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Sample records for p-selectin sialylated ligands

  1. CD24, a mucin-type glycoprotein, is a ligand for P-selectin on human tumor cells.

    Science.gov (United States)

    Aigner, S; Sthoeger, Z M; Fogel, M; Weber, E; Zarn, J; Ruppert, M; Zeller, Y; Vestweber, D; Stahel, R; Sammar, M; Altevogt, P

    1997-05-01

    P-selectin (CD62P) is a Ca2+-dependent endogenous lectin that can be expressed by vascular endothelium and platelets. The major ligand for P-selectin on leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). P-selectin can also bind to carcinoma cells, but the nature of the ligand(s) on these cells is unknown. Here we investigated the P-selectin binding to a breast and a small cell lung carcinoma cell line that are negative for PSGL-1. We report that CD24, a mucin-type glycosylphosphatidylinositol-linked cell surface molecule on human neutrophils, pre B lymphocytes, and many tumors can promote binding to P-selectin. Latex beads coated with purified CD24 from the two carcinoma cell lines but also neutrophils could bind specifically to P-selectin-IgG. The binding was dependent on divalent cations and was abolished by treatment with O-sialoglycoprotein endopeptidase but not endoglycosidase F or sialidase. The beads were stained with a monoclonal antibody (MoAb) to CD57 (HNK-1 carbohydrate epitope) but did not react with MoAbs against the sialylLe(x/a) epitope. The carcinoma cells and CD24-beads derived from these cells could bind to activated platelets or P-selectin transfected Chinese hamster ovary cells (P-CHO) in a P-selectin-dependent manner and this binding was blocked by soluble CD24. Transfection of human adenocarcinoma cells with CD24 enhanced the P-selectin-dependent binding to activated platelets. Treatment of the carcinoma cells or the CD24 transfectant with phosphatidylinositol-specific phospholipase C reduced CD24 expression and P-selectin-IgG binding concomitantly. These results establish a role of CD24 as a novel ligand for P-selectin on tumor cells. The CD24/P-selectin binding pathway could be important in the dissimination of tumor cells by facilitating the interaction with platelets or endothelial cells.

  2. P-selectin glycoprotein ligand-1 in T cells.

    Science.gov (United States)

    Abadier, Michael; Ley, Klaus

    2017-05-01

    We review P-selectin glycoprotein ligand-1 (PSGL-1) as a selectin and chemokine-binding adhesion molecule. PSGL-1 is widely studied in neutrophils. Here, we focus on T cells, because PSGL-1 was recently described as a major immunomodulatory molecule during viral infection. PSGL-1 also plays a crucial role in T-cell homeostasis by binding to lymphoid chemokines, and can induce tolerance by enhancing the functions of regulatory T cells. PSGL-1 was originally described as a leukocyte ligand for P-selectin, but it is actually a ligand for all selectins (P-, L- and E-selectin), binds chemokines, activates integrins and profoundly affects T-cell biology. It has been shown recently that PSGL-1 can modulate T cells during viral infection by acting as a negative regulator for T-cell functions. Absence of PSGL-1 promotes effector CD4 and CD8 T-cell differentiation and prevents T-cell exhaustion. Consistent with this, tumor growth was significantly reduced in PSGL-1-deficient mice because of an enhanced number of effector T cells together with reduced levels of inhibitory receptors that induce T-cell exhaustion. PSGL-1 is the best-studied selectin ligand and has become a posterchild of versatility in leukocyte adhesion, inflammation and immunology. The direct involvement of PSGL-1 in T-cell biology suggests that it might be a drug target. Indeed, PSGL-1 has been tested in some clinical trials and recently, PSGL-1 blockers were proposed as a potential cotherapy in cancer immunotherapy.

  3. Bromelain decreases neutrophil interactions with P-selectin, but not E-selectin, in vitro by proteolytic cleavage of P-selectin glycoprotein ligand-1.

    Directory of Open Access Journals (Sweden)

    Jessica M Banks

    Full Text Available Stem bromelain, a cysteine protease isolated from pineapples, is a natural anti-inflammatory treatment, yet its mechanism of action remains unclear. Curious as to whether bromelain might affect selectin-mediated leukocyte rolling, we studied the ability of bromelain-treated human neutrophils to tether to substrates presenting immobilized P-selectin or E-selectin under shear stress. Bromelain treatment attenuated P-selectin-mediated tethering but had no effect on neutrophil recruitment on E-selectin substrates. Flow cytometric analysis of human neutrophils, using two antibodies against distinct epitopes within the P-selectin glycoprotein ligand-1 (PSGL-1 active site, revealed that bromelain cleaves PSGL-1 to remove one of two sites required for P-selectin binding, while leaving the region required for E-selectin binding intact. These findings suggest one molecular mechanism by which bromelain may exert its anti-inflammatory effects is via selective cleavage of PSGL-1 to reduce P-selectin-mediated neutrophil recruitment.

  4. Bromelain decreases neutrophil interactions with P-selectin, but not E-selectin, in vitro by proteolytic cleavage of P-selectin glycoprotein ligand-1.

    Science.gov (United States)

    Banks, Jessica M; Herman, Christine T; Bailey, Ryan C

    2013-01-01

    Stem bromelain, a cysteine protease isolated from pineapples, is a natural anti-inflammatory treatment, yet its mechanism of action remains unclear. Curious as to whether bromelain might affect selectin-mediated leukocyte rolling, we studied the ability of bromelain-treated human neutrophils to tether to substrates presenting immobilized P-selectin or E-selectin under shear stress. Bromelain treatment attenuated P-selectin-mediated tethering but had no effect on neutrophil recruitment on E-selectin substrates. Flow cytometric analysis of human neutrophils, using two antibodies against distinct epitopes within the P-selectin glycoprotein ligand-1 (PSGL-1) active site, revealed that bromelain cleaves PSGL-1 to remove one of two sites required for P-selectin binding, while leaving the region required for E-selectin binding intact. These findings suggest one molecular mechanism by which bromelain may exert its anti-inflammatory effects is via selective cleavage of PSGL-1 to reduce P-selectin-mediated neutrophil recruitment.

  5. Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function

    Directory of Open Access Journals (Sweden)

    Schapira Marc

    2007-09-01

    Full Text Available Abstract Background P-selectin glycoprotein ligand-1 (PSGL-1 plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog and examined mammalian PSGL-1 interactions with human selectins. Results A signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250–280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR. Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P-selectin

  6. The use of surface immobilization of P-selectin glycoprotein ligand-1 on mesenchymal stem cells to facilitate selectin mediated cell tethering and rolling

    Science.gov (United States)

    Lo, Chi Y.; Antonopoulos, Aristotelis; Dell, Anne; Haslam, Stuart M.; Lee, Techung; Neelamegham, Sriram

    2013-01-01

    Mesenchymal stem/stromal cells (MSCs) are an important candidate for cell-based therapy since they can be easily isolated and expanded, secrete beneficial paracrine factors, and differentiate into multiple lineages. Since the endothelium at sites of injury and inflammation often express adhesion molecules belonging to the selectin family, methods to endow MSCs with selectin-ligands can enhance the efficacy of cell delivery and tissue engraftment. Here, we describe a construct 19Fc[FUT7+], where the first 19 amino acids of the pan-selectin ligand PSGL-1 (P-selectin glycoprotein ligand-1) was fused to a human IgG1. When expressed in HEK293T cells over-expressing the α(1,3)fucosyltransferase FUT7, 19Fc[FUT7+] is decorated by a core-2 sialyl Lewis-X sialofucosylated O-glycan. The non-covalent coupling of this protein onto MSC surface using palmitated protein G (PPG) enhanced cell binding to E- and P-selectin under hydrodynamic shear, without altering MSC multipotency. MSCs functionalized with 19Fc[FUT7+] were captured/tethered onto stimulated endothelial cell monolayers at wall shear stresses up to 4 dyn/cm2. Once captured, the cells rolled robustly up to the highest shear stress tested, 10 dyn/cm2. Unlike previous work where MSCs could only be captured onto selectin-bearing substrates at low or no-flow conditions, the current work presents a ‘glycan engineering’ strategy to enable leukocyte-like capture and rolling. PMID:23891082

  7. Completely enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Zeng, S.; Gutiérrez Gallego, R.; Dinter, A.; Malissard, M.; Kamerling, J.P.; Berger, E.G.

    1999-01-01

    Sialyl Lewis x (sLex) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLex-oligosaccharide

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

    Science.gov (United States)

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

    2009-02-01

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

  9. Redistribution of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated neutrophils: a role of lipid microdomains.

    Science.gov (United States)

    Itoh, Saotomo; Susuki, Chie; Takeshita, Kana; Nagata, Kisaburo; Tsuji, Tsutomu

    2007-06-01

    P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like cell adhesion molecule expressed on leukocyte plasma membranes and involved in platelet-leukocyte and endothelium-leukocyte interactions. The treatment of neutrophils with a low concentration of IL-8 induced the redistribution of PSGL-1 to one end of the cell to form a cap-like structure. We investigated the role of lipid microdomains in the redistribution of PSGL-1 and its effect on the adhesive characteristics of IL-8-treated neutrophils. The redistribution of PSGL-1 induced by IL-8 was inhibited by cholesterol-perturbing agents such as methyl-beta-cyclodextrin and filipin. Sucrose density gradient centrifugation analysis revealed that PSGL-1 was enriched in a low-density fraction together with the GM1 ganglioside after solubilization of the cell membranes with a nonionic detergent, Brij 58. However, when Triton X-100 was used for the solubilization, PSGL-1 was no longer recovered in the low-density fraction, although GM1 ganglioside remained in the low-density fraction. Furthermore, immunofluorescence microscopic observation demonstrated that the localization of PSGL-1 differed from that of GM1 ganglioside, suggesting that PSGL-1 is associated with a microdomain distinct from that containing the GM1 ganglioside. Treatment of neutrophils with IL-8 increased the formation of microaggregates composed of neutrophils and activated platelets, and this treatment also enhanced reactive oxygen species production in neutrophils induced by the cross-linking of PSGL-1 with antibodies. These results suggest that the association of PSGL-1 with lipid microdomains is essential for its redistribution induced by IL-8 stimulation and that the redistribution modulates neutrophil functions mediated by interactions with P-selectin.

  10. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

    KAUST Repository

    Abu Samra, Dina Bashir Kamil

    2015-06-29

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Protective effects of oligosaccharides in P-selectin-dependent lung injury.

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    Mulligan, M S; Paulson, J C; De Frees, S; Zheng, Z L; Lowe, J B; Ward, P A

    1993-07-08

    Neutrophil recruitment into tissues is a multistep process involving sequential engagement of adhesion molecules, including selectins (E,P,L), which are reactive with oligosaccharides, and the family of beta 2 integrins which are reactive with endothelial intercellular adhesion molecules. These processes result in the initial rolling of leukocytes along the endothelial surfaces, followed by the firm attachment of leukocytes to the endothelium. The intravenous infusion of cobra venom factor into rats results in acute lung injury that is neutrophil-dependent, oxygen radical mediated and P-selectin-dependent. Here we report that infusion of sialyl-Lewis X, a ligand for P-selectin, dramatically reduced lung injury and diminished the tissue accumulation of neutrophils, whereas irrelevant oligosaccharides had no such effects. These results suggest that sialyl-Lewis X carbohydrates may be used as a new strategy for anti-inflammatory therapy.

  12. An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

    Directory of Open Access Journals (Sweden)

    Amal J. Ali

    2017-05-01

    Full Text Available Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1 and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL. Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

  13. An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

    KAUST Repository

    Ali, Amal J.

    2017-05-03

    Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL). Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

  14. Development of an autoimmune syndrome affecting the skin and internal organs in P-selectin glycoprotein ligand 1 leukocyte receptor-deficient mice.

    Science.gov (United States)

    Pérez-Frías, A; González-Tajuelo, R; Núñez-Andrade, N; Tejedor, R; García-Blanco, M J; Vicente-Rabaneda, E; Castañeda, S; Gamallo, C; Silván, J; Esteban-Villafruela, A; Cubero-Rueda, L; García-García, C; Muñoz-Calleja, C; García-Diez, A; Urzainqui, A

    2014-11-01

    To define and characterize the progression of the spontaneous autoimmune disease that develops in mice in the absence of the leukocyte adhesion receptor P-selectin glycoprotein ligand 1 (PSGL-1). Skin-resident immune cells from PSGL-1-deficient mice and C57BL/6 control mice of different ages were isolated and analyzed by flow cytometry. Biochemical parameters were analyzed in mouse serum and urine, and the presence of serum autoantibodies was investigated. Skin and internal organs were extracted, and their structure was analyzed histologically. Skin-resident innate and adaptive immune cells from PSGL-1(-/-) mice had a proinflammatory phenotype with an imbalanced T effector cell:Treg cell ratio. Sera from PSGL-1(-/-) mice had circulating autoantibodies commonly detected in connective tissue-related human autoimmune diseases. Biochemical and histologic analysis of skin and internal organs revealed skin fibrosis and structural and functional abnormalities in the lungs and kidneys. Furthermore, PSGL-1(-/-) mice exhibited vascular alterations, showing loss of dermal vessels, small vessel medial layer remodeling in the lungs and kidneys, and ischemic processes in the kidney that promote renal infarcts. Our study demonstrates that immune system overactivation due to PSGL-1 deficiency triggers an autoimmune syndrome with characteristics similar to systemic sclerosis, including skin fibrosis, vascular alterations, and systemic organ involvement. These results suggest that PSGL-1 expression contributes to the maintenance of the homeostasis of the immune system and could act as a barrier for autoimmunity in mice. Copyright © 2014 by the American College of Rheumatology.

  15. Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules.

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    Asako, H; Kurose, I; Wolf, R; DeFrees, S; Zheng, Z L; Phillips, M L; Paulson, J C; Granger, D N

    1994-04-01

    The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.

  16. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets.

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    Palabrica, T; Lobb, R; Furie, B C; Aronovitz, M; Benjamin, C; Hsu, Y M; Sajer, S A; Furie, B

    1992-10-29

    The glycoprotein P-selectin is a cell adhesion molecule of stimulated platelets and endothelial cells, which mediates the interaction of these cells with neutrophils and monocytes. It is a membrane component of cell storage granules, and is a member of the selectin family which includes E-selectin and L-selectin. P-selectin recognizes both lineage-specific carbohydrate ligands on monocytes and neutrophils, including the Lewis x antigen, sialic acid, and a protein component. In inflammation and thrombosis, P-selectin may mediate the interaction of leukocytes with platelets bound in the region of tissue injury and with stimulated endothelium. To evaluate the role of P-selectin in platelet-leukocyte adhesion in vivo, the accumulation of leukocytes within an experimental thrombus was explored in an arteriovenous shunt model in baboons. A Dacron graft implanted within an arteriovenous shunt is thrombogenic, accumulating platelets and fibrin within its lumen. These bound platelets express P-selectin. Here we show that antibody inhibition of leukocyte binding to P-selectin expressed on platelets immobilized on the graft blocks leukocyte accumulation and inhibits the deposition of fibrin within the thrombus. These results indicate that P-selectin is an important adhesion molecule on platelets, mediating platelet-leukocyte binding in vivo, that the presence of leukocytes in thrombi is mediated by P-selectin, and that these leukocytes promote fibrin deposition.

  17. Differential localization of P-selectin and von Willebrand factor during megakaryocyte maturation

    DEFF Research Database (Denmark)

    Zingariello, M; Fabucci, M E; Bosco, D

    2010-01-01

    Willebrand factor are two proteins present in the alpha-granules that recognize P-selectin glycoprotein ligand on neutrophils and collagen in the subendothelial matrix. These proteins may play an important role in determining the differential release of the alpha-granule contents in response to external....... These observations support the hypothesis that P-selectin and von Willebrand factor may ensure differential release of the alpha-granule content in response to external stimuli....

  18. Targeting P-Selectin by Gallium-68–Labeled Fucoidan Positron Emission Tomography for Noninvasive Characterization of Vulnerable Plaques: Correlation With In Vivo 17.6T MRI

    National Research Council Canada - National Science Library

    Li, Xiang; Bauer, Wolfgang; Israel, Ina; Kreissl, Michael C; Weirather, Johannes; Richter, Dominik; Bauer, Elisabeth; Herold, Volker; Jakob, Peter; Buck, Andreas; Frantz, Stefan; Samnick, Samuel

    2014-01-01

    .... We proposed a new approach for noninvasive in vivo characterization of P-selectin on active plaques based on Ga-Fucoidan, which is a polysaccharidic ligand of P-selectin with a nanomolar affinity...

  19. Differing patterns of P-selectin expression in lung injury

    DEFF Research Database (Denmark)

    Bless, N M; Tojo, S J; Kawarai, H

    1998-01-01

    -selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of 125I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression...

  20. Peptides from multiple regions of the lectin domain of P-selectin inhibiting neutrophil adhesion.

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    Heavner, G A; Falcone, M; Kruszynski, M; Epps, L; Mervic, M; Riexinger, D; McEver, R P

    1993-11-01

    The selectins are a family of three structurally related glycoproteins that are integral components of leukocyte adhesion to the vascular endothelium. Their involvement in the recruitment and extravasation of neutrophils is critical in mounting an inflammatory reaction. The carbohydrate nature of the selectin ligands suggests that the binding regions of the selectins are contained within the lectin-like domains of the selectins. The synthesis and evaluation for inhibition of selectin binding of overlapping peptides of the lectin and adjacent EGF-like domains of P-selectin have been used to identify small peptides that completely inhibit P-selectin-dependent neutrophil adhesion. These peptides span a region of more than 100 amino acids and may define the carbohydrate recognition domain of P-selectin.

  1. Differing Patterns of P-Selectin Expression in Lung Injury

    Science.gov (United States)

    Bless, Nicolas M.; Tojo, Shinichiro J.; Kawarai, Hiroko; Natsume, Yasuhiro; Lentsch, Alex B.; Padgaonkar, Vaishalee A.; Czermak, Boris J.; Schmal, Hagen; Friedl, Hans P.; Ward, Peter A.

    1998-01-01

    Using two models of acute lung inflammatory injury in rats (intrapulmonary deposition of immunoglobulin G immune complexes and systemic activation of complement after infusion of purified cobra venom factor), we have analyzed the requirements and patterns for upregulation of lung vascular P-selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of 125I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression was sustained for the next 7 hours, in striking contrast to the pattern of P-selectin expression in the cobra venom factor model, in which upregulation was very transient (within the 1st hour). In the immune complex model, injury and neutrophil accumulation were P-selectin dependent. Upregulation of P-selectin was dependent on an intact complement system, and the presence of blood neutrophils was susceptible to the antioxidant dimethyl sulfoxide and required C5a but not tumor necrosis factor α. In contrast, in the cobra venom factor model, upregulation of P-selectin, which is C5a dependent, was also dimethyl sulfoxide sensitive but neutrophil independent. Different mechanisms that may explain why upregulation of lung vascular P-selectin is either transient or sustained are discussed. PMID:9777942

  2. Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo.

    Directory of Open Access Journals (Sweden)

    Andrew D Westmuckett

    Full Text Available We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1 is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST activity.Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6 or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6 which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.

  3. Selectin ligands and tumor-associated carbohydrate structures: specificities of alpha 2,3-sialyltransferases in the assembly of 3'-sialyl-6-sialyl/sulfo Lewis a and x, 3'-sialyl-6'-sulfo Lewis x, and 3'-sialyl-6-sialyl/sulfo blood group T-hapten.

    Science.gov (United States)

    Chandrasekaran, E V; Jain, R K; Larsen, R D; Wlasichuk, K; Matta, K L

    1995-03-07

    The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 48 T-haptens was 5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 40 units of Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl

  4. Sialyl Lewis(x) (sLe(x)) and an sLe(x) mimetic, CGP69669A, disrupt E-selectin-dependent leukocyte rolling in vivo.

    Science.gov (United States)

    Norman, K E; Anderson, G P; Kolb, H C; Ley, K; Ernst, B

    1998-01-15

    Leukocyte rolling is the earliest observable even in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewis(x) (sLe(x)) is required for function. Despite findings that sLe(x) inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin-dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNF alpha)-stimulated mouse cremaster is increased following treatment with either sLe(x) or the sLe(x)-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti-P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin-dependent) rolling is unaffected by either sLe(x) or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.

  5. P-selectin, carcinoma metastasis and heparin: novel mechanistic connections with therapeutic implications

    Directory of Open Access Journals (Sweden)

    Varki A.

    2001-01-01

    Full Text Available Metastasis is a multistep cascade initiated when malignant cells penetrate the tissue surrounding the primary tumor and enter the bloodstream. Classic studies indicated that blood platelets form complexes around tumor cells in the circulation and facilitate metastases. In other work, the anticoagulant drug heparin diminished metastasis in murine models, as well is in preliminary human studies. However, attempts to follow up the latter observation using vitamin K antagonists failed, indicating that the primary mechanism of heparin action was unrelated to its anticoagulant properties. Other studies showed that the overexpression of sialylated fucosylated glycans in human carcinomas is associated with a poor prognosis. We have now brought all these observations together into one mechanistic explanation, which has therapeutic implications. Carcinoma cells expressing sialylated fucosylated mucins can interact with platelets, leukocytes and endothelium via the selectin family of cell adhesion molecules. The initial organ colonization of intravenously injected carcinoma cells is attenuated in P-selectin-deficient mice, in mice receiving tumor cells pretreated with O-sialoglycoprotease (to selectively remove mucins from cell surfaces, or in mice receiving a single dose of heparin prior to tumor cell injection. In each case, we found that formation of a platelet coating on cancer cells was impeded, allowing increased access of leukocytes to the tumor cells. Several weeks later, all animals showed a decrease in the extent of established metastasis, indicating a long-lasting effect of the short-term intervention. The absence of obvious synergism amongst the three treatments suggests that they all act via a common pathway. Thus, a major mechanism of heparin action in cancer may be inhibition of P-selectin-mediated platelet coating of tumor cells during the initial phase of the metastatic process. We therefore suggest that heparin use in cancer be re

  6. Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib

    Directory of Open Access Journals (Sweden)

    Barbara Muz

    2015-01-01

    Full Text Available Multiple myeloma (MM is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1 play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

  7. P-selectin in preterm infants suffering necrotizing enterocolitis ...

    African Journals Online (AJOL)

    Objective: To study P-selectin, a possible cause of NEC, in the blood of preterm infants. Study design: Twenty-four consecutive preterms, clinically suspected or proven to have NEC, were enrolled in this pilot study. Their weight ranged from 1 to 2.3 Kg (mean ±SD: 1.7±0.5 Kg), age ranged from 2 to 21 days (mean ±SD: ...

  8. Role of Soluble P-Selectin Among Type 2 Diabetic Patients with and ...

    African Journals Online (AJOL)

    ROC curve analysis for hsCRP and sP-selectin indicated that, sP-selectin had higher sensitivity and specificity than hsCRP in diabetic patient with coronary artery disease. In conclusion, measurement of soluble P-selectin seems more helpful marker of impending coronary artery insult in diabetic patients and had higher ...

  9. Plasma substance P and soluble P-selectin as biomarkers of β ...

    African Journals Online (AJOL)

    Samia A. Ebeid

    2013-09-19

    Sep 19, 2013 ... Abstract Background: Hypercoagulability in thalassemia especially in thalassemia major has emerged as a complication of the disease. There is evidence of increased platelet aggregation and increased proportion of platelets expressing P-selectin in thalassemia. P-selectin is a cell adhesion molecule ...

  10. Plasma substance P and soluble P-selectin as biomarkers of β ...

    African Journals Online (AJOL)

    Background: Hypercoagulab ility in thalassemia especially in thalassemia major has emerged as a complication of the disease. There is evidence of increased platelet aggregation and increased proportion of platelets expressing P-selectin in thalassemia. P-selectin is a cell adhesion molecule which plays a key role in ...

  11. Fetal wound healing using a genetically modified murine model: the contribution of P-selectin

    Science.gov (United States)

    During early gestation, fetal wounds heal with paucity of inflammation and absent scar formation. P-selectin is an adhesion molecule that is important for leukocyte recruitment to injury sites. We used a murine fetal wound healing model to study the specific contribution of P-selectin to scarless wo...

  12. Value of plasma P-selectin for vascular complications in liver transplantation.

    Science.gov (United States)

    Fábrega, E; Casafont, F; Merino, J; de la Peña, J; Crespo, J; Amado, J A; Pons-Romero, F

    1996-06-01

    Recent data suggest that plasma P-selectin, an adhesion molecule, may be a clinically useful marker for thrombosis. Hepatic vessel thrombosis is one of the most serious complications following liver transplantation. To assess the contribution of the soluble P-selectin to this complication, we measured plasma P-selectin levels in 32 orthotopic liver transplantations, pre-, intra-, and post-operatively. We found that levels of circulating P-selectin were not different between cirrhotic patients and healthy subjects. Of the 32 patients, 8 had vascular complications. We found a significant increase in the plasma P-selectin concentration in the thrombotic group in early postoperative period compared with the non-thrombotic group (p < 0.002). It measurement may facilitate the diagnosis of thrombosis in the early postoperative period after liver transplantation, and therefore the management of these patients.

  13. Clinical and genetic correlates of soluble P-selectin in the community.

    Science.gov (United States)

    Lee, D S; Larson, M G; Lunetta, K L; Dupuis, J; Rong, J; Keaney, J F; Lipinska, I; Baldwin, C T; Vasan, R S; Benjamin, E J

    2008-01-01

    P-selectin is a cell adhesion molecule that is involved in atherogenesis, and soluble concentrations of this biomarker reflect cardiovascular risk. However, the clinical correlates and genetic characterization of soluble P-selectin have not been clearly elucidated. To describe clinical and genetic correlates of circulating P-selectin in the community. In Framingham Heart Study Offspring (European descent) and Omni (ethnic/racial minority) participants, we examined the association of cardiovascular risk factors with soluble P-selectin concentrations. In Offspring participants, we evaluated heritability, linkage and association of 29 SELP single-nucleotide polymorphisms (SNPs) with adjusted P-selectin concentrations. In multivariable analysis of 3,690 participants (54% women, mean age 60 +/- 10 years), higher log-transformed P-selectin concentrations were inversely associated with female sex and hormone replacement therapy, and positively associated with age, ethnic/racial minority status, cigarette smoking, waist circumference, systolic blood pressure, fasting glucose, and total/high-density lipoprotein cholesterol and triglyceride concentrations. Clinical factors explained 10.4% of the interindividual variability in P-selectin concentrations. In 571 extended pedigrees (n = 1,841) with >or= 2 phenotyped members per family, multivariable-adjusted heritability was 45.4 +/- 5.8%. Among the SELP SNPs examined, a non-synonymous SNP (rs6136) encoding a threonine-to-proline substitution at position 715 was highly significantly associated with decreased P-selectin concentrations (P = 5.2 x 10(-39)), explaining 9.7% of variation after adjustment for clinical factors. Multiple clinical factors and an SNP in the SELP gene were significantly associated with circulating P-selectin concentrations. One SNP in SELP explained significant variation in circulating P-selectin concentrations, even after accounting for known clinical correlates.

  14. Crystal Structures of the Staphylococcal Toxin SSL5 in Complex With Sialyl-Lewis X Reveal a Conserved Binding Site That Shares Common Features With Viral And Bacterial Sialic Acid-Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Baker, H.M.; Basu, I.; Chung, M.C.; Caradoc-Davies, T.; Fraser, J.D.; Baker, E.N.

    2009-06-02

    Staphylococcus aureus is a significant human pathogen. Among its large repertoire of secreted toxins is a group of staphylococcal superantigen-like proteins (SSLs). These are homologous to superantigens but do not have the same activity. SSL5 is shown here to bind to human granulocytes and to the cell surface receptors for human IgA (Fc alphaRI) and P-selectin [P-selectin glycoprotein ligand-1 (PSGL-1)] in a sialic acid (Sia)-dependent manner. Co-crystallization of SSL5 with the tetrasaccharide sialyl Lewis X (sLe(X)), a key determinant of PSGL-1 binding to P-selectin, led to crystal structures of the SSL5-sLe(X) complex at resolutions of 1.65 and 2.75 A for crystals at two pH values. In both structures, sLe(X) bound to a specific site on the surface of the C-terminal domain of SSL5 in a conformation identical with that bound by P-selectin. Conservation of the key carbohydrate binding residues indicates that this ability to bind human glycans is shared by a substantial subgroup of the SSLs, including SSL2, SSL3, SSL4, SSL5, SSL6, and SSL11. This indicates that the ability to target human glycans is an important property of this group of toxins. Structural comparisons also showed that the Sia binding site in SSL5 contains a substructure that is shared by other Sia binding proteins from bacteria as well as viruses and represents a common binding motif.

  15. CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin.

    Science.gov (United States)

    Doyle, Emily L; Ridger, Victoria; Ferraro, Francesco; Turmaine, Mark; Saftig, Paul; Cutler, Daniel F

    2011-10-13

    The activation of endothelial cells is critical to initiating an inflammatory response. Activation induces the fusion of Weibel-Palade Bodies (WPB) with the plasma membrane, thus transferring P-selectin and VWF to the cell surface, where they act in the recruitment of leukocytes and platelets, respectively. CD63 has long been an established component of WPB, but the functional significance of its presence within an organelle that acts in inflammation and hemostasis was unknown. We find that ablating CD63 expression leads to a loss of P-selectin-dependent function: CD63-deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant reduction in both leukocyte rolling and recruitment and we show a failure of leukocyte extravasation in a peritonitis model. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin.

  16. Assembly of multimeric von Willebrand factor directs sorting of P-selectin

    NARCIS (Netherlands)

    Hop, C.; Guilliatt, A.; Daly, M.; de Leeuw, H. P.; Brinkman, H. J.; Peake, I. R.; van Mourik, J. A.; Pannekoek, H.

    2000-01-01

    We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis

  17. Synthesis and biological activities of three sulfated sialyl Le(x) ganglioside analogues for clarifying the real carbohydrate ligand structure of L-selectin.

    Science.gov (United States)

    Komba, S; Ishida, H; Kiso, M; Hasegawa, A

    1996-11-01

    Sulfated sialyl Le(x) ganglioside analogues at C-6 of D-galactose, N-acetyl-D-glucosamine, and of both D-galactose and N-acetyl-D-glucosamine residues have been synthesized, in order to clarify the structure of the real carbohydrate ligand of L-selectin. Coupling of the suitably protected N-acetyl-D-glucosaminyl-beta (1-->3)-lactose derivatives 13 and 16 with the sialyl alpha(2-->3)-D-galactopyranosyl trichloroacetimidates 10 and 12 (glycosyl donors), via glycosylation of 2-(trimethylsilyl)ethyl 4,6-O-benzylidene-beta-D-galactopyranoside (1) with the phenyl 2-thioglycoside derivative (2) of N-acetylneuraminic acid (Neu5Ac) using N-iodosuccinimide/TfOH, O-benzoylation, removal of the benzylidene group affording 5, selective 6-O-levulinoylation, O-benzoylation, removal of the 2-(trimethylsilyl)ethyl group, and imidate formation, or via O-acetylation of 5, removal of the 2-trimethylsilyl)ethyl group, then imidate formation, gave the pentasaccharides 18-20. The glycosylation of the pentasaccharide acceptors (21-23) derived from 18-20 by removal of the 4-methoxybenzyl group, with phenyl 1-thioglycoside derivative 27 of L-fucose using dimethyl(methylthio)sulfonium triflate (DMTST) afforded the corresponding hexasaccharides 28-30, which were transformed in good yields, via reductive removal of their benzyl groups, O-acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, imidate formation, coupling with (2S,3R,4E)-2-azido-O-benzoyl-4-octadecene-1,3-diol (35) in the presence of boron trifluoride etherate, selective reduction of the azido group, coupling with octadecanoic acid, selective removal of the levulinoyl groups, treatment with sulfur trioxide-pyridine complex, then removal of the protecting groups, into the desired sulfated sialyl Le(x) ganglioside analogues 50-52.

  18. Melanoma cell metastasis via P-selectin-mediated activation of acid sphingomyelinase in platelets.

    Science.gov (United States)

    Becker, Katrin Anne; Beckmann, Nadine; Adams, Constantin; Hessler, Gabriele; Kramer, Melanie; Gulbins, Erich; Carpinteiro, Alexander

    2017-01-01

    Metastatic dissemination of cancer cells is one of the hallmarks of malignancy and accounts for approximately 90 % of human cancer deaths. Within the blood vasculature, tumor cells may aggregate with platelets to form clots, adhere to and spread onto endothelial cells, and finally extravasate to form metastatic colonies. We have previously shown that sphingolipids play a central role in the interaction of tumor cells with platelets; this interaction is a prerequisite for hematogenous tumor metastasis in at least some tumor models. Here we show that the interaction between melanoma cells and platelets results in rapid and transient activation and secretion of acid sphingomyelinase (Asm) in WT but not in P-selectin-deficient platelets. Stimulation of P-selectin resulted in activation of p38 MAPK, and inhibition of p38 MAPK in platelets prevented the secretion of Asm after interaction with tumor cells. Intravenous injection of melanoma cells into WT mice resulted in multiple lung metastases, while in P-selectin-deficient mice pulmonary tumor metastasis and trapping of tumor cells in the lung was significantly reduced. Pre-incubation of tumor cells with recombinant ASM restored trapping of B16F10 melanoma cells in the lung in P-selectin-deficient mice. These findings indicate a novel pathway in tumor metastasis, i.e., tumor cell mediated activation of P-selectin in platelets, followed by activation and secretion of Asm and in turn release of ceramide and tumor metastasis. The data suggest that p38 MAPK acts downstream from P-selectin and is necessary for the secretion of Asm.

  19. Role of Soluble P-Selectin Among Type 2 Diabetic Patients with and ...

    African Journals Online (AJOL)

    Type 2 diabetes mellitus (T2DM) is associated with a marked increase in the risk of coronary heart disease. Platelets play a significant role in coronary artery disease (CAD). P-selectin is a component of the platelet granule membrane that is expressed on the platelet surface membrane and shed into the plasma as ...

  20. Early Dynamics of P-selectin and Interleukin 6 Predicts Outcomes in Ischemic Stroke

    DEFF Research Database (Denmark)

    Pusch, Gabriella; Debrabant, Birgit; Molnar, Tihamer

    2015-01-01

    to poststroke infection, death, and functional outcome, and assessed the ability of the models to predict each outcome. RESULTS: Interleukin 6 (IL-6) levels and change of IL-6 concentrations by 72 hours correlated with the size of tissue damage indicated by S100B titers. Levels of IL-6 and P-selectin at 72...

  1. Association between circulating levels of P-selectins and burden of ...

    African Journals Online (AJOL)

    We tested the hypothesis that, in the acute phase of ST-segment elevation myocardial infarction (STEMI), the circulating level of P-selectin (PS) is predictive of angiographic morphologic features that indicate burden of thrombus formation in the infarct-related artery (IRA). One hundred and ninety-five consecutive patients ...

  2. Structure and function of the selectin ligand PSGL-1

    Directory of Open Access Journals (Sweden)

    R.D. Cummings

    1999-05-01

    Full Text Available P-selectin glycoprotein ligand-1 (PSGL-1 is a dimeric mucin-like 120-kDa glycoprotein on leukocyte surfaces that binds to P- and L-selectin and promotes cell adhesion in the inflammatory response. The extreme amino terminal extracellular domain of PSGL-1 is critical for these interactions, based on site-directed mutagenesis, blocking monoclonal antibodies, and biochemical analyses. The current hypothesis is that for high affinity interactions with P-selectin, PSGL-1 must contain O-glycans with a core-2 branched motif containing the sialyl Lewis x antigen (NeuAca2®3Galß1®4[Fuca1®3]GlcNAcß1®R. In addition, high affinity interactions require the co-expression of tyrosine sulfate on tyrosine residues near the critical O-glycan structure. This review addresses the biochemical evidence for this hypothesis and the evidence that PSGL-1 is an important in vivo ligand for cell adhesion.

  3. Differential expression of genes encoding CD30L and P-selectin in cattle with Johne's disease: Progress toward a diagnostic gene expression signature

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Grell, S. N.; Heegaard, Peter M. H.

    2006-01-01

    a unique gene expression signature for paraTB infection. In the present study, non-stimulated leukocytes isolated from 10 sub-clinical paraTB infected cows were examined for genes being expressed at significantly different levels than in similar cells from control cows with the same herd background. We...... of the seven genes selected for qRT-PCR, CD30 ligand (CD30L) and P-selectin were consistently differentially expressed in freshly isolated leukocytes from paraTB infected and control animals of both breeds of cattle. Although further work is clearly needed to develop a more complete gene expression signature...

  4. Platelet and endothelial cell P-selectin are required for host defense against Klebsiella pneumoniae-induced pneumosepsis.

    Science.gov (United States)

    de Stoppelaar, S F; Van't Veer, C; Roelofs, J J T H; Claushuis, T A M; de Boer, O J; Tanck, M W T; Hoogendijk, A J; van der Poll, T

    2015-06-01

    Sepsis is associated with activation of platelets and endothelial cells accompanied by enhanced P-selectin surface expression. Both platelet- and endothelial P-selectin have been associated with leukocyte recruitment and induction of inflammatory alterations. Klebsiella (K.) pneumoniae is a common human sepsis pathogen, particularly in the context of pneumonia. Wild-type (WT) and P-selectin-deficient (Selp(-/-) ) mice or bone marrow chimeric mice were infected with K. pneumoniae via the airways to induce pneumosepsis. Mice were sacrificed during early (12 h after infection) or late-stage (44 h) sepsis for analyses, or followed in a survival study. Selp(-/-) mice displayed 10-1000-fold higher bacterial burdens in the lungs, blood and distant organs during late-stage sepsis. P-selectin deficiency did not influence leukocyte recruitment to the lungs, but was associated with decreased platelet-monocyte complexes and increased cytokine release. Bone marrow transfer studies revealed a role for both platelet and endothelial cell P-selectin as mice deficient in platelet or endothelial cell P-selectin displayed an intermediate phenotype in bacterial loads and survival compared with full wild-type or full knockout control mice. Both platelet and endothelial cell P-selectin contribute to host defense during Klebsiella pneumosepsis. © 2015 International Society on Thrombosis and Haemostasis.

  5. Glycopeptide Analogues of PSGL-1 Inhibit P-Selectin In Vitro and In Vivo

    OpenAIRE

    Krishnamurthy, Venkata R.; Sardar, Mohammed Y. R.; Yu, Ying; Song, Xuezheng; Haller, Carolyn; Dai, Erbin; Wang, Xiacong; Hanjaya-Putra, Donny; Sun, Lijun; Morikis, Vasilios; Simon, Scott I.; Woods, Robert; Cummings, Richard D.; Chaikof, Elliot L.

    2015-01-01

    Blockade of P-selectin/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis, and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan bu...

  6. Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase

    Directory of Open Access Journals (Sweden)

    Lubor Borsig

    2011-05-01

    Full Text Available Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation.

  7. Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. II. Tether flow terminated by P-selectin dissociation from PSGL-1.

    Science.gov (United States)

    Heinrich, Volkmar; Leung, Andrew; Evans, Evan

    2005-03-01

    We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip most often by one precipitous step of P-selectin:PSGL-1 dissociation. Analyzing the initial elastic response and membrane unbinding from the cytoskeleton in our companion article I, we focus in this article on the regime of tether extrusion that nearly always occurred before release of the extracellular adhesion bond at pulling speeds > or =1 microm/s. The force during tether growth appeared to approach a plateau at long times. Examined over a large range of pulling speeds up to 150 microm/s, the plateau force exhibited a significant shear thinning as indicated by a weak power-law dependence on pulling speed, f(infinity) = 60 pN(nu(pull)/microm/s)(0.25). Using this shear-thinning response to describe the viscous element in a nonlinear Maxwell-like fluid model, we show that a weak serial-elastic component with a stiffness of approximately 0.07 pN/nm provides good agreement with the time course of the tether force approach to the plateau under constant pulling speed.

  8. In Vivo Tracking of Platelets: Circulating Degranulated Platelets Rapidly Lose Surface P-Selectin but Continue to Circulate and Function

    National Research Council Canada - National Science Library

    Michelson, A

    1995-01-01

    To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo...

  9. Levels of platelet-derived microparticles and soluble p-selectin in patients of acute myocardial infarction (case control study).

    Science.gov (United States)

    Hameed, Aisha; Rubab, Zille; Abbas Rizvi, Syed Khizar; Hussain, Shabbir; Latif, Waqas; Mohsin, Shahida

    2017-07-01

    TTo measure levels of platelet-derived microparticles and soluble P-selectin in patients of acute myocardial infarction and their comparison with healthy controls. This case-control study was conducted in Department of Haematology, University of Health Sciences Lahore from April to September 2013, and comprised patients of acute myocardial infarction in group 1 and healthy controls in group 2. Platelet-derived microparticles and soluble P-selectin were measured by enzyme-linked immunosorbent assay. SPSS21 was used for data analysis. Of the 80 participants, 50(62.5%) were patients and 30(37.5%) were controls. The mean levels of platelet-derived microparticles and soluble P-selectin were significantly higher in group 1 compared to group 2 (45.70±10.30 vs 10.60±0.96, and 51.46±9.30 vs 9.16±1.04, respectively) (pderived microparticles and soluble P-selectin in three intervals after acute myocardial infarction (p>0.05). Although levels of platelet-derived microparticles and soluble P-selectin did not correlate to creatinekinase-myocardial band levels (p>0.05), but there was a trend of significant correlation with cardiac troponin T (pderived microparticles and soluble P-selectin can be used as novel early diagnostic marker of acute myocardial infarction.

  10. A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins

    Directory of Open Access Journals (Sweden)

    Reeja Maria Cherian

    2015-08-01

    Full Text Available Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b, CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1, core 3 (B3GNT6, core 4 (GCNT1 and B3GNT6, or extended core 1 (B3GNT3 chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc or type 2 (Galb4GlcNAc outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

  11. Development of Polymer Microcapsules Functionalized with Fucoidan to Target P-Selectin Overexpressed in Cardiovascular Diseases.

    Science.gov (United States)

    Li, Bo; Juenet, Maya; Aid-Launais, Rachida; Maire, Murielle; Ollivier, Véronique; Letourneur, Didier; Chauvierre, Cédric

    2017-02-01

    New tools for molecular imaging and targeted therapy for cardiovascular diseases are still required. Herein, biodegradable microcapsules (MCs) made of polycyanoacrylate and polysaccharide and functionalized with fucoidan (Fuco-MCs) are designed as new carriers to target arterial thrombi overexpressing P-selectin. Physicochemical characterizations demonstrated that microcapsules have a core-shell structure and that fucoidan is present onto the surface of Fuco-MCs. Furthermore, their sizes range from 2 to 6 µm and they are stable on storage over 30 d at 4 °C. Flow cytometry experiments evidenced the binding of Fuco-MCs for human activated platelets as compared to MCs (mean fluorescence intensity: 12 008 vs. 9, p microcapsules reveal excellent compatibility with 3T3 cells in cytotoxicity assay. One hour after intravenous injection of microcapsules, histological analysis revealed that Fuco-MCs are localized in the rat abdominal aortic aneurysm thrombotic wall and that the binding in the healthy aorta is low. In conclusion, these microcapsules appear as promising carriers for targeting of tissues characterized by P-selectin overexpression and for their molecular imaging or treatment. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Interleukin-2 regulatory effect on P-selectin and interleukin-8 production in patients with chronic renal failure.

    Science.gov (United States)

    Abou-Shousha, Seham A; Youssef, Amany I

    2006-01-01

    Patients with chronic renal failure (CRF) show a clinical state of immunodysfunction that occurs in both humoral and cellular immunity as well as inflammatory response. In this study, we investigated the mononuclear-endothelial cells (MCs/ECs) interaction and the possible protective role of IL-2 as the main T lymphocyte activator in CRF patients. The levels of soluble P-selectin (sP-selectin) and interleukin-8 (IL-8) as the two main mediators of MCs/ECs interaction were measured in IL-2 supplemented and non-supplemented peripheral blood mononuclear cells (PBMCs) supernatant of CRF patients. The obtained results were correlated with those of sex and age matched controls. Significantly higher levels of sP-selectin and IL-8 were detected in both IL-2 supplemented and non-supplemented PBMCs culture supernatant of CRF patients than controls (P = 0.000). Those levels were significantly lower in IL-2 supplemented PBMCs culture supernatant than non-supplemented ones of both CRF (P = 0.000) (for both mediators) and normal control groups (P = 0.01, P= 0.04 for sP-selectin and IL-8 respectively). The higher sP-selectin in CRF indicates impairment of MCs/ECs interaction that may be resulted from blockade of P-selectin receptors on PBMCs by P-selectin molecules shedded from ECs to plasma and bind to PBMCs in vivo. The elevated IL-8 level in PBMCs of CRF reflect the imbalance of Thl/Th2 ratio and subsequent impairment of cellular immunity in those patients. The lower level of both sP-selectin and IL-8 in IL-2 supplemented PBMCs supernatant than in non-supplemented one seemed to be due to the IL-2 induced proliferation of Th1 lymphocytes yielding newly in vitro formed T cells which do not carry P-selectin as well as relative increase of Th1/Th2 ratio in both normal and CRF groups. Thus, IL-2 may improve the MCs/ECs interaction and correct the Th1/Th2 ratio in CRF providing a novel promising therapeutic approach to improve the immuno-pathological condition of those patients.

  13. Contribution of the CR domain to P-selectin lectin domain allostery by regulating the orientation of the EGF domain.

    Directory of Open Access Journals (Sweden)

    Shouqin Lü

    Full Text Available The allostery of P-selectin has been studied extensively with a focus on the Lec and EGF domains, whereas the contribution of the CR domain remains unclear. Here, molecular dynamics simulations (MDS combined with homology modeling were preformed to investigate the impact of the CR domain on P-selectin allostery. The results indicated that the CR domain plays a role in the allosteric dynamics of P-selectin in two ways. First, the CR1 domain tends to stabilize the low affinity of P-selectin during the equilibration processes with the transition inhibition from the S1 to S1' state by restraining the extension of the bent EGF orientation, or with the relaxation acceleration of the S2 state by promoting the bending of the extended EGF orientation. Second, the existence of CR domain increases intramolecular extension prior to complex separation, increasing the time available for the allosteric shift during forced dissociation with a prolonged bond duration. These findings further our understanding of the structure-function relationship of P-selectin with the enriched micro-structural bases of the CR domain.

  14. Specificity analysis of three clonal and five non-clonal alpha 1,3-L-fucosyltransferases with sulfated, sialylated, or fucosylated synthetic carbohydrates as acceptors in relation to the assembly of 3'-sialyl-6'-sulfo Lewis x (the L-selectin ligand) and related complex structures.

    Science.gov (United States)

    Chandrasekaran, E V; Jain, R K; Larsen, R D; Wlasichuk, K; DiCioccio, R A; Matta, K L

    1996-07-09

    Unique specificities of the cloned alpha 1,3-L-fucosyltransferases (FTs), FT III (Lewis type), FT IV (myeloid type), and FT V (plasma type), and the alpha 1,3-FTs of Colo 205 (colon carcinoma), HL 60 (myeloid), B142 (lymphoid), EKVX (lung carcinoma), and calf mesenteric lymph nodes (CMLN) were discerned with sulfated, sialylated, and/or fucosylated Gal beta 1,3/4GlcNAc beta-based acceptor moieties. (a) FT V was 1.0-, 20.8-, and 4.6-fold active in forming Lewis x, Lewis y, and 3'-alpha-galactosyl Lewis x, respectively. (b) FT III and FT V formed approximately 4-fold 3'-sulfo Lewis x, as compared to 3'-sialyl Lewis x. (c) FT IV showed great efficiency in forming 3'-sulfo Lewis x (249%) and Lewis x (345%) in mucin-type branched chains. (d) FT III, FT IV, and FT V formed 19%, 62%, and 47% 6-sulfo Lewis x as compared to Lewis x. (e) 6'-Sulfo Lewis x and 3'-sialyl-6'-sulfo Lewis x (GLYCAM ligand) were not synthesized from their immediate precursors by FT III, FT IV, or FT V. (f) FT III, FT IV, and FT V were 311%, 9%, and 188% active, respectively, with 2'-fucosyl lactose but were not active with 2'- fucosyl-6'-sulfo lactose. (g) FT III and FT V were 7.0- and 0.5-fold active in forming Lewis a as compared to Lewis x, whereas, FT IV was inactive. (h) FT III was -2.0-fold more active in forming 3'-alpha-galactosyl Lewis a than Lewis b. (i) FT III synthesized 6-sialyl Lewis a (40% efficiency as compared to Lewis a) from 6-sialyl type 1. (j) FT III did not act on 6'-sulfo or 6'-sialyl type 1 but was 106% and 22% active with 3'-sulfo and 6-sulfo type 1, respectively. (k) The Colo 205 FT activities with type 1 compounds almost paralleled that of FT III except for the low activity (9%) with Gal beta 1,3(NeuAc alpha 2, 6)GlcNAc beta-O-Bn, but with type 2 considerable differences between Colo 205 FT and FT III were noticed. (l) The alpha 1,3-FTs of CMLN, HL60, B142, and EKVX were 1.2-1.7 times active with Fuc alpha 1,2Gal beta 1,4GlcNAc beta- O-pNP and Gal alpha 1,3Gal beta 1

  15. HuEP5C7 as a humanized monoclonal anti-E/P-selectin neurovascular protective strategy in a blinded placebo-controlled trial of nonhuman primate stroke.

    Science.gov (United States)

    Mocco, J; Choudhri, Tanvir; Huang, Judy; Harfeldt, Elisabeth; Efros, Lyubov; Klingbeil, Corine; Vexler, Vladimir; Hall, William; Zhang, Yuan; Mack, William; Popilskis, Sulli; Pinsky, David J; Connolly, E Sander

    2002-11-15

    Although inhibiting interaction of beta(2) integrins with cognate immunoglobulin class adhesion receptor ligands is an effective neuroprotective strategy in small mammal models of stroke, the strategy has failed in human trials. A completely different antiadhesion receptor strategy was therefore rigorously tested in a model that may more closely approximate human reperfused stroke. Early leukoadhesive events in postischemic cerebral microvessels are mediated by upregulated selectin-class adhesion receptors on endothelial cells. Therefore, a blocking antibody prepared against common P- and E-selectin epitopes was humanized to suppress complement activation and tested in a reperfused hemispheric stroke model in Papio anubis (baboon). Histological examination of postischemic cerebral microvessels revealed a strong upregulation of E-and P-selectin expression. Placebo-blinded administration of the humanized anti-human E- and P-selectin monoclonal antibody (HuEP5C7, 20 mg/kg IV, n=9; placebo, n=9) immediately after the onset of 1 hour of temporary ischemia resulted in trends showing reduced polymorphonuclear leukocyte (PMN) infiltration into ischemic cortex, reduced infarct volumes (by 41%), improved neurological score (by 35%), and improved ability to self-care (by 39%). Importantly, there was no evidence of systemic complement activation, immune suppression, or pathological coagulopathy associated with this therapy. These data suggest that a humanized anti-E/P-selectin antibody approach is safe and may be effective as a clinical treatment for human stroke.

  16. P-Selectin (CD62P) Expression in Liver Tissue of Biliary Atresia: A New Perspective in Etiopathogenesis.

    Science.gov (United States)

    Sira, Mostafa M; Sira, Ahmad M; Ehsan, Nermine A; Mosbeh, Asmaa

    2015-11-01

    The etiology of biliary atresia (BA) is still elusive. Inflammation plays a key role in bile duct and liver injury. The recruitment and accumulation of inflammatory cells is largely dependent on adhesion molecules. We aimed to investigate P-selectin (CD62P) expression in liver tissue in patients with BA compared with other neonatal cholestatic disorders. The study included 63 infants with neonatal cholestasis in 2 groups: BA group (n = 32) and non-BA group (n = 31) with non-BA cholestatic disorders as controls. Demographic, clinical, laboratory, ultrasonographic, and histopathological parameters were collected. P-selectin immunostaining was performed. Immunostaining in bile duct epithelium, cellular infiltrate, and vascular endothelial cells were scored as positive or negative. The frequency of P-selectin-positive endothelium, platelets, and bile duct epithelium was significantly higher in the BA group (72%, 72%, and 63%, respectively) than in the non-BA group (32%, 16%, and 13%, respectively) with P of 0.002, thrombocytosis versus 25% in the controls (P = 0.001 for both). The significant expression of P-selectin in endothelium, platelets, and bile duct epithelium in patients with BA suggests a potential role for this adhesion molecule in the pathogenesis of this devastating neonatal hepatic disorder. It further suggests that platelets in BA are activated and may have a role in the inflammatory process in BA.

  17. Isolation and characterization of N-feruloyltyramine as the P-selectin expression suppressor from garlic (Allium sativum)

    Science.gov (United States)

    Because garlic (Allium sativum) is believed to have positive health effects on cardiovascular disease, the screening of isolated fractions from a garlic extract against cardiovascular disease related-processes should help identify active compounds. Both P-selectin expression suppressing activity ag...

  18. The significance of diminished sTWEAK and P-selectin content in platelets of patients with pulmonary arterial hypertension.

    Science.gov (United States)

    Kazimierczyk, Remigiusz; Błaszczak, Piotr; Kowal, Krzysztof; Jasiewicz, Małgorzata; Knapp, Małgorzata; Szpakowicz, Anna; Ptaszyńska-Kopczyńska, Katarzyna; Sobkowicz, Bożena; Waszkiewicz, Ewa; Grzywna, Ryszard; Musial, Włodzimierz J; Kamiński, Karol A

    2017-12-01

    Pulmonary arterial hypertension (PAH) is a progressive disease characterized by proliferative changes in pulmonary arteries. There is growing evidence suggesting that soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and P-selectin could be involved in PAH development and progression. Here we investigate whether circulating platelets may be a source of sTWEAK and contribute to diminished availability of sTWEAK and P-selectin in PAH patients. We have prospectively enrolled two independent study groups of stable patients with confirmed PAH and age matched controls: derivation (10 PAH; 15 controls) and validation (20 PAH; 12 controls). P-selectin and sTWEAK concentrations were measured in platelet-poor plasma and platelet lysate. To avoid procedural bias, in each group we employed different protocols for platelet isolation. Consistently, both in derivation and validation groups PAH patients presented significantly lower sTWEAK content in platelets than control group with no significant differences in plasma levels. Similarly, patients presented comparable to controls plasma P-selectin concentrations and lower concentration in platelet lysate. Kaplan-Meier analysis revealed that patients with low platelet sTWEAK/total protein concentration ratio had more frequently detoriation of PAH in the follow-up (16.51 ± 3.32 months), log-rank test, p = .03. Patients diagnosed with pulmonary arterial hypertension present diminished sTWEAK and P-selectin storage capacity in platelets. Thrombocytes appear to be a major source of sTWEAK that could be released upon local injury and its decreased availability could have an impact on pathophysiology and prognosis in PAH. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Critical role of P-selectin-dependent leukocyte recruitment in endotoxin-induced intestinal barrier dysfunction in mice.

    OpenAIRE

    Mangell, Peter; Röme, Andrada; Wang, Yusheng; Schramm, R; Jeppsson, Bengt; Thorlacius, Henrik

    2007-01-01

    Objective: To define the importance of leukocyte recruitment in endotoxin-induced gut permeability. Materials and methods: 31 male C57BL/6 mice were challenged with lipopolysaccharide (LPS). Ileal permeability was measured in Ussing chambers and leukocyte-endothelium interactions studied with intravital fluorescence microscopy after 18 h. Results: LPS caused a clear-cut increase in leukocyte accumulation and intestinal permeability. Immunoneutralisation of P-selectin not only reduced leukocyt...

  20. Detection of early stage atherosclerotic plaques using PET and CT fusion imaging targeting P-selectin in low density lipoprotein receptor-deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Ikuko, E-mail: nakamuri@riken.jp [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Department of Cardiovascular Medicine, Saga University, Saga (Japan); Hasegawa, Koki [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Department of Pathology and Experimental Medicine, Kumamoto University, Kumamoto (Japan); Wada, Yasuhiro [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Hirase, Tetsuaki; Node, Koichi [Department of Cardiovascular Medicine, Saga University, Saga (Japan); Watanabe, Yasuyoshi, E-mail: yywata@riken.jp [RIKEN Center for Molecular Imaging Science, Kobe (Japan)

    2013-03-29

    Highlights: ► P-selectin regulates leukocyte recruitment as an early stage event of atherogenesis. ► We developed an antibody-based molecular imaging probe targeting P-selectin for PET. ► This is the first report on successful PET imaging for delineation of P-selectin. ► P-selectin is a candidate target for atherosclerotic plaque imaging by clinical PET. -- Abstract: Background: Sensitive detection and qualitative analysis of atherosclerotic plaques are in high demand in cardiovascular clinical settings. The leukocyte–endothelial interaction mediated by an adhesion molecule P-selectin participates in arterial wall inflammation and atherosclerosis. Methods and results: A {sup 64}Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated anti-P-selectin monoclonal antibody ({sup 64}Cu-DOTA-anti-P-selectin mAb) probe was prepared by conjugating an anti-P-selectin monoclonal antibody with DOTA followed by {sup 64}Cu labeling. Thirty-six hours prior to PET and CT fusion imaging, 3 MBq of {sup 64}Cu-DOTA-anti-P-selectin mAb was intravenously injected into low density lipoprotein receptor-deficient Ldlr-/- mice. After a 180 min PET scan, autoradiography and biodistribution of {sup 64}Cu-DOTA-anti-P-selectin monoclonal antibody was examined using excised aortas. In Ldlr-/- mice fed with a high cholesterol diet for promotion of atherosclerotic plaque development, PET and CT fusion imaging revealed selective and prominent accumulation of the probe in the aortic root. Autoradiography of aortas that demonstrated probe uptake into atherosclerotic plaques was confirmed by Oil red O staining for lipid droplets. In Ldlr-/- mice fed with a chow diet to develop mild atherosclerotic plaques, probe accumulation was barely detectable in the aortic root on PET and CT fusion imaging. Probe biodistribution in aortas was 6.6-fold higher in Ldlr-/- mice fed with a high cholesterol diet than in those fed with a normal chow diet. {sup 64}Cu-DOTA-anti-P-selectin m

  1. The influence of propofol on P-selectin expression and nitric oxide production in re-oxygenated human umbilical vein endothelial cells.

