to assess the current use of heparinized saline and bolus doses of heparin in non-neurological interventional radiology and to determine whether consensus could be reached to produce guidance for heparin use during arterial vascular intervention. Methods: An interactive electronic questionnaire was distributed to members of the British Society of Interventional Radiology regarding their current practice in the use, dosage, and timing of heparin boluses and heparinized flushing solutions.ResultsA total of 108 completed questionnaires were received. More than 80% of respondents used heparinized saline with varying concentrations; the most prevalent was 1,000 IU/l (international units of heparin per liter) and 5,000 IU/l. Fifty-one percent of interventionalists use 3,000 IU as their standard bolus dose; however, the respondents were split regarding the timing of bolus dose with ∼60% administering it after arterial access is obtained and 40% after crossing the lesion. There was no consensus on altering dose according to body weight, and only 4% monitored clotting parameters. Conclusions: There seems to be some coherence among practicing interventionalists regarding heparin administration. We hypothesize that heparinized saline should be used at a recognized standard concentration of 1,000 IU/l as a flushing concentration in all arterial vascular interventions and that 3,000 IU bolus is considered the standard dose for straightforward therapeutic procedures and 5000 IU for complex, crural, and endovascular aneurysm repair work. The bolus should be given after arterial access is obtained to allow time for optimal anticoagulation to be achieved by the time of active intervention and stenting. Further research into clotting abnormalities following such interventional procedures would be an interesting quantifiable follow-up to this initial survey of opinions and practice.

  19. Vitamin K3 disrupts the microtubule networks by binding to tubulin: a novel mechanism of its antiproliferative activity.

    Science.gov (United States)

    Acharya, Bipul R; Choudhury, Diptiman; Das, Amlan; Chakrabarti, Gopal

    2009-07-28

    Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

  20. Studies on binding mechanism between carotenoids from sea buckthorn and thermally treated α-lactalbumin

    Science.gov (United States)

    Dumitraşcu, Loredana; Ursache, Florentina Mihaela; Stănciuc, Nicoleta; Aprodu, Iuliana

    2016-12-01

    Sea buckthorn is a natural food ingredient rich in bioactive compounds such as carotenoids, tocopherols, sterols, flavonoids, lipids, vitamins, tannins and minerals. Herein, fluorescence and UV-vis techniques were used to study the interaction of heat treated α-lactalbumin (α-LA) with carotenoids from sea buckthorn berries extract (CSB) and β-carotene. Further atomic level details on the interaction between α-LA and β-carotene were obtained by means of molecular modelling techniques. The quenching rate constants, binding constants, and number of binding sites were calculated in the presence of CSB. The emission spectral studies revealed that, CSB have the ability to bind α-LA and form a ground state complex via static quenching process. Maximum degree of quenching was reached at 100 °C, where β-carotene and CSB quenched the Trp fluorescence of α-LA by 56% and 47%, respectively. In order to reveal the interaction between CSB and α-LA, the thermodynamic parameters were determined from the van't Hoff plot based on the temperature dependence of the binding constant. In agreement with the in silico observations, the thermodynamic parameters enabled us to consider that the association between α-LA and β-carotene is a spontaneous process driven by enthalpy, dominated mainly by the van der Waals interaction, but hydrophobic interactions might also be considered. The interaction between CSB and α-LA was further confirmed by UV-vis absorption spectra, where a blue shift of position was noticed at higher temperature suggesting the complex formation. The results provided here supply a better understanding of the binding of CSB to α-LA, which can be further exploited in designing new healthy food applications.

  1. Capillary electrophoresis of heparin and other glycosaminoglycans using a polyamine running electrolyte

    International Nuclear Information System (INIS)

    Loegel, Thomas N.; Trombley, John D.; Taylor, Richard T.; Danielson, Neil D.

    2012-01-01

    Highlights: ► Ethylenediamine is likely acting as an ion-pairing agent. ► Oversulfated chondroitin sulfate is last peak instead of first peak. ► There is about a factor of five improved detectability with a 12.5 min analysis time. ► Use of a 50 μm ID capillary is possible. - Abstract: This study involves the use of polyamines as potential resolving agents for the capillary electrophoresis (CE) of glycosaminoglycans (GAGs), specifically heparin, dermatan sulfate, chondroitin sulfate, over-sulfated chondroitin sulfate (OSCS), and hyaluronan. All of the compounds can be separated from each other with the exception of chondroitin sulfate and hyaluronan. Using optimization software, the final run conditions are found to be 200 mM ethylenediamine and 45.5 mM phosphate as the electrolyte with −14 V applied across a 50 μm ID × 24.5 cm fused silica capillary at 15 °C. The ion migration order, with OSCS as the last instead of the first peak, is in contrast to previous reports using either a high molarity TRIS or lithium phosphate run buffer with narrower bore capillaries. Total analysis time is 12. 5 min and the relative standard deviation of the heparin migration time is about 2.5% (n = 5). The interaction mechanism between selected polyamines and heparin is explored using conductivity measurements in addition to CE experiments to show that an ion-pairing mechanism is likely.

  2. 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 evaluate the thromboprophylactic use of low molecular weight heparins (LMWHs), publications from 27 orthopaedic trials and 35 studies of patients undergoing general or gynaecological surgery were scrutinized and subjected to a partial meta-analysis. In orthopaedic surgery, LMWHs were superior...... 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...

  3. 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......). A reduced number of patients needed blood transfusions in the RxDx group, although this was not statistically significant (19% vs. 38%, respectively; p = .13). Initial heparin dose was significantly reduced in the RxDx group (250 mg; range, 100-375 mg) compared with the control group (300 mg; range, 200...

  4. Effects of the repeated administration of adenosine and heparin on myocardial perfusion in patients with chronic stable angina pectoris.

    Science.gov (United States)

    Barron, H V; Sciammarella, M G; Lenihan, K; Michaels, A D; Botvinick, E H

    2000-01-01

    The mechanism by which ischemia stimulates angiogenesis is unknown. Adenosine is released during myocardial ischemia and may be a mediator of this process. Experimental data suggest that heparin may enhance this effect. The purpose of this open-labeled, placebo-controlled trial was to determine whether repeated intravenous administration of adenosine and heparin could mimic physiologic angiogenesis and reduce the amount of exercise-induced myocardial ischemia in patients with coronary artery disease. Subjects with chronic stable angina refractory to conventional medical therapy and not suitable for revascularization received either adenosine (140 microg/kg/min for 6 minutes) and heparin (10,000 U bolus), (n = 14), or placebo, (n = 7) daily for 10 days. All patients underwent baseline and follow-up exercise testing with thallium-201 single-photon emission computed tomography myocardial perfusion imaging. A semiquantitative assessment of the extent and severity of the perfusion abnormalities was calculated by 2 blinded investigators. There was no significant change in exercise duration or in the peak heart rate systolic blood pressure product associated with adenosine and heparin compared with placebo treatment. There was, however, a 9% reduction in the extent (60.6 +/- 4.0 vs 54.9 +/- 4.1, p = 0.03) and a 14% improvement in severity (41.5 +/- 3.2 vs 35.7 +/- 2.9, p = 0.01) of the myocardial perfusion abnormalities seen in patients who received adenosine and heparin compared with placebo. Thus, in this pilot study, repeated administration of adenosine and heparin reduced the amount of exercise-induced ischemia in patients with chronic stable angina refractory to conventional treatment.

  5. Results of the HepZero study comparing heparin-grafted membrane and standard care show that heparin-grafted dialyzer is safe and easy to use for heparin-free dialysis

    NARCIS (Netherlands)

    Laville, Maurice; Dorval, Marc; Ros, Joan Fort; Fay, Renaud; Cridlig, Joelle; Nortier, Joelle L.; Juillard, Laurent; Debska-Slizien, Alicja; Lorente, Loreto Fernandez; Thibaudin, Damien; Franssen, Casper; Schulz, Michael; Moureau, Frederique; Loughraieb, Nathalie; Rossignol, Patrick

    2014-01-01

    Heparin is used to prevent clotting during hemodialysis, but heparin-free hemodialysis is sometimes needed to decrease the risk of bleeding. The HepZero study is a randomized, multicenter international controlled open-label trial comparing no-heparin hemodialysis strategies designed to assess

  6. Free energies of binding from large-scale first-principles quantum mechanical calculations: application to ligand hydration energies.

    Science.gov (United States)

    Fox, Stephen J; Pittock, Chris; Tautermann, Christofer S; Fox, Thomas; Christ, Clara; Malcolm, N O J; Essex, Jonathan W; Skylaris, Chris-Kriton

    2013-08-15

    Schemes of increasing sophistication for obtaining free energies of binding have been developed over the years, where configurational sampling is used to include the all-important entropic contributions to the free energies. However, the quality of the results will also depend on the accuracy with which the intermolecular interactions are computed at each molecular configuration. In this context, the energy change associated with the rearrangement of electrons (electronic polarization and charge transfer) upon binding is a very important effect. Classical molecular mechanics force fields do not take this effect into account explicitly, and polarizable force fields and semiempirical quantum or hybrid quantum-classical (QM/MM) calculations are increasingly employed (at higher computational cost) to compute intermolecular interactions in free-energy schemes. In this work, we investigate the use of large-scale quantum mechanical calculations from first-principles as a way of fully taking into account electronic effects in free-energy calculations. We employ a one-step free-energy perturbation (FEP) scheme from a molecular mechanical (MM) potential to a quantum mechanical (QM) potential as a correction to thermodynamic integration calculations within the MM potential. We use this approach to calculate relative free energies of hydration of small aromatic molecules. Our quantum calculations are performed on multiple configurations from classical molecular dynamics simulations. The quantum energy of each configuration is obtained from density functional theory calculations with a near-complete psinc basis set on over 600 atoms using the ONETEP program.

  7. Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding

    Science.gov (United States)

    Newcomer, Rebecca L.; Fraser, LaTasha C.R.; Teschke, Carolyn M.; Alexandrescu, Andrei T.

    2015-01-01

    The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC’ couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC’ H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. PMID:26682823

  8. Unraveling the Molecular Mechanism of Benzothiophene and Benzofuran scaffold merged compounds binding to anti-apoptotic Myeloid cell leukemia 1.

    Science.gov (United States)

    Marimuthu, Parthiban; Singaravelu, Kalaimathy

    2018-05-10

    Myeloid cell leukemia 1 (Mcl1), is an anti-apoptotic member of the Bcl-2 family proteins, has gained considerable importance due to its overexpression activity prevents the oncogenic cells to undergo apoptosis. This overexpression activity of Mcl1 eventually develops strong resistance to a wide variety of anticancer agents. Therefore, designing novel inhibitors with potentials to elicit higher binding affinity and specificity to inhibit Mcl1 activity is of greater importance. Thus, Mcl1 acts as an attractive cancer target. Despite recent experimental advancement in the identification and characterization of Benzothiophene and Benzofuran scaffold merged compounds the molecular mechanisms of their binding to Mcl1 are yet to be explored. The current study demonstrates an integrated approach -pharmacophore-based 3D-QSAR, docking, Molecular Dynamics (MD) simulation and free-energy estimation- to access the precise and comprehensive effects of current inhibitors targeting Mcl1 together with its known activity values. The pharmacophore -ANRRR.240- based 3D-QSAR model from the current study provided high confidence (R 2 =0.9154, Q 2 =0.8736, and RMSE=0.3533) values. Furthermore, the docking correctly predicted the binding mode of highly active compound 42. Additionally, the MD simulation for docked complex under explicit-solvent conditions together with free-energy estimation exhibited stable interaction and binding strength over the time period. Also, the decomposition analysis revealed potential energy contributing residues -M231, M250, V253, R265, L267, and F270- to the complex stability. Overall, the current investigation might serve as a valuable insight, either to (i) improve the binding affinity of the current compounds or (ii) discover new generation anti-cancer agents that can effectively downregulate Mcl1 activity.

  9. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2010-01-01

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations

  10. Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Jin [Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Science, Wuhan University, Wuhan 430071 (China); Wang, Ying [Department of Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Su, Ke [Department of Nephrology, Renmin Hospital of Wuhan University, Wuhan 430060 (China); Liu, Min [Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Science, Wuhan University, Wuhan 430071 (China); Hu, Peng-Chao [Department of Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Ma, Tian; Li, Jia-Xi [Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Science, Wuhan University, Wuhan 430071 (China); Wei, Lei [Department of Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Zheng, Zhongliang, E-mail: biochem@whu.edu.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Yang, Fang, E-mail: fang-yang@whu.edu.cn [Department of Physiology, School of Medicine, Wuhan University, Wuhan 430071 (China)

    2014-10-01

    Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERβ expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17β-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17β-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis. - Graphical abstract: RTV directly binds to ERα and Leu536 in the hydrophobic core of ligand binding domain is essential for the interaction. - Highlights: • RTV increases the thickness of rat coronary artery wall and foam cell formation. • RTV downregulates the expression of ERα and ERβ. • RTV inhibits ERα promoter activity. • RTV directly binds to ERα and the key amino acid is Leu536. • RTV inhibits the nuclear translocation of ERα and GPER.

  11. A mechanism-based binding model for the population pharmacokinetics and pharmacodynamics of omalizumab

    Science.gov (United States)

    Hayashi, Naoto; Tsukamoto, Yuko; Sallas, William M; Lowe, Philip J

    2007-01-01

    Aim Omalizumab, a humanized IgG monoclonal antibody that binds to human immunoglobulin E (IgE), interrupts the allergic cascade in asthmatic patients. The aim was to compare simultaneously drug exposure and IgE biomarker responses in Japanese and White patient populations. Methods An instantaneous equilibrium drug–ligand binding and turnover population model was built from 202 Japanese patients. A posterior predictive evaluation for the steady-state distributions of omalizumab and IgE was then carried out against 531 White patients. Results The mean parameters estimated from the Japanese patients were as follows: omalizumab clearance 7.32 ± 0.153 ml h−1, IgE clearance 71.0 ± 4.68 ml h−1 and the difference between that for omalizumab and the complex 5.86 ± 0.920 ml h−1, the volume of distribution for omalizumab and IgE 5900 ± 107 ml, and that for the complex 3630 ± 223 ml, the rate of IgE production 30.3 ± 2.04 µg h−1. Half-lives of IgG (23 days) and IgE (2.4 days) were close to previous reports. The dissociation constant for binding, 1.07 nM, was similar to in vitro values. Clearance and volume of distribution for omalizumab varied with bodyweight, whereas the clearance and rate of production of IgE were predicted accurately by baseline IgE. Overall, these covariates explained much of the interindividual variability. Conclusions The predictiveness of the Japanese model was confirmed by Monte-Carlo simulations for a White population, also providing evidence that the pharmacokinetics of omalizumab and IgE were similar in these two populations. Furthermore, the model enabled the estimation of not only omalizumab disposition parameters, but also the binding with and the rate of production, distribution and elimination of its target, IgE. PMID:17096680

  12. Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

    International Nuclear Information System (INIS)

    Xiang, Jin; Wang, Ying; Su, Ke; Liu, Min; Hu, Peng-Chao; Ma, Tian; Li, Jia-Xi; Wei, Lei; Zheng, Zhongliang; Yang, Fang

    2014-01-01

    Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERβ expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17β-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17β-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis. - Graphical abstract: RTV directly binds to ERα and Leu536 in the hydrophobic core of ligand binding domain is essential for the interaction. - Highlights: • RTV increases the thickness of rat coronary artery wall and foam cell formation. • RTV downregulates the expression of ERα and ERβ. • RTV inhibits ERα promoter activity. • RTV directly binds to ERα and the key amino acid is Leu536. • RTV inhibits the nuclear translocation of ERα and GPER

  13. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms.

    Science.gov (United States)

    Durocher, D; Taylor, I A; Sarbassova, D; Haire, L F; Westcott, S L; Jackson, S P; Smerdon, S J; Yaffe, M B

    2000-11-01

    Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.

  14. C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

    Directory of Open Access Journals (Sweden)

    Christopher J Brown

    Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

  15. Adsorption performance and mechanism in binding of Reactive Red 4 by coke waste

    International Nuclear Information System (INIS)

    Won, Sung Wook; Wu Guiping; Ma Hui; Liu Qiong; Yan Yao; Cui Longzhe; Liu Chengfu; Yun, Yeoung-Sang

    2006-01-01

    The protonated coke waste was used as a new type of adsorbent for the removal of Reactive Red 4. To identify the binding sites in the protonated coke waste, the waste was potentiometrically titrated. As a result, four types of functional groups were present in the waste, which was confirmed by FT-IR analysis. Among functional groups, primary amine groups (-NH 2 ) were likely the binding sites for anionic Reactive Red 4. It was also found that sulfonate, carboxyl and phosphonate groups played a role in electrostatic interference with the binding of dye molecules. The maximum adsorption capacities of the coke waste were 70.3 ± 11.1 and 24.9 ± 1.8 mg/g at pH 1 and 2, respectively. Kinetic study showed a pseudo-first-order rate of adsorption with respect to the solution. The uptake of Reactive Red 4 was not significantly affected by the high concentration of salts. These results of adsorption performance indicate the coke waste as a potentially economical adsorbent for dye removal

  16. Binding mechanisms for histamine and agmatine ligands in plasmid deoxyribonucleic acid purifications.

    Science.gov (United States)

    Sousa, Ângela; Pereira, Patrícia; Sousa, Fani; Queiroz, João A

    2014-10-31

    Histamine and agmatine amino acid derivatives were immobilized into monolithic disks, in order to combine the specificity and selectivity of the ligand with the high mass transfer and binding capacity offered by monolithic supports, to purify potential plasmid DNA biopharmaceuticals. Different elution strategies were explored by changing the type and salt concentration, as well as the pH, in order to understand the retention pattern of different plasmids isoforms The pVAX1-LacZ supercoiled isoform was isolated from a mixture of pDNA isoforms by using NaCl increasing stepwise gradient and also by ammonium sulfate decreasing stepwise gradient, in both histamine and agmatine monoliths. Acidic pH in the binding buffer mainly strengthened ionic interactions with both ligands in the presence of sodium chloride. Otherwise, for histamine ligand, pH values higher than 7 intensified hydrophobic interactions in the presence of ammonium sulfate. In addition, circular dichroism spectroscopy studies revealed that the binding and elution chromatographic conditions, such as the combination of high ionic strength with extreme pH values can reversibly influence the structural stability of the target nucleic acid. Therefore, ascending sodium chloride gradients with pH manipulation can be preferable chromatographic conditions to be explored in the purification of plasmid DNA biopharmaceuticals, in order to avoid the environmental impact of ammonium sulfate. Copyright © 2014. Published by Elsevier B.V.

  17. Structural insights into the binding mechanism of IDO1 with hydroxylamidine based inhibitor INCB14943

    International Nuclear Information System (INIS)

    Wu, You; Xu, Tingting; Liu, Jinsong; Ding, Ke; Xu, Jinxin

    2017-01-01

    IDO1 (indoleamine 2, 3-dioxygenase 1), a well characterized immunosuppressive enzyme, has attracted growing attention as a potential target for cancer immunotherapy. Hydroxylamidine compounds INCB024360 and INCB14943 (INCB024360 analogue) are highly effective IDO1 inhibitors. INCB024360 is undergoing clinical trials for treatment of various types of human cancer. Here, we determined the co-crystal structure of IDO1 and INCB14943, and elucidate the detailed binding mode. INCB14943 binds to heme iron in IDO1 protein through the oxime nitrogen. Further analysis also reveals that a halogen bonding interaction between the chlorine atom (3-Cl) of INCB14943 and the sulphur atom of C129 significantly improves the inhibition activity against IDO1. Comparing with the other reported inhibitors, the oxime nitrogen and halogen bond interaction are identified as the unique features of INCB14943 among the IDO1 inhibitors. Thus, our study provides novel insights into the interaction between a small molecule inhibitor INCB14943 and IDO1 protein. The structural information will facilitate future IDO1 inhibitor design. - Highlights: • This is the first co-crystal structure of IDO1 with hydroxylamidine compound. • INCB14943 binds to heme iron through oxime nitrogen instead of imidazole nitrogen. • Halogen bond interaction with C129 is another unique feature of INCB14943.

  18. Mechanism of μ-conotoxin PIIIA binding to the voltage-gated Na+ channel NaV1.4.

    Directory of Open Access Journals (Sweden)

    Rong Chen

    Full Text Available Several subtypes of voltage-gated Na+ (NaV channels are important targets for pain management. μ-Conotoxins isolated from venoms of cone snails are potent and specific blockers of different NaV channel isoforms. The inhibitory effect of μ-conotoxins on NaV channels has been examined extensively, but the mechanism of toxin specificity has not been understood in detail. Here the known structure of μ-conotoxin PIIIA and a model of the skeletal muscle channel NaV1.4 are used to elucidate elements that contribute to the structural basis of μ-conotoxin binding and specificity. The model of NaV1.4 is constructed based on the crystal structure of the bacterial NaV channel, NaVAb. Six different binding modes, in which the side chain of each of the basic residues carried by the toxin protrudes into the selectivity filter of NaV1.4, are examined in atomic detail using molecular dynamics simulations with explicit solvent. The dissociation constants (Kd computed for two selected binding modes in which Lys9 or Arg14 from the toxin protrudes into the filter of the channel are within 2 fold; both values in close proximity to those determined from dose response data for the block of NaV currents. To explore the mechanism of PIIIA specificity, a double mutant of NaV1.4 mimicking NaV channels resistant to μ-conotoxins and tetrodotoxin is constructed and the binding of PIIIA to this mutant channel examined. The double mutation causes the affinity of PIIIA to reduce by two orders of magnitude.

  19. Clinical effects of low-molecular-weight heparin combined with ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research August 2016; 15 (8): 1787-1792 ... Keywords: Acute pancreatitis, Low-molecular-weight heparin, Multiple organ function syndrome,. APACHE II score ... mediators by lowering the expression of.

  20. Thrombolytic treatment for acute ischemic cerebral stroke: intraarterial urokinase infusion vs. intravenous heparin and urokinase infusion

    International Nuclear Information System (INIS)

    Ko, Gi Young; Suh, Dae Chul; Lee, Jae Hong; Kim, Jun Hyoung; Choi, Choong Gon; Lee, Ho Kyu; Lee, Myoung Chong

    1996-01-01

    To evaluate the efficacy and limitation of intra-arterial urokinase (IAUK) infusion for treatment of acute cerebral stroke. Twenty-seven acute cerebral stroke patients treated with IAUK infusion within six hours of stroke onset were reviewed. All patients showed normal initial brain findings on CT. In 21 patients, urokinase(5-15 x 10 5 IU) was administered through a microcatheter placed into or proximal to occluded segment. Mechanical disruption of thrombus by guidewire was performed in 17 patients. Angiographic and clinical responses and complications after IAUK infusion, were evaluated and the results were compared with those of intravenous heparin(N=19) and urokinase infusion(N=19). Complete or partial angiographic recanalization of occluded segment was found in 18 patients (67%), and neurologic improvement was followed in 14 patients(52%). The degree of improvement on the stroke scale score after IAUK infusion was statistically more significant(p<0.05) than that shown after intravenous heparin and urokinase infusion. Complications after IAUK infusion were large(15%) and small amount intracerebral hemorrhage(15%), contrast leakage into brain parenchyma(11%), and gastrointestinal bleeding(4%). Between the IAVK and the intravenous urokinase infusion group, differences in extent and types of complications were statistically insignificant, but were significantly higher in those two groups than in the intravenous heparin infusion group. IAUK infusion may be effective for the treatment of acute cerebral stroke

  1. Intrinsically disordered caldesmon binds calmodulin via the “buttons on a string” mechanism

    Directory of Open Access Journals (Sweden)

    Sergei E. Permyakov

    2015-09-01

    Full Text Available We show here that chicken gizzard caldesmon (CaD and its C-terminal domain (residues 636–771, CaD136 are intrinsically disordered proteins. The computational and experimental analyses of the wild type CaD136 and series of its single tryptophan mutants (W674A, W707A, and W737A and a double tryptophan mutant (W674A/W707A suggested that although the interaction of CaD136 with calmodulin (CaM can be driven by the non-specific electrostatic attraction between these oppositely charged molecules, the specificity of CaD136-CaM binding is likely to be determined by the specific packing of important CaD136 tryptophan residues at the CaD136-CaM interface. It is suggested that this interaction can be described as the “buttons on a charged string” model, where the electrostatic attraction between the intrinsically disordered CaD136 and the CaM is solidified in a “snapping buttons” manner by specific packing of the CaD136 “pliable buttons” (which are the short segments of fluctuating local structure condensed around the tryptophan residues at the CaD136-CaM interface. Our data also show that all three “buttons” are important for binding, since mutation of any of the tryptophans affects CaD136-CaM binding and since CaD136 remains CaM-buttoned even when two of the three tryptophans are mutated to alanines.

  2. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    Science.gov (United States)

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

  3. A repetitive DNA element regulates expression of the Helicobacter pylori sialic acid binding adhesin by a rheostat-like mechanism.

    Directory of Open Access Journals (Sweden)

    Anna Åberg

    2014-07-01

    Full Text Available During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR, which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.

  4. Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

    OpenAIRE

    Kahvejian, Avak; Svitkin, Yuri V.; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

    2005-01-01

    Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5′-end of the mRNA to promote the recruitment of the ribosome. Although the 3′ poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for ...

  5. Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively

    OpenAIRE

    Nowak, Jakub Stanislaw; Hobor, Fruzsina; Downie Ruiz Velasco, Angela; Choudhury, Nila Roy; Heikel, Gregory; Kerr, Alastair; Ramos, Andres; Michlewski, Gracjan

    2017-01-01

    Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3’-5’ exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We presen...

  6. Computational revelation of binding mechanisms of inhibitors to endocellular protein tyrosine phosphatase 1B using molecular dynamics simulations.

    Science.gov (United States)

    Yan, Fangfang; Liu, Xinguo; Zhang, Shaolong; Su, Jing; Zhang, Qinggang; Chen, Jianzhong

    2017-11-06

    Endocellular protein tyrosine phosphatase 1B (PTP1B) is one of the most promising target for designing and developing drugs to cure type-II diabetes and obesity. Molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) and solvated interaction energy methods were applied to study binding differences of three inhibitors (ID: 901, 941, and 968) to PTP1B, the calculated results show that the inhibitor 901 has the strongest binding ability to PTP1B among the current inhibitors. Principal component (PC) analysis was also carried out to investigate the conformational change of PTP1B, and the results indicate that the associations of inhibitors with PTP1B generate a significant effect on the motion of the WPD-loop. Free energy decomposition method was applied to study the contributions of individual residues to inhibitor bindings, it is found that three inhibitors can generate hydrogen bonding interactions and hydrophobic interactions with different residues of PTP1B, which provide important forces for associations of inhibitors with PTP1B. This research is expected to give a meaningfully theoretical guidance to design and develop of effective drugs curing type-II diabetes and obesity.

  7. The role of the Zn(II binding domain in the mechanism of E. coli DNA topoisomerase I

    Directory of Open Access Journals (Sweden)

    Tse-Dinh Yuk-Ching

    2002-05-01

    Full Text Available Abstract Background Escherichia coli DNA topoisomerase I binds three Zn(II with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain. The 67 kDa N-terminal domain (Top67 has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA. We analyzed the role of the ZD domain in the enzyme mechanism. Results Addition of purified ZD domain to Top67 partially restored the relaxation activity, demonstrating that covalent linkage between the two domains is not necessary for removal of negative supercoils from DNA. The two domains had similar affinities to ssDNA. However, only Top67 could bind dsDNA with high affinity. DNA cleavage assays showed that the Top67 had the same sequence and structure selectivity for DNA cleavage as the intact enzyme. DNA rejoining also did not require the presence of the ZD domain. Conclusions We propose that during relaxation of negatively supercoiled DNA, Top67 by itself can position the active site tyrosine near the junction of double-stranded and single-stranded DNA for cleavage. However, the interaction of the ZD domain with the passing single-strand of DNA, coupled with enzyme conformational change, is needed for removal of negative supercoils.

  8. Conformational and mechanical changes of DNA upon transcription factor binding detected by a QCM and transmission line model.

    Science.gov (United States)

    de-Carvalho, Jorge; Rodrigues, Rogério M M; Tomé, Brigitte; Henriques, Sílvia F; Mira, Nuno P; Sá-Correia, Isabel; Ferreira, Guilherme N M

    2014-04-21

    A novel quartz crystal microbalance (QCM) analytical method is developed based on the transmission line model (TLM) algorithm to analyze the binding of transcription factors (TFs) to immobilized DNA oligoduplexes. The method is used to characterize the mechanical properties of biological films through the estimation of the film dynamic shear moduli, G and G, and the film thickness. Using the Saccharomyces cerevisiae transcription factor Haa1 (Haa1DBD) as a biological model two sensors were prepared by immobilizing DNA oligoduplexes, one containing the Haa1 recognition element (HRE(wt)) and another with a random sequence (HRE(neg)) used as a negative control. The immobilization of DNA oligoduplexes was followed in real time and we show that DNA strands initially adsorb with low or non-tilting, laying flat close to the surface, which then lift-off the surface leading to final film tilting angles of 62.9° and 46.7° for HRE(wt) and HRE(neg), respectively. Furthermore we show that the binding of Haa1DBD to HRE(wt) leads to a more ordered and compact film, and forces a 31.7° bending of the immobilized HRE(wt) oligoduplex. This work demonstrates the suitability of the QCM to monitor the specific binding of TFs to immobilized DNA sequences and provides an analytical methodology to study protein-DNA biophysics and kinetics.

  9. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Jaslyn E. M. M. [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark); Midtgaard, Søren Roi [University of Copenhagen, Universitetsparken 5, 2100 Copenhagen (Denmark); Gysel, Kira [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark); Thygesen, Mikkel B.; Sørensen, Kasper K.; Jensen, Knud J. [University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Stougaard, Jens; Thirup, Søren; Blaise, Mickaël, E-mail: mickael.blaise@cpbs.cnrs.fr [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark)

    2015-03-01

    The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

  10. Advanced nanocarriers based on heparin and its derivatives for cancer management.

    Science.gov (United States)

    Yang, Xiaoye; Du, Hongliang; Liu, Jiyong; Zhai, Guangxi

    2015-02-09

    To obtain a satisfying anticancer effect, rationally designed nanocarriers are intensively studied. In this field, heparin and its derivatives have been widely attempted recently as potential component of nanocarriers due to their unique biological and physiochemical features, especially the anticancer activity. This review focuses on state-of-the-art nanocarriers with heparin/heparin derivatives as backbone or coating material. At the beginning, the unique advantages of heparin used in cancer nanotechnology are discussed. After that, different strategies of heparin chemical modification are reviewed, laying the foundation of developing various nanocarriers. Then a systematic summary of diverse nanoparticles with heparin as component is exhibited, involving heparin-drug conjugate, polymeric nanoparticles, nanogels, polyelectrolyte complex nanoparticles, and heparin-coated organic and inorganic nanoparticles. The application of these nanoparticles in various novel cancer therapy (containing targeted therapy, magnetic therapy, photodynamic therapy, and gene therapy) will be highlighted. Finally, future challenges and opportunities of heparin-based biomaterials in cancer nanotechnology are discussed.

  11. Towards understanding the E. coli PNP binding mechanism and FRET absence between E. coli PNP and formycin A.

    Science.gov (United States)

    Prokopowicz, Małgorzata; Greń, Bartosz; Cieśla, Joanna; Kierdaszuk, Borys

    2017-11-01

    The aim of this study is threefold: (1) augmentation of the knowledge of the E. coli PNP binding mechanism; (2) explanation of the previously observed 'lack of FRET' phenomenon and (3) an introduction of the correction (modified method) for FRET efficiency calculation in the PNP-FA complexes. We present fluorescence studies of the two E. coli PNP mutants (F159Y and F159A) with formycin A (FA), that indicate that the aromatic amino acid is indispensable in the nucleotide binding, additional hydroxyl group at position 159 probably enhances the strength of binding and that the amino acids pair 159-160 has a great impact on the spectroscopic properties of the enzyme. The experiments were carried out in hepes and phosphate buffers, at pH7 and 8.3. Two methods, a conventional and a modified one, that utilizes the dissociation constant, for calculations of the energy transfer efficiency (E) and the acceptor-to-donor distance (r) between FA and the Tyr (energy donor) were employed. Total difference spectra were calculated for emission spectra (λ ex 280nm, 295nm, 305nm and 313nm) for all studied systems. Time-resolved techniques allowed to conclude the existence of a specific structure formed by amino acids at positions 159 and 160. The results showed an unexpected pattern change of FRET in the mutants, when compared to the wild type enzyme and a probable presence of a structure created between 159 and 160 residue, that might influence the binding efficiency. Additionally, we confirmed the indispensable role of the modification of the FRET efficiency (E) calculation on the fraction of enzyme saturation in PNP-FA systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study

    DEFF Research Database (Denmark)

    da Graça Thrige, D; Buur, J R; Jørgensen, Flemming Steen

    1997-01-01

    cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between...... water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations...... the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular...

  13. Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili: Evidence for a Novel and Heterospecific Probiotic Mechanism.

    Science.gov (United States)

    Tytgat, Hanne L P; Douillard, François P; Reunanen, Justus; Rasinkangas, Pia; Hendrickx, Antoni P A; Laine, Pia K; Paulin, Lars; Satokari, Reetta; de Vos, Willem M

    2016-10-01

    Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections. Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of

  14. Surface binding sites (SBSs), mechanism and regulation of enzymes degrading amylopectin and α-limit dextrins

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Cockburn, Darrell; Nielsen, Jonas W.

    2013-01-01

    into barley seed α-amylase 1 (AMY1) and limit dextrinase (LD) includes i. kinetics of bi-exponential amylopectin hydrolysis by AMY1, one reaction having low Km (8 μg/mL) and high kcat (57 s-1) and the other high Km (97 μg/mL) and low kcat (23 s-1). β-Cyclodextrin (β-CD) inhibits the first reaction by binding...... to an SBS (SBS2) on domain C with Kd = 70 μM, which for the SBS2 Y380A mutant increases to 1.4 mM. SBS2 thus has a role in the fast, high-affinity component of amylopectin degradation. ii. The N-terminal domain of LD, the debranching enzyme in germinating seeds, shows distant structural similarity...

  15. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo; Zhu, Bin; Hamdan, Samir; Richardson, Charles C.

    2010-01-01

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical

  16. Converging ligand-binding free energies obtained with free-energy perturbations at the quantum mechanical level.

    Science.gov (United States)

    Olsson, Martin A; Söderhjelm, Pär; Ryde, Ulf

    2016-06-30

    In this article, the convergence of quantum mechanical (QM) free-energy simulations based on molecular dynamics simulations at the molecular mechanics (MM) level has been investigated. We have estimated relative free energies for the binding of nine cyclic carboxylate ligands to the octa-acid deep-cavity host, including the host, the ligand, and all water molecules within 4.5 Å of the ligand in the QM calculations (158-224 atoms). We use single-step exponential averaging (ssEA) and the non-Boltzmann Bennett acceptance ratio (NBB) methods to estimate QM/MM free energy with the semi-empirical PM6-DH2X method, both based on interaction energies. We show that ssEA with cumulant expansion gives a better convergence and uses half as many QM calculations as NBB, although the two methods give consistent results. With 720,000 QM calculations per transformation, QM/MM free-energy estimates with a precision of 1 kJ/mol can be obtained for all eight relative energies with ssEA, showing that this approach can be used to calculate converged QM/MM binding free energies for realistic systems and large QM partitions. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  17. Converging ligand‐binding free energies obtained with free‐energy perturbations at the quantum mechanical level

    Science.gov (United States)

    Olsson, Martin A.; Söderhjelm, Pär

    2016-01-01

    In this article, the convergence of quantum mechanical (QM) free‐energy simulations based on molecular dynamics simulations at the molecular mechanics (MM) level has been investigated. We have estimated relative free energies for the binding of nine cyclic carboxylate ligands to the octa‐acid deep‐cavity host, including the host, the ligand, and all water molecules within 4.5 Å of the ligand in the QM calculations (158–224 atoms). We use single‐step exponential averaging (ssEA) and the non‐Boltzmann Bennett acceptance ratio (NBB) methods to estimate QM/MM free energy with the semi‐empirical PM6‐DH2X method, both based on interaction energies. We show that ssEA with cumulant expansion gives a better convergence and uses half as many QM calculations as NBB, although the two methods give consistent results. With 720,000 QM calculations per transformation, QM/MM free‐energy estimates with a precision of 1 kJ/mol can be obtained for all eight relative energies with ssEA, showing that this approach can be used to calculate converged QM/MM binding free energies for realistic systems and large QM partitions. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. PMID:27117350

  18. Quantum mechanics capacitance molecular mechanics modeling of core-electron binding energies of methanol and methyl nitrite on Ag(111) surface.

    Science.gov (United States)

    Löytynoja, T; Li, X; Jänkälä, K; Rinkevicius, Z; Ågren, H

    2016-07-14

    We study a newly devised quantum mechanics capacitance molecular mechanics (QMCMM) method for the calculation of core-electron binding energies in the case of molecules adsorbed on metal surfaces. This yet untested methodology is applied to systems with monolayer of methanol/methyl nitrite on an Ag(111) surface at 100 K temperature. It was found out that the studied C, N, and O 1s core-hole energies converge very slowly as a function of the radius of the metallic cluster, which was ascribed to build up of positive charge on the edge of the Ag slab. Further analysis revealed that an extrapolation process can be used to obtain binding energies that deviated less than 0.5 eV against experiments, except in the case of methanol O 1s where the difference was as large as 1.8 eV. Additional QM-cluster calculations suggest that the latter error can be connected to the lack of charge transfer over the QM-CMM boundary. Thus, the results indicate that the QMCMM and QM-cluster methods can complement each other in a holistic picture of molecule-adsorbate core-ionization studies, where all types of intermolecular interactions are considered.

  19. In vitro anticoagulation monitoring of low-molecular-weight heparin

    Institute of Scientific and Technical Information of China (English)

    WANG Jian-qi; SHI Xu-bo; YANG Jin-gang; HU Da-yi

    2009-01-01

    Background Although low-molecular-weight heparin has replaced unfractionated heparin to become the primary anticoagulation drug for treatment of acute coronary syndrome, there is no convenient bedside monitoring method. We explored the best laboratory monitoring method of low-molecular-weight heparins (enoxapadn, dalteparin, and nadroparin) by use of the Sonoclot coagulation analyzer to monitor the activated clotting time.Methods Atotal of 20 healthy volunteers were selected and 15 ml of fasting venous blood samples were collected and incubated. Four coagulants, kaolin, diatomite, glass bead, and magnetic stick, were used to determine the activated clotting time of the low-molecular-weight heparins at different in vitro anti-Xa factor concentrations. A correlation analysis was made to obtain the regression equation. The activated clotting time of the different low-molecular-weight heparins with the same anti-Xa factor concentration was monitored when the coagulant glass beads were applied. Results The activated clotting time measured using the glass beads, diatomite, kaolin, and magnetic stick showed a linear correlation with the concentration of nadroparin (r= 0.964, 0.966, 0.970, and 0.947, respectively). The regression equation showed that the linear slopes of different coagulants were significantly different (glass beads 230.03 s/IU,diatomite 89.91 s/IU, kaolin 50.87 s/IU, magnetic stick could not be calculated). When the concentration of the anti-Xa factor was the same for different low-molecular-weight heparins, the measured activated clotting time was different after the application of the glass bead coagulant.Conclusions The glass bead coagulant is most feasible for monitoring the in vitro anticoagulation activity of nadroparin.The different effects of different low-molecular-weight heparins on the activated clotting time may be related to the different anti-Ila activities.

  20. New insights into the binding and catalytic mechanisms of Bacillus thuringiensis lactonase: insights into B. thuringiensis AiiA mechanism.

    Directory of Open Access Journals (Sweden)

    Marc N Charendoff

    Full Text Available The lactonase enzyme (AiiA produced by Bacillus thuringiensis serves to degrade autoinducer-1 (AI-1 signaling molecules in what is an evolved mechanism by which to compete with other bacteria. Bioassays have been previously performed to determine whether the AI-1 aliphatic tail lengths have any effect on AiiA's bioactivity, however, data to date are conflicting. Additionally, specific residue contributions to the catalytic activity of AiiA provide for some interesting questions. For example, it has been proposed that Y194 serves to provide an oxyanion hole to AI-1 which is curious given the fact the substrate spans two Zn(2+ ions. These ions might conceivably provide enough charge to promote both ligand stability and the carbonyl activation necessary to drive a nucleophilic attack. To investigate these questions, multiple molecular dynamics simulations were performed across a family of seven acylated homoserine lactones (AHL along with their associated intermediate and product states. Distance analyses and interaction energy analyses were performed to investigate current bioassay data. Our simulations are consistent with experimental studies showing that AiiA degrades AHLs in a tail length independent manner. However, the presence of the tail is required for activity. Also, the putative oxyanion hole function of Y194 toward the substrate is not observed in any of the reactant or product state simulation trajectories, but does seem to show efficacy in stabilizing the intermediate state. Last, we argue through ionization state analyses, that the proton shuttling necessary for catalytic activity might be mediated by both water and substrate-based intra-molecular proton transfer. Based on this argument, an alternate catalytic mechanism is proposed.

  1. Veno-venous bypass without systemic heparinization using a centrifugal pump: a blind comparison of a heparin bonded circuit versus a non heparin bonded circuit

    NARCIS (Netherlands)

    van der Hulst, V. P.; Henny, C. P.; Moulijn, A. C.; Engbers, G.; ten Cate, H.; Gründeman, P. F.; Klopper, P. J.

    1989-01-01

    Veno-venous bypass without the use of systemic heparinization has recently become of increasing interest for application during liver transplantation and surgery on the large abdominal veins. However, possible adverse effects on blood components as demonstrated by means of hematologic and hemostatic

  2. Pharmacokinetics of heparin and related polysaccharides

    International Nuclear Information System (INIS)

    Boneu, B.; Dol, F.; Caranobe, C.; Sie, P.; Houin, G.

    1989-01-01

    The pharmacodynamic profile of standard heparin (SH), a low molecular weight derivative (CY 216) and of dermatan sulfate (DS), a new potential antithrombotic drug, was investigated in the rabbit over a large range of doses. After bolus i.v. injection of low doses, the biological activity of SH disappeared exponentially; however, its half-life was prolonged when the dose injected increased, and over 158 micrograms/kg (100 anti-factor Xa U/kg) the biological activity disappeared as a concave-convex curve. CY 216 disappeared more slowly than SH at low doses but faster than SH at higher doses. More than 90% of the DS biological activity present 1 minute after the i.v. injection disappeared exponentially without dose-dependent effects. Increasing doses of the three drugs were then delivered for 5 h under continuous infusions. Below 500 micrograms/kg/h the DS and CY 216 plateau concentrations were higher than that of SH while above this dose the SH concentration was higher than that of DS and CY 216. These observations may be explained by the results of pharmacokinetics experiments where 125 I-labeled compounds were delivered by bolus i.v. injection in association with increasing doses of their unlabeled counterparts. For SH there was a 10-fold difference between the half-life of the lower dose (32 micrograms/kg or 5 anti-factor Xa U/kg) and that of the higher dose (3200 micrograms/kg); it was demonstrated that the half-life of SH continuously shortened as its plasma concentration decreased. In contrast the CY 216 and DS half-lives were very close, independent of the dose delivered, and therefore longer than that of SH at low doses and shorter than that of SH at higher doses

  3. Potentiation of C1-esterase inhibitor by heparin and interactions with C1s protease as assessed by surface plasmon resonance.