    LENUS (Irish Health Repository)

    Corcoran, T B

    2012-02-03

    BACKGROUND: Reperfusion injury is characterized by free radical production and endothelial inflammation. Neutrophils mediate much of the end-organ injury that occurs, requiring P-selectin-mediated neutrophil-endothelial adhesion, and this is associated with decreased endothelial nitric oxide production. Propofol has antioxidant properties in vitro which might abrogate this inflammation. METHODS: Cultured human umbilical vein endothelial cells were exposed to 20 h of hypoxia and then returned to normoxic conditions. Cells were treated with saline, Diprivan 5 microg\\/l or propofol 5 microg\\/l for 4 h after re-oxygenation and were then examined for P-selectin expression and supernatant nitric oxide concentrations for 24 h. P-selectin was determined by flow cytometry, and culture supernatant nitric oxide was measured as nitrite. RESULTS: In saline-treated cells, a biphasic increase in P-selectin expression was demonstrated at 30 min (P = 0.01) and 4 h (P = 0.023) after re-oxygenation. Propofol and Diprivan prevented these increases in P-selectin expression (P < 0.05). Four hours after re-oxygenation, propofol decreased endothelial nitric oxide production (P = 0.035). CONCLUSION: This is the first study to demonstrate an effect of propofol upon endothelial P-selectin expression. Such an effect may be important in situations of reperfusion injury such as cardiac transplantation and coronary artery bypass surgery. We conclude that propofol attenuates re-oxygenation-induced endothelial inflammation in vitro.

  2. Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

    DEFF Research Database (Denmark)

    Evans, Evan; Heinrich, Volkmar; Leung, Andrew

    2005-01-01

    , and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane...... unbinding forces for rates of loading (force/time) in the elastic regime from 240 pN/s to 38,000 pN/s, we discovered that the force distributions agreed well with the theory for kinetically limited failure of a weak bond. The kinetic rate for membrane unbinding was found to increase as an exponential...

  3. Effect of thrombopoietin receptor agonists on markers of coagulation and P-selectin in patients with immune thrombocytopenia

    DEFF Research Database (Denmark)

    Garabet, Lamya; Ghanima, Waleed; Monceyron Jonassen, Christine

    2018-01-01

    Thrombopoietin-receptor-agonists (TPO-RA) are effective treatments of immune thrombocytopenia (ITP). Previous long-term TPO-RA clinical trials have shown that thrombotic events occurred in 6% of TPO-RA-treated ITP patients. To explore the increased risk of thrombosis, the effects of TPO......-RA on markers of coagulation and P-selectin were studied. The study comprised two ITP cohorts and controls. Cohort 1 included 26 patients with sequential samples acquired before and during treatment with TPO-RA. Cohort 2 included a single sample in 18 patients on TPO-RA for more than one year. Thrombin...... patients in cohort 1. Significantly higher levels of F1+2, D-dimer, and PAI-1 were found in ITP patients before TPO-RA treatment and in patients on long-term TPO-RA treatment than in controls. Pre-treatment levels of sP-selectin did not differ from controls. Analysis of longitudinal trends showed...

  4. Molecular Characterization of the Interactions between Vascular Selectins and Glycoprotein Ligands on Human Hematopoietic Stem/Progenitor Cells

    KAUST Repository

    Abusamra, Dina

    2016-12-01

    The human bone marrow vasculature constitutively expresses both E-selectin and P-selectin where they interact with the cell-surface glycan moiety, sialyl Lewis x, on circulating hematopoietic stem/progenitor cells (HSPCs) to mediate the essential tethering/rolling step. Although several E-selectin glycoprotein ligands (E-selLs) have been identified, the importance of each E-selL on human HSPCs is debatable and requires additional methodologies to advance their specific involvement. The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E

  5. Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products

    Science.gov (United States)

    Fuentes, Eduardo; Palomo, Iván

    2013-01-01

    Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids) have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists. PMID:24324520

  6. Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products

    Directory of Open Access Journals (Sweden)

    Eduardo Fuentes

    2013-01-01

    Full Text Available Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists.

  7. C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression

    Science.gov (United States)

    Scotland, Ramona S.; Cohen, Marc; Foster, Paul; Lovell, Matthew; Mathur, Anthony; Ahluwalia, Amrita; Hobbs, Adrian J.

    2005-10-01

    The multifaceted process of immune cell recruitment to sites of tissue injury is key to the development of an inflammatory response and involved in the pathogenesis of numerous cardiovascular disorders. We recently identified C-type natriuretic peptide (CNP) as an important endothelium-derived mediator that regulates vascular tone and protects against myocardial ischemia/reperfusion injury. Herein, we investigated whether CNP inhibits leukocyte recruitment and platelet aggregation and thereby exerts a potential antiinflammatory influence on the blood vessel wall. We assessed the effects of CNP on leukocyte-endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in animals with high basal leukocyte activation (endothelial nitric oxide synthase knockout mice, eNOS-/-) or under acute inflammatory conditions (induced by interleukin-1 or histamine). CNP suppressed basal leukocyte rolling in eNOS-/- mice in a rapid, reversible, and concentration-dependent manner. These effects of CNP were mimicked by the selective natriuretic peptide receptor-C agonist cANF4-23. CNP also suppressed leukocyte rolling induced by IL-1 or histamine, inhibited platelet-leukocyte interactions, and prevented thrombin-induced platelet aggregation of human blood. Furthermore, analysis of human umbilical vein endothelial cells, leukocytes, and platelets revealed that CNP selectively attenuates expression of P-selectin. Thus, CNP is a modulator of acute inflammation in the blood vessel wall characterized by leukocyte and platelet activation. These antiinflammatory effects appear to be mediated, at least in part, via suppression of P-selectin expression. These observations suggest that endothelial CNP might maintain an anti-atherogenic influence on the blood vessel wall and represent a target for therapeutic intervention in inflammatory cardiovascular disorders. endothelium | natriuretic peptide receptor type C | atherosclerosis | thrombosis

  8. Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro

    Directory of Open Access Journals (Sweden)

    Gröschel Werner

    2004-04-01

    Full Text Available Abstract Background The effect of non-steroidal anti-inflammatory drugs (NSAIDs for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated. Methods Whole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay. Results There were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol. Conclusions Accordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets.

  9. Soluble P-selectin level correlates with acetylsalicylic acid but not with clopidogrel response in patients with stable coronary artery disease after a percutaneous coronary intervention.

    Science.gov (United States)

    Kaufmann, Jan; Wellnhofer, Ernst; Kappert, Kai; Urban, Daniel; Meyborg, Heike; Hauptmann, Tobias; Müller, Aline; Meixner, Martin; Graf, Kristof; Fleck, Eckart; Stawowy, Philipp

    2013-06-01

    Impaired response to dual antiplatelet therapy is associated with worse cardiovascular outcome. Besides antiplatelet effects, there is evidence that both clopidogrel and acetylsalicylic acid (ASA) have anti-inflammatory properties. However, little is known about the relationship between platelet function and inflammation under dual antiplatelet therapy in patients with stable coronary artery disease. The purpose of the study was to investigate the correlation of platelet function with soluble (s)P-selectin and soluble (s)CD40L in patients undergoing elective percutaneous coronary intervention. Poor response to ASA and clopidogrel could lead to increased levels of inflammatory markers. A total of 148 patients were included. Eighty percent of the patients were on 100 mg ASA and all patients were clopidogrel naive. They underwent percutaneous coronary intervention and received a loading dose of 600 mg clopidogrel. Platelet function was assessed by light transmittance aggregometry (LTA) and vasodilator-stimulated phosphoprotein analysis at baseline, 24 h after loading, and after 1 month of maintenance therapy, respectively. Plasma levels of sP-selectin and sCD40L were measured. To classify low responders to clopidogrel, patients were screened for genetic variants determining clopidogrel absorption and metabolization. sP-selectin levels correlated with LTA findings after stimulation with arachidonic acid (P=0.012). Further, in addition to decreased platelet reactivity observed on LTA, lower sP-selectin levels were seen in patients under ASA therapy (P=0.004). CYP2C19*2 allele carriers had a higher platelet reactivity after clopidogrel loading measured by adenosine diphosphate-induced aggregation in LTA (P=0.008) and vasodilator-stimulated phosphoprotein phosphorylation (P=0.035); however, there was no difference in the inflammatory markers. Multiple regression analysis showed that variables significantly related to sP-selectin plasma levels were sCD40L (Pacid (P<0

  10. Elevated maternal serum sP-selectin levels in preeclamptic pregnancies with and without intrauterine fetal growth restriction, but not in normotensive pregnancies complicated by isolated IUGR.

    Science.gov (United States)

    Laskowska, Marzena; Laskowska, Katarzyna; Oleszczuk, Jan

    2013-02-15

    The aim of this study was to show differences of maternal serum sP-selectin levels in pregnancies complicated by intrauterine fetal growth restriction (IUGR) in the course of preeclampsia and to compare the results with normotensive pregnant women with isolated IUGR. These studies were also conducted on preeclamptic pregnancies with appropriate-for-gestational-age weight infants and on the control normotensive pregnant women. The study was carried out on 55 patients with pregnancy complicated by fetal growth restriction in the course of preeclampsia, 70 normotensive patients with pregnancies complicated by isolated IUGR, 39 preeclamptic patients with appropriate-for-gestational-age weight fetuses and 54 healthy normotensive pregnant patients with normal fetal growth. Maternal serum levels of sP-selectin were determined using the enzyme-linked immunosorbent assay. Levels of sP-selectin were higher in women with pregnancy complicated by preeclampsia with and without IUGR; whereas, in the group of normotensive pregnant women with isolated fetal growth restriction, serum sP-selectin levels tended to be lower than in the control subjects, but this difference was not statistically significant. The mean values were 192.05±70.96 ng/mL in the IUGR group, 293.18±222.92 ng/mL in the PI group, 379.78±353.13 ng/mL in the P group and 227.96±134.04 ng/mL in the healthy controls (p<0.001*). Our findings may suggest that the elevated level of the soluble P-selectin is associated with preeclampsia, and that it may confirm the presence of platelet and endothelial activation, the presence of the hypercoagulant state and may be due to the systemic inflammatory response in this serious pregnancy disorder.

  11. Soluble P-selectin and matrix metalloproteinase 2 levels are elevated in patients with diastolic dysfunction independent of glucose metabolism disorder or coronary artery disease.

    Science.gov (United States)

    Füth, Reiner; Dinh, Wilfried; Nickl, Werner; Bansemir, Lars; Barroso, Michael Coll; Bufe, Alexander; Sause, Armin; Scheffold, Thomas; Krahn, Thomas; Ellinghaus, Peter; Lankisch, Mark

    2009-01-01

    The development of diastolic dysfunction (DDF) is multifactorial. Possible mechanisms include metabolic disturbances, myocardial fibrosis, chronic inflammation and endothelial dysfunction. Recognizing early stages of DDF may help to identify patients at risk of developing symptomatic DDF. Therefore, biomarkers reflecting pathophysiological changes within the myocardium were investigated in patients with DDF. Seventy-seven patients submitted for coronary angiography with stable or suspected coronary artery disease (CAD) were consecutively enrolled. Those without known diabetes mellitus (DM) underwent a standardized oral glucose tolerance test. Echocardiography for the diagnosis of DDF was performed according to the European Society of Cardiology. Matrix metalloproteinase 2 (MMP-2) and soluble P-selectin (sP-selectin) serum concentrations were analyzed using the ELISA technique. A total of 36% of patients had DM and 74% had CAD. The prevalence of DDF was higher in patients with DM (89% versus 74%) and CAD (84% versus 53%) (PDDF in patients with DM was more severe with a significantly lower mitral annulus velocity of 6.5 cm/s versus 7.8 cm/s (PDDF showed significantly higher sP-selectin (140.3 mug/L versus 107.6 mug/L, PDDF. There was a significant correlation between sP-selectin and MMP-2 (P=0.01), independent of the diagnosis of DM or CAD. sP-selectin as a marker for platelet hyperactivity, inflammation and endothelial dysfunction, and MMP-2 as a marker for extracellular matrix turnover were significantly elevated in patients with DDF. This elevation was independent of coexisting DM or CAD. This observation may help to identify and monitor patients with DDF.

  12. Effect of marathon run and air travel on pre- and post-run soluble d-dimer, microparticle procoagulant activity, and p-selectin levels.

    Science.gov (United States)

    Parker, Beth A; Augeri, Amanda L; Capizzi, Jeffrey A; Ballard, Kevin D; Kupchak, Brian R; Volek, Jeffrey S; Troyanos, Christopher; Kriz, Peter; D'Hemecourt, Pierre; Thompson, Paul D

    2012-05-15

    D-dimer, microparticles, and p-selectin are venous thrombotic risk markers. Elevated p-selectin is associated with increased cardiovascular events. We examined the effects of exercise and air travel on the markers of vascular risk in marathon runners. Forty-one persons participating in the 114th Boston Marathon (April 19, 2010) were divided into travel (n = 23) and nontravel "control" (n = 18) groups according to whether they lived more than a 4-hour plane flight or less than a 2-hour car trip from Boston. The subjects provided venous blood samples the day before, immediately after, and after returning home the day after the marathon. The blood was analyzed for soluble d-dimer, microparticle procoagulant activity, and p-selectin. D-dimer levels increased more before to immediately after (142 ± 83 to 387 ± 196 ng/mL) in the travel group than in the controls (85 ± 26 to 233 ± 95 ng/mL; p = 0.02). Moreover, 6 travel subjects versus 0 controls had d-dimer values >500 ng/mL after returning home the day after the marathon, the clinical threshold for excluding venous thrombosis (p = 0.03). P-selectin increased with exercise (p marathon (r(2) = 0.16, p = 0.01). In conclusion, the combination of exercise and travel increases venous and arterial thrombotic risk. Moreover, the p-selectin levels at rest and after exercise were greater with age. These results might explain the reports of venous thrombosis with air travel after athletic events and the reports of cardiac events in older participants running marathons. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. In vitro effect of anti-β2 glycoprotein I antibodies on P-selectin expression, a marker of platelet activation

    Directory of Open Access Journals (Sweden)

    A. Hoxha

    2012-03-01

    Full Text Available Antiphospholipid antibodies (aPL associated with thromboembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS. Beta2glycoprotein I (β2GPI is the main target antigen for aPL, but the pathogenic role of anti-β2GPI antibodies (aβ2GPI is still unclear. Some authors assume they play a role in activating platelets. We evaluated the effects of aβ2GPI antibodies on platelet P-selectin expression. Aβ2GPI antibodies in the plasma of a pregnant APS patient were isolated by affinity chromatography at two different stages (catastrophic and quiescent of the disease. Gel filtered platelets (100 x 109/L from healthy volunteers were incubated with β2-GPI (20 µg/mL and with different concentrations (5. 25 and 50 µg/mL of aβ2GPI antibodies. P-selectin surface expression on platelets was assessed by flow cytometry using a specific fluorescent antibody directed against P-selectin. Aβ2GPI antibodies induced platelet activation only in the presence of thrombin receptor activator for peptide 6 (TRAP-6, a platelet agonist, at a subthreshold concentration. Aβ2GPI antibody enhancement on platelet surface P-selectin expression was stronger in the catastrophic than in the quiescent phase of the disease (47 vs 15%. TRAP-6 dependent platelet activation by aβ2GPI antibodies is consistent with the “two hit” pathogenetic hypothesis for thrombosis. Aβ2GPI antibodies induce higher platelet P-selectin expression during the active rather than the acute phases.

  14. DEFINITION OF ACTIVATED THROMBOCYTE NUMBER WITH ANTIBODIES FOR ACTIVATED FIBRINOGEN AND P-SELECTIN IN PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA AND ANTIAGGREGATION DRUG EFFECT

    Directory of Open Access Journals (Sweden)

    Samo Zver

    2004-12-01

    Full Text Available Background. Essential thrombocythemia (ET is a chronic myeloproliferative disease with a platelet count within the range of 400–2000 × 109/L. Higher percentage of platelets in the circulation of patients with ET express also activation markers on their membranes. Two of such markers are P-selectin and activated fibrinogen on platelet membranes. Because of frequent thrombembolic and also bleeding related complications, treatment of ET is mandatory. Patients whose platelet count is less than 1000 × 109/L and who did not suffer any thrombembolic complication during the course of the disease, are ussually treated with an antiaggregation drug, acetylsalicylic acid 100 mg/daily orally. Clopidogrel is an adenosyn-di-phosphate (ADP receptor antagonist in platelets. There is no routine clinical data about clopidogrel treatment in the patients with ET and only sporadic case reports can be find in the literature.Patients and methods. In our clinical study we compared antiaggregational effects of acetylsalicylic acid and clopidogrel, by measuring the P-selectin level and activated fibrinogen expression on platelet membranes.There were 35 ET patients included, within the age range between 21 and 78 years and with platelet counts within 451–952 × 109/L. None of the patients did suffer any thrombembolic complication during the course of the disease. During the sequential 14 day periods, patients received acetylsalicylic acid 100 mg/daily orally, followed by clopidogrel 75 mg/daily orally and ultimativelly, together acetylsalicylic acid 100 mg/daily orally plus clopidogrel 75 mg/daily orally. After each fourteen days period the level of P-selectin and activated fibrinogen activated platelets were determined with monoclonal antibodies on flow cytometer. Statistical evaluation was calculated on the difference of average values between the two small, independent pair groups with the t-test.Results. When the patients stopped with acetylsalicylic acid and

  15. Time course of soluble P-selectin and von Willebrand factor levels in trauma patients: a prospective observational study.

    Science.gov (United States)

    Tang, Ning; Yin, Shiyu; Sun, Ziyong; Pan, Yingying

    2013-09-14

    Coagulopathy often develops in patients with serious trauma and is correlated with the clinical outcome. The contribution of platelet activity and endothelial dysfunction to trauma-induced coagulopathy remain to be defined. The purpose of this study was to investigate the time courses of soluble P-selectin (sPsel, an index of platelet activation) and von Willebrand factor (VWF, an index of endothelial dysfunction) in trauma patients and elucidate their relationship to coagulation parameter levels, the presence of coagulopathy, and patient outcome. This prospective observational study, which took place in a university hospital intensive care unit (ICU), included 82 severely injured trauma patients. The sPsel, VWF antigen, protein C, and factor VII levels were measured and routine coagulation tests were performed upon admission to ICU and daily within the first week. The 30-day mortality rate was also determined. Thirty-seven (45.1%) patients developed coagulopathy upon admission to the ICU, and the 30-day mortality rate was 20.7% (n = 17). Both the admission sPsel and VWF levels were lower in patients with coagulopathy than in those without (p trauma patients in the ICU, lower levels of sPsel and VWF on admission were associated with the presence of coagulopathy and might not predict a better outcome. An increase in the VWF level at the end of the first week after admission to ICU was associated with increased 30-day mortality.

  16. Amyloid-β peptide on sialyl-Lewis(X-selectin-mediated membrane tether mechanics at the cerebral endothelial cell surface.

    Directory of Open Access Journals (Sweden)

    Sholpan Askarova

    Full Text Available Increased deposition of amyloid-β peptide (Aβ at the cerebral endothelial cell (CEC surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB in Alzheimer's disease (AD. In this study, quantitative immunofluorescence microscopy (QIM and atomic force microscopy (AFM with cantilevers biofunctionalized by sialyl-Lewis(x (sLe(x were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe(x and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf , and produced a bimodal population of Fmtf , suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf . In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.

  17. Distamycin A Inhibits HMGA1-Binding to the P-Selectin Promoter and Attenuates Lung and Liver Inflammation during Murine Endotoxemia

    Science.gov (United States)

    Baron, Rebecca M.; Lopez-Guzman, Silvia; Riascos, Dario F.; Macias, Alvaro A.; Layne, Matthew D.; Cheng, Guiying; Harris, Cailin; Chung, Su Wol; Reeves, Raymond; von Andrian, Ulrich H.; Perrella, Mark A.

    2010-01-01

    Background The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes (“enhanceosomes”) that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. Objectives To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. Methodology/Principal Findings Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-κB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-κB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. Conclusions/Significance We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight

  18. Distamycin A inhibits HMGA1-binding to the P-selectin promoter and attenuates lung and liver inflammation during murine endotoxemia.

    Directory of Open Access Journals (Sweden)

    Rebecca M Baron

    2010-05-01

    Full Text Available The architectural transcription factor High Mobility Group-A1 (HMGA1 binds to the minor groove of AT-rich DNA and forms transcription factor complexes ("enhanceosomes" that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI. AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs, such as Distamycin A (Dist A, interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs.To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules.Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-kappaB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-kappaB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo.We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting

  19. Escherichia coli fimbriae recognizing sialyl galactosides.

    OpenAIRE

    Korhonen, T K; Väisänen-Rhen, V; Rhen, M; Pere, A; Parkkinen, J; Finne, J

    1984-01-01

    Fimbriae recognizing sialyl galactosides (S fimbriae) were purified from an Escherichia coli strain. The S fimbriae were morphologically identical to type 1 and P fimbriae of E. coli and showed a hemagglutination that was abolished when erythrocytes were treated with neuraminidase. Hemagglutination by the purified fimbriae was inhibited by orosomucoid but not by its desialylated derivative. Of the oligosaccharides tested, sialyl-(alpha 2-3)-lactose and sialyl-(alpha 2-3)-N-acetyllactosamine h...

  20. Activation of platelets by in vitro whole blood contact with materials: increases in microparticle, procoagulant activity, and soluble P-selectin blood levels.

    Science.gov (United States)

    Gemmell, C H

    2001-01-01

    Non-adherent platelets and plasma were analyzed for evidence of platelet activation after whole blood contact with materials under conditions of low shear for one hour at 37 degrees C. The contact involved adding heparinized whole blood to small diameter tubes that were connected to two arms extending from a rocking platform. For all surfaces (polyethylene, polypropylene, Silastic, PVA hydrogel) tested there was strong evidence of platelet activation in the bulk blood: platelet-derived microparticles, procoagulant platelet membranes and soluble P-selectin levels. Flow cytometric quantification of microparticles (MPs) was highly sensitive and entailed the direct determination of microparticle concentrations as opposed to the traditional quantification of microparticle percentages (relative to total number of MPs and platelets). Whole blood contact with polypropylene surfaces led to the greatest drops in bulk platelet counts and also to the lowest increases in microparticle concentrations. Flow cytometry was also used to assess procoagulant levels (annexin V binding) within a light scatter region known to contain platelets and some large microparticles. All surfaces were noted to generate a significant procoagulant population that was, based on forward light scatter, mostly very small platelets or large microparticles. In contrast, most of the P-selectin positive platelets were averaged sized. Lastly. all surfaces generated soluble P-selectin levels that were approximately double the level (25 ng ml(-1)) noted in the resting whole blood samples. In addition to our previous reports, these findings support the observation that there is strong evidence of platelet activation in the bulk that we anticipate will ultimately lead to more relevant in vitro testing of the compatibility of platelets towards materials.

  1. Gene deletion of P-Selectin and ICAM-1 does not inhibit neutrophil infiltration into peritoneal cavity following cecal ligation-puncture

    Directory of Open Access Journals (Sweden)

    Hess Karen

    2004-07-01

    Full Text Available Abstract Background Neutrophil infiltration is one of the critical cellular components of an inflammatory response during peritonitis. The adhesion molecules, P-selectin and intercellular adhesion molecule (ICAM-1, mediate neutrophil-endothelial cell interactions and the subsequent neutrophil transendothelial migration during the inflammatory response. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy, suggesting that the length of injury might be a critical factor in neutrophil infiltration. Therefore, the objective of this study was to determine the role of P-selectin and ICAM-1 in neutrophil infiltration into the peritoneal cavity during early and late phases of peritonitis. Methods Peritonitis was induced in both male wild-type and P-selectin/ICAM-1 double deficient (P/I null mice by cecal ligation-puncture (CLP. Peripheral blood and peritoneal lavage were collected at 6 and 24 hours after CLP. The total leukocyte and neutrophil contents were determined, and neutrophils were identified with the aid of in situ immunohistochemical staining. Comparisons between groups were made by applying ANOVA and student t-test analysis. Results CLP induced a severe inflammatory response associated with a significant leukopenia in both wild-type and P/I null mice. Additionally, CLP caused a significant neutrophil infiltration into the peritoneal cavity that was detected in both groups of mice. However, neutrophil infiltration in the P/I null mice at 6 hours of CLP was significantly lower than the corresponding wild-type mice, which reached a similar magnitude at 24 hours of CLP. In contrast, in peritonitis induced by intraperitoneal inoculation of 2% glycogen, no significant difference in neutrophil infiltration was observed between the P/I null and wild-type mice at 6 hours of peritonitis. Conclusions The data suggest that alternative adhesion pathway(s independent of P-selectin and ICAM

  2. In vitro effect of anti-β2 glycoprotein I antibodies on P-selectin expression, a marker of platelet activation

    OpenAIRE

    Hoxha, A.; M. Tonello; E. Falcinelli; Giannini, S.; A. Ruffatti; A. Bontadi; P. Gresele; L. Punzi

    2012-01-01

    Antiphospholipid antibodies (aPL) associated with thromboembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS). Beta2glycoprotein I (β2GPI) is the main target antigen for aPL, but the pathogenic role of anti-β2GPI antibodies (aβ2GPI) is still unclear. Some authors assume they play a role in activating platelets. We evaluated the effects of aβ2GPI antibodies on platelet P-selectin expression. Aβ2GPI antibodies in the plasma of a pregnant APS patie...

  3. Automated synthesis of sialylated oligosaccharides

    Directory of Open Access Journals (Sweden)

    Davide Esposito

    2012-09-01

    Full Text Available Sialic acid-containing glycans play a major role in cell-surface interactions with external partners such as cells and viruses. Straightforward access to sialosides is required in order to study their biological functions on a molecular level. Here, automated oligosaccharide synthesis was used to facilitate the preparation of this class of biomolecules. Our strategy relies on novel sialyl α-(2→3 and α-(2→6 galactosyl imidates, which, used in combination with the automated platform, provided rapid access to a small library of conjugation-ready sialosides of biological relevance.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nygaard, Gyrid [Proteomic Unit at University of Bergen (PROBE), University of Bergen, Bergen (Norway); Department of Biomedicine, University of Bergen, Bergen (Norway); Herfindal, Lars; Kopperud, Reidun [Department of Biomedicine, University of Bergen, Bergen (Norway); Aragay, Anna M. [Department of Biomedicine, University of Bergen, Bergen (Norway); Molecular Biology Institute of Barcelona (IBMB, CSIC), Barcelona (Spain); Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune [Department of Biomedicine, University of Bergen, Bergen (Norway); Selheim, Frode, E-mail: Frode.Selheim@biomed.uib.no [Proteomic Unit at University of Bergen (PROBE), University of Bergen, Bergen (Norway); Department of Biomedicine, University of Bergen, Bergen (Norway)

    2014-07-04

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

  5. Standardized flavonoid-rich Eugenia jambolana seed extract retards in vitro and in vivo LDL oxidation and expression of VCAM-1 and P-selectin in atherogenic rats.

    Science.gov (United States)

    Jadeja, Ravirajsinh N; Thouaojam, Menaka C; Sankhari, Jayantha M; Jain, Mahendra; Devkar, Ranjitsinh V; Ramachandran, A V

    2012-03-01

    The present inventory evaluates anti-atherogenic potential of flavonoid-rich Eugenia jambolana seed extract (EJSE) against in vitro low-density lipoprotein (LDL) oxidation, foam cell formation, and atherogenic (ATH) diet-induced experimental atherosclerosis in rats. EJSE was able to prevent in vitro LDL oxidation and oxidized LDL-induced macrophage foam cell formation. Also, EJSE supplementation to ATH rats significantly minimized increment in serum markers of LDL oxidation. The ex vivo oxidation indices were also minimized in LDL of EJSE-treated animals. Microscopic evaluation of thoracic aorta of ATH + EJSE rats recorded minimal evidence of atheromatous plaque formation, accumulation of lipid laden macrophages, calcium deposition, and expression of cell adhesion molecules (vascular cell adhesion molecule-1 and P-selectin). This is the first scientific report that demonstrates anti-atherogenic potential of EJSE and warrants further evaluation at clinical level.

  6. Relevance of angiopoietin-2 and soluble P-selectin levels in patients with pulmonary arterial hypertension receiving combination therapy with oral treprostinil: a FREEDOM-C2 biomarker substudy

    Science.gov (United States)

    Schermuly, Ralph; Seeger, Werner; Rao, Youlan; Ghofrani, Hossein A.; Gall, Henning

    2016-01-01

    Abstract Studies have suggested roles for angiopoietin-2 (Ang-2) and soluble P-selectin (sP-selectin) as biomarkers of disease severity and treatment response in pulmonary arterial hypertension (PAH), but additional data are required for validation. We evaluated these biomarkers using data from FREEDOM-C2, in which patients with PAH receiving stable monotherapy or combination therapy were randomized to receive additional treatment with oral treprostinil (up-titrated from 0.25 mg twice daily) or placebo for 16 weeks. Biomarker analysis was optional in FREEDOM-C2. We measured plasma Ang-2 and sP-selectin levels at baseline and at week 16, and we assessed their association with predefined outcomes (6-minute walk distance [6MWD] change from baseline >40 m, 6MWD >380 m, functional class I/II, and/or N-terminal pro-brain natriuretic peptide [NT-proBNP] <1,800 pg/mL at week 16) using Spearman correlation, receiver operating characteristics, and logistic regression. Biomarker data were available for 83 of 157 and 95 of 153 patients in the oral treprostinil and placebo groups, respectively. In the oral treprostinil group, baseline Ang-2 levels correlated with week 16 NT-proBNP levels (P < 0.0001). Baseline Ang-2 ≥12 ng/mL was associated with a reduced likelihood of having NT-proBNP <1,800 pg/mL at week 16 (multivariate odds ratio: 0.08; 95% confidence interval: 0.02–0.32). However, Ang-2 showed no significant association with the other assessed outcomes, and sP-selectin was not associated or correlated with any of the outcomes. These data suggest that Ang-2 and sP-selectin are not associated with response to oral treprostinil in patients already receiving stable PAH therapy. Trial registration: Clinicaltrials.gov identifier NCT00887978. PMID:28090293

  7. Localization of T and B Lymphocytes to the White Pulp of the Spleen is Independent of L-, E-, and P-Selectin

    Directory of Open Access Journals (Sweden)

    Mitchell H. Grayson

    2003-01-01

    Full Text Available T and B cell interactions are thought to be of prime importance in the generation of a humoral immune response. These interactions are thought to take place in the secondary lymphoid organs. The largest of which is the spleen. While the pathways involved in lymphocyte migration into other secondary lymphoid organs have been unraveled, very little is understood about T and B cell migration to the spleen. We report that adoptively transferred T lymphocytes appear more rapidly within the lymphoid compartment of the spleen than do B lymphocytes. Indeed, half of the transferred T lymphocytes in the spleen appear within the white pulp by 1.4 hours. B lymphocytes take nearly 4.3 hours to achieve the same level of accumulation. In addition, T lymphocyte arrival is fucoidan sensitive, while B cells are not affected by this polysaccharide. Finally, we show that neither L-, E-, or P-selectin appears to play a significant role in the accumulation of lymphocytes in the white pulp.

  8. The human megakaryocytic cell line UT-7/TPO responds to platelet agonists with intracellular Ca2+ elevation and P-selectin expression.

    Science.gov (United States)

    Kawaguchi, Tatsuya; Hashimoto, Ryuji; Nawa, Katsuhiko; Yokota, Hiroshi

    2011-05-01

    Megakaryocytes have several signal transduction cascades that are similar, but not identical to platelet activation signals. In order to understand platelet signals in detail, it is useful to compare the similarities and/or differences between platelets and megakaryocytes. We evaluated platelet activation signals related to three kinds of Gq protein-coupled receptors using the megakaryocytic cell line UT-7/TPO. It was found that UT-7/TPO responded to thrombin, resulting in a continuous elevation of the [Ca2+]i (intracellular Ca2+) and P-selectin expression on the surface of the cells. Activation of integrin αIIbβ3 and thromboxane generation was not detected by any of the three stimulations. Taken together, although strong [Ca2+]i elevation by thrombin stimulation caused further P-selection expression, we could detect [Ca2+]i elevation, which is thought to be the individual signals through the thrombin, thromboxane A2 or ADP receptor, without considering the secondary signalling caused by αIIbβ3 activation and the arachidonic acid cascade using UT-7/TPO.

  9. Sialyl Lewis x expression in cervical scrapes of premalignant lesions

    Indian Academy of Sciences (India)

    In the present study we determined the expression profiles of the glycosidic antigens Tn, sialyl Tn (sTn), Lewis a (Lea), sialyl Lewis a (sLea), Lewis x (Lex) and sialyl Lewis x (sLex) in cervical scrapes with cytological diagnoses of normal, low-grade squamous intraepithelial lesions (LGSIL) and high-grade squamous ...

  10. Escherichia coli fimbriae recognizing sialyl galactosides.

    Science.gov (United States)

    Korhonen, T K; Väisänen-Rhen, V; Rhen, M; Pere, A; Parkkinen, J; Finne, J

    1984-08-01

    Fimbriae recognizing sialyl galactosides (S fimbriae) were purified from an Escherichia coli strain. The S fimbriae were morphologically identical to type 1 and P fimbriae of E. coli and showed a hemagglutination that was abolished when erythrocytes were treated with neuraminidase. Hemagglutination by the purified fimbriae was inhibited by orosomucoid but not by its desialylated derivative. Of the oligosaccharides tested, sialyl-(alpha 2-3)-lactose and sialyl-(alpha 2-3)-N-acetyllactosamine had the strongest inhibitory activities. It was concluded that S fimbriae have the strongest affinity for (alpha 2-3)-linked sialyl galactosides. In the enzyme-linked immunosorbent assay, the hyperimmune serum to the S fimbriae reacted strongly with the homologous antigen but not with type 1, P, or nonhemagglutinating KS71C fimbriae of E. coli. Analogously, the hyperimmune sera to the other E. coli fimbriae did not react with the purified S fimbriae. The immunoprecipitation assay showed that S fimbriae on different E. coli serotypes shared immunological cross-reactivity.

  11. Agent-based model of therapeutic adipose-derived stromal cell trafficking during ischemia predicts ability to roll on P-selectin.

    Directory of Open Access Journals (Sweden)

    Alexander M Bailey

    2009-02-01

    , or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 microm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.

  12. Agent-based model of therapeutic adipose-derived stromal cell trafficking during ischemia predicts ability to roll on P-selectin.

    Science.gov (United States)

    Bailey, Alexander M; Lawrence, Michael B; Shang, Hulan; Katz, Adam J; Peirce, Shayn M

    2009-02-01

    vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 microm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.

  13. Selected immunological changes in patients with Goeckerman's therapy TNF-alpha, sE-selectin, sP-selectin, sICAM-1 and IL-8

    Energy Technology Data Exchange (ETDEWEB)

    Borska, L.; Fiala, Z.; Krejsek, J.; Andrys, C.; Vokurkova, D.; Hamakova, K.; Kremlacek, J.; Ettler, K. [Charles University, Hradec Kralove (Czech Republic). Faculty of Medicine

    2006-07-01

    Psoriasis is one of the most frequent inflammatory skin diseases in which abnormal individual immune reactivity plays an important role. The aim of the present study was to describe selected immunological changes, concerning pro-inflammatory cytokines (TNF-alpha, IL-8) and adhesion molecules (sE-selectin, sP-selectin, sICAM-1), in 56 patients cured by Goeckerman's therapy (GT). GT includes dermal application of crude coal tar (containing polycyclic aromatic hydrocarbons) and exposure to UV radiation.

  14. Associations of combined polymorphisms of the platelet membrane glycoproteins Ia and IIIa and the platelet-endothelial cell adhesion molecule-1 and P-Selectin genes with IVF implantation failures.

    Science.gov (United States)

    Vlachadis, Nikolaos; Tsamadias, Vasileios; Vrachnis, Nikolaos; Kaparos, Georgios; Vitoratos, Nikolaos; Kouskouni, Evaggelia; Economou, Emmanuel

    2017-04-01

    The aim of the study was to investigate the combined impact of the genetic heterogeneity of the glycoproteins Ia (GpIa) and IIIa (GpIIIa) and the platelet-endothelial cell adhesion molecule-1 (PECAM-1) and P-Selectin genes on IVF embryo transfer implantation failures (IVF-ET failures). Sixty nulligravida women with previous IVF-ET failures and 60 fertile controls were genotyped for the GpIa-C807T, GpIIIa-PlA1/PA2, PECAM-1-C373G (Leu125Val) and P-Selectin-A37674C (Thr715Pro) polymorphisms by pyrosequencing. Compared with wild-type combined homozygotes, carriers of combinations of risk alleles in two gene loci were at significantly increased risk for IVF-ET failure, whereas carriers of the combination of GpIa-807T, GpIIIa-PlA2 and PECAM-1-373G alleles had OR = 52.50 (95%CI: 4.05-680.95, p IVF-ET failures especially for younger women and provided a genetic risk score with good diagnostic accuracy in the prediction of IVF-ET failures.

  15. Towards In Vivo Imaging of Cancer Sialylation

    Directory of Open Access Journals (Sweden)

    Ivan Martinez-Duncker

    2011-01-01

    Full Text Available In vivo assessment of tumor glucose catabolism by positron emission tomography (PET has become a highly valued study in the medical management of cancer. Emerging technologies offer the potential to evaluate in vivo another aspect of cancer carbohydrate metabolism related to the increased anabolic use of monosaccharides like sialic acid (Sia. Sia is used for the synthesis of sialylated oligosaccharides in the cell surface that in cancer cells are overexpressed and positively associated to malignancy and worse prognosis because of their role in invasion and metastasis. This paper addresses the key points of the different strategies that have been developed to image Sia expression in vivo and the perspectives to translate it from the bench to the bedside where it would offer the clinician highly valued complementary information on cancer carbohydrate metabolism that is currently unavailable in vivo.

  16. Galectin-8-N-domain Recognition Mechanism for Sialylated and Sulfated Glycans*

    Science.gov (United States)

    Ideo, Hiroko; Matsuzaka, Tsutomu; Nonaka, Takamasa; Seko, Akira; Yamashita, Katsuko

    2011-01-01

    Galectin-8 has much higher affinity for 3′-O-sulfated or 3′-O-sialylated glycoconjugates and a Lewis X-containing glycan than for oligosaccharides terminating in Galβ1→3/4GlcNAc, and this specificity is mainly attributed to the N-terminal carbohydrate recognition domain (N-domain, CRD) (Ideo, H., Seko, A., Ishizuka, I., and Yamashita, K. (2003) Glycobiology 13, 713–723). In this study, we elucidated the crystal structures of the human galectin-8-N-domain (-8N) in the absence or presence of 4 ligands. The apo molecule forms a dimer, which is different from the canonical 2-fold symmetric dimer observed for galectin-1 and -2. In a galectin-8N-lactose complex, the lactose-recognizing amino acids are highly conserved among the galectins. However, Arg45, Gln47, Arg59, and the long loop region between the S3 and S4 β-strands are unique to galectin-8N. These amino acids directly or indirectly interact with the sulfate or sialic acid moieties of 3′-sialyl- and 3′-sulfolactose complexed with galectin-8N. Furthermore, in the LNF-III-galectin-8N complex, van der Waals interactions occur between the α1–3-branched fucose and galactose and between galactose and Tyr141, and these interactions increase the affinity toward galectin-8N. Based on the findings of these x-ray crystallographic analyses, a mutagenesis study using surface plasmon resonance showed that Arg45, Gln47, and Arg59 of galectin-8N are indispensable and coordinately contribute to the strong binding of galectins-8N to sialylated and sulfated oligosaccharides. Arg59 is the most critical amino acid for binding in the S3–S4 loop region. PMID:21288902

  17. Galectin-8-N-domain recognition mechanism for sialylated and sulfated glycans.

    Science.gov (United States)

    Ideo, Hiroko; Matsuzaka, Tsutomu; Nonaka, Takamasa; Seko, Akira; Yamashita, Katsuko

    2011-04-01

    Galectin-8 has much higher affinity for 3'-O-sulfated or 3'-O-sialylated glycoconjugates and a Lewis X-containing glycan than for oligosaccharides terminating in Galβ1→3/4GlcNAc, and this specificity is mainly attributed to the N-terminal carbohydrate recognition domain (N-domain, CRD) (Ideo, H., Seko, A., Ishizuka, I., and Yamashita, K. (2003) Glycobiology 13, 713-723). In this study, we elucidated the crystal structures of the human galectin-8-N-domain (-8N) in the absence or presence of 4 ligands. The apo molecule forms a dimer, which is different from the canonical 2-fold symmetric dimer observed for galectin-1 and -2. In a galectin-8N-lactose complex, the lactose-recognizing amino acids are highly conserved among the galectins. However, Arg(45), Gln(47), Arg(59), and the long loop region between the S3 and S4 β-strands are unique to galectin-8N. These amino acids directly or indirectly interact with the sulfate or sialic acid moieties of 3'-sialyl- and 3'-sulfolactose complexed with galectin-8N. Furthermore, in the LNF-III-galectin-8N complex, van der Waals interactions occur between the α1-3-branched fucose and galactose and between galactose and Tyr(141), and these interactions increase the affinity toward galectin-8N. Based on the findings of these x-ray crystallographic analyses, a mutagenesis study using surface plasmon resonance showed that Arg(45), Gln(47), and Arg(59) of galectin-8N are indispensable and coordinately contribute to the strong binding of galectins-8N to sialylated and sulfated oligosaccharides. Arg(59) is the most critical amino acid for binding in the S3-S4 loop region.

  18. Sialylation regulates brain structure and function.

    Science.gov (United States)

    Yoo, Seung-Wan; Motari, Mary G; Susuki, Keiichiro; Prendergast, Jillian; Mountney, Andrea; Hurtado, Andres; Schnaar, Ronald L

    2015-07-01

    Every cell expresses a molecularly diverse surface glycan coat (glycocalyx) comprising its interface with its cellular environment. In vertebrates, the terminal sugars of the glycocalyx are often sialic acids, 9-carbon backbone anionic sugars implicated in intermolecular and intercellular interactions. The vertebrate brain is particularly enriched in sialic acid-containing glycolipids termed gangliosides. Human congenital disorders of ganglioside biosynthesis result in paraplegia, epilepsy, and intellectual disability. To better understand sialoglycan functions in the nervous system, we studied brain anatomy, histology, biochemistry, and behavior in mice with engineered mutations in St3gal2 and St3gal3, sialyltransferase genes responsible for terminal sialylation of gangliosides and some glycoproteins. St3gal2/3 double-null mice displayed dysmyelination marked by a 40% reduction in major myelin proteins, 30% fewer myelinated axons, a 33% decrease in myelin thickness, and molecular disruptions at nodes of Ranvier. In part, these changes may be due to dysregulation of ganglioside-mediated oligodendroglial precursor cell proliferation. Neuronal markers were also reduced up to 40%, and hippocampal neurons had smaller dendritic arbors. Young adult St3gal2/3 double-null mice displayed impaired motor coordination, disturbed gait, and profound cognitive disability. Comparisons among sialyltransferase mutant mice provide insights into the functional roles of brain gangliosides and sialoglycoproteins consistent with related human congenital disorders. © FASEB.

  19. Immunologic changes in TNF-alpha, sE-selectin, sP-selectin, sICAM-1, and IL-8 in pediatric patients treated for psoriasis with the Goeckerman regimen

    Energy Technology Data Exchange (ETDEWEB)

    Borska, L.; Fiala, Z.; Krejsek, J.; Andrys, C.; Vokurkova, D.; Hamakova, K.; Kremlacek, J.; Ettler, K. [Charles University of Prague, Hradec Kralove (Czech Republic). Faculty of Medicine

    2007-11-15

    Psoriasis is a chronic inflammatory skin disease which is often manifested during childhood. The present study investigated changes in the serum levels of proinflammatory cytokines and soluble forms of adhesion molecules in children with psoriasis. The observed patient group of 26 children was treated with the Goeckerman regimen. This therapy combines dermal application of crude coal tar with ultraviolet radiation. The Psoriasis Area Severity Index decreased significantly after treatment by with the Goeckerman regimen (p < 0.001). Serum levels of the proinflammatory cytokine TNF-alpha and adhesion molecules sICAM-1, sP-selectin and sE-selectin decreased after the Goeckerman regimen. The TNF-alpha and sICAM-1 decreased significantly (p < 0.05). Our findings support the complex role of these immune parameters in the immunopathogenesis of psoriasis in children. The serum level of IL-8 increased after the Goeckerman regimen. This fact indicates that the chemokine pathway of IL-8 activity could be modulated by this treatment, most likely by polycyclic aromatic hydrocarbons.

  20. Expression of Lewisa, Sialyl Lewisa, Lewisx, Sialyl Lewisx, Antigens as Prognostic Factors in Patients with Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Tohru Nakagoe

    2000-01-01

    Full Text Available BACKGROUND: Altered expression of blood group-related carbohydrate antigens such as sialyl Lewis (Lex antigen in tumours is associated with tumour progression behaviour and subsequent prognosis. However, the prognostic value of the expression of Le-related antigens in colorectal tumours remains unclear.

  1. ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

    DEFF Research Database (Denmark)

    Marcos, Nuno T; Bennett, Eric P; Gomes, Joana

    2011-01-01

    NAc-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl......Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6Gal......NAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues....

  2. Expression and Purification of Glycosyltransferases in Pichia Pastoris: Towards Improving the Migration of Stem Cells by Enhancing Surface Expression of Sialyl Lewis X

    KAUST Repository

    Al-Amoodi, Asma S.

    2017-05-01

    Recruitment of circulating cells towards target sites is primarily dependent on E-selectin receptor/ligand adhesive interactions. Glycosyltransferase (GTs) are involved in the creation of E-selectin ligands. A sialofucosylated terminal tetrasaccharide like glycan structure known as sialyl Lewis x (sLex), is the most recognized ligand by selectins. This structure is found on the surface of cancer cells and leukocytes but is often absent on the surface of many adult stem cell populations. In order to synthesize sLex, GTs must be endogenously expressed and remain active within the cells. Generally, these stem cells express terminal sialylated lactosamine structures on their glycoproteins which require the addition of alpha-(1,3)-fucose to be converted into an E-selectin ligand. There are a number of fucosyltransferases (FUTs) that are able to modify terminal lactosamine structures to create sLex such as FUT6. In this work we focused on expressing and purifying active recombinant FUTs as a tool to help create sLex structures on the surface of adult stem cells in order to enhance their migration.

  3. Differences in sialyl-Tn antigen expression between keratoacanthomas and cutaneous squamous cell carcinomas.

    Science.gov (United States)

    Jensen, P; Clausen, O P; Bryne, M

    1999-04-01

    Keratoacanthoma and squamous cell carcinoma are common skin tumors, especially in immunosuppressed transplant recipients, but the distinction between these two types of epidermal neoplasia may be difficult. Sialyl-Tn (Sia-GalNAc-O-Ser/Thr) is a cell surface carbohydrate associated with hyperplasia in squamous epithelium, and correlated with poor prognosis in several human adenocarcinomas. Twenty-seven keratoacanthomas and 29 cutaneous squamous cell carcinomas were examined for the expression of sialyl-Tn and of the Ki67 epitope, the latter a marker for cell proliferation. By immunohistochemistry, basaloid tumor cells at the periphery of tumor nests showed some degree of sialyl-Tn expression in 16 keratoacanthomas (59%), while only three squamous cell carcinomas (10%) showed sialyl-Tn-positive basaloid tumor cells (p<0.001). Keratinized, differentiated tumor cells were more often sialyl-Tn-positive in keratoacanthomas (89%) than in squamous cell carcinomas (31%) (p<0.001). A striking sialyl-Tn-positivity in the basal cell layer was found in a border zone directly adjacent to most tumors of both types (88 and 88%). By immunohistochemical examination of parallel sections and by double immunofluorescence, sialyl-Tn antigen expression was primarily seen in cells that did not express Ki67, although some overlap was present. Keratoacanthomas from transplant recipients did not differ in sialyl-Tn expression compared to those from non-immunosuppressed patients. The results indicate that sialyl-Tn expression is not directly related to cell proliferation, but rather to cellular features of post-mitotic cells, and that sialyl-Tn is not associated with a malignant phenotype. Sialyl-Tn may be linked to tumor regression, as seen in keratoacanthomas.

  4. The porcine paramyxovirus LPM specifically recognizes sialyl (alpha 2,3) lactose-containing structures.

    Science.gov (United States)

    Reyes-Leyva, J; Hernández-Jáuregui, P; Montaño, L F; Zenteno, E

    1993-01-01

    The porcine paramyxovirus LPM recognizes alpha, but not beta, anomers of sialic acid containing structures, specifically sialyl (alpha 2,3) lactose. The virus specificity is directed to the sialyl residue and to the C'4 axial OH and the C'6 CH2OH of the galactose present in this structure.

  5. ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

    DEFF Research Database (Denmark)

    Marcos, Nuno T; Bennett, Eric Paul; Gomes, Joana

    2011-01-01

    NAc-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl......-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we......NAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues....

  6. A Pasteurella multocida sialyltransferase displaying dual trans-sialidase activities for production of 3'-sialyl and 6'-sialyl glycans.

    Science.gov (United States)

    Guo, Yao; Jers, Carsten; Meyer, Anne S; Arnous, Anis; Li, Haiying; Kirpekar, Finn; Mikkelsen, Jørn D

    2014-01-20

    This study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose as the acceptor, resulting in production of both 3'-sialyllactose and 6'-sialyllactose. This is the first study reporting α-2,6-trans-sialidase activity of this sialyltransferase (EC 2.4.99.1 and 2.4.99.4). A response surface design was used to evaluate the effects of three reaction parameters (pH, temperature, and lactose concentration) on enzymatic production of 3'- and 6'-sialyllactoses using 5% (w/v) casein glycomacropeptide (equivalent to 9mM bound sialic acid) as the donor. The maximum yield of 3'-sialyllactose (2.75±0.35mM) was achieved at a reaction condition with pH 6.4, 40°C, 100mM lactose after 6h; and the largest concentration of 6'-sialyllactose (3.33±0.38mM) was achieved under a condition with pH 5.4, 40°C, 100mM lactose after 8h. 6'-sialyllactose was presumably formed from α-2,3 bound sialic acid in the casein glycomacropeptide as well as from 3'-sialyllactose produced in the reaction. The kcat/Km value for the enzyme using 3'-sialyllactose as the donor for 6'-sialyllactose synthesis at pH 5.4 and 40°C was determined to be 23.22±0.7M(-1)s(-1). Moreover, the enzyme was capable of catalyzing the synthesis of both 3'- and 6'-sialylated galactooligosaccharides, when galactooligosaccharides served as acceptors. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Sialylation of Campylobacter jejuni lipo-oligosaccharides: impact on phagocytosis and cytokine production in mice.

    Directory of Open Access Journals (Sweden)

    Ruth Huizinga

    Full Text Available Guillain-Barré syndrome (GBS is a post-infectious polyradiculoneuropathy, frequently associated with antecedent Campylobacter jejuni (C. jejuni infection. The presence of sialic acid on C. jejuni lipo-oligosaccharide (LOS is considered a risk factor for development of GBS as it crucially determines the structural homology between LOS and gangliosides, explaining the induction of cross-reactive neurotoxic antibodies. Sialylated C. jejuni are recognised by TLR4 and sialoadhesin; however, the functional implications of these interactions in vivo are unknown.In this study we investigated the effects of bacterial sialylation on phagocytosis and cytokine secretion by mouse myeloid cells in vitro and in vivo. Using fluorescently labelled GM1a/GD1a ganglioside-mimicking C. jejuni strains and corresponding (Cst-II-mutant control strains lacking sialic acid, we show that sialylated C. jejuni was more efficiently phagocytosed in vitro by BM-MΦ, but not by BM-DC. In addition, LOS sialylation increased the production of IL-10, IL-6 and IFN-β by both BM-MΦ and BM-DC. Subsequent in vivo experiments revealed that sialylation augmented the deposition of fluorescent bacteria in splenic DC, but not macrophages. In addition, sialylation significantly amplified the production of type I interferons, which was independent of pDC.These results identify novel immune stimulatory effects of C. jejuni sialylation, which may be important in inducing cross-reactive humoral responses that cause GBS.

  8. Sialylation of Campylobacter jejuni lipo-oligosaccharides: impact on phagocytosis and cytokine production in mice.

    Science.gov (United States)

    Huizinga, Ruth; Easton, Alistair S; Donachie, Anne M; Guthrie, Jim; van Rijs, Wouter; Heikema, Astrid; Boon, Louis; Samsom, Janneke N; Jacobs, Bart C; Willison, Hugh J; Goodyear, Carl S

    2012-01-01

    Guillain-Barré syndrome (GBS) is a post-infectious polyradiculoneuropathy, frequently associated with antecedent Campylobacter jejuni (C. jejuni) infection. The presence of sialic acid on C. jejuni lipo-oligosaccharide (LOS) is considered a risk factor for development of GBS as it crucially determines the structural homology between LOS and gangliosides, explaining the induction of cross-reactive neurotoxic antibodies. Sialylated C. jejuni are recognised by TLR4 and sialoadhesin; however, the functional implications of these interactions in vivo are unknown. In this study we investigated the effects of bacterial sialylation on phagocytosis and cytokine secretion by mouse myeloid cells in vitro and in vivo. Using fluorescently labelled GM1a/GD1a ganglioside-mimicking C. jejuni strains and corresponding (Cst-II-mutant) control strains lacking sialic acid, we show that sialylated C. jejuni was more efficiently phagocytosed in vitro by BM-MΦ, but not by BM-DC. In addition, LOS sialylation increased the production of IL-10, IL-6 and IFN-β by both BM-MΦ and BM-DC. Subsequent in vivo experiments revealed that sialylation augmented the deposition of fluorescent bacteria in splenic DC, but not macrophages. In addition, sialylation significantly amplified the production of type I interferons, which was independent of pDC. These results identify novel immune stimulatory effects of C. jejuni sialylation, which may be important in inducing cross-reactive humoral responses that cause GBS.

  9. Sialylated IgG-Fc: a novel biomarker of chronic inflammatory demyelinating polyneuropathy.

    Science.gov (United States)

    Wong, Anna Hiu Yi; Fukami, Yuki; Sudo, Makoto; Kokubun, Norito; Hamada, Shinsuke; Yuki, Nobuhiro

    2016-03-01

    Sialylation in Fc portion of IgG plays a crucial role in the pathogenesis of autoimmune diseases and the working mechanism of intravenous immunoglobulin (IVIG). We aim to test whether IgG-Fc sialylation is a biomarker of disease activity for chronic inflammatory demyelinating polyneuropathy (CIDP). By using specific lectins for sialylation, galactosylation and agalactosylation, lectin-enzyme assay and lectin blotting with pretreatment of IgG degradating enzyme of Streptococcus pyogenes were performed to compare the glycosylation levels of serum IgG-Fc (1) between patients of untreated CIDP (n=107) and normal control subjects (n=27), (2) among patients with untreated CIDP of different clinical severities and (3) before and after IVIG treatment of patients with CIDP (n=12). Sialylation and galactosylation of IgG-Fc were significantly reduced in patients with CIDP than normal control subjects (p=0.003 and 0.033, respectively), whereas agalactosylation was increased in CIDP (p=0.21). Ratios of sialylated/agalactosylated IgG-Fc levels were significantly reduced in CIDP (pdisease severity (p=0.044). After IVIG treatment, levels of sialylated IgG-Fc significantly increased (p=0.003). Sialylation of IgG-Fc is reduced in CIDP. Its level correlated with clinical severity and increased after IVIG treatment. Sialylated as well as ratio of sialylated/agalactosylated IgG-Fc could be new measures to monitor the disease severity and treatment status in CIDP. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  10. Quantitative enzymatic production of sialylated galactooligosaccharides with an engineered sialidase from Trypanosoma rangeli

    DEFF Research Database (Denmark)

    Zeuner, Birgitte; Holck, Jesper; Perna, Valentina

    2016-01-01

    molecules, providing the novelty point that GOS molecules are sialylated independently of their size by Tr13. Using CGMP, GOS, and Tr13, the production of gram-scale quantities of sialyl-GOS was achieved in 20L volume reactions. Compared to the benchmark transsialidase from pathogenic Trypanosoma cruzi...... be produced from κ(kappa)-casein glycomacropeptide (CGMP), a sialylated side stream component from cheese-making, by sialidase-catalyzed transsialylation. Using a rationally designed mutant of the sialidase from Trypanosoma rangeli, Tr13, with enhanced transsialylation activity, six different GOS preparations...

  11. Influence of the Length of the Lipooligosaccharide α Chain on Its Sialylation in Neisseria meningitidis

    OpenAIRE

    Tsai, Chao-Ming; Kao, George; Zhu, Peixuan

    2002-01-01

    The sialylation of lipooligosaccharide (LOS) in Neisseria meningitidis plays a role in the resistance of the organism to killing by normal human serum. The length of the α chain extending out from the heptose I [Hep (I)] moiety of LOS influenced sialylation of N. meningitidis LOS in vitro and in vivo. The α chain required a terminal Gal and a trisaccharide or longer oligosaccharide to serve as an acceptor for sialylation. The disaccharide lactose (Galβ1-4Glc) in the α chain of immunotype L8 L...

  12. Aberrant sialylation of granulocyte membranes in chronic myelogenous leukemia.

    Science.gov (United States)

    Baker, M A; Taub, R N; Whelton, C H; Hindenburg, A

    1984-05-01

    Peripheral blood granulocytes from patients with chronic myelogenous leukemia (CML) were studied for accessibility of membrane sialic acid and galactose residues to sodium borohydride-3H radiolabeling after oxidation with sodium metaperiodate (PI/B3H4) or with galactose oxidase (GO/B3H4). Granulocytes from untreated patients with chronic myelogenous leukemia showed increased radiolabeling with PI/B3H4, and decreased labeling with GO/B3H4 when compared to normal granulocytes. Granulocytes from leukemic patients receiving chemotherapy showed normal labeling patterns. Gel electrophoresis of membrane extracts showed that the changes in PI/B3H4 and GO/B3H4 reactivity of CML cells were distributed over all membrane protein bands. Our data suggest that CML granulocyte membrane proteins are aberrantly sialylated, with decreased accessibility of galactose residues, and that these changes may be reversed by clinical drug treatment.

  13. Cigarette smoking alters sialylation in the Fallopian tube of women, with implications for the pathogenesis of ectopic pregnancy.

    Science.gov (United States)

    Nio-Kobayashi, Junko; Abidin, Hazirah B Z; Brown, Jeremy K; Iwanaga, Toshihiko; Horne, Andrew W; Duncan, W Colin

    2016-12-01

    Sialylation creates a negative charge on the cell surface that can interfere with blastocyst implantation. For example, α2,6-sialylation on terminal galactose, catalyzed by the sialyltransferase ST6GAL1, inhibits the binding of galectin-1, a β-galactoside-binding lectin. We recently reported the potential involvement of galectin-1 and -3 in the pathogenesis of tubal ectopic pregnancy; however, the precise role of galectins and their ligand glycoconjugates remain unclear. Here, we investigated the expression of the genes encoding α2,3- and α2,6-galactoside sialyltransferases (ST3GAL1-6 and ST6GAL1-2) and the localization of sialic acids in the Fallopian tube of women with or without ectopic implantation. ST6GAL1 expression was higher in the mid-secretory phase than the proliferative phase of non-pregnant women (P < 0.0001), whereas ST6GAL1 (P < 0.0001), ST3GAL3 (P = 0.0029), ST3GAL5 (P = 0.0089), and ST3GAL6 (P = 0.0018) were all lower in Fallopian tubes with ectopic implantations. α2,3- and α2,6-sialic acids, however, both remained enriched on the surface of Fallopian tube epithelium. Cigarette smoking, a major risk factor for tubal ectopic pregnancy, was associated with reduced mid-secretory-phase expression of ST6GAL1 (P = 0.0298), but elevated expression of ST3GAL5 (P = 0.0006), an enzyme known to be involved in ciliogenesis. Indeed, sialic acid-containing ciliated inclusion cysts, which are associated with abnormal ciliogenesis, were observed within the epithelium at a higher frequency in women who smoked (P = 0.0177), suggesting that abnormal ciliogenesis is associated with smoking. Thus, cigarette smoking alters sialylation in the Fallopian tube epithelium, and is potentially a source of decreased tubal transport and increased receptivity for blastocyst in the human Fallopian tube. Mol. Reprod. Dev. 83: 1083-1091, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Sialyl alpha(2-->3) lactose clusters using carbosilane dendrimer core scaffolds as influenza hemagglutinin blockers.

    Science.gov (United States)

    Oka, Hiroyuki; Onaga, Tomotsune; Koyama, Tetsuo; Guo, Chao-Tan; Suzuki, Yasuo; Esumi, Yasuaki; Hatano, Ken; Terunuma, Daiyo; Matsuoka, Koji

    2008-08-01

    An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with sialyl alpha(2-->3) lactose (Neu5Acalpha(2-->3)Galbeta(1-->4)Glcbeta1-->) moieties was accomplished. The results of a preliminary study on biological responses against influenza virus hemagglutinin, using the sialyl lactose clusters showed unique biological activities on the basis of the structure-activity relationship according to the carbosilane scaffolds.

  15. Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase.

    Science.gov (United States)

    Holck, Jesper; Larsen, Dorte M; Michalak, Malwina; Li, Haiying; Kjærulff, Louise; Kirpekar, Finn; Gotfredsen, Charlotte H; Forssten, Sofia; Ouwehand, Arthur C; Mikkelsen, Jørn D; Meyer, Anne S

    2014-03-25

    A Trypanosoma cruzi trans-sialidase (E.C. 3.2.1.18) was cloned into Pichia pastoris and expressed. The pH and temperature optimum of the enzyme was determined as pH 5.7 and 30°C. Using casein glycomacropeptide (CGMP) and lactose as sialyl-donor and acceptor respectively, the optimal donor/acceptor ratio for the trans-sialidase catalysed 3'-sialyllactose production was found to be 1:4. Quantitative amounts of 3'-sialyllactose were produced from CGMP and lactose at a yield of 40mg/g CGMP. The 3'-sialyllactose obtained exerted a stimulatory effect on selected probiotic strains, including different Bifidobacterium strains in single culture fermentations. The trans-sialidase also catalysed the transfer of sialic acid from CGMP to galacto-oligosaccharides (GOS) and to the human milk oligosaccharide (HMO) backbone lacto-N-tetraose (LNT) to produce 3'-sialyl-GOS, including doubly sialylated GOS products, and 3'-sialyl-LNT, respectively. This work thus provides proof of the concept of producing 3'-sialyllactose and potentially other sialylated HMOs as well as sialylated GOS enzymatically by trans-sialidase activity, while at the same time providing valorisation of CGMP, a co-processing product from cheese manufacture. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. New derivatives of reducing oligosaccharides and their use in enzymatic reactions: efficient synthesis of sialyl Lewis a and sialyl dimeric Lewis x glycoconjugates.

    Science.gov (United States)

    Bårström, M; Bengtsson, M; Blixt, O; Norberg, T

    2000-10-06

    The reducing oligosaccharides lactose and lacto-N-tetraose were reductively aminated with benzyloxycarbonylaminoaniline and sodium cyanoborohydride, followed by treatment of the resulting secondary amines with acetic anhydride. The resulting N-acetyl-N-(4-benzyloxycarbonylaminophenyl)-1-amino-1-deoxyaldi tol oligosaccharide derivatives were subjected to stepwise enzymatic elongation with various glycosyltransferases/nucleotide sugars. Purification of the products after each enzymatic step was conveniently performed by solid-phase extraction on silica gel C-18 cartridges. Two oligosaccharide derivatives (with sialyl Lewis a and sialyl dimeric Lewis x structures) were prepared. Conversion of the obtained derivatives into neoglycoproteins by the sequence hydrogenolysis, thiophosgene treatment, and protein coupling was carried out.

  17. Potential ligands for cell adhesion molecules in human milk.

    Science.gov (United States)

    Schwertmann, A; Rudloff, S; Kunz, C

    1996-01-01

    In this study, glycoproteins and oligosaccharides with sialyl Lewis a, sialyl Lewis x, Lewis x, and Lewis y epitopes were isolated by ultracentrifugation and fast-protein liquid chromatography from human milk of mothers with term or preterm infants. The identification of these epitopes on whey proteins was achieved by monoclonal antibodies and lectins after Western blotting. Lactose-derived oligosaccharides were characterized by high-performance thin-layer chromatography and high-pH anion-exchange chromatography with pulsed amperometric detection. These carbohydrate epitopes which are potential ligands for selections are not of cellular origin but appear in the soluble fraction of milk. Here, they are present as lactose-derived oligosaccharides (molecular weight 100 kD). Lewis antigens might represent another category of protective nonimmunological substances in human milk with the potential to influence inflammatory processes in human milk fed infants.

  18. Spatial screening of hemagglutinin on Influenza A virus particles: Sialyl-LacNAc displays on DNA and PEG scaffolds reveal the requirements for bivalency enhanced interactions with weak monovalent binders.

    Science.gov (United States)

    Bandlow, Victor; Liese, Susanne; Lauster, Daniel; Ludwig, Kai; Netz, Roland R; Herrmann, Andreas; Seitz, Oliver

    2017-10-20

    Attachment of the Influenza A virus onto host cells involves multivalent interactions between virus surface hemagglutinin (HA) and sialoside-containing glyco ligands. Despite the development of extremely powerful multivalent bind-ers of the Influenza virus and other viruses, comparably little is known about the optimal spacing of HA ligands, which ought to bridge binding sites within or across the trimeric HA molecules. To explore the criteria for ligand economical high affinity binding, we systematically probed distance-affinity relationships by means of two differently behaving scaffold types based on i) flexible polyethylene glycol (PEG) conjugates and ii) rigid self-assembled DNA·PNA complexes. The bivalent scaffolds presented two sialyl-LacNAc ligands in 23-101 Å distance. A combined analysis of binding by means of microscale thermophoresis measurements and statistical mechanics models exposed the inherent limitations of PEG-based spacers. Given the distance requirements of HA, the flexibility of PEG scaffolds is too high to raise the effective concentration of glyco ligands above a value that allows interactions with the low affinity binding site. By contrast, spatial screening with less flexible, self-assembled peptide nucleic acid (PNA)·DNA complexes uncovered a well-defined and, surprisingly, bimodal distance-affinity relationship for interactions of the Influenza A virus HA with bivalent displays of the natural sialyl-LacNAc ligand. Optimal constructs conferred 10(3)-fold binding enhancements with only two ligands. We discuss the existence of secondary binding sites and shine light on the preference for intramolecular rather than intermolecular recognition of HA trimers on the virus surface.

  19. Generation of biologically active multi-sialylated recombinant human EPOFc in plants.

    Directory of Open Access Journals (Sweden)

    Alexandra Castilho

    Full Text Available Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO. Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i branching (ii β1,4-galactosylation as well as for the (iii synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.

  20. Pseudomonas aeruginosa pyocyanin modulates mucin glycosylation with sialyl-Lewis(x) to increase binding to airway epithelial cells.

    Science.gov (United States)

    Jeffries, Jayme L; Jia, Jing; Choi, Woosuk; Choe, Shawn; Miao, Jinfeng; Xu, Ying; Powell, Rebecca; Lin, Jingjun; Kuang, Zhizhou; Gaskins, H Rex; Lau, Gee W

    2016-07-01

    Cystic fibrosis (CF) patients battle life-long pulmonary infections with the respiratory pathogen Pseudomonas aeruginosa (PA). An overabundance of mucus in CF airways provides a favorable niche for PA growth. When compared with that of non-CF individuals, mucus of CF airways is enriched in sialyl-Lewis(x), a preferred binding receptor for PA. Notably, the levels of sialyl-Lewis(x) directly correlate with infection severity in CF patients. However, the mechanism by which PA causes increased sialylation remains uncharacterized. In this study, we examined the ability of PA virulence factors to modulate sialyl-Lewis(x) modification in airway mucins. We found pyocyanin (PCN) to be a potent inducer of sialyl-Lewis(x) in both mouse airways and in primary and immortalized CF and non-CF human airway epithelial cells. PCN increased the expression of C2/4GnT and ST3Gal-IV, two of the glycosyltransferases responsible for the stepwise biosynthesis of sialyl-Lewis(x), through a tumor necrosis factor (TNF)-α-mediated phosphoinositol-specific phospholipase C (PI-PLC)-dependent pathway. Furthermore, PA bound more efficiently to airway epithelial cells pre-exposed to PCN in a flagellar cap-dependent manner. Importantly, antibodies against sialyl-Lewis(x) and anti-TNF-α attenuated PA binding. These results indicate that PA secretes PCN to induce a favorable environment for chronic colonization of CF lungs by increasing the glycosylation of airway mucins with sialyl-Lewis(x).

  1. Histochemical identification of sialylated glycans in Xenopus laevis testis

    Science.gov (United States)

    Valbuena, Galder; Alonso, Edurne; Ubago, María Martínez; Madrid, Juan Francisco; Díaz-Flores, Lucio; Sáez, Francisco José

    2012-01-01

    Carbohydrate chains of glycoprotein and glycosphingolipids are highly diverse molecules involved in many cell functions, including cell recognition, adhesion and signalling. Sialylated glycans are of special interest because the terminal position of sialic acid (NeuAc) in glycans linked by different ways to subterminal monosaccharides has been shown to be involved in several biological processes, as occurs with gangliosides, which have been reported as being essential in spermatogenesis in mammals. Some glycan-binding proteins, the lectins, which specifically recognize glycan sequences, have been extensively used to characterize tissue and cell carbohydrates by means of cytochemical techniques. The aim of the present work was to determine the presence of NeuAc by means of histochemical techniques in the testis of Xenopus laevis, an animal model widely used in cell and molecular biology research. However, considering that some NeuAc-binding lectins are capable of binding to N-acetylglucosamine (GlcNAc), other GlcNAc-binding lectins were also assayed. The results showed that NeuAc is mainly expressed in the interstitium, and only a weak labelling in the male germ cells was observed. Most NeuAc was located in O-linked oligosaccharides, but some masked NeuAc in N-glycans were identified in primary and secondary spermatogonia and spermatocytes. By contrast, GlcNAc was widely expressed in all germ cell types. Deglycosylative pre-treatments suggest that both N- and O-glycans and/or glycolipids could be responsible for this labelling. In addition, GlcNAc in O-linked oligosaccharides has been identified in spermatogonial cells. The acrosome of spermatids was always negative. Variations of glycan expression have been found in different cell types, suggesting that glycosylation is modified during spermatogenetic development. PMID:22881213

  2. Response of high-risk of recurrence/progression bladder tumours expressing sialyl-Tn and sialyl-6-T to BCG immunotherapy

    OpenAIRE

    Lima, Luís; Severino, Paulo; Silva, Mariana; Miranda, Andreia; Tavares, Ana; Pereira, Sofia; Fernandes, Elisabete; Cruz, Ricardo; Amaro, Teresina; Reis, Celso; Dall'Olio, Fabio; Amado, Francisco; Videira, Paula; Santos, Lúcio; Ferreira, José Alexandre

    2013-01-01

    Background: High risk of recurrence/progression bladder tumours is treated with Bacillus Calmette-Guérin (BCG) immunotherapy after complete resection of the tumour. Approximately 75% of these tumours express the uncommon carbohydrate antigen sialyl-Tn (Tn), a surrogate biomarker of tumour aggressiveness. Such changes in the glycosylation of cell-surface proteins influence tumour microenvironment and immune responses that may modulate treatment outcome and the course of disease. The aim of thi...

  3. Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation

    DEFF Research Database (Denmark)

    Yin, Bojiao; Gao, Yuan; Chung, Cheng-yu

    2015-01-01

    -glycosylation of recombinant erythropoietin (rEPO), a human α2,6-sialyltransferase (ST6Gal1) was expressed in Chinese hamster ovary (CHO-K1) cells. Sialylation increased on both EPO and CHO cellular proteins as observed by SNA lectin analysis, and HPLC profiling revealed that the sialic acid content of total glycans on EPO...

  4. Intravenous immunoglobulin G (IVIg): Properties of dimeric and sialylated IgG

    NARCIS (Netherlands)

    Guhr, T.

    2013-01-01

    This thesis describes the properties of the IgG dimer and sialylated IgG fraction in IVIg. Both fractions have been suggested to contribute to the immune modulating effects of IVIg in the treatment of autoimmune and inflammatory diseases. Taken together we can conclude that the IgG dimer fraction is

  5. T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.

    Science.gov (United States)

    Hess, Constanze; Winkler, André; Lorenz, Alexandra K; Holecska, Vivien; Blanchard, Véronique; Eiglmeier, Susanne; Schoen, Anna-Lena; Bitterling, Josephine; Stoehr, Alexander D; Petzold, Dominique; Schommartz, Tim; Mertes, Maria M M; Schoen, Carolin T; Tiburzy, Ben; Herrmann, Anne; Köhl, Jörg; Manz, Rudolf A; Madaio, Michael P; Berger, Markus; Wardemann, Hedda; Ehlers, Marc

    2013-09-01

    Antigen-specific Abs are able to enhance or suppress immune responses depending on the receptors that they bind on immune cells. Recent studies have shown that pro- or antiinflammatory effector functions of IgG Abs are also regulated through their Fc N-linked glycosylation patterns. IgG Abs that are agalactosylated (non-galactosylated) and asialylated are proinflammatory and induced by the combination of T cell-dependent (TD) protein antigens and proinflammatory costimulation. Sialylated IgG Abs, which are immunosuppressive, and Tregs are produced in the presence of TD antigens under tolerance conditions. T cell-independent (TI) B cell activation via B cell receptor (BCR) crosslinking through polysaccharides or via BCR and TLR costimulation also induces IgG Abs, but the Fc glycosylation state of these Abs is unknown. We found in mouse experiments that TI immune responses induced suppressive sialylated IgGs, in contrast to TD proinflammatory Th1 and Th17 immune responses, which induced agalactosylated and asialylated IgGs. Transfer of low amounts of antigen-specific sialylated IgG Abs was sufficient to inhibit B cell activation and pathogenic immune reactions. These findings suggest an immune regulatory function for TI immune responses through the generation of immunosuppressive sialylated IgGs and may provide insight on the role of TI immune responses during infection, vaccination, and autoimmunity.

  6. Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase

    DEFF Research Database (Denmark)

    Holck, Jesper; Larsen, Dorte Møller; Michalak, Malwina

    2014-01-01

    A Trypanosoma cruzi trans-sialidase (E.C. 3.2.1.18) was cloned into Pichia pastoris and expressed. The pH and temperature optimum of the enzyme was determined as pH 5.7 and 30°C. Using casein glycomacropeptide (CGMP) and lactose as sialyl-donor and acceptor respectively, the optimal donor...

  7. T cell–independent B cell activation induces immunosuppressive sialylated IgG antibodies

    Science.gov (United States)

    Hess, Constanze; Winkler, André; Lorenz, Alexandra K.; Holecska, Vivien; Blanchard, Véronique; Eiglmeier, Susanne; Schoen, Anna-Lena; Bitterling, Josephine; Stoehr, Alexander D.; Petzold, Dominique; Schommartz, Tim; Mertes, Maria M.M.; Schoen, Carolin T.; Tiburzy, Ben; Herrmann, Anne; Köhl, Jörg; Manz, Rudolf A.; Madaio, Michael P.; Berger, Markus; Wardemann, Hedda; Ehlers, Marc

    2013-01-01

    Antigen-specific Abs are able to enhance or suppress immune responses depending on the receptors that they bind on immune cells. Recent studies have shown that pro- or antiinflammatory effector functions of IgG Abs are also regulated through their Fc N-linked glycosylation patterns. IgG Abs that are agalactosylated (non-galactosylated) and asialylated are proinflammatory and induced by the combination of T cell–dependent (TD) protein antigens and proinflammatory costimulation. Sialylated IgG Abs, which are immunosuppressive, and Tregs are produced in the presence of TD antigens under tolerance conditions. T cell–independent (TI) B cell activation via B cell receptor (BCR) crosslinking through polysaccharides or via BCR and TLR costimulation also induces IgG Abs, but the Fc glycosylation state of these Abs is unknown. We found in mouse experiments that TI immune responses induced suppressive sialylated IgGs, in contrast to TD proinflammatory Th1 and Th17 immune responses, which induced agalactosylated and asialylated IgGs. Transfer of low amounts of antigen-specific sialylated IgG Abs was sufficient to inhibit B cell activation and pathogenic immune reactions. These findings suggest an immune regulatory function for TI immune responses through the generation of immunosuppressive sialylated IgGs and may provide insight on the role of TI immune responses during infection, vaccination, and autoimmunity. PMID:23979161

  8. Synthesis of dimeric lactose and dimeric (sialyl) Lewis(X) glycolipids.

    Science.gov (United States)

    Gege, Christian; Schmidt, Richard R

    2002-06-12

    To investigate structural requirements for the homophilic interaction between carbohydrates on planar model membranes, divalent derivatives with enforced proximity between the two carbohydrate epitopes (lactose, Lewis(X), and sialyl Lewis(X)) were synthesized by use of a dimeric membrane anchor as scaffold.

  9. Synthesis of oxime-linked mucin mimics containing thetumor-related TN and sialyl TN antigens

    Energy Technology Data Exchange (ETDEWEB)

    Marcaurelle, Lisa A.; Shin, Youngsook; Goon, Scarlett; Bertozzi,Carolyn R.

    2001-08-21

    The synthesis of oxime-linked mucin mimics was accomplished via the incorporation of multiple ketone residues into a peptide followed by reaction with aminooxy sugars corresponding to the tumor-related T{sub N} and sialyl T{sub N} (ST{sub N}) antigens.

  10. Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG after lectin fractionation.

    Directory of Open Access Journals (Sweden)

    Fabian Käsermann

    Full Text Available It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1 or acidified lactose (E2 were analyzed for total IgG, F(ab'(2 and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2; again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10% of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

  11. Enzymatic sialylation of IgA1 O-glycans: implications for studies of IgA nephropathy.

    Directory of Open Access Journals (Sweden)

    Kazuo Takahashi

    Full Text Available Patients with IgA nephropathy (IgAN have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal-deficient N-acetylgalactosamine (GalNAc with or without N-acetylneuraminic acid (NeuAc. We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1 of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal than α2,6-sialylated O-glycans (NeuAc attached to GalNAc. As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc, we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a Gal

  12. Structural characterization of sialylated glycoforms of H. influenzae by electrospray mass spectrometry: fragmentation of protonated and sodiated O-deacylated lipopolysaccharides.

    Science.gov (United States)

    Li, Jianjun; Dzieciatkowska, Monika; Hood, Derek W; Cox, Andrew D; Schweda, Elke K H; Moxon, E Richard; Richards, James C

    2007-01-01

    Sialylated lipopolysaccharide (LPS) glycoforms from Haemophilus influenzae were characterized by tandem mass spectrometry using a new generation hyphenated mass spectrometer which combines a triple quadrupole and a linear ion trap (Q-Trap). The fragmentation of both protonated and sodiated molecular ions from O-deacylated LPS (LPS-OH) obtained in MS(2) experiments in the positive mode was studied. The MS(2) spectra of protonated ions provided unambiguous evidence for the presence and sequence of sialylated lactosamine present in lacto-N-neotetraose oligosaccharide extensions but not for sialyl-lactose structures whilst fragmentation of sodiated adducts, [M+Na](+), afforded information diagnostic of mono- and disialylated lactose extensions. To study this we used a highly sialylated LPS from a H. influenzae strain capable of sialyl-lactose expression only. We then applied the method to the H. influenzae genome strain, Rd, in which glycoforms containing both sialyl-lactose and sialyl-lacto-N-neotetraose were detected from diagnostic B-ions at m/z 638.2 ([Neu5Ac(1) Hex(2)+Na](+)) and 657.2 ([Neu5Ac(1) Hex(1) HexNAc(1)+H](+)). Unique fragmentation patterns provided the locations and sequences of these oligosaccharide extensions. This is the first time both sialylated lactose and sialylated lacto-N-neotetraose units have been detected and characterized by tandem mass spectrometry in the same molecule. This methodology is of general applicability for determination of common sialylated oligosaccharide extension in bacterial LPS.

  13. CMP-Sialic Acid Synthetase: The Point of Constriction in the Sialylation Pathway.

    Science.gov (United States)

    Sellmeier, Melanie; Weinhold, Birgit; Münster-Kühnel, Anja

    2015-01-01

    Sialoglycoconjugates form the outermost layer of animal cells and play a crucial role in cellular communication processes. An essential step in the biosynthesis of sialylated glycoconjugates is the activation of sialic acid to the monophosphate diester CMP-sialic acid. Only the activated sugar is transported into the Golgi apparatus and serves as a substrate for the linkage-specific sialyltransferases. Interference with sugar activation abolishes sialylation and is embryonic lethal in mammals. In this chapter we focus on the enzyme catalyzing the activation of sialic acid, the CMP-sialic acid synthetase (CMAS), and compare the enzymatic properties of CMASs isolated from different species. Information concerning the reaction mechanism and active site architecture is included. Moreover, the unusual nuclear localization of vertebrate CMASs as well as the biotechnological application of bacterial CMAS enzymes is addressed.

  14. A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.

    Science.gov (United States)

    Yin, Bojiao; Wang, Qiong; Chung, Cheng-Yu; Bhattacharya, Rahul; Ren, Xiaozhi; Tang, Juechun; Yarema, Kevin J; Betenbaugh, Michael J

    2017-08-01

    A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu 3 ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well. For example, addition of 200-300 µM 1,3,4-O-Bu 3 ManNAc resulted in >40% increase in final sialic acid content of recombinant EPO, while natural ManNAc at ∼100 times higher concentration of 20 mM produced a less profound change in EPO sialylation. Collectively, these results indicate that butyrate-derivatization of ManNAc improves the capacity of cells to incorporate exogenous ManNAc into the sialic acid biosynthetic pathway and thereby increase sialylation of recombinant EPO and other glycoproteins. This study establishes 1,3,4-O-Bu 3 ManNAc as a novel chemical supplement to improve glycoprotein quality and sialylation levels at concentrations orders of magnitude lower than alternative approaches. Biotechnol. Bioeng. 2017;114: 1899-1902. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Characterization of two novel sialyl N-acetyllactosaminyl nucleotides separated from ovine colostrum

    OpenAIRE

    Sasaki, Masashi; Nakamura, Tadashi; Hirayama, Kentaro; Fukuda, Kenji; Saito, Tadao; Urashima, Tadasu; Asakuma, Sadaki

    2016-01-01

    The milk/colostrum of some mammalian species is known to contain sugar nucleotides including uridine diphosphate (UDP) oligosaccharides in addition to lactose and milk oligosaccharides, but the detailed structures of these UDP oligosaccharides have not so far been clarified. In this study we isolated two UDP-sialyl N-acetyllactosamines from ovine colostrum and characterized them using H-1-NMR and MALDITOFMS spectroscopies. Their structures were found to be Neu5Gc(alpha 2 - 3) Gal (beta 1 - 4)...

  16. Pseudomonas aeruginosa mucoid strain 8830 binds glycans containing the sialyl-Lewis x epitope.

    Science.gov (United States)

    Xia, Baoyun; Sachdev, Goverdhan P; Cummings, Richard D

    2007-01-01

    Pseudomonas aeruginosa infection of patients with cystic fibrosis (CF) is a leading cause of their morbidity and mortality. Pathogenesis is initiated in part by molecular interactions of P. aeruginosa with carbohydrate residues in airway mucins that accumulate in the lungs of patients with this disease. To explore the nature of the glycans recognized by a stable, mucoid, alginate-producing strain P. aeruginosa 8830 we generated a genetically modified Pa8830 expressing green fluorescent protein (Pa3380-GFP). We tested its binding to a panel of glycolipids and neoglycolipids in which selected glycans were covalently attached to dipalmitoyl phosphatidylethanolamine and analyzed on silica gel surfaces. Among all glycans tested, Pa8830-GFP bound best to sialyl-Le(x)-containing glycan NeuAc(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R and bound weakly to H-type blood group Fucalpha1-2Galbeta1-4GlcNAc-R, sialyl-lactose, and Le(x), and exhibited little binding toward non-fucosylated derivatives. Interestingly, while Pa8830-GFP bound to the glycosphingolipid asialoGM1, it did not appear to bind to a wide variety of other glycosphingolipids including GM1, GM2, asialoGM2, and sulfatide. These results indicate that P. aeruginosa 8830 has preferential binding to sialyl-Le(x)-containing glycans and has weak recognition of related fucose- and sialic acid-containing glycans. The finding that Pa8830 binds sialyl-Le(x)-containing glycans, which occur at increased levels in mucins from CF patients, is consistent with studies of other strains of P. aeruginosa and further suggests that such glycans on CF mucins contribute to disease pathogenesis.

  17. Saturation transfer difference NMR and computational modeling of a sialoadhesin-sialyl lactose complex.

    Science.gov (United States)

    Bhunia, Anirban; Jayalakshmi, V; Benie, Andrew J; Schuster, Oliver; Kelm, Sørge; Rama Krishna, N; Peters, Thomas

    2004-01-22

    The siglecs are a family of I-type lectins binding to sialic acids on the cell surface. Sialoadhesin (siglec-1) is expressed at much higher levels in inflammatory macrophages and specifically binds to alpha-2,3-sialylated N-acetyl lactosamine residues of glycan chains. The terminal disaccharide alpha-D-Neu5Ac-(2-->3)-beta-D-Gal is thought to be the main epitope recognized by sialoadhesin. To understand the basis of this biological recognition reaction we combined NMR experiments with a molecular modeling study. We employed saturation transfer difference (STD) NMR experiments to characterize the binding epitope of alpha-2,3-sialylated lactose, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-D-Glc 1 to sialoadhesin at atomic resolution. The experimental results were compared to a computational docking model and to X-ray data of a complex of sialyl lactose and sialoadhesin. The data reveal that sialoadhesin mainly recognizes the N-acetyl neuraminic acid and a small part of the galactose moiety of 1. The crystal structure of a complex of sialoadhesin with sialyl lactose 1 was used as a basis for a modeling study using the FlexiDock algorithm. The model generated was very similar to the original crystal structure. Therefore, the X-ray data were used to predict theoretical STD values utilizing the CORCEMA-STD protocol. The good agreement between experimental and theoretical STD values indicates that a combined modeling/STD NMR approach yields a reliable structural model for the complex of sialoadhesin with alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-D-Glc 1 in aqueous solution.

  18. Sialyl Lewisxexpression at the invasive front as a predictive marker of liver recurrence in stage II colorectal cancer.

    Science.gov (United States)

    Yamadera, Masato; Shinto, Eiji; Tsuda, Hitoshi; Kajiwara, Yoshiki; Naito, Yoshihisa; Hase, Kazuo; Yamamoto, Junji; Ueno, Hideki

    2018-01-01

    Sialyl Lewis x (SLX) is a carbohydrate ligand for endothelial selectin that participates in cell adhesion, proliferation and scattering. It plays an important role in cancer cell adhesion to vascular endothelial cells, leading to hematogenous metastasis. The prognostic significance of SLX expression level at the invasive front in patients with stage II colorectal cancer (CRC) was examined. A total of 209 patients with stage II CRC curatively resected between 1997 and 2000 were enrolled. The preoperative serum SLX levels measured by radioimmunoassay and SLX immunoexpression levels at the invasive front, and at the non-invasive frontal region determined by tissue microarray were analyzed. SLX expression at the invasive front was positively associated with tumor invasion depth (P=0.007) and tumor budding grade (P=0.038). Disease-free survival curves differed between the high and low SLX-expression groups (5-year survival rates, 77.0 and 89.7%, respectively; P=0.036). Liver cancer recurrence was more frequent in the high-expression group than in the low-expression group (15.9 and 2.4%; P=0.002). Multivariate analysis revealed that its expression (hazard ratio, 5.26; P=0.015) and venous invasion (hazard ratio, 4.14; P=0.040) were independent predictive markers of liver cancer recurrence. Neither the preoperative serum SLX level nor SLX expression at the non-invasive frontal region showed any association with histopathological features or disease-free survival. SLX expression level at the invasive front is a promising marker for identifying patients with stage II CRC with a high risk of liver cancer recurrence.

  19. A Pasteurella multocida sialyltransferase displaying dual trans-sialidase activities for production of 3′-sialyl and 6′-sialyl glycans

    DEFF Research Database (Denmark)

    Guo, Yao; Jers, Carsten; Meyer, Anne S.

    2014-01-01

    tThis study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)--d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose asthe......, andlactose concentration) on enzymatic production of 3- and 6-sialyllactoses using 5% (w/v) casein glyco-macropeptide (equivalent to 9 mM bound sialic acid) as the donor. The maximum yield of 3-sialyllactose(2.75 ± 0.35 mM) was achieved at a reaction condition with pH 6.4, 40◦C, 100 mM lactose after 6 h......; andthe largest concentration of 6-sialyllactose (3.33 ± 0.38 mM) was achieved under a condition with pH5.4, 40◦C, 100 mM lactose after 8 h. 6-sialyllactose was presumably formed from -2,3 bound sialic acidin the casein glycomacropeptide as well as from 3-sialyllactose produced in the reaction. The kcat...

  20. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation

    Directory of Open Access Journals (Sweden)

    Haitham A. Badr

    2015-12-01

    Full Text Available This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, “Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show sialylated glycans intracellularly. The impact of sialic acid supplementation under nutrient deprivation was demonstrated by measuring levels of expression and sialylation of two markers, EGFR1 and MUC1. This Data in Brief article complements the main manuscript by providing detailed instructions and representative results for cell-level imaging and Western blot analyses of changes in sialylation during nutrient deprivation and sialic acid supplementation. These methods can be readily generalized for the study of many types of glycosylation and various glycoprotein markers through the appropriate selection of fluorescently-labeled lectins.

  1. Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine kappa-Casein-Derived Glycomacropeptide as the Donor Substrate

    NARCIS (Netherlands)

    Wilbrink, Maarten H.; ten Kate, Geert A.; van Leeuwen, Sander S.; Sanders, Peter; Sallomons, Erik; Hage, Johannes A.; Dijkhuizen, Lubbert; Kamerling, Johannis P.

    2014-01-01

    trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(alpha 2-3) Gal(beta 1-x)-glycans (sialo-glycans) to Gal(beta 1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)(n)Glc oligosaccharide mixtures (GOS) using

  2. Up-regulation of the oligosaccharide sialyl LewisX: a new prognostic parameter in metastatic prostate cancer.

    Science.gov (United States)

    Jørgensen, T; Berner, A; Kaalhus, O; Tveter, K J; Danielsen, H E; Bryne, M

    1995-05-01

    Metastatic prostate cancer has an unpredictable long-term prognosis. At present, there are few specific predictors to indicate the outcome for the individual patient. We have studied immunoreactivity for type-2 carbohydrate structures, known to be involved in various cell adhesion processes, in patients with metastatic prostate cancer. One group of patients (n = 26) did not progress within 3 years after orchiectomy, while another group of patients (n = 33) progressed within 1 year following castration and survived less than 2 years. Among the parameters studied, sialyl LewisX carbohydrate up-regulation was the only variable showing significant association with poor prognosis (P < 0.01). Sialyl LewisX discriminated between these two outcome groups with 71% predictability and 96% specificity. Our results indicate that up-regulation of sialyl LewisX is associated with hormonal-resistant, aggressive disease. This prognostic marker may, therefore, have an important role in selecting proper treatment for patients with metastatic prostate cancer.

  3. Characterization of the specific interaction between sialoadhesin and sialylated Campylobacter jejuni lipooligosaccharides.

    Science.gov (United States)

    Heikema, Astrid P; Bergman, Mathijs P; Richards, Hannah; Crocker, Paul R; Gilbert, Michel; Samsom, Janneke N; van Wamel, Willem J B; Endtz, Hubert P; van Belkum, Alex

    2010-07-01

    In Campylobacter jejuni-induced Guillain-Barré syndrome (GBS), molecular mimicry between C. jejuni lipooligosaccharide (LOS) and host gangliosides leads to the production of cross-reactive antibodies directed against the peripheral nerves of the host. Currently, the presence of surface exposed sialylated LOS in C. jejuni is the single known bacterial pathogenesis factor associated with the development of GBS. Using a unique, well-characterized strain collection, we demonstrate that GBS-associated C. jejuni strains bind preferentially to sialoadhesin (Sn, Siglec-1, or CD169), a sialic acid receptor found on a subset of macrophages. In addition, using a whole-cell enzyme-linked immunosorbent assay (ELISA), C. jejuni strains with sialylated LOS bound exclusively to soluble Sn. Mass spectrometry revealed that binding was sialic acid-linkage specific with a preference for alpha(2,3)-linked sialic acid attached to the terminal galactose of the LOS chain as seen in the gangliosides GD1a, GM1b, and GM3. This molecular interaction was also related to functional consequences as a GBS-associated C. jejuni strain that bound Sn in a whole-cell ELISA adhered to surface-expressed Sn of Sn-transfected CHO cells but was unable to adhere to wild-type CHO cells. Moreover, a sialic acid-negative mutant of the same C. jejuni strain was unable to bind Sn-transfected CHO cells. This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor (P = 0.014). Moreover, because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in GBS disease.

  4. Neoplastic transformation of the thyroid gland is accompanied by changes in cellular sialylation.

    Science.gov (United States)

    Babál, Pavel; Janega, Pavol; Cerná, Andrea; Kholová, Ivana; Brabencová, Eva

    2006-01-01

    Cancer of the thyroid gland is one of the most common endocrine diseases. Histological evaluation is often complicated by difficulty in distinguishing between benign and malignant lesions. Abnormal glycosylation of cell structures, including changes in sialylation, is a feature of the neoplastic transformation process. The aim of this study was to evaluate associations between neoplastic changes in the thyroid gland and changes in sialylation, with reference to its terminal linkage type. Lectin histochemistry using three sialic acid-binding lectins: Tritrichomonas mobilensis lectin (TML), which recognizes sialic acid without linkage preference; Maackia amurensis leukoagglutinin (MAL), which preferentially binds alpha-2,3-linked sialic acid; and Sambucus nigra agglutinin (SNA), which preferentially binds alpha-2,6-linked sialic acid, were used for detection of sialylated glycoconjugates in 50 human thyroid gland specimens. These included papillary, follicular, oncocytic, medullary and anaplastic carcinomas, follicular adenomas and benign follicular and parenchymatous goiter. The luminal surface of follicular cells in normal thyroid glands, adenomas and goiters showed weak or absent labelling for sialic acid. Malignant transformation of the gland was accompanied by an increase of sialic acid positivity on follicular epithelial cells, especially of alpha-2,3-linked sialic acid. Strong luminal positivity for sialic acid was found in papillary carcinomas, whereas moderate positivity was seen in follicular carcinomas. Inconsistent, weak positivity for sialic acid was documented in medullary and anaplastic carcinomas. Increased membrane sialic acid on thyroid gland cells may be an important diagnostic pathological finding, that could be useful in distinction of malignant from benign thyroid lesions, especially with respect to aspiration cytology diagnostics.

  5. Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans

    DEFF Research Database (Denmark)

    Jers, Carsten; Michalak, Malwina; Larsen, Dorte Møller

    2014-01-01

    This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used...... to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197–203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous...

  6. Enrichment of sialylated IgG by lectin fractionation does not enhance the efficacy of immunoglobulin G in a murine model of immune thrombocytopenia.

    Directory of Open Access Journals (Sweden)

    Theresa Guhr

    Full Text Available Intravenous immunoglobulin G (IVIg is widely used against a range of clinical symptoms. For its use in immune modulating therapies such as treatment of immune thrombocytopenic purpura high doses of IVIg are required. It has been suggested that only a fraction of IVIg causes this anti immune modulating effect. Recent studies indicated that this fraction is the Fc-sialylated IgG fraction. The aim of our study was to determine the efficacy of IVIg enriched for sialylated IgG (IVIg-SA⁺ in a murine model of passive immune thrombocytopenia (PIT. We enriched IVIg for sialylated IgG by Sambucus nigra agglutinin (SNA lectin fractionation and determined the degree of sialylation. Analysis of IVIg-SA⁺ using a lectin-based ELISA revealed that we enriched predominantly for Fab-sialylated IgG, whereas we did not find an increase in Fc-sialylated IgG. Mass spectrometric analysis confirmed that Fc sialylation did not change after SNA lectin fractionation. The efficacy of sialylated IgG was measured by administering IVIg or IVIg-SA⁺ 24 hours prior to an injection of a rat anti-mouse platelet mAb. We found an 85% decrease in platelet count after injection of an anti-platelet mAb, which was reduced to a 70% decrease by injecting IVIg (p<0.01. In contrast, IVIg-SA⁺ had no effect on the platelet count. Serum levels of IVIg and IVIg-SA⁺ were similar, ruling out enhanced IgG clearance as a possible explanation. Our results indicate that SNA lectin fractionation is not a suitable method to enrich IVIg for Fc-sialylated IgG. The use of IVIg enriched for Fab-sialylated IgG abolishes the efficacy of IVIg in the murine PIT model.

  7. Effect of alpha 2,6 sialylation on integrin-mediated adhesion of breast cancer cells to fibronectin and collagen IV.

    Science.gov (United States)

    Yuan, Ye; Wu, Larry; Shen, Siqi; Wu, Shiyong; Burdick, Monica M

    2016-03-15

    To determine the role of sialylation on α5β1 and α2β1 integrins in the regulation of adhesion between breast cancer cells and extracellular matrix (ECM). Static cell adhesion assays were performed to quantify avidity of breast cancer cells to ECM. The effects of sialidases on α2,6 sialylation was assessed by flow cytometry using biotin conjugated Sambucus nigra lectin. Lectin affinity assays were used to determine expression of α2,6 sialylated integrins. Cell migration and invasion were investigated by wound healing and transwell invasion assays. α2, α5 and β1 integrins had considerable α2,6 sialylation on MDA-MB-231 cells, whereas signals from MCF-7 cells were undetectable. Cleavage of α2,6 sialylation increased adhesion of MDA-MB-231 cells to ECM, while adhesion of MCF-7 cells was unaffected, consistent with the latter's lack of endogenous α2,6 sialylated surface integrins. Neither surface expression of α2β1 and α5β1 integrins, nor activated β1 integrin, changed in MDA-MB-231 cells after sialidase treatment. However, sialidase treatment did not have significant impact on migration or invasion of MDA-MB-231 cells. Cell adhesion is an important early step of cancer metastasis, yet the roles of sialylation in regulating integrin-mediated breast cancer cell adhesion in comparison to migration and invasion are not well-understood. Our data suggest desialylation of α2,6-sialylated integrins increases adhesion, but not migration or invasion, of MDA-MB-231 cells to ECM without altering integrin expression. It should be considered that α2,6 sialylation may play different roles in regulating cell adhesion of different cancer cells when developing potential therapeutics targeting α2,6 sialylation. Published by Elsevier Inc.

  8. CD43 functions as a ligand for E-Selectin on activated T cells.

    Science.gov (United States)

    Matsumoto, Masanori; Atarashi, Kazuyuki; Umemoto, Eiji; Furukawa, Yuko; Shigeta, Akiko; Miyasaka, Masayuki; Hirata, Takako

    2005-12-15

    E-selectin, an inducible cell adhesion molecule expressed on endothelial cells, mediates the rolling on endothelium of leukocytes expressing E-selectin ligands, such as neutrophils and activated T cells. Although previous studies using mice lacking P-selectin glycoprotein ligand-1 (PSGL-1) have indicated that PSGL-1 on Th1 cells functions as an E-selectin ligand, the molecular nature of E-selectin ligands other than PSGL-1 remains unknown. In this study, we show that a 130-kDa glycoprotein was precipitated by an E-selectin-IgG chimera from mouse Th1 cells. This protein was cleaved by O-sialoglycoprotein endopeptidase and required sialic acid for E-selectin binding. The mAb 1B11, which recognizes the 130-kDa glycoform of CD43, recognized the 130-kDa band in the E-selectin-IgG precipitate. In addition, immunoprecipitation of the E-selectin-IgG precipitate with 1B11 depleted the 130-kDa protein, further confirming its identity as CD43. CD43 was also precipitated with E-selectin-IgG from cultured human T cells. E-selectin-dependent cell rolling on CD43 was observed under flow conditions using a CD43-IgG chimera generated in Chinese hamster ovary cells expressing alpha-1,3-fucosyltransferase VII and a core 2 beta-1,6-N-acetylglucosaminyltransferase. These results suggest that CD43, when modified by a specific set of glycosyltranferases, can function as an E-selectin ligand and therefore potentially mediate activated T cell migration into inflamed sites.

  9. Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Su Pei-Yi

    2012-08-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 is a major causative agent of hand-foot-and-mouth disease (HFMD, and infection of EV71 to central nerve system (CNS may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear. Results We demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, Maackia amurensis (MAA and Sambucus Nigra (SNA, could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation. Conclusions In this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells.

  10. Sialylation of Porphyromonas gingivalis LPS and its effect on bacterial-host interactions.

    Science.gov (United States)

    Zaric, Svetislav S; Lappin, Mark J; Fulton, Catherine R; Lundy, Fionnuala T; Coulter, Wilson A; Irwin, Christopher R

    2017-04-01

    Porphyromonas gingivalis produces different LPS isoforms with significant structural variations of their lipid A and O-antigen moieties that can affect its pro-inflammatory and bone-resorbing potential. We show here, for the first time, that P. gingivalis LPS isolated from W83 strain is highly sialylated and possesses significantly reduced inflammatory potential compared with less sialylated ATCC 33277 strain LPS. Nevertheless, the reduction in the endotoxin activity is not mediated by the presence of sialic acid LPS moieties as the sialic acid-free LPS produced by the mutant W83 strain exhibits a similar inflammatory potential to the wild type strain. Furthermore, our findings suggest that the interaction between the sialic acid LPS moieties and the inhibitory CD33 receptor is prevented by endogenously expressed sialic acid on the surface of THP-1 cells that cannot be out-competed by sialic acid containing P. gingivalis LPS. The present study also highlights the importance of endogenous sialic acid as a 'self-associated molecular pattern' and CD33 receptors in modulation of innate immune response as human gingival fibroblasts, which do not express CD33 receptors, and desialylated THP-1 cells have both been found to have much higher spontaneous IL-8 production than naïve THP-1 cells.

  11. Drift of the sialyl-linkage specific recognition of the sialidase of influenza B virus isolates.

    Science.gov (United States)

    Xu, G; Suzuki, T; Hanagata, G; Deya, E; Kiso, M; Hasegawa, A; Suzuki, Y

    1993-03-01

    Sialyl-linkage specificity of the sialidase of influenza B viruses isolated in different years from 1940 through 1990 (B/Lee/40,B/Setagaya/3/56,B/Tokyo/7/66,B/Kagoshima/1/68, B/Gifu/2/73, B/Kanagawa/3/76, B/Ibaraki/2/85, B/Yamagata/16/88, and B/Bangkok/163/90) was studied with N-acetylneuraminyl (alpha 2-3)- and (alpha 2-6)-lactoses, GM3 gangliosides containing the same sialyl-oligosaccharide sequences as sialyllactose, and also with type I and type II lacto-series gangliosides carrying Neu5Ac alpha 2-3Gal and NeuAc alpha 2-6Gal linkages as substrates. From an examination of up to nine strains, the sialidases of all viruses preferentially hydrolyze substrates with Neu5Ac alpha 2-3Gal linkage rather than the Neu5Ac alpha 2-6Gal linkage. It was found that the sialidase activity toward Neu5Ac alpha 2-6Gal linkage relative to Neu5Ac alpha 2-3Gal linkage is increased in later strains, whether sialyllactose or ganglioside is used as the substrate. These results suggested that the sialidase of influenza B virus isolates has shown a drift in linkage specificity which correlates with the year of isolation.

  12. Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.

    Science.gov (United States)

    Mabashi-Asazuma, Hideaki; Shi, Xianzong; Geisler, Christoph; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2013-02-01

    Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

  13. Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells

    NARCIS (Netherlands)

    Perdicchio, Maurizio; Cornelissen, Lenneke A. M.; Streng-Ouwehand, Ingeborg; Engels, Steef; Verstege, Marleen I.; Boon, Louis; Geerts, Dirk; van Kooyk, Yvette; Unger, Wendy W. J.

    2016-01-01

    The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector

  14. Cloning and characterization of sialidases with 2-6' and 2-3' sialyl lactose specificity from Pasteurella multocida.

    Science.gov (United States)

    Mizan, S; Henk, A; Stallings, A; Maier, M; Lee, M D

    2000-12-01

    Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene, nanH, from a fowl cholera isolate of P. multocida. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3' sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3' and 2-6' sialyl lactose, while NanH demonstrated activity only on 2-3' sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8' sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces.

  15. Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells

    NARCIS (Netherlands)

    M. Perdicchio (Maurizio); L.A.M. Cornelissen (Lenneke A.M.); I. Streng-Ouwehand (Ingeborg); S. Engels (Steef); M.I. Verstege (Marleen I.); L. Boon (Louis); D. Geerts (Dirk); Y. van Kooyk (Yvette); W.W. Unger (Wendy)

    2016-01-01

    textabstractThe increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation

  16. Active-Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP-Neu5Ac.

    Science.gov (United States)

    Schmölzer, Katharina; Eibinger, Manuel; Nidetzky, Bernd

    2017-08-04

    Sialyltransferases of the GT-80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans-sialylation, and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild-type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3- but also the α2,6-sialylated product (21 %). The H85N variant was virtually inactive in trans-sialylation but showed almost the same CMP-Neu5Ac hydrolase activity as wild-type. The competition between sialyl transfer and hydrolysis in the conversion of CMP-Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m-1 for the H85N variant, compared to 17 000 m-1 for the wild-type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP-Neu5Ac in the reaction. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Diverse inflammatory cytokines induce selectin ligand expression on murine CD4 T cells via p38 alpha MAP kinase1

    Science.gov (United States)

    Ebel, Mark E.; Awe, Olufolakemi; Kaplan, Mark H.; Kansas, Geoffrey S.

    2015-01-01

    Selectins are glycan-binding adhesion molecules which mediate the initial steps of leukocyte recognition of endothelium. Cytokines control numerous aspects of CD4 T helper differentiation, but how cytokines control induction of ligands for E- and P-selectin on T helper subsets remains poorly understood. Among 20 cytokines that affect T helper cell differentiation, we identified six, IL-12, IL-18, IL-27, IL-9, IL-25 and TGFβ1, that induce expression of selectin ligands on murine CD4 T cells above the low levels associated with TCR engagement. Collectively, these six cytokines could potentially account for selectin ligand expression on all of the currently defined non-sessile T helper lineages, including Th1, Th2, Th9, Th17 and Treg. Induction of selectin ligand expression by each of these six cytokines was almost completely inhibited by pharmacologic inhibition of p38 MAPK, but not other MAPKs, or by conditional genetic deletion of p38 alpha MAPK. Analysis of the expression of key glycosyltransferase genes revealed that p38 alpha signaling was selectively required for induction of Fut7 and Gcnt1, but not for induction of St3gal4 or St3gal6. Constitutively active MKK6, an immediate upstream activator of p38 MAPK, induced selectin ligand expression equivalent to that of cytokines, and this induction was completely dependent on expression of p38 alpha. Our results identify the repertoire of cytokines responsible for selectin ligand induction on CD4 T cells and provide a mechanistic link between T helper development and T cell migration. PMID:25941329

  18. Synthesis and biological evaluation of a new sialyl Lewis X mimetic derived from lactose.

    Science.gov (United States)

    Chervin, Stephanie M; Lowe, John B; Koreeda, Masato

    2002-08-09

    A sialyl Lewis X (sLe(x)) mimetic compound, 2-(trimethylsilyl)ethyl 3-O-carboxymethyl-beta-D-galactopyranosyl-(1-->4)-[alpha-L-fucosyl-(1-->6)]-beta-D-glucopyranoside (2a), has been synthesized in 14 steps from D-lactose. This synthesis features the use of the activated glycosylating donor, lactosyl iodide, in a Koenigs-Knorr sequence, the regioselective derivatization at the C-3 position of the galactose moiety, and the stereoselective construction of a fucose-alpha(1-->6)-lactose linkage. The mimetic was tested for its ability to inhibit human polymorphonuclear leukocyte (hPMNL) adhesion to immobilized recombinant human E-selectin under shear stress conditions.

  19. Characterization of two novel sialyl N-acetyllactosaminyl nucleotides separated from ovine colostrum.

    Science.gov (United States)

    Sasaki, Masashi; Nakamura, Tadashi; Hirayama, Kentaro; Fukuda, Kenji; Saito, Tadao; Urashima, Tadasu; Asakuma, Sadaki

    2016-10-01

    The milk/colostrum of some mammalian species is known to contain sugar nucleotides including uridine diphosphate (UDP) oligosaccharides in addition to lactose and milk oligosaccharides, but the detailed structures of these UDP oligosaccharides have not so far been clarified. In this study we isolated two UDP-sialyl N-acetyllactosamines from ovine colostrum and characterized them using (1)H-NMR and MALDI-TOFMS spectroscopies. Their structures were found to be Neu5Gc(α2-3)Gal(β1-4)GlcNAcα1-UDP and Neu5Gc(α2-6)Gal(β1-4)GlcNAcα1-UDP.

  20. Analysis of sialyl oligosaccharides by high-performance liquid chromatography-electrospray ionisation-mass spectrometry with differentiation of alpha2-3 and alpha2-6 sialyl linkages.

    Science.gov (United States)

    Pan, Geoffrey Ge; Melton, Laurence D

    2005-06-10

    A method for analysing sialyl oligosaccharides from bovine colostrum using high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS) is described. Under positive ionisation mode, mass spectra of alpha2-3 and alpha2-6 linkages were different, and the former produced a prominent B2 (or B3 in disialyl lactose) mass fragment. This fragment was absent from mass spectra with alpha2-6 linkages. Two sialyl oligosaccharides, which have not been reported previously, were tentatively identified. One comprises a N-acetyl neuraminic acid (Neu5Ac), two hexoses (Hex), and one N-acetyl hexosamine (HexNAc) residue ((Neu5Ac)1 (Hex)2 (HexNAc)1), and the other comprises one Neu5Ac and one Hex residue ((Neu5Ac)1(Hex)1).

  1. PANP is a novel O-glycosylated PILR{alpha} ligand expressed in neural tissues

    Energy Technology Data Exchange (ETDEWEB)

    Kogure, Amane [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); Shiratori, Ikuo [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Wang, Jing [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); Lanier, Lewis L. [Department of Microbiology and Immunology and the Cancer Research Institute, University of California San Francisco, San Francisco, CA 94143 (United States); Arase, Hisashi, E-mail: arase@biken.osaka-u.ac.jp [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); JST CREST, Saitama 332-0012 (Japan)

    2011-02-18

    Research highlights: {yields} A Novel molecule, PANP, was identified to be a PILR{alpha} ligand. {yields} Sialylated O-glycan structures on PANP were required for PILR{alpha} recognition. {yields} Transcription of PANP was mainly observed in neural tissues. {yields} PANP seems to be involved in immune regulation as a ligand for PILR{alpha}. -- Abstract: PILR{alpha} is an immune inhibitory receptor possessing an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain enabling it to deliver inhibitory signals. Binding of PILR{alpha} to its ligand CD99 is involved in immune regulation; however, whether there are other PILR{alpha} ligands in addition to CD99 is not known. Here, we report that a novel molecule, PILR-associating neural protein (PANP), acts as an additional ligand for PILR{alpha}. Transcription of PANP was mainly observed in neural tissues. PILR{alpha}-Ig fusion protein bound cells transfected with PANP and the transfectants stimulated PILR{alpha} reporter cells. Specific O-glycan structures on PANP were found to be required for PILR recognition of this ligand. These results suggest that PANP is involved in immune regulation as a ligand of the PILR{alpha}.

  2. Analysis of glycoprotein E-selectin ligANDs on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

    KAUST Repository

    Merzaban, Jasmeen S.

    2011-06-09

    Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin-/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved byWestern blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand- 1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL."E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL\\'s contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. © 2011 by The American Society of Hematology.

  3. Sialylation of Glycosylphosphatidylinositol (GPI) Anchors of Mammalian Prions Is Regulated in a Host-, Tissue-, and Cell-specific Manner*

    Science.gov (United States)

    Katorcha, Elizaveta; Srivastava, Saurabh; Klimova, Nina; Baskakov, Ilia V.