    Science.gov (United States)

    Rajabi, Mohsen; Struble, Evi; Zhou, Zhaohua; Karnaukhova, Elena

    2012-01-01

    Human C1-esterase inhibitor (C1-INH) is a multifunctional plasma protein with a wide range of inhibitory and non-inhibitory properties, mainly recognized as a key down-regulator of the complement and contact cascades. The potentiation of C1-INH by heparin and other glycosaminoglycans (GAGs) regulates a broad spectrum of C1-INH activities in vivo both in normal and disease states. SCOPE OF RESEARCH: We have studied the potentiation of human C1-INH by heparin using Surface Plasmon Resonance (SPR), circular dichroism (CD) and a functional assay. To advance a SPR for multiple-unit interaction studies of C1-INH we have developed a novel (consecutive double capture) approach exploring different immobilization and layout. Our SPR experiments conducted in three different design versions showed marked acceleration in C1-INH interactions with complement protease C1s as a result of potentiation of C1-INH by heparin (from 5- to 11-fold increase of the association rate). Far-UV CD studies suggested that heparin binding did not alter C1-INH secondary structure. Functional assay using chromogenic substrate confirmed that heparin does not affect the amidolytic activity of C1s, but does accelerate its consumption due to C1-INH potentiation. This is the first report that directly demonstrates a significant acceleration of the C1-INH interactions with C1s due to heparin by using a consecutive double capture SPR approach. The results of this study may be useful for further C-INH therapeutic development, ultimately for the enhancement of current C1-INH replacement therapies. Published by Elsevier B.V.

  4. Evidence of vanillin binding to CAMKIV explains the anti-cancer mechanism in human hepatic carcinoma and neuroblastoma cells.

    Science.gov (United States)

    Naz, Huma; Tarique, Mohd; Khan, Parvez; Luqman, Suaib; Ahamad, Shahzaib; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2018-01-01

    Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr kinase family, and is associated with different types of cancer and neurodegenerative diseases. Vanillin is a natural compound, a primary component of the extract of the vanilla bean which possesses varieties of pharmacological features including anti-oxidant, anti-inflammatory, anti-bacterial and anti-tumor. Here, we have investigated the binding mechanism and affinity of vanillin to the CAMKIV which is being considered as a potential drug target for cancer and neurodegenerative diseases. We found that vanillin binds strongly to the active site cavity of CAMKIV and stabilized by a large number of non-covalent interactions. We explored the utility of vanillin as anti-cancer agent and found that it inhibits the proliferation of human hepatocyte carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells in a dose-dependent manner. Furthermore, vanillin treatment resulted into the significant reduction in the mitochondrial membrane depolarization and ROS production that eventually leads to apoptosis in HepG2 and SH-SY5Y cancer cells. These findings may offer a novel therapeutic approach by targeting the CAMKIV using natural product and its derivative with a minimal side effect.

  5. Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively.

    Science.gov (United States)

    Nowak, Jakub Stanislaw; Hobor, Fruzsina; Downie Ruiz Velasco, Angela; Choudhury, Nila Roy; Heikel, Gregory; Kerr, Alastair; Ramos, Andres; Michlewski, Gracjan

    2017-03-01

    Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing. © 2017 Nowak et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  6. Binding mechanisms of DNA/RNA nucleobases adsorbed on graphene under charging: first-principles van der Waals study

    Science.gov (United States)

    Gürel, Hikmet Hakan; Salmankurt, Bahadır

    2017-06-01

    Graphene is a 2D material that has attracted much attention due to its outstanding properties. Because of its high surface area and unique chemical and physical properties, graphene is a good candidate for biological applications. For this reason, a deep understanding of the mechanism of interaction of graphene with biomolecules is required. In this study, theoretical investigation of van der Waals effects has been conducted using density functional theory. Here we show that the order of the binding energies of five nucleobases with graphene is G  >  A  >  T  >  C  >   U. This trend is in good agreement with most of the theoretical and experimental data. Also, the effects of charging on the electronic and structural properties of the graphene-nucleubase systems are studied for the first time. We show that the binding energy can be changed by adding or removing an electron from the system. The results presented in this work provide fundamental insights into the quantum interactions of DNA with carbon-based nanostructures and will be useful for developments in biotechnology and nanotechnology.

  7. The intracellular uptake and protracted release of exogenous heparins by cultured endothelial cells

    International Nuclear Information System (INIS)

    Hiebert, L.M.; McDuffie, N.M.

    1989-01-01

    Heparins from bovine or porcine sources were fed in media for 48 hrs to cultured porcine aortic and human umbilical vein endothelial cells. Heparin was found in pericellular and cellular fractions after extraction by chemical methods and 125 I radiolabelled heparins were recovered when radiolabelled heparin was included in the feed. Even after washing and media changes heparin was detected in media and cell fractions up to 6 days post feeding. Metachromatic vacuoles within cells were demonstrated histologically up to 7 days post feeding after staining with toluidine blue. This is the first report of protracted internalization of exogenous heparin by cultured endothelial cells with concurrent prolonged release of the heparin to the media. This clearly demonstrates that the endothelium plays an important role in the distribution and metabolism of heparin

  8. Angiogenesis alteration by defibrotide: implications for its mechanism of action in severe hepatic veno-occlusive disease.

    Science.gov (United States)

    Benimetskaya, Luba; Wu, Sijian; Voskresenskiy, Anatoliy M; Echart, Cinara; Zhou, Jin-Feng; Shin, Joongho; Iacobelli, Massimo; Richardson, Paul; Ayyanar, Kanyalakshmi; Stein, C A

    2008-11-15

    Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity. This binding occurred in a length- and concentration-dependent manner. DF can mobilize proangiogenic factors such as bFGF from their depot or storage sites on bovine corneal endothelial matrix. However, these molecules do not interfere with high-affinity binding of bFGF to FGFR1 IIIc but can replace heparin as a required cofactor for binding and hence cellular mitogenesis. DF also protects bFGF against digestion by trypsin and chymotrypsin and from air oxidation. In addition, DF binds to collagen I with low nanomolar affinity and can promote human microvascular endothelial cell-1 (HMEC-1) cell mitogenesis and tubular morphogenesis in three-dimensional collagen I gels. Thus, our data suggest that DF may provide a stimulus to the sinusoidal endothelium of a liver that has suffered a severe angiotoxic event, thus helping to ameliorate the clinical sVOD/MOF syndrome.

  9. Quantitation of heparosan with heparin lyase III and spectrophotometry.

    Science.gov (United States)

    Huang, Haichan; Zhao, Yingying; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

    2014-02-15

    Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Heparin-Based Nanoparticles: An Overview of Their Applications

    Directory of Open Access Journals (Sweden)

    Maria del Pilar Rodriguez-Torres

    2018-01-01

    Full Text Available This review deals with nanoparticles synthesized using heparin. Such nanoparticles have been widely studied since a long time ago, obtaining satisfactory outcomes. An outstanding aspect of these nanoparticles is that they possess good biocompatible characteristics, and since heparin is produced in the human body within the mast cells, this makes these nanoparticles useful for future applications like imaging, disease and cancer treatment, and antibacterial activity. They can also be used for applications that are not oriented directly to the medical and biological areas such as in the case of analyte detection in aqueous solution, although such studies are very few. These nanoparticles synthesis is mainly through wet chemistry methods, using heparin that could have been modified or not.

  11. The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

    Science.gov (United States)

    Vivancos, Julien; Spinner, Lara; Mazubert, Christelle; Charlot, Florence; Paquet, Nicolas; Thareau, Vincent; Dron, Michel; Nogué, Fabien; Charon, Céline

    2012-03-01

    The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

  12. Using docking and alchemical free energy approach to determine the binding mechanism of eEF2K inhibitors and prioritizing the compound synthesis.

    Science.gov (United States)

    Wang, Qiantao; Edupuganti, Ramakrishna; Tavares, Clint D J; Dalby, Kevin N; Ren, Pengyu

    2015-01-01

    A-484954 is a known eEF2K inhibitor with submicromolar IC50 potency. However, the binding mechanism and the crystal structure of the kinase remains unknown. Here, we employ a homology eEF2K model, docking and alchemical free energy simulations to probe the binding mechanism of eEF2K, and in turn, guide the optimization of potential lead compounds. The inhibitor was docked into the ATP-binding site of a homology model first. Three different binding poses, hypothesis 1, 2, and 3, were obtained and subsequently applied to molecular dynamics (MD) based alchemical free energy simulations. The calculated relative binding free energy of the analogs of A-484954 using the binding pose of hypothesis 1 show a good correlation with the experimental IC50 values, yielding an r (2) coefficient of 0.96 after removing an outlier (compound 5). Calculations using another two poses show little correlation with experimental data, (r (2) of less than 0.5 with or without removing any outliers). Based on hypothesis 1, the calculated relative free energy suggests that bigger cyclic groups, at R1 e.g., cyclobutyl and cyclopentyl promote more favorable binding than smaller groups, such as cyclopropyl and hydrogen. Moreover, this study also demonstrates the ability of the alchemical free energy approach in combination with docking and homology modeling to prioritize compound synthesis. This can be an effective means of facilitating structure-based drug design when crystal structures are not available.

  13. Molecular binding mechanisms of aqueous cadmium and lead to siderophores, bacteria and mineral surfaces

    Science.gov (United States)

    Mishra, Bhoopesh

    Recent studies have shown that diverse groups of bacteria adsorb metals to similar extents and uptake can be modeled using a universal adsorption model. In this study, XAFS has been used to resolve whether binding sites determined for single species systems are responsible for adsorption in more complex natural bacterial assemblages. Results obtained from a series of XAFS experiments on pure Gram positive and Gram negative bacterial strains and consortia of bacteria as a function of pH and Cd loading suggests that every bacterial strain has a complex physiology and they are all slightly different from each other. Nevertheless from the metal adsorption chemistry point of view, the main difference between them lies in the site ratio of three fundamental sites only - carboxyl, phosphoryl and sulfide. Two completely different consortia of bacteria (obtained from natural river water, and soil system with severe organic contamination) were successfully modeled in the pH range 3.4--7.8 using the EXAFS models developed for single species systems. Results thus obtained can potentially have very high impact on the modeling of the complex bacterial systems in realistic geological settings, leading to further refinement and development of robust remediation strategies for metal contamination at macroscopic level. In another study, solution speciation of Pb and Cd with DFO-B has been examined using a combination of techniques (ICP, TOC, thermodynamic modeling and XAFS). Results indicate that Pb does not complex with DFO-B at all until about pH 3.5, but forms a totally caged structure at pH 7.5. At intermediate pH conditions, mixture of species (one and two hydroxamate groups complexed) is formed. Cd on the other hand, does not complex until pH 5, forms intermediate complexes at pH 8 and is totally chelated at pH 9. Further studies were conducted for Pb sorption to mineral surface kaolinite with and without DFO-B. In the absence of DFO-B, results suggest outer sphere and inner

  14. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2011-01-01

    Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

  15. 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.

  16. Histones Differentially Modulate the Anticoagulant and Profibrinolytic Activities of Heparin, Heparin Derivatives, and Dabigatran.

    Science.gov (United States)

    Ammollo, Concetta Tiziana; Semeraro, Nicola; Carratù, Maria Rosaria; Colucci, Mario; Semeraro, Fabrizio

    2016-02-01

    The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  17. Capillary electrophoresis of heparin and other glycosaminoglycans using a polyamine running electrolyte

    Energy Technology Data Exchange (ETDEWEB)

    Loegel, Thomas N.; Trombley, John D.; Taylor, Richard T. [Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056 (United States); Danielson, Neil D., E-mail: danielnd@muohio.edu [Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056 (United States)

    2012-11-13

    Highlights: Black-Right-Pointing-Pointer Ethylenediamine is likely acting as an ion-pairing agent. Black-Right-Pointing-Pointer Oversulfated chondroitin sulfate is last peak instead of first peak. Black-Right-Pointing-Pointer There is about a factor of five improved detectability with a 12.5 min analysis time. Black-Right-Pointing-Pointer Use of a 50 {mu}m ID capillary is possible. - Abstract: This study involves the use of polyamines as potential resolving agents for the capillary electrophoresis (CE) of glycosaminoglycans (GAGs), specifically heparin, dermatan sulfate, chondroitin sulfate, over-sulfated chondroitin sulfate (OSCS), and hyaluronan. All of the compounds can be separated from each other with the exception of chondroitin sulfate and hyaluronan. Using optimization software, the final run conditions are found to be 200 mM ethylenediamine and 45.5 mM phosphate as the electrolyte with -14 V applied across a 50 {mu}m ID Multiplication-Sign 24.5 cm fused silica capillary at 15 Degree-Sign C. The ion migration order, with OSCS as the last instead of the first peak, is in contrast to previous reports using either a high molarity TRIS or lithium phosphate run buffer with narrower bore capillaries. Total analysis time is 12. 5 min and the relative standard deviation of the heparin migration time is about 2.5% (n = 5). The interaction mechanism between selected polyamines and heparin is explored using conductivity measurements in addition to CE experiments to show that an ion-pairing mechanism is likely.

  18. The effect of different forms of heparin on point-of-care blood gas ...

    African Journals Online (AJOL)

    and heparin vacutainers on blood gas and electrolyte analysis and ... This prospective, cross-sectional study took place in the ED of a ... the effect of two concentrations of liquid heparin and the use of heparin vacutainers on the reliability of blood gas ... Germany) and (iv) a 2 mL plastic syringe (BD) washed with 5 000 IU/.

  19. Coexistence of Antiphospholipid Syndrome and Heparin-Induced Thrombocytopenia in a Patient with Recurrent Venous Thromboembolism

    Directory of Open Access Journals (Sweden)

    Samuel Adediran

    2017-01-01

    Full Text Available Heparin-induced thrombocytopenia (HIT is a prothrombotic adverse drug reaction in which heparin forms complexes with platelet factor 4 forming neoantigens that are recognized by autoantibodies. Antiphospholipid syndrome (APS is similar to HIT in that it is mediated by autoantibodies that are also prothrombotic. We present a case of rare coexistence of antiphospholipid antibody syndrome and heparin-induced thrombocytopenia.

  20. A genome-wide association study of heparin-induced thrombocytopenia using an electronic medical record

    DEFF Research Database (Denmark)

    Karnes, Jason H; Cronin, Robert M; Rollin, Jerome

    2015-01-01

    Heparin-induced thrombocytopenia (HIT) is an unpredictable, potentially catastrophic adverse effect of heparin treatment resulting from an immune response to platelet factor 4 (PF4)/heparin complexes. No genome-wide evaluations have been performed to identify potential genetic influences on HIT. ...

  1. Biosorption behaviors of uranium (VI) from aqueous solution by sunflower straw and insights of binding mechanism

    International Nuclear Information System (INIS)

    Lian Ai; Xuegang Luo; Xiaoyan Lin; Sizhao Zhang

    2013-01-01

    Uranium (VI)-containing water has been recognized as a potential longer-term radiological health hazard. In this work, the sorptive potential of sunflower straw for U (VI) from aqueous solution was investigated in detail, including the effect of initial solution pH, adsorbent dosage, temperature, contact time and initial U (VI) concentration. A dose of 2.0 g L -1 of sunflower straw in an initial U (VI) concentration of 20 mg L -1 with an initial pH of 5.0 and a contact time of 10 h resulted in the maximum U (VI) uptake (about 6.96 mg g -1 ) at 298 K. The isotherm adsorption data was modeled best by the nonlinear Langmuir-Freundlich equation. The equilibrium sorption capacity of sunflower straw was observed to be approximately seven times higher than that of coconut-shell activated carbon as 251.52 and 32.37 mg g -1 under optimal conditions, respectively. The positive enthalpy and negative free energy suggested the endothermic and spontaneous nature of sorption, respectively. The kinetic data conformed successfully to the pseudo-second-order equation. Furthermore, energy dispersive X-ray, fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy demonstrated that U (VI) adsorption onto sunflower straw was predominantly controlled by ion exchange as well as complexation mechanism. The study revealed that sunflower straw could be exploited for uranium remediation of aqueous streams as a promising adsorbent. (author)

  2. Structural mechanism underlying capsaicin binding and activation of TRPV1 ion channel

    Science.gov (United States)

    Cheng, Wei; Yang, Wei; Yu, Peilin; Song, Zhenzhen; Yarov-Yarovoy, Vladimir; Zheng, Jie

    2015-01-01

    Capsaicin bestows spiciness by activating TRPV1 channel with exquisite potency and selectivity. Capsaicin-bound channel structure was previously resolved by cryo-EM at 4.2-to-4.5 Å resolution, however important details required for mechanistic understandings are unavailable: capsaicin was registered as a small electron density, reflecting neither its chemical structure nor specific ligand-channel interactions. We obtained the missing atomic-level details by iterative computation, which were confirmed by systematic site-specific functional tests. We observed that the bound capsaicin takes “tail-up, head-down” configurations. The vanillyl and amide groups form specific interactions to anchor its bound position, while the aliphatic tail may sample a range of conformations, making it invisible in cryo-EM images. Capsaicin stabilizes the open state by “pull-and-contact” interactions between the vanillyl group and the S4-S5 linker. Our study provided a structural mechanism for the agonistic function of capsaicin and its analogs, and demonstrated an effective approach to obtain atomic level information from cryo-EM structures. PMID:26053297

  3. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    Science.gov (United States)

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  4. 12500 E heparin and 12500 E of a semisynthetic heparin analogue (SSHA) in preventing thrombosis during radiotherapy of gynaecological carcinomas

    International Nuclear Information System (INIS)

    Hilscher, T.M.

    1983-01-01

    The effects of 12500 E calcium heparin given once daily were contrasted with those seen under daily treatment with 12500 E of a semisynthetic heparin analogue (SSHA) and evaluated using iodine-125-labelled fibrinogen. The study included 80 patients, who were randomly assigned to the two treatment groups on a 1:1 basis. The findings revealed here led to the conclusion that both drugs, administered once daily by the subcutaneous route, were effective in preventing the occurrence of thrombosis during radiation treatment of gynaecological tumours. (orig./MG) [de

  5. Revealing vilazodone's binding mechanism underlying its partial agonism to the 5-HT1A receptor in the treatment of major depressive disorder.

    Science.gov (United States)

    Zheng, Guoxun; Xue, Weiwei; Yang, Fengyuan; Zhang, Yang; Chen, Yuzong; Yao, Xiaojun; Zhu, Feng

    2017-11-01

    It has been estimated that major depressive disorder (MDD) will become the second largest global burden among all diseases by 2030. Various types of drugs, including selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and serotonin receptor partial agonist/reuptake inhibitors (SPARIs), have been approved and become the primary or first-line medications prescribed for MDD. SPARI was expected to demonstrate more enhanced drug efficacy and a rapid onset of action as compared to SSRI and SNRI. As one of the most famous SPARIs, vilazodone was approved by the FDA for the treatment of MDD. Because of the great clinical importance of vilazodone, its binding mechanism underlying its partial agonism to the 5-HT 1A receptor (5-HT 1A R) could provide valuable information to SPARIs' drug-like properties. However, this mechanism has not been reported to date; consequently, the rational design of new efficacious SPARI-based MDD drugs is severely hampered. To explore the molecular mechanism of vilazodone, an integrated computational strategy was adopted in this study to reveal its binding mechanism and prospective structural feature at the agonist binding site of 5-HT 1A R. As a result, 22 residues of this receptor were identified as hotspots, consistently favoring the binding of vilazodone and its analogues, and a common binding mechanism underlying their partial agonism to 5-HT 1A R was, therefore, discovered. Moreover, three main interaction features between vilazodone and 5-HT 1A R have been revealed and schematically summarized. In summary, this newly identified binding mechanism will provide valuable information for medicinal chemists working in the field of rational design of novel SPARIs for MDD treatment.

  6. Theoretical Characterization of the Spectral Density of the Water-Soluble Chlorophyll-Binding Protein from Combined Quantum Mechanics/Molecular Mechanics Molecular Dynamics Simulations.

    Science.gov (United States)

    Rosnik, Andreana M; Curutchet, Carles

    2015-12-08

    Over the past decade, both experimentalists and theorists have worked to develop methods to describe pigment-protein coupling in photosynthetic light-harvesting complexes in order to understand the molecular basis of quantum coherence effects observed in photosynthesis. Here we present an improved strategy based on the combination of quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations and excited-state calculations to predict the spectral density of electronic-vibrational coupling. We study the water-soluble chlorophyll-binding protein (WSCP) reconstituted with Chl a or Chl b pigments as the system of interest and compare our work with data obtained by Pieper and co-workers from differential fluorescence line-narrowing spectra (Pieper et al. J. Phys. Chem. B 2011, 115 (14), 4042-4052). Our results demonstrate that the use of QM/MM MD simulations where the nuclear positions are still propagated at the classical level leads to a striking improvement of the predicted spectral densities in the middle- and high-frequency regions, where they nearly reach quantitative accuracy. This demonstrates that the so-called "geometry mismatch" problem related to the use of low-quality structures in QM calculations, not the quantum features of pigments high-frequency motions, causes the failure of previous studies relying on similar protocols. Thus, this work paves the way toward quantitative predictions of pigment-protein coupling and the comprehension of quantum coherence effects in photosynthesis.

  7. Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

    Directory of Open Access Journals (Sweden)

    Christos eLamprakis

    2015-07-01

    Full Text Available We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution, to investigate interaction and binding of α-tochoperol transfer protein (α-TTP to phosphatidylinositol phosphate lipids (PIPs. Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP / PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein / ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED phenotypes. Specifically, R221 is main residue responsible for the stabilisation of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.

  8. A Two-pronged Binding Mechanism of IgG to the Neonatal Fc Receptor Controls Complex Stability and IgG Serum Half-life

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Schoch, Angela; Larraillet, Vincent

    2017-01-01

    The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism...... half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct...

  9. Acid-citrate-dextrose compared with heparin in the preparation of in vivo/in vitro technetium-99m red blood cells

    International Nuclear Information System (INIS)

    Porter, W.C.; Dees, S.M.; Freitas, J.E.; Dworkin, H.J.