    2016-01-01

    Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of the prion protein or PrPC. PrPC is posttranslationally modified with N-linked glycans and a sialylated glycosylphosphatidylinositol (GPI) anchor. Conformational conversion of PrPC gives rise to glycosylated and GPI-anchored PrPSc. The question of the sialylation status of GPIs within PrPSc has been controversial. Previous studies that examined scrapie brains reported that both sialo- and asialo-GPIs were present in PrPSc, with the majority being asialo-GPIs. In contrast, recent work that employed cultured cells claimed that only PrPC with sialylo-GPIs could be recruited into PrPSc, whereas PrPC with asialo-GPIs inhibited conversion. To resolve this controversy, we analyzed the sialylation status of GPIs within PrPSc generated in the brain, spleen, or cultured N2a or C2C12 myotube cells. We found that recruiting PrPC with both sialo- and asialo-GPIs is a common feature of PrPSc. The mixtures of sialo- and asialo-GPIs were observed in PrPSc universally regardless of prion strain as well as host, tissue, or type of cells that produced PrPSc. Remarkably, the proportion of sialo- versus asialo-GPIs was found to be controlled by host, tissue, and cell type but not prion strain. In summary, this study found no strain-specific preferences for selecting PrPC with sialo- versus asialo-GPIs. Instead, this work suggests that the sialylation status of GPIs within PrPSc is regulated in a cell-, tissue-, or host-specific manner and is likely to be determined by the specifics of GPI biosynthesis. PMID:27317661

  4. Milk oligosaccharide sialyl(α2,3)lactose activates intestinal CD11c+ cells through TLR4

    OpenAIRE

    Kurakevich, Ekaterina; Hennet, Thierry; Hausmann, Martin; Rogler, Gerhard; Borsig, Lubor

    2013-01-01

    Breast milk provides all nutrients required for the growth and development of the newborn child. In addition to energy source, milk contains other biomolecules, such as oligosaccharides, which contribute to the intestinal colonization through microbiota and development of mucosal immunity. This study shows that specific milk oligosaccharides stimulate intestinal immune responses through binding and activation of dendritic cells. We demonstrate that the milk oligosaccharide sialyl(α2,3)lactose...

  5. Inhibition of adhesion and metastasis of HepG2 hepatocellular carcinoma cells in vitro by DNA aptamer against sialyl Lewis X.

    Science.gov (United States)

    Wang, Xiao-Kang; Peng, Yan; Tao, Hao-Ran; Zhou, Fen-Fang; Zhang, Chi; Su, Fei; Wang, Shi-Pei; Liu, Qing; Xu, Li-Hua; Pan, Xue-Kai; Xie, Wei; Feng, Mao-Hui

    2017-06-01

    The sialyl Lewis X (SLe x ) antigen encoded by the FUT7 gene is the ligand of endotheliam-selectin (E-selectin). The combination of SLe x antigen and E-selectin represents an important way for malignant tumor metastasis. In the present study, the effect of the SLe x -binding DNA aptamer on the adhesion and metastasis of hepatocellular carcinoma HepG2 cells in vitro was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence staining were conducted to detect the expression of FUT7 at both transcriptional and translational levels. The SLe x expression in HepG2 cells treated with different concentrations of SLe x -binding DNA aptamer was detected by flow cytometry. Besides, the adhesion, migration, and invasion of HepG2 cells were measured by cell adhesion assay, and the Transwell migration and invasion assay. The results showed that the FUT7 expression was up-regulated at both mRNA and protein levels in HepG2 cells. SLe x -binding DNA aptamer could significantly decrease the expression of SLe x in HepG2 cells. The cell adhesion assay revealed that the SLe x -binding DNA aptamer could effectively inhibit the interactions between E-selectin and SLe x in the HepG2 cells. Additionally, SLe x -binding DNA aptamers at 20 nmol/L were found to have the similar effect to the monoclonal antibody CSLEX-1. The Transwell migration and invasion assay revealed that the number of penetrating cells on the down-side of Transwell membrane was significantly less in cells treated with 5, 10, 20 nmol/L SLe x -binding DNA aptamer than those in the negative control group (Paptamer could significantly inhibit the in vitro adhesion, migration, and invasion of HepG2 cells, suggesting that the SLe x -binding DNA aptamer may be used as a potential molecular targeted drug against metastatic hepatocellular carcinoma.

  6. Evaluation of Receptor-Ligand Mechanisms of Dual-Targeted Particles to an Inflamed Endothelium.

    Science.gov (United States)

    Fromen, Catherine A; Fish, Margaret B; Zimmerman, Anthony; Adili, Reheman; Holinstat, Michael; Eniola-Adefeso, Omolola

    2016-03-01

    Vascular-targeted carriers (VTCs) are designed as leukocyte mimics, decorated with ligands that target leukocyte adhesion molecules (LAMs) and facilitate adhesion to diseased endothelium. VTCs require different design considerations than other targeted particle therapies; adhesion of VTCs in regions with dynamic blood flow requires multiple ligand-receptor (LR) pairs that provide particle adhesion and disease specificity. Despite the ultimate goal of leukocyte mimicry, the specificity of multiple LAM-targeted VTCs remains poorly understood, especially in physiological environments. Here, we investigate particle binding to an inflamed mesentery via intravital microscopy using a series of particles with well-controlled ligand properties. We find that the total number of sites of a single ligand can drive particle adhesion to the endothelium, however, combining ligands that target multiple LR pairs provides a more effective approach. Combining sites of sialyl Lewis A (sLeA) and anti-intercellular adhesion molecule-1 (aICAM), two adhesive molecules, resulted in ~3-7-fold increase of adherent particles at the endothelium over single-ligand particles. At a constant total ligand density, a particle with a ratio of 75% sLeA: 25% aICAM resulted in more than 3-fold increase over all over other ligand ratios tested in our in vivo model. Combined with in vitro and in silico data, we find the best dual-ligand design of a particle is heavily dependent on the surface expression of the endothelial cells, producing better adhesion with more particle ligand for the lesser-expressed receptor. These results establish the importance of considering LR-kinetics in intelligent VTC ligand design for future therapeutics.

  7. Role of sialylation in determining the pharmacokinetics of neutrophil inhibitory factor (NIF) in the Fischer 344 rat.

    Science.gov (United States)

    Webster, R; Taberner, J; Edgington, A; Guhan, S; Varghese, J; Feeney, H; Blocker, L; Jezequel, S G

    1999-11-01

    1. Recombinant neutrophil inhibitory factor (NIF) is a glycoprotein. Its amino acid sequence remains constant and has a molecular weight of 28.9 kD. However, approximately 40% of the total molecular weight consists of glycans with variable structure. 2. The pharmacokinetics of 11 different NIF batches with varying extents and patterns of sialylation have been investigated in the Fischer 344 rat following intravenous administration. These data indicate that reducing the extent of NIF sialylation reduces the half-life of the molecule due to an increase in the systemic clearance. Also, an increase in the number of unsialylated or neutral glycans may increase the volume of distribution of NIF, although this effect is marginal. 3. Isolated perfused rat liver (IPRL) investigations have shown that sialylated NIF has a low hepatic extraction (NIF has an extraction that is > 20-fold higher. Co-administration of asialo NIF with asialo fetuin (a protein cleared by hepatic asialoglycoprotein receptor (possibly galactose)-mediated uptake reduced the hepatic extraction of asialo NIF. 4. These data suggest that NIF molecules that have free sugar moieties (possibly galactose) interact with an asialoglycoprotein receptor (possibly galactose-mediated) in the liver (parenchymal cells/hepatocytes). Interaction with this receptor leads to cellular internalization and degradation.

  8. Sialylation of prion protein controls the rate of prion amplification, the cross-species barrier, the ratio of PrPSc glycoform and prion infectivity.

    Science.gov (United States)

    Katorcha, Elizaveta; Makarava, Natallia; Savtchenko, Regina; D'Azzo, Alessandra; Baskakov, Ilia V

    2014-09-01

    The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP(C)) into the disease-associated, transmissible form (PrP(Sc)). Pr(PC) is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP(C) and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrP(C) glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP(Sc) glycoform ratio. The current study demonstrates that undersialylated PrP(C) is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrP(C). As a result, PMCAb-derived PrP(Sc) was less sialylated than brain-derived PrP(Sc). A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP(C) using sialidase was found to increase the rate of PrP(Sc) amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP(C) reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP(C) was also found to control PrP(Sc) glycoform ratio. A decrease in Pr(PC) sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP(Sc). 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP(C) differed from that of spleen-derived PrP(C). Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP(C), suggesting that Neu1 is not responsible for desialylation of Pr

  9. Sialylation of prion protein controls the rate of prion amplification, the cross-species barrier, the ratio of PrPSc glycoform and prion infectivity.

    Directory of Open Access Journals (Sweden)

    Elizaveta Katorcha

    2014-09-01

    Full Text Available The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP(C into the disease-associated, transmissible form (PrP(Sc. Pr(PC is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP(C and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrP(C glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP(Sc glycoform ratio. The current study demonstrates that undersialylated PrP(C is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb at the expense of oversialylated PrP(C. As a result, PMCAb-derived PrP(Sc was less sialylated than brain-derived PrP(Sc. A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP(C using sialidase was found to increase the rate of PrP(Sc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP(C reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP(C was also found to control PrP(Sc glycoform ratio. A decrease in Pr(PC sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP(Sc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP(C differed from that of spleen-derived PrP(C. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP(C, suggesting that Neu1 is not responsible for desialylation of Pr

  10. The Association of Human Apolipoprotein C-III Sialylation Proteoforms with Plasma Triglycerides.

    Directory of Open Access Journals (Sweden)

    Hussein N Yassine

    Full Text Available Apolipoprotein C-III (apoC-III regulates triglyceride (TG metabolism. In plasma, apoC-III exists in non-sialylated (apoC-III0a without glycosylation and apoC-III0b with glycosylation, monosialylated (apoC-III1 or disialylated (apoC-III2 proteoforms. Our aim was to clarify the relationship between apoC-III sialylation proteoforms with fasting plasma TG concentrations.In 204 non-diabetic adolescent participants, the relative abundance of apoC-III plasma proteoforms was measured using mass spectrometric immunoassay.Compared with the healthy weight subgroup (n = 16, the ratios of apoC-III0a, apoC-III0b, and apoC-III1 to apoC-III2 were significantly greater in overweight (n = 33 and obese participants (n = 155. These ratios were positively correlated with BMI z-scores and negatively correlated with measures of insulin sensitivity (Si. The relationship of apoC-III1 / apoC-III2 with Si persisted after adjusting for BMI (p = 0.02. Fasting TG was correlated with the ratio of apoC-III0a / apoC-III2 (r = 0.47, p<0.001, apoC-III0b / apoC-III2 (r = 0.41, p<0.001, apoC-III1 / apoC-III2 (r = 0.43, p<0.001. By examining apoC-III concentrations, the association of apoC-III proteoforms with TG was driven by apoC-III0a (r = 0.57, p<0.001, apoC-III0b (r = 0.56. p<0.001 and apoC-III1 (r = 0.67, p<0.001, but not apoC-III2 (r = 0.006, p = 0.9 concentrations, indicating that apoC-III relationship with plasma TG differed in apoC-III2 compared with the other proteoforms.We conclude that apoC-III0a, apoC-III0b, and apoC-III1, but not apoC- III2 appear to be under metabolic control and associate with fasting plasma TG. Measurement of apoC-III proteoforms can offer insights into the biology of TG metabolism in obesity.

  11. Changes in the sialylation and sulfation of secreted thyrotropin in congenital hypothyroidism

    Energy Technology Data Exchange (ETDEWEB)

    Gyves, P.W.; Gesundheit, N.; Thotakura, N.R.; Stannard, B.S.; DeCherney, G.S.; Weintraub, B.D. (National Institutes of Health, Bethesda, MD (USA))

    1990-05-01

    The authors have examined the oligosaccharide structure of secreted thyrotropin (TSH) in perinatal and mature rats with congenital primary hypothyroidism. Rat pituitaries from euthyroid control animals and those rendered hypothyroid by methimazole treatment were incubated with ({sup 3}H)glucosamine in vitro. Secreted TSH was purified, and oligosaccharides were enzymatically released and characterized by anion-exchange HPLC. In perinatal hypothyroid animals compared with control animals, oligosaccharides from TSH {alpha} and {beta} subunits contained more species with three or more negative charges. Moreover, perinatal hypothyroid animals demonstrated a dramatic increase in the ratio of sialylated to sulfated species within oligosaccharides of the same negative charge. In mature hypothyroid 9-week-old animals compared with control animals, changes were less pronounced, suggesting that endocrine regulation of oligosaccharide structure is dependent upon the maturational state of the animal. Together, these data provide direct evidence and characterization of specific changes in the structure of a secreted pituitary glycoprotein hormone occurring as a result of in vivo endocrine alterations during early development. Moreover, they provide a potential structural basis to explain the delayed clearance of both TSH and the gonadotropins with end-organ deficiency, which may have important implications for the in vivo biological activities of these hormones.

  12. Synthesis and molecular tumbling properties of sialyl Lewis X and derived neoglycolipids.

    Science.gov (United States)

    Gege, Christian; Geyer, Armin; Schmidt, Richard R

    2002-06-03

    The sialyl Lewis X (sLeX) epitope has become a prominent target for biological studies because of its role in inflammation through binding to selectins. This epitope is located at the terminal end in glycosphingolipids and a lactose unit serves as spacer to the ceramide moiety. This paper focuses on the influence of the spacer structure and spacer length in regard to the mobility of the sLeX epitope. To this end sLex neoglycolipids 1a-c, with one, two, or three lactose units as spacer between the sLeX tetrasaccharide epitope and the membrane anchor, were synthesized. The synthetic strategy was also applied to the synthesis of the corresponding Lewis X (LeX) derivatives. The glycolipids were inserted in model membranes, and the tumbling frequencies of the sLex tetrasaccharide epitopes were then analysed by NMR spectroscopy. A nonaethylene glycol spacer decouples the carbohydrate moiety from the membrane mobility while (oligo-)lactoses act as more rigid distance keepers between the Lewis epitope and the surface of the membrane. Quantification of the different degrees of decoupling was possible by analysis of rotational correlation times.

  13. Oligomeric Amyloid-β Peptide on Sialylic Lewisx–Selectin Bonding at Cerebral Endothelial Surface

    Directory of Open Access Journals (Sweden)

    Sholpan Askarova

    2014-12-01

    Full Text Available Introduction: Alzheimer’s disease (AD is a chronic neurodegenerative disorder, which affects approximately 10% of the population aged 65 and 40% of people over the age 80. Currently, AD is on the list of diseases with no effective treatment. Thus, the study of molecular and cellular mechanisms of AD progression is of high scientific and practical importance. In fact, dysfunction of the blood-brain barrier (BBB plays an important role in the onset and progression of the disease. Increased deposition of amyloid b peptide (Aβ in cerebral vasculature and enhanced transmigration of monocytes across the BBB are frequently observed in AD brains and are some of the pathological hallmarks of the diseases. Since the transmigration of monocytes across the BBB is both a mechanical and a biochemical process, the expression of adhesion molecules and mechanical properties of endothelial cells are the critical factors that require investigation.Methods: Because of recent advances in the biological applications of atomic force microscopy (AFM, we applied AFM with cantilever tips bio-functionalized by sLex in combination with the advanced immunofluorescent microscopy (QIM to study the direct effects of Aβ42 oligomers on the selectins expression, actin polymerization, and cellular mechanical and adhesion properties in cerebral endothelial cells (mouse bEnd3 line and primary human CECs and find a possible way to attenuate these effects. Results: QIM results showed that Aβ42 increased the expressions of P-selectin on the cell surface and enhanced actin polymerization. Consistent with our QIM results, AFM data showed that Aβ42 increased the probability of cell adhesion with sLex-coated cantilever and cell stiffness. These effects were counteracted by lovstatin, a cholesterol-lowering drug.  Surprisingly, the apparent rupture force of sLex-selectin bonding was significantly lower after treatment with Aβ42, as compared with the control (i.e. no treatment

  14. Synthesis and Functional Characterization of Novel Sialyl LewisX Mimic-Decorated Liposomes for E-selectin-Mediated Targeting to Inflamed Endothelial Cells.

    Science.gov (United States)

    Chantarasrivong, Chanikarn; Ueki, Akiharu; Ohyama, Ryutaro; Unga, Johan; Nakamura, Shinya; Nakanishi, Isao; Higuchi, Yuriko; Kawakami, Shigeru; Ando, Hiromune; Imamura, Akihiro; Ishida, Hideharu; Yamashita, Fumiyoshi; Kiso, Makoto; Hashida, Mitsuru

    2017-05-01

    Sialyl LewisX (sLeX) is a natural ligand of E-selectin that is overexpressed by inflamed and tumor endothelium. Although sLeX is a potential ligand for drug targeting, synthesis of the tetrasaccharide is complicated with many reaction steps. In this study, structurally simplified novel sLeX analogues were designed and linked with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG) for E-selectin-mediated liposomal delivery. The sLeX structural simplification strategies include (1) replacement of the Gal-GlcNAc disaccharide unit with lactose to reduce many initial steps and (2) substitution of neuraminic acid with a negatively charged group, i.e., 3'-sulfo, 3'-carboxymethyl (3'-CM), or 3'-(1-carboxy)ethyl (3'-CE). While all the liposomes developed were similar in particle size and charge, the 3'-CE sLeX mimic liposome demonstrated the highest uptake in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs), being even more potent than native sLeX-decorated liposomes. Inhibition studies using antiselectin antibodies revealed that their uptake was mediated primarily by overexpressed E-selectin on inflamed HUVECs. Molecular dynamics simulations were performed to gain mechanistic insight into the E-selectin binding differences among native and mimic sLeX. The terminally branched methyl group of the 3'-CE sLeX mimic oriented and faced the bulk hydrophilic solution during E-selectin binding. Since this state is entropically unfavorable, the 3'-CE sLeX mimic molecule might be pushed toward the binding pocket of E-selectin by a hydrophobic effect, leading to a higher probability of hydrogen-bond formation than native sLeX and the 3'-CM sLeX mimic. This corresponded with the fact that the 3'-CE sLeX mimic liposome exhibited much greater uptake than the 3'-CM sLeX mimic liposome.

  15. Ultrasensitive electrochemical biosensor based on graphite oxide, Prussian blue, and PTC-NH2 for the detection of α2,6-sialylated glycans in human serum.

    Science.gov (United States)

    Gao, Liuliu; He, Junlin; Xu, Wailan; Zhang, Jing; Hui, Junmin; Guo, Yanlei; Li, Wenjuan; Yu, Chao

    2014-12-15

    α2,6-Sialylated glycans are crucial molecular targets for cancer diagnosis and clinical research. In this work, a novel ultrasensitive electrochemical biosensor was fabricated based on a graphite oxide (GO), Prussian blue (PB), and PTC-NH2 (an ammonolysis product of 3,4,9,10-perylenetetracarboxylic dianhydride) nanocomposite for the selective detection of α2,6-sialylated glycans. To increase the sensitivity of the electrochemical biosensor, gold nanoparticles (GNPs) were immobilized on a GO-PB-PTC-NH2 modified glassy carbon electrode (GCE). Sambucus nigra agglutinins (SNAs), which specifically bind with α2,6-sialylated glycans, were covalently immobilized on GNPs for the sensitive detection of α2,6-sialylated glycans in serum. This proposed method can be applied to human serum, and it worked well over a broad linear range (0.1 pg mL(-1)-500 ng mL(-1)) with detection limits of 0.03 pg mL(-1). Moreover, recovery of the spiked samples ranged from 100.2% to 105.0%, suggesting that this excellent electrochemical biosensor can be used for the practical detection of α2,6-sialylated glycans. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. α-2,3-sialyltransferase expression level impacts the kinetics of lipooligosaccharide sialylation, complement resistance, and the ability of Neisseria gonorrhoeae to colonize the murine genital tract.

    Science.gov (United States)

    Lewis, Lisa A; Gulati, Sunita; Burrowes, Elizabeth; Zheng, Bo; Ram, Sanjay; Rice, Peter A

    2015-02-03

    Neisseria meningitidis and Neisseria gonorrhoeae modify the terminal lacto-N-neotetraose moiety of their lipooligosaccharide (LOS) with sialic acid. N. gonorrhoeae LOS sialylation blocks killing by complement, which is mediated at least in part by enhanced binding of the complement inhibitor factor H (FH). The role of LOS sialylation in resistance of N. meningitidis to serum killing is less well defined. Sialylation in each species is catalyzed by the enzyme LOS α-2,3-sialyltransferase (Lst). Previous studies have shown increased Lst activity in N. gonorrhoeae compared to N. meningitidis due to an ~5-fold increase in lst transcription. Using isogenic N. gonorrhoeae strains engineered to express gonococcal lst from either the N. gonorrhoeae or N. meningitidis lst promoter, we show that decreased expression of lst (driven by the N. meningitidis promoter) reduced LOS sialylation as determined by less incorporation of tritium-labeled cytidine monophospho-N-acetylneuraminic acid (CMP-NANA; the donor molecule for sialic acid). Diminished LOS sialylation resulted in reduced rates of FH binding and increased pathway activation compared to N. gonorrhoeae promoter-driven lst expression. The N. meningitidis lst promoter generated sufficient Lst to sialylate N. gonorrhoeae LOS in vivo, and the level of sialylation after 24 h in the mouse genital tract was sufficient to mediate resistance to human serum ex vivo. Despite demonstrable LOS sialylation in vivo, gonococci harboring the N. meningitidis lst promoter were outcompeted by those with the N. gonorrhoeae lst promoter during coinfection of the vaginal tract of estradiol-treated mice. These data highlight the importance of high lst expression levels for gonococcal pathogenesis. Neisseria gonorrhoeae has become resistant to nearly every therapeutic antibiotic used and is listed as an "urgent threat" by the Centers for Disease Control and Prevention. Novel therapies are needed to combat drug

  17. Alteration of the sialylation pattern and memory deficits by injection of Aβ(25-35) into the hippocampus of rats.

    Science.gov (United States)

    Limón, Ilhuicamina Daniel; Ramírez, Eleazar; Díaz, Alfonso; Mendieta, Liliana; Mayoral, Miguel Ángel; Espinosa, Blanca; Guevara, Jorge; Zenteno, Edgar

    2011-05-09

    Sialic acid in glycoconjugates participates in important cellular functions associated with normal development, growth, and communication. Therefore we evaluated the sialylation pattern and memory deficits caused by the injection of Aβ((25-35)) into the hippocampus (Hp) of rats. The eight-arm maze spatial-learning and memory test indicated that the injection of Aβ((25-35)) into subfield CA1 of the Hp impaired both learning and memory. The sialylation pattern was examined using sialic acid-specific lectins. Our results showed that Maackia amurensis agglutinin (MAA, specific for Neu5Acα2,3Gal) showed reactivity in the CA1 and dentate gyrus (DG) subfields of the Hp mainly in the group injected with vehicle, whereas Macrobrachium rosenbergii lectin (MRL, specific for Neu5,9,7Ac) and Sambucus nigra agglutinin (SNA, specific for Neu5Acα2,6Gal-GalNAc) had increased reactivity in the CA1 and DG subfields of the Hp in the Aβ((25-35))-injected group. The staining pattern of the antibody specific for polysialic acid (a linear homopolymer of α-2,8-linked sialic acid) increased in the CA1 and DG subfields of the Hp of the Aβ((25-35)) group compared to the control group. Our results suggest that injection of Aβ((25-35)) causes impairment in spatial memory and alters the sialylation pattern in response to compensatory reorganization and-or sprouting of dendrites and axons of the surviving neurons. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Enzymatic synthesis of a 6-sialyl lactose analogue using a pH-responsive water-soluble polymer support.

    Science.gov (United States)

    Wang, Wenjun; Li, Lei; Jin, Chen; Niu, Yujie; Li, Sen; Ma, Ji; Li, Linfeng; Liu, Yu; Cai, Li; Zhao, Wei; Wang, Peng George

    2011-09-01

    The Letter describes a strategy for the enzymatic synthesis of glycans based on a pH-responsive water-soluble polymer. In neutral condition, the polymer is water-soluble and convenient for in-solution enzymatic synthesis, whereas in acidic condition (pH lower than 4.0), the polymer disconnects with the product and becomes insoluble, which can be easily removed. A 6-Sialyl lactose analogue was synthesized as a model reaction using this approach. Copyright © 2011. Published by Elsevier Ltd.

  19. Pro-inflammatory State in Monoclonal Gammopathy of Undetermined Significance and in Multiple Myeloma Is Characterized by Low Sialylation of Pathogen-Specific and Other Monoclonal Immunoglobulins

    Directory of Open Access Journals (Sweden)

    Adrien Bosseboeuf

    2017-10-01

    Full Text Available Multiple myeloma (MM and its pre-cancerous stage monoclonal gammopathy of undetermined significance (MGUS allow to study immune responses and the chronology of inflammation in the context of blood malignancies. Both diseases are characterized by the production of a monoclonal immunoglobulin (mc Ig which for subsets of MGUS and MM patients targets pathogens known to cause latent infection, a major cause of inflammation. Inflammation may influence the structure of both polyclonal (pc Ig and mc Ig produced by malignant plasma cells via the sialylation of Ig Fc fragment. Here, we characterized the sialylation of purified mc and pc IgGs from 148 MGUS and MM patients, in comparison to pc IgGs from 46 healthy volunteers. The inflammatory state of patients was assessed by the quantification in serum of 40 inflammation-linked cytokines, using Luminex technology. While pc IgGs from MGUS and MM patients showed heterogeneity in sialylation level, mc IgGs from both MGUS and MM patients exhibited a very low level of sialylation. Furthermore, mc IgGs from MM patients were less sialylated than mc IgGs from MGUS patients (p < 0.01, and mc IgGs found to target an infectious pathogen showed a lower level of sialylation than mc IgGs of undetermined specificity (p = 0.048. Regarding inflammation, 14 cytokines were similarly elevated with a p value < 0.0001 in MGUS and in MM compared to healthy controls. MM differed from MGUS by higher levels of HGF, IL-11, RANTES and SDF-1-α (p < 0.05. MGUS and MM patients presenting with hyposialylated pc IgGs had significantly higher levels of HGF, IL-6, tumor necrosis factor-α, TGF-β1, IL-17, and IL-33 compared to patients with hyper-sialylated pc IgGs (p < 0.05. In MGUS and in MM, the degree of sialylation of mc and pc IgGs and the levels of four cytokines important for the anti-microbial response were correlated, either positively (IFN-α2, IL-13 or negatively (IL-17, IL-33. Thus in MGUS as in MM

  20. Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

    OpenAIRE

    Wilbrink, Maarten H.; ten Kate, Geert A.; van Leeuwen, Sander S.; Sanders, Peter; Sallomons, Erik; Hage, Johannes A.; Dijkhuizen, Lubbert; Kamerling, Johannis P.

    2014-01-01

    trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(β1-x)-glycans (sialo-glycans) to Gal(β1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)nGlc oligosaccharide mixtures (GOS) using bovine κ-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3′-galactosyl-lactose (β3′-GL), 4′-galactosyl-lactose (β4′-GL), and ...

  1. Multi-ligand nanoparticles for targeted drug delivery to the injured vascular wall

    Science.gov (United States)

    Kona, Soujanya

    Pathological conditions like coronary artery disease, acute myocardial infarction, stroke, and peripheral artery diseases as well as cardiovascular interventions used in the treatment of coronary artery diseases such as angioplasty and stenting damage/injure the blood vessel wall, leading to inflamed or activated endothelial cells that have been implicated in events leading to thrombosis, inflammation, and restenosis. Oral administration of anti-coagulant and anti-inflammatory drugs causes systemic toxicity, bleeding, patient incompliance, and inadequate amounts of drugs at the injured area. Though drug-eluting stents have shown therapeutic benefits, complications such as in-stent restenosis and late thrombosis still remain and are a cause for concern. Rapid growth in the field of nanotechnology and nanoscience in recent years has paved the way for new targeted and controlled drug delivery strategies. In this perspective, the development of biodegradable nanoparticles for targeted intracellular drug delivery to the inflamed endothelial cells may offer an improved avenue for treatment of cardiovascular diseases. The major objective of this research was to develop "novel multi-ligand nanoparticles," as drug carriers that can efficiently target and deliver therapeutic agents to the injured/inflamed vascular cells under dynamic flow conditions. Our approach mimics the natural binding ability of platelets to injured/activated endothelial cells through glycoprotein Ib (GPIb) bound to P-selectin expressed on inflamed endothelial cells and to the subendothelium through GPIb binding to von Willebrand factor (vWF) deposited onto the injured vascular wall. Our design also exploits the natural cell membrane translocation ability of the internalizing cell peptide - trans-activating transcriptor (TAT) to enhance the nanoparticle uptake by the targeted cells. Our hypothesis is that these multi-ligand nanoparticles would show an increased accumulation at the injury site since GPIb

  2. Profiles of carbohydrate ligands associated with adsorbed proteins on self-assembled monolayers of defined chemistries.

    Science.gov (United States)

    Shankar, Sucharita P; Chen, Inn Inn; Keselowsky, Benjamin G; García, Andrés J; Babensee, Julia E

    2010-03-15

    Conserved protein-carbohydrate-lipid pathogen-associated molecular patterns (PAMPs) interact with cells of the innate immune system to mediate antigen recognition and internalization and activation of immune cells. We examined if analogous "biomaterial-associated molecular patterns" composed of proteins, specifically their carbohydrate modifications, existed on biomaterials, which can play a role in mediating the innate immune response to biomaterials. To probe for these carbohydrates in the adsorbed protein layer, as directed by the underlying biomaterial chemistry, self-assembled monolayers (SAMs) presenting -CH(3), -OH, -COOH, or -NH(2) were preincubated with serum/plasma, and the presence of carbohydrate ligands of C-type lectin receptors (CLRs) was investigated using lectin probes in an enzyme-linked lectin assay (ELLA). Presentation of CLR ligands was detected on control tissue culture polystyrene (TCPS). Absorbances of mannose or N-acetylglucosamine increased with decreasing incubating serum concentration, whereas absorbances of sialylated epitopes or fucose remained unchanged. Absorbances of alpha-galactose or N-acetylgalactosamine decreased with decreasing incubating serum concentration; beta-galactose was undetectable. Among SAM endgroups, preincubation with 10% serum resulted in differential presentation of CLR ligands: higher alpha-galactose on COOH SAMs than NH(2) or CH(3) SAMs, highest complex mannose on NH(2) SAMs, and higher complex mannose on OH SAMs than CH(3) SAMs. Least sialylated groups were detected on CH(3) SAMs. In summary, biomaterial chemistry may regulate protein adsorption and hence unique presentation of associated carbohydrates. The ultimate goal is to identify the effects of protein glycosylations associated with biomaterials in stimulating innate immune responses. (c) 2009 Wiley Periodicals, Inc.

  3. Identification of sialyl oligosaccharides including an oligosaccharide nucleotide in colostrum of an addax (Addax nasomaculatus) (Subfamily Antelopinae).

    Science.gov (United States)

    Ganzorig, Khuukhenbaatar; Asakawa, Takuya; Sasaki, Masashi; Saito, Tadao; Suzuki, Isao; Fukuda, Kenji; Urashima, Tadasu

    2018-01-01

    Mammalian milk/colostrum usually contains milk oligosaccharides along with the predominant lactose. Although milk oligosaccharides of a variety of Bovidae species including cow, sheep and goat have been characterized, those of the addax, an Antelopinae species of the Bovidae, have not as yet been clarified. In this study, several sialyl oligosaccharides were purified from a sample of addax colostrum and characterized as follows: Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(β1-4)Glc, Neu5Gc(α2-8)Neu5Gc(α2-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-4)Glc, Neu5Ac(α2-6)Gal(β1-4)GlcNAc, Neu5Gc(α2-3)Gal(β1-4)Glc, Neu5Gc(α2-6)Gal(β1-4)Glc, Neu5Gc(α2-6)Gal(β1-4)GlcNAc. In addition, an oligosaccharide nucleotide Neu5Gc(α2-6)Gal(β1-4)GlcNAcα1-UDP was characterized. Molecular species of a variety of sialyl oligosaccharides found in milk and colostrum of these Bovidae were compared. © 2017 Japanese Society of Animal Science.

  4. Chemoenzymatic synthesis and enzymatic analysis of 8-modified cytidine monophosphate-sialic acid and sialyl lactose derivatives.

    Science.gov (United States)

    Morley, Thomas J; Withers, Stephen G

    2010-07-14

    The sialic acids found on eukaryotic glycans have remarkably diverse core structures, with a range of modifications at C5, C7, C8 and C9. These carbohydrates have been found to play key roles in cell-cell interactions within eukaryotes and often serve as the initial site of attachment for viruses and bacteria. Consequently simple changes to the structures of the sialic acids can result in profoundly different and often opposing biological effects. Of particular importance are modifications at the 8-position. These include O-acetylation, carried out by an acetyl transferase, and particularly polysialylation, catalyzed by a polysialyltransferase. As part of a structural and mechanistic study of sialyltransferases and polysialyltransferases, access was needed to sialic acid-containing oligosaccharides that are modified at the 8-position of the sialic acid to render this center non-nucleophilic. The free 8-modified sialic acid analogues were synthesized using a concise, divergent chemical synthetic approach, and each was converted to its cytidine monophosphate (CMP) sugar donor form using a bacterial CMP-sialic acid synthetase. The transfer of each of the modified donors to lactose by each of two sialyltransferases was investigated, and kinetic parameters were determined. These yielded insights into the roles of interactions occurring at that position during enzymatic sialyl transfer. A transferase from Campylobacter jejuni was identified as the most suitable for the enzymatic coupling and utilized to synthesize the 8''-modified sialyl lactose trisaccharides in multimilligram amounts.

  5. A sialylation study of mouse brain gangliosides by MALDI a-TOF and o-TOF mass spectrometry.

    Science.gov (United States)

    Zarei, Mostafa; Bindila, Laura; Souady, Jamal; Dreisewerd, Klaus; Berkenkamp, Stefan; Müthing, Johannes; Peter-Katalinić, Jasna

    2008-06-01

    Matrix-assisted laser desorption/ionization (MALDI) process of sialoglycoconjugates is generally accompanied by different levels of cleavage of sialic acid residues and/or by dehydration, and decarboxylation reactions. Quantitative densitometry of the mouse brain ganglioside (MBG) components separated by high-performance thin layer chromatography (HPTLC) and evidenced by orcinol staining was a basis to verify the ganglioside composition pattern with respect to the relative abundances of individual components in the mixture. A systematic mass spectrometry (MS) sialylation analysis has been carried out to evaluate the feasibility of an axial time-of-flight (a-TOF) MS, equipped with a vacuum MALDI source and an orthogonal-TOF (o-TOF) instrument with an ion source operated at about 1 mbar of N(2). Besides, the esterification by one methyl group of the carboxyl group in sialic acid to increase the stability of the ganglioside species for MALDI MS analysis has been tested and the yield of intact ganglioside species and of the neutral loss of water and carbon dioxide estimated. For the sialylation analysis of native ganglioside mixtures the MALDI o-TOF analysis with 6-azo-2-thiothymine/diammonium citrate (ATT/DAC) as a matrix appears as an optimal approach for ganglioside profiling.

  6. Improvement of trans-sialylation versus hydrolysis activity of an engineered sialidase from Trypanosoma rangeli by use of co-solvents

    DEFF Research Database (Denmark)

    Zeuner, Birgitte; Riisager, Anders; Mikkelsen, Jørn Dalgaard

    2014-01-01

    hexafluorophosphate), were examined as co-solvents for the improvement of the synthesis versus hydrolysis ratio in the trans-sialylation of lactose, catalysed by an engineered sialidase from Trypanosoma rangeli. The use of 25 % (v/v) t-butanol as co-solvent significantly increased 3'-sialyllactose production by 40...

  7. Characterization of sialylated and fucosylated glycopeptides of beta2-glycoprotein I by a combination of HILIC LC and MALDI MS/MS

    DEFF Research Database (Denmark)

    Kondo, Akira; Thaysen-Andersen, Morten; Hjernø, Karin

    2010-01-01

    were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and tri-antennary complex type glycans, were characterized at three N-glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of beta2-GPI. Further exploration of the complementary nature...

  8. Influence of monoclonal anti-Lewis b, anti-H type 1, and anti-sialyl Lewis x antibodies on binding of Helicobacter pylori to MUC1 mucin.

    Science.gov (United States)

    Radziejewska, I; Leszczyńska, K; Borzym-Kluczyk, M

    2014-01-01

    To assess the influence of monoclonal anti-Lewis b, anti-H type 1, and anti-sialyl Lewis x addition on interactions of sugar structures of MUC1 mucin with Helicobacter pylori. The investigations were carried out on gastric juices of 11 patients and 12 H. pylori strains. The levels of Lewis b and sialyl Lewis x antigens on MUC1 were assessed by sandwich ELISA tests. Anti-Lewis b, anti-H type 1 or anti-sialyl Lewis x monoclonal antibodies were added to MUC1 to determine whether the adhesion activities of H. pylori isolates to examined mucin would be affected. Binding of bacteria to MUC1 was assessed by ELISA test. Clear inhibitory effect of examined antibodies was revealed in 6 of 12 examined H. pylori isolates independently on babA2 status. In the rest of strains this effect was negligible. We confirmed participation of Lewis b, H type 1 and also sialyl Lewis x of MUC1 mucin in interactions with H. pylori independently on babA genopositivity. Not full inhibition and a lack of this effect in some strains suggest an existence of other mechanisms of H. pylori adherence to mucin.

  9. Cloning and Characterization of Sialidases with 2-6′ and 2-3′ Sialyl Lactose Specificity from Pasteurella multocida†

    Science.gov (United States)

    Mizan, Shaikh; Henk, Adam; Stallings, Amy; Maier, Marie; Lee, Margie D.

    2000-01-01

    Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene, nanH, from a fowl cholera isolate of P. multocida. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3′ sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3′ and 2-6′ sialyl lactose, while NanH demonstrated activity only on 2-3′ sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8′ sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces. PMID:11092845

  10. 360-MHz 1H nuclear-magnetic-resonance spectroscopy of sialyl-oligosaccharides from patients with sialidosis (mucolipidosis I and II)

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Dorland, L.; Haverkamp, J.; Strecker, G.; Michalski, J.-C.; Fournet, b.; Spik, G.; Montreuil, J.

    1978-01-01

    360-MHz proton nuclear magnetic resonance spectra were recorded of 10 sialyl-oligosaccharides isolated form urine of sialidosis patients. Their structures are related to the complex aspareagine-linked glydan chains of glycoproteins. By correlation of these spectra and comparison with spectra of

  11. Treatment response in Kawasaki disease is associated with sialylation levels of endogenous but not therapeutic intravenous immunoglobulin G.

    Directory of Open Access Journals (Sweden)

    Shohei Ogata

    Full Text Available Although intravenous immunoglobulin (IVIG is highly effective in Kawasaki disease (KD, mechanisms are not understood and 10-20% of patients are treatment-resistant, manifesting a higher rate of coronary artery aneurysms. Murine models suggest that α2-6-linked sialic acid (α2-6Sia content of IVIG is critical for suppressing inflammation. However, pro-inflammatory states also up-regulate endogenous levels of β-galactoside:α2-6 sialyltransferase-I (ST6Gal-I, the enzyme that catalyzes addition of α2-6Sias to N-glycans. We asked whether IVIG failures correlated with levels of α2-6Sia on infused IVIG or on the patient's own endogenous IgG.We quantified levels of α2-6Sia in infused IVIG and endogenous IgG from 10 IVIG-responsive and 10 resistant KD subjects using multiple approaches. Transcript levels of ST6GAL1, in patient whole blood and B cell lines were evaluated by RT-PCR. Plasma soluble (sST6Gal-I levels were measured by ELISA.There was no consistent difference in median sialylation levels of infused IVIG between groups. However, α2-6Sia levels in endogenous IgG, ST6GAL1 transcript levels, and ST6Gal-I protein in serum from IVIG-resistant KD subjects were lower than in responsive subjects at both pre-treatment and one-year time points (p <0.001, respectively.Our data indicate sialylation levels of therapeutic IVIG are unrelated to treatment response in KD. Rather, lower sialylation of endogenous IgG and lower blood levels of ST6GALI mRNA and ST6Gal-I enzyme predict therapy resistance. These differences were stable over time, suggesting a genetic basis. Because IVIG-resistance increases risk of coronary artery aneurysms, our findings have important implications for the identification and treatment of such individuals.

  12. Human surfactant protein A2 gene mutations impair dimmer/trimer assembly leading to deficiency in protein sialylation and secretion.

    Directory of Open Access Journals (Sweden)

    Yi Song

    Full Text Available Surfactant protein A2 (SP-A2 plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA, a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2.

  13. Ligand modeling and design

    Energy Technology Data Exchange (ETDEWEB)

    Hay, B.P. [Pacific Northwest National Lab., Richland, WA (United States)

    1997-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used in the cost-effective removal of specific radionuclides from nuclear waste streams. Organic ligands with metal ion specificity are critical components in the development of solvent extraction and ion exchange processes that are highly selective for targeted radionuclides. The traditional approach to the development of such ligands involves lengthy programs of organic synthesis and testing, which in the absence of reliable methods for screening compounds before synthesis, results in wasted research effort. The author`s approach breaks down and simplifies this costly process with the aid of computer-based molecular modeling techniques. Commercial software for organic molecular modeling is being configured to examine the interactions between organic ligands and metal ions, yielding an inexpensive, commercially or readily available computational tool that can be used to predict the structures and energies of ligand-metal complexes. Users will be able to correlate the large body of existing experimental data on structure, solution binding affinity, and metal ion selectivity to develop structural design criteria. These criteria will provide a basis for selecting ligands that can be implemented in separations technologies through collaboration with other DOE national laboratories and private industry. The initial focus will be to select ether-based ligands that can be applied to the recovery and concentration of the alkali and alkaline earth metal ions including cesium, strontium, and radium.

  14. MUC1 and the simple mucin-type antigens: Tn and Sialyl-Tn are differently expressed in salivary gland acini and ducts from the submandibular gland, the vestibular folds, and the soft palate

    DEFF Research Database (Denmark)

    Kirkeby, Svend; Moe, Dennis; Jensen, Allan Bardow

    2010-01-01

    Autopsies of the submandibular gland, the vestibular folds and the soft palate from 65-87 old humans were examined to record the immunohistochemical expression of MUC1 and the simple mucin-type antigens Tn and Sialyl-Tn.......Autopsies of the submandibular gland, the vestibular folds and the soft palate from 65-87 old humans were examined to record the immunohistochemical expression of MUC1 and the simple mucin-type antigens Tn and Sialyl-Tn....

  15. Ligand modeling and design

    Energy Technology Data Exchange (ETDEWEB)

    Hay, B. [Pacific Northwest Lab., Richland, WA (United States)

    1996-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used tin applications for the cost-effective removal of specific radionuclides from nuclear waste streams.

  16. Sialylation by ?-galactoside ?-2,6-sialyltransferase and N-glycans regulate cell adhesion and invasion in human anaplastic large cell lymphoma

    OpenAIRE

    Suzuki, Osamu; ABE, MASAFUMI; HASHIMOTO Yuko

    2015-01-01

    The interaction between cell surface glycans and extracellular matrix (ECM) including galectins is known to be closely associated with tumor cell adhesion, invasion and metastasis. We analyzed the roles of cell surface sialylation or glycosylation in galectin or ECM-mediated cell adhesion and invasion of human malignant lymphoma cells. Neuraminidase from Arthrobacter ureafaciens (AU) treatment resulted in reduction of cell adhesion to galectin-8 in human anaplastic large cell lymphoma (H-ALCL...

  17. Sialylation of Campylobacter jejuni endotoxin promotes dendritic cell-mediated B cell responses through CD14-dependent production of IFN-β and TNF-α.

    Science.gov (United States)

    Huizinga, Ruth; van Rijs, Wouter; Bajramovic, Jeffrey J; Kuijf, Mark L; Laman, Jon D; Samsom, Janneke N; Jacobs, Bart C

    2013-12-01

    Campylobacter jejuni is the most common bacterial cause of human gastroenteritis and often precedes development of Guillain-Barré syndrome (GBS), a life-threatening paralytic disease. The incorporation of the carbohydrate sialic acid into C. jejuni lipooligosaccharides (LOS) is associated with increased severity of gastroenteritis and with induction of GBS; however, the underlying mechanisms remain completely unknown. In this study, we demonstrate that sialic acids in C. jejuni endotoxin enhance the rapid production of IFN-β and TNF-α by human dendritic cells (DCs). Using neutralizing Abs and receptors it was shown that these DC-derived cytokines promote the proliferation of human mucosal B cells in a T cell-independent manner. The production of both IFN-β and TNF-α by DCs in response to LOS requires CD14, and the amplified response of DCs to sialylated C. jejuni LOS is CD14 dependent. Together, these results indicate that sialylation of C. jejuni LOS increases DC activation and promotes subsequent B cell responses through CD14-driven production of IFN-β and TNF-α. This enhanced DC/B cell response may explain the increased pathogenicity of sialylated C. jejuni and may be key to the initiation of B cell-mediated autoimmunity in GBS.

  18. Increased Sialylation of Anti-Thomsen-Friedenreich Antigen (CD176 Antibodies in Patients with Gastric Cancer: A Diagnostic and Prognostic Potential

    Directory of Open Access Journals (Sweden)

    Oleg Kurtenkov

    2014-01-01

    Full Text Available Aim. To study whether alterations in the sialylation of antibodies (Ab specific to the Thomsen-Friedenreich (TF glycotope have a diagnostic and prognostic potential in gastric cancer. Methods. Serum samples were taken from patients with gastric carcinoma (n=142 and controls (n=61. The level of TF-specific antibodies and their sialylation was detected using ELISA with synthetic TF-polyacrylamide conjugate as antigen and sialic acid-specific Sambucus nigra agglutinin (SNA. Results. The level of TF-specific IgM was significantly decreased in cancer compared with controls (P≤0.001. Cancer patients showed a higher level of SNA binding to anti-TF IgM and IgA (P≤0.001 irrespective of disease stage, tumor morphology, and gender. Changes in the SNA/Ab index demonstrated moderate sensitivity (66–71% and specificity (60–73% for stomach cancer. The best diagnostic accuracy (100% was achieved in 29% patients with high SNA binding and low anti-TF IgM level. This subset of patients demonstrated the poorest survival. Conclusion. Our findings are the first evidence that the increased sialylation of TF-specific Abs combined with a low level of anti-TF IgM is strongly linked to gastric cancer and patients survival, which can be used as a novel biomarker for cancer detection and prognosis.

  19. Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.

    Science.gov (United States)

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R; George, Henry J; Brooks, Jeanne; Kayser, Kevin J; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers.

  20. Plasma substance P and soluble P-selectin as biomarkers of β ...

    African Journals Online (AJOL)

    Samia A. Ebeid

    2013-09-19

    Sep 19, 2013 ... molecule which plays a key role in hemostasis and thrombosis, mediating platelet rolling and gen- erating procoagulant molecules. Substance P is one of the tachykinins which constitute a family of neuropeptides. It now appears that platelets contain substance P which is released upon stimulation.

  1. Association between circulating levels of P-selectins and burden of ...

    African Journals Online (AJOL)

    Patients in the HBTF group had a significantly lower prevalence of post-PCI thrombolysis in myocardial infarction (TIMI) grade-3 flow than that of the LBTF group (84.52% vs 93.69%, p = 0.037). Compared with the LBTF group, the HBTF group had higher peak values of the myocardial band (MB) fraction of creatine kinase ...

  2. Association between circulating levels of P-selectins and burden of ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Lin et al., 1984; Carlos and. Harlan, 1998; Frenette et al., 1995; Gamble et al., 1990). Studies have indicated that PS mediates the accu- mulation of leukocytes, which in turn promotes fibril deposition (Palabrica et al., 1992).

  3. The prognostic significance of Galectin-3 and P-Selectin in ...

    African Journals Online (AJOL)

    Bladder carcinoma is one of the most common malignancies in urology. The most common type of the bladder cancer is transitional cell carcinoma (TCC). TCC of bladder has a recurrence rate of more than 50%. Therefore, it is important to find some indicators that can predict for recurrence or the development of metastasis.

  4. Design and synthesis of glycoprotein-based multivalent glyco-ligands for influenza hemagglutinin and human galectin-3.

    Science.gov (United States)

    Wang, Helen; Huang, Wei; Orwenyo, Jared; Banerjee, Aditi; Vasta, Gerardo R; Wang, Lai-Xi

    2013-04-01

    We report a facile synthesis of glycoprotein-based glyco-ligands and their binding with influenza hemagglutinin and human galectin-3. Human serum albumin (HSA) was used as the scaffold and an Asn-linked complex type N-glycan prepared from chicken eggs was used as the glycan building block. It was found that Cu(I)-catalyzed alkyne-azide cycloaddition reaction (click chemistry) between the alkyne-labeled glycan and the azide-tagged HSA led to an efficient formation of the glycoconjugates. The density of glycan ligands on the protein scaffold was readily varied by changing the molar ratios of the two reactants. Binding studies indicated that the sialylated and desialylated multivalent glycoligands could selectively bind to influenza hemagglutinin and human galectin-3, respectively, with high affinity. In the two glycan-lectin interactions, a clear multivalent effect was observed. Moreover, a cell-based assay showed that the synthetic multivalent glyco-ligands could efficiently inhibit the attachment of galectin-3 to human prostate cancer and lung cancer cell lines. This study suggests that the synthetic glycoprotein-based glyco-ligands can be useful for different applications, including blocking the function of galectin-3 in cancer metastasis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Galactosyl-lactose sialylation using Trypanosoma cruzi trans-sialidase as the biocatalyst and bovine κ-casein-derived glycomacropeptide as the donor substrate.

    Science.gov (United States)

    Wilbrink, Maarten H; ten Kate, Geert A; van Leeuwen, Sander S; Sanders, Peter; Sallomons, Erik; Hage, Johannes A; Dijkhuizen, Lubbert; Kamerling, Johannis P

    2014-10-01

    trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(β1-x)-glycans (sialo-glycans) to Gal(β1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)nGlc oligosaccharide mixtures (GOS) using bovine κ-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3'-galactosyl-lactose (β3'-GL), 4'-galactosyl-lactose (β4'-GL), and 6'-galactosyl-lactose (β6'-GL). This prebiotic GOS is prepared from lactose by incubation with suitable β-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25°C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (α2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (α2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of β3'-GL, β4'-GL, and β6'-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated β3'-GL and β4'-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of β6'-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the Vmax and kcat values were higher in the case of lactose. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

    Science.gov (United States)

    Wilbrink, Maarten H.; ten Kate, Geert A.; van Leeuwen, Sander S.; Sanders, Peter; Sallomons, Erik; Hage, Johannes A.; Kamerling, Johannis P.

    2014-01-01

    trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(β1-x)-glycans (sialo-glycans) to Gal(β1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)nGlc oligosaccharide mixtures (GOS) using bovine κ-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3′-galactosyl-lactose (β3′-GL), 4′-galactosyl-lactose (β4′-GL), and 6′-galactosyl-lactose (β6′-GL). This prebiotic GOS is prepared from lactose by incubation with suitable β-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25°C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (α2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (α2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of β3′-GL, β4′-GL, and β6′-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated β3′-GL and β4′-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of β6′-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the Vmax and kcat values were higher in the case of lactose. PMID:25063655

  7. Sialylation of Campylobacter jejuni lipo-oligosaccharides is associated with severe gastro-enteritis and reactive arthritis.

    Science.gov (United States)

    Mortensen, Ninell P; Kuijf, Mark L; Ang, C Wim; Schiellerup, Peter; Krogfelt, Karen A; Jacobs, Bart C; van Belkum, Alex; Endtz, Hubert Ph; Bergman, Mathijs P

    2009-10-01

    We used various genotyping methods to identify bacterial genetic markers for development of arthritic symptoms following Campylobacter enteritis. We genotyped a collection of population derived Campylobacter strains, with detailed information on clinical characteristics, including arthritic symptoms. Besides using whole genome screening methods, we focused on the lipo-oligosaccharide (LOS) gene locus in which marker genes for developing post-Campylobacter neurological disease are present. Patients with arthritic symptoms were more frequently infected with Campylobacter jejuni strains with a class A LOS locus. We also found that patients who were infected with a C. jejuni strain containing sialic acid-positive LOS (class A, B or C) more frequently had bloody diarrhoea and a longer duration of symptoms. Furthermore, the IgM antibody response against Campylobacter was stronger in patients with a sialic acid containing LOS. Ganglioside auto-antibodies were observed in a small number of patients following infection with a class C strain. We conclude that sialylation of C. jejuni LOS is not only a risk factor for development of post-infectious symptoms, but is also associated with increased severity of enteric disease.

  8. Dehydroepiandrosterone (DHEA) inhibition of monocyte binding by vascular endothelium is associated with sialylation of neural cell adhesion molecule.

    Science.gov (United States)

    Curatola, Anna-Maria; Huang, Kui; Naftolin, Frederick

    2012-01-01

    Adhesion of monocytes to vascular endothelium is necessary for atheroma formation. This adhesion requires binding of endothelial neural cell adhesion molecule (NCAM) to monocyte NCAM. NCAM:NCAM binding is blocked by sialylation of NCAM (polysialylated NCAM; PSA-NCAM). Since estradiol (E2) and dihydrotestosterone (DHT) induced PSA-NCAM and decreased monocyte adhesion, in consideration of possible clinical applications we tested whether their prohormone dehydroepiandrosterone (DHEA) has similar effects. (1) DHEA was administered to cultured human coronary artery endothelial cells (HCAECs) from men and women. Monocyte binding was assessed using fluorescence-labeled monocytes. (2) HCEACs were incubated with E2, DHT, DHEA alone, or with trilostane, fulvestrant or flutamide. Expression of PSA-NCAM was assessed by immunohistochemistry and Western blotting. Dehydroepiandrosterone inhibited monocyte adhesion to HCAECs by ≥50% (P DHEA's inhibition of monocyte binding appeared to be gender dependent. The DHEA-induced expression of PSA-NCAM was completely blocked by trilostane. In these preliminary in vitro studies, DHEA increased PSA-NCAM expression and inhibited monocyte binding in an estrogen- and androgen receptor-dependent manner. Dehydroepiandrosteroneappears to act via its end metabolites, E2 and DHT. Dehydroepiandrosterone could furnish clinical prevention against atherogenesis and arteriosclerosis.

  9. Sialylation of neurites inhibits complement-mediated macrophage removal in a human macrophage-neuron co-culture system

    Science.gov (United States)

    Linnartz-Gerlach, Bettina; Schuy, Christine; Shahraz, Anahita; Tenner, Andrea J.; Neumann, Harald

    2015-01-01

    The complement system has been implicated in the removal of dysfunctional synapses and neurites during development and in disease processes in the mouse, but it is unclear how far the mouse data can be transferred to humans. Here, we co-cultured macrophages derived from human THP1 monocytes and neurons derived from human induced pluripotent stem cells, to study the role of the complement system in a human model. Components of the complement system were expressed by the human macrophages and human neuronal culture, while receptors of the complement cascade were expressed by human macrophages as shown via gene transcript analysis and flow cytometry. We mimicked pathological conditions leading to an altered glycocalyx by treatment of human neurons with sialidases. Desialylated human neurites were opsonized by the complement component C1q. Furthermore, human neurites with an intact sialic acid cap remained untouched, while desialylated human neurites were removed and ingested by human macrophages. While blockage of the complement receptor 1 (CD35) had no effect, blockage of CD11b as part of the complement receptor 3 (CR3) reversed the effect on macrophage phagocytosis of desialylated human neurites. Data demonstrate that in the human system sialylation of the neuronal glycocalyx serves as an inhibitory flag for complement binding and CR3 mediated phagocytosis by macrophages. PMID:26257016

  10. Milk oligosaccharide sialyl(α2,3)lactose activates intestinal CD11c+ cells through TLR4.

    Science.gov (United States)

    Kurakevich, Ekaterina; Hennet, Thierry; Hausmann, Martin; Rogler, Gerhard; Borsig, Lubor

    2013-10-22

    Breast milk oligosaccharides shape the intestinal environment by affecting mucosal immunity and bacterial colonization. To clarify the role of milk oligosaccharide sialyl(α2,3)lactose (3SL) in intestinal physiology and disease, we investigated colitis development in Il10(-/-) mice exposed to normal or 3SL-deficient milk during lactation. Onset and progression of intestinal inflammation were delayed in Il10(-/-) mice deficient for the α2,3 sialyltransferase 4 (ST3GAL4) responsible for 3SL biosynthesis. The proinflammatory role of 3SL was confirmed by showing that oral supplementation of newborn Il10(-/-);St3gal4(-/-) mice with 3SL increased colitis severity. Conversely, fostering of newborn Il10(-/-) mice to lactating St3gal4(-/-) mothers reduced colitis severity. 3SL directly stimulated mesenteric lymph node CD11c(+) dendritic cells and induced production of cytokines required for expansion of TH1 and TH17 T cells. The stimulatory effect of 3SL was attenuated in Tlr4-deficient CD11c(+) cells, demonstrating that 3SL induces inflammation through Toll-like receptor 4 (TLR4) signaling. Thus, 3SL directly modulates mucosal immunity, which increases susceptibility to colitis.

  11. Co-overexpression of Mgat1 and Mgat4 in CHO cells for production of highly sialylated albumin-erythropoietin.

    Science.gov (United States)

    Cha, Hyun-Myoung; Lim, Jin-Hyuk; Yeon, Jung-Heum; Hwang, Jeong-Min; Kim, Dong-Il

    2017-08-01

    Terminal sialic acids on N-glycan of recombinant human erythropoietin are very important for in vivo half-life, as this glycoprotein has three N-glycosylation sites. N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) catalyze the formation of a glycan antennary structure. These enzymes display different reaction kinetics for a common substrate and generally show low expression in Chinese hamster ovary (CHO) cells. Therefore, genetic control of Mgat expression is an effective method to increase sialic acid contents by enhancing glycan antennarity. To produce highly sialylated albumin-erythropoietin (Alb-EPO), we co-overexpressed the Mgat1 and Mgat4 genes in CHO cells and determined the optimal ratio of Mgat1:Mgat4 gene expression. All transfected cell lines showed increased gene expression of Mgat4, including Mgat1 overexpressing cell line. Sialic acid content of Alb-EPO was highest in co-transfected cells with excess Mgat4 gene, and these cells showed a higher tri- and tetra-antennary structure than control cells. Based on these results, we suggest that co-transfection of the Mgat1 and Mgat4 genes at a ratio of 2:8 is optimal for extension of antennary structures. Also, regulation of Mgat gene expression in the glycan biosynthesis pathway can be a novel approach to increase the terminal sialic acids of N-glycans. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Sterically demanding iminopyridine ligands

    NARCIS (Netherlands)

    Irrgang, Torsten; Keller, Sandra; Maisel, Heidi; Kretschmer, Winfried; Kempe, Rhett

    Two sterically demanding iminopyridine ligands, (2,6-diisopropylphenyl)[6-(2,4,6-triisopropylphenyl)pyridin-2-ylmeth- ylene]amine and (2,6-diisopropylphenyl)]6-(2,6-dimethylphenyl)pyridin-2-ylmethylene]amine, were prepared by a two-step process: first, condensation of 6-bromopyridine-2-carbaldehyde

  13. Metal-ligand cooperation at tethered pi-ligands

    NARCIS (Netherlands)

    Verhoeven, Dide G. A.; Moret, Marc-Etienne

    2016-01-01

    Metal–ligand cooperativity in homogeneous catalysis is emerging as a powerful tool for the design of efficient transition-metal catalysts. This perspective highlights recent advances in the use of neutral π-coordinating ligands, tethered to a transition-metal center by other donor ligands, as

  14. Staphylococcal SSL5 inhibits leukocyte activation by chemokines and anaphylatoxins.

    Science.gov (United States)

    Bestebroer, Jovanka; van Kessel, Kok P M; Azouagh, Hafida; Walenkamp, Annemiek M; Boer, Ingrid G J; Romijn, Roland A; van Strijp, Jos A G; de Haas, Carla J C

    2009-01-08

    Staphylococcus aureus secretes several virulence factors modulating immune responses. Staphylococcal superantigen-like (SSL) proteins are a family of 14 exotoxins with homology to superantigens, but with generally unknown function. Recently, we showed that SSL5 binds to P-selectin glycoprotein ligand 1 dependently of sialyl Lewis X and inhibits P-selectin-dependent neutrophil rolling. Here, we show that SSL5 potently and specifically inhibits leukocyte activation by anaphylatoxins and all classes of chemokines. SSL5 inhibited calcium mobilization, actin polymerization, and chemotaxis induced by chemokines and anaphylatoxins but not by other chemoattractants. Antibody competition experiments showed that SSL5 targets several chemokine and anaphylatoxin receptors. In addition, transfection studies showed that SSL5 binds glycosylated N-termini of all G protein-coupled receptors (GPCRs) but only inhibits stimuli of protein nature that require the receptor N-terminus for activation. Furthermore, SSL5 increased binding of chemokines to cells independent of chemokine receptors through their common glycosaminoglycan-binding site. Importance of glycans was shown for both GPCR and chemokine binding. Thus, SSL5 is an important immunomodulatory protein of S aureus that targets several crucial, initial stages of leukocyte extravasation. It is therefore a potential new antiinflammatory compound for diseases associated with chemoattractants and their receptors and disorders characterized by excessive recruitment of leukocytes.

  15. Primary structure determination of five sialylated oligosaccharides derived from bronchial- mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAcα(2→3)Galα(1→4)[Fucα(1→3)]GlcNAcα(1→.) structural element revealed by 500-MHz 1H NMR spectroscopy

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Lamblin, G.; Boersma, A.; Klein, A.; Roussel, P.; Halbeek, H. van

    1984-01-01

    The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were

  16. Reduced Mucin-7 (Muc7) Sialylation and Altered Saliva Rheology in Sjögren's Syndrome Associated Oral Dryness*

    Science.gov (United States)

    Chaudhury, Nayab M. A.; Proctor, Gordon B.; Karlsson, Niclas G.; Carpenter, Guy H.; Flowers, Sarah A.

    2016-01-01

    Sjögren's syndrome is a chronic autoimmune disorder characterized by lymphocytic infiltration and hypofunction of salivary and lacrimal glands. This loss of salivary function leads to oral dryness, impaired swallowing and speech, and increased infection and is associated with other autoimmune diseases and an increased risk of certain cancers. Despite the implications of this prevalent disease, diagnosis currently takes years, partly due to the diversity in patient presentation. Saliva is a complicated biological fluid with major constituents, including heavily glycosylated mucins MUC5B and MUC7, important for its viscoelastic and hydrating and lubricating properties. This study investigated Sjögren's patient's perception of dryness (bother index questionnaires) along with the rheological, protein composition, and glycan analysis of whole mouth saliva and the saliva on the mucosal surface (residual mucosal saliva) to understand the properties that most affect patient wellbeing. Sjögren's patients exhibited a statistically significant reduction in residual mucosal saliva, salivary flow rate, and extensional rheology, spinnbarkeit (stringiness). Although the concentration of mucins MUC5B and MUC7 were similar between patients and controls, a comparison of protein Western blotting and glycan staining identified a reduction in mucin glycosylation in Sjögren's, particularly on MUC7. LC-MS/MS analysis of O-glycans released from MUC7 by β-elimination revealed that although patients had an increase in core 1 sulfation, the even larger reduction in sialylation resulted in a global decline of charged glycans. This was primarily due to the loss of the extended core 2 disialylated structure, with and without fucosylation. A decrease in the extended, fucosylated core 2 disialylated structure on MUC7, residual mucosal wetness, and whole mouth saliva flow rate appeared to have a negative and cumulative effect on the perception of oral dryness. The observed changes in MUC7

  17. Biochemical and biophysical characterization of the sialyl-/hexosyltransferase synthesizing the meningococcal serogroup W135 heteropolysaccharide capsule.

    Science.gov (United States)

    Romanow, Angela; Haselhorst, Thomas; Stummeyer, Katharina; Claus, Heike; Bethe, Andrea; Mühlenhoff, Martina; Vogel, Ulrich; von Itzstein, Mark; Gerardy-Schahn, Rita

    2013-04-26

    Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. Crucial virulence determinants of pathogenic Nm strains are the polysaccharide capsules that support invasion by hindering complement attack. In NmW-135 and NmY the capsules are built from the repeating units (→ 6)-α-D-Gal-(1 → 4)-α-Neu5Ac-(2 →)n and (→ 6)-α-D-Glc-(1 → 4)-α-Neu5Ac-(2 →)n, respectively. These unusual heteropolymers represent unique examples of a conjugation between sialic acid and hexosyl-sugars in a polymer chain. Moreover, despite the various catalytic strategies needed for sialic acid and hexose transfer, single enzymes (SiaDW-135/Y) have been identified to form these heteropolymers. Here we used SiaDW-135 as a model system to delineate structure-function relationships. In size exclusion chromatography active SiaDW-135 migrated as a monomer. Fold recognition programs suggested two separate glycosyltransferase domains, both containing a GT-B-fold. Based on conserved motifs predicted folds could be classified as a hexosyl- and sialyltransferase. To analyze enzyme properties and interplay of the two identified glycosyltransferase domains, saturation transfer difference NMR and mutational studies were carried out. Simultaneous and independent binding of UDP-Gal and CMP-Sia was seen in the absence of an acceptor as well as when the catalytic cycle was allowed to proceed. Enzyme variants with only one functionality were generated by site-directed mutagenesis and shown to complement each other in trans when combined in an in vitro test system. Together the data strongly suggests that SiaDW-135 has evolved by fusion of two independent ancestral genes encoding sialyl- and galactosyltransferase activity.

  18. The solution conformation of sialyl-alpha (2----6)-lactose studied by modern NMR techniques and Monte Carlo simulations.

    Science.gov (United States)

    Poppe, L; Stuike-Prill, R; Meyer, B; van Halbeek, H

    1992-03-01

    We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used [1H,1H] NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear [1H,13C] NOEs, (c) isotope effects of O1H/O2H hydroxyl groups on 13C chemical shifts, and (d) long-range heteronuclear scalar couplings across glycosidic bonds. We have used this approach to study the trisaccharide sialyl-alpha (2----6)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the alpha (2----6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with phi angles at -60 degrees or 180 degrees and of the 3 staggered rotamers of the omega angle with a predominant gt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution.

  19. ST6Gal-I sialyltransferase promotes tumor necrosis factor (TNF)-mediated cancer cell survival via sialylation of the TNF receptor 1 (TNFR1) death receptor.

    Science.gov (United States)

    Holdbrooks, Andrew T; Britain, Colleen M; Bellis, Susan L

    2018-02-02

    Activation of the tumor necrosis factor receptor 1 (TNFR1) death receptor by TNF induces either cell survival or cell death. However, the mechanisms mediating these distinct outcomes remain poorly understood. In this study, we report that the ST6Gal-I sialyltransferase, an enzyme up-regulated in numerous cancers, sialylates TNFR1 and thereby protects tumor cells from TNF-induced apoptosis. Using pancreatic and ovarian cancer cells with ST6Gal-I knockdown or overexpression, we determined that α2-6 sialylation of TNFR1 had no effect on early TNF-induced signaling events, including the rapid activation of NF-κB, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt (occurring within 15 min). However, upon extended TNF treatment (6-24 h), cells with high ST6Gal-I levels exhibited resistance to TNF-induced apoptosis, as indicated by morphological evidence of cell death and decreased activation of caspases 8 and 3. Correspondingly, at these later time points, high ST6Gal-I expressers displayed sustained activation of the survival molecules Akt and NF-κB. Additionally, extended TNF treatment resulted in the selective enrichment of clonal variants with high ST6Gal-I expression, further substantiating a role for ST6Gal-I in cell survival. Given that TNFR1 internalization is known to be essential for apoptosis induction, whereas survival signaling is initiated by TNFR1 at the plasma membrane, we examined TNFR1 localization. The α2-6 sialylation of TNFR1 was found to inhibit TNF-induced TNFR1 internalization. Thus, by restraining TNFR1 at the cell surface via sialylation, ST6Gal-I acts as a functional switch to divert signaling toward survival. These collective findings point to a novel glycosylation-dependent mechanism that regulates the cellular response to TNF and may promote cancer cell survival within TNF-rich tumor microenvironments. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Soluble CD40 Ligand in Aspirin-Treated Patients Undergoing Cardiac Catheterization.

    Directory of Open Access Journals (Sweden)

    Thomas Gremmel

    Full Text Available Plasma soluble CD40 ligand (sCD40L is mainly generated by cleavage of CD40L from the surface of activated platelets, and therefore considered a platelet activation marker. Although the predictive value of sCD40L for ischemic events has been demonstrated in patients with acute coronary syndromes (ACS, studies on the association of sCD40L with cardiovascular outcomes in lower risk populations yielded heterogeneous results. We therefore sought to investigate factors influencing sCD40L levels, and the predictive value of sCD40L for long-term ischemic events in unselected, aspirin-treated patients undergoing cardiac catheterization. sCD40L was determined by a commercially available enzyme-linked immunosorbent assay in 682 consecutive patients undergoing cardiac catheterization. Two-year follow-up data were obtained from 562 patients. Dual antiplatelet therapy with aspirin and clopidogrel was associated with significantly lower levels of sCD40L and lower platelet surface expressions of P-selectin and activated GPIIb/IIIa compared to aspirin monotherapy (all p≤0.01. Hypertension was linked to lower plasma concentrations of sCD40L, whereas female sex, increasing high-sensitivity C-reactive protein, and hematocrit were associated with higher sCD40L concentrations (all p<0.05. sCD40L levels were similar in patients without and with the primary endpoint in the overall study population (p = 0.4. Likewise, sCD40L levels did not differ significantly between patients without and with the secondary endpoints (both p≥0.4. Similar results were obtained when only patients with angiographically-proven coronary artery disease (n = 459, stent implantation (n = 205 or ACS (n = 125 were analyzed. The adjustment for differences in patient characteristics by multivariate regression analyses did not change the results. ROC curve analyses did not reveal cut-off values for sCD40L for the prediction of the primary or secondary endpoints. In conclusion, plasma sCD40L

  1. Glutamate receptor ligands

    DEFF Research Database (Denmark)

    Guldbrandt, Mette; Johansen, Tommy N; Frydenvang, Karla Andrea

    2002-01-01

    Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA.......g., IC(50) = 300 microM for (2R,4S)-4-methyl-AA (5d)]. The two unsaturated analogs (S)- (7a) and (R)-(E)-Delta(4)-5-methyl-AA (7b) turned out to be a weak AMPA receptor agonist and a weak mixed NMDA/AMPA receptor antagonist, respectively....

  2. AMPA receptor ligands

    DEFF Research Database (Denmark)

    Strømgaard, Kristian; Mellor, Ian

    2004-01-01

    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR), subtype of the ionotropic glutamate receptors (IGRs), mediate fast synaptic transmission in the central nervous system (CNS), and are involved in many neurological disorders, as well as being a key player...... polyamines are known to modulate the function of these receptors in vivo. In this study, recent developments in the medicinal chemistry of polyamine-based ligands are given, particularly focusing on the use of solid-phase synthesis (SPS) as a tool for the facile generation of libraries of polyamine toxin...

  3. Silver nanoparticle plasmonic enhanced förster resonance energy transfer (FRET) imaging of protein-specific sialylation on the cell surface.

    Science.gov (United States)

    Zhao, Tingbi; Li, Ting; Liu, Yang

    2017-07-20

    A large amount of proteins are post-translationally modified with a sialic acid terminal oligosaccharide, and sialylation directly affects the function of glycoproteins and adjusts relevant biological processes. Herein, we developed a method for imaging analysis of protein-specific sialylation on the cell surface via silver nanoparticle (AgNPs) plasmonic enhanced Förster resonance energy transfer (FRET). In this strategy, the target monosaccharide was labelled with the FRET acceptor of Cy5 via bioorthogonal chemistry. In addition, aptamer linked AgNPs were combined with the Cy3 fluorophore by DNA hybridization as the FRET donor probe, which could be conjugated to the target glycoprotein based on specific aptamer-protein recognition. The Cy5 fluorescence signal was obtained under the Cy3 excitation wavelength via FRET. Moreover, the FRET fluorescence signal was obviously enhanced owing to the plasmonic effect of AgNPs at an appropriate distance to Cy3 on the cell surface. Hence, the protein-specific sialic acids were detected with high contrast. The results showed that the AgNP plasmonic enhanced FRET method was not only superior to the bare FRET method but also can be used to evaluate the expression of sialoglycoproteins in different cell types under pharmacological treatments. The AgNP plasmonic enhanced FRET method provides a valuable tool in the research of glycan metabolism biological processes, the active site of glycoproteins and drug screening.

  4. Reduced Mucin-7 (Muc7) Sialylation and Altered Saliva Rheology in Sjögren's Syndrome Associated Oral Dryness.

    Science.gov (United States)

    Chaudhury, Nayab M A; Proctor, Gordon B; Karlsson, Niclas G; Carpenter, Guy H; Flowers, Sarah A

    2016-03-01

    Sjögren's syndrome is a chronic autoimmune disorder characterized by lymphocytic infiltration and hypofunction of salivary and lacrimal glands. This loss of salivary function leads to oral dryness, impaired swallowing and speech, and increased infection and is associated with other autoimmune diseases and an increased risk of certain cancers. Despite the implications of this prevalent disease, diagnosis currently takes years, partly due to the diversity in patient presentation. Saliva is a complicated biological fluid with major constituents, including heavily glycosylated mucins MUC5B and MUC7, important for its viscoelastic and hydrating and lubricating properties. This study investigated Sjögren's patient's perception of dryness (bother index questionnaires) along with the rheological, protein composition, and glycan analysis of whole mouth saliva and the saliva on the mucosal surface (residual mucosal saliva) to understand the properties that most affect patient wellbeing. Sjögren's patients exhibited a statistically significant reduction in residual mucosal saliva, salivary flow rate, and extensional rheology, spinnbarkeit (stringiness). Although the concentration of mucins MUC5B and MUC7 were similar between patients and controls, a comparison of protein Western blotting and glycan staining identified a reduction in mucin glycosylation in Sjögren's, particularly on MUC7. LC-MS/MS analysis of O-glycans released from MUC7 by β-elimination revealed that although patients had an increase in core 1 sulfation, the even larger reduction in sialylation resulted in a global decline of charged glycans. This was primarily due to the loss of the extended core 2 disialylated structure, with and without fucosylation. A decrease in the extended, fucosylated core 2 disialylated structure on MUC7, residual mucosal wetness, and whole mouth saliva flow rate appeared to have a negative and cumulative effect on the perception of oral dryness. The observed changes in MUC7

  5. Metal-ligand cooperation at tethered π-ligands.

    Science.gov (United States)

    Verhoeven, Dide G A; Moret, Marc-Etienne

    2016-10-12

    Metal-ligand cooperativity in homogeneous catalysis is emerging as a powerful tool for the design of efficient transition-metal catalysts. This perspective highlights recent advances in the use of neutral π-coordinating ligands, tethered to a transition-metal center by other donor ligands, as cooperative reaction centers. The state-of-the-art organometallic complexes, including π-coordinating ligands originating from C[double bond, length as m-dash]C, C[double bond, length as m-dash]E (E = O, N) and boron containing moieties, are described here, with special attention on their specific reactivity. Geometric and electronic aspects of ligand design and their influence on the coordination mode and reactivity of the π-system are discussed.

  6. Carbohydrate antigen expression in primary tumors, metastatic lesions, and serous effusions from patients diagnosed with epithelial ovarian carcinoma: evidence of up-regulated Tn and Sialyl Tn antigen expression in effusions.

    Science.gov (United States)

    Davidson, B; Berner, A; Nesland, J M; Risberg, B; Kristensen, G B; Tropé, C G; Bryne, M

    2000-09-01

    The object of this study was the investigation of carbohydrate antigen expression in malignant epithelial cells and benign mesothelial cells in serous effusions from patients diagnosed with epithelial ovarian carcinomas. In addition, to compare antigen expression in carcinoma cells in effusions with those of corresponding primary tumors and metastatic lesions. Sections from 63 malignant effusions from ovarian carcinoma patients and 15 reactive effusions were immunohistochemically stained, using 5 monoclonal antibodies for Lewis(y), Sialyl Lewis(x), Tn, and Sialyl Tn antigens. Tissue sections (n = 97) from corresponding primary ovarian carcinomas and metastatic lesions, as well as from 12 malignant mesotheliomas, were additionally stained using the above panel. Staining for the 4 antigens was seen in carcinoma cells in serous effusions in the majority of cases (range = 71% to 85%). In contrast, immunoreactivity was detected in mesothelial cells in only 6% to 23% of the specimens studied (P < .001 for all 5 markers). With the exception of B3 antibody against Lewis(y) antigen, malignant mesotheliomas stained negative, infrequently showing focal immunoreactivity. An up-regulation of Tn and Sialyl Tn expression was detected in carcinoma cells in effusions when compared with both primary tumors (P < .003 and P < .007, respectively) and metastatic lesions (P < .034 and .041, respectively). Cancer-associated carbohydrate antigens can thus be used as an adjunct in the differentiation between malignant epithelial and reactive mesothelial cells. Ovarian carcinoma cells in effusions show up-regulation of Tn and Sialyl Tn, possibly representing a transient phenotypic alteration facilitating metastasis.

  7. Macrocyclic G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, M C; Ulven, Trond

    2010-01-01

    are macrocyclic structures which have been modeled after the natural product telomestatin or from porphyrin-based ligands discovered in the late 1990s. These two structural classes of G-quadruplex ligands are reviewed here with special attention to selectivity and structure-activity relationships, and with focus...

  8. The ruminant parasite Haemonchus contortus expresses an alpha1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens.

    Science.gov (United States)

    DeBose-Boyd, R A; Nyame, A K; Jasmer, D P; Cummings, R D

    1998-08-01

    Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize alpha1,3-fucosylated GlcNAc and terminal beta-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal beta-Gal, and the glycoconjugates lack the Lewis x (Le(x)) antigen or other related fucose-containing antigens, such as sialylated Le(x), Le(a), Le(b) Le(y), or H-type 1. Direct assays of parasite extracts demonstrate the presence of an alpha1,3-fucosyltransferase (alpha1,3FT) and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT), but not beta1,4-galactosyltransferase. The H. contortus alpha1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galalpha1-->4GlcNAcbeta1-->3Galbeta1-->4Glc to form lacto-N-fucopentaose III Galbeta1-->4[Fuca1-->3]GlcNAc beta1-->3Galbeta1-4GIc, which contains the Le(x) antigen, and the acceptor lacdiNAc (LDN) GalNAcbeta1-->4GlcNAc to form GalNAc beta1-->4[Fualpha1-->3]GlcNAc. The alpha1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 microM and a Vmax of 10.8 nmol-mg(-1) h(-1). The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The alpha1,3FT acts best with type-2 glycan acceptors (Galbeta1-->4GlcNAcbeta1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus alpha1,3FT can synthesize the Le(x) antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.

  9. P53 and cancer-associated sialylated glycans are surrogate markers of cancerization of the bladder associated with Schistosoma haematobium infection.

    Science.gov (United States)

    Santos, Júlio; Fernandes, Elisabete; Ferreira, José Alexandre; Lima, Luís; Tavares, Ana; Peixoto, Andreia; Parreira, Beatriz; Correia da Costa, José Manuel; Brindley, Paul J; Lopes, Carlos; Santos, Lúcio L

    2014-12-01

    Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers. Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%), cell-surface cancer-associated glycan sialyl-Tn (sTn) and sialyl-Lewisa/x (sLea/sLex), involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed in the healthy urothelium. This

  10. P53 and cancer-associated sialylated glycans are surrogate markers of cancerization of the bladder associated with Schistosoma haematobium infection.

    Directory of Open Access Journals (Sweden)

    Júlio Santos

    2014-12-01

    Full Text Available Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers.Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%, cell-surface cancer-associated glycan sialyl-Tn (sTn and sialyl-Lewisa/x (sLea/sLex, involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed in the healthy urothelium

  11. A factor in animal tissues that causes sialyl residues of mucoproteins to react as free sialic acid in the Warren assay.

    Science.gov (United States)

    Brown, C R; Srivastava, P N; Hartree, E F

    1970-06-01

    1. The mucin of the Cowper's gland of the boar is a sialomucoprotein similar to submaxillary-gland mucin. When a solution of either mucin has been incubated for 5min or less with a particulate fraction from homogenized uterine endometriumplus-myometrium of the rabbit, 10-20% of sialyl residues (N-acetylneuraminic acid) give a positive Warren reaction for free N-acetylneuraminic acid. The particulate fraction is devoid of neuraminidase and no free (diffusible) N-acetylneuraminic acid appears during incubation. The factor that catalyses the formation of directreading non-diffusible N-acetylneuraminic acid occurs also in liver, kidney and intestinal mucosa of the rabbit. The factor is present in very small (;microsomal') particles and has not yet been solubilized. Homogenates of boar Cowper's gland contain both factor and mucin; thus direct-reading non-diffusible N-acetylneuraminic acid appears when such homogenates are stored. 2. Under optimum conditions 1mg of uterine protein catalyses the formation of 0.05-0.1mumol of direct-reading non-diffusible N-acetylneuraminic acid/min. This activity is considerably higher than the neuraminidase activities of comparable homogenates of animal tissues or of liver lysosomes. The factor is thermostable and its activity shows little variation within (i) the pH range 3-10, (ii) the temperature range 20-37 degrees C. Activity is inhibited strongly by 2,2'-bipyridyl and by ammonium pyrrolidine dithiocarbamate but is unaffected by EDTA. Its action can be simulated by low concentrations of Fe(2+). From this it may be inferred that the factor is a protein-bound from of bivalent iron. A number of pure iron-containing proteins and haemoproteins were completely inactive. The following substrates were not sources of direct-reading non-diffusible N-acetylneuraminic acid: methoxyneuraminic acid, sialyl-lactose, brain gangliosides, and sialoproteins in which N-acetylneuraminic acid is linked to galactose residues. 3. It is proposed that the

  12. Synthesis of divalent ligands of β-thio- and β-N-galactopyranosides and related lactosides and their evaluation as substrates and inhibitors of Trypanosoma cruzi trans-sialidase

    Directory of Open Access Journals (Sweden)

    María Emilia Cano

    2014-12-01

    Full Text Available In this work we describe the synthesis of mono- and divalent β-N- and β-S-galactopyranosides and related lactosides built on sugar scaffolds and their evaluation as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS. This enzyme catalyzes the transfer of sialic acid from an oligosaccharidic donor in the host, to parasite βGalp terminal units and it has been demonstrated that it plays an important role in the infection. Herein, the enzyme was also tested as a tool for the chemoenzymatic synthesis of sialic acid containing glycoclusters. The transfer reaction of sialic acid was performed using a recombinant TcTS and 3’-sialyllactose as sialic acid donor, in the presence of the acceptor having βGalp non reducing ends. The products were analyzed by high performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD. The ability of the different S-linked and N-linked glycosides to inhibit the sialic acid transfer reaction from 3’-sialyllactose to the natural substrate N-acetyllactosamine, was also studied. Most of the substrates behaved as good acceptors and moderate competitive inhibitors. A di-N-lactoside showed to be the strongest competitive inhibitor among the compounds tested (70% inhibition at equimolar concentration. The usefulness of the enzymatic trans-sialylation for the preparation of sialylated ligands was assessed by performing a preparative sialylation of a divalent substrate, which afforded the monosialylated compound as main product, together with the disialylated glycocluster.

  13. Syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl alpha(2-->3) lactose moieties as inhibitors for human influenza viruses.

    Science.gov (United States)

    Oka, Hiroyuki; Onaga, Tomotsune; Koyama, Tetsuo; Guo, Chao-Tan; Suzuki, Yasuo; Esumi, Yasuaki; Hatano, Ken; Terunuma, Daiyo; Matsuoka, Koji

    2009-08-01

    A series of carbosilane dendrimers uniformly functionalized with sialyl lactose moieties (Neu5Ac alpha2-->3Gal beta1-->4Glc) was systematically synthesized, and biological evaluations for anti-influenza virus activity using the glycodendrimers were performed. The results suggested that the glycodendrimers had unique biological activities depending on the form of their core frame, and Dumbbell(1)6-amide type glycodendrimer 7 showed particularly strong inhibitory activities against human influenza viruses [A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2)]. The results suggested that the structure-activity relationship (SAR) on the glycolibrary against various influenza viruses was observed, and dumbbell-shaped dendrimers as supporting carbohydrate moieties were found to be the most suitable core scaffolds in this study.

  14. Two novel mixed-ligand complexes containing organosulfonate ligands.

    Science.gov (United States)

    Li, Mingtian; Huang, Jun; Zhou, Xuan; Fang, Hua; Ding, Liyun

    2008-07-01

    The structures reported herein, viz. bis(4-aminonaphthalene-1-sulfonato-kappaO)bis(4,5-diazafluoren-9-one-kappa(2)N,N')copper(II), [Cu(C(10)H(8)NO(3)S)(2)(C(11)H(6)N(2)O)(2)], (I), and poly[[[diaquacadmium(II)]-bis(mu-4-aminonaphthalene-1-sulfonato)-kappa(2)O:N;kappa(2)N:O] dihydrate], {[Cd(C(10)H(8)NO(3)S)(2)(H(2)O)(2)].2H(2)O}(n), (II), are rare examples of sulfonate-containing complexes where the anion does not fulfill a passive charge-balancing role, but takes an active part in coordination as a monodentate and/or bridging ligand. Monomeric complex (I) possesses a crystallographic inversion center at the Cu(II) atom, and the asymmetric unit contains one-half of a Cu atom, one complete 4-aminonaphthalene-1-sulfonate (ans) ligand and one 4,5-diazafluoren-9-one (DAFO) ligand. The Cu(II) atom has an elongated distorted octahedral coordination geometry formed by two O atoms from two monodentate ans ligands and by four N atoms from two DAFO molecules. Complex (II) is polymeric and its crystal structure is built up by one-dimensional chains and solvent water molecules. Here also the cation (a Cd(II) atom) lies on a crystallographic inversion center and adopts a slightly distorted octahedral geometry. Each ans anion serves as a bridging ligand linking two Cd(II) atoms into one-dimensional infinite chains along the [010] direction, with each Cd(II) center coordinated by four ans ligands via O and N atoms and by two aqua ligands. In both structures, there are significant pi-pi stacking interactions between adjacent ligands and hydrogen bonds contribute to the formation of two- and three-dimensional networks.

  15. Transforming growth factor beta receptor 2 (TGFBR2 changes sialylation in the microsatellite unstable (MSI Colorectal cancer cell line HCT116.

    Directory of Open Access Journals (Sweden)

    Jennifer Lee

    Full Text Available Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs. About 15% of CRC show the microsatellite instability (MSI phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the transforming growth factor beta receptor 2 (TGFBR2 gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox-inducible expression of a single copy wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE. In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor N-acetyl-D-mannosamine (ManNAc revealed a significant decline (∼30% of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.

  16. Transforming growth factor beta receptor 2 (TGFBR2) changes sialylation in the microsatellite unstable (MSI) Colorectal cancer cell line HCT116.

    Science.gov (United States)

    Lee, Jennifer; Ballikaya, Seda; Schönig, Kai; Ball, Claudia R; Glimm, Hanno; Kopitz, Juergen; Gebert, Johannes

    2013-01-01

    Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs). About 15% of CRC show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the transforming growth factor beta receptor 2 (TGFBR2) gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox)-inducible expression of a single copy wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor N-acetyl-D-mannosamine (ManNAc) revealed a significant decline (∼30%) of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.

  17. Correcting ligands, metabolites, and pathways

    NARCIS (Netherlands)

    Ott, M.A.; Vriend, G.

    2006-01-01

    BACKGROUND: A wide range of research areas in bioinformatics, molecular biology and medicinal chemistry require precise chemical structure information about molecules and reactions, e.g. drug design, ligand docking, metabolic network reconstruction, and systems biology. Most available databases,

  18. FAS Ligand Triggers Pulmonary Silicosis

    Science.gov (United States)

    Borges, Valéria M.; Falcão, Haroldo; Leite-Júnior, José H.; Alvim, Luciana; Teixeira, Gerlinde P.; Russo, Momtchilo; Nóbrega, Alberto F.; Lopes, Marcela F.; Rocco, Patricia M.; Davidson, Wendy F.; Linden, Rafael; Yagita, Hideo; Zin, Walter A.; DosReis, George A.

    2001-01-01

    We investigated the role of Fas ligand in murine silicosis. Wild-type mice instilled with silica developed severe pulmonary inflammation, with local production of tumor necrosis factor (TNF)-α, and interstitial neutrophil and macrophage infiltration in the lungs. Strikingly, Fas ligand–deficient generalized lymphoproliferative disease mutant (gld) mice did not develop silicosis. The gld mice had markedly reduced neutrophil extravasation into bronchoalveolar space, and did not show increased TNF-α production, nor pulmonary inflammation. Bone marrow chimeras and local adoptive transfer demonstrated that wild-type, but not Fas ligand–deficient lung macrophages recruit neutrophils and initiate silicosis. Silica induced Fas ligand expression in lung macrophages in vitro and in vivo, and promoted Fas ligand–dependent macrophage apoptosis. Administration of neutralizing anti-Fas ligand antibody in vivo blocked induction of silicosis. Thus, Fas ligand plays a central role in induction of pulmonary silicosis. PMID:11457890

  19. Anti-selectin therapy for the treatment of inflammatory diseases.

    Science.gov (United States)

    Rossi, Barbara; Constantin, Gabriela

    2008-06-01

    Leukocyte migration into the tissues represents a key process in the pathogenesis of inflammatory diseases. Data obtained in clinical trials have convincingly shown that inhibition of leukocyte migration into the target organs represents an effective therapeutic approach for diseases in which inflammation has a noxious effect. Leukocyte tethering and rolling are the earliest steps of leukocyte adhesion cascade in inflamed vessels. Selectins are type I transmembrane glycoproteins that bind sialylated carbohydrate structures in a calcium-dependent manner and are involved in the tethering and rolling of leukocytes under physiological and pathological conditions. Three selectins have been identified: L-, P- and E-selectin. Current understanding of the glycosylation-dependent selectin function reveals a complex role for selectins and their ligands during inflammatory diseases. Among selectin ligands, mucin P-selectin glycoprotein ligand-1 (PSGL-1) binds all three selectins and has a well-documented role in organ targeting during inflammation in animal models. However, although inhibition of selectins and their ligands in animal models of inflammatory diseases has proven the validity of this approach in vivo, only a limited number of anti-selectin drugs have been tested in humans. Recent results obtained in clinical trials for asthma and psoriasis show that, although very challenging, the development of selectin antagonists holds concrete promise for the therapy of inflammatory diseases.

  20. Why mercury prefers soft ligands

    Energy Technology Data Exchange (ETDEWEB)

    Riccardi, Demian M [ORNL; Guo, Hao-Bo [ORNL; Gu, Baohua [ORNL; Parks, Jerry M [ORNL; Summers, Anne [University of Georgia, Athens, GA; Miller, S [University of California, San Francisco; Liang, Liyuan [ORNL; Smith, Jeremy C [ORNL

    2013-01-01

    Mercury (Hg) is a major global pollutant arising from both natural and anthropogenic sources. Defining the factors that determine the relative affinities of different ligands for the mercuric ion, Hg2+, is critical to understanding its speciation, transformation, and bioaccumulation in the environment. Here, we use quantum chemistry to dissect the relative binding free energies for a series of inorganic anion complexes of Hg2+. Comparison of Hg2+ ligand interactions in the gaseous and aqueous phases shows that differences in interactions with a few, local water molecules led to a clear periodic trend within the chalcogenide and halide groups and resulted in the well-known experimentally observed preference of Hg2+ for soft ligands such as thiols. Our approach establishes a basis for understanding Hg speciation in the biosphere.

  1. Identification of the homing molecules that escort pluripotent stem cells-derived hematopoietic stem cells to their niches and human activated T-cells to inflammatory sites.

    KAUST Repository

    Ali, Amal

    2017-12-01

    Hematopoietic cells exploit the multistep paradigm of cell migration to ultimately enable them to perform their function. This process is dictated by the ability of adhesion molecules on the circulating hematopoietic cells to find their counter-receptors on endothelial cells. Of those molecules, the selectin family and their respective ligands induce the initial transient interactions between circulating cells and the opposing endothelium. In this thesis, I focused on studying E-selectin mediated cellular migration in two hematopoietic cell types, namely human hematopoietic stem and progenitor cells (HSPCs) and human T-lymphocytes. HSPCs derived from pluripotent sources theoretically offers a novel, unlimited source for hematopoietic stem cell transplantation therapy. In vitro pluripotent stem cell derived- hematopoietic stem/progenitor cells (ES/iPS-HSPCs) behave much like somatic HSPCs in that they exhibit clonal expansion and multilineage hematopoietic capacity. However, unlike somatic sources, ES/iPS-HSPCs do not give rise to effective hematopoietic repopulation, which may be due to insufficient HSPCs homing to the bone marrow. HSPCs exploit E- and P-selectin to home and engraft into bone marrow niches. Thus, one of my objectives in this thesis was to study the expression of E-selectin ligands associated with ES/iPS-HSPCs. I showed that ES/iPS-HSPCs lack functional E-selectin ligand(s). In an effort to enhance the interaction between Eselectin and ES/iPS-HSPCs, we decorated the cell surface with sialyl-Lewis x (sLex) using the ex-vivo glycan engineering technology. However, this decoration did not improve the engraftment capacity of ES/iPS-HSPCs, in vivo. Induction of E-selectin expression during inflammation is key to recruitment of immune cells and therefore I also focused on analyzing the expression of E-selectin ligands on activated human T-cells. I identified several novel glycoproteins that may function as E-selectin ligands. Specifically, I compared the

  2. 脑心通胶囊对老年人2型糖尿病颈动脉内膜中层厚度和血浆β血小板球蛋白P选择素及纤溶酶原激活物抑制剂1的影响%The effect of Naoxintong capsule treatment on carotid artery intima-media thickness, serum beta thromboglobulin,P-selectin and plasminogen activator inhibitor-1 in elderly type 2 diabetic patients with subclinical atherosclerotic vascular disease

    Institute of Scientific and Technical Information of China (English)

    王俊; 程森华; 杨晓翠; 孙国香; 徐更华; 王益波

    2017-01-01

    目的 探讨脑心通胶囊治疗老年2型糖尿病血管病变患者对其颈动脉内膜中层厚度(IMT)、血浆β-血小板球蛋白(β-TG)、P-选择素(CD62p)和纤溶酶原激活物抑制剂-1(PAI-1)的影响.方法 回顾性研究,选择医院2014年6月至2015年5月收治的110例老年2型糖尿病血管病变患者为研究对象.数字抽签随机分为观察组和对照组(各55例),入选患者均给予常规治疗,观察组患者同时给予脑心通胶囊治疗.对比分析两组患者血浆β-TG、CD62p和PAI-1水平变化. 结果 治疗前,两组患者颈动脉IMT、PAI-1、β-TG和CD62p水平比较,差异无统计学意义(均P>0.05).治疗后,观察组与对照组患者比较,颈动脉IMT(1.31±0.26)mm比(1.44±0.26)mm,差异有统计学意义(t=4.058,P<0.05);观察组患者血浆PAI-1、β-TG和CD62p水平与对照组比较,PAI-1(2.23±0.48)μg/L比(2.56±0.61)μg/L、β TG(29.76±10.24) μg/L比(35.98±10.35)μg/L,CD62p(162.3±21.5)ng/L比(176.96±20.3)ng/L,差异有统计学意义(t值分别为3.965、11.293、14.624,均P<0.05). 结论 脑心通胶囊治疗2型糖尿病亚临床血管病变患者有利于减少颈动脉内膜中层厚度,降低血小板释放活性,减轻患者机体氧化应激反应,从而减轻老年2型糖尿病患者血管病变.%Objective To investigate the effects of Naoxintong Capsule treatment on the carotid artery intima media thickness(IMT),plasma beta thromboglobulin(beta TG),P-selectin(CD62p)and plasminogen activator inhibitor 1 (PAI 1)in elderly patients with type 2 diabetes mellitus and subclinical atherosclerotic vascular disease.Methods In retrospective study,110 cases of elderly patients with type 2 diabetic vascular diseases were selected and admitted as study subjects from June 2014 to May 2015.They were randomized into observation group and control group(n=55,each).All patients were given routine treatment.The patients in the observation group were treated with Naoxintong Capsule.The levels of

  3. Rosetta Ligand docking with flexible XML protocols.

    Science.gov (United States)

    Lemmon, Gordon; Meiler, Jens

    2012-01-01

    RosettaLigand is premiere software for predicting how a protein and a small molecule interact. Benchmark studies demonstrate that 70% of the top scoring RosettaLigand predicted interfaces are within 2Å RMSD from the crystal structure [1]. The latest release of Rosetta ligand software includes many new features, such as (1) docking of multiple ligands simultaneously, (2) representing ligands as fragments for greater flexibility, (3) redesign of the interface during docking, and (4) an XML script based interface that gives the user full control of the ligand docking protocol.

  4. THERMODYNAMIC ASSESSMENT OF ANIONIC LIGANDS ...

    African Journals Online (AJOL)

    DJFLEX

    2010-06-30

    Jun 30, 2010 ... ion. This brings about repulsion between the adsorbent and positively charged ion and consequently a reduction in the amount of heavy metal adsorbed at lower pH. (Lee, 1999). The effectiveness of the anionic ligands in the removal of heavy metal ions from aqueous solution seems to be low for the EDTA.

  5. Sialic acid and sialyl-lactose glyco-conjugates: design, synthesis and binding assays to lectins and swine influenza H1N1 virus.

    Science.gov (United States)

    Zevgiti, Stella; Zabala, Juliana Gonzalez; Darji, Ayub; Dietrich, Ursula; Panou-Pomonis, Eugenia; Sakarellos-Daitsiotis, Maria

    2012-01-01

    The terminal parts of the influenza hemagglutinin (HA) receptors α2,6- and α2,3-sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC(4) ), to formulate human and avian receptor mimics, respectively. SOC(4) , formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N(ε) H(2) groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC(4) -glyco-conjugate bearing two copies of the α2,6-sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6-linkage. SOC(4) -glyco-conjugate bearing two copies of the α2,3-sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its well-known α2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC(4) -glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC(4) [(Ac)(2) ,(3'SL-Aoa)(2) ]-NH(2) 5 and Ac-SOC(4) [(Ac)(2) ,(6'SL-Aoa)(2) ]-NH(2) 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

  6. Leukocyte rolling on P-selectin: a three-dimensional numerical study of the effect of cytoplasmic viscosity.

    Science.gov (United States)

    Khismatullin, Damir B; Truskey, George A

    2012-04-18

    Rolling leukocytes deform and show a large area of contact with endothelium under physiological flow conditions. We studied the effect of cytoplasmic viscosity on leukocyte rolling using our three-dimensional numerical algorithm that treats leukocyte as a compound droplet in which the core phase (nucleus) and the shell phase (cytoplasm) are viscoelastic fluids. The algorithm includes the mechanical properties of the cell cortex by cortical tension and considers leukocyte microvilli that deform viscoelastically and form viscous tethers at supercritical force. Stochastic binding kinetics describes binding of adhesion molecules. The leukocyte cytoplasmic viscosity plays a critical role in leukocyte rolling on an adhesive substrate. High-viscosity cells are characterized by high mean rolling velocities, increased temporal fluctuations in the instantaneous velocity, and a high probability for detachment from the substrate. A decrease in the rolling velocity, drag, and torque with the formation of a large, flat contact area in low-viscosity cells leads to a dramatic decrease in the bond force and stable rolling. Using values of viscosity consistent with step aspiration studies of human neutrophils (5-30 Pa·s), our computational model predicts the velocities and shape changes of rolling leukocytes as observed in vitro and in vivo. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Leukocyte Rolling on P-Selectin: A Three-Dimensional Numerical Study of the Effect of Cytoplasmic Viscosity

    Science.gov (United States)

    Khismatullin, Damir B.; Truskey, George A.

    2012-01-01

    Rolling leukocytes deform and show a large area of contact with endothelium under physiological flow conditions. We studied the effect of cytoplasmic viscosity on leukocyte rolling using our three-dimensional numerical algorithm that treats leukocyte as a compound droplet in which the core phase (nucleus) and the shell phase (cytoplasm) are viscoelastic fluids. The algorithm includes the mechanical properties of the cell cortex by cortical tension and considers leukocyte microvilli that deform viscoelastically and form viscous tethers at supercritical force. Stochastic binding kinetics describes binding of adhesion molecules. The leukocyte cytoplasmic viscosity plays a critical role in leukocyte rolling on an adhesive substrate. High-viscosity cells are characterized by high mean rolling velocities, increased temporal fluctuations in the instantaneous velocity, and a high probability for detachment from the substrate. A decrease in the rolling velocity, drag, and torque with the formation of a large, flat contact area in low-viscosity cells leads to a dramatic decrease in the bond force and stable rolling. Using values of viscosity consistent with step aspiration studies of human neutrophils (5–30 Pa·s), our computational model predicts the velocities and shape changes of rolling leukocytes as observed in vitro and in vivo. PMID:22768931

  8. Circulating microparticles and plasma levels of soluble E- and P-selectins in patients with systemic sclerosis

    DEFF Research Database (Denmark)

    Iversen, Lars; Østergaard, O; Ullman, S

    2013-01-01

    Microparticles (MPs) may be involved in the pathogenesis of systemic sclerosis (SSc), which includes vasculopathy, endothelial cell activation, and coagulation activation. Circulating MPs from SSc patients were characterized and their relationship with soluble markers of vascular activation...

  9. -Pincer Ligand Family through Ligand Post-Modification

    KAUST Repository

    Huang, Mei-Hui

    2017-10-02

    A series of air-stable nickel complexes containing triazine-based PN3P-pincer ligands were synthesized and fully characterized. Complex 3 contains a de-aromatized central triazine ring from the deprotonation of one of the N–H arms. With a post-modification strategy, the Me-PN3P*NiCl complex (3) could be converted into a new class of diimine–traizine PN3P-pincer nickel complexes.

  10. Tumor targeting via integrin ligands

    Directory of Open Access Journals (Sweden)

    Udaya Kiran eMarelli

    2013-08-01

    Full Text Available Selective and targeted delivery of drugs to tumors is a major challenge for an effective cancer therapy and also to overcome the side effects associated with current treatments. Overexpression of various receptors on tumor cells is a characteristic structural and biochemical aspect of tumors and distinguishes them from physiologically normal cells. This abnormal feature is therefore suitable for selectively directing anticancer molecules to tumors by using ligands that can preferentially recognize such receptors. Several subtypes of integrin receptors that are crucial for cell adhesion, cell signaling, cell viability and motility have been shown to have an upregulated expression on cancer cells. Thus, ligands that recognize specific integrin subtypes represent excellent candidates to be conjugated to drugs or drug carrier systems and be targeted to tumors. In this regard, integrins recognizing the RGD cell adhesive sequence have been extensively targeted for tumor specific drug delivery. Here we review key recent examples on the presentation of RGD-based integrin ligands by means of distinct drug delivery systems, and discuss the prospects of such therapies to specifically target tumor cells.

  11. Expression of sialyl-Tn sugar antigen in bladder cancer cells affects response toBacillus Calmette Guérin(BCG) and to oxidative damage.

    Science.gov (United States)

    Severino, Paulo F; Silva, Mariana; Carrascal, Mylene; Malagolini, Nadia; Chiricolo, Mariella; Venturi, Giulia; Astolfi, Annalisa; Catera, Mariangela; Videira, Paula A; Dall'Olio, Fabio

    2017-08-15

    The sialyl-Tn (sTn) antigen is an O -linked carbohydrate chain aberrantly expressed in bladder cancer (BC), whose biosynthesis is mainly controlled by the sialyltransferase ST6GALNAC1. Treatment with Bacillus Calmette-Guérin (BCG) is the most effective adjuvant immunotherapy for superficial BC but one third of the patients fail to respond. A poorly understood correlation between the expression of sTn and BC patient's response to BCG was previously observed. By analyzing tumor tissues, we showed that patients with high ST6GALNAC1 and IL-6 mRNA expression were BCG responders. To investigate the role of sTn in BC cell biology and BCG response, we established the cell lines MCR sTn and MCR Nc by retroviral transduction of the BC cell line MCR with the ST6GALNAC1 cDNA or with an empty vector, respectively. Compared with MCR Nc , BCG-stimulated MCR sTn secreted higher levels of IL-6 and IL-8 and their secretome induced a stronger IL-6, IL-1β, and TNFα secretion by macrophages, suggesting the induction of a stronger inflammatory response. Transcriptomic analysis of MCR Nc and MCR sTn revealed that ST6GALNAC1 /sTn expression modulates hundreds of genes towards a putative more malignant phenotype and down-regulates several genes maintaining genomic stability. Consistently, MCR sTn cells displayed higher H 2 O 2 sensitivity. In MCR sTn ,, BCG challenge induced an increased expression of several regulatory non coding RNA genes. These results indicate that the expression of ST6GALNAC1 /sTn improves the response to BCG therapy by inducing a stronger macrophage response and alters gene expression towards malignancy and genomic instability, increasing the sensitivity of BC cells to the oxidizing agents released by BCG.

  12. Tyrosine sulfation of the amino terminus of PSGL-1 is critical for enterovirus 71 infection.

    Directory of Open Access Journals (Sweden)

    Yorihiro Nishimura

    Full Text Available Enterovirus 71 (EV71 is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1 as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1-P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. To elucidate the molecular basis of the PSGL-1-EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1-selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1-EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes.

  13. Selectins mediate small cell lung cancer systemic metastasis.

    Directory of Open Access Journals (Sweden)

    Franziska Heidemann

    Full Text Available Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181. However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.

  14. Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile.

    Science.gov (United States)

    Lee, Jong Hyun; Jeong, Yeong Ran; Kim, Yeon-Gu; Lee, Gyun Min

    2017-08-01

    To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (q Fc ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After 3 days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. Biotechnol. Bioeng

  15. Ligand photo-isomerization triggers conformational changes in iGluR2 ligand binding domain.

    Directory of Open Access Journals (Sweden)

    Tino Wolter

    Full Text Available Neurological glutamate receptors bind a variety of artificial ligands, both agonistic and antagonistic, in addition to glutamate. Studying their small molecule binding properties increases our understanding of the central nervous system and a variety of associated pathologies. The large, oligomeric multidomain membrane protein contains a large and flexible ligand binding domains which undergoes large conformational changes upon binding different ligands. A recent application of glutamate receptors is their activation or inhibition via photo-switchable ligands, making them key systems in the emerging field of optochemical genetics. In this work, we present a theoretical study on the binding mode and complex stability of a novel photo-switchable ligand, ATA-3, which reversibly binds to glutamate receptors ligand binding domains (LBDs. We propose two possible binding modes for this ligand based on flexible ligand docking calculations and show one of them to be analogues to the binding mode of a similar ligand, 2-BnTetAMPA. In long MD simulations, it was observed that transitions between both binding poses involve breaking and reforming the T686-E402 protein hydrogen bond. Simulating the ligand photo-isomerization process shows that the two possible configurations of the ligand azo-group have markedly different complex stabilities and equilibrium binding modes. A strong but slow protein response is observed after ligand configuration changes. This provides a microscopic foundation for the observed difference in ligand activity upon light-switching.

  16. Imaging and treatment of malignant metastatic tumors by using radiation-sensitive, immunolabeled liquid-core microcapsules

    Science.gov (United States)

    Harada, Satoshi; Ehara, Shigeru; Ishii, Keizo; Sato, Takahiro; Kouka, Masashi; Kamiya, Tomihiro; Sera, Koichiro; Goto, Shyoko

    2014-01-01

    In this study, two types of microcapsules were designed: (1) computed tomography (CT)-detectable anti-αvβ3 (E[c(RGDfK)]2) microcapsules, containing P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1), for the observation of metastases through αvβ3-antigen-antibody accumulation; and (2) metastasis-targeting microcapsules that upon irradiation release anticancer drugs with high affinity for P-selectin. These microcapsules were tested on C3He/N mice with MM48 tumors undergoing two radiotherapy sessions.

  17. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    G protein-coupled receptors (GPCRs) belong to a large superfamily of membrane receptors mediating a variety of physiological functions. As such they are attractive targets for drug therapy. However, it remains a challenge to develop subtype selective GPCR ligands due to the high conservation...... sites. Computational studies on ligand binding to GPCRs have revealed transient, low-affinity binding sites, termed metastable binding sites. Metastable binding sites may provide a new source of allosteric binding sites that could be exploited in the design of bitopic ligands. Unlike the bitopic ligands...

  18. Clinical Use of PPARγ Ligands in Cancer

    Directory of Open Access Journals (Sweden)

    Jennifer L. Hatton

    2008-01-01

    Full Text Available The role of PPARγ in adipocyte differentiation has fueled intense interest in the function of this steroid nuclear receptor for regulation of malignant cell growth and differentiation. Given the antiproliferative and differentiating effects of PPARγ ligands on liposarcoma cells, investigation of PPARγ expression and ligand activation in other solid tumors such as breast, colon, and prostate cancers ensued. The anticancer effects of PPARγ ligands in cell culture and rodent models of a multitude of tumor types suggest broad applicability of these agents to cancer therapy. This review focuses on the clinical use of PPARγ ligands, specifically the thiazolidinediones, for the treatment and prevention of cancer.

  19. Macrocyclic ligands for uranium complexation

    Energy Technology Data Exchange (ETDEWEB)

    Potts, K.T.

    1991-04-01

    A highly preorganized 24-macrocycle containing biuret, thiobiuret and pyridine subunits has been prepared by high dilution ring-closure procedures. Intermediate products to this macrocycle have been utilized to extend this synthetic route to include further representatives where solubility and stability will be influenced by substituent variation. A 1:1 complex has been formed from uranyl acetate and a quinquepyridine derivative, this representing a new type of ligand for the uranyl ion. A very convenient synthetic procedure that will allow the incorporation of these macrocycles into polymeric systems has been developed for the introduction of a vinyl substituent into the 4-position of the pyridine ring. Using triflate, vinyltributyltin and Pd{sup 0} chemistry, this procedure should make a variety of substituted 4-vinylpyridines available for the first time. 3 refs.

  20. Ligand-binding kinetics on histamine receptors

    NARCIS (Netherlands)

    Bosma, Reggie; Mocking, T.A.M.; Leurs, R.; Vischer, H.F.

    2017-01-01

    Equilibrium-binding affinities of ligands for a drug target do not always accurately reflect the success of drug candidates in the clinic. Affinity-based predictions concerning competitive antagonism on the target will only be accurate if equilibrium binding of both ligands is allowed. Unless

  1. Ligand based pharmacophore modelling of anticancer histone ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Jun 21, 2010 ... deacetylases (HDAC) have emerged as an important class of anticancer agents. Various side effects like myocardium damage and ... calculated through the software Jmol. The proposed model has been .... The study was carried out using the software Ligand Scout (version. 2.03[i2_001]). Ligand Scout is a ...

  2. Simple tertiary phosphines to hexaphosphane ligands: Syntheses ...

    Indian Academy of Sciences (India)

    Abstract. Designing efficient phosphorus-based ligands to make catalysts for homogeneous catalysis has been a great challenge for chemists. Despite a plethora of phosphorus ligands ranging from simple tertiary phosphines to polyphosphines are known, the enthusiasm to generate new ones is mainly due to the demand.

  3. Organotellurium ligands – designing and complexation reactions

    Indian Academy of Sciences (India)

    Unknown

    Abstract. A variety of tellurium ligands has been designed and studied for their complexation reactions in the last decade. Of these hybrid telluroethers, halotellurium ligands and polytellurides are the most notable ones. RTe–and polytelluride ions have also been used to design clusters. Ligation of ditelluroethers and ...

  4. CXCR4 Ligands : The Next Big Hit?

    NARCIS (Netherlands)

    Walenkamp, Annemiek M. E.; Lapa, Constantin; Herrmann, Ken; Wester, Hans-Juergen

    2017-01-01

    The G protein-coupled protein receptor C-X-C chemokine receptor 4 (CXCR4) is an attractive target for cancer diagnosis and treatment, as it is overexpressed in many solid and hematologic cancers. Binding of its ligand, C-X-C chemokine ligand 12 (CXCL12), results in receptor internalization and

  5. Flexible ligand docking using a genetic algorithm

    Science.gov (United States)

    Oshiro, C. M.; Kuntz, I. D.; Dixon, J. Scott

    1995-04-01

    Two computational techniques have been developed to explore the orientational and conformational space of a flexible ligand within an enzyme. Both methods use the Genetic Algorithm (GA) to generate conformationally flexible ligands in conjunction with algorithms from the DOCK suite of programs to characterize the receptor site. The methods are applied to three enzyme-ligand complexes: dihydrofolate reductase-methotrexate, thymidylate synthase-phenolpthalein and HIV protease-thioketal haloperidol. Conformations and orientations close to the crystallographically determined structures are obtained, as well as alternative structures with low energy. The potential for the GA method to screen a database of compounds is also examined. A collection of ligands is evaluated simultaneously, rather than docking the ligands individually into the enzyme.

  6. A High-Throughput Technique Reveals the Load- and Site Density-Dependent Kinetics of E-Selectin

    Science.gov (United States)

    Snook, Jeremy H.; Guilford, William H.