    1983-01-01

    Red blood cells labeled in vivo/in vitro with Tc-99m (Tc-99m RBC) were prepared in a series of 21 patients and two normal volunteers. In each subject both heparin and acid-citrate-dextrose (ACD) solutions were used to label tandem blood samples. The immediate preinjection binding efficiency (BE) was then determined. In each of the 23 studies, the ACD preparation yielded superior BE. The average BE was 93.47% (+/- 3.78) with ACD and 87.23% (+/- 4.29) with heparin. With the ACD method the effect of carrier Tc-99 may be as great as a 24% reduction in BE observed when initial eluates from long-ingrowth-time generators were used. Improved image quality with minimal renal and urinary-bladder activity results with ACD labeling. It is concluded that the use of ACD results in superior RBC labeling with less nontarget activity relative to heparin and is preferred over heparin for preparing in vivo/in vitro Tc-99m RBC

  10. Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies.

    Science.gov (United States)

    Kumorek, Marta; Kubies, Dana; Filová, Elena; Houska, Milan; Kasoju, Naresh; Mázl Chánová, Eliška; Matějka, Roman; Krýslová, Markéta; Bačáková, Lucie; Rypáček, František

    2015-01-01

    In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm(2). The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm(2)) than on surfaces with a higher concentration of FGF-2 (120 ng/cm(2)).

  11. Cyclic Voltammetry of Biopolymer Heparin at PVC Plasticized Liquid Membrane

    Czech Academy of Sciences Publication Activity Database

    Samec, Zdeněk; Trojánek, Antonín; Langmaier, Jan; Samcová, E.

    2003-01-01

    Roč. 5, - (2003), s. 867-870 ISSN 1388-2481 R&D Projects: GA ČR GA203/04/0424 Institutional research plan: CEZ:AV0Z4040901 Keywords : cyclic voltammetry * PVC plasticized liquit membrane * heparin Subject RIV: CG - Electrochemistry Impact factor: 2.300, year: 2003

  12. 21 CFR 864.5680 - Automated heparin analyzer.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated heparin analyzer. 864.5680 Section 864.5680 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Automated and Semi-Automated Hematology Devices § 864...

  13. Clinical effects of low-molecular-weight heparin combined with ...

    African Journals Online (AJOL)

    Purpose: To explore the clinical effects of low-molecular-weight heparin (LMWH) combined with ulinastatin (UTI) in children with acute pancreatitis. Methods: In total, 560 patients with severe acute pancreatitis treated at Binzhou People's Hospital, Shandong, China, from April 2012 to June 2014 were enrolled in this study.

  14. Alternative method for determination of contaminated heparin using chiral recognition.

    Science.gov (United States)

    Szekely, J; Collins, M; Currie, C A

    2014-05-15

    Since 2008 a significant amount of work has focused on the development of methods to analyze contaminated heparin. This work focuses on utilizing heparin's ability to serve as a chiral selector as a means for determining contamination. Specifically, the effect of contamination on the separation of pheniramine and chloroquine enantiomers was explored. Separations were conducted using heparin contaminated with chondroitin sulfate at varying levels. For each pair of enantiomers, electrophoretic mobility and resolution were calculated. For pheniramine enantiomers, an increase in contamination leads to a decrease in the electrophoretic mobility and resolution. A linear relationship between contamination level and electrophoretic mobility of the pheniramine enantiomers was observed for the entire contamination range. A linear relationship was also found between contamination level and resolution of the enantiomers between 0 and 70 percent contamination. For the separation of chloroquine enantiomers, it was found that at low levels of contamination, the resolution of enantiomers was increased due to the secondary interaction between the chloroquine enantiomers and the chondroitin sulfate. Results of this study illustrate the potential of using chiral recognition as a means to determine heparin contamination as well as the improvement of the chiral resolution of chloroquine with the additional of low levels of chondroitin sulfate A. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe.

    Science.gov (United States)

    Mehta, Pramod Kumar; Lee, Hyeri; Lee, Keun-Hyeung

    2017-05-15

    The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R 2 =0.99) in aqueous solutions, 2.98nM (R 2 =0.98) in 1% serum samples, and 3.43nM (R 2 =0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. 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.

  17. Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

    Science.gov (United States)

    Kahvejian, Avak; Svitkin, Yuri V; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

    2005-01-01

    Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

  18. Endocytosis of ABCG2 drug transporter caused by binding of 5D3 antibody: trafficking mechanisms and intracellular fate.

    Science.gov (United States)

    Studzian, Maciej; Bartosz, Grzegorz; Pulaski, Lukasz

    2015-08-01

    ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Mechanism-based population pharmacokinetic modelling in diabetes: vildagliptin as a tight binding inhibitor and substrate of dipeptidyl peptidase IV

    Science.gov (United States)

    Landersdorfer, Cornelia B; He, Yan-Ling; Jusko, William J

    2012-01-01

    AIMS To assess the pharmacokinetics of vildagliptin at different doses and build a mechanism-based population model that simultaneously describes vildagliptin pharmacokinetics and its effects on DPP-4 activity based on underlying physiology and biology. METHODS Vildagliptin concentrations and DPP-4 activity vs. time from 13 type 2 diabetic patients after oral vildagliptin 10, 25 or 100 mg and placebo twice daily for 28 days were co-modelled. NONMEM VI and S-ADAPT were utilized for population modelling. RESULTS A target-mediated drug disposition (TMDD) model accounting for capacity-limited high affinity binding of vildagliptin to DPP-4 in plasma and tissues had good predictive performance. Modelling the full time course of the vildagliptin-DPP-4 interaction suggested parallel vildagliptin dissociation from DPP-4 by a slow first-order process and hydrolysis by DPP-4 to an inactive metabolite as a disposition mechanism. Due to limited amounts of DPP-4, vildagliptin concentrations increased slightly more than dose proportionally. This newly proposed model and the parameter estimates are supported by published in vitro studies. Mean parameter estimates (inter-individual coefficient of variation) were: non-saturable clearance 36 l h−1 (25%), central volume of distribution 22 l (37%), half-life of dissociation from DPP-4 1.1 h (94%) and half-life of hydrolysis 6.3 h (81%). CONCLUSIONS Vildagliptin is both an inhibitor and substrate for DPP-4. By utilizing the TMDD approach, slow dissociation of vildagliptin from DPP-4 was found in patients and the half-life of hydrolysis by DPP-4 estimated. This model can be used to predict DPP-4 inhibition effects of other dosage regimens and be modified for other DPP-4 inhibitors to differentiate their properties. PMID:22442826

  20. Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement

    KAUST Repository

    Jiang, Hanlun

    2015-07-16

    Argonaute (Ago) proteins and microRNAs (miRNAs) are central components in RNA interference, which is a key cellular mechanism for sequence-specific gene silencing. Despite intensive studies, molecular mechanisms of how Ago recognizes miRNA remain largely elusive. In this study, we propose a two-step mechanism for this molecular recognition: selective binding followed by structural re-arrangement. Our model is based on the results of a combination of Markov State Models (MSMs), large-scale protein-RNA docking, and molecular dynamics (MD) simulations. Using MSMs, we identify an open state of apo human Ago-2 in fast equilibrium with partially open and closed states. Conformations in this open state are distinguished by their largely exposed binding grooves that can geometrically accommodate miRNA as indicated in our protein-RNA docking studies. miRNA may then selectively bind to these open conformations. Upon the initial binding, the complex may perform further structural re-arrangement as shown in our MD simulations and eventually reach the stable binary complex structure. Our results provide novel insights in Ago-miRNA recognition mechanisms and our methodology holds great potential to be widely applied in the studies of other important molecular recognition systems.

  1. Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement

    KAUST Repository

    Jiang, Hanlun; Sheong, Fu Kit; Zhu, Lizhe; Gao, Xin; Bernauer, Julie; Huang, Xuhui

    2015-01-01

    Argonaute (Ago) proteins and microRNAs (miRNAs) are central components in RNA interference, which is a key cellular mechanism for sequence-specific gene silencing. Despite intensive studies, molecular mechanisms of how Ago recognizes miRNA remain largely elusive. In this study, we propose a two-step mechanism for this molecular recognition: selective binding followed by structural re-arrangement. Our model is based on the results of a combination of Markov State Models (MSMs), large-scale protein-RNA docking, and molecular dynamics (MD) simulations. Using MSMs, we identify an open state of apo human Ago-2 in fast equilibrium with partially open and closed states. Conformations in this open state are distinguished by their largely exposed binding grooves that can geometrically accommodate miRNA as indicated in our protein-RNA docking studies. miRNA may then selectively bind to these open conformations. Upon the initial binding, the complex may perform further structural re-arrangement as shown in our MD simulations and eventually reach the stable binary complex structure. Our results provide novel insights in Ago-miRNA recognition mechanisms and our methodology holds great potential to be widely applied in the studies of other important molecular recognition systems.

  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. Polarizable molecular mechanics studies of Cu(I)/Zn(II) superoxide dismutase: bimetallic binding site and structured waters.

    Science.gov (United States)

    Gresh, Nohad; El Hage, Krystel; Perahia, David; Piquemal, Jean-Philip; Berthomieu, Catherine; Berthomieu, Dorothée

    2014-11-05

    The existence of a network of structured waters in the vicinity of the bimetallic site of Cu/Zn-superoxide dismutase (SOD) has been inferred from high-resolution X-ray crystallography. Long-duration molecular dynamics (MD) simulations could enable to quantify the lifetimes and possible interchanges of these waters between themselves as well as with a ligand diffusing toward the bimetallic site. The presence of several charged or polar ligands makes it necessary to resort to second-generation polarizable potentials. As a first step toward such simulations, we benchmark in this article the accuracy of one such potential, sum of interactions between fragments Ab initio computed (SIBFA), by comparisons with quantum mechanics (QM) computations. We first consider the bimetallic binding site of a Cu/Zn-SOD, in which three histidines and a water molecule are bound to Cu(I) and three histidines and one aspartate are bound to Zn(II). The comparisons are made for different His6 complexes with either one or both cations, and either with or without Asp and water. The total net charges vary from zero to three. We subsequently perform preliminary short-duration MD simulations of 296 waters solvating Cu/Zn-SOD. Six representative geometries are selected and energy-minimized. Single-point SIBFA and QM computations are then performed in parallel on model binding sites extracted from these six structures, each of which totals 301 atoms including the closest 28 waters from the Cu metal site. The ranking of their relative stabilities as given by SIBFA is identical to the QM one, and the relative energy differences by both approaches are fully consistent. In addition, the lowest-energy structure, from SIBFA and QM, has a close overlap with the crystallographic one. The SIBFA calculations enable to quantify the impact of polarization and charge transfer in the ranking of the six structures. Five structural waters, which connect Arg141 and Glu131, are endowed with very high dipole moments

  4. QSAR, docking, dynamic simulation and quantum mechanics studies to explore the recognition properties of cholinesterase binding sites.

    Science.gov (United States)

    Correa-Basurto, J; Bello, M; Rosales-Hernández, M C; Hernández-Rodríguez, M; Nicolás-Vázquez, I; Rojo-Domínguez, A; Trujillo-Ferrara, J G; Miranda, René; Flores-Sandoval, C A

    2014-02-25

    A set of 84 known N-aryl-monosubstituted derivatives (42 amides: series 1 and 2, and 42 imides: series 3 an 4, from maleic and succinic anhydrides, respectively) that display inhibitory activity toward both acetylcholinesterase and butyrylcholinesterase (ChEs) was considered for Quantitative structure-activity relationship (QSAR) studies. These QSAR studies employed docking data from both ChEs that were previously submitted to molecular dynamics (MD) simulations. Donepezil and galanthamine stereoisomers were included to analyze their quantum mechanics properties and for validating the docking procedure. Quantum parameters such as frontier orbital energies, dipole moment, molecular volume, atomic charges, bond length and reactivity parameters were measured, as well as partition coefficients, molar refractivity and polarizability were also analyzed. In order to evaluate the obtained equations, four compounds: 1a (4-oxo-4-(phenylamino)butanoic acid), 2a ((2Z)-4-oxo-4-(phenylamino)but-2-enoic acid), 3a (2-phenylcyclopentane-1,3-dione) and 4a (2-phenylcyclopent-4-ene-1,3-dione) were employed as independent data set, using only equations with r(m(test))²>0.5. It was observed that residual values gave low value in almost all series, excepting in series 1 for compounds 3a and 4a, and in series 4 for compounds 1a, 2a and 3a, giving a low value for 4a. Consequently, equations seems to be specific according to the structure of the evaluated compound, that means, series 1 fits better for compound 1a, series 3 or 4 fits better for compounds 3a or 4a. Same behavior was observed in the butyrylcholinesterase (BChE). Therefore, obtained equations in this QSAR study could be employed to calculate the inhibition constant (Ki) value for compounds having a similar structure as N-aryl derivatives described here. The QSAR study showed that bond lengths, molecular electrostatic potential and frontier orbital energies are important in both ChE targets. Docking studies revealed that

  5. Possible Insecticidal Mechanisms Mediated by Immune-Response-Related Cry-Binding Proteins in the Midgut Juice of Plutella xylostella and Spodoptera exigua.

    Science.gov (United States)

    Lu, Keyu; Gu, Yuqing; Liu, Xiaoping; Lin, Yi; Yu, Xiao-Qiang

    2017-03-15

    Cry toxins are insecticidal toxin proteins produced by a spore-forming Gram-positive bacterium Bacillus thuringiensis. Interactions between the Cry toxins and the receptors from midgut brush border membrane vesicles (BBMVs), such as cadherin, alkaline phosphatase, and aminopeptidase, are key steps for the specificity and insecticidal activity of Cry proteins. However, little is known about the midgut juice proteins that may interfere with Cry binding to the receptors. To validate the hypothesis that there exist Cry-binding proteins that can interfere with the insecticidal process of Cry toxins, we applied Cry1Ab1-coupled Sepharose beads to isolate Cry-binding proteins form midgut juice of Plutella xylostella and Spodoptera exigua. Trypsin-like serine proteases and Dorsal were found to be Cry1Ab1-binding proteins in the midgut juice of P. xylostella. Peroxidase-C (POX-C) was found to be the Cry1Ab1-binding protein in the midgut juice of S. exigua. We proposed possible insecticidal mechanisms of Cry1Ab1 mediated by the two immune-related proteins: Dorsal and POX-C. Our results suggested that there exist, in the midgut juice, Cry-binding proteins, which are different from BBMV-specific receptors.

  6. Autocrine effect of DHT on FGF signaling and cell proliferation in LNCaP cells: role of heparin/heparan-degrading enzymes.

    Science.gov (United States)

    Kassen, A E; Sensibar, J A; Sintich, S M; Pruden, S J; Kozlowski, J M; Lee, C

    2000-07-01

    LNCaP cells are androgen-sensitive human prostate cancer cells. They are characterized by a bell-shaped growth curve in response to increasing doses of dihydrotestosterone (DHT) in culture. At a low concentration of DHT (0.1 nM), these cells show an increase in proliferation, but their growth is arrested at a high concentration (100 nM) of DHT. Results of our previous study demonstrated that the inhibitory effect of DHT at a high concentration was mediated through the action of TGF-beta1. The objective of the present study was to elucidate the mechanism of the proliferative effect of DHT in LNCaP cells. METHODS AND RESULTS DHT stimulated LNCaP proliferation only when cells were cultured in the presence of serum. In serum-free cultures, the characteristic DHT-induced proliferation was not observed. The addition of neutralizing antibody against FGF-2 (basic fibroblast growth factor) was able to inhibit this DHT-induced proliferation. These results suggest that the proliferative effect of DHT was mediated through the action of FGF-2. However, results of the reverse transcriptase polymerase chain reaction indicated that LNCaP cells did not express FGF-2 message. As a result, the source of FGF-2 in these cultures must be the serum supplemented in the culture media. FGF-2 can bind to heparin sulfate chains within the extracellular matrix (ECM). In cultures treated with exogenous heparin, the proliferative effect of DHT was abolished. These results led to the development of the hypothesis that DHT treatment mediates the release of FGF-2 entrapped in the ECM through increased heparinase activity. The addition of heparinase to cultures of LNCaP cells, in the absence of DHT, was able to stimulate cell proliferation. Moreover, 0.1 nM DHT caused a significant increase in heparinase activity. These results provide a possible mechanism for DHT action in LNCaP cells. In the absence of DHT, FGF-2 in culture was trapped in the extracellular matrix and was not available to interact

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

    International Nuclear Information System (INIS)

    Wang, Xue; Liu, Tao; Chen, Yuan; Zhang, Kun; Maitz, Manfred F.; Pan, Changjiang; Chen, Junying; Huang, Nan

    2014-01-01

    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

  8. Sensitive and selective turn off-on fluorescence detection of heparin based on the energy transfer platform using the BSA-stabilized Au nanoclusters/amino-functionalized graphene oxide hybrids.

    Science.gov (United States)

    Lan, Jing; Zou, Hong Yan; Wang, Qiang; Zeng, Ping; Li, Yuan Fang; Huang, Cheng Zhi

    2016-12-01

    An ultra-sensitive and selective turn off-on fluorescence detection of heparin based on the energy transfer in the BSA-stabilized gold nanoclusters/amino-functionalized graphene oxide (BSA-AuNCs/NH 2 -GO) hybrids was successfully realized. The BSA-AuNCs containing amounts of carboxyl groups could be absorbed on the surface of NH 2 -GO through the electrostatic interaction, which resulted in the fluorescence quenching of BSA-AuNCs with high efficiency. However, heparin, possessing high density of negative charge, could compete with BSA-AuNCs to bind NH 2 -GO and block the energy transfer from BSA-AuNCs to NH 2 -GO. The fluorescence recovery of BSA-AuNCs was closely related to the amount of heparin and there was a good linear relationship between fluorescence recovery of BSA-AuNCs and heparin over the range of 100ng/mL to 30μg/mL with a detection limit of 40ng/mL. What's more, the fluorescence assay was successfully applied for heparin sensing in human serums and intracellular imaging. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. The regulatory role of heparin on c-Met signaling in hepatocellular carcinoma cells.

    Science.gov (United States)

    İşcan, Evin; Güneş, Aysim; Korhan, Peyda; Yılmaz, Yeliz; Erdal, Esra; Atabey, Neşe

    2017-06-01

    The role of heparin as an anticoagulant is well defined; however, its role in tumorigenesis and tumor progression is not clear yet. Some studies have shown that anticoagulant treatment in cancer patients improve overall survival, however, recent clinical trials have not shown a survival benefit in cancer patients receiving heparin treatment. In our previous studies we have shown the inhibitory effects of heparin on Hepatocyte Growth Factor (HGF)-induced invasion and migration in hepatocellular carcinoma (HCC) cells. In this study, we showed the differential effects of heparin on the behaviors of HCC cells based on the presence or absence of HGF. In the absence of HGF, heparin activated HGF/c-Met signaling and promoted motility and invasion in HCC cells. Heparin treatment led to c-Met receptor dimerization and activated c-Met signaling in an HGF independent manner. Heparin-induced c-Met activation increased migration and invasion through ERK1/2, early growth response factor 1 (EGR1) and Matrix Metalloproteinases (MMP) axis. Interestingly, heparin modestly decreased the proliferation of HCC cells by inhibiting activatory phosphorylation of Akt. The inhibition of c-Met signaling reversed heparin-induced increase in motility and invasion and, proliferation inhibition. Our study provides a new perspective into the role of heparin on c-Met signaling in HCC.

  10. Molecular Dynamics Simulation Study on the Binding and Stabilization Mechanism of Antiprion Compounds to the "Hot Spot" Region of PrPC.

    Science.gov (United States)

    Zhou, Shuangyan; Liu, Xuewei; An, Xiaoli; Yao, Xiaojun; Liu, Huanxiang

    2017-11-15

    Structural transitions in the prion protein from the cellular form, PrP C , into the pathological isoform, PrP Sc , are regarded as the main cause of the transmissible spongiform encephalopathies, also known as prion diseases. Hence, discovering and designing effective antiprion drugs that can inhibit PrP C to PrP Sc conversion is regarded as a promising way to cure prion disease. Among several strategies to inhibit PrP C to PrP Sc conversion, stabilizing the native PrP C via specific binding is believed to be one of the valuable approaches and many antiprion compounds have been reported based on this strategy. However, the detailed mechanism to stabilize the native PrP C is still unknown. As such, to unravel the stabilizing mechanism of these compounds to PrP C is valuable for the further design and discovery of antiprion compounds. In this study, by molecular dynamics simulation method, we investigated the stabilizing mechanism of several antiprion compounds on PrP C that were previously reported to have specific binding to the "hot spot" region of PrP C . Our simulation results reveal that the stabilization mechanism of specific binding compounds can be summarized as (I) to stabilize both the flexible C-terminal of α2 and the hydrophobic core, such as BMD42-29 and GN8; (II) to stabilize the hydrophobic core, such as J1 and GJP49; (III) to stabilize the overall structure of PrP C by high binding affinity, as NPR-056. In addition, as indicated by the H-bond analysis and decomposition analysis of binding free energy, the residues N159 and Q160 play an important role in the specific binding of the studied compounds and all these compounds interact with PrP C in a similar way with the key interacting residues L130 in the β1 strand, P158, N159, Q160, etc. in the α1-β2 loop, and H187, T190, T191, etc. in the α2 C-terminus although the compounds have large structural difference. As a whole, our obtained results can provide some insights into the specific binding

  11. Voltammetric determination of heparin based on its interaction with ...

    African Journals Online (AJOL)

    ... 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.

  12. Protein interactions with quaternized chitosan/heparin multilayers

    Czech Academy of Sciences Publication Activity Database

    Kumorek, Marta M.; Kubies, Dana; Riedel, Tomáš

    2016-01-01

    Roč. 65, Suppl. 2 (2016), S253-S261 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LQ1604 Institutional support: RVO:61389013 Keywords : heparin * chitosan * protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.461, year: 2016 http://www.biomed.cas.cz/physiolres/pdf/65%20Suppl%202/65_S253.pdf

  13. The cytotoxic effect of spiroflavanone derivatives, their binding ability to human serum albumin (HSA) and a DFT study on the mechanism of their synthesis

    Science.gov (United States)

    Budzisz, Elzbieta; Paneth, Piotr; Geromino, Inacrist; Muzioł, Tadeusz; Rozalski, Marek; Krajewska, Urszula; Pipiak, Paulina; Ponczek, Michał B.; Małecka, Magdalena; Kupcewicz, Bogumiła

    2017-06-01

    This paper examines the cytotoxic effect of nine compounds with spiropyrazoline structures, and determines the reaction mechanism between diazomethane and selected benzylideneflavanones, their lipophilicity, and their binding ability to human serum albumin. The cytotoxic effect was determined on two human leukaemia cell lines (HL-60 and NALM-6) and melanoma WM-115 cells, as well as on normal human umbilical vein endothelial cells (HUVEC). The highest cytotoxicity was exhibited by compound B7: it was found to have an IC50 of less than 10 μM for all three cancer cell lines, with five to 12-fold lower sensitivity against normal cells (HUVEC). All the compounds exhibit comparable affinity energy in human serum albumin binding (from -8.1 to -8.6 kcal mol-1) but vary in their binding sites depending on the substituent. X-ray crystallography of two derivatives confirmed their synthetic pathway, and their structures were carefully examined.