    2013-01-01

    The kinetics of bond rupture between receptors and ligand are critically dependent on applied mechanical force. Force spectroscopy of single receptor-ligand pairs to measure kinetics is a laborious and time-consuming process that is generally performed using individual force probes and making one measurement at a time when typically hundreds of measurements are needed. A high-throughput approach is thus desirable. We report here a magnetic bond puller that provides high-throughput measurements of single receptor-ligand bond kinetics. Electromagnets are used to apply pN tensile and compressive forces to receptor-coated magnetic microspheres while monitoring their contact with a ligand-coated surface. Bond lifetimes and the probability of forming a bond are measured via videomicroscopy, and the data are used to determine the load dependent rates of bond rupture and bond formation. The approach is simple, customizable, relatively inexpensive, and can make dozens of kinetic measurements simultaneously. We used the device to investigate how compressive and tensile forces affect the rates of formation and rupture, respectively, of bonds between E-selectin and sialyl Lewisa (sLea), a sugar on P-selectin glycoprotein ligand-1 to which selectins bind. We confirmed earlier findings of a load-dependent rate of bond formation between these two molecules, and that they form a catch-slip bond like other selectin family members. We also make the novel observation of an “ideal” bond in a highly multivalent system of this receptor-ligand pair. PMID:24511329

  7. Correcting ligands, metabolites, and pathways

    Directory of Open Access Journals (Sweden)

    Vriend Gert

    2006-11-01

    Full Text Available Abstract Background A wide range of research areas in bioinformatics, molecular biology and medicinal chemistry require precise chemical structure information about molecules and reactions, e.g. drug design, ligand docking, metabolic network reconstruction, and systems biology. Most available databases, however, treat chemical structures more as illustrations than as a datafield in its own right. Lack of chemical accuracy impedes progress in the areas mentioned above. We present a database of metabolites called BioMeta that augments the existing pathway databases by explicitly assessing the validity, correctness, and completeness of chemical structure and reaction information. Description The main bulk of the data in BioMeta were obtained from the KEGG Ligand database. We developed a tool for chemical structure validation which assesses the chemical validity and stereochemical completeness of a molecule description. The validation tool was used to examine the compounds in BioMeta, showing that a relatively small number of compounds had an incorrect constitution (connectivity only, not considering stereochemistry and that a considerable number (about one third had incomplete or even incorrect stereochemistry. We made a large effort to correct the errors and to complete the structural descriptions. A total of 1468 structures were corrected and/or completed. We also established the reaction balance of the reactions in BioMeta and corrected 55% of the unbalanced (stoichiometrically incorrect reactions in an automatic procedure. The BioMeta database was implemented in PostgreSQL and provided with a web-based interface. Conclusion We demonstrate that the validation of metabolite structures and reactions is a feasible and worthwhile undertaking, and that the validation results can be used to trigger corrections and improvements to BioMeta, our metabolite database. BioMeta provides some tools for rational drug design, reaction searches, and

  8. Chemistry of marine ligands and siderophores.

    Science.gov (United States)

    Vraspir, Julia M; Butler, Alison

    2009-01-01

    Marine microorganisms are presented with unique challenges to obtain essential metal ions required to survive and thrive in the ocean. The production of organic ligands to complex transition metal ions is one strategy to both facilitate uptake of specific metals, such as iron, and to mitigate the potential toxic effects of other metal ions, such as copper. A number of important trace metal ions are complexed by organic ligands in seawater, including iron, cobalt, nickel, copper, zinc, and cadmium, thus defining the speciation of these metal ions in the ocean. In the case of iron, siderophores have been identified and structurally characterized. Siderophores are low molecular weight iron-binding ligands produced by marine bacteria. Although progress has been made toward the identity of in situ iron-binding ligands, few compounds have been identified that coordinate the other trace metals. Deciphering the chemical structures and production stimuli of naturally produced organic ligands and the organisms they come from is fundamental to understanding metal speciation and bioavailability. The current evidence for marine ligands, with an emphasis on siderophores, and discussion of the importance and implications of metal-binding ligands in controlling metal speciation and cycling within the world's oceans are presented.

  9. Naïve T cells re-distribute to the lungs of selectin ligand deficient mice.

    Directory of Open Access Journals (Sweden)

    John R Harp

    2010-06-01

    Full Text Available Selectin mediated tethering represents one of the earliest steps in T cell extravasation into lymph nodes via high endothelial venules and is dependent on the biosynthesis of sialyl Lewis X (sLe(x ligands by several glycosyltransferases, including two fucosyltransferases, fucosyltransferase-IV and -VII. Selectin mediated binding also plays a key role in T cell entry to inflamed organs.To understand how loss of selectin ligands (sLe(x influences T cell migration to the lung, we examined fucosyltransferase-IV and -VII double knockout (FtDKO mice. We discovered that FtDKO mice showed significant increases (approximately 5-fold in numbers of naïve T cells in non-inflamed lung parenchyma with no evidence of induced bronchus-associated lymphoid tissue. In contrast, activated T cells were reduced in inflamed lungs of FtDKO mice following viral infection, consistent with the established role of selectin mediated T cell extravasation into inflamed lung. Adoptive transfer of T cells into FtDKO mice revealed impaired T cell entry to lymph nodes, but selective accumulation in non-lymphoid organs. Moreover, inhibition of T cell entry to the lymph nodes by blockade of L-selectin, or treatment of T cells with pertussis toxin to inhibit chemokine dependent G-coupled receptor signaling, also resulted in increased T cells in non-lymphoid organs. Conversely, inhibition of T cell egress from lymph nodes using FTY720 agonism of S1P1 impaired T cell migration into non-lymphoid organs.Taken together, our results suggest that impaired T cell entry into lymph nodes via high endothelial venules due to genetic deficiency of selectin ligands results in the selective re-distribution and accumulation of T cells in non-lymphoid organs, and correlates with their increased frequency in the blood. Re-distribution of T cells into organs could potentially play a role in the initiation of T cell mediated organ diseases.

  10. Coordinate unsaturation with fluorinated ligands

    Energy Technology Data Exchange (ETDEWEB)

    Rack, J.L.; Hurlburt, P.K.; Anderson, O.P.; Strauss, S.H. [Colorado State Univ., Ft. Collins, CO (United States)

    1993-12-31

    The preparation and characterization of Zn(OTeF{sub 5}){sub 2} has resulted in a model compound with which to explore the concept of coordinative unsaturation. The coordination of solvents of varying donicity and dielectric constant to the Zn(II) ions in Zn(OTeF{sub 5}){sub 2} was studied by vapor phase monometry, NMR and IR spectroscopy, conductimetry, and X-Ray crystallography. The structures of [Zn(C{sub 6}H{sub 5}NO{sub 2}){sub 2}(OTeF{sub 5})2]2 and Zn(C{sub 6}H{sub 5}NO{sub 2}){sub 3}(OTEF{sub 5}){sub 2} demonstrate the electronic flexibility of some weakly coordinating solvents in that nitrobenzene can function as either an {eta}{sup 1}O or {eta}{sup 2}O,O`-ligand. The dependence of the number of bound solvent molecules and the degree of OTeF{sub 5}{minus} dissociation on solvent donor number and dielectric constant will be presented.

  11. Ligand based pharmacophore modelling of anticancer histone ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Jun 21, 2010 ... for designing the pharmacophore onto the set of 70 compounds of three different classes and two subclasses. ... More effective anticancer drug production with novel modes of action .... with Ligand Scout using default settings.

  12. Controlling Signal Transduction with Synthetic Ligands

    Science.gov (United States)

    Spencer, David M.; Wandless, Thomas J.; Schreiber, Stuart L.; Crabtree, Gerald R.

    1993-11-01

    Dimerization and oligomerization are general biological control mechanisms contributing to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. Cell permeable, synthetic ligands were devised that can be used to control the intracellular oligomerization of specific proteins. To demonstrate their utility, these ligands were used to reduce intracellular oligomerization of cell surface receptors that lacked their transmembrane and extracellular regions but contained intracellular signaling domains. Addition of these ligands to cells in culture resulted in signal transmission and specific target gene activation. Monomeric forms of the ligands blocked the pathway. This method of ligandregulated activation and termination of signaling pathways has the potential to be applied wherever precise control of a signal transduction pathway is desired.

  13. Organometallic chemistry of chiral diphosphazane ligands ...

    Indian Academy of Sciences (India)

    Unknown

    Organometallic chemistry of chiral diphosphazane ligands: Synthesis and structural characterisation. KANNAN RAGHURAMAN, SWADHIN K MANDAL,. T S VENKATAKRISHNAN, SETHARAMPATTU S. KRISHNAMURTHY* and MUNIRATHINAM NETHAJI. Department of Inorganic and Physical Chemistry, Indian Institute of ...

  14. EGFR Activation by Spatially Restricted Ligands

    National Research Council Canada - National Science Library

    Goodrich, Jennifer S

    2005-01-01

    ...) activity has been associated with an increased prognosis of breast cancer. During oogenesis in Drosophila melanogaster, local EGFR activation by the spatially restricted TGF alpha-like ligand, Gurken (Grk...

  15. Asymmetric hydrogenation using monodentate phosphoramidite ligands

    NARCIS (Netherlands)

    Minnaard, Adriaan J.; Feringa, Ben L.; Lefort, Laurent; De Vries, Johannes G.

    2007-01-01

    Monodentate phosphoramidites are excellent ligands for Rh-catalyzed asymmetric hydrogenations of substituted olefins. Enantioselectivities between 95 and 99% were obtained in the asymmetric hydrogenation of protected alpha- and beta-dehydroamino acids and esters, itaconic acid and esters, aromatic

  16. Ligand Interface Chemistry of Lead Chalcogenide Nanocrystals

    Science.gov (United States)

    Liu, Kun; Bealing, Clive; Hennig, Richard; Hennig Group Team

    2013-03-01

    Lead chalcogenides nanocrystals (NCs) have shown promise in photovoltaic applications. Surface chemistry is one of the most important, yet least understood aspects of NC synthesis and functionalization controlling their properties. For example, an incomplete surface passivation could create trap states that enhance undesired exciton recombination. Clever choices of ligands ensure that neighboring NCs are electronically coupled while maintaining their quantum size effects. These two barriers limiting performance of NC solar cells illustrate the importance of fundamental studies for the interaction between ligands and NC surfaces. We use density functional theory to determine the binding sites and energies of ligands commonly used in NC synthesis and functionalization. Specifically we study amine-, carboxyl-, and thiol-ligands on different PbSe and PbS surfaces. For methylamine ligands we find a similar binding energy on the (100) and (111) facet while carboxylic acid ligands strongly prefer the (111) facet leading to different effective NC shapes. This work was supported in part by Award No. KUS-C1-018-02, made by King Abdullah University of Science and Technology (KAUST) and by the Energy Materials Center at Cornell (EMC2) funded by the U.S. Department of Energy under Award Number DE-SC0001086

  17. Designer TGFβ superfamily ligands with diversified functionality.

    Directory of Open Access Journals (Sweden)

    George P Allendorph

    Full Text Available Transforming Growth Factor--beta (TGFβ superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs, and Bone Morphogenetic Proteins (BMPs, are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer, to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.

  18. Construction of dinuclear complexes using multidentate ligands

    Energy Technology Data Exchange (ETDEWEB)

    Sampson, C.L

    2000-04-01

    This work details the synthesis of novel copper(I), copper(II), nickel(II) and zinc(II) dinuclear complexes. Attempts have been made to control the co-ordination architectures of the metal centres by using bis-bidentate and tridentate chelating N,S- and N-donor ligands to generate dinuclear systems. The ligands were both symmetrically and asymmetrically disubstituted pyridazine-based and pyridine-based ligands consisting of a mixture of N-only and mixed N,S-donors. The study using the pyridazine-based ligands continues previous research in our group using 3,6-bis disubstituted pyridazine-based ligands to form complexes with copper(l) and copper(II). The pyridazine-based ligands have been seen to be bis-bidentate upon co-ordination of copper. The pyridazine-based ligands could be envisaged to generate dinuclear complexes by directly bridging between two metal ions. This study involved the formation of copper(l), nickel(II) and zinc(II) complexes with these ligands. The structural properties of two particular complexes have been explored using X-ray crystallography and spectroscopic techniques. Pyridine-based ligands have also been used previously in our group as tridentate chelating ligands. They have been seen to form dinuclear complexes with copper(I) and copper(II) when reacted with an additional bridging ligand e.g. 4,4'-bipyridine. This provides an alternative method for generating dinuclear complexes. Chapter 1 presents an introduction to the area of supramolecular chemistry from which we can learn the principles of polymer formation and them 'in reverse' to generate discrete dinuclear systems. Chapter 2 details the synthesis of the pyridazine and pyridine-based ligands including a detailed nmr study of the ligands. Since the ligands were synthesised using cyclic thioamides as terminal groups it has been found that thiol-thione tautomerisation occurred during synthesis giving rise to two possible ligand conformations. The nmr study has been used

  19. Semiconductor Quantum Dots with Photoresponsive Ligands.

    Science.gov (United States)

    Sansalone, Lorenzo; Tang, Sicheng; Zhang, Yang; Thapaliya, Ek Raj; Raymo, Françisco M; Garcia-Amorós, Jaume

    2016-10-01

    Photochromic or photocaged ligands can be anchored to the outer shell of semiconductor quantum dots in order to control the photophysical properties of these inorganic nanocrystals with optical stimulations. One of the two interconvertible states of the photoresponsive ligands can be designed to accept either an electron or energy from the excited quantum dots and quench their luminescence. Under these conditions, the reversible transformations of photochromic ligands or the irreversible cleavage of photocaged counterparts translates into the possibility to switch luminescence with external control. As an alternative to regulating the photophysics of a quantum dot via the photochemistry of its ligands, the photochemistry of the latter can be controlled by relying on the photophysics of the former. The transfer of excitation energy from a quantum dot to a photocaged ligand populates the excited state of the species adsorbed on the nanocrystal to induce a photochemical reaction. This mechanism, in conjunction with the large two-photon absorption cross section of quantum dots, can be exploited to release nitric oxide or to generate singlet oxygen under near-infrared irradiation. Thus, the combination of semiconductor quantum dots and photoresponsive ligands offers the opportunity to assemble nanostructured constructs with specific functions on the basis of electron or energy transfer processes. The photoswitchable luminescence and ability to photoinduce the release of reactive chemicals, associated with the resulting systems, can be particularly valuable in biomedical research and can, ultimately, lead to the realization of imaging probes for diagnostic applications as well as to therapeutic agents for the treatment of cancer.

  20. LigandRFs: random forest ensemble to identify ligand-binding residues from sequence information alone

    KAUST Repository

    Chen, Peng

    2014-12-03

    Background Protein-ligand binding is important for some proteins to perform their functions. Protein-ligand binding sites are the residues of proteins that physically bind to ligands. Despite of the recent advances in computational prediction for protein-ligand binding sites, the state-of-the-art methods search for similar, known structures of the query and predict the binding sites based on the solved structures. However, such structural information is not commonly available. Results In this paper, we propose a sequence-based approach to identify protein-ligand binding residues. We propose a combination technique to reduce the effects of different sliding residue windows in the process of encoding input feature vectors. Moreover, due to the highly imbalanced samples between the ligand-binding sites and non ligand-binding sites, we construct several balanced data sets, for each of which a random forest (RF)-based classifier is trained. The ensemble of these RF classifiers forms a sequence-based protein-ligand binding site predictor. Conclusions Experimental results on CASP9 and CASP8 data sets demonstrate that our method compares favorably with the state-of-the-art protein-ligand binding site prediction methods.

  1. Characterization of quinoxolinol salen ligands as selective ligands for chemosensors for uranium

    Energy Technology Data Exchange (ETDEWEB)

    DeVore, Michael A. II; Kerns, Spencer A.; Gorden, Anne E.V. [Department of Chemistry and Biochemistry, Auburn University, Auburn, AL (United States)

    2015-12-15

    Quinoxalinol salen ligands have been characterized as selective ligands for the rapid identification of uranyl. The absorption maximum of ligand 1 presented a hypsochromic (blue) shift with the addition of UO{sub 2}{sup 2+} (as the acetate salt), and a bathochromic (red) shift in the presence of Cu{sup 2+} or Co{sup 2+} acetate salts, resulting in distinct, visible color changes for all three metal ions. The absorption maximum of ligand 2 was not observed to change with the addition of UO{sub 2}{sup 2+}; however, it does present a bathochromic shift with the addition of Cu{sup 2+}, and a hypsochromic shift with Co{sup 2+} added. Using TDDFT calculations, it was demonstrated that the hypsochromic shift for UO{sub 2}{sup 2+} ion complexation with ligand 1 is caused by a ligand-to-metal charge transfer, while the bathochromic shift observed with Cu{sup 2+} ion addition was caused by a metal-to-ligand charge transfer. Finally, it was found that the addition of Cu{sup 2+} (as metal salts) to either ligand resulted in rapid, complete quenching of the ligand fluorescence. (Copyright copyright 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  2. Fully Flexible Docking of Medium Sized Ligand Libraries with RosettaLigand.

    Directory of Open Access Journals (Sweden)

    Samuel DeLuca

    Full Text Available RosettaLigand has been successfully used to predict binding poses in protein-small molecule complexes. However, the RosettaLigand docking protocol is comparatively slow in identifying an initial starting pose for the small molecule (ligand making it unfeasible for use in virtual High Throughput Screening (vHTS. To overcome this limitation, we developed a new sampling approach for placing the ligand in the protein binding site during the initial 'low-resolution' docking step. It combines the translational and rotational adjustments to the ligand pose in a single transformation step. The new algorithm is both more accurate and more time-efficient. The docking success rate is improved by 10-15% in a benchmark set of 43 protein/ligand complexes, reducing the number of models that typically need to be generated from 1000 to 150. The average time to generate a model is reduced from 50 seconds to 10 seconds. As a result we observe an effective 30-fold speed increase, making RosettaLigand appropriate for docking medium sized ligand libraries. We demonstrate that this improved initial placement of the ligand is critical for successful prediction of an accurate binding position in the 'high-resolution' full atom refinement step.

  3. Synthesis and characterization of mixed ligand chiral nanoclusters

    KAUST Repository

    Guven, Zekiye P.

    2016-06-22

    Chiral mixed ligand silver nanoclusters were synthesized in the presence of a chiral and an achiral ligand. While the chiral ligand led mostly to the formation of nanoparticles, the presence of the achiral ligand drastically increased the yield of nanoclusters with enhanced chiral properties. © 2016 The Royal Society of Chemistry.

  4. Immobilisation of ligands by radio-derivatized polymers; Immobilisering av ligander med radioderiverte polymerer

    Energy Technology Data Exchange (ETDEWEB)

    Varga, J.M.; Fritsch, P.

    1995-01-30

    The invention relates to radio-derivatized polymers and a method of producing them by contacting non-polymerizable conjugands with radiolysable polymers in the presence of irradiation. The resulting radio-derivatized polymers can be further linked with ligand of organic or inorganic nature to immobilize such ligands. 2 figs., 5 tabs.

  5. A new class of PN3-pincer ligands for metal–ligand cooperative catalysis

    KAUST Repository

    Li, Huaifeng

    2014-12-01

    Work on a new class of PN3-pincer ligands for metal-ligand cooperative catalysis is reviewed. While the field of the pyridine-based PN3-transition metal pincer complexes is still relatively young, many important applications of these complexes have already emerged. In several cases, the PN3-pincer complexes for metal-ligand cooperative catalysis result in significantly improved or unprecedented activities. The synthesis and coordination chemistry of PN3-pincer ligands are briefly summarized first to cover the synthetic routes for their preparation, followed by a focus review on their applications in catalysis. A specific emphasis is placed on the later section about the role of PN3-pincer ligands\\' dearomatization-rearomatization steps during the catalytic cycles. The mechanistic insights from density functional theory (DFT) calculations are also discussed.

  6. PPARα Ligands as Antitumorigenic and Antiangiogenic Agents

    Directory of Open Access Journals (Sweden)

    Ambra Pozzi

    2008-01-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs belong to the nuclear receptor family of ligand-activated transcription factors. This subfamily is composed of three members—PPARα, PPARδ, and PPARγ—that differ in their cell and tissue distribution as well as in their target genes. PPARα is abundantly expressed in liver, brown adipose tissue, kidney, intestine, heart, and skeletal muscle; and its ligands have been used to treat diseases such as obesity and diabetes. The recent finding that members of the PPAR family, including the PPARα, are expressed by tumor and endothelial cells together with the observation that PPAR ligands regulate cell growth, survival, migration, and invasion, suggested that PPARs also play a role in cancer. In this review, we focus on the contribution of PPARα to tumor and endothelial cell functions and provide compelling evidence that PPARα can be viewed as a new class of ligand activated tumor “suppressor” gene with antiangiogenic and antitumorigenic activities. Given that PPAR ligands are currently used in medicine as hypolipidemic drugs with excellent tolerance and limited toxicity, PPARα activation might offer a novel and potentially low-toxic approach for the treatment of tumor-associated angiogenesis and cancer.

  7. A screening cascade to identify ERβ ligands.

    Science.gov (United States)

    Filgueira, Carly S; Benod, Cindy; Lou, Xiaohua; Gunamalai, Prem S; Villagomez, Rosa A; Strom, Anders; Gustafsson, Jan-Åke; Berkenstam, Anders L; Webb, Paul

    2014-01-01

    The establishment of effective high throughput screening cascades to identify nuclear receptor (NR) ligands that will trigger defined, therapeutically useful sets of NR activities is of considerable importance. Repositioning of existing approved drugs with known side effect profiles can provide advantages because de novo drug design suffers from high developmental failure rates and undesirable side effects which have dramatically increased costs. Ligands that target estrogen receptor β (ERβ) could be useful in a variety of diseases ranging from cancer to neurological to cardiovascular disorders. In this context, it is important to minimize cross-reactivity with ERα, which has been shown to trigger increased rates of several types of cancer. Because of high sequence similarities between the ligand binding domains of ERα and ERβ, preferentially targeting one subtype can prove challenging. Here, we describe a sequential ligand screening approach comprised of complementary in-house assays to identify small molecules that are selective for ERβ. Methods include differential scanning fluorimetry, fluorescence polarization and a GAL4 transactivation assay. We used this strategy to screen several commercially-available chemical libraries, identifying thirty ERβ binders that were examined for their selectivity for ERβ versus ERα, and tested the effects of selected ligands in a prostate cancer cell proliferation assay. We suggest that this approach could be used to rapidly identify candidates for drug repurposing.

  8. Effects of PPARγ Ligands on Leukemia

    Directory of Open Access Journals (Sweden)

    Yoko Tabe

    2012-01-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs and retinoic acid receptors (RARs, members of the nuclear receptor superfamily, are transcription factors that regulate a variety of important cellular functions. PPARs form heterodimers retinoid X receptor (RXR, an obligate heterodimeric partner for other nuclear receptors. Several novel links between retinoid metabolism and PPAR responses have been identified, and activation of PPAR/RXR expression has been shown to increase response to retinoids. PPARγ has emerged as a key regulator of cell growth and survival, whose activity is modulated by a number of synthetic and natural ligands. While clinical trials in cancer patients with thiazolidinediones (TZD have been disappointing, novel structurally different PPARγ ligands, including triterpenoids, have entered clinical arena as therapeutic agents for epithelial and hematopoietic malignancies. Here we shall review the antitumor advances of PPARγ, alone and in combination with RARα ligands in control of cell proliferation, differentiation, and apoptosis and their potential therapeutic applications in hematological malignancies.

  9. Flexible Ligand Docking Using Evolutionary Algorithms

    DEFF Research Database (Denmark)

    Thomsen, Rene

    2003-01-01

    The docking of ligands to proteins can be formulated as a computational problem where the task is to find the most favorable energetic conformation among the large space of possible protein–ligand complexes. Stochastic search methods such as evolutionary algorithms (EAs) can be used to sample large...... search spaces effectively and is one of the commonly used methods for flexible ligand docking. During the last decade, several EAs using different variation operators have been introduced, such as the ones provided with the AutoDock program. In this paper we evaluate the performance of different EA...... settings such as choice of variation operators, population size, and usage of local search. The comparison is performed on a suite of six docking problems previously used to evaluate the performance of search algorithms provided with the AutoDock program package. The results from our investigation confirm...

  10. Mechanism and functional impact of CD40 ligand-induced von Willebrand factor release from endothelial cells.

    Science.gov (United States)

    Möller, Kerstin; Adolph, Oliver; Grünow, Jennifer; Elrod, Julia; Popa, Miruna; Ghosh, Subhajit; Schwarz, Manuel; Schwale, Chrysovalandis; Grässle, Sandra; Huck, Volker; Bruehl, Claus; Wieland, Thomas; Schneider, Stefan W; Nobiling, Rainer; Wagner, Andreas H; Hecker, Markus

    2015-05-01

    Co-stimulation via CD154 binding to CD40, pivotal for both innate and adaptive immunity, may also link haemostasis to vascular remodelling. Here we demonstrate that human platelet-bound or recombinant soluble CD154 (sCD154) elicit the release from and tethering of ultra-large (UL) von Willebrand factor (vWF) multimers to the surface of human cultured endothelial cells (ECs) exposed to shear stress. This CD40-mediated ULVWF multimer release from the Weibel-Palade bodies was triggered by consecutive activation of TRAF6, the tyrosine kinase c-Src and phospholipase Cγ1 followed by inositol-1,4,5 trisphosphate-mediated calcium mobilisation. Subsequent exposure to human washed platelets caused ULVWF multimer-platelet string formation on the EC surface in a shear stress-dependent manner. Platelets tethered to these ULVWF multimers exhibited P-selectin on their surface and captured labelled monocytes from the superfusate. When exposed to shear stress and sCD154, native ECs from wild-type but not CD40 or vWF-deficient mice revealed a comparable release of ULVWF multimers to which murine washed platelets rapidly adhered, turning P-selectin-positive and subsequently capturing monocytes from the perfusate. This novel CD154-provoked ULVWF multimer-platelet string formation at normal to fast flow may contribute to vascular remodelling processes requiring the perivascular or intravascular accumulation of pro-inflammatory macrophages such as arteriogenesis or atherosclerosis.

  11. Isotherm of ligand adsorption on DNA at multiplicative noise.

    Science.gov (United States)

    Arakelyan, Valeri; Harutyunyan, Sos; Andriasyan, Vardan; Arakelyan, Hakob

    2012-01-01

    Fluctuations of the number of ligands adsorbed on macromolecules are considered in the case when the number of ligands in the solution fluctuates under the action of fluctuations of the external medium (external noise). For the case of small filling, the multiplicative type of stochastic differential equation is obtained, describing the time variation of the number of ligands adsorbed on macromolecules. The isotherm of adsorption of ligands on DNA is obtained. It is shown that at small ligand concentrations, for some relations between adsorption parameters and the intensity of the external noise, no macromolecule adsorption of ligands takes place.

  12. Ligand Exchange Kinetics of Environmentally Relevant Metals

    Energy Technology Data Exchange (ETDEWEB)

    Panasci, Adele Frances [Univ. of California, Davis, CA (United States)

    2014-07-15

    The interactions of ground water with minerals and contaminants are of broad interest for geochemists but are not well understood. Experiments on the molecular scale can determine reaction parameters (i.e. rates of ligand exchange, activation entropy, activation entropy, and activation volume) that can be used in computations to gain insight into reactions that occur in natural groundwaters. Experiments to determine the rate of isotopic ligand exchange for three environmentally relevant metals, rhodium (Rh), iron (Fe), and neptunium (Np), are described. Many environmental transformations of metals (e.g. reduction) in soil occur at trivalent centers, Fe(III) in particular. Contaminant ions absorb to mineral surfaces via ligand exchange, and the reversal of this reaction can be dangerous, releasing contaminants into the environment. Ferric iron is difficult to study spectroscopically because most of its complexes are paramagnetic and are generally reactive toward ligand exchange; therefore, Rh(III), which is diamagnetic and less reactive, was used to study substitution reactions that are analogous to those that occur on mineral oxide surfaces. Studies on both Np(V) and Np(VI) are important in their own right, as 237Np is a radioactive transuranic element with a half-life of 2 million years.

  13. Supramolecular architectures constructed using angular bipyridyl ligands

    CERN Document Server

    Barnett, S A

    2003-01-01

    This work details the synthesis and characterization of a series of coordination frameworks that are formed using bidentate angular N-donor ligands. Pyrimidine was reacted with metal(ll) nitrate salts. Reactions using Cd(NO sub 3) sub 2 receive particular focus and the analogous reactions using the linear ligand, pyrazine, were studied for comparison. In all cases, two-dimensional coordination networks were prepared. Structural diversity is observed for the Cd(ll) centres including metal-nitrate bridging. In contrast, first row transition metal nitrates form isostructural one-dimensional chains with only the bridging N-donor ligands generating polymeric propagation. The angular ligand, 2,4-bis(4-pyridyl)-1,3,5-triazine (dpt), was reacted with Cd(NO sub 3) sub 2 and Zn(NO sub 3) sub 2. Whereas Zn(NO sub 3) sub 2 compounds exhibit solvent mediated polymorphism, a range of structures were obtained for the reactions with Cd(NO sub 3) sub 2 , including the first example of a doubly parallel interpenetrated 4.8 sup...

  14. Flexible Ligand Docking Using Differential Evolution

    DEFF Research Database (Denmark)

    Thomsen, René

    2003-01-01

    Molecular docking of biomolecules is becoming an increasingly important part in the process of developing new drugs, as well as searching compound databases for promising drug candidates. The docking of ligands to proteins can be formulated as an optimization problem where the task is to find...

  15. A versatile dinucleating ligand containing sulfonamide groups

    DEFF Research Database (Denmark)

    Sundberg, Jonas; Witt, Hannes; Cameron, Lisa

    2014-01-01

    Copper, iron, and gallium coordination chemistries of the new pentadentate bis-sulfonamide ligand 2,6-bis(N-2-pyridylmethylsulfonamido)-4-methylphenol (psmpH3) were investigated. PsmpH3 is capable of varying degrees of deprotonation, and notably, complexes containing the fully trideprotonated...

  16. Noncalcemic actions of vitamin D receptor ligands.

    Science.gov (United States)

    Nagpal, Sunil; Na, Songqing; Rathnachalam, Radhakrishnan

    2005-08-01

    1alpha,25-Dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the active metabolite of vitamin D(3), is known for the maintenance of mineral homeostasis and normal skeletal architecture. However, apart from these traditional calcium-related actions, 1,25-(OH)(2)D(3) and its synthetic analogs are being increasingly recognized for their potent antiproliferative, prodifferentiative, and immunomodulatory activities. These actions of 1,25-(OH)(2)D(3) are mediated through vitamin D receptor (VDR), which belongs to the superfamily of steroid/thyroid hormone nuclear receptors. Physiological and pharmacological actions of 1,25-(OH)(2)D(3) in various systems, along with the detection of VDR in target cells, have indicated potential therapeutic applications of VDR ligands in inflammation (rheumatoid arthritis, psoriatic arthritis), dermatological indications (psoriasis, actinic keratosis, seborrheic dermatitis, photoaging), osteoporosis (postmenopausal and steroid-induced osteoporosis), cancers (prostate, colon, breast, myelodysplasia, leukemia, head and neck squamous cell carcinoma, and basal cell carcinoma), secondary hyperparathyroidism, and autoimmune diseases (systemic lupus erythematosus, type I diabetes, multiple sclerosis, and organ transplantation). As a result, VDR ligands have been developed for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. Furthermore, encouraging results have been obtained with VDR ligands in clinical trials of prostate cancer and hepatocellular carcinoma. This review deals with the molecular aspects of noncalcemic actions of vitamin D analogs that account for the efficacy of VDR ligands in the above-mentioned indications.

  17. CHELATING LIGANDS: ENHANCERS OF QUALITY AND PURITY ...

    African Journals Online (AJOL)

    Nwokem et al.

    ABSTRACT. The quality of biogas depends largely on the percentage of methane and hydrogen sulphide gas present. High concentration of hydrogen sulphide results in low quality biogas. This work employed the use of chelating ligands in scrubbing hydrogen sulphide gas while improving the yield of methane gas.

  18. Ligand sphere conversions in terminal carbide complexes

    DEFF Research Database (Denmark)

    Morsing, Thorbjørn Juul; Reinholdt, Anders; Sauer, Stephan P. A.

    2016-01-01

    Metathesis is introduced as a preparative route to terminal carbide complexes. The chloride ligands of the terminal carbide complex [RuC(Cl)2(PCy3)2] (RuC) can be exchanged, paving the way for a systematic variation of the ligand sphere. A series of substituted complexes, including the first...... demonstrates that details of the coordination geometry affect the carbide chemical shift equally as much as variations in the nature of the auxiliary ligands. Furthermore, the kinetics of formation of the sqaure pyramidal dicyano complex, trans-[RuC(CN)2(PCy3)2], from RuC has been examined and the reaction...... found to be quite sluggish and of first order in both RuC and cyanide with a rate constant of k = 0.0104(6) M–1 s–1. Further reaction with cyanide leads to loss of the carbide ligand and formation of trans-[Ru(CN)4(PCy3)2]2–, which was isolated and structurally characterized as its PPh4+ salt....

  19. Simple tertiary phosphines to hexaphosphane ligands: Syntheses ...

    Indian Academy of Sciences (India)

    In this context, we have developed new synthetic methodologies for making unusual inorganic ring systems containing trivalent phosphorus centres, novel phosphorus-based multidentate and hybrid ligands and explored their rich transition metal chemistry and catalytic applications. We have also fine tuned a few existing ...

  20. Ammonia formation by metal-ligand cooperative hydrogenolysis of a nitrido ligand

    Science.gov (United States)

    Askevold, Bjorn; Nieto, Jorge Torres; Tussupbayev, Samat; Diefenbach, Martin; Herdtweck, Eberhardt; Holthausen, Max C.; Schneider, Sven

    2011-07-01

    Bioinspired hydrogenation of N2 to ammonia at ambient conditions by stepwise nitrogen protonation/reduction with metal complexes in solution has experienced remarkable progress. In contrast, the highly desirable direct hydrogenation with H2 remains difficult. In analogy to the heterogeneously catalysed Haber-Bosch process, such a reaction is conceivable via metal-centred N2 splitting and unprecedented hydrogenolysis of the nitrido ligands to ammonia. We report the synthesis of a ruthenium(IV) nitrido complex. The high nucleophilicity of the nitrido ligand is demonstrated by unusual N-C coupling with π-acidic CO. Furthermore, the terminal nitrido ligand undergoes facile hydrogenolysis with H2 at ambient conditions to produce ammonia in high yield. Kinetic and quantum chemical examinations of this reaction suggest cooperative behaviour of a phosphorus-nitrogen-phosphorus pincer ligand in rate-determining heterolytic hydrogen splitting.

  1. Quantum.Ligand.Dock: protein-ligand docking with quantum entanglement refinement on a GPU system.

    Science.gov (United States)

    Kantardjiev, Alexander A

    2012-07-01

    Quantum.Ligand.Dock (protein-ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein-ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments-the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein-ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html.

  2. Role of Lanthanide-Ligand bonding in the magnetization relaxation ...

    Indian Academy of Sciences (India)

    Ligand bonding. Our calculations transpire comparatively improved Single-Ion Magnet (SIM) behaviour for carbene analogues due to the more axially compressed trigonal prismatic ligand environment. Furthermore, our detailed Mulliken charge, ...

  3. Fluorescent GPCR ligands as new tools in pharmacology.

    Science.gov (United States)

    Kuder, Kamil; Kieć-Kononowicz, Katarzyna

    2008-01-01

    The expansion of fluorescent techniques for studying the ligand-receptor interaction resulted in a burst of the novel fluorescent ligands development. The discovery of the ligand, that is of high affinity to the receptor and whose localization could be easily visualized, even on the single cell level, gave the researchers a strong impulse to investigate that field of GPCR ligands. Moreover, paying attention to the "non pharmacological" advantages of these ligands, as well as the techniques to be used, fluorescent ligands are becoming treated more seriously, as the ligands themselves, and as novel, useful tools for studying GPCRs. Herein, we review results described in the literature, starting from the year 2000, in the field of the fluorescent GPCR small, non-peptide ligands according to the affinity to the selected receptors (histamine, adenosine, adrenergic, cannabinoid, muscarinie, neuropeptide Y and serotonine) as well as the fluorophores that have been used to tag the molecules.

  4. Ligand-Mediated Coating of Liposomes with Human Serum Albumin.

    Science.gov (United States)

    Sato, Hikari; Nakhaei, Elnaz; Kawano, Takahito; Murata, Masaharu; Kishimura, Akihiro; Mori, Takeshi; Katayama, Yoshiki

    2018-02-13

    Coating liposome surfaces with human serum albumin (HSA) can improve the colloidal stability and prevent opsonization. HSA coating via specific binding with alkyl ligands is promising because although the ligand-mediated coating is relatively stable it can spontaneously exchange with fresh HSA. However, to achieve surface coating with HSA, multiple hydrophobic ligands must be exposed to an aqueous medium prior to binding with HSA. This presents a challenge, as hydrophobic ligands tend to be buried in the liposomal membrane. Here we present the first HSA modification of liposome surfaces via alkyl ligands. We found that a relatively short alkyl ligand, or a long alkyl ligand with a terminal carboxylate, could be exposed on the liposome surface without causing aggregation of the liposomes and these ligands could subsequently bind HSA. The resulting HSA-coated liposomes were as inert as conventional PEGylated liposomes in terms of macrophage recognition.

  5. GLIDA: GPCR-ligand database for chemical genomic drug discovery

    OpenAIRE

    Okuno, Yasushi; Yang, Jiyoon; Taneishi, Kei; Yabuuchi, Hiroaki; Tsujimoto, Gozoh

    2005-01-01

    G-protein coupled receptors (GPCRs) represent one of the most important families of drug targets in pharmaceutical development. GPCR-LIgand DAtabase (GLIDA) is a novel public GPCR-related chemical genomic database that is primarily focused on the correlation of information between GPCRs and their ligands. It provides correlation data between GPCRs and their ligands, along with chemical information on the ligands, as well as access information to the various web databases regarding GPCRs. Thes...

  6. AutoSite: an automated approach for pseudo-ligands prediction—from ligand-binding sites identification to predicting key ligand atoms

    Science.gov (United States)

    Ravindranath, Pradeep Anand; Sanner, Michel F.

    2016-01-01

    Motivation: The identification of ligand-binding sites from a protein structure facilitates computational drug design and optimization, and protein function assignment. We introduce AutoSite: an efficient software tool for identifying ligand-binding sites and predicting pseudo ligand corresponding to each binding site identified. Binding sites are reported as clusters of 3D points called fills in which every point is labelled as hydrophobic or as hydrogen bond donor or acceptor. From these fills AutoSite derives feature points: a set of putative positions of hydrophobic-, and hydrogen-bond forming ligand atoms. Results: We show that AutoSite identifies ligand-binding sites with higher accuracy than other leading methods, and produces fills that better matches the ligand shape and properties, than the fills obtained with a software program with similar capabilities, AutoLigand. In addition, we demonstrate that for the Astex Diverse Set, the feature points identify 79% of hydrophobic ligand atoms, and 81% and 62% of the hydrogen acceptor and donor hydrogen ligand atoms interacting with the receptor, and predict 81.2% of water molecules mediating interactions between ligand and receptor. Finally, we illustrate potential uses of the predicted feature points in the context of lead optimization in drug discovery projects. Availability and Implementation: http://adfr.scripps.edu/AutoDockFR/autosite.html Contact: sanner@scripps.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27354702

  7. Integrating structural and mutagenesis data to elucidate GPCR ligand binding

    DEFF Research Database (Denmark)

    Munk, Christian; Harpsøe, Kasper; Hauser, Alexander S

    2016-01-01

    G protein-coupled receptors (GPCRs) represent the largest family of human membrane proteins, as well as drug targets. A recent boom in GPCR structural biology has provided detailed images of receptor ligand binding sites and interactions on the molecular level. An ever-increasing number of ligands...... elucidate new GPCR ligand binding sites, and ultimately design drugs with tailored pharmacological activity....

  8. New mixed ligand complexes of ruthenium (II) that incorporate a ...

    Indian Academy of Sciences (India)

    New mixed ligand complexes of ruthenium(II) that incorporate a modified phenanthroline ligand: Synthesis, spectral characterization and DNA binding. S Murali C V ... Fulltext. Click here to view fulltext PDF ... Ruthenium(II) complexes; modified phenanthroline ligand; spectroscopy; DNA binding; fluorescence enhancement.

  9. Novel peptide ligand with high binding capacity for antibody purification

    DEFF Research Database (Denmark)

    Lund, L. N.; Gustavsson, P. E.; Michael, R.

    2012-01-01

    ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1 M NaOH. Here, we present a novel synthetic peptide...

  10. Page 1 ASSESSMENT OF BINDING ENERGIES AND LIGAND ...

    African Journals Online (AJOL)

    The successive binding energies of up to six water ligands to ferric ion and the reduction in the gross binding energies by ligand-ligand repulsion are determined by electrostatic interaction. The gross binding energy is found to be both geometry and coordination nuitber dependent: whereas it increases with number of ...

  11. Crystallization and preliminary X-ray diffraction studies of the carbohydrate-recognition domain of SIGN-R1, a receptor for microbial polysaccharides and sialylated antibody on splenic marginal zone macrophages.

    Science.gov (United States)

    Silva-Martin, Noella; Schauer, Joseph D; Park, Chae Gyu; Hermoso, Juan A

    2009-12-01

    SIGN-R1, or CD209b, is a mouse C-type lectin receptor that is expressed at high levels on macrophages in lymphoid tissues, especially within the marginal zone of the spleen. SIGN-R1 can bind and mediate the uptake of various microbial polysaccharides, including dextrans, lipopolysaccharides and pneumococcal capsular polysaccharides. It has been shown that SIGN-R1 mediates the clearance of encapsulated pneumococcus, complement fixation via binding C1q independent of antibody and innate resistance to pneumococcal infection. Recently, SIGN-R1 has also been demonstrated to bind sialylated antibody and mediate its activity to suppress autoimmunity. The carbohydrate-recognition domain (CRD) of SIGN-R1 has been cloned and overexpressed in a soluble secretory form in mammalian Chinese hamster ovary (CHO) cells. The CRD protein of SIGN-R1 was purified from CHO cell-culture supernatant and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 2 M ammonium sulfate in 0.1 M bis-tris pH 5.5. Single crystals, which belonged to the monoclinic space group C2 with unit-cell parameters a = 146.72, b = 92.77, c = 77.06 A, beta = 121.66 degrees , allowed the collection of a full X-ray data set to a maximum resolution of 1.87 A.

  12. Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers.

    Science.gov (United States)

    Jarillo, Javier; Morín, José A; Beltrán-Heredia, Elena; Villaluenga, Juan P G; Ibarra, Borja; Cao, Francisco J

    2017-01-01

    Ligands binding to polymers regulate polymer functions by changing their physical and chemical properties. This ligand regulation plays a key role in many biological processes. We propose here a model to explain the mechanical, thermodynamic, and kinetic properties of the process of binding of small ligands to long biopolymers. These properties can now be measured at the single molecule level using force spectroscopy techniques. Our model performs an effective decomposition of the ligand-polymer system on its covered and uncovered regions, showing that the elastic properties of the ligand-polymer depend explicitly on the ligand coverage of the polymer (i.e., the fraction of the polymer covered by the ligand). The equilibrium coverage that minimizes the free energy of the ligand-polymer system is computed as a function of the applied force. We show how ligands tune the mechanical properties of a polymer, in particular its length and stiffness, in a force dependent manner. In addition, it is shown how ligand binding can be regulated applying mechanical tension on the polymer. Moreover, the binding kinetics study shows that, in the case where the ligand binds and organizes the polymer in different modes, the binding process can present transient shortening or lengthening of the polymer, caused by changes in the relative coverage by the different ligand modes. Our model will be useful to understand ligand-binding regulation of biological processes, such as the metabolism of nucleic acid. In particular, this model allows estimating the coverage fraction and the ligand mode characteristics from the force extension curves of a ligand-polymer system.

  13. Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers.

    Directory of Open Access Journals (Sweden)

    Javier Jarillo

    Full Text Available Ligands binding to polymers regulate polymer functions by changing their physical and chemical properties. This ligand regulation plays a key role in many biological processes. We propose here a model to explain the mechanical, thermodynamic, and kinetic properties of the process of binding of small ligands to long biopolymers. These properties can now be measured at the single molecule level using force spectroscopy techniques. Our model performs an effective decomposition of the ligand-polymer system on its covered and uncovered regions, showing that the elastic properties of the ligand-polymer depend explicitly on the ligand coverage of the polymer (i.e., the fraction of the polymer covered by the ligand. The equilibrium coverage that minimizes the free energy of the ligand-polymer system is computed as a function of the applied force. We show how ligands tune the mechanical properties of a polymer, in particular its length and stiffness, in a force dependent manner. In addition, it is shown how ligand binding can be regulated applying mechanical tension on the polymer. Moreover, the binding kinetics study shows that, in the case where the ligand binds and organizes the polymer in different modes, the binding process can present transient shortening or lengthening of the polymer, caused by changes in the relative coverage by the different ligand modes. Our model will be useful to understand ligand-binding regulation of biological processes, such as the metabolism of nucleic acid. In particular, this model allows estimating the coverage fraction and the ligand mode characteristics from the force extension curves of a ligand-polymer system.

  14. A ligand's view of target similarity

    DEFF Research Database (Denmark)

    Garland, Stephen L; Gloriam, David E

    2011-01-01

    shows with several examples how focusing on the binding site(s) has a clear advantage when it comes to establishing sequence-correlated pharmacological profiles. By organizing and comparing sequence and structural data it is possible to "borrow" SAR from similar targets to increase the speed of lead......-finding and, potentially, to produce ligands for previously intractable receptors. Sequence motifs correlated with ligands can be applied in the design of target-specific focused libraries that are both efficient and cost-effective and should provide increased hit-rates over diversity screening. Furthermore......, in the optimization phase, the binding motif approach offers the possibility to identify quickly the most likely off-target candidates to be chosen for selectivity screening, as well as potentially characterizing those pockets which may best be exploited for improved selectivity....

  15. Sigma-2 receptor ligands QSAR model dataset

    Directory of Open Access Journals (Sweden)

    Antonio Rescifina

    2017-08-01

    Full Text Available The data have been obtained from the Sigma-2 Receptor Selective Ligands Database (S2RSLDB and refined according to the QSAR requirements. These data provide information about a set of 548 Sigma-2 (σ2 receptor ligands selective over Sigma-1 (σ1 receptor. The development of the QSAR model has been undertaken with the use of CORAL software using SMILES, molecular graphs and hybrid descriptors (SMILES and graph together. Data here reported include the regression for σ2 receptor pKi QSAR models. The QSAR model was also employed to predict the σ2 receptor pKi values of the FDA approved drugs that are herewith included.

  16. Isolation and characterization from porcine serum of a soluble sulfotransferase responsible for 6-O-sulfation of the galactose residue in 2'-fucosyllactose: implications in the synthesis of the ligand for L-selectin.

    Science.gov (United States)

    Huynh, Q K; Shailubhai, K; Boddupalli, H; Yu, H H; Broschat, K O; Jacob, G S

    1999-07-01

    A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) to 2'-fucosyllactose (2'-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 microM for PAPS and 15.38 mM for 2'-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl LewisX (sLeX). Results indicated that 3'-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6'-sialyllactose and 6'-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2'-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin.

  17. Hydrogen bonding penalty upon ligand binding.

    Directory of Open Access Journals (Sweden)

    Hongtao Zhao

    Full Text Available Ligand binding involves breakage of hydrogen bonds with water molecules and formation of new hydrogen bonds between protein and ligand. In this work, the change of hydrogen bonding energy in the binding process, namely hydrogen bonding penalty, is evaluated with a new method. The hydrogen bonding penalty can not only be used to filter unrealistic poses in docking, but also improve the accuracy of binding energy calculation. A new model integrated with hydrogen bonding penalty for free energy calculation gives a root mean square error of 0.7 kcal/mol on 74 inhibitors in the training set and of 1.1 kcal/mol on 64 inhibitors in the test set. Moreover, an application of hydrogen bonding penalty into a high throughput docking campaign for EphB4 inhibitors is presented, and remarkably, three novel scaffolds are discovered out of seven tested. The binding affinity and ligand efficiency of the most potent compound is about 300 nM and 0.35 kcal/mol per non-hydrogen atom, respectively.

  18. Ligand mediated evolution of size dependent magnetism in cobalt nanoclusters.

    Science.gov (United States)

    Hartmann, Michael J; Millstone, Jill E; Häkkinen, Hannu

    2018-02-07

    We use density functional theory to model the impact of a ligand shell on the magnetic properties of CoN (15 ≤ N ≤ 55) nanoclusters. We study three different ligand shells on each nanocluster core size, each known to have different electronic interactions with the surface: pure Cl ligand shells (X-type), pure PH3 ligand shells (L-type), and two component ligand shells with mixtures of Cl and PH3 ligands. The simulations show that the identity, arrangement, and total coverage of the ligand shell controls the distribution of local magnetic moments across the CoN core. On the surface of an unpassivated CoN nanocluster, the Co-Co coordination number (CN) is known to determine the local magnetic moments. Upon the introduction of a ligand, the Co-Co CN remains important, however the nature of the metal-ligand bond changes the extent to which increasing Co-Co CN quenches magnetism. Further, we identify an additional and significant long-range impact on local magnetic moments (LMM) from the PH3 ligand shells. Thus, we establish important design principles of magnetic nanoclusters, where ligand shell chemistry mediates the distribution of LMMs across a CoNLM nanocluster, allowing a route to rational design of specific magnetic properties.

  19. Separation of tryptophan enantiomers by ligand-exchange chromatography with novel chiral ionic liquids ligand.

    Science.gov (United States)

    Qing, Haiqun; Jiang, Xinyu; Yu, Jingang

    2014-03-01

    Chiral ionic liquids (CILs) with amino acids as cations have been applied as novel chiral ligands coordinated with Cu(2+) to separate tryptophan enantiomers in ligand exchange chromatography. Four kinds of amino acid ionic liquids, including [L-Pro][CF3COO], [L-Pro][NO3], [L-Pro]2[SO4], and [L-Phe][CF3COO] were successfully synthesized and used for separation of tryptophan enantiomers. To optimize the separation conditions, [L-Pro][CF3COO] was selected as the model ligand. Some factors influencing the efficiency of chiral separation, such as copper ion concentration, CILs concentration, methanol ratio (methanol/H2O, v/v), and pH, were investigated. The obtained optimal separation conditions were as follows: 8.0 mmol/L Cu(OAc)2, 4.0 mmol/L [L-Pro][CF3COO], and 20% (v/v) methanol at pH 3.6. Under the optimum conditions, acceptable enantioseparation of tryptophan enantiomers could be observed with a resolution of 1.89. The results demonstrate the good applicability of CILs with amino acids as cations for chiral separation. Furthermore, a comparative study was also conducted for exploring the mechanism of the CILs as new ligands in ligand exchange chromatography. © 2014 Wiley Periodicals, Inc.

  20. Amino acid ionic liquids as chiral ligands in ligand-exchange chiral separations.

    Science.gov (United States)

    Liu, Qian; Wu, Kangkang; Tang, Fei; Yao, Lihua; Yang, Fei; Nie, Zhou; Yao, Shouzhuo

    2009-09-28

    Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1-alkyl-3-methylimidazolium L-proline (L-Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers-dl-phenylalanine (dl-Phe), dl-histidine (dl-His), dl-tryptophane (dl-Trp), and dl-tyrosine (dl-Tyr)-were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R(s))=3.26-10.81 for HPLC; R(s)=1.34-4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L-Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L-Pro are consequently attached to the support surface, thus inducing an ion-exchange type of retention for the dl-enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand-exchange-based chiral separation. It also reveals the tremendous application potential of this new type of task-specific ILs.

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

    Science.gov (United States)

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

    1999-10-01

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

  2. ProBiS-ligands: a web server for prediction of ligands by examination of protein binding sites.

    Science.gov (United States)

    Konc, Janez; Janežič, Dušanka

    2014-07-01

    The ProBiS-ligands web server predicts binding of ligands to a protein structure. Starting with a protein structure or binding site, ProBiS-ligands first identifies template proteins in the Protein Data Bank that share similar binding sites. Based on the superimpositions of the query protein and the similar binding sites found, the server then transposes the ligand structures from those sites to the query protein. Such ligand prediction supports many activities, e.g. drug repurposing. The ProBiS-ligands web server, an extension of the ProBiS web server, is open and free to all users at http://probis.cmm.ki.si/ligands. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Characterizing common substructures of ligands for GPCR protein subfamilies.

    Science.gov (United States)

    Erguner, Bekir; Hattori, Masahiro; Goto, Susumu; Kanehisa, Minoru

    2010-01-01

    The G-protein coupled receptor (GPCR) superfamily is the largest class of proteins with therapeutic value. More than 40% of present prescription drugs are GPCR ligands. The high therapeutic value of GPCR proteins and recent advancements in virtual screening methods gave rise to many virtual screening studies for GPCR ligands. However, in spite of vast amounts of research studying their functions and characteristics, 3D structures of most GPCRs are still unknown. This makes target-based virtual screenings of GPCR ligands extremely difficult, and successful virtual screening techniques rely heavily on ligand information. These virtual screening methods focus on specific features of ligands on GPCR protein level, and common features of ligands on higher levels of GPCR classification are yet to be studied. Here we extracted common substructures of GPCR ligands of GPCR protein subfamilies. We used the SIMCOMP, a graph-based chemical structure comparison program, and hierarchical clustering to reveal common substructures. We applied our method to 850 GPCR ligands and we found 53 common substructures covering 439 ligands. These substructures contribute to deeper understanding of structural features of GPCR ligands which can be used in new drug discovery methods.

  4. (Ligand intermediates in metal-catalyzed reactions)

    Energy Technology Data Exchange (ETDEWEB)

    1992-01-01

    This report consists of sections on sigma bond complexes of alkenes, a new carbon-hydrogen bond activation reaction of alkene complexes, carbon-hydrogen bond migrations in alkylidene complexes, carbon- hydrogen bond migrations in alkyne complexes, synthesis, structure and reactivity of C{sub x} complexes, synthesis and reactivity of alcohol and ether complexes, new catalysts for the epimerization of secondary alcohols; carbon-hydrogen bond activation in alkoxide complexes, pi/sigma equilibria in metal/O=CXX' complexes, and other hydrocarbon ligands; miscellaneous.(WET)

  5. Computer-aided design of GPCR ligands.

    Science.gov (United States)

    Gutiérrez-de-Terán, Hugo; Keränen, Henrik; Azuaje, Jhonny; Rodríguez, David; Åqvist, Johan; Sotelo, Eddy

    2015-01-01

    The recent availability of several GPCR crystal structures now contributes decisively to the perspective of structure-based ligand design. In this context, computational approaches are extremely helpful, particularly if properly integrated in drug design projects with cooperation between computational and medicinal chemistry teams. Here, we present the pipelines used in one such project, devoted to the design of novel potent and selective antagonists for the different adenosine receptors. The details of the computational strategies are described, and particular attention is given to explain how these procedures can effectively guide the synthesis of novel chemical entities.

  6. Increased accuracy of ligand sensing by receptor internalization

    Science.gov (United States)

    Aquino, Gerardo; Endres, Robert G.

    2010-02-01

    Many types of cells can sense external ligand concentrations with cell-surface receptors at extremely high accuracy. Interestingly, ligand-bound receptors are often internalized, a process also known as receptor-mediated endocytosis. While internalization is involved in a vast number of important functions for the life of a cell, it was recently also suggested to increase the accuracy of sensing ligand as the overcounting of the same ligand molecules is reduced. Here we show, by extending simple ligand-receptor models to out-of-equilibrium thermodynamics, that internalization increases the accuracy with which cells can measure ligand concentrations in the external environment. Comparison with experimental rates of real receptors demonstrates that our model has indeed biological significance.

  7. Ligands of Therapeutic Utility for the Liver X Receptors

    Directory of Open Access Journals (Sweden)

    Rajesh Komati

    2017-01-01

    Full Text Available Liver X receptors (LXRs have been increasingly recognized as a potential therapeutic target to treat pathological conditions ranging from vascular and metabolic diseases, neurological degeneration, to cancers that are driven by lipid metabolism. Amidst intensifying efforts to discover ligands that act through LXRs to achieve the sought-after pharmacological outcomes, several lead compounds are already being tested in clinical trials for a variety of disease interventions. While more potent and selective LXR ligands continue to emerge from screening of small molecule libraries, rational design, and empirical medicinal chemistry approaches, challenges remain in minimizing undesirable effects of LXR activation on lipid metabolism. This review provides a summary of known endogenous, naturally occurring, and synthetic ligands. The review also offers considerations from a molecular modeling perspective with which to design more specific LXRβ ligands based on the interaction energies of ligands and the important amino acid residues in the LXRβ ligand binding domain.

  8. Prediction of GPCR-Ligand Binding Using Machine Learning Algorithms

    Directory of Open Access Journals (Sweden)

    Sangmin Seo

    2018-01-01

    Full Text Available We propose a novel method that predicts binding of G-protein coupled receptors (GPCRs and ligands. The proposed method uses hub and cycle structures of ligands and amino acid motif sequences of GPCRs, rather than the 3D structure of a receptor or similarity of receptors or ligands. The experimental results show that these new features can be effective in predicting GPCR-ligand binding (average area under the curve [AUC] of 0.944, because they are thought to include hidden properties of good ligand-receptor binding. Using the proposed method, we were able to identify novel ligand-GPCR bindings, some of which are supported by several studies.

  9. Predicting Nanocrystal Shape through Consideration of Surface-Ligand Interactions

    KAUST Repository

    Bealing, Clive R.

    2012-03-27

    Density functional calculations for the binding energy of oleic acid-based ligands on Pb-rich {100} and {111} facets of PbSe nanocrystals determine the surface energies as a function of ligand coverage. Oleic acid is expected to bind to the nanocrystal surface in the form of lead oleate. The Wulff construction predicts the thermodynamic equilibrium shape of the PbSe nanocrystals. The equilibrium shape is a function of the ligand surface coverage, which can be controlled by changing the concentration of oleic acid during synthesis. The different binding energy of the ligand on the {100} and {111} facets results in different equilibrium ligand coverages on the facets, and a transition in the equilibrium shape from octahedral to cubic is predicted when increasing the ligand concentration during synthesis. © 2012 American Chemical Society.

  10. The autoxidation activity of new mixed-ligand manganese and iron complexes with tripodal ligands

    NARCIS (Netherlands)

    van Gorkum, R.; Berding, J.; Tooke, D.M.; Spek, A.L.|info:eu-repo/dai/nl/156517566; Reedijk, J.; Bouwman, E.

    2007-01-01

    The activity of new manganese and iron complexes of dianionic tripodal ligands in the autoxidation of ethyl linoleate (EL) is reported. EL consumption rates were monitored using time-resolved FTIR and the degree of oligomerisation was determined by SEC. Almost all complexes showed the same trend in

  11. Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

    Science.gov (United States)

    Ruff, Karen M.

    2014-01-01

    The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform. PMID:25246650

  12. Does the ligand-biopolymer equilibrium binding constant depend on the number of bound ligands?

    Science.gov (United States)

    Beshnova, Daria A; Lantushenko, Anastasia O; Evstigneev, Maxim P

    2010-11-01

    Conventional methods, such as Scatchard or McGhee-von Hippel analyses, used to treat ligand-biopolymer interactions, indirectly make the assumption that the microscopic binding constant is independent of the number of ligands, i, already bound to the biopolymer. Recent results on the aggregation of aromatic molecules (Beshnova et al., J Chem Phys 2009, 130, 165105) indicated that the equilibrium constant of self-association depends intrinsically on the number of molecules in an aggregate due to loss of translational and rotational degrees of freedom on formation of the complex. The influence of these factors on the equilibrium binding constant for ligand-biopolymer complexation was analyzed in this work. It was shown that under the conditions of binding of "small" molecules, these factors can effectively be ignored and, hence, do not provide any hidden systematic error in such widely-used approaches, such as the Scatchard or McGhee-von Hippel methods for analyzing ligand-biopolymer complexation. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 932-935, 2010.

  13. New chiral ligand exchange capillary electrophoresis system with chiral amino amide ionic liquids as ligands.

    Science.gov (United States)

    Jiang, Junfang; Mu, Xiaoyu; Qiao, Juan; Su, Yuan; Qi, Li

    2017-12-01

    Using chiral amino amide ionic liquids as the ligands, a new chiral ligand exchange capillary electrophoresis method with Cu(II) as the central ion was constructed for enantioseparation of labeled D,L-amino acids. The effects of key parameters, including pH value of the running buffer, the ratio of Cu(II) to chiral amino amide ionic liquids, the concentration of complexes based on Cu(II)-chiral amino amide ionic liquids were investigated. It has been observed that eight pairs of labeled D,L-amino acids could be baseline-separated with a running buffer of 15.0mM ammonium acetate, 10.0mM Cu(II) and 20.0mML-phenylalaninamide based ionic liquid at pH 5.0. The quantitation of D,L-amino acids was conducted and good linearity (r2 ≥ 0.964) was obtained. Furthermore, an assay for determining the enantiomeric purity of D,L-amino acids was developed and the possible enantiorecognition mechanism was discussed briefly. The results indicated that the chiral amino amide ionic liquids could play the role of ligands in chiral ligand exchange capillary electrophoresis system and exhibit great potential in chiral analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Quasielastic neutron scattering study of POSS ligand dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Jalarvo, Niina H [ORNL; Tyagi, Madhusudan [NIST Center for Neutron Research (NCRN), Gaithersburg, MD; Crawford, Michael [DuPont Experimental Station

    2015-01-01

    Polyoligosilsesquioxanes are molecules having cage-like structures composed of silicon and oxygen. These molecules can have a wide variety of functional ligands attached to them. Depending on the nature of the ligand, interesting properties and applications are found. In this work we present results from quasielastic neutron scattering measurements of four different POSS molecules that illustrate the presence of strong coupling between the ligand dynamics and the POSS crystal structures.

  15. Do organic ligands affect calcite dissolution rates?

    Science.gov (United States)

    Oelkers, Eric H.; Golubev, Sergey V.; Pokrovsky, Oleg S.; Bénézeth, Pascale

    2011-04-01

    Steady state Iceland-spar calcite dissolution rates were measured at 25 °C in aqueous solutions containing 0.1 M NaCl and up to 0.05 M dissolved bicarbonate at pH from 7.9 to 9.1 in the presence of 13 distinct dissolved organic ligands in mixed-flow reactors. The organic ligands considered in this study include those most likely to be present in either (1) aquifers at the conditions pertinent to CO 2 sequestration or (2) soil/early diagenetic environments: acetate, phthalate, citrate, EDTA 4-, succinate, D-glucosaminate, L-glutamate, D-gluconate, 2,4-dihydroxybenzoate, 3,4-dihydroxybenzoate, fumarate, malonate, and gallate. Results show that the presence of exopolysaccharides, and analogs of microbial cell envelopes: alginate, lichen extract, humic acid, pectin, and gum xanthan. In no case did the presence of <100 ppm of these organics change calcite dissolution rates by more than a factor of 2.5. Results obtained in this study suggest that the presence of aqueous organic anions negligibly affects calcite forward dissolution rates in most natural environments. Some effect on calcite reactivity may be observed, however, by the presence of organic anions if they change substantially the chemical affinity of the fluid with respect to calcite.

  16. Singular Value Decomposition and Ligand Binding Analysis

    Directory of Open Access Journals (Sweden)

    André Luiz Galo

    2013-01-01

    Full Text Available Singular values decomposition (SVD is one of the most important computations in linear algebra because of its vast application for data analysis. It is particularly useful for resolving problems involving least-squares minimization, the determination of matrix rank, and the solution of certain problems involving Euclidean norms. Such problems arise in the spectral analysis of ligand binding to macromolecule. Here, we present a spectral data analysis method using SVD (SVD analysis and nonlinear fitting to determine the binding characteristics of intercalating drugs to DNA. This methodology reduces noise and identifies distinct spectral species similar to traditional principal component analysis as well as fitting nonlinear binding parameters. We applied SVD analysis to investigate the interaction of actinomycin D and daunomycin with native DNA. This methodology does not require prior knowledge of ligand molar extinction coefficients (free and bound, which potentially limits binding analysis. Data are acquired simply by reconstructing the experimental data and by adjusting the product of deconvoluted matrices and the matrix of model coefficients determined by the Scatchard and McGee and von Hippel equation.

  17. Superior serum half life of albumin tagged TNF ligands

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, Nicole [Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wuerzburg, Roentgenring 11, 97070 Wuerzburg (Germany); Schneider, Britta; Pfizenmaier, Klaus [Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart (Germany); Wajant, Harald, E-mail: harald.wajant@mail.uni-wuerzburg.de [Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wuerzburg, Roentgenring 11, 97070 Wuerzburg (Germany)

    2010-06-11

    Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1 h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24 h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.

  18. Design of targeting ligands in medicinal inorganic chemistry.

    Science.gov (United States)

    Storr, Tim; Thompson, Katherine H; Orvig, Chris

    2006-06-01

    This tutorial review will highlight recent advances in medicinal inorganic chemistry pertaining to the use of multifunctional ligands for enhanced effect. Ligands that adequately bind metal ions and also include specific targeting features are gaining in popularity due to their ability to enhance the efficacy of less complicated metal-based agents. Moving beyond the traditional view of ligands modifying reactivity, stabilizing specific oxidation states, and contributing to substitution inertness, we will discuss recent work involving metal complexes with multifunctional ligands that target specific tissues, membrane receptors, or endogenous molecules, including enzymes.

  19. A response calculus for immobilized T cell receptor ligands

    DEFF Research Database (Denmark)

    Andersen, P S; Menné, C; Mariuzza, R A

    2001-01-01

    To address the molecular mechanism of T cell receptor (TCR) signaling, we have formulated a model for T cell activation, termed the 2D-affinity model, in which the density of TCR on the T cell surface, the density of ligand on the presenting surface, and their corresponding two-dimensional affinity...... determine the level of T cell activation. When fitted to T cell responses against purified ligands immobilized on plastic surfaces, the 2D-affinity model adequately simulated changes in cellular activation as a result of varying ligand affinity and ligand density. These observations further demonstrated...

  20. Ligand diffusion in proteins via enhanced sampling in molecular dynamics.

    Science.gov (United States)

    Rydzewski, J; Nowak, W

    2017-12-01

    Computational simulations in biophysics describe the dynamics and functions of biological macromolecules at the atomic level. Among motions particularly important for life are the transport processes in heterogeneous media. The process of ligand diffusion inside proteins is an example of a complex rare event that can be modeled using molecular dynamics simulations. The study of physical interactions between a ligand and its biological target is of paramount importance for the design of novel drugs and enzymes. Unfortunately, the process of ligand diffusion is difficult to study experimentally. The need for identifying the ligand egress pathways and understanding how ligands migrate through protein tunnels has spurred the development of several methodological approaches to this problem. The complex topology of protein channels and the transient nature of the ligand passage pose difficulties in the modeling of the ligand entry/escape pathways by canonical molecular dynamics simulations. In this review, we report a methodology involving a reconstruction of the ligand diffusion reaction coordinates and the free-energy profiles along these reaction coordinates using enhanced sampling of conformational space. We illustrate the above methods on several ligand-protein systems, including cytochromes and G-protein-coupled receptors. The methods are general and may be adopted to other transport processes in living matter. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Ultrafast heme-ligand recombination in truncated hemoglobin HbO from Mycobacterium tuberculosis: A ligand cage

    Science.gov (United States)

    Jasaitis, Audrius; Ouellet, Hugues; Lambry, Jean-Christophe; Martin, Jean-Louis; Friedman, Joel M.; Guertin, Michel; Vos, Marten H.

    2012-03-01

    Truncated hemoglobin HbO from Mycobacterium tuberculosis displays very slow exchange of diatomic ligands with its environment. Using femtosecond spectroscopy, we show that upon photoexcitation, ligands rebind with unusual speed and efficiency. Only ˜1% O2 can escape from the heme pocket and less than 1% NO. Most remarkably, CO rebinding occurs for 95%, predominantly in 1.2 ns. The general CO rebinding properties are unexpectedly robust against changes in the interactions with close by aromatic residues Trp88 (G8) and Tyr36 (CD1). Molecular dynamics simulations of the CO complex suggest that interactions of the ligand with structural water molecules as well as its rotational freedom play a role in the high reactivity of the ligand and the heme. The slow exchange of ligands between heme and environment may result from a combination of hindered ligand access to the heme pocket by the network of distal aromatic residues, and low escape probability from the pocket.

  2. Constitutive and ligand-induced TCR degradation

    DEFF Research Database (Denmark)

    von Essen, Marina; Bonefeld, Charlotte Menné; Siersma, Volkert

    2004-01-01

    divergent models for TCR down-regulation and degradation have been suggested. The aims of this study were to determine the rate constants for constitutive and ligand-induced TCR degradation and to determine whether the TCR subunits segregate or are processed as an intact unit during TCR down...... to the lysosomes. Similar results were obtained in studies of primary human Vbeta8+ T cells stimulated with superantigen. Based on these results, the simplest model for TCR internalization, sorting, and degradation is proposed.......Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several...

  3. Ligand interactions with galactose oxidase: mechanistic insights.

    Science.gov (United States)

    Whittaker, M M; Whittaker, J W

    1993-01-01

    Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme. PMID:8386015

  4. From three-dimensional GPCR structure to rational ligand discovery

    NARCIS (Netherlands)

    Kooistra, A.J.; Leurs, R.; de Esch, I.J.P.; de Graaf, C.

    2014-01-01

    This chapter will focus on G protein-coupled receptor structure-based virtual screening and ligand design. A generic virtual screening workflow and its individual elements will be introduced, covering amongst others the use of experimental data to steer the virtual screening process, ligand binding

  5. GLIDA: GPCR-ligand database for chemical genomic drug discovery.

    Science.gov (United States)

    Okuno, Yasushi; Yang, Jiyoon; Taneishi, Kei; Yabuuchi, Hiroaki; Tsujimoto, Gozoh

    2006-01-01

    G-protein coupled receptors (GPCRs) represent one of the most important families of drug targets in pharmaceutical development. GPCR-LIgand DAtabase (GLIDA) is a novel public GPCR-related chemical genomic database that is primarily focused on the correlation of information between GPCRs and their ligands. It provides correlation data between GPCRs and their ligands, along with chemical information on the ligands, as well as access information to the various web databases regarding GPCRs. These data are connected with each other in a relational database, allowing users in the field of GPCR-related drug discovery to easily retrieve such information from either biological or chemical starting points. GLIDA includes structure similarity search functions for the GPCRs and for their ligands. Thus, GLIDA can provide correlation maps linking the searched homologous GPCRs (or ligands) with their ligands (or GPCRs). By analyzing the correlation patterns between GPCRs and ligands, we can gain more detailed knowledge about their interactions and improve drug design efforts by focusing on inferred candidates for GPCR-specific drugs. GLIDA is publicly available at http://gdds.pharm.kyoto-u.ac.jp:8081/glida. We hope that it will prove very useful for chemical genomic research and GPCR-related drug discovery.

  6. Role of ligands in controlling the regioselectivity in ruthenium ...

    Indian Academy of Sciences (India)

    Keywords. Enol esters; DFT; regio-selectivity; ligands; ruthenium(II) catalyst. Abstract. Density functional studies are performed to understand the role of chelating bi-phosphine ligands [(Ph2P(CH2)mPPh2); m=1–4] in modulating the regio-selectivity of benzoic acid addition to 1-hexyne, in presence of ruthenium(II) catalyst ...

  7. Nickel(II) complexes containing ONS donor ligands: Synthesis ...

    Indian Academy of Sciences (India)

    that the complexes possess a distorted octahedral geometry with the ligand coordinating in a uni-negative tri- dentate ONS fashion. The catalytic activity of complexes towards some C–C ... gen, nitrogen and sulfur was carried out using Vario EL. III Elemental analyzer at SAIF - Cochin India. The IR spectra of the ligand and ...

  8. Mixed-Ligand Complexes Of Nickel (II) With 2-Acetylpyridine ...

    African Journals Online (AJOL)

    The preparation and spectral properties of five nickel (II) mixed-ligands complexes (Ni [2-Actsc.Y]CI2), derived from 2-acetylpyridinethiosermicarbazones and some nitrogen/sulphur monodentate ligands such as thiophene, ammonia, picoline, pyridine and aniline are described. The complexes have been characterized on ...

  9. Iron(III) complexes of certain tetradentate phenolate ligands as ...

    Indian Academy of Sciences (India)

    complexes of tripodal phenolate ligands containing N3O and N2O2 donor sets represent the metal binding re- gion of the iron proteins. ... 1,2-CTD enzymes; functional and structural models; Fe(III) complexes; phenolate ligands; intradiol cleavage. 1. ..... nolates are equatorial in 5, while they are mixed axial–equatorial in the ...

  10. Ligand-modified metal clusters for gas separation and purification

    Science.gov (United States)

    Okrut, Alexander; Ouyang, Xiaoying; Runnebaum, Ron; Gates, Bruce C.; Katz, Alexander

    2017-02-21

    Provided is an organic ligand-bound metal surface that selects one gaseous species over another. The species can be closely sized molecular species having less than 1 Angstrom difference in kinetic diameter. In one embodiment, the species comprise carbon monoxide and ethylene. Such organic ligand-bound metal surfaces can be successfully used in gas phase separations or purifications, sensing, and in catalysis.

  11. Biological Activities of Recombinant Liver X Receptor â- Ligand ...

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  12. Kinetics and Mechanism of the Ligand Exchange Reaction Between ...

    African Journals Online (AJOL)

    NICO

    Visible spectrophotometry is used to study the kinetics of ligand exchange in the system Ni(salpn)/H2salen with or without triethylamine (NEt3) and ... coordination chemistry.4,5 However, the kinetics of polydentate ligands exchange ..... chlorides and tetraaza Schiff bases: synthesis and characterization of some novel tin(IV) ...

  13. Proteome-wide covalent ligand discovery in native biological systems.

    Science.gov (United States)

    Backus, Keriann M; Correia, Bruno E; Lum, Kenneth M; Forli, Stefano; Horning, Benjamin D; González-Páez, Gonzalo E; Chatterjee, Sandip; Lanning, Bryan R; Teijaro, John R; Olson, Arthur J; Wolan, Dennis W; Cravatt, Benjamin F

    2016-06-23

    Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

  14. Technetium radiodiagnostic fatty acids derived from bisamide bisthiol ligands

    Science.gov (United States)

    Jones, Alun G.; Lister-James, John; Davison, Alan

    1988-05-24

    A bisamide-bisthiol ligand containing fatty acid substituted thiol useful for producing Tc-labelled radiodiagnostic imaging agents is described. The ligand forms a complex with the radionuclide .sup.99m Tc suitable for administration as a radiopharmaceutical to obtain images of the heart for diagnosis of myocardial disfunction.

  15. Biological Activities of Recombinant Liver X Receptor β- Ligand ...

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, high- quality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  16. Improved ligand geometries in crystallographic refinement using AFITT in PHENIX.

    Science.gov (United States)

    Janowski, Pawel A; Moriarty, Nigel W; Kelley, Brian P; Case, David A; York, Darrin M; Adams, Paul D; Warren, Gregory L

    2016-09-01

    Modern crystal structure refinement programs rely on geometry restraints to overcome the challenge of a low data-to-parameter ratio. While the classical Engh and Huber restraints work well for standard amino-acid residues, the chemical complexity of small-molecule ligands presents a particular challenge. Most current approaches either limit ligand restraints to those that can be readily described in the Crystallographic Information File (CIF) format, thus sacrificing chemical flexibility and energetic accuracy, or they employ protocols that substantially lengthen the refinement time, potentially hindering rapid automated refinement workflows. PHENIX-AFITT refinement uses a full molecular-mechanics force field for user-selected small-molecule ligands during refinement, eliminating the potentially difficult problem of finding or generating high-quality geometry restraints. It is fully integrated with a standard refinement protocol and requires practically no additional steps from the user, making it ideal for high-throughput workflows. PHENIX-AFITT refinements also handle multiple ligands in a single model, alternate conformations and covalently bound ligands. Here, the results of combining AFITT and the PHENIX software suite on a data set of 189 protein-ligand PDB structures are presented. Refinements using PHENIX-AFITT significantly reduce ligand conformational energy and lead to improved geometries without detriment to the fit to the experimental data. For the data presented, PHENIX-AFITT refinements result in more chemically accurate models for small-molecule ligands.

  17. Polymerization catalysts containing electron-withdrawing amide ligands

    Science.gov (United States)

    Watkin, John G.; Click, Damon R.

    2002-01-01

    The present invention describes methods of making a series of amine-containing organic compounds which are used as ligands for group 3-10 and lanthanide metal compounds. The ligands have electron-withdrawing groups bonded to them. The metal compounds, when combined with a cocatalyst, are catalysts for the polymerization of olefins.

  18. The Evaluation of Novel Camphor-derived Pyridyl Ligands as ...

    African Journals Online (AJOL)

    NJD

    2009-03-03

    Mar 3, 2009 ... catalysts in the asymmetric Diels-Alder reaction.19–21 These ligands have proven to be reasonably successful in terms of selectivity with enantiomeric excesses in the 80 and low 90 % range. Herein we report the evaluation of a series of recently reported2,3 camphor-derived pyridyl ligands 4-9 (Fig.

  19. Heterogenization of Some PNP Ligands for the Oligomerization of ...

    African Journals Online (AJOL)

    NICO

    Bis(diphenylphosphino)amine ligands were supported on Merrifield's resin and tested in catalytic ethylene oligomerization reactions with a ... When comparing the homogeneous ligands with their heterogeneous counterparts, the latter showed a four-fold ..... Practice, 3rd edn., Springer-Verlag, Berlin, 2001. 38 J. Emsley and ...

  20. Models of protein-ligand crystal structures: trust, but verify

    Science.gov (United States)

    Deller, Marc C.; Rupp, Bernhard

    2015-09-01

    X-ray crystallography provides the most accurate models of protein-ligand structures. These models serve as the foundation of many computational methods including structure prediction, molecular modelling, and structure-based drug design. The success of these computational methods ultimately depends on the quality of the underlying protein-ligand models. X-ray crystallography offers the unparalleled advantage of a clear mathematical formalism relating the experimental data to the protein-ligand model. In the case of X-ray crystallography, the primary experimental evidence is the electron density of the molecules forming the crystal. The first step in the generation of an accurate and precise crystallographic model is the interpretation of the electron density of the crystal, typically carried out by construction of an atomic model. The atomic model must then be validated for fit to the experimental electron density and also for agreement with prior expectations of stereochemistry. Stringent validation of protein-ligand models has become possible as a result of the mandatory deposition of primary diffraction data, and many computational tools are now available to aid in the validation process. Validation of protein-ligand complexes has revealed some instances of overenthusiastic interpretation of ligand density. Fundamental concepts and metrics of protein-ligand quality validation are discussed and we highlight software tools to assist in this process. It is essential that end users select high quality protein-ligand models for their computational and biological studies, and we provide an overview of how this can be achieved.

  1. The Evaluation of Novel Camphor-derived Pyridyl Ligands as ...

    African Journals Online (AJOL)

    A series of camphor-derived pyridyl ligands were evaluated in the Diels-Alder reaction of 3-acryloyl-2-oxazolidinone 1 with cyclopentadiene 2. High yields with good endo:exo selectivity, but only moderate enantioselectivities (43 % ee), were obtained. The structures of the copper (II) complexes of the ligands were ...

  2. Receptor-ligand binding assays : Technologies and applications

    NARCIS (Netherlands)

    de Jong, LAA; Uges, DRA; Franke, JP; Bischoff, R; A.A. de Jong, Lutea; Piet Franke, Rainer

    2005-01-01

    Receptor-ligand interactions play a crucial role in biological systems and their measurement forms an important part of modem pharmaceutical development. Numerous assay formats are available that can be used to screen and quantify receptor ligands. In this review, we give an overview over both

  3. Role of Lanthanide-Ligand bonding in the magnetization relaxation ...

    Indian Academy of Sciences (India)

    examples studied. Keywords. Lanthanides; magnetic anisotropy; ligand field environment; QTAIM and wiberg bond index analysis; single ion magnets. 1. Introduction ... their isomeric pyrazole {M(BpMe)3}ligands-based lan- thanide analogues ... from ANO-RCC basis library included in MOLCAS 8.0 suite. The active space ...

  4. THERMODYNAMICS OF PROTEIN-LIGAND INTERACTIONS AND THEIR ANALYSIS

    Directory of Open Access Journals (Sweden)

    Rummi Devi Saini

    2017-11-01

    Full Text Available Physiological processes are controlled mainly by intermolecular recognition mechanisms which involve protein–protein and protein–ligand interactions with a high specificity and affinity to form a specific complex. Proteins being an important class of macromolecules in biological systems, it is important to understand their actions through binding to other molecules of proteins or ligands. In fact, the binding of low molecular weight ligands to proteins plays a significant role in regulating biological processes such as cellular metabolism and signal transmission. Therefore knowledge of the protein–ligand interactions and the knowledge of the mechanisms involved in the protein-ligand recognition and binding are key in understanding biology at molecular level which will facilitate the discovery, design, and development of drugs. In this review, the mechanisms involved in protein–ligand binding, the binding kinetics, thermodynamic concepts and binding driving forces are discussed. Thermodynamic mechanisms involved in a few important protein-ligand binding are described. Various spectroscopic, non-spectroscopic and computational method for analysis of protein–ligand binding are also discussed.

  5. The autoxidation activity of new mixed-ligand manganese and iron complexes with tripodal ligands

    OpenAIRE

    van Gorkum, R.; Berding, J.; Tooke, D.M.; Spek, A.L.; Reedijk, J.; Bouwman, E.

    2007-01-01

    The activity of new manganese and iron complexes of dianionic tripodal ligands in the autoxidation of ethyl linoleate (EL) is reported. EL consumption rates were monitored using time-resolved FTIR and the degree of oligomerisation was determined by SEC. Almost all complexes showed the same trend in the autoxidation of EL. After a short induction time, the reaction started at a relatively high constant rate; later, this rate changes to a lower rate, which was again constant and on average was ...

  6. Dynamic control of chirality in phosphine ligands for enantioselective catalysis.

    Science.gov (United States)

    Zhao, Depeng; Neubauer, Thomas M; Feringa, Ben L

    2015-03-25

    Chirality plays a fundamental role in biology and chemistry and the precise control of chirality in a catalytic conversion is a key to modern synthesis most prominently seen in the production of pharmaceuticals. In enantioselective metal-based catalysis, access to each product enantiomer is commonly achieved through ligand design with chiral bisphosphines being widely applied as privileged ligands. Switchable phosphine ligands, in which chirality is modulated through an external trigger signal, might offer attractive possibilities to change enantioselectivity in a catalytic process in a non-invasive manner avoiding renewed ligand synthesis. Here we demonstrate that a photoswitchable chiral bisphosphine based on a unidirectional light-driven molecular motor, can be used to invert the stereoselectivity of a palladium-catalysed asymmetric transformation. It is shown that light-induced changes in geometry and helicity of the switchable ligand enable excellent selectivity towards the racemic or individual enantiomers of the product in a Pd-catalysed desymmetrization reaction.

  7. HDX reveals unique fragment ligands for the vitamin D receptor.

    Science.gov (United States)

    Carson, Matthew W; Zhang, Jun; Chalmers, Michael J; Bocchinfuso, Wayne P; Holifield, Karol D; Masquelin, Thierry; Stites, Ryan E; Stayrook, Keith R; Griffin, Patrick R; Dodge, Jeffery A

    2014-08-01

    Modulation of the vitamin D receptor (VDR) with a ligand has the potential to be useful for the oral treatment of osteoporosis. One component of our lead generation strategy to identify synthetic ligands for VDR included a fragment based drug design approach. Screening of ligands in a VDR fluorescence polarization assay and a RXR/VDR conformation sensing assay resulted in the identification of multiple fragment hits (lean >0.30). These fragment scaffolds were subsequently evaluated for interaction with the VDR ligand binding domain using hydrogen-deuterium exchange (HDX) mass spectrometry. Significant protection of H/D exchange was observed for some fragments in helixes 3, 7, and 8 of the ligand binding domain, regions which are similar to those seen for the natural hormone VD3. The fragments appear to mimic the A-ring of VD3 thereby providing viable starting points for synthetic expansion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Transporters of ligands for essential metal ions in plants.

    Science.gov (United States)

    Haydon, Michael J; Cobbett, Christopher S

    2007-01-01

    Essential metals are required for healthy plant growth but can be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalization. However, very little metal in plants is thought to exist as free ions. A number of small, organic molecules have been implicated in metal ion homeostasis as metal ion ligands to facilitate uptake and transport of metal ions with low solubility and also as chelators implicated in sequestration for metal tolerance and storage. Ligands for a number of essential metals have been identified and proteins involved in the transport of these ligands and of metal-ligand complexes have been characterized. Here we review recent advances in understanding the role of mugineic acid, nicotianamine, organic acids (citrate and malate), histidine and phytate as ligands for iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and nickel (Ni) in plants, and the proteins identified as their transporters.

  9. Automated identification of crystallographic ligands using sparse-density representations

    Energy Technology Data Exchange (ETDEWEB)

    Carolan, C. G.; Lamzin, V. S., E-mail: victor@embl-hamburg.de [European Molecular Biology Laboratory (EMBL), c/o DESY, Notkestrasse 85, 22603 Hamburg (Germany)

    2014-07-01

    A novel procedure for identifying ligands in macromolecular crystallographic electron-density maps is introduced. Density clusters in such maps can be rapidly attributed to one of 82 different ligands in an automated manner. A novel procedure for the automatic identification of ligands in macromolecular crystallographic electron-density maps is introduced. It is based on the sparse parameterization of density clusters and the matching of the pseudo-atomic grids thus created to conformationally variant ligands using mathematical descriptors of molecular shape, size and topology. In large-scale tests on experimental data derived from the Protein Data Bank, the procedure could quickly identify the deposited ligand within the top-ranked compounds from a database of candidates. This indicates the suitability of the method for the identification of binding entities in fragment-based drug screening and in model completion in macromolecular structure determination.

  10. Polypharmacology: in silico methods of ligand design and development.

    Science.gov (United States)

    McKie, Samuel A

    2016-04-01

    How to design a ligand to bind multiple targets, rather than to a single target, is the focus of this review. Rational polypharmacology draws on knowledge that is both broad ranging and hierarchical. Computer-aided multitarget ligand design methods are described according to their nested knowledge level. Ligand-only and then receptor-ligand strategies are first described; followed by the metabolic network viewpoint. Subsequently strategies that view infectious diseases as multigenomic targets are discussed, and finally the disease level interpretation of medicinal therapy is considered. As yet there is no consensus on how best to proceed in designing a multitarget ligand. The current methodologies are bought together in an attempt to give a practical overview of how polypharmacology design might be best initiated.

  11. Mathematical model for determining the binding constants between immunoglobulins, bivalent ligands, and monovalent ligands.

    Science.gov (United States)

    Mack, Eric T; Cummings, Linda; Perez-Castillejos, Raquel

    2011-02-01

    This paper analyzes the equilibria between immunoglobulins (R(2)), homo-bifunctional ligands (L(2)), monovalent ligands (I), and their complexes. We present a mathematical model that can be used to estimate the concentration of each species present in a mixture of R(2), L(2), and I, given the initial conditions defining the total concentration of R(2), L(2), I, and four dissociation constants (K(d)(inter), K(d)(intra), K(d)(mono), and α). This model is based on fewer assumptions than previous models and can be used to describe exactly a broad range of experimental conditions. A series of curves illustrates the dependence of the equilibria upon the total concentrations of receptors and ligands, and the dissociation constants. We provide a set of guidelines for the design and analysis of experiments with a focus on estimating the binding constants from experimental binding isotherms. Two analytical equations relate the conditions for maximum aggregation in this system to the binding constants. This model is a tool to quantify the binding of immunoglobulins to antigens and a guide to understanding and predicting the experimental data of assays and techniques that employ immunoglobulins.

  12. Developing ligands for palladium(II)-catalyzed C-H functionalization: intimate dialogue between ligand and substrate.

    Science.gov (United States)

    Engle, Keary M; Yu, Jin-Quan

    2013-09-20

    Homogeneous transition-metal-catalyzed reactions are indispensable to all facets of modern chemical synthesis. It is thus difficult to imagine that for much of the early 20th century, the reactivity and selectivity of all known homogeneous metal catalysts paled in comparison to their heterogeneous and biological counterparts. In the intervening decades, advances in ligand design bridged this divide, such that today some of the most demanding bond-forming events are mediated by ligand-supported homogeneous metal species. While ligand design has propelled many areas of homogeneous catalysis, in the field of Pd(II)-catalyzed C-H functionalization, suitable ligand scaffolds are lacking, which has hampered the development of broadly practical transformations based on C-H functionalization logic. In this Perspective, we offer an account of our research employing three ligand scaffolds, mono-N-protected amino acids, 2,6-disubstituted pyridines, and 2,2'-bipyridines, to address challenges posed by several synthetically versatile substrate classes. Drawing on this work, we discuss principles of ligand design, such as the need to match a ligand to a particular substrate class, and how ligand traits such as tunability and modularity can be advantageous in reaction discovery.

  13. Developing Ligands for Palladium(II)-Catalyzed C–H Functionalization: Intimate Dialogue between Ligand and Substrate

    Science.gov (United States)

    Engle, Keary M.; Yu, Jin-Quan

    2013-01-01

    Homogeneous transition metal–catalyzed reactions are indispensable to all facets of modern chemical synthesis. It is thus difficult to imagine that for much of the early 20th century, the reactivity and selectivity of all known homogeneous metal catalysts paled in comparison to their heterogeneous and biological counterparts. In the intervening decades, advances in ligand design bridged this divide, such that today some of the most demanding bond-forming events are mediated by ligand-supported homogeneous metal species. While ligand design has propelled many areas of homogeneous catalysis, in the field of Pd(II)-catalyzed C–H functionalization, suitable ligand scaffolds are lacking, which has hampered the development of broadly practical transformations based on C–H functionalization logic. In this review, we offer an account of our research employing three ligand scaffolds, mono-N-protected amino acids, 2,6-disubstituted pyridines, and 2,2′-bipyridines, to address challenges posed by several synthetically versatile substrate classes. Drawing on this work, we discuss principles of ligand design, such as the need to match a ligand to a particular substrate class, and how ligand traits such as tunability and modularity can be advantageous in reaction discovery. PMID:23565982

  14. Regulation of the O-glycan-type Sialyl-Lewis X (sLex) Bio-synthesis Pathway during Cell Transformation Programs: Epithelial-Mesenchymal Transition (EMT) and Molecular Subtypes in Breast Carcinoma and Human T Cell Activation

    KAUST Repository

    AbuElela, Ayman

    2017-12-01

    During tumor progression and development of distant metastases, a subset of cancer cells undergoes transformation programs, such as epithelial-mesenchymal transition (EMT), to acquire enhanced migratory attributes to commence the metastatic cascade with the intension of achieving an active cell adhesion molecule-mediated organ-specific homing. Similarly, naive T cells reform the assemblage of their surface adhesion molecules during differentiation to activated T cells in order to successfully home to sites of inflammation and other extra-lymphoid organs for surveillance purposes. Sialyl-Lewis X (sLex) is well-known for mediating the homing of epithelial circulating tumor cellss (CTCs) and activated T cells to target sites through the interaction with endothelial selectins. Since glycan structures are not directly encoded by the genome, their expression is dependent on the glycosyltransferase (GT) expression and activity. Yet, the modulation of GTs during breast cancer transformation and in different molecular subtypes is still unknown. In addition, although the regulation of GTs during T cell activation is well-understood, the regulation at the epigenetic level is lacking. O-glycan-type sLex expression and E-selectin binding under static and flow conditions varies among molecular subtypes of breast cancer and upon the induction of EMT which is linked to the expression patterns of GTs. GTs displayed a significant prognostic value of in the association with the patients\\' survival profiles and in the ability to predict the breast cancer molecular subtypes from the expression data of a random patient sample. Also, GTs were able to differentiate between tumor and their normal counterparts as well as cancer types and glioblastoma subtypes. On the other hand, we studied the regulation of GTs in human CD4+ memory T cells compared to the naive cells at the epigenetic level. Memory T cell subsets demonstrated differential chromatin accessibility and histone marks within

  15. Gas adsorption and gas mixture separations using mixed-ligand MOF material

    Science.gov (United States)

    Hupp, Joseph T [Northfield, IL; Mulfort, Karen L [Chicago, IL; Snurr, Randall Q [Evanston, IL; Bae, Youn-Sang [Evanston, IL

    2011-01-04

    A method of separating a mixture of carbon dioxiode and hydrocarbon gas using a mixed-ligand, metal-organic framework (MOF) material having metal ions coordinated to carboxylate ligands and pyridyl ligands.

  16. Substituted biurets as uranophilic ligands: A facile DMSO-induced conversion of a 1:1 into a 2:1 uranyl-ligand complex

    Energy Technology Data Exchange (ETDEWEB)

    Potts, K.T.; O' Brien, J.J.; Tham, F.S. (Rensselaer Polytechnic Inst., Troy, NY (United States))

    1990-01-01

    1,5-Bis(6-(1-ethoxycarbonyl-3-thioureido)-2-pyridindiyl)biuret and uranyl acetate gave a crystalline 1:1 uranyl-ligand complex which, on crystallization from DMSO, underwent rearrangement to a crystalline 2:1 uranyl-ligand complex and a stoichiometric amount of the uncomplexed ligand. Spectral characteristics of these ligands and their uranyl complexes together with single crystal x-ray data for the uranyl-ligand complexes are described.

  17. Regulation mechanisms of the FLT3-ligand after irradiation; Mecanismes de regulation du FLT3-ligand apres irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Prat-Lepesant, M

    2005-06-15

    The hematopoietic compartment is one of the most severely damaged after chemotherapy, radiotherapy or accidental irradiations. Whatever its origin, the resulting damage to the bone marrow remains difficult to evaluate. Thus, it would be of great interest to get a biological indicator of residual hematopoiesis in order to adapt the treatment to each clinical situation. Recent results indicated that the plasma Flt3 ligand concentration was increased in patients suffering from either acquired or induced aplasia, suggesting that Flt3 ligand might be useful as a biological indicator of bone marrow status. We thus followed in a mouse model as well as in several clinical situations the variations in plasma Flt3 ligand concentration, after either homogeneous or heterogeneous irradiations. These variations were correlated to the number of hematopoietic progenitors and to other parameters such as duration and depth of pancytopenia. The results indicated that the concentration of Flt3 ligand in the blood reflects the bone marrow status, and that the follow-up of plasma Flt3 ligand concentration could give predictive information about the bone marrow function and the duration and severity of pancytopenia and thrombocytopenia. Nevertheless, the clinical use of Flt3 ligand as a biological indicator of bone marrow damage require the knowledge of the mechanisms regulating the variations in plasma Flt3 ligand concentration. We thus developed a study in the mouse model. The results indicated that the variations in plasma Flt3 ligand variations were not solely due to a balance between its production by lymphoid cells and its consumption by hematopoietic cells. Moreover, we showed that T lymphocytes are not the main regulator of plasma Flt3 ligand concentration as previously suggested, and that other cell types, possibly including bone marrow stromal cells, might be strongly implicated. These results also suggest that the Flt3 ligand is a main systemic regulator of hematopoiesis

  18. Structural Analysis of Chemokine Receptor–Ligand Interactions

    Science.gov (United States)

    2017-01-01

    This review focuses on the construction and application of structural chemokine receptor models for the elucidation of molecular determinants of chemokine receptor modulation and the structure-based discovery and design of chemokine receptor ligands. A comparative analysis of ligand binding pockets in chemokine receptors is presented, including a detailed description of the CXCR4, CCR2, CCR5, CCR9, and US28 X-ray structures, and their implication for modeling molecular interactions of chemokine receptors with small-molecule ligands, peptide ligands, and large antibodies and chemokines. These studies demonstrate how the integration of new structural information on chemokine receptors with extensive structure–activity relationship and site-directed mutagenesis data facilitates the prediction of the structure of chemokine receptor–ligand complexes that have not been crystallized. Finally, a review of structure-based ligand discovery and design studies based on chemokine receptor crystal structures and homology models illustrates the possibilities and challenges to find novel ligands for chemokine receptors. PMID:28165741

  19. Ligand-controlled Iron-catalyzed Cross Coupling of Benzylic Chlorides with Aryl Grignard Reagents

    National Research Council Canada - National Science Library

    Kawamura, Shintaro; Nakamura, Masaharu

    2013-01-01

    .... Although electron-deficient ligands promoted the reductive homocoupling of benzylic halides, electron-rich ligands effectively promoted the desired cross-coupling reaction to afford the corresponding...

  20. Competitive antagonism of AMPA receptors by ligands of different classes

    DEFF Research Database (Denmark)

    Hogner, Anders; Greenwood, Jeremy R; Liljefors, Tommy

    2003-01-01

    Ionotropic glutamate receptors (iGluRs) constitute a family of ligand-gated ion channels that are essential for mediating fast synaptic transmission in the central nervous system. This study presents a high-resolution X-ray structure of the competitive antagonist (S)-2-amino-3-[5-tert-butyl-3......-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO) in complex with the ligand-binding core of the receptor. Comparison with the only previous structure of the ligand-binding core in complex with an antagonist, 6,7-dinitro-2,3-quinoxalinedione (DNQX) (Armstrong, N.; Gouaux, E. Neuron 2000, 28, 165-181), reveals...

  1. Optimal Overlay of Ligands with Flexible Bonds Using Differential Evolution

    DEFF Research Database (Denmark)

    Kristensen, Thomas Greve; Pedersen, Christian Storm

    2009-01-01

    spatial alignment of a set of active ligands taking the flexibility of chemical bonds into account. We present two implementations of our method. One using Differential Evolution (DE) for numerical optimization, and one using the Nelder-Mead method for numerical optimization. We investigate the quality......When designing novel drugs, the need arise to screen large databases of drug candidates (small synthesizable chemical structures) for structures that resemble active ligands, i.e. small chemical structures that are known to react with the target protein. If several active ligands are known one...

  2. Synthesis of symmetrical and non-symmetrical bivalent neurotransmitter ligands

    DEFF Research Database (Denmark)

    Stuhr-Hansen, Nicolai; Andersen, Jacob; Thygesen, Mikkel Boas

    2016-01-01

    A novel procedure for synthesis of bivalent neurotransmitter ligands was developed by reacting O-benzyl protected N-nosylated dopamine and serotonin with alkyl- or PEG-linked diols under Fukuyama-Mitsunobu conditions in the presence of DIAD/PPh3 generating three different bivalent neurotransmitter...... ligands in a one-pot reaction. The methodol. establishes a facile route towards bivalent neurotransmitter ligands, and libraries of in total 40 sym. and non-sym. bivalent and monovalent dopamine and serotonin compds. linked through alkyl or PEG spacers of varying length were prepd. Interestingly...

  3. Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands

    Science.gov (United States)

    Costanzi, Stefano; Tikhonova, Irina G.; Harden, T. Kendall; Jacobson, Kenneth A.

    2009-11-01

    Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered.

  4. Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands.

    Science.gov (United States)

    Costanzi, Stefano; Tikhonova, Irina G; Harden, T Kendall; Jacobson, Kenneth A

    2009-11-01

    Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered.

  5. Ligand-biased ensemble receptor docking (LigBEnD): a hybrid ligand/receptor structure-based approach

    Science.gov (United States)

    Lam, Polo C.-H.; Abagyan, Ruben; Totrov, Maxim

    2017-09-01

    Ligand docking to flexible protein molecules can be efficiently carried out through ensemble docking to multiple protein conformations, either from experimental X-ray structures or from in silico simulations. The success of ensemble docking often requires the careful selection of complementary protein conformations, through docking and scoring of known co-crystallized ligands. False positives, in which a ligand in a wrong pose achieves a better docking score than that of native pose, arise as additional protein conformations are added. In the current study, we developed a new ligand-biased ensemble receptor docking method and composite scoring function which combine the use of ligand-based atomic property field (APF) method with receptor structure-based docking. This method helps us to correctly dock 30 out of 36 ligands presented by the D3R docking challenge. For the six mis-docked ligands, the cognate receptor structures prove to be too different from the 40 available experimental Pocketome conformations used for docking and could be identified only by receptor sampling beyond experimentally explored conformational subspace.

  6. Residual ligand entropy in the binding of p-substituted benzenesulfonamide ligands to bovine carbonic anhydrase II.

    Science.gov (United States)

    Stöckmann, Henning; Bronowska, Agnieszka; Syme, Neil R; Thompson, Gary S; Kalverda, Arnout P; Warriner, Stuart L; Homans, Steve W

    2008-09-17

    In studies on the thermodynamics of ligand-protein interactions, it is often assumed that the configurational and conformational entropy of the ligand is zero in the bound state (i.e., the ligand is rigidly fixed in the binding pocket). However, there is little direct experimental evidence for this assumption, and in the case of binding of p-substituted benzenesulfonamide inhibitors to bovine carbonic anhydrase II (BCA II), the observed thermodynamic binding signature derived from isothermal titration calorimetry experiments leads indirectly to the conclusion that a considerable degree of residual entropy remains in the bound ligand. Specifically, the entropy of binding increases with glycine chain length n, and strong evidence exists that this thermodynamic signature is not driven by solvent reorganization. By use of heteronuclear (15)N NMR relaxation measurements in a series (n = 1-6) of (15)N-glycine-enriched ligands, we find that the observed thermodynamic binding signature cannot be explained by residual ligand dynamics in the bound state, but rather results from the indirect influence of ligand chain length on protein dynamics.

  7. Investigation of the enantioselectivity of tetramethylammonium L-hydroxyproline ionic liquid as a novel chiral ligand in ligand-exchange CE and ligand-exchange MEKC.

    Science.gov (United States)

    Liu, Ruijuan; Du, Yingxiang; Chen, Jiaquan; Zhang, Qi; Du, Shuaijing; Feng, Zijie

    2015-01-01

    Chiral ionic liquids (ILs) have drawn more and more attention in separation science; however, only a few papers focused on the application of chiral ILs as chiral ligands in LE-CE. In this article, a novel amino acid ionic liquid (AAIL), tetramethylammonium L-hydroxyproline ([TMA][L-OH-Pro]), was first applied as a chiral ligand to evaluate its enantioselectivity towards several aromatic amino acids in ligand-exchange capillary electrophoresis (LE-CE) and ligand-exchange micellar electrokinetic capillary chromatography (LE-MEKC). In the LE-CE system, excellent separations were achieved for tryptophan (Rs = 3.03) and 3, 4-dihydroxyphenylalanine (DOPA) (Rs = 4.35). Several parameters affecting the enantioseparation were systematically investigated, including AAIL concentration, type and concentration of central metal ion, buffer pH, as well as applied voltage. The optimum separation was obtained with 60 mM AAIL containing 30 mM Cu (II) at pH 4.5. Additionally, an LE-MEKC system was established to further study the enantioselectivity of [TMA][L-OH-Pro] towards selected analytes. As observed, the separations of the enantiomers of tryptophan, phenylalanine, and histidine were all improved compared to the LE-CE system. The results indicated that the application of AAILs as chiral ligands is a promising method in chiral separation science. © 2014 Wiley Periodicals, Inc.

  8. Norovirus Binding to Ligands Beyond Histo-Blood Group Antigens

    Science.gov (United States)

    Almand, Erin A.; Moore, Matthew D.; Jaykus, Lee-Ann

    2017-01-01

    Histo-blood group antigens (HBGAs) are commonly accepted as the cellular receptors for human norovirus. However, some human noroviruses have been found not to bind any HBGA ligand, suggesting potential additional co-factors. Some ligands have been found to bind noroviruses and have the potential to be additional cellular receptors/attachment factors for human norovirus or inhibitors of the HBGA interaction. The studies identifying these mostly characterize different chemical, human, food, or bacterial components and their effect on norovirus binding and infection, although the mechanism of interaction is unknown in many cases. This review seeks to supplement the already well-covered HBGA-norovirus literature by covering non-HBGA human norovirus ligands and inhibitors to provide investigators with a more comprehensive view of norovirus ligands. PMID:29312233

  9. Characterization of Ligand-Induced Endocytosis of EGF-Receptors

    National Research Council Canada - National Science Library

    Schmid, Sandra

    1997-01-01

    Under the auspices of this training Fellowship (May 9, 1994-November 9, 1997) I have undertaken studies on the molecular mechanisms that govern ligand-induced endocytosis of epidermal growth factor receptors (EGFR...

  10. Unique advantages of organometallic supporting ligands for uranium complexes

    Energy Technology Data Exchange (ETDEWEB)

    Diaconescu, Paula L. [Univ. of California, Los Angeles, CA (United States); Garcia, Evan [Univ. of California, Los Angeles, CA (United States)

    2014-05-31

    The objective of our research project was to study the reactivity of uranium complexes supported by ferrocene-based ligands. In addition, this research provides training of graduate students as the next generation of actinide scientists.

  11. (TIQ) Thiazole and Oxazoline Ligands for Asymmetric Henry Reactions

    African Journals Online (AJOL)

    NICO

    TIQ) backbone were synthesized. Their application in the catalytic asymmetric Henry reaction was investigated with comparison to a corresponding TIQ oxazoline ligand. The Cu(II)-oxazoline complex was more reactive and furnished moderate ...

  12. Tetrapyrroles as Endogenous TSPO Ligands in Eukaryotes and Prokaryotes: Comparisons with Synthetic Ligands

    Directory of Open Access Journals (Sweden)

    Leo Veenman

    2016-06-01

    Full Text Available The 18 kDa translocator protein (TSPO is highly 0conserved in eukaryotes and prokaryotes. Since its discovery in 1977, numerous studies established the TSPO’s importance for life essential functions. For these studies, synthetic TSPO ligands typically are applied. Tetrapyrroles present endogenous ligands for the TSPO. Tetrapyrroles are also evolutionarily conserved and regulate multiple functions. TSPO and tetrapyrroles regulate each other. In animals TSPO-tetrapyrrole interactions range from effects on embryonic development to metabolism, programmed cell death, response to stress, injury and disease, and even to life span extension. In animals TSPOs are primarily located in mitochondria. In plants TSPOs are also present in plastids, the nuclear fraction, the endoplasmic reticulum, and Golgi stacks. This may contribute to translocation of tetrapyrrole intermediates across organelles’ membranes. As in animals, plant TSPO binds heme and protoporphyrin IX. TSPO-tetrapyrrole interactions in plants appear to relate to development as well as stress conditions, including salt tolerance, abscisic acid-induced stress, reactive oxygen species homeostasis, and finally cell death regulation. In bacteria, TSPO is important for switching from aerobic to anaerobic metabolism, including the regulation of photosynthesis. As in mitochondria, in bacteria TSPO is located in the outer membrane. TSPO-tetrapyrrole interactions may be part of the establishment of the bacterial-eukaryote relationships, i.e., mitochondrial-eukaryote and plastid-plant endosymbiotic relationships.

  13. A Pipeline To Enhance Ligand Virtual Screening: Integrating Molecular Dynamics and Fingerprints for Ligand and Proteins.

    Science.gov (United States)

    Spyrakis, Francesca; Benedetti, Paolo; Decherchi, Sergio; Rocchia, Walter; Cavalli, Andrea; Alcaro, Stefano; Ortuso, Francesco; Baroni, Massimo; Cruciani, Gabriele

    2015-10-26

    The importance of taking into account protein flexibility in drug design and virtual ligand screening (VS) has been widely debated in the literature, and molecular dynamics (MD) has been recognized as one of the most powerful tools for investigating intrinsic protein dynamics. Nevertheless, deciphering the amount of information hidden in MD simulations and recognizing a significant minimal set of states to be used in virtual screening experiments can be quite complicated. Here we present an integrated MD-FLAP (molecular dynamics-fingerprints for ligand and proteins) approach, comprising a pipeline of molecular dynamics, clustering and linear discriminant analysis, for enhancing accuracy and efficacy in VS campaigns. We first extracted a limited number of representative structures from tens of nanoseconds of MD trajectories by means of the k-medoids clustering algorithm as implemented in the BiKi Life Science Suite ( http://www.bikitech.com [accessed July 21, 2015]). Then, instead of applying arbitrary selection criteria, that is, RMSD, pharmacophore properties, or enrichment performances, we allowed the linear discriminant analysis algorithm implemented in FLAP ( http://www.moldiscovery.com [accessed July 21, 2015]) to automatically choose the best performing conformational states among medoids and X-ray structures. Retrospective virtual screenings confirmed that ensemble receptor protocols outperform single rigid receptor approaches, proved that computationally generated conformations comprise the same quantity/quality of information included in X-ray structures, and pointed to the MD-FLAP approach as a valuable tool for improving VS performances.

  14. Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

    Science.gov (United States)

    Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

    2012-01-01

    The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

  15. Metal-Ligand Multiple Bonds in High-Spin Complexes

    OpenAIRE

    King, Evan

    2012-01-01

    The chemistry of late first row transition metals supported by dipyrromethane and dipyrromethene ligands bearing sterically bulky substituents was explored. Transition metal complexes (Mn, Fe, Co, Ni, Zn) of the dipyrromethane ligand 1,9-dimesityl-5,5-dimethyldipyrromethane (dpma) were prepared. Structural and magnetic characterization (SQUID, EPR) of the bis-pyridine adducts \\((dpma)Mn(py)_2\\), \\((dpma)Fe(py)_2\\), and \\((dpma)Co(py)_2\\) showed each tetrahedral divalent ion to be high-spin, w...

  16. Cyclometallated iridium and platinum complexes with noninnocent ligands.

    Science.gov (United States)

    Hirani, Bhavna; Li, Jian; Djurovich, Peter I; Yousufuddin, Muhammed; Oxgaard, Jonas; Persson, Petter; Wilson, Scott R; Bau, Robert; Goddard, William A; Thompson, Mark E

    2007-05-14

    The electronic properties of the cyclometalated (CwedgeN) complexes of iridium and platinum metals with a catechol ligand have been studied experimentally and computationally. The synthesis and characterization of (p-tolylpyridine)Ir(3,5-di-tert-butylcatechol) (abbreviated Ir-sq) and (2,4-diflorophenylpyridine)Pt(3,5-di-tert-butylcatechol) (abbreviated Pt-sq) are reported along with their structural, spectral, and electrochemical properties. Reaction of the 3,5-di-tert-butylcatechol (DTBCat) ligand with the prepared cyclometalated metal complex was carried out in air in the presence of a base. The resulting complexes are air stable and are paramagnetic with the unpaired electron residing mainly on the catechol ligand. The bond lengths obtained from X-ray structure analysis and the theoretical results suggest the semiquinone form of the catechol ligand. Low-energy, intense (approximately 10(3) M-1 cm-1) transitions are observed in the visible to near-infrared region (600-700 nm) of the absorption spectra of the metal complexes. Electrochemically, the complexes exhibit a reversible reduction of the semiquinone form to the catechol form of the ligand and an irreversible oxidation to the unstable quinone form of the ligand. The noninnocent catechol ligand plays a significant role in the electronic properties of the metal complexes. Density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations on the two open-shell molecules provide the ground-state and excited-state energies of the molecular orbitals involved in the observed low-energy transitions. The spin density in the two complexes resides mainly on the catechol ligand. The intense transition arises from excitation of the beta electron from a HOMO-n (n = 1 or 2 here) to the LUMO, rather than from the excitation of the unpaired alpha electron.

  17. Increased CD40 ligand in patients with acute anterior uveitis

    DEFF Research Database (Denmark)

    Øgard, Carsten; Sørensen, Torben Lykke; Krogh, Erik

    2005-01-01

    The inflammatory response in acute anterior uveitis (AU) is believed to be primarily mediated by autoreactive T-cells. We wanted to evaluate whether the T-cell activation marker CD40 ligand is involved in the AU immunopathogenesis.......The inflammatory response in acute anterior uveitis (AU) is believed to be primarily mediated by autoreactive T-cells. We wanted to evaluate whether the T-cell activation marker CD40 ligand is involved in the AU immunopathogenesis....

  18. Synthesis and Characterization of PEGylated Toll Like Receptor 7 Ligands

    OpenAIRE

    Chan, Michael; Hayashi, Tomoko; Mathewson, Richard D.; Yao, Shiyin; Gray, Christine; Tawatao, Rommel; Kalenian, Kevin; Zhang, Yanmei; Hayashi, Yuki; Lao, Fitzgerald S.; Cottam, Howard B.; Carson, Dennis A.

    2011-01-01

    Toll like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid or polyethylene glycol (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in exi...

  19. Reaction chemistry and ligand exchange at cadmium selenide nanocrystal surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Owen, Jonathan; Park, Jungwon; Trudeau, Paul-Emile; Alivisatos, A. Paul

    2008-12-02

    Chemical modification of nanocrystal surfaces is fundamentally important to their assembly, their implementation in biology and medicine, and greatly impacts their electrical and optical properties. However, it remains a major challenge owing to a lack of analytical tools to directly determine nanoparticle surface structure. Early nuclear magnetic resonance (NMR) and X-ray photoelectron spectroscopy (XPS) studies of CdSe nanocrystals prepared in tri-n-octylphosphine oxide (1) and tri-n-octylphosphine (2), suggested these coordinating solvents are datively bound to the particle surface. However, assigning the broad NMR resonances of surface-bound ligands is complicated by significant concentrations of phosphorus-containing impurities in commercial sources of 1, and XPS provides only limited information about the nature of the phosphorus containing molecules in the sample. More recent reports have shown the surface ligands of CdSe nanocrystals prepared in technical grade 1, and in the presence of alkylphosphonic acids, include phosphonic and phosphinic acids. These studies do not, however, distinguish whether these ligands are bound datively, as neutral, L-type ligands, or by X-type interaction of an anionic phosphonate/phosphinate moiety with a surface Cd{sup 2+} ion. Answering this question would help clarify why ligand exchange with such particles does not proceed generally as expected based on a L-type ligand model. By using reagents with reactive silicon-chalcogen and silicon-chlorine bonds to cleave the ligands from the nanocrystal surface, we show that our CdSe and CdSe/ZnS core-shell nanocrystal surfaces are likely terminated by X-type binding of alkylphosphonate ligands to a layer of Cd{sup 2+}/Zn{sup 2+} ions, rather than by dative interactions. Further, we provide spectroscopic evidence that 1 and 2 are not coordinated to our purified nanocrystals.

  20. Reversible Size Control of Silver Nanoclusters via Ligand-exchange

    KAUST Repository

    Bootharaju, Megalamane Siddaramappa

    2015-05-21

    The properties of atomically monodisperse noble metal nanoclusters (NCs) are intricately intertwined with their precise molecular formula. The vast majority of size-specific NC syntheses start from the reduction of the metal salt and thiol ligand mixture. Only in gold was it recently shown that ligand-exchange could induce the growth of NCs from one atomically precise species to another; a process of yet unknown reversibility. Here, we present a process for the ligand-exchange-induced growth of atomically precise silver NCs, in a biphasic liquid-liquid system, which is particularly of interest because of its complete reversibility and ability to occur at room temperature. We explore this phenomenon in-depth using Ag35(SG)18 [SG= glutathionate] and Ag44(4-FTP)30 [4-FTP= 4-fluorothiophenol] as model systems. We show that the ligand-exchange conversion of Ag35(SG)18 into Ag44(4-FTP)30 is rapid (< 5 min) and direct, while the reverse process proceeds slowly through intermediate cluster sizes. We adapt a recently developed theory of reverse Ostwald ripening to model the NCs’ interconvertibility. The model’s predictions are in good agreement with the experimental observations, and they highlight the importance of small changes in the ligand-metal binding energy in determining the final equilibrium NC size. Based on the insight provided by this model, we demonstrated experimentally that by varying the choice of ligands, ligand-exchange can be used to obtain different sized NCs. The findings in this work establish ligand-exchange as a versatile tool for tuning cluster sizes.