  14. Heparin defends against the toxicity of circulating histones in sepsis.

    Science.gov (United States)

    Wang, Feifei; Zhang, Naipu; Li, Biru; Liu, Lanbo; Ding, Lei; Wang, Ying; Zhu, Yimin; Mo, Xi; Cao, Qing

    2015-06-01

    Although circulating histones were demonstrated as major mediators of death in septic mice models, their roles in septic patients are not clarified. The present study sought to evaluate the clinical relevance of the circulating histone levels in septic children, and the antagonizing effects of heparin on circulating histones. Histone levels in the plasma of septic children were significantly higher than healthy controls, and positively correlated with disease severity. Histone treatment could activate NF-κB pathway of the endothelial cells and induce the secretion of large amount of cytokines that further amplify inflammation, subsequently leading to organ damage. Co-injection of low dose heparin with lethal dose histones could protect mouse from organ damage and death by antagonizing circulating histones, and similar effects were also observed in other septic models. Collectively, these findings indicated that circulating histones might serve as key factors in the pathogenesis of sepsis and their levels in plasma might be a marker for disease progression and prognosis. Furthermore, low dose heparin might be an effective therapy to hamper sepsis progression and reduce the mortality.

  15. Interfacial binding of bee venom secreted phospholipase A2 to membranes occurs predominantly by a nonelectrostatic mechanism.

    Science.gov (United States)

    Bollinger, James G; Diraviyam, Karthikeyan; Ghomashchi, Farideh; Murray, Diana; Gelb, Michael H

    2004-10-26

    The secreted phospholipase A(2) from bee venom (bvPLA(2)) contains a membrane binding surface composed mainly of hydrophobic residues and two basic residues that come in close contact with the membrane. Previous studies have shown that the mutant in which these two basic residues (K14 and R23) as well as three other nearby basic residues were collectively changed to glutamate (charge reversal), like wild-type enzyme, binds with high affinity to anionic phospholipid vesicles. In the present study, we have measured the equilibrium constants for the interaction of wild-type bvPLA(2), the charge-reversal mutant (bvPLA(2)-E5), and the mutant in which the five basic residues were changed to neutral glutamine (bvPLA(2)-Q5) with phosphatidylcholine (PC) vesicles containing various amounts of the anionic phosphatidylserine (PS). Remarkably, bvPLA(2)-E5 with an anionic membrane binding surface binds more tightly to vesicles as the mole percent of PS is increased. Computational studies predict that this is due to a significant upward shift in the pK(a) of E14 (and to some extent E23) when the enzyme binds to PC/PS vesicles such that the carboxylate of the glutamate side chain near the membrane surface undergoes protonation. The experimental pH dependence of vesicle binding supports this prediction. bvPLA(2)-E5 binds more weakly to PS/PC vesicles than does wild-type enzyme due to electrostatic protein-vesicle repulsion coupled with the similar energetics of desolvation of basic residues and glutamates that accompanies enzyme-vesicle contact. Studies with bvPLA(2)-Q5 show that only a small fraction of the total bvPLA(2) interfacial binding energy ( approximately 10%) is due to electrostatics.

  16. 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.

  17. The potent opioid agonist, (+)-cis-3-methylfentanyl binds pseudoirreversibly to the opioid receptor complex in vitro and in vivo: Evidence for a novel mechanism of action

    Energy Technology Data Exchange (ETDEWEB)

    Band, L.; Xu, Heng; Bykov, V.; Rothman, R.B.; Kim, Chongho; Newman, A.; Jacobson, A.E.; Rice, K.C. (NIDDK, Bethesda, MD (USA)); Greig, N. (NIA, Bethesda, MD (USA))

    1990-01-01

    The present study demonstrates that pretreatment of rat brain membranes with (+)-cis-3-methylfentanyl ((+)-cis-MF), followed by extensive washing of the membranes, produces a wash-resistant decreasing in the binding of ({sup 3}H)-(D-ala{sup 2}, D-leu{sup 5})enkephalin to the d binding site of the opioid receptor complex ({delta}{sub cx} binding site). Intravenous administration of (+)-cis-MF (50 {mu}g/kg) to rats produced a pronounced catalepsy and also produced a wash-resistant masking of {delta}{sub cx} and {mu} binding sites in membranes prepared 120 min post-injection. Administration of 1 mg/kg i.v. of the opioid antagonist, 6-desoxy-6{beta}-fluoronaltrexone (cycloFOXY), 100 min after the injection of (+)-cis-MF (20 min prior to the preparation of membranes) completely reversed the catatonia and restored masked {delta}{sub cx} binding sites to control levels. This was not observed with (+)-cycloFOXY. The implications of these and other findings for the mechanism of action of (+)-cis-MF and models of the opioid receptors are discussed.

  18. Coarse-grained/molecular mechanics of the TAS2R38 bitter taste receptor: experimentally-validated detailed structural prediction of agonist binding.

    Directory of Open Access Journals (Sweden)

    Alessandro Marchiori

    Full Text Available Bitter molecules in humans are detected by ∼25 G protein-coupled receptors (GPCRs. The lack of atomic resolution structure for any of them is complicating an in depth understanding of the molecular mechanisms underlying bitter taste perception. Here, we investigate the molecular determinants of the interaction of the TAS2R38 bitter taste receptor with its agonists phenylthiocarbamide (PTC and propylthiouracil (PROP. We use the recently developed hybrid Molecular Mechanics/Coarse Grained (MM/CG method tailored specifically for GPCRs. The method, through an extensive exploration of the conformational space in the binding pocket, allows the identification of several residues important for agonist binding that would have been very difficult to capture from the standard bioinformatics/docking approach. Our calculations suggest that both agonists bind to Asn103, Phe197, Phe264 and Trp201, whilst they do not interact with the so-called extra cellular loop 2, involved in cis-retinal binding in the GPCR rhodopsin. These predictions are consistent with data sets based on more than 20 site-directed mutagenesis and functional calcium imaging experiments of TAS2R38. The method could be readily used for other GPCRs for which experimental information is currently lacking.

  19. Qualitative and Quantitative Analysis of Heparin during Precipitation by Near-Infrared Spectroscopy

    OpenAIRE

    Lian Li; Jinfeng Wang; Hengchang Zang; Hui Zhang; Wei Jiang; Shang Chen; Fengshan Wang

    2016-01-01

    Heparin is a glycosaminoglycan (GAG) that plays an important role in the blood coagulation system. Its quality is of great importance, so it is necessary to develop a fast analytical method during the manufacture process to analyse the quality of heparin produced. In this study, the heparin contents of 80 samples collected from five batches during the precipitation process were analysed using nearinfrared (NIR) spectroscopy and a chemometrics approach. This was done in order to improve the ef...

  20. From Farm to Pharma: An Overview of Industrial Heparin Manufacturing Methods.

    Science.gov (United States)

    van der Meer, Jan-Ytzen; Kellenbach, Edwin; van den Bos, Leendert J

    2017-06-21

    The purification of heparin from offal is an old industrial process for which commercial recipes date back to 1922. Although chemical, chemoenzymatic, and biotechnological alternatives for this production method have been published in the academic literature, animal-tissue is still the sole source for commercial heparin production in industry. Heparin purification methods are closely guarded industrial secrets which are not available to the general (scientific) public. However by reviewing the academic and patent literature, we aim to provide a comprehensive overview of the general methods used in industry for the extraction of heparin from animal tissue.

  1. 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.

  2. Evidence-based algorithm for heparin dosing before cardiopulmonary bypass. Part 1: Development of the algorithm.

    Science.gov (United States)

    McKinney, Mark C; Riley, Jeffrey B

    2007-12-01

    The incidence of heparin resistance during adult cardiac surgery with cardiopulmonary bypass has been reported at 15%-20%. The consistent use of a clinical decision-making algorithm may increase the consistency of patient care and likely reduce the total required heparin dose and other problems associated with heparin dosing. After a directed survey of practicing perfusionists regarding treatment of heparin resistance and a literature search for high-level evidence regarding the diagnosis and treatment of heparin resistance, an evidence-based decision-making algorithm was constructed. The face validity of the algorithm decisive steps and logic was confirmed by a second survey of practicing perfusionists. The algorithm begins with review of the patient history to identify predictors for heparin resistance. The definition for heparin resistance contained in the algorithm is an activated clotting time 450 IU/kg heparin loading dose. Based on the literature, the treatment for heparin resistance used in the algorithm is anti-thrombin III supplement. The algorithm seems to be valid and is supported by high-level evidence and clinician opinion. The next step is a human randomized clinical trial to test the clinical procedure guideline algorithm vs. current standard clinical practice.

  3. Identification of a novel structure in heparin generated by potassium permanganate oxidation

    Science.gov (United States)

    Beccati, Daniela; Roy, Sucharita; Yu, Fei; Gunay, Nur Sibel; Capila, Ishan; Lech, Miroslaw; Linhardt, Robert J.; Venkataraman, Ganesh

    2012-01-01

    The worldwide heparin contamination crisis in 2008 led health authorities to take fundamental steps to better control heparin manufacture, including implementing appropriate analytical and bio-analytical methods to ensure production and release of high quality heparin sodium product. Consequently, there is an increased interest in the identification and structural elucidation of unusually modified structures that may be present in heparin. Our study focuses on the structural elucidation of species that give rise to a signal observed at 2.10 ppm in the N-acetyl region of the 1H NMR spectrum of some pharmaceutical grade heparin preparations. Structural elucidation experiments were carried out using homonuclear (COSY, TOSCY and NOESY) and heteronuclear (HSQC, HSQC-DEPT, HMQC-COSY, HSQC-TOCSY, and HMBC) 2D NMR spectroscopy on both heparin as well as heparin-like model compounds. Our results identify a novel type of oxidative modification of the heparin chain that results from a specific step in the manufacturing process used to prepare heparin. PMID:25147414

  4. Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

    Energy Technology Data Exchange (ETDEWEB)

    Shyy, Y.J.; Tian, G.; Tsai, M.D.

    1987-10-06

    Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

  5. Binding of 99mTc-labelled polyclonal human immunoglobulin to bacteria as a mechanism for scintigraphic detection of infection

    International Nuclear Information System (INIS)

    Calame, W.; Furth, R. van

    1991-01-01

    The aim of the present study was to determine whether 99m Tc-labelled polyclonal human immunoglobulin ( 99m Tc-HIG) binds to bacteria in vitro as well as in vivo. In vitro, the binding of 99m Tc-HIG to various gram-positive and gram-negative bacteria was determined. In vivo, mice were infected with Staphylococcus aureus Cowan I (protein A rich) or S. aureus EMS (protein A deficient) in a tigh muscle and then 99m Tc-HIG or 99m Tc-labelled human serum albumin ( 99m Tc-HSA) was administered; scintigrams were made 1, 4 and 18 h later. In vitro binding of 99m Tc-HIG to bacteria was higher for gram-positive than for gram-negative forms. A positive correlation was found between the protein A content and the degree of binding to S. aureus. This was also found in vivo. The accumulation of 99m Tc-HIG at the site of infection was significantly (P 99m Tc-HSA, for both strains of S. aureus. It is concluded that vascular permeability cannot fully explain the accumulation of 99m Tc-HIG at the site of infection and that binding of 99m Tc-HIG to bacteria plays a role in this respect. (orig.)

  6. Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Gerlach, Lars-Ole; Jakobsen, Janus S

    2004-01-01

    , respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp(262) and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build......-up in the rather distinct CXCR3 receptor, for which the compound normally had no effect. Introduction of only a Glu at position VII:06 and the removal of a neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in affinity of AMD3100 to within 10-fold of its affinity in CXCR4. We conclude...

  7. Influences of binding to dissolved organic matter on hydrophobic organic compounds in a multi-contaminant system: Coefficients, mechanisms and ecological risks

    International Nuclear Information System (INIS)

    Li, Yi-Long; He, Wei; Liu, Wen-Xiu; Kong, Xiang-Zhen; Yang, Bin; Yang, Chen; Xu, Fu-Liu

    2015-01-01

    The complexation flocculation (CF) method was successfully employed to identify binding coefficients (K_d_o_c) of specific organic contaminants to dissolved organic matter (DOM, often indicated by dissolved organic carbon, DOC) in a multi-contaminant hydrophobic organic contaminant (HOC) system. K_d_o_c values were obtained for most of the evaluated 33 HOCs, indicating the feasibility and applicability of the CF method in a multi-contaminant system. Significant positive correlations were observed between binding coefficients and octanol–water partition coefficients (K_o_w) for organic halogen compounds, such as polybrominated diphenyl ethers (PBDEs) (R"2 = 0.95, p < 0.05) and organic chlorine pesticides (OCPs) (methoxychlor excluded, R"2 = 0.82, p < 0.05). The positive correlations identified between the lgK_d_o_c and lgBCF (bioconcentration factor) for PBDEs and OCPs, as well as the negative correlation observed for polycyclic aromatic hydrocarbons (PAHs), indicated that different binding or partition mechanisms between PAHs and organic halogen compounds exist. These differences further result in discriminative competition partitions of HOCs between DOM and organisms. Assuming that only freely dissolved HOCs are bioconcentrative, the results of DOM-influenced bioconcentration factor (BCF_D_O_M) and DOM-influenced lowest observed effect level (LOEL_D_O_M) indicate that the ecological risk of HOCs is decreased by DOM. - Highlights: • Complexing-flocculation is viable in measuring K_d_o_c in a multi-polluted system. • The binding mechanisms between PAHs and organic halogens were different. • DOM should be considered when assessing ecological risk of HOCs in natural ecosystem. - Assuming only freely dissolved HOCs are effective, bioconcentration factors and ecological risks of HOCs are decreased by dissolved organic matter via binding.

  8. Computational Studies of a Mechanism for Binding and Drug Resistance in the Wild Type and Four Mutations of HIV-1 Protease with a GRL-0519 Inhibitor

    Directory of Open Access Journals (Sweden)

    Guodong Hu

    2016-05-01

    Full Text Available Drug resistance of mutations in HIV-1 protease (PR is the most severe challenge to the long-term efficacy of HIV-1 PR inhibitor in highly active antiretroviral therapy. To elucidate the molecular mechanism of drug resistance associated with mutations (D30N, I50V, I54M, and V82A and inhibitor (GRL-0519 complexes, we have performed five molecular dynamics (MD simulations and calculated the binding free energies using the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA method. The ranking of calculated binding free energies is in accordance with the experimental data. The free energy spectra of each residue and inhibitor interaction for all complexes show a similar binding model. Analysis based on the MD trajectories and contribution of each residues show that groups R2 and R3 mainly contribute van der Waals energies, while groups R1 and R4 contribute electrostatic interaction by hydrogen bonds. The drug resistance of D30N can be attributed to the decline in binding affinity of residues 28 and 29. The size of Val50 is smaller than Ile50 causes the residue to move, especially in chain A. The stable hydrophobic core, including the side chain of Ile54 in the wild type (WT complex, became unstable in I54M because the side chain of Met54 is flexible with two alternative conformations. The binding affinity of Ala82 in V82A decreases relative to Val82 in WT. The present study could provide important guidance for the design of a potent new drug resisting the mutation inhibitors.

  9. Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters

    DEFF Research Database (Denmark)

    Stolzenberg, Sebastian; Quick, Matthias; Zhao, Chunfeng

    2015-01-01

    -related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications...... with global conformational changes that are critical for the transport mechanism. That the AIN between the Na+ binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function....

  10. Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Harris, Nicholas; Koppel, Juraj; Zsila, Ferenc; Juhas, Stefan; Il'kova, Gabriela; Kogan, Faina Yurgenzon; Lahmy, Orly; Wildbaum, Gizi; Karin, Nathan; Zhuk, Regina; Gregor, Paul

    2016-04-01

    Elucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease. The glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo. Circular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo. RX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE. RX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

  11. Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Tanaka Hall, Traci M. [NIH; (UNC); (Columbia)

    2014-08-06

    Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

  12. Kinetic Mechanism of Uracil Phosphoribosyltransferase from Escherichia coli and Catalytic Importance of the Conserved Proline in the PRPP Binding Site

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Jensen, Kaj Frank

    1999-01-01

    Phosphoribosyltransferases catalyze the formation of nucleotides from a nitrogenous base and 5-phosphoribosyl-a-1-pyrophosphate (PRPP). These enzymes and the PRPP synthases resemble each other in a short homologous sequence of 13 amino acid residues which has been termed the PRPP binding site and...

  13. Mechanisms of Intentional Binding and Sensory Attenuation: The Role of Temporal Prediction, Temporal Control, Identity Prediction, and Motor Prediction

    Science.gov (United States)

    Hughes, Gethin; Desantis, Andrea; Waszak, Florian

    2013-01-01

    Sensory processing of action effects has been shown to differ from that of externally triggered stimuli, with respect both to the perceived timing of their occurrence (intentional binding) and to their intensity (sensory attenuation). These phenomena are normally attributed to forward action models, such that when action prediction is consistent…

  14. Selection, Identification, and Binding Mechanism Studies of an ssDNA Aptamer Targeted to Different Stages of E. coli O157:H7.

    Science.gov (United States)

    Zou, Ying; Duan, Nuo; Wu, Shijia; Shen, Mofei; Wang, Zhouping

    2018-06-06

    Enterohemorrhagic Escherichia coli O157:H7 ( E. coli O157:H7) is known as an important food-borne pathogen related to public health. In this study, aptamers which could bind to different stages of E. coli O157:H7 (adjustment phase, log phase, and stationary phase) with high affinity and specificity were obtained by the whole cell-SELEX method through 14 selection rounds including three counter-selection rounds. Altogether, 32 sequences were obtained, and nine families were classified to select the optimal aptamer. To analyze affinity and specificity by flow cytometer, an ssDNA aptamer named Apt-5 was picked out as the optimal aptamer that recognizes different stages of E. coli O157:H7 specifically with the K d value of 9.04 ± 2.80 nM. In addition, in order to study the binding mechanism, target bacteria were treated by proteinase K and trypsin, indicating that the specific binding site is not protein on the cell membrane. Furthermore, when we treated E. coli O157:H7 with EDTA, the result showed that the binding site might be lipopolysaccharide (LPS) on the outer membrane of E. coli O157:H7.

  15. Mechanisms of Zn(II) binded to collagen and its effect on the capacity of eco-friendly Zn-Cr combination tanning system.

    Science.gov (United States)

    Cao, Shan; Liu, Bing; Cheng, Baozhen; Lu, Fuping; Wang, Yanping; Li, Yu

    2017-01-05

    The eco-friendly combination tanning process has been developed to reduce chromium in existing researches, which is based on zinc tanning agents. This can be considered as a less-chrome substitute for current tanning process. To gain deeper understanding of the binding mechanisms of zinc-collagen interaction, which are affected by tanning pH, experiments have been carried out. Analysis in this paper reveals how chemical bonds from the collagen's main function groups combine with zinc. XPS and NIR data was analyzed for further understanding of where the zinc binding sites lie on collagen fibers at different pH. The results indicate that high pH is helpful to amino-binding sites while low pH promotes carboxyl-binding sites on collagen fibers. Furthermore, from the effect of Zinc-chrome combination tanning, we can see that the new method reduces the chromium dosage in tanning process compared to the conventional chrome tanning method. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Binding of the cSH3 domain of Grb2 adaptor to two distinct RXXK motifs within Gab1 docker employs differential mechanisms.

    Science.gov (United States)

    McDonald, Caleb B; Seldeen, Kenneth L; Deegan, Brian J; Bhat, Vikas; Farooq, Amjad

    2011-01-01

    A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3, and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 3(10) -helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease. Copyright © 2010 John Wiley & Sons, Ltd.

  17. A conserved mechanism of autoinhibition for the AMPK kinase domain: ATP-binding site and catalytic loop refolding as a means of regulation

    International Nuclear Information System (INIS)

    Littler, Dene R.; Walker, John R.; Davis, Tara; Wybenga-Groot, Leanne E.; Finerty, Patrick J. Jr; Newman, Elena; Mackenzie, Farell; Dhe-Paganon, Sirano

    2010-01-01

    A 1.9 Å resolution crystal structure of the isolated kinase domain from the α2 subunit of human AMPK, the first from a multicellular organism, is presented. The AMP-activated protein kinase (AMPK) is a highly conserved trimeric protein complex that is responsible for energy homeostasis in eukaryotic cells. Here, a 1.9 Å resolution crystal structure of the isolated kinase domain from the α2 subunit of human AMPK, the first from a multicellular organism, is presented. This human form adopts a catalytically inactive state with distorted ATP-binding and substrate-binding sites. The ATP site is affected by changes in the base of the activation loop, which has moved into an inhibited DFG-out conformation. The substrate-binding site is disturbed by changes within the AMPKα2 catalytic loop that further distort the enzyme from a catalytically active form. Similar structural rearrangements have been observed in a yeast AMPK homologue in response to the binding of its auto-inhibitory domain; restructuring of the kinase catalytic loop is therefore a conserved feature of the AMPK protein family and is likely to represent an inhibitory mechanism that is utilized during function

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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

  19. Vitamin K antagonists or low-molecular-weight heparin for the long term treatment of symptomatic venous thromboembolism

    NARCIS (Netherlands)

    van der Heijden, J. F.; Hutten, B. A.; Büller, H. R.; Prins, M. H.

    2002-01-01

    BACKGROUND: People with venous thromboembolism are generally treated for five days with intravenous unfractionated heparin or subcutaneous low-molecular-weight heparin followed by three months of vitamin K antagonists treatment. Treatment with vitamin K antagonists requires regular laboratory

  20. Cerebral venous thrombosis due to cryptogenic organising pneumopathy with antiphospholipid syndrome worsened by heparin-induced thrombocytopenia.

    Science.gov (United States)

    Hsieh, J; Kuzmanovic, I; Vargas, M I; Momjian-Mayor, I

    2013-07-09

    Cerebral venous thrombosis (CVT) has usually been ascribed to prothrombotic conditions, oral contraceptives, pregnancy, malignancy, infection, head injury or mechanical precipitants. The case reported here illustrates two rare causes of CVT observed in the same patient: the presence of antiphospholipid antibodies associated with an asymptomatic cryptogenic organising pneumopathy (COP) which were considered the origin of the venous cerebral thrombosis and heparin-induced thrombocytopenia (HIT) which was responsible for the worsening of the thrombosis observed a few days after the introduction of treatment. Moreover, we provide here additional positive experience in the treatment of both, CVT and HIT, by fondaparinux with bridging to warfarin given their successful evolution under this anticoagulant option.

  1. Assessment of HIT Antibody Complex in Hip Fracture Patients Receiving Enoxaparin or Unfractionated Heparin

    DEFF Research Database (Denmark)

    Griffin, Justin W; Hopkinson, William J; Rud-Lassen, Michael

    2011-01-01

    of antiheparin-PF4 antibodies and a greater prevalence of immunoglobulin G (IgG) subtype. Heparin and enoxaparin are capable of generating heparin-induced thrombocytopenia (HIT) antibodies in elderly patients undergoing orthopedic surgery but perhaps not to the same extent. When comparing low...

  2. Heparin induced alterations in clearance and distribution of blood-borne microparticles following operative trauma.

    Science.gov (United States)

    Saba, T M; Antikatzides, T G

    1979-04-01

    The influence of systemic heparin administration on the vascular clearance and tissue distribution of blood-borne microparticles was evaluated in normal rats and rats after operation (laparotomy plus intestinal manipulation) utilizing an (131)I- colloid which is phagocytized by the reticuloendothelial system (RES). Intravenous heparin administration (100 USP/100g body weight) into normal animals three minutes prior to colloid injection (50 mg/lOOg) induced a significant increase in pulmonary localization of the microparticles as compared to nonheparinized control rats, while hepatic and splenic uptake were decreased. Surgical trauma decreased hepatic RE uptake and increased pulmonary localization of the microparticles when injected systemically at 60 minutes postsurgery. Heparin administration 60 minutes after surgery and three minutes prior to colloid injection, magnified the increased pulmonary localization response with an associated further depression of the RES. The ability of heparin to alter both RE clearance function and lung localization of microparticles was dose dependent and a function of the interval between heparin administration and systemic particulate infusion. Thus, low dose heparin administration was capable of stimulating RE activity while heparin in doses of excess of 50 USP units/lOOg body weight decreased RE function. These findings suggest that the functional state of the hepatic RE system can be greatly affected in a dose-dependent manner by systemic heparin administration which may influence distribution of blood-borne microparticles.

  3. Covalently bound conjugates of albumin and heparin: Synthesis, fractionation and characterization

    NARCIS (Netherlands)

    Hennink, Wim E.; Feijen, Jan; Ebert, Charles D.; Kim, Sung Wan

    1983-01-01

    Covalently bound conjugates of human serum albumin and heparin were prepared as compounds which could improve the blood-compatibility of polymer surfaces either by preadsorption or by covalent coupling of the conjugates onto blood contacting surfaces. The conjugates (10–16 weight % of heparin) were

  4. Safety of low-molecular-weight heparin in pregnancy: a systematic review

    NARCIS (Netherlands)

    Sanson, B. J.; Lensing, A. W.; Prins, M. H.; Ginsberg, J. S.; Barkagan, Z. S.; Lavenne-Pardonge, E.; Brenner, B.; Dulitzky, M.; Nielsen, J. D.; Boda, Z.; Turi, S.; Mac Gillavry, M. R.; Hamulyák, K.; Theunissen, I. M.; Hunt, B. J.; Büller, H. R.

    1999-01-01

    Unfractionated heparin (UFH) remains the anticoagulant of choice during pregnancy. Low-molecular-weight heparins (LMWH) are an attractive alternative to UFH due to their logistic advantages and their association with a lower incidence of osteoporosis and HIT. We reviewed all published clinical

  5. Poly(vinyl alcohol)-heparin hydrogels as sensor catheter membranes

    NARCIS (Netherlands)

    Brinkman, E.; van der Does, L.; Bantjes, A.

    1991-01-01

    Poly(vinyl alcohol)-heparin hydrogels with varying water content were synthesized for use as sensor catheter membranes. Films were cast from aqueous mixtures of poly(viny) alcohol) (PVA), a photosensitive cross-linker p-diazonium diphenyl amine polymer (PA), glutaraldehyde (GA) and heparin. After

  6. Design of a new type of coating for the controlled release of heparin

    NARCIS (Netherlands)

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

    1997-01-01

    Thrombus formation at the surface of blood contacting devices can be prevented by local release of heparin. Preferably, the release rate should be constant for prolonged periods of time. The minimum heparin release rate to achieve thromboresistance will be different for various applications and

  7. Anti-Platelet Factor 4/Heparin Antibody Formation Occurs Endogenously and at Unexpected High Frequency in Polycythemia Vera

    Directory of Open Access Journals (Sweden)

    Sara C. Meyer

    2017-01-01

    Full Text Available Background. Myeloproliferative neoplasms (MPN encounter thromboses due to multiple known risk factors. Heparin-induced thrombocytopenia (HIT is a thrombotic syndrome mediated by anti-platelet factor 4 (PF4/heparin antibodies with undetermined significance for thrombosis in MPN. We hypothesized that anti-PF4/heparin Ab might occur in MPN and promote thrombosis. Methods. Anti-PF4/heparin antibodies were analyzed in 127 MPN patients including 76 PV and 51 ET. Screening, validation testing, and isotype testing of anti-PF4/heparin Ab were correlated with disease characteristics. Results. Anti-PF4/heparin antibodies were detected in 21% of PV and 12% of ET versus 0.3–3% in heparin-exposed patients. Validation testing confirmed anti-PF4/heparin immunoglobulins in 15% of PV and 10% of ET. Isotype testing detected 9.2% IgG and 5.3% IgM in PV and exclusively IgM in ET. IgG-positive PV patients encountered thromboses in 57.1% suggesting anti-PF4/heparin IgG may contribute to higher risk for thrombosis in MPN. Overall, 45% of PV patients experienced thromboses with 11.8% positive for anti-PF4/heparin IgG versus 7.1% in PV without thrombosis. Conclusion. Anti-PF4/heparin antibodies occur endogenously and more frequently in MPN than upon heparin exposure. Thrombotic risk increases in anti-PF4/heparin IgG-positive PV reflecting potential implications and calling for larger, confirmatory cohorts. Anti-PF4/heparin IgG should be assessed upon thrombosis in PV to facilitate avoidance of heparin in anti-PF4/heparin IgG-positive PV.

  8. α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

    Science.gov (United States)

    Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

    2014-01-01

    Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

  9. Prevention of iron- and copper-mediated DNA damage by catecholamine and amino acid neurotransmitters, L-DOPA, and curcumin: metal binding as a general antioxidant mechanism.

    Science.gov (United States)

    García, Carla R; Angelé-Martínez, Carlos; Wilkes, Jenna A; Wang, Hsiao C; Battin, Erin E; Brumaghim, Julia L

    2012-06-07

    Concentrations of labile iron and copper are elevated in patients with neurological disorders, causing interest in metal-neurotransmitter interactions. Catecholamine (dopamine, epinephrine, and norepinephrine) and amino acid (glycine, glutamate, and 4-aminobutyrate) neurotransmitters are antioxidants also known to bind metal ions. To investigate the role of metal binding as an antioxidant mechanism for these neurotransmitters, L-dihydroxyphenylalanine (L-DOPA), and curcumin, their abilities to prevent iron- and copper-mediated DNA damage were quantified, cyclic voltammetry was used to determine the relationship between their redox potentials and DNA damage prevention, and UV-vis studies were conducted to determine iron and copper binding as well as iron oxidation rates. In contrast to amino acid neurotransmitters, catecholamine neurotransmitters, L-DOPA, and curcumin prevent significant iron-mediated DNA damage (IC(50) values of 3.2 to 18 μM) and are electrochemically active. However, glycine and glutamate are more effective at preventing copper-mediated DNA damage (IC(50) values of 35 and 12.9 μM, respectively) than L-DOPA, the only catecholamine to prevent this damage (IC(50) = 73 μM). This metal-mediated DNA damage prevention is directly related to the metal-binding behaviour of these compounds. When bound to iron or copper, the catecholamines, amino acids, and curcumin significantly shift iron oxidation potentials and stabilize Fe(3+) over Fe(2+) and Cu(2+) over Cu(+), a factor that may prevent metal redox cycling in vivo. These results highlight the disparate antioxidant activities of neurotransmitters, drugs, and supplements and highlight the importance of considering metal binding when identifying antioxidants to treat and prevent neurodegenerative disorders.

  10. Direct binding of microRNA-21 pre-element with Regorafenib: An alternative mechanism for anti-colorectal cancer chemotherapy?

    Science.gov (United States)

    Chen, Xiaobing; Xie, Bojian; Cao, Liang; Zhu, Feng; Chen, Beibei; Lv, Huifang; Fan, Xingxing; Han, Lili; Bie, Liangyu; Cao, Xinguang; Shen, Xiaokun; Cao, Feilin

    2017-05-01

    The Regorafenib is a broad-spectrum kinase inhibitor that has been approved to treat colorectal cancer (CRC). However, evidences have shown that the agent is also implicated in drug interaction with microRNA-21 (miR-21), an oncogenic miRNA which plays a key role in resisting programmed cell death in CRC cells. Here, we supposed that, instead of kinase inhibition, Regorafenib can directly bind to and then stabilize miR-21 pre-element, thus preventing RNase Dicer-meditated cleavage of the pre-element to mature miR-21. In order to verify the notion, an in silico-in vitro integrated investigation of the direct intermolecular interaction between Regorafenib and miR-21 pre-element was performed by using active pocket identification, RNA-ligand docking, molecular dynamics (MD) simulation, binding energetic analysis, and fluorescence-based assay. It was revealed that the Regorafenib can bind at the major groove-like stem region of miR-21 pre-element through three geometrically satisfactory hydrogen bonds (H-bonds) as well as a number of hydrophobic forces and π-π stacking, conferring strong specificity and high stability to the RNA-ligand complex system (K d =0.73μM). Separate inversion mutation of two base pairs (G6C, C12G) and (A13U, U4A) that are involved in the H-bonding can considerably impair the affinity of Regorafenib to miR-21 pre-element, with K d increase to 27 and 96μM, respectively. All these supported that Regorafenib can directly bind to miR-21 pre-element at molecular level and the binding mode can be properly modeled by using the proposed integrated strategy. This study would provide a potential, alternative mechanism for anti-colorectal cancer chemotherapy with Regorafenib. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. A Nexus between Theory and Experiment: Non-Empirical Quantum Mechanical Computational Methodology Applied to Cucurbit[n]uril⋅Guest Binding Interactions.

    Science.gov (United States)

    Hostaš, Jiří; Sigwalt, David; Šekutor, Marina; Ajani, Haresh; Dubecký, Matúš; Řezáč, Jan; Zavalij, Peter Y; Cao, Liping; Wohlschlager, Christian; Mlinarić-Majerski, Kata; Isaacs, Lyle; Glaser, Robert; Hobza, Pavel

    2016-11-21

    A training set of eleven X-ray structures determined for biomimetic complexes between cucurbit[n]uril (CB[7 or 8]) hosts and adamantane-/diamantane ammonium/aminium guests were studied with DFT-D3 quantum mechanical computational methods to afford ΔG calcd binding energies. A novel feature of this work is that the fidelity of the BLYP-D3/def2-TZVPP choice of DFT functional was proven by comparison with more accurate methods. For the first time, the CB[n]⋅guest complex binding energy subcomponents [for example, ΔE dispersion , ΔE electrostatic , ΔG solvation , binding entropy (-TΔS), and induced fit E deformation(host) , E deformation(guest) ] were calculated. Only a few weeks of computation time per complex were required by using this protocol. The deformation (stiffness) and solvation properties (with emphasis on cavity desolvation) of cucurbit[n]uril (n=5, 6, 7, 8) isolated host molecules were also explored by means of the DFT-D3 method. A high ρ 2 =0.84 correlation coefficient between ΔG exptl and ΔG calcd was achieved without any scaling of the calculated terms (at 298 K). This linear dependence was utilized for ΔG calcd predictions of new complexes. The nature of binding, including the role of high energy water molecules, was also studied. The utility of introduction of tethered [-(CH 2 ) n NH 3 ] + amino loops attached to N,N-dimethyl-adamantane-1-amine and N,N,N',N'-tetramethyl diamantane-4,9-diamine skeletons (both from an experimental and a theoretical perspective) is presented here as a promising tool for the achievement of new ultra-high binding guests to CB[7] hosts. Predictions of not yet measured equilibrium constants are presented herein. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. The mechanism of reduced IgG/IgE-binding of β-lactoglobulin by pulsed electric field pretreatment combined with glycation revealed by ECD/FTICR-MS.

    Science.gov (United States)

    Yang, Wenhua; Tu, Zongcai; Wang, Hui; Zhang, Lu; Kaltashov, Igor A; Zhao, Yunlong; Niu, Chendi; Yao, Honglin; Ye, Wenfeng

    2018-01-24

    Bovine β-lactoglobulin (β-Lg) is a major allergen existing in milk and causes about 90% of IgE-mediated cow's milk allergies. Previous studies showed that pulsed electric field (PEF) treatment could partially unfold the protein, which may contribute to the improvement of protein glycation. In this study, the effect of PEF pretreatment combined with glycation on the IgG/IgE-binding ability and the structure of β-Lg was investigated. The result showed that PEF pretreatment combined with glycation significantly reduced the IgG and IgE binding abilities, which was attributed to the changes of secondary and tertiary structure and the increase in glycation sites and degree of substitution per peptide (DSP) value determined by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD/FTICR-MS). Unexpectedly, glycation sites (K47, K91 and K135) added by two mannose molecules were identified in glycated β-Lg with PEF pretreatment. Moreover, the results indicated that PEF pretreatment at 25 kV cm -1 for 60 μs promoted the reduction of IgG/IgE-binding capacity by increasing the glycation degree of β-Lg, whereas single PEF treatment under the same conditions markedly enhanced the IgG/IgE-binding ability by partially unfolding the structure of β-Lg. The results suggested that ECD/FTICR-MS could help us to understand the mechanism of reduction in the IgG/IgE-binding of β-Lg by structural characterization at the molecular level. Therefore, PEF pretreatment combined with glycation may provide an alternative method for β-Lg desensitization.

  13. Binding of influenza A virus NS1 protein to the inter-SH2 domain of p85 suggests a novel mechanism for phosphoinositide 3-kinase activation.

    Science.gov (United States)

    Hale, Benjamin G; Batty, Ian H; Downes, C Peter; Randall, Richard E

    2008-01-18

    Influenza A virus NS1 protein stimulates host-cell phosphoinositide 3-kinase (PI3K) signaling by binding to the p85beta regulatory subunit of PI3K. Here, in an attempt to establish a mechanism for this activation, we report further on the functional interaction between NS1 and p85beta. Complex formation was found to be independent of NS1 RNA binding activity and is mediated by the C-terminal effector domain of NS1. Intriguingly, the primary direct binding site for NS1 on p85beta is the inter-SH2 domain, a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. In vitro kinase activity assays, together with protein binding competition studies, reveal that NS1 does not displace p110 from the inter-SH2 domain, and indicate that NS1 can form an active heterotrimeric complex with PI3K. In addition, it was established that residues at the C terminus of the inter-SH2 domain are essential for mediating the interaction between p85beta and NS1. Equivalent residues in p85alpha have previously been implicated in the basal inhibition of p110. However, such p85alpha residues were unable to substitute for those in p85beta with regards NS1 binding. Overall, these data suggest a model by which NS1 activates PI3K catalytic activity by masking a normal regulatory element specific to the p85beta inter-SH2 domain.

  14. Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid; Rapoport, Tom A. (UMASS, MED); (Harvard-Med)

    2017-03-12

    The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

  15. Heparinization of alimentation solutions administered through peripheral veins in premature infants: a controlled study.

    Science.gov (United States)

    Alpan, G; Eyal, F; Springer, C; Glick, B; Goder, K; Armon, J

    1984-09-01

    A randomized controlled study was done to determine whether the addition of heparin (1 U/mL) to peripheral intravenous alimentation solutions would affect the incidence of phlebitis and duration of patency of intravenous catheters in premature infants. Twenty-two-gauge Teflon catheters were uniformly used. One hundred five catheters infused with heparin were placed in 13 infants, and 122 catheters were placed in the control group of 13 infants. The time, nature, and incidence of complications were noted for each infusion site. Infusion of heparin was found to double the duration of patency of intravenous catheters and to reduce significantly the incidence of phlebitis. No complications related to the administration of heparin were noted. Heparinization of intravenous alimentation solutions should therefore be considered in premature infants as a means of reducing the work load and incidence of complications associated with peripheral lines.

  16. Heparin-Induced Cardiac Tamponade and Life-Threatening Hyperkalemia in a Patient with Chronic Hemodialysis

    Directory of Open Access Journals (Sweden)

    Ho-Ming Su

    2005-03-01

    Full Text Available Heparin, a commonly used anticoagulant agent, is frequently used in patients undergoing hemodialysis. As with most medications, heparin has a significant side effect profile. Two of its most important side effects, major bleeding and hyperkalemia, may be devastating without immediate diagnosis and treatment. Major bleeding such as gastrointestinal, genitourinary or intracranial bleeding is occasionally encountered and rarely neglected. However, heparin-induced cardiac tamponade is rarely encountered and may be easily overlooked. Another side effect, heparin-induced hyperkalemia, an unusual but well-described side effect, is frequently forgotten until life-threatening arrhythmia has occurred. We report a case involving a 40-year-old male patient with uremia, who had received heparin for 10 days for deep vein thrombosis in the left lower extremity. Hemopericardium with cardiac tamponade and life-threatening hyperkalemia were both noted in this patient.

  17. The US regulatory and pharmacopeia response to the global heparin contamination crisis.

    Science.gov (United States)

    Szajek, Anita Y; Chess, Edward; Johansen, Kristian; Gratzl, Gyöngyi; Gray, Elaine; Keire, David; Linhardt, Robert J; Liu, Jian; Morris, Tina; Mulloy, Barbara; Nasr, Moheb; Shriver, Zachary; Torralba, Pearle; Viskov, Christian; Williams, Roger; Woodcock, Janet; Workman, Wesley; Al-Hakim, Ali

    2016-06-09

    The contamination of the widely used lifesaving anticoagulant drug heparin in 2007 has drawn renewed attention to the challenges that are associated with the characterization, quality control and standardization of complex biological medicines from natural sources. Heparin is a linear, highly sulfated polysaccharide consisting of alternating glucosamine and uronic acid monosaccharide residues. Heparin has been used successfully as an injectable antithrombotic medicine since the 1930s, and its isolation from animal sources (primarily porcine intestine) as well as its manufacturing processes have not changed substantially since its introduction. The 2007 heparin contamination crisis resulted in several deaths in the United States and hundreds of adverse reactions worldwide, revealing the vulnerability of a complex global supply chain to sophisticated adulteration. This Perspective discusses how the US Food and Drug Administration (FDA), the United States Pharmacopeial Convention (USP) and international stakeholders collaborated to redefine quality expectations for heparin, thus making an important natural product better controlled and less susceptible to economically motivated adulteration.

  18. Interactions of oversulfated chondroitin sulfate (OSCS) from different sources with unfractionated heparin.

    Science.gov (United States)

    Gray, Angel; Litinas, Evangelos; Jeske, Walter; Fareed, Jawed; Hoppensteadt, Debra

    2012-01-01

    In 2008, oversulfated chondroitin sulfate (OSCS) was identified as the main contaminant in recalled heparin. Oversulfated chondroitin sulfate can be prepared from bovine (B), porcine (P), shark (Sh), or skate (S) origin and may produce changes in the antithrombotic, bleeding, and hemodynamic profile of heparins. This study examines the interactions of various OSCSs on heparin in animal models of thrombosis and bleeding, as well as on the anticoagulant and antiprotease effects in in vitro assays. Mixtures of 70% unfractionated heparin (UFH) with 30% OSCS from different sources were tested. In the in vitro activated partial thromboplastin time (aPTT) assay, all contaminant mixtures showed a decrease in clotting times. In addition, a significant increase in bleeding time compared to the control (UFH/saline) was observed. In the thrombosis model, no significant differences were observed. The OSCSs significantly increased anti-Xa activity in ex vivo blood samples. These results indicate that various sources of OSCS affect the hemostatic properties of heparin.

  19. Inhibitor mechanisms in the S1 binding site of the dopamine transporter defined by multi-site molecular tethering of photoactive cocaine analogs.

    Science.gov (United States)

    Krout, Danielle; Pramod, Akula Bala; Dahal, Rejwi Acharya; Tomlinson, Michael J; Sharma, Babita; Foster, James D; Zou, Mu-Fa; Boatang, Comfort; Newman, Amy Hauck; Lever, John R; Vaughan, Roxanne A; Henry, L Keith

    2017-10-15

    Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2β position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site

  20. Insight into PreImplantation Factor (PIF* mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP through essential RIKP [corrected] binding site.

    Directory of Open Access Journals (Sweden)

    Eytan R Barnea

    Full Text Available Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control. Murine embryo (d10 lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS. In silico evaluation examined binding of PIF to critical targets, using mutation analysis.PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90, co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a

  1. 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.

  2. Intravitreal low molecular weight heparin in PVR surgery.

    Directory of Open Access Journals (Sweden)

    Kumar Atul

    2003-01-01

    Full Text Available Purpose: To evaluate the efficacy of low molecular weight heparin (LMWH in prevention of postoperative fibrin formation following vitreoretinal surgery with proliferative vitreoretinopathy (PVR. Material and Methods: Thirty consecutive patients of retinal detachment with advanced PVR were enrolled in the study. They were randomised to study and control groups (n = 15 each. Study group patients received vitreoretinal surgery with 5 IU/cc of LMWH in vitrectomy infusion fluid. The control group patients received vitroretinal surgery without heparin in the infusion fluid. Patients were followed up at 1 week, 1 month and 3 months after surgery. Postoperative bleeding, media clarity, best-corrected visual acuity and success of the surgery at the end of 3 months were compared between the two groups. Results: At each follow-up visit, the study group showed a better media clarity, which was statistically significant ( P = 0.0042. The study group had a 50% better chance of retinal reattachment compared to the control group. Five patients had intraoperative bleeding in the study group (33% compared to 3 patients in the control group (20%. Conclusion: Use of intravitreal LMWH prevents postoperative fibrin formation and is beneficial in repair of retinal detachments with PVR.

  3. 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.

    2013-01-01

    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. PMID:24099708

  4. An RNA editing/dsRNA binding-independent gene regulatory mechanism of ADARs and its clinical implication in cancer.

    Science.gov (United States)

    Qi, Lihua; Song, Yangyang; Chan, Tim Hon Man; Yang, Henry; Lin, Chi Ho; Tay, Daryl Jin Tai; Hong, HuiQi; Tang, Sze Jing; Tan, Kar Tong; Huang, Xi Xiao; Lin, Jaymie Siqi; Ng, Vanessa Hui En; Maury, Julien Jean Pierre; Tenen, Daniel G; Chen, Leilei

    2017-10-13

    Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by Adenosine DeAminases acting on double-stranded RNA(dsRNA) (ADAR), occurs predominantly in the 3' untranslated regions (3'UTRs) of spliced mRNA. Here we uncover an unanticipated link between ADARs (ADAR1 and ADAR2) and the expression of target genes undergoing extensive 3'UTR editing. Using METTL7A (Methyltransferase Like 7A), a novel tumor suppressor gene with multiple editing sites at its 3'UTR, we demonstrate that its expression could be repressed by ADARs beyond their RNA editing and double-stranded RNA (dsRNA) binding functions. ADARs interact with Dicer to augment the processing of pre-miR-27a to mature miR-27a. Consequently, mature miR-27a targets the METTL7A 3'UTR to repress its expression level. In sum, our study unveils that the extensive 3'UTR editing of METTL7A is merely a footprint of ADAR binding, and there are a subset of target genes that are equivalently regulated by ADAR1 and ADAR2 through their non-canonical RNA editing and dsRNA binding-independent functions, albeit maybe less common. The functional significance of ADARs is much more diverse than previously appreciated and this gene regulatory function of ADARs is most likely to be of high biological importance beyond the best-studied editing function. This non-editing side of ADARs opens another door to target cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    International Nuclear Information System (INIS)

    Canales-Mayordomo, Angeles; Fayos, Rosa; Angulo, Jesus; Ojeda, Rafael; Martin-Pastor, Manuel; Nieto, Pedro M.; Martin-Lomas, Manuel; Lozano, Rosa; Gimenez-Gallego, Guillermo; Jimenez-Barbero, Jesus

    2006-01-01

    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)

  6. 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)

  7. A new mechanism to render clinical isolates of Escherichia coli non-susceptible to imipenem: substitutions in the PBP2 penicillin-binding domain.

    Science.gov (United States)

    Aissa, Nejla; Mayer, Noémie; Bert, Fréderic; Labia, Roger; Lozniewski, Alain; Nicolas-Chanoine, Marie-Hélène

    2016-01-01

    So far, two types of mechanism are known to be involved in carbapenem non-susceptibility of Escherichia coli clinical isolates: reduced outer membrane permeability associated with production of ESBLs and/or overproduction of class C β-lactamases; and production of carbapenemases. Non-susceptibility to only imipenem observed in two clinical isolates suggested a new mechanism, described in the present study. The ST was determined for the two isolates of E. coli (strains LSNy and VSBj), and their chromosomal region encoding the penicillin-binding domain of PBP2 was amplified, sequenced and then used for recombination experiments in E. coli K12 C600. Antibiotic MICs were determined using the Etest method. Strains LSNy and VSBj, which displayed ST23 and ST345, respectively, showed amino acid substitutions in their PBP2 penicillin-binding domain. Substitution Ala388Ser located in motif 2 (SXD) was common to the two strains. Two additional substitutions (Ala488Thr and Leu573Val) located outside the two other motifs were identified in strain LSNy, whereas another one (Thr331Pro) located in motif 1 was identified in strain VSBj. Recombination experiments to reproduce non-susceptibility to imipenem in E. coli K12 C600 were not successful when only the common substitution was transferred, whereas recombination with DNA fragments including either the three substitutions (strain LSNy) or the two substitutions (strain VSBj) were successful. Substitution of amino acids in the penicillin-binding domain of PBP2 is a new mechanism by which E. coli clinical isolates specifically resist imipenem. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. A peptide of heparin cofactor II inhibits endotoxin-mediated shock and invasive Pseudomonas aeruginosa infection.

    Directory of Open Access Journals (Sweden)

    Martina Kalle

    Full Text Available Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.

  9. An antimicrobial helix A-derived peptide of heparin cofactor II blocks endotoxin responses in vivo.

    Science.gov (United States)

    Papareddy, Praveen; Kalle, Martina; Singh, Shalini; Mörgelin, Matthias; Schmidtchen, Artur; Malmsten, Martin

    2014-05-01

    Host defense peptides are key components of the innate immune system, providing multi-facetted responses to invading pathogens. Here, we describe that the peptide GKS26 (GKSRIQRLNILNAKFAFNLYRVLKDQ), corresponding to the A domain of heparin cofactor II (HCII), ameliorates experimental septic shock. The peptide displays antimicrobial effects through direct membrane disruption, also at physiological salt concentration and in the presence of plasma and serum. Biophysical investigations of model lipid membranes showed the antimicrobial action of GKS26 to be mirrored by peptide incorporation into, and disordering of, bacterial lipid membranes. GKS26 furthermore binds extensively to bacterial lipopolysaccharide (LPS), as well as its endotoxic lipid A moiety, and displays potent anti-inflammatory effects, both in vitro and in vivo. Thus, for mice challenged with ip injection of LPS, GKS26 suppresses pro-inflammatory cytokines, reduces vascular leakage and infiltration in lung tissue, and normalizes coagulation. Together, these findings suggest that GKS26 may be of interest for further investigations as therapeutic against severe infections and septic shock. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. A peptide of heparin cofactor II inhibits endotoxin-mediated shock and invasive Pseudomonas aeruginosa infection.

    Science.gov (United States)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; van der Plas, Mariena J A; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

    2014-01-01

    Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII) has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.

  11. Indicine N-oxide binds to tubulin at a distinct site and inhibits the assembly of microtubules: a mechanism for its cytotoxic activity.

    Science.gov (United States)

    Appadurai, Prakash; Rathinasamy, Krishnan

    2014-02-10

    Indicine N-oxide, a pyrrolizidine alkaloid present in the plant Heliotropium indicum had shown promising cytotoxic activity in various tumor models. The compound exhibited severe toxicity to hepatocytes and bone marrow cells. The present work was aimed to evaluate the molecular mechanism of the toxicity of indicine N-oxide. We found that indicine N-oxide inhibited the proliferation of various cancer cell lines in a concentration dependent manner with IC50 ranging from 46 to 100 μM. At the half maximal inhibitory concentration it blocked the cell cycle progression at mitosis without significantly altering the organization of the spindle and interphase microtubules. The toxicities of the compound at higher concentrations are attributed to its severe depolymerizing effect on both the interphase and spindle microtubules. Binding studies using purified goat brain tubulin indicated that indicine N-oxide binds to tubulin at a distinct site not shared by colchicine or taxol. It decreased the polymer mass of both purified tubulin and MAP-rich tubulin. It was found to induce cleavage of DNA using pUC18 plasmid. The interactions of indicine N-oxide on DNA were also confirmed by computational analysis; which predicted its binding site at the minor groove of DNA. These studies bring to light that the toxicities of indicine N-oxide were due to its DNA damaging effects and depolymerization of microtubules. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. 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.

  13. Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

    Science.gov (United States)

    Liu, Mingming; Huang, Rong; Weisman, Adam; Yu, Xiaoyang; Lee, Shih-Hui; Chen, Yalu; Huang, Chao; Hu, Senhua; Chen, Xiuhua; Tan, Wenfeng; Liu, Fan; Chen, Hao; Shea, Kenneth J

    2018-05-24

    We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R 1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280 FRGSAQGIEGS 290 , 368 RRPFNIGINNQQ 379 and 436 FRSGFSNSSVSIIR 449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368 RRPFNIGINNQQ 379 . This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865 NITIHITDTNNK 876 , a specific recognition site in insect receptor Bt-R 1 , to 368 RRPFNIGINNQQ 379 . The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368 RRPFNIGINNQQ 379 . This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

  14. Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases*

    Science.gov (United States)

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H.; Allain, Fabrice

    2010-01-01

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells. PMID:19940140

  15. Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

    Science.gov (United States)

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H; Allain, Fabrice

    2010-01-15

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

  16. Quantitative determination of heparin levels in serum with microtiter plate-format optode

    International Nuclear Information System (INIS)

    Kim, Sung Bae; Kang, Tae Young; Cha, Geun Sig; Nam, Hakhyun

    2006-01-01

    A new assay method has been developed for the quantitative determination of heparin in serum using a microtiter plate-format optode (MPO). Heparin and proton in physiological sample are favorably co-extracted into the solvent polymeric optode membrane containing both cationic lipophilic additive, tridodecylmethyl ammonium chloride (TDMAC), and proton-selective ionophore, 3-hydroxy-4-(4-nitrophenylazo)-phenyloctadecanoate (ETH 2412), resulting in the absorbance change of the membrane to varying heparin levels. The optimized MPO composition contains low polymer-to-plasticizer ratio compared to those of conventional ion-selective optodes or electrodes, i.e., poly(vinyl chloride) (20.0)/dioctylsebacate (76.3)/ETH 2412 (1.7)/TDMAC (1.0) (wt.%): it resulted in a quantitative response to heparin from 0 to 15 unit/mL in serum with high sensitivity. The heparin-protamine titration on the MPO could provide rapid and precise determination of heparin. It was shown that the heparin levels in serum sample could be determined from the rate of absorbance change over time (ΔA/Δt); this method was more effective than the direct absorbance measurement in minimizing the interferences from color and turbidity of serum samples. MPO has been developed as a high throughput and convenient disposable sensing device, and may find a wide application in the determination of polyions and charged macromolecules

  17. Heparin modulates human intestinal smooth muscle (HISM) cell proliferation and matrix production

    International Nuclear Information System (INIS)

    Graham, M.; Perr, H.; Drucker, D.E.; Diegelmann, R.F.

    1986-01-01

    (HISM) cell proliferation and collagen production may play a role in the pathogenesis of intestinal stricture in Crohn's disease. The present studies were performed to evaluate the effects of heparin, a known modulator of vascular smooth muscle cells, on HISM cell proliferation and collagen production. Heparin (100 μg/ml) was added daily to HISM cell cultures for cell proliferation studies and for 24 hours at various time points during culture for collagen synthesis studies. Collagen synthesis was determined by the uptake of 3 H proline into collagenase-sensitive protein. Heparin completely inhibited cell proliferation for 7 days, after which cell numbers increased but at a slower rate than controls. Cells released from heparin inhibition demonstrated catch-up growth to control levels. Collagen production was significantly inhibited by 24 hours exposure to heparin but only at those times during culture when collagen synthesis was maximal (8 to 12 days). Non-collagen protein synthesis was inhibited by heparin at all time points during culture. Heparin through its modulation of HISM cells may play an important role in the control of the extracellular matrix of the intestinal wall

  18. Nerve growth factor loaded heparin/chitosan scaffolds for accelerating peripheral nerve regeneration.

    Science.gov (United States)

    Li, Guicai; Xiao, Qinzhi; Zhang, Luzhong; Zhao, Yahong; Yang, Yumin

    2017-09-01

    Artificial chitosan scaffolds have been widely investigated for peripheral nerve regeneration. However, the effect was not as good as that of autologous grafts and therefore could not meet the clinical requirement. In the present study, the nerve growth factor (NGF) loaded heparin/chitosan scaffolds were fabricated via electrostatic interaction for further improving nerve regeneration. The physicochemical properties including morphology, wettability and composition were measured. The heparin immobilization, NGF loading and release were quantitatively and qualitatively characterized, respectively. The effect of NGF loaded heparin/chitosan scaffolds on nerve regeneration was evaluated by Schwann cells culture for different periods. The results showed that the heparin immobilization and NGF loading did not cause the change of bulk properties of chitosan scaffolds except for morphology and wettability. The pre-immobilization of heparin in chitosan scaffolds could enhance the stability of subsequently loaded NGF. The NGF loaded heparin/chitosan scaffolds could obviously improve the attachment and proliferation of Schwann cells in vitro. More importantly, the NGF loaded heparin/chitosan scaffolds could effectively promote the morphology development of Schwann cells. The study may provide a useful experimental basis to design and develop artificial implants for peripheral nerve regeneration and other tissue regeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Chitosan-capped gold nanoparticles for selective and colorimetric sensing of heparin

    International Nuclear Information System (INIS)

    Chen, Zhanguang; Wang, Zhen; Chen, Xi; Xu, Haixiong; Liu, Jinbin

    2013-01-01

    In this contribution, novel chitosan-stabilized gold nanoparticles (AuNPs) were prepared by mixing chitosan with citrate-reductive AuNPs under appropriate conditions. The as-prepared chitosan-stabilized AuNPs were positively charged and highly stably dispersed in aqueous solution. They exhibited weak resonance light scattering (RLS) intensity and a wine red color. In addition, the chitosan-stabilized AuNPs were successfully utilized as novel sensitive probes for the detection of heparin for the first time. It was found that the addition of heparin induced a strong increase of RLS intensity for AuNPs and the color change from red to blue. The increase in RLS intensity and the color change of chitosan-stabilized AuNPs caused by heparin allowed the sensitive detection of heparin in the range of 0.2–60 μM (∼6.7 U/mL). The detection limit for heparin is 0.8 μM at a signal-to-noise ratio of 3. The present sensor for heparin detection possessed a low detection limit and wide linear range. Additionally, the proposed method was also applied to the detection of heparin in biological media with satisfactory results

  20. P-selectin- and heparanase-dependent antimetastatic activity of non-anticoagulant heparins.

    Science.gov (United States)

    Hostettler, Nina; Naggi, Annamaria; Torri, Giangiacomo; Ishai-Michaeli, Riva; Casu, Benito; Vlodavsky, Israel; Borsig, Lubor

    2007-11-01

    Vascular cell adhesion molecules, P- and L-selectins, facilitate metastasis of cancer cells in mice by mediating interactions with platelets, endothelium, and leukocytes. Heparanase is an endoglycosidase that degrades heparan sulfate of extracellular matrix, thereby promoting tumor invasion and metastasis. Heparin is known to efficiently attenuate metastasis in different tumor models. Here we identified modified, nonanticoagulant species of heparin that specifically inhibit selectin-mediated cell-cell interactions, heparanase enzymatic activity, or both. We show that selective inhibition of selectin interactions or heparanase with specific heparin derivatives in mouse models of MC-38 colon carcinoma and B16-BL6 melanoma attenuates metastasis. Selectin-specific heparin derivatives attenuated metastasis of MC-38 carcinoma, but heparanase-specific derivatives had no effect, in accordance with the virtual absence of heparanase activity in these cells. Heparin derivatives had no further effect on metastasis in mice deficient in P- and L-selectin, indicating that selectins are the primary targets of heparin antimetastatic activity. Selectin-specific and heparanase-specific derivatives attenuated metastasis of B16-BL6 melanomas to a similar extent. When mice were injected with a derivative containing both heparanase and selectin inhibitory activity, no additional attenuation of metastasis could be observed. Thus, selectin-specific heparin derivatives efficiently attenuated metastasis of both tumor cell types whereas inhibition of heparanase led to reduction of metastasis only in tumor cells producing heparanase.

  1. Characterization of currently marketed heparin products: key tests for LMWH quality assurance.

    Science.gov (United States)

    Ye, Hongping; Toby, Timothy K; Sommers, Cynthia D; Ghasriani, Houman; Trehy, Michael L; Ye, Wei; Kolinski, Richard E; Buhse, Lucinda F; Al-Hakim, Ali; Keire, David A

    2013-11-01

    During the 2007-2008 heparin crisis it was found that the United States Pharmacopeia (USP) testing monograph for heparin sodium or low molecular weight heparins did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS). In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to detect not only the contaminant OSCS, but also to improve assurance of quality and purity of these drug products. The USP monographs for the low molecular weight heparins (LMWHs) approved for use in the United States (dalteparin, tinzaparin and enoxaparin) are also undergoing revision to include many of the same tests used for heparin sodium, including; one-dimensional (1D) 500 MHz (1)H NMR, SAX-HPLC, percent galactosamine in total hexosamine and anticoagulation time assays with purified Factor IIa or Factor Xa. These tests represent orthogonal approaches for heparin identification, measurement of bioactivity and for detection of process impurities or contaminants in these drug products. Here we describe results from a survey of multiple lots from three types of LMWHs in the US market which were collected after the 2009 heparin sodium monograph revision. In addition, innovator and generic versions of formulated enoxaparin products purchased in 2011 are compared using these tests and found to be highly similar within the discriminating power of the assays applied. Published by Elsevier B.V.

  2. Chitosan-capped gold nanoparticles for selective and colorimetric sensing of heparin

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhanguang, E-mail: kqlu@stu.edu.cn; Wang, Zhen; Chen, Xi [Shantou University, Department of Chemistry (China); Xu, Haixiong [Shantou Central Hospital, Affiliated Shantou Hospital of SUN YAT-SEN University (China); Liu, Jinbin [University of Texasat Dallas, Department of Chemistry (United States)

    2013-09-15

    In this contribution, novel chitosan-stabilized gold nanoparticles (AuNPs) were prepared by mixing chitosan with citrate-reductive AuNPs under appropriate conditions. The as-prepared chitosan-stabilized AuNPs were positively charged and highly stably dispersed in aqueous solution. They exhibited weak resonance light scattering (RLS) intensity and a wine red color. In addition, the chitosan-stabilized AuNPs were successfully utilized as novel sensitive probes for the detection of heparin for the first time. It was found that the addition of heparin induced a strong increase of RLS intensity for AuNPs and the color change from red to blue. The increase in RLS intensity and the color change of chitosan-stabilized AuNPs caused by heparin allowed the sensitive detection of heparin in the range of 0.2-60 {mu}M ({approx}6.7 U/mL). The detection limit for heparin is 0.8 {mu}M at a signal-to-noise ratio of 3. The present sensor for heparin detection possessed a low detection limit and wide linear range. Additionally, the proposed method was also applied to the detection of heparin in biological media with satisfactory results.

  3. Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples.

    Science.gov (United States)

    Alban, Susanne; Lühn, Susanne; Schiemann, Simone; Beyer, Tanja; Norwig, Jochen; Schilling, Claudia; Rädler, Oliver; Wolf, Bernhard; Matz, Magnus; Baumann, Knut; Holzgrabe, Ulrike

    2011-01-01

    The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides (1)H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180 IU/mg. However, also more than 50% of the uncontaminated samples had interpretation of the results.

  4. Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides.

    Science.gov (United States)

    Singh, Shalini; Papareddy, Praveen; Kalle, Martina; Schmidtchen, Artur; Malmsten, Martin

    2013-11-01

    Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure-activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics. Copyright © 2013. Published by Elsevier B.V.

  5. Community nurse resource implications for a change in heparin prophylaxis policy

    Directory of Open Access Journals (Sweden)

    Parker Martyn J.

    2015-01-01

    Full Text Available Introduction: A review was undertaken for a consecutive series of hip fracture patients for the year before and then after a change in low dose heparin prophylaxis policy. Patients and methods: For the first year heparin was administered in hospital for a maximum of 14 days only. Patients sent home before this time were not discharged taking heparin. For the second year heparin was administered as recommended by NICE guidelines for 28 days from admission regardless of whether the patient was discharged. Results: For the first year 486 patients were treated with a mean of 10.4 doses of heparin per patient. For the second year 465 patients were treated with a mean of 24.3 doses per patient. In total an extra 6,464 doses of heparin were administered. 33.8% of patients were unable to administer their heparin at home therefore a district nurse administered 2,284 of these doses of subcutaneous heparin at the patient’s home. The increased cost associated with the change in policy was estimated to be £161 per patient, with over 90% of this increase being incurred by the district nurse expense. If applied nationally for the England, using extended heparin prophylaxis for hip fracture patients would cost in excess of 12 million pounds each year. Conclusion: Whilst the necessity for and duration of thromboembolic prophylaxis for these patients remains undetermined, there is a need to re-evaluate the cost effectiveness of the current recommendations for hip fracture patients.

  6. Heparin induced thrombocytopenia type ii and myocardial infarction: Two case reports

    Directory of Open Access Journals (Sweden)

    Antonijević Nebojša

    2004-01-01

    Full Text Available Heparin-induced thrombocytopenia (HIT type II is an acquired thrombophylic state and life-threatening immune complication of a heparin treatment mainly clinically manifested by marked thrombocytopenia, frequently by arterial and venous thrombosis, and sometimes by skin changes. Functional assay as heparin aggregation test and 14C-serotonin release assays are used in diagnostics as well as antigen assays of which detection tests for heparin-platelet factor 4 antibodies are most frequently used. Considering the fact that there is no single reliable assays for HIT II detection available, sometimes it is necessary to combine both of the above-mentioned types of assays. We present the case of a 57-year-old patient with an acute anterior myocardial infarction with cardiac insufficiency of III and IV degree according to Killip, recurrent ventricular fibrillation and diabetes mellitus type II developing thrombocytopenia to 37x10 9/l accompanied with typical skin changes. The diagnosis was confirmed by the heparin aggregation test. The second patient aged 70 undergoing the treatment for anteroseptal myocardial infarction and reinfarction of the inferior wall complicated by a cardiogenic shock and acute right bundle branch block developed thrombocytopenia 59x10 9/I on the third day of the heparin therapy, with the remark that he had received a heparin therapy during the first infarction as well. Antibodies against heparin-platelet factor 4 were detected by particle gel ID-HPF4 immunoassay. In both patients, the disease had a lethal outcome despite all then available therapeutic measures applied. Further on we discuss advantages of certain types of tests, a therapy doctrine, need for urgent therapeutic measures, inclusive of the administration of anitithrombins, avoidance of harmful procedures like low-molecular-weight heparins administration and prophylactic platelet transfusion as well as preventive measures.

  7. [Thrombocytopenia induced by type II heparin and myocardial infarct: 2 case reports].

    Science.gov (United States)

    Antonijević, Nabojsa; Stanojević, Milica; Perunicić, Jovan; Djokić, Milan; Miković, Danijla; Kovac, Mirjana; Miljić, Predrag; Milosević, Rajko; Terzić, Branka; Vasiljević, Zorana

    2004-01-01

    Heparin-induced thrombocytopenia (HIT) type II is an acquired thrombophylic state and life-threatening immune complication of a heparin treatment mainly clinically manifested by marked thrombocytopenia, frequently by arterial and venous thrombosis, and sometimes by skin changes. Functional assay as heparin aggregation test and 14C-serotonin release assays are used in diagnostics as well as antigen assays of which detection tests for heparin-platelet factor 4 antibodies are most frequently used. Considering the fact that there is no single reliable assays for HIT II detection available, sometimes it is necessary to combine both of the above-mentioned types of assays. We present the case of a 57-year-old patient with an acute anterior myocardial infarction with cardiac insufficiency of III and IV degree according to Killip, recurrent ventricular fibrillation and diabetes mellitus type II developing thrombocytopenia to 37 x 10(9)/l accompanied with typical skin changes. The diagnosis was confirmed by the heparin aggregation test. The second patient aged 70 undergoing the treatment for anteroseptal myocardial infarction and reinfarction of the inferior wall complicated by a cardiogenic shock and acute right bundle branch block developed thrombocytopenia 59 x 10(9)/l on the third day of the heparin therapy, with the remark that he had received a heparin therapy during the first infarction as well. Antibodies against heparin-platelet factor 4 were detected by particle gel ID-HPF4 immuno-assay. In both patients, the disease had a lethal outcome despite all then available therapeutic measures applied. Further on we discuss advantages of certain types of tests, a therapy doctrine, need for urgent therapeutic measures, inclusive of the administration of antithrombins, avoidance of harmful procedures like low-molecular-weight heparins administration and prophylactic platelet transfusion as well as preventive measures.

  8. On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

    International Nuclear Information System (INIS)

    Du, Z.; Boyer, P.D.

    1990-01-01

    Washed chloroplast thylakoid membranes upon exposure to [ 3 H]ADP retain in tightly bound [ 3 H]ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg 2+ results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound [ 3 H]ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg 2+ - and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22 degree C and of about 15 s at 37 degree C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg 2+ and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound [ 3 H]ADP parallels the onset of ATPase activity, although some [ 3 H]ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound [ 3 H]ADP being at a catalytic site and being replaced as this Mg 2+ - and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P i binding site of the enzyme-ADP-Mg 2+ complex to give a form more readily activated by ATP binding at an alternative site

  9. Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection.

    Science.gov (United States)

    Pernigo, Stefano; Fukuzawa, Atsushi; Beedle, Amy E M; Holt, Mark; Round, Adam; Pandini, Alessandro; Garcia-Manyes, Sergi; Gautel, Mathias; Steiner, Roberto A

    2017-01-03

    The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  10. Computational study on the inhibitor binding mode and allosteric regulation mechanism in hepatitis C virus NS3/4A protein.

    Directory of Open Access Journals (Sweden)

    Weiwei Xue

    Full Text Available HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state, while the truncated apo protein adopts an open conformation (active state. Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors.

  11. Structure and function of A41, a vaccinia virus chemokine binding protein.

    Directory of Open Access Journals (Sweden)

    Mohammad W Bahar

    2008-01-01

    Full Text Available The vaccinia virus (VACV A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI, and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM chemokine-chemokine receptor interactions.

  12. Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells.

    Science.gov (United States)

    Feyerabend, Thorsten B; Li, Jin-Ping; Lindahl, Ulf; Rodewald, Hans-Reimer

    2006-04-01

    Biosynthesis of heparin, a mast cell-derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) residues. We have generated Hsepi-null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O-sulfation pattern.

  13. 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

    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......). A reduced number of patients needed blood transfusions in the RxDx group, although this was not statistically significant (19% vs. 38%, respectively; p = .13). Initial heparin dose was significantly reduced in the RxDx group (250 mg; range, 100-375 mg) compared with the control group (300 mg; range, 200...

  14. Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates.

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    Full Text Available Heparan sulfate/heparin class of proteoglycans (HSPG have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9 and human parechovirus 1 (HPeV-1 are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54 CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9 had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%, heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.

  15. Recent developments in separation of low molecular weight heparin anticoagulants.

    Science.gov (United States)

    Sadowski, Radosław; Gadzała-Kopciuch, Renata; Buszewski, Bogusław

    2017-10-05

    The general function of anticoagulants is to prevent blood clotting and growing of the existing clots in blood vessels. In recent years, there has been a significant improvement in developing methods of prevention as well as pharmacologic and surgical treatment of thrombosis. For over the last two decades, low molecular weight heparins (LMWHs) have found their application in the antithrombotic diseases treatment. These types of drugs are widely used in clinical therapy. Despite the biological and medical importance of LMWHs, they have not been completely characterized in terms of their chemical structure. Due to both, the structural complexity of these anticoagulants and the presence of impurities, their structural characterization requires the employment of advanced analytical techniques. Since separation techniques play the key role in these endeavors, this review will focus on the presentation of recent developments in the separation of LMWH anticoagulants. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Hyaluronan- and heparin-reduced silver nanoparticles with antimicrobial properties

    Science.gov (United States)

    Kemp, Melissa M; Kumar, Ashavani; Clement, Dylan; Ajayan, Pulickel; Mousa, Shaker

    2009-01-01

    Aims Silver nanoparticles exhibit unique antibacterial properties that make these ideal candidates for biological and medical applications. We utilized a clean method involving a single synthetic step to prepare silver nanoparticles that exhibit antimicrobial activity. Materials & methods These nanoparticles were prepared by reducing silver nitrate with diaminopyridinylated heparin (DAPHP) and hyaluronan (HA) polysaccharides and tested for their efficacy in inhibiting microbial growth. Results & discussion The resulting silver nanoparticles exhibit potent antimicrobial activity against Staphylococcus aureus and modest activity against Escherichia coli. Silver–HA showed greater antimicrobial activity than silver–DAPHP, while silver–glucose nanoparticles exhibited very weak antimicrobial activity. Neither HA nor DAPHP showed activity against S. aureus or E. coli. Conclusion These results suggest that DAPHP and HA silver nanoparticles have potential in antimicrobial therapeutic applications. PMID:19505245

  17. Obstetric outcome with low molecular weight heparin therapy during pregnancy.

    LENUS (Irish Health Repository)

    Donnelly, J

    2012-01-01

    This was a prospective study of women attending a combined haematology\\/obstetric antenatal clinic in the National Maternity Hospital (2002-2008). Obstetric outcome in mothers treated with low molecular weight heparin (LMWH) was compared to the general obstetric population of 2006. There were 133 pregnancies in 105 women. 85 (63.9%) received prophylactic LMWH and 38 (28.6%) received therapeutic LMWH in pregnancy. 10 (7.5%) received postpartum prophylaxis only. The perinatal mortality rate was 7.6\\/1000 births. 14 (11.3%) women delivered preterm which is significantly higher than the hospital population rate (5.7%, p<0.05). Despite significantly higher labour induction rates (50% vs 29.2% p<0.01), there was no difference in CS rates compared to the general hospital population (15.4% vs 18.9%, NS). If carefully managed, these high-risk women can achieve similar vaginal delivery rates as the general obstetric population.

  18. Genome-Wide Progesterone Receptor Binding: Cell Type-Specific and Shared Mechanisms in T47D Breast Cancer Cells and Primary Leiomyoma Cells

    Science.gov (United States)

    Huang, Lei; Owen, Jonas K.; Xie, Anna; Navarro, Antonia; Monsivais, Diana; Coon V, John S.; Kim, J. Julie; Dai, Yang; Bulun, Serdar E.

    2012-01-01

    Background Progesterone, via its nuclear receptor (PR), exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma) and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. Principal Findings ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs), whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2), a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. Conclusions Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in uterine fibroids and

  19. Engineering the mechanical and biological properties of nanofibrous vascular grafts for in situ vascular tissue engineering.

    Science.gov (United States)

    Henry, Jeffrey J D; Yu, Jian; Wang, Aijun; Lee, Randall; Fang, Jun; Li, Song

    2017-08-17

    Synthetic small diameter vascular grafts have a high failure rate, and endothelialization is critical for preventing thrombosis and graft occlusion. A promising approach is in situ tissue engineering, whereby an acellular scaffold is implanted and provides stimulatory cues to guide the in situ remodeling into a functional blood vessel. An ideal scaffold should have sufficient binding sites for biomolecule immobilization and a mechanical property similar to native tissue. Here we developed a novel method to blend low molecular weight (LMW) elastic polymer during electrospinning process to increase conjugation sites and to improve the mechanical property of vascular grafts. LMW elastic polymer improved the elasticity of the scaffolds, and significantly increased the amount of heparin conjugated to the micro/nanofibrous scaffolds, which in turn increased the loading capacity of vascular endothelial growth factor (VEGF) and prolonged the release of VEGF. Vascular grafts were implanted into the carotid artery of rats to evaluate the in vivo performance. VEGF treatment significantly enhanced endothelium formation and the overall patency of vascular grafts. Heparin coating also increased cell infiltration into the electrospun grafts, thus increasing the production of collagen and elastin within the graft wall. This work demonstrates that LMW elastic polymer blending is an approach to engineer the mechanical and biological property of micro/nanofibrous vascular grafts for in situ vascular tissue engineering.

  20. On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

    1990-01-16

    Washed chloroplast thylakoid membranes upon exposure to ({sup 3}H)ADP retain in tightly bound ({sup 3}H)ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg{sup 2+} results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound ({sup 3}H)ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg{sup 2+}- and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22{degree}C and of about 15 s at 37{degree}C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg{sup 2+} and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound ({sup 3}H)ADP parallels the onset of ATPase activity, although some ({sup 3}H)ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound ({sup 3}H)ADP being at a catalytic site and being replaced as this Mg{sup 2+}- and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P{sub i} binding site of the enzyme-ADP-Mg{sup 2+} complex to give a form more readily activated by ATP binding at an alternative site.

  1. Mechanism of Coomassie Brilliant Blue G-250 binding to cetyltrimethylammonium bromide: an interference with the Bradford assay.

    Science.gov (United States)

    Aminian, Mahdi; Nabatchian, Fariba; Vaisi-Raygani, Asad; Torabi, Mojgan

    2013-03-15

    The Bradford protein assay is a popular method because of its rapidity, sensitivity, and relative specificity. This method is subject to some interference by nonprotein compounds. In this study, we describe the interference of cetyltrimethylammonium bromide (CTAB) with the Bradford assay. This interference is based on the interaction of Coomassie Brilliant Blue G-250 (CBB) with this cationic detergent. This study suggests that both electrostatic and hydrophobic interactions are involved in the interaction of CTAB and CBB. The anionic and neutral forms of CBB bind to CTAB by electrostatic attraction, which accelerates hydrophobic interactions of these CBB forms and the hydrophobic tail of CTAB. Consequently, the hydrophobic regions of the dominant free cationic form of CBB dye compete for the tail of CTAB with two other forms of the dye and gradually displace the primary hydrophobic interactions and rearrange the primary CBB-CTAB complex. This interaction of CTAB and CBB dye produces a primary 650-nm-absorbing complex that then gradually rearranges to a complex that shows an absorbance shoulder at 800-950 nm. This study conclusively shows a strong response of CBB to CTAB that causes a time-dependent and nearly additive interference with the Bradford assay. This study also may promote an application of CBB for CTAB quantification. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Forecast of actin-binding proteins as the oncotarget in osteosarcoma - a review of mechanism, diagnosis and therapy.

    Science.gov (United States)

    Fu, Yucheng; Yu, Wei; Cai, Hongliu; Lu, Anwei

    2018-01-01

    Osteosarcoma (OS) is the most common bone malignant tumor with a high rate of lung metastasis and principally emerges in children and adolescents. Although neoadjuvant chemotherapy is widely used around the world, a high rate of chemoresistance occurs and frequently generates a poor prognosis. Therefore, finding a new appropriate prognostic marker for OS is a valuable research direction, which will give patients a better chance to receive proper therapy. Actin-binding proteins (ABPs) are a group of proteins that interact with actin cytoskeleton and play a crucial role in the regulation of the cell motility and morphology in eukaryotes. Meanwhile, ABPs also act as a bridge between the cytomembrane and nucleus, which transmit the outside-in and inside-out signals in cytoplasm. Furthermore, ABPs alter the dynamic structure of actin and regulate the invasion and metastasis of cancer. Hence, ABPs have a wide application in predicting the prognosis, and may be new targets, in tumor therapy. This review focuses on a series of ABPs and discusses their modulatory functions. It provides a new insight into the classification of ABPs' functions in the process of invasion and metastasis in OS and illuminates the potential ability in predicting the prognosis of OS patients.

  3. A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase

    NARCIS (Netherlands)

    Wijma, HJ; Jeuken, LJC; Verbeet, MP; Armstrong, FA; Canters, GW

    2006-01-01

    The homotrimeric copper-containing nitrite reductase ( NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of

  4. Qualitative and quantitative analysis of heparin and low molecular weight heparins using size exclusion chromatography with multiple angle laser scattering/refractive index and inductively coupled plasma/mass spectrometry detectors.

    Science.gov (United States)

    Ouyang, Yilan; Zeng, Yangyang; Yi, Lin; Tang, Hong; Li, Duxin; Linhardt, Robert J; Zhang, Zhenqing

    2017-11-03

    Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. Low molecular weight heparins (LMWHs), heparins partially depolymerized using different processes, are widely used as clinical anticoagulants. Qualitative molecular weight (MW) and quantitative mass content analysis are two important factors that contribute to LMWH quality control. Size exclusion chromatography (SEC), relying on multiple angle laser scattering (MALS)/refractive index (RI) detectors, has been developed for accurate analysis of heparin MW in the absence of standards. However, the cations, which ion-pair with the anionic polysaccharide chains of heparin and LMWHs, had not been considered in previous reports. In this study, SEC with MALS/RI and inductively coupled plasma/mass spectrometry detectors were used in a comprehensive analytical approach taking both anionic polysaccharide and ion-paired cations heparin products. This approach was also applied to quantitative analysis of heparin and LMWHs. Full profiles of MWs and mass recoveries for three commercial heparin/LMWH products, heparin sodium, enoxaparin sodium and nadroparin calcium, were obtained and all showed higher MWs than previously reported. This important improvement more precisely characterized the MW properties of heparin/LMWHs and potentially many other anionic polysaccharides. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Mechanisms of pancreatic beta-cell growth and regeneration

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    1989-01-01

    Information about the mechanism of beta-cell growth and regeneration may be obtained by studies of insulinoma cells. In the present study the growth and function of the rat insulinoma cell lines RINm5F and 5AH were evaluated by addition of serum, hormones, and growth factors. It was found...... of insulin mRNA content showed that the insulinoma cells only contained about 2% of that of normal rat beta-cells. These results are discussed in relation to the role of growth factors, oncogenes, and differentiation in the growth and regeneration of beta-cells....... that transferrin is the only obligatory factor whereas growth hormone, epidermal growth factor, fibroblast growth factor, and TRH had modulating effects. A heat-labile heparin binding serum factor which stimulated thymidine incorporation but not cell proliferation was demonstrated in human serum. Measurements...

  6. Effects of Sildenafil Citrate and Heparin Treatments on Placental Cell Morphology in a Murine Model of Pregnancy Loss.

    Science.gov (United States)

    Luna, Rayana Leal; Vasconcelos, Anne Gabrielle; Nunes, Ana Karolina Santana; de Oliveira, Wilma Helena; Barbosa, Karla Patricia de Sousa; Peixoto, Christina Alves

    2016-01-01

    Lipopolysaccharide (LPS) injections during pregnancy are well established as models for pregnancy complications, including fetal growth restriction (FGR), thrombophilia, preterm labor and abortion. Indeed, inflammation, as induced by LPS injection has been described as a pivotal factor in cases of miscarriage related to placental tissue damage. The phosphodiesterase-5 inhibitor sildenafil (Viagra®) is currently used to treat FGR cases in women, while low-molecular weight heparin (Fragmin®) is a standard treatment for recurrent miscarriage (RM). However, the pathways and cellular dynamics involved in RM are not completely understood. The aim of this study was to evaluate the protective effect of sildenafil and dalteparin in a mouse model of LPS-induced abortion. Histopathology, ultrastructural analysis and immunofluorescence for P-selectin were studied in two different placental cell types: trophoblast cells and labyrinth endothelial cells. Treatment with sildenafil either alone or in combination with heparin showed the best response against LPS-induced injury during pregnancy. In conclusion, our results support the use of these drugs as future therapeutic agents that may protect the placenta against inflammatory injury in RM events. Analyses of the ultrastructure and placental immunophysiology are important to understand the mechanism underlying RM. These findings may spark future studies and aid in the development of new therapies in cases of RM. © 2016 S. Karger AG, Basel.

  7. Preparation of tin -heparin complex to be applied for myocardial infarct diagnosis

    International Nuclear Information System (INIS)

    Badi, J. M.; Al-Azzawi, H. A.; Resen, H. M.; Abed, I. G.; Owiad, H.; Manji, A. N.

    2012-12-01

    Tin-heparin complex has been prepared (liquid form) to be labeled with technetium-99 can be applied for diagnosis of myocardial infarcts vascular diseases and deep vein thrombosis. The preparation contents are 0.1mg tin chloride dehydrate and 1250 1.U of heparin. The results of the pH effect on the labeling yield indicated that high percentage of labeling yield (96.1%) was obtained in the optimal pH (5.50). The obtained results showed that the quantity of reducing agent (tin chloride dehydrate) and chelating agent (heparin) has no effect on the labeling yield. Results of radio analytical studies by paper chromatography technique wear confirmed by data obtained by Gel chromatography column scanning techniques. These techniques showed the high labeling yield of the tin-heparin complex. The persistence of high labeling yield for 8 hours is a good indication for its stability and efficiency for radio diagnosis examination in nuclear medicine centers. (Author)

  8. Distribution of heparin-/sup 67/Ga in tumor-bearing rats

    Energy Technology Data Exchange (ETDEWEB)

    Hiraki, T; Ando, A; Sanada, S [Kanazawa Univ. (Japan). School of Paramedicine; Ando, I; Hisada, K

    1976-06-01

    Heparin is a kind of acidic mucopolysaccharide. The distribution of heparin-/sup 67/Ga complex in tumor-bearing rats was investigated by administering it to rats into which Yoshida sarcoma had been transplanted subcutaneously. These rats were sacrificed at 10 minutes, 30 minutes, 1 hour and 3 hours after injection. Radioactivity of the tumor, blood, muscle, liver, kidney, spleen and urine were measured with a well-type scintillation counter. Retention values in these organs and excretion rates in the urine were calculated. Excretion rates (%/dose) of heparin-/sup 67/Ga in 10 min., 30 min., 1 hour, and 3 hours were 38.2%, 67.5%, 79.5% and 78.0%, respectively. From these facts, it was thought that heparin-/sup 67/Ga complex was not suitable for tumor scanning, but that this compound might be a suitable agent for the renal function test.

  9. Bilateral adrenal haemorrhage associated with heparin-induced thrombocytopaenia during treatment of Fournier gangrene.

    Science.gov (United States)

    Tattersall, Timothy Lee; Thangasamy, Isaac A; Reynolds, Jamie

    2014-10-14

    We present a case of bilateral adrenal haemorrhage (BAH) associated with heparin-induced thrombocytopaenia (HIT) in a 61-year-old man admitted to hospital for the treatment of Fournier's gangrene. He presented to hospital with scrotal swelling and fever, and developed spreading erythaema and a gangrenous scrotum. His scrotum was surgically debrided and intravenous broad-spectrum antibiotics were administered. Unfractionated heparin was given postoperatively for venous thromboembolism prophylaxis. The patient deteriorated clinically 8-11 days postoperatively with delirium, chest pain and severe hypertension followed by hypotension and thrombocytopaenia. Abdominal CT scan revealed bilateral adrenal haemorrhage. Antibodies to the heparin-platelet factor 4 complex were present. HIT-associated BAH was diagnosed and heparin was discontinued. Intravenous bivalirudin and hydrocortisone were started, with rapid improvement in clinical status. BAH is a rare complication of HIT and should be considered in the postoperative patient with unexplained clinical deterioration. 2014 BMJ Publishing Group Ltd.

  10. Docking based 3d-QSAR studies applied at the BRAF inhibitors to understand the binding mechanism

    International Nuclear Information System (INIS)

    Mahmood, U.; Haq, Z.U.

    2011-01-01

    BRAF is a great therapeutic target in a wide variety of human cancers. It is the member of Ras Activating Factor (RAF) family of serine/throenine kinase. The mutated form of the BRAF has diverted all the attention towards itself because of increase severity and elevated kinase activity. The RAF signal transduction cascade is a conserved protein pathway that is involved in cell cycle progression and apoptosis. The ERK regulates phosphorylation of different proteins either in cytosol or in nucleus but disorders in ERK signaling pathway cause mutation in BRAF. This cascade in these cells may provide sel