  1. Synthesis of Copper Nanoparticles Coated with Nitrogen Ligands

    OpenAIRE

    Rubén Sierra-Ávila; Marissa Pérez-Alvarez; Gregorio Cadenas-Pliego; Carlos Alberto Ávila-Orta; Rebeca Betancourt-Galindo; Enrique Jiménez-Regalado; Rosa Martha Jiménez-Barrera; Juan Guillermo Martínez-Colunga

    2014-01-01

    The synthesis of copper nanoparticles was studied by wet chemical methods using copper sulfate pentahydrate (CuSO4·5H2O) and nitrogen ligands allylamine (AAm) and polyallylamine (PAAm) as stabilizers. The results suggest that the use of these ligands leads to the exclusive formation of metallic copper nanoparticles (Cu-NPs). The use of partially crosslinked polyallylamine (PAAmc) leads to nanoparticles (NPs) with low yields and high coating content, while linear PAAm leads to NPs with high yi...

  2. Discovery of GPCR ligands for probing signal transduction pathways.

    Science.gov (United States)

    Brogi, Simone; Tafi, Andrea; Désaubry, Laurent; Nebigil, Canan G

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven integral transmembrane proteins that are the primary targets of almost 30% of approved drugs and continue to represent a major focus of pharmaceutical research. All of GPCR targeted medicines were discovered by classical medicinal chemistry approaches. After the first GPCR crystal structures were determined, the docking screens using these structures lead to discovery of more novel and potent ligands. There are over 360 pharmaceutically relevant GPCRs in the human genome and to date about only 30 of structures have been determined. For these reasons, computational techniques such as homology modeling and molecular dynamics simulations have proven their usefulness to explore the structure and function of GPCRs. Furthermore, structure-based drug design and in silico screening (High Throughput Docking) are still the most common computational procedures in GPCRs drug discovery. Moreover, ligand-based methods such as three-dimensional quantitative structure-selectivity relationships, are the ideal molecular modeling approaches to rationalize the activity of tested GPCR ligands and identify novel GPCR ligands. In this review, we discuss the most recent advances for the computational approaches to effectively guide selectivity and affinity of ligands. We also describe novel approaches in medicinal chemistry, such as the development of biased agonists, allosteric modulators, and bivalent ligands for class A GPCRs. Furthermore, we highlight some knockout mice models in discovering biased signaling selectivity.

  3. Discovery of GPCR ligands for probing signal transduction pathways

    Science.gov (United States)

    Brogi, Simone; Tafi, Andrea; Désaubry, Laurent; Nebigil, Canan G.

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven integral transmembrane proteins that are the primary targets of almost 30% of approved drugs and continue to represent a major focus of pharmaceutical research. All of GPCR targeted medicines were discovered by classical medicinal chemistry approaches. After the first GPCR crystal structures were determined, the docking screens using these structures lead to discovery of more novel and potent ligands. There are over 360 pharmaceutically relevant GPCRs in the human genome and to date about only 30 of structures have been determined. For these reasons, computational techniques such as homology modeling and molecular dynamics simulations have proven their usefulness to explore the structure and function of GPCRs. Furthermore, structure-based drug design and in silico screening (High Throughput Docking) are still the most common computational procedures in GPCRs drug discovery. Moreover, ligand-based methods such as three-dimensional quantitative structure–selectivity relationships, are the ideal molecular modeling approaches to rationalize the activity of tested GPCR ligands and identify novel GPCR ligands. In this review, we discuss the most recent advances for the computational approaches to effectively guide selectivity and affinity of ligands. We also describe novel approaches in medicinal chemistry, such as the development of biased agonists, allosteric modulators, and bivalent ligands for class A GPCRs. Furthermore, we highlight some knockout mice models in discovering biased signaling selectivity. PMID:25506327

  4. Complexation of cobalt by natural ligands in freshwater

    Energy Technology Data Exchange (ETDEWEB)

    Qian, J.; Xue, H.B. [Swiss Federal Inst. for Environmental Science and Technology, Kastanienbaum (Switzerland); Sigg, L.; Albrecht, A. [Swiss Federal Inst. for Environmental Science and Technology, Duebendorf (Switzerland)

    1998-07-15

    Ligand exchange with dimethylglyoxime (DMG) and differential pulse cathodic stripping voltammetry (DPCSV) was applied to determine free Co(II) ionic concentrations [Co{sup 2+}] and Co(II) complexation by natural organic ligands in freshwater. [Co{sup 2+}] and the complexing parameters were calculated on the basis of the thermodynamic equilibria between added and natural ligands. The optimal working conditions were carefully evaluated in synthetic solutions and in natural freshwater samples. The results from samples collected at various sites from the Aare and Rhine Rivers and the Biel and Lucerne Lakes, Switzerland, are presented. The total dissolved Co concentrations are in the range of 0.5--6.5 nM, and [Co{sup 2+}] is in the range of 0.05--0.5 nM. Organic complexes of Co are predominant in most samples with 80--96% of total dissolved Co. A one-ligand model could well fit the titrations of freshwater samples with Co(II) (using the FITEQL program). The conditional stability constants of the natural ligands were computed to be log K = 9.5--11.6 (pH 8.0 {+-} 0.1), and the ligand concentrations were [L] = 1.4--7.6 nM. The reliability and applicability of the technique and the implications of the results are discussed.

  5. Ligand binding analysis and screening by chemical denaturation shift.

    Science.gov (United States)

    Schön, Arne; Brown, Richard K; Hutchins, Burleigh M; Freire, Ernesto

    2013-12-01

    The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (≥60°C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Ligand- and receptor-based docking with LiBELa

    Science.gov (United States)

    dos Santos Muniz, Heloisa; Nascimento, Alessandro S.

    2015-08-01

    Methodologies on molecular docking are constantly improving. The problem consists on finding an optimal interplay between the computational cost and a satisfactory physical description of ligand-receptor interaction. In pursuit of an advance in current methods we developed a mixed docking approach combining ligand- and receptor-based strategies in a docking engine, where tridimensional descriptors for shape and charge distribution of a reference ligand guide the initial placement of the docking molecule and an interaction energy-based global minimization follows. This hybrid docking was evaluated with soft-core and force field potentials taking into account ligand pose and scoring. Our approach was found to be competitive to a purely receptor-based dock resulting in improved logAUC values when evaluated with DUD and DUD-E. Furthermore, the smoothed potential as evaluated here, was not advantageous when ligand binding poses were compared to experimentally determined conformations. In conclusion we show that a combination of ligand- and receptor-based strategy docking with a force field energy model results in good reproduction of binding poses and enrichment of active molecules against decoys. This strategy is implemented in our tool, LiBELa, available to the scientific community.

  7. Ligand- and receptor-based docking with LiBELa.

    Science.gov (United States)

    dos Santos Muniz, Heloisa; Nascimento, Alessandro S

    2015-08-01

    Methodologies on molecular docking are constantly improving. The problem consists on finding an optimal interplay between the computational cost and a satisfactory physical description of ligand-receptor interaction. In pursuit of an advance in current methods we developed a mixed docking approach combining ligand- and receptor-based strategies in a docking engine, where tridimensional descriptors for shape and charge distribution of a reference ligand guide the initial placement of the docking molecule and an interaction energy-based global minimization follows. This hybrid docking was evaluated with soft-core and force field potentials taking into account ligand pose and scoring. Our approach was found to be competitive to a purely receptor-based dock resulting in improved logAUC values when evaluated with DUD and DUD-E. Furthermore, the smoothed potential as evaluated here, was not advantageous when ligand binding poses were compared to experimentally determined conformations. In conclusion we show that a combination of ligand- and receptor-based strategy docking with a force field energy model results in good reproduction of binding poses and enrichment of active molecules against decoys. This strategy is implemented in our tool, LiBELa, available to the scientific community.

  8. Ligand-target interactions: what can we learn from NMR?

    Science.gov (United States)

    Carlomagno, Teresa

    2005-01-01

    The conformation of the ligand in complex with a macromolecular target can be studied by nuclear magnetic resonance (NMR) in solution for both tightly and weakly forming complexes. In the weak binding regime (k(off) > 10(4) Hz), the structure of the bound ligand is accessible also for very large complexes (>100 kDa), which are not amenable to NMR studies in the tight binding regime. Here I review the state-of-the-art NMR methodology used for screening ligands and for the structural investigation of bound ligand conformations, in both tight and weak binding regimes. The advantages and disadvantages of each approach are critically described. The NMR methodology used to investigate transiently forming complexes has expanded considerably in the past few years, opening new possibilities for a detailed description of ligand-target interactions. Novel methods for the determination of the bound ligand conformation, in particular transferred cross-correlated relaxation, are thoroughly reviewed, and their advantages with respect to established methodology are discussed, using the epothilone-tubulin complex as a primary example.

  9. A General Strategy for Ligand Exchange on Upconversion Nanoparticles.

    Science.gov (United States)

    Kong, Wei; Sun, Tianying; Chen, Bing; Chen, Xian; Ai, Fujin; Zhu, Xiaoyue; Li, Mingyu; Zhang, Wenjun; Zhu, Guangyu; Wang, Feng

    2017-01-17

    Lanthanide-doped upconversion nanoparticles with a suitable surface coating are appealing for biomedical applications. Because high-quality upconversion nanoparticles are typically prepared in an organic solvent and passivated by hydrophobic oleate ligands, a convenient and reliable method for the surface modification of upconversion nanoparticles is thus highly desired to satisfy downstream biological investigations. In this work, we describe a facile and versatile strategy for displacing native oleate ligands on upconversion nanoparticles with a diversity of hydrophilic molecules. The ligand-exchange procedure involves the removal of original oleate ligands followed by the attachment of new ligands in a separate step. The successful coating of relevant ligands was confirmed by Fourier transform infrared spectroscopy, thermogravimetry analysis, and ζ-potential measurement. The surface-modified nanoparticles display high stability and good biocompatibility, as revealed by electron microscopy, photoluminescence spectroscopy, and cytotoxicity assessment. Our study demonstrates that functional biomolecules such as biotin can be directly immobilized on the nanoparticle surface using this approach for the quick and effective detection of streptavidin.

  10. Predicting Efficient Antenna Ligands for Tb(III) Emission

    Energy Technology Data Exchange (ETDEWEB)

    Samuel, Amanda P.S.; Xu, Jide; Raymond, Kenneth

    2008-10-06

    A series of highly luminescent Tb(III) complexes of para-substituted 2-hydroxyisophthalamide ligands (5LI-IAM-X) has been prepared (X = H, CH{sub 3}, (C=O)NHCH{sub 3}, SO{sub 3}{sup -}, NO{sub 2}, OCH{sub 3}, F, Cl, Br) to probe the effect of substituting the isophthalamide ring on ligand and Tb(III) emission in order to establish a method for predicting the effects of chromophore modification on Tb(III) luminescence. The energies of the ligand singlet and triplet excited states are found to increase linearly with the {pi}-withdrawing ability of the substituent. The experimental results are supported by time-dependent density functional theory (TD-DFT) calculations performed on model systems, which predict ligand singlet and triplet energies within {approx}5% of the experimental values. The quantum yield ({Phi}) values of the Tb(III) complex increases with the triplet energy of the ligand, which is in part due to the decreased non-radiative deactivation caused by thermal repopulation of the triplet. Together, the experimental and theoretical results serve as a predictive tool that can be used to guide the synthesis of ligands used to sensitize lanthanide luminescence.

  11. Ligands turning around in the midst of protein conformers: the origin of ligand-protein mating. A NMR view.

    Science.gov (United States)

    Pertinhez, T A; Spisni, A

    2011-01-01

    Protein-ligand binding is a puzzling process. Many theories have been devised since the pioneering key-and-lock hypothesis based on the idea that both the protein and the ligand have a rigid single conformation. Indeed, molecular motion is the essence of the universe. Consequently, not only proteins are characterized by an extraordinary conformational freedom, but ligands too can fluctuate in a rather vast conformational space. In this scenario, the quest to understand how do they match is fascinating. Recognizing that the inherent dynamics of molecules is the key factor controlling the success of the binding and, subsequently, their chemical/biological function, here we present a view of this process from the NMR stand point. A description of the most relevant NMR parameters that can provide insights, at atomic level, on the mechanisms of protein-ligand binding is provided in the final section.

  12. The use of fake ligands from computational solvent mapping in ligand and structure-based virtual screening.

    Science.gov (United States)

    Hall, David R; Enyedy, Istvan J

    2016-10-01

    Virtual screening selects compounds that resemble a known modulator or compounds that fit into the binding site of a target protein. Computational solvent mapping defines important chemical features for binding to a target protein. Results/methodology: We have tested the ability to use solvent mapping for generating a 'fake' ligand that is a negative image of the binding site. We used this fake ligand as a query for the program ROCS and to define the search space of the docking programs FRED and HYBRID. The fake ligands perform comparably to or better than the ligands from crystal structures across a set of ten targets. Thus, the approach is suitable for guiding virtual screening and hit-to-lead optimization.

  13. PPARγ ligand production is tightly linked to clonal expansion during initiation of adipocyte differentiation

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Koefoed Petersen, Rasmus; Feddersen, Søren

    2014-01-01

    Adipocyte differentiation is orchestrated by the ligand-activated nuclear receptor PPAR. Endogenous ligands comprise oxidized derivatives of arachidonic acid and structurally similar PUFAs. Although expression of PPAR peaks in mature adipocytes, ligands are produced primarily at the onset...... expansion for PPAR ligand production at the onset of adipocyte differentiation....

  14. Ligand-based virtual screening under partial shape constraints

    Science.gov (United States)

    von Behren, Mathias M.; Rarey, Matthias

    2017-04-01

    Ligand-based virtual screening has proven to be a viable technology during the search for new lead structures in drug discovery. Despite the rapidly increasing number of published methods, meaningful shape matching as well as ligand and target flexibility still remain open challenges. In this work, we analyze the influence of knowledge-based sterical constraints on the performance of the recently published ligand-based virtual screening method mRAISE. We introduce the concept of partial shape matching enabling a more differentiated view on chemical structure. The new method is integrated into the LBVS tool mRAISE providing multiple options for such constraints. The applied constraints can either be derived automatically from a protein-ligand complex structure or by manual selection of ligand atoms. In this way, the descriptor directly encodes the fit of a ligand into the binding site. Furthermore, the conservation of close contacts between the binding site surface and the query ligand can be enforced. We validated our new method on the DUD and DUD-E datasets. Although the statistical performance remains on the same level, detailed analysis reveal that for certain and especially very flexible targets a significant improvement can be achieved. This is further highlighted looking at the quality of calculated molecular alignments using the recently introduced mRAISE dataset. The new partial shape constraints improved the overall quality of molecular alignments especially for difficult targets with highly flexible or different sized molecules. The software tool mRAISE is freely available on Linux operating systems for evaluation purposes and academic use (see http://www.zbh.uni-hamburg.de/raise).

  15. Ligand-based virtual screening under partial shape constraints.

    Science.gov (United States)

    von Behren, Mathias M; Rarey, Matthias

    2017-04-01

    Ligand-based virtual screening has proven to be a viable technology during the search for new lead structures in drug discovery. Despite the rapidly increasing number of published methods, meaningful shape matching as well as ligand and target flexibility still remain open challenges. In this work, we analyze the influence of knowledge-based sterical constraints on the performance of the recently published ligand-based virtual screening method mRAISE. We introduce the concept of partial shape matching enabling a more differentiated view on chemical structure. The new method is integrated into the LBVS tool mRAISE providing multiple options for such constraints. The applied constraints can either be derived automatically from a protein-ligand complex structure or by manual selection of ligand atoms. In this way, the descriptor directly encodes the fit of a ligand into the binding site. Furthermore, the conservation of close contacts between the binding site surface and the query ligand can be enforced. We validated our new method on the DUD and DUD-E datasets. Although the statistical performance remains on the same level, detailed analysis reveal that for certain and especially very flexible targets a significant improvement can be achieved. This is further highlighted looking at the quality of calculated molecular alignments using the recently introduced mRAISE dataset. The new partial shape constraints improved the overall quality of molecular alignments especially for difficult targets with highly flexible or different sized molecules. The software tool mRAISE is freely available on Linux operating systems for evaluation purposes and academic use (see http://www.zbh.uni-hamburg.de/raise ).

  16. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid

    OpenAIRE

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular f...

  17. Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy.

    Science.gov (United States)

    Lamblin, G; Boersma, A; Klein, A; Roussel, P; van Halbeek, H; Vliegenthart, J F

    1984-07-25

    The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.

  18. Copper(II)-mediated transformation of a tridentate non-innocent ligand into a tetradentate salen-type innocent ligand.

    Science.gov (United States)

    Ghorai, Samir; Mukherjee, Chandan

    2014-12-01

    A non-innocent ligand, H4L(CH2NH2), was synthesized by introducing a -CH2NH2 group at the ortho carbon atom to the aniline moiety of 2-anilino-4,6-di-tert-butylphenol. The new ligand was characterized by IR and NMR spectroscopy and mass spectrometry techniques. Upon treatment with CuCl2⋅2H2O, this non-innocent ligand provided a mononuclear four-coordinate salen-type Cu(II) complex by complete modification of the ligand backbone. The complex was characterized by IR spectroscopy, mass spectrometry, X-ray single-crystal diffraction, electron paramagnetic resonance (EPR) spectroscopy, and UV/Vis/near-IR spectroscopy techniques. X-ray crystallographic analysis showed an asymmetric environment around the Cu(II) center with a small (≈12°) twist between the two biting planes. Analysis of the X-band EPR spectrum also supported the asymmetric environment and also indicated the presence of an unpaired electron on the dx2-y2 orbital. The UV/Vis/near-IR spectrum showed strong absorption bands for metal-to-ligand charge transfer and ligand-to-metal charge transfer along with a Cu(II) -centered d-d transition. Mechanistic investigation of the formation of complex 1 indicated that modification of the ligand backbone proceeded through ligand-centered amine to imine oxidation as well as through C-N bond-breaking processes. During these processes, 3,5-di-tert-butyl-1,2-benzoquinone and 2-aminobenzylidene were produced. Ammonia, generated in situ through hydrolysis of the imine to the aldehyde, reacted with 3,5-di-tert-butyl-1,2-benzoquinone to form the corresponding 3,5-di-tert-butyl-1,2-iminobenzoquinone moiety, which upon two-electron reduction in the reaction medium formed 3,5-di-tert-butyl-1,2-aminophenol. This aminophenol underwent condensation with the H2L5 ligand that was formed by self-condensation of two molecules of 2-aminobenzaldehyde and provided the modified ligand backbone. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Preference for bridging versus terminal ligands in magnesium dimers.

    Science.gov (United States)

    Lioe, Hadi; White, Jonathan M; O'Hair, Richard A J

    2011-06-01

    Magnesium dimers play important roles in inorganic and organometallic chemistry. This study evaluates the inherent bridging ability of a range of different ligands in magnesium dimers. In the first part, the Cambridge Structural Database is interrogated to establish the frequency of different types of ligands found in bridging versus terminal positions in two key structural motifs: one in which there are two bridging ligands (the D(2h) "Mg(2)(μ-X(2))" structure); the other in which there are three bridging ligands (the C(3v) "Mg(2)(μ-X(3))" structure). The most striking finding from the database search is the overwhelming preference for magnesium dimers possessing two bridging ligands. The most common bridging ligands are C-, N-, and O-based. In the second part, DFT calculations (at the B3LYP/6-311+G(d) level of theory) are carried out to examine a wider range of structural types for dimers consisting of the stoichiometries Mg(2)Cl(3)R and Mg(2)Cl(2)R(2), where R = CH(3), SiH(3), NH(2), PH(2), OH, SH, CH(2)CH(3), CH=CH(2), C≡CH, Ph, OAc, F and Br. Consistent with the database search, the most stable magnesium dimers are those that contain two bridging ligands. Furthermore, it was demonstrated that the electronic effect of the bridging ligands is important in influencing the stability of the magnesium dimers. The preference for a bridging ligand, which reflects its ability to stabilize a magnesium dimer, follows the order: OH > NH(2) > C≡CH > SH > Ph > Br > PH(2) = CH=CH(2) > CH(2)CH(3) > CH(3) > SiH(3). Finally, the role that the ether solvent Me(2)O has on the stability of isomeric Mg(2)Cl(2)Me(2) dimers was studied. It was found that the first solvent molecule stabilizes the dimers, while the second solvent molecule can either have a stabilizing or destabilizing effect, depending on the isomer structure.

  20. An efficient computational method for calculating ligand binding affinities.

    Directory of Open Access Journals (Sweden)

    Atsushi Suenaga

    Full Text Available Virtual compound screening using molecular docking is widely used in the discovery of new lead compounds for drug design. However, the docking scores are not sufficiently precise to represent the protein-ligand binding affinity. Here, we developed an efficient computational method for calculating protein-ligand binding affinity, which is based on molecular mechanics generalized Born/surface area (MM-GBSA calculations and Jarzynski identity. Jarzynski identity is an exact relation between free energy differences and the work done through non-equilibrium process, and MM-GBSA is a semimacroscopic approach to calculate the potential energy. To calculate the work distribution when a ligand is pulled out of its binding site, multiple protein-ligand conformations are randomly generated as an alternative to performing an explicit single-molecule pulling simulation. We assessed the new method, multiple random conformation/MM-GBSA (MRC-MMGBSA, by evaluating ligand-binding affinities (scores for four target proteins, and comparing these scores with experimental data. The calculated scores were qualitatively in good agreement with the experimental binding affinities, and the optimal docking structure could be determined by ranking the scores of the multiple docking poses obtained by the molecular docking process. Furthermore, the scores showed a strong linear response to experimental binding free energies, so that the free energy difference of the ligand binding (ΔΔG could be calculated by linear scaling of the scores. The error of calculated ΔΔG was within ≈ ± 1.5 kcal.mol(-1 of the experimental values. Particularly, in the case of flexible target proteins, the MRC-MMGBSA scores were more effective in ranking ligands than those generated by the MM-GBSA method using a single protein-ligand conformation. The results suggest that, owing to its lower computational costs and greater accuracy, the MRC-MMGBSA offers efficient means to rank the ligands, in

  1. Insights into Protein–Ligand Interactions: Mechanisms, Models, and Methods

    Science.gov (United States)

    Du, Xing; Li, Yi; Xia, Yuan-Ling; Ai, Shi-Meng; Liang, Jing; Sang, Peng; Ji, Xing-Lai; Liu, Shu-Qun

    2016-01-01

    Molecular recognition, which is the process of biological macromolecules interacting with each other or various small molecules with a high specificity and affinity to form a specific complex, constitutes the basis of all processes in living organisms. Proteins, an important class of biological macromolecules, realize their functions through binding to themselves or other molecules. A detailed understanding of the protein–ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein-ligand recognition and binding will also facilitate the discovery, design, and development of drugs. In the present review, first, the physicochemical mechanisms underlying protein–ligand binding, including the binding kinetics, thermodynamic concepts and relationships, and binding driving forces, are introduced and rationalized. Next, three currently existing protein-ligand binding models—the “lock-and-key”, “induced fit”, and “conformational selection”—are described and their underlying thermodynamic mechanisms are discussed. Finally, the methods available for investigating protein–ligand binding affinity, including experimental and theoretical/computational approaches, are introduced, and their advantages, disadvantages, and challenges are discussed. PMID:26821017

  2. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  3. Cloud Computing for Protein-Ligand Binding Site Comparison

    Directory of Open Access Journals (Sweden)

    Che-Lun Hung

    2013-01-01

    Full Text Available The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  4. Cloud Computing for Protein-Ligand Binding Site Comparison

    Science.gov (United States)

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  5. A motion planning approach to flexible ligand binding.

    Science.gov (United States)

    Singh, A P; Latombe, J C; Brutlag, D L

    1999-01-01

    Most computational models of protein-ligand interactions consider only the energetics of the final bound state of the complex and do not examine the dynamics of the ligand as it enters the binding site. We have developed a novel technique for studying the dynamics of protein-ligand interactions based on motion planning algorithms from the field of robotics. Our algorithm uses electrostatic and van der Waals potentials to compute the most energetically favorable path between any given initial and goal ligand configurations. We use probabilistic motion planning to sample the distribution of possible paths to a given goal configuration and compute an energy-based "difficulty weight" for each path. By statistically averaging this weight over several randomly generated starting configurations, we compute the relative difficulty of entering and leaving a given binding configuration. This approach yields details of the energy contours around the binding site and can be used to characterize and predict good binding sites. Results from tests with three protein-ligand complexes indicate that our algorithm is able to detect energy barriers around the true binding site that distinguish this site from other predicted low-energy binding sites.

  6. Synthesis, structure characterization and biological activity of selected metal complexes of sulfonamide Schiff base as a primary ligand and some mixed ligand complexes with glycine as a secondary ligand

    Science.gov (United States)

    Sharaby, Carmen M.; Amine, Mona F.; Hamed, Asmaa A.

    2017-04-01

    The current work reports synthesis of metal complexes and mixed ligand complexes of a novel sulfonamide Schiff base ligand (HL) resulted from the condensation of sulfametrole [N‧-(4-methoxy-1,2,5-thiadiazol-3-yl]sulfanilamide and acetyl-acetone as a primary ligand and glycine as a secondary ligand. The metal complexes and mixed ligand complexes of HL Schiff base ligand were synthesized and characterized using different physicochemical studies as elemental analyses, mass spectra, conductivity measurement, IR spectra, 1H NMR spectra, UV-vis Spectra, solid reflectance, magnetic susceptibility, thermal analyses (TGA and DTA) and their microbial and anticancer activities. The spectroscopic data of the complexes suggest their 1:2(L1:M) complex structures and 1:2:2(L1:L2:M) mixed ligand complex structures, where L1 = HL and L2 = glycine. Also, the spectroscopic studies suggested the octahedral structure for all complexes. The synthesized Schiff base, its metal and mixed ligand complexes were screened for their bacterial, antifungal and anticancer activity. The activity data show that the metal complexes and mixed ligand complexes exhibited promising microbial and anticancer activities than their parent HL Schiff base ligand, also the data show that the mixed ligand complexes more effective than the metal complexes.

  7. Activation of Neuropeptide FF Receptors by Kisspeptin Receptor Ligands

    Science.gov (United States)

    2010-01-01

    Kisspeptin is a member of the RFamide neuropeptide family that is implicated in gonadotropin secretion. Because kisspeptin-GPR54 signaling is implicated in the neuroendocrine regulation of reproduction, GPR54 ligands represent promising therapeutic agents against endocrine secretion disorders. In the present study, the selectivity profiles of GPR54 agonist peptides were investigated for several GPCRs, including RFamide receptors. Kisspeptin-10 exhibited potent binding and activation of neuropeptide FF receptors (NPFFR1 and NPFFR2). In contrast, short peptide agonists bound with much lower affinity to NPFFRs while showing relatively high selectivity toward GPR54. The possible localization of secondary kisspeptin targets was also demonstrated by variation in the levels of GnRH release from the median eminence and the type of GPR54 agonists used. Negligible affinity of the reported NPFFR ligands to GPR54 was observed and indicates the unidirectional cross-reactivity between both ligands. PMID:24900254

  8. Structure and properties of a new rigid tripodal oxime ligand

    Science.gov (United States)

    Premužić, Dejan; Filarowski, Aleksander; Hołyńska, Małgorzata

    2017-05-01

    Synthesis and properties of a new rigid tripodal oxime ligand are reported. The ligand is a derivative of phloroglucine with three carboxime arms alternate with hydroxyl groups and three intramolecular Osbnd H⋯N hydrogen bonds. Intermolecular Osbnd H⋯O hydrogen bonds result in formation of a characteristic three-dimensional network. The structure of the compound is further analyzed and confirmed by NMR, IR and MS-techniques. DFT calculations were employed to accomplish the analysis of both the hydrogen bonding and the conformational state of the ligand under study. Additionally, the study addresses the way the conformational state and metal-replacement influence the tautomeric equilibrium OH⋯N ⇆ O-⋯+HN in the studied moieties.

  9. Evaluation of macrocyclic hydroxyisophthalamide ligands as chelators for zirconium-89.

    Directory of Open Access Journals (Sweden)

    Nikunj B Bhatt

    Full Text Available The development of bifunctional chelators (BFCs for zirconium-89 immuno-PET applications is an area of active research. Herein we report the synthesis and evaluation of octadentate hydroxyisophthalamide ligands (1 and 2 as zirconium-89 chelators. While both radiometal complexes could be prepared quantitatively and with excellent specific activity, preparation of 89Zr-1 required elevated temperature and an increased reaction time. 89Zr-1 was more stable than 89Zr-2 when challenged in vitro by excess DTPA or serum proteins and in vivo during acute biodistribution studies. Differences in radiometal complex stability arise from structural changes between the two ligand systems, and suggest further ligand optimization is necessary to enhance 89Zr chelation.

  10. Ligand Binding Thermodynamics in Drug Discovery: Still a Hot Tip?

    Science.gov (United States)

    Geschwindner, Stefan; Ulander, Johan; Johansson, Patrik

    2015-08-27

    The use of ligand binding thermodynamics has been proposed as a potential success factor to accelerate drug discovery. However, despite the intuitive appeal of optimizing binding enthalpy, a number of factors complicate routine use of thermodynamic data. On a macroscopic level, a range of experimental parameters including temperature and buffer choice significantly influence the observed thermodynamic signatures. On a microscopic level, solute effects, structural flexibility, and cooperativity lead to nonlinear changes in enthalpy. This multifactorial character hides essential enthalpy contributions of intermolecular contacts, making them experimentally nonobservable. In this perspective, we present three case studies, reflect on some key factors affecting thermodynamic signatures, and investigate their relation to the hydrophobic effect, enthalpy-entropy compensation, lipophilic ligand efficiency, and promiscuity. The studies highlight that enthalpy and entropy cannot be used as direct end points but can together with calculations increase our understanding of ligand binding and identify interesting outliers that do not behave as expected.

  11. Coordination chemistry of N-heterocyclic nitrenium-based ligands.

    Science.gov (United States)

    Tulchinsky, Yuri; Kozuch, Sebastian; Saha, Prasenjit; Mauda, Assaf; Nisnevich, Gennady; Botoshansky, Mark; Shimon, Linda J W; Gandelman, Mark

    2015-05-04

    Comprehensive studies on the coordination properties of tridentate nitrenium-based ligands are presented. N-heterocyclic nitrenium ions demonstrate general and versatile binding abilities to various transition metals, as exemplified by the synthesis and characterization of Rh(I) , Rh(III) , Mo(0) , Ru(0) , Ru(II) , Pd(II) , Pt(II) , Pt(IV) , and Ag(I) complexes based on these unusual ligands. Formation of nitrenium-metal bonds is unambiguously confirmed both in solution by selective (15) N-labeling experiments and in the solid state by X-ray crystallography. The generality of N-heterocyclic nitrenium as a ligand is also validated by a systematic DFT study of its affinity towards all second-row transition and post-transition metals (Y-Cd) in terms of the corresponding bond-dissociation energies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. The Parathyroid Hormone Family of Ligands and Receptors

    Directory of Open Access Journals (Sweden)

    Damian G. D'Souza

    2015-07-01

    Full Text Available The PTH family of ligands and receptors have a wide range of vital functions from calcium homeostasis to tissue and bone development from the embryo to adult. This family has undergone whole genome duplication events predating vertebrate evolution, indicating more primitive and ancient functions other than skeletal development. The N-terminal region of the ligands, have been widely studied by biophysical and functional analysis, resulting in the discovery of key characteristics essential for ligand-receptor activation being elucidated. Multi-substituted amino acid analogs with differential binding affinities and either antagonistic or agonistic signalling potencies have been created based on these findings allowing for improvement on potential therapies affected by the PTH system in skeletal and embryonic development. The PTH family has diversely evolved to cover a wide range of pivotal pathways crucial to growth and development throughout all animal life.

  13. Ligands of histamine receptors modulate acid-sensing ion channels.

    Science.gov (United States)

    Shteinikov, V Y; Korosteleva, A S; Tikhonova, T B; Potapieva, N N; Tikhonov, D B

    2017-09-02

    Recently we found that synthetic compounds containing amino group linked to hydrophobic or aromatic moiety are potent modulators of the proton-gated channels (ASICs). These structures have clear similarity with ligands of histamine receptors. We have also demonstrated that histamine potentiates homomeric ASIC1a by shifting its activation dependence to less acidic conditions. In the present work the action of a series of histamine receptors ligands on recombinant ASIC1a and ASIC2a was characterized. Two types of action were found for ASIC1a. 1-methylhistamine, N-alpha-methylhistamine, dimaprit and thioperamide caused significant potentiation, which was pH-dependent and voltage-independent. The H4R antagonist A943931 caused inhibition, which is likely due to voltage-dependent pore block. ASIC2a were virtually insensitive to the drugs tested. We conclude that ligands of histamine receptors should also be considered as ASIC modulators. Copyright © 2017. Published by Elsevier Inc.

  14. Thiolate ligands for synthesis of water-soluble gold clusters.

    Science.gov (United States)

    Ackerson, Christopher J; Jadzinsky, Pablo D; Kornberg, Roger D

    2005-05-11

    Water-soluble monolayer-protected gold clusters (MPCs) have been an object of investigation by many research groups since their first syntheses were reported in 1998 and 1999. The basic requirements for a ligand to form a monolayer protecting a gold cluster were established some time ago for alkanethiolate MPCs, but there has been no such information published for water-soluble MPCs. We identify 6 new ligands capable of forming water-soluble MPCs, as well as 22 water-soluble ligands that fail to form MPCs. Our findings contribute not only to the definition of the requirements for MPC formation but also to the variety of MPCs available for applications in chemistry and biology.

  15. Xanthene and Xanthone Derivatives as G-Quadruplex Stabilizing Ligands

    Directory of Open Access Journals (Sweden)

    Alessandro Altieri

    2013-10-01

    Full Text Available Following previous studies on anthraquinone and acridine-based G-quadruplex ligands, here we present a study of similar aromatic cores, with the specific aim of increasing G-quadruplex binding and selectivity with respect to duplex DNA. Synthesized compounds include two and three-side chain xanthone and xanthene derivatives, as well as a dimeric “bridged” form. ESI and FRET measurements suggest that all the studied molecules are good G-quadruplex ligands, both at telomeres and on G-quadruplex forming sequences of oncogene promoters. The dimeric compound and the three-side chain xanthone derivative have been shown to represent the best compounds emerging from the different series of ligands presented here, having also high selectivity for G-quadruplex structures with respect to duplex DNA. Molecular modeling simulations are in broad agreement with the experimental data.

  16. Ligand screening by saturation-transfer difference (STD) NMR spectroscopy.

    Energy Technology Data Exchange (ETDEWEB)

    Krishnan, V V

    2005-04-26

    NMR based methods to screen for high-affinity ligands have become an indispensable tool for designing rationalized drugs, as these offer a combination of good experimental design of the screening process and data interpretation methods, which together provide unprecedented information on the complex nature of protein-ligand interactions. These methods rely on measuring direct changes in the spectral parameters, that are often simpler than the complex experimental procedures used to study structure and dynamics of proteins. The goal of this review article is to provide the basic details of NMR based ligand-screening methods, with particular focus on the saturation transfer difference (STD) experiment. In addition, we provide an overview of other NMR experimental methods and a practical guide on how to go about designing and implementing them.

  17. The thermodynamic principles of ligand binding in chromatography and biology

    DEFF Research Database (Denmark)

    Mollerup, Jørgen

    2007-01-01

    In chromatography, macromolecules do not adsorb in the traditional sense of the word but bind to ligands that are covalently bonded to the surface of the porous bead. Therefore, the adsorption must be modelled as a process where protein molecules bind to the immobilised ligands. The paper discusses...... but it is also observed in chromatography due to protein-protein interactions. Retention measurements on P-lactoglobulin A demonstrate this. A discussion of salt effects on hydrophobic interactions in precipitation and chromatography of proteins concludes the paper. (c) 2007 Elsevier B.V. All rights reserved....... the general thermodynamic principles of ligand binding. Models of the multi-component adsorption in ion-exchange and hydrophobic chromatography, HIC and RPLC, are developed. The parameters in the models have a well-defined physical significance. The models are compared to the Langmuir model...

  18. Patients with acute coronary syndromes express enhanced CD40 ligand/CD154 on platelets.

    Science.gov (United States)

    Garlichs, C D; Eskafi, S; Raaz, D; Schmidt, A; Ludwig, J; Herrmann, M; Klinghammer, L; Daniel, W G; Schmeisser, A

    2001-12-01

    To investigate whether CD40L/CD154 on platelets and soluble CD40L/CD154 may play a role in the inflammatory process of acute coronary syndromes. Observational study in a university hospital. 15 patients with acute myocardial infarction, 25 patients with unstable angina, 15 patients with stable angina, and 12 controls. CD40L/CD154 on platelets, P-selectin/CD62P on platelets, soluble CD40L/CD154 serum concentrations. Mean (SD) CD40L/CD154 expression on platelets was 6.2 (2.8) MFI (mean fluorescence intensity) in the infarct group, 11 (3.3) MFI in the unstable angina group (p angina group (p angina), and 3.2 (1.0) MFI in the controls (p angina; NS v stable angina). Soluble CD40L/CD154 concentration was 5.2 (1.1) ng/ml in the infarct group, 4.2 (0.7) ng/ml in the unstable angina group (p angina group (p angina), and 3.0 (0.5) ng/ml in the controls (p angina; NS v stable angina). At a six months follow up, there was lower expression of CD40L/CD154 on platelets in patients with unstable angina (12.3 (3.6) v 3.8 (1.2) MFI, p angina who needed redo coronary angioplasty (PTCA) or who had recurrence of angina were characterised by increased CD40L/CD154 expression on platelets compared with the remainder of the study group (recurrence of angina: 12.7 (3.2) v 9.7 (1.6) MFI, p angina and myocardial infarction. These findings suggest that CD40-CD40L/CD154 interactions may play a pathogenic role in triggering and propagation of acute coronary syndromes.

  19. Control of Energy Flow Dynamics between Tetracene Ligands and PbS Quantum Dots by Size Tuning and Ligand Coverage

    Energy Technology Data Exchange (ETDEWEB)

    Kroupa, Daniel M. [National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States; Arias, Dylan H. [National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States; Blackburn, Jeffrey L. [National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States; Carroll, Gerard M. [National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States; Granger, Devin B. [Department; Anthony, John E. [Department; Beard, Matthew C. [National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States; Johnson, Justin C. [National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States

    2018-01-08

    We have prepared a series of samples with the ligand 6,13-bistri(iso-propyl)silylethynyl tetracene 2-carboxylic acid (TIPS-Tc-COOH) attached to PbS quantum dot (QD) samples of three different sizes in order to monitor and control the extent and time scales of energy flow after photoexcitation. Fast energy transfer (~1 ps) to the PbS QD occurs upon direct excitation of the ligand for all samples. The largest size QD maintains the microsecond exciton lifetime characteristic of the as-prepared oleate terminated PbS QDs. However, two smaller QD sizes with lowest exciton energies similar to or larger than the TIPS-Tc-COO- triplet energy undergo energy transfer between QD core and ligand triplet on nanosecond to microsecond timescales. For the intermediate size QDs in particular, energy can be recycled many times between ligand and core, but the triplet remains the dominant excited species at long times, living for ~3 us for fully exchanged QDs and up to 30 us for partial ligand exchange, which is revealed as a method for controlling the triplet lifetime. A unique upconverted luminescence spectrum is observed that results from annihilation of triplets after exclusive excitation of the QD core.

  20. Complexation of trivalent americium and lanthanides with terdentate 'N' donor ligands: the role of rigidity in the ligand structure.

    Science.gov (United States)

    Bhattacharyya, Arunasis; Gadly, Trilochan; Pathak, Priyanath; Ghosh, Sunil K; Mohapatra, Manoj; Ghanty, Tapan K; Mohapatra, Prasanta K

    2014-08-28

    A systematic study on the Ln(3+) complexation behaviour with two terdentate 'N' donor ligands of varying structural rigidity, viz. 5,6-dimethyl-(1,2,4)-triazinylbipyridine (Me2TBipy) and 5,6-dimethyl-(1,2,4)-triazinylphenanthroline (Me2TPhen), is performed in the present work by UV-Vis spectrophotometry, time resolved fluorescence spectroscopy (TRFS) and electrospray ionization mass spectrometric (ESI-MS) studies. These studies indicate the formation of a 1 : 1 complex of La(3+), 1 : 2 complexes of Eu(3+) and Er(3+) with both the ligands. Density functional theoretical (DFT) study is carried out to determine the solution phase structure of the Eu(3+) complex considering the species (from UV-Vis spectrophotometry) and C2v site symmetry around the Eu(3+) ion (from TRFS study). Me2TPhen is found to be a stronger complexing ligand as compared to Me2TBipy irrespective of the Ln(3+) ions. The solid state crystal structure of the La(3+) complex of Me2TPhen is determined using the single crystal X-ray diffraction (SCXRD) technique. The complexation of the trivalent Am(3+) ion is also studied with both these ligands using UV-Vis spectrophotometric titrations which show the formation of 1 : 2 complexes with higher complexation constant values as compared to all the Ln(3+) ions studied, indicating the selectivity of these ligands for the trivalent actinides over the lanthanides.

  1. Methods for Identifying Ligands that Target Nucleic Acid Molecules and Nucleic Acid Structural Motifs

    Science.gov (United States)

    Disney, Matthew D. (Inventor); Childs-Disney, Jessica L. (Inventor)

    2017-01-01

    Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particular ligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; and identifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

  2. Systematic study of ligand structures of metal oxide EUV nanoparticle photoresists

    KAUST Repository

    Jiang, Jing

    2015-03-19

    Ligand stabilized metal oxide nanoparticle resists are promising candidates for EUV lithography due to their high sensitivity for high-resolution patterning and high etching resistance. As ligand exchange is responsible for the patterning mechanism, we systematically studied the influence of ligand structures of metal oxide EUV nanoparticles on their sensitivity and dissolution behavior. ZrO2 nanoparticles were protected with various aromatic ligands with electron withdrawing and electron donating groups. These nanoparticles have lower sensitivity compared to those with aliphatic ligands suggesting the structures of these ligands is more important than their pka on resist sensitivity. The influence of ligand structure was further studied by comparing the nanoparticles’ solubility for a single type ligand to mixtures of ligands. The mixture of nanoparticles showed improved pattern quality. © (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

  3. Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials.

    Science.gov (United States)

    Benitez, Patrick L; Mascharak, Shamik; Proctor, Amy C; Heilshorn, Sarah C

    2016-01-01

    While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer

  4. Contrasting roles for TLR ligands in HIV-1 pathogenesis.

    Directory of Open Access Journals (Sweden)

    Beda Brichacek

    2010-09-01

    Full Text Available The first line of a host's response to various pathogens is triggered by their engagement of cellular pattern recognition receptors (PRRs. Binding of microbial ligands to these receptors leads to the induction of a variety of cellular factors that alter intracellular and extracellular environment and interfere directly or indirectly with the life cycle of the triggering pathogen. Such changes may also affect any coinfecting microbe. Using ligands to Toll-like receptors (TLRs 5 and 9, we examined their effect on human immunodeficiency virus (HIV-1 replication in lymphoid tissue ex vivo. We found marked differences in the outcomes of such treatment. While flagellin (TLR5 agonist treatment enhanced replication of CC chemokine receptor 5 (CCR 5-tropic and CXC chemokine receptor 4 (CXCR4-tropic HIV-1, treatment with oligodeoxynucleotide (ODN M362 (TLR9 agonist suppressed both viral variants. The differential effects of these TLR ligands on HIV-1 replication correlated with changes in production of CC chemokines CCL3, CCL4, CCL5, and of CXC chemokines CXCL10, and CXCL12 in the ligand-treated HIV-1-infected tissues. The nature and/or magnitude of these changes were dependent on the ligand as well as on the HIV-1 viral strain. Moreover, the tested ligands differed in their ability to induce cellular activation as evaluated by the expression of the cluster of differentiation markers (CD 25, CD38, CD39, CD69, CD154, and human leukocyte antigen D related (HLA-DR as well as of a cell proliferation marker, Ki67, and of CCR5. No significant effect of the ligand treatment was observed on apoptosis and cell death/loss in the treated lymphoid tissue ex vivo. Our results suggest that binding of microbial ligands to TLRs is one of the mechanisms that mediate interactions between coinfected microbes and HIV-1 in human tissues. Thus, the engagement of appropriate TLRs by microbial molecules or their mimetic might become a new strategy for HIV therapy or prevention.

  5. Designer ligands: The search for metal ion selectivity

    Directory of Open Access Journals (Sweden)

    Perry T. Kaye

    2011-03-01

    Full Text Available The paper reviews research conducted at Rhodes University towards the development of metal-selective ligands. The research has focused on the rational design, synthesis and evaluation of novel ligands for use in the formation of copper complexes as biomimetic models of the metalloenzyme, tyrosinase, and for the selective extraction of silver, nickel and platinum group metal ions in the presence of contaminating metal ions. Attention has also been given to the development of efficient, metal-selective molecular imprinted polymers.

  6. Coordination chemistry of poly(thioether)borate ligands.

    Science.gov (United States)

    Riordan, Charles G

    2010-08-01

    This review traces the development and application of the tris(thioether)borate ligands, tripodal ligands with highly polarizable thioether donors. Areas of emphasis include the basic coordination chemistry of the mid-to-late first row transition metals (Fe, Ni, Co, Cu), and the role of the thioether substituent in directing complex formation, the modeling of zinc thiolate protein active sites, high-spin organo-iron and organo-cobalt chemistry, the preparation of monovalent complexes of Fe, Co and Ni, and dioxygen and sulfur activation by monovalent nickel complexes.

  7. Synthesis of Oxovanadium(IV Complexes with Tetraaza Coordinating Ligands

    Directory of Open Access Journals (Sweden)

    Sanjay Singh

    2013-01-01

    Full Text Available Oxovanadium(IV complexes of the type [VO(mac]SO4 (where mac = tetraaza macrocyclic ligands derived by condensation of thenil with 1,4-diaminobenzene or 3,4-diaminopyridine and their reaction with β-diketones have been prepared using vanadyl ion as kinetic template. The prepared macrocyclic complexes were characterized by elemental analyses, molar conductance, magnetic moments, and infrared, electronic, and electron spin resonance data. From the spectroscopic studies, five-coordinate square-pyramidal geometry for the VO2+ complexes have been proposed wherein derived ligands act as tetradentate chelating agents.

  8. Force loading explains spatial sensing of ligands by cells

    Science.gov (United States)

    Oria, Roger; Wiegand, Tina; Escribano, Jorge; Elosegui-Artola, Alberto; Uriarte, Juan Jose; Moreno-Pulido, Cristian; Platzman, Ilia; Delcanale, Pietro; Albertazzi, Lorenzo; Navajas, Daniel; Trepat, Xavier; García-Aznar, José Manuel; Cavalcanti-Adam, Elisabetta Ada; Roca-Cusachs, Pere

    2017-12-01

    Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin–ligand bonds are separated by more than a few tens of nanometres. It has thus been suggested that a crosslinking ‘adaptor’ protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model, in which individual integrin–ECM bonds—the molecular clutches—respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow

  9. Coordination chemistry of poly(thioether)borate ligands

    Science.gov (United States)

    Riordan, Charles G.

    2010-01-01

    This review traces the development and application of the tris(thioether)borate ligands, tripodal ligands with highly polarizable thioether donors. Areas of emphasis include the basic coordination chemistry of the mid-to-late first row transition metals (Fe, Ni, Co, Cu), and the role of the thioether substituent in directing complex formation, the modeling of zinc thiolate protein active sites, high-spin organo-iron and organo-cobalt chemistry, the preparation of monovalent complexes of Fe, Co and Ni, and dioxygen and sulfur activation by monovalent nickel complexes. PMID:20607091

  10. Carbohydrate Analogue Microarrays for Identification of Lectin-Selective Ligands.

    Science.gov (United States)

    Hyun, Ji Young; Park, Cheol Wan; Liu, Yanna; Kwon, Daeun; Park, Seong-Hyun; Park, Sungjin; Pai, Jaeyoung; Shin, Injae

    2017-06-19

    Fifty-five mono- and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin-selective ligands. The microarray study showed that two disaccharide analogues, 28' and 44', selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28' and 44' selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Development of catalysts and ligands for enantioselective gold catalysis.

    Science.gov (United States)

    Wang, Yi-Ming; Lackner, Aaron D; Toste, F Dean

    2014-03-18

    During the past decade, the use of Au(I) complexes for the catalytic activation of C-C π-bonds has been investigated intensely. Over this time period, the development of homogeneous gold catalysis has been extraordinarily rapid and has yielded a host of mild and selective methods for the formation of carbon-carbon and carbon-heteroatom bonds. The facile formation of new bonds facilitated by gold naturally led to efforts toward rendering these transformations enantioselective. In this Account, we survey the development of catalysts and ligands for enantioselective gold catalysis by our research group as well as related work by others. We also discuss some of our strategies to address the challenges of enantioselective gold(I) catalysis. Early on, our work with enantioselective gold-catalyzed transformations focused on bis(phosphinegold) complexes derived from axially chiral scaffolds. Although these complexes were highly successful in some reactions like cyclopropanation, the careful choice of the weakly coordinating ligand (or counterion) was necessary to obtain high levels of enantioselectivity for the case of allene hydroamination. These counterion effects led us to use the anion itself as a source of chirality, which was successful in the case of allene hydroalkoxylation. In general, these tactics enhance the steric influence around the reactive gold center beyond the two-coordinate ligand environment. The use of binuclear complexes allowed us to use the second gold center and its associated ligand (or counterion) to exert a further steric influence. In a similar vein, we employed a chiral anion (in place of or in addition to a chiral ligand) to move the chiral information closer to the reactive center. In order to expand the scope of reactions amenable to enantioselective gold catalysis to cycloadditions and other carbocyclization processes, we also developed a new class of mononuclear phosphite and phosphoramidite ligands to supplement the previously widely

  12. Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes.

    Science.gov (United States)

    Da Veiga, Cyrielle; Mezher, Joelle; Dumas, Philippe; Ennifar, Eric

    2016-01-01

    The success rate of nucleic acids/ligands co-crystallization can be significantly improved by performing preliminary biophysical analyses. Among suitable biophysical approaches, isothermal titration calorimetry (ITC) is certainly a method of choice. ITC can be used in a wide range of experimental conditions to monitor in real time the formation of the RNA- or DNA-ligand complex, with the advantage of providing in addition the complete binding profile of the interaction. Following the ITC experiment, the complex is ready to be concentrated for crystallization trials. This chapter describes a detailed experimental protocol for using ITC as a tool for monitoring RNA/small molecule binding, followed by co-crystallization.

  13. Synthesis of multifunctional polyvinylsaccharide containing controllable amounts of biospecific ligands.

    Science.gov (United States)

    Korzhikov, V; Roeker, S; Vlakh, E; Kasper, C; Tennikova, T

    2008-03-01

    In the present study, the attempt to synthesize a multibiofunctional polymeric vector to be used for construction of composite scaffolds for bone tissue engineering has been undertaken. The polymers based on 2-deoxy-2-methacrylamido- d-glucose were functionalized by a growth factor (BMP-2), GRGDSP peptide, and poly( l-lysine) using aldehyde chemistry. The covalent modification process was quantitatively studied, and a polymer conjugate containing all these ligands was formed. In addition, the impacts of coupled ligands toward the adsorption of polymers on the commercial mineral macroporous matrix Sponceram used in cell culture applications were studied.

  14. Luminescent cyclometallated iridium(III) complexes having acetylide ligands

    Science.gov (United States)

    Thompson, Mark E.; Bossi, Alberto; Djurovich, Peter Ivan

    2014-09-02

    The present invention relates to phosphorescent (triplet-emitting) organometallic materials. The phosphorescent materials of the present invention comprise Ir(III)cyclometallated alkynyl complexes for use as triplet light-emitting materials. The Ir(III)cyclometallated alkynyl complexes comprise at least one cyclometallating ligand and at least one alkynyl ligand bonded to the iridium. Also provided is an organic light emitting device comprising an anode, a cathode and an emissive layer between the anode and the cathode, wherein the emissive layer comprises a Ir(III)cyclometallated alkynyl complex as a triplet emitting material.

  15. Tear lipocalins bind a broad array of lipid ligands.

    Science.gov (United States)

    Glasgow, B J; Abduragimov, A R; Farahbakhsh, Z T; Faull, K F; Hubbell, W L

    1995-05-01

    To identify the native ligands of tear lipocalins, tear proteins were separated by size exclusion chromatography and the lipid content in the major protein fractions identified. Lipids extracted from native tears and purified tear lipocalins comigrated with fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol on thin layer chromatograms. Abundant stearic and palmitic acids as well as cholesterol, and lesser amounts of lauric acid were specifically identified in extracts of purified lipocalins by gas chromatography-mass spectroscopy. A preliminary study of the ligand-protein interaction was carried out using nitroxide spin-labeled lipids.

  16. Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Graham M West

    Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

  17. Ionic liquids with amino acids as cations: novel chiral ligands in chiral ligand-exchange capillary electrophoresis.

    Science.gov (United States)

    Mu, Xiaoyu; Qi, Li; Zhang, Haizhi; Shen, Ying; Qiao, Juan; Ma, Huimin

    2012-08-15

    Ionic liquids (ILs) with L-proline (L-Pro) as cations have been developed for the novel chiral ligands coordinated with Cu(II) in chiral ligand exchange capillary electrophoresis (CLE-CE). Four kinds of amino acid ionic liquids (AAILs), including [L-Pro][CF(3)COO], [L-Pro][NO(3)], [L-Pro][BF(4)] and [L-Pro(2)][SO(4)], were successfully synthesized. Among them, [L-Pro][CF(3)COO] was selected as the model ligand to optimize the separation conditions. The influences of AAIL concentration, pH, and methanol concentration on efficiency of chiral separation were investigated. Then it has been testified that the optimal buffer solution consisted of 25.0mM Cu(Ac)(2), 50.0 mM AAIL and 20% (v/v) methanol at pH 4.0. The interesting thing is well enantioresolution could be observed with [L-Pro][CF(3)COO] as the new chiral ligand and nine pairs of labeled D,L-AAs were successfully separated with the resolution ranging from 0.93 to 6.72. Meanwhile, the baseline separation of labeled D,L-AAs could be achieved with the other three kinds of AAILs as ligands. The results have demonstrated the good applicability of AAILs with AAs as cations for chiral separation in CLE-CE system. In addition, comparative study was also conducted for exploring the mechanism of the AAILs as new ligands in CLE-CE. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Synthesis, structure and reactivity of rare-earth metal complexes containing anionic phosphorus ligands.

    Science.gov (United States)

    Li, Tianshu; Kaercher, Sabrina; Roesky, Peter W

    2014-01-07

    A comprehensive review of structurally characterized rare-earth metal complexes containing anionic phosphorus ligands is presented. Since rare-earth elements form hard ions and phosphorus is considered as a soft ligand, the rare-earth metal phosphorus coordination is regarded as a less favorite combination. Three classes of phosphorus ligands, (1) the monoanionic organophosphide ligands (PR2(-)) bearing one negative charge on the phosphorus atom; (2) the dianionic phosphinidene (PR(2-)) and P(3-) ligands; and (3) the pure inorganic polyphosphide ligands (Pn(x-)), are included here. Particular attention has been paid to the synthesis, structure, and reactivity of the rare-earth metal phosphides.

  19. Tunable Phosphoramidite Ligands for Asymmetric Hydrovinylation: Ligands par excellence for Generation of All-Carbon Quaternary Centers

    Science.gov (United States)

    Smith, Craig R.; Lim, Hwan Jung; Zhang, Aibin; RajanBabu, T. V.

    2009-01-01

    1-Alkylstyrenes undergo efficient hydrovinylation (addition of ethylene) in the presence of a Ni-catalyst prepared from [(allyl)NiBr]2, Na+ [BAr4]− (Ar = 3,5-bis-trifluromethylphenyl), and a phosphoramidite ligand giving products in excellent yields and enantioselectivities. In many cases phosphoramidites derived from achiral 2,2′-biphenol are almost as good as ligands derived from the more expensive enantiopure 2,2′-binaphthols. The hydrovinylation products, which carry two versatile latent functionalities, an aryl and a vinyl group, are potentially useful for the synthesis of several important natural products containing benzylic all-carbon quaternary centers. PMID:19763244

  20. Fas Ligand Expression in Lynch Syndrome-Associated Colorectal Tumours

    NARCIS (Netherlands)

    Koornstra, Jan J.; de Jong, Steven; Boersma-van Eck, Wietske; Zwart, Nynke; Hollema, Harry; de Vries, Elisabeth G. E.; Kleibeuker, Jan H.

    Fas Ligand (FasL) expression by cancer cells may contribute to tumour immune escape via the Fas counterattack against tumour-infiltrating lymphocytes (TILs). Whether this plays a role in colorectal carcinogenesis in Lynch syndrome was examined studying FasL expression, tumour cell apoptosis and

  1. Synthesis of meta-substituted monodentate phosphinite ligands and ...

    Indian Academy of Sciences (India)

    Synthesis of meta-substituted phosphinite ligands 3,3' (methoxyphosphanediyl) bis (N,Ndiethylaniline) (4a) and methoxybis(3-methoxyphenyl)phosphane (4b), in high yields, has been demonstrated. Typical phosphorus chemical shift between 110–120 ppm, appearance of methoxy protons and corresponding carbon, ...

  2. Biosensors engineered from conditionally stable ligand-binding domains

    Science.gov (United States)

    Church, George M.; Feng, Justin; Mandell, Daniel J.; Baker, David; Fields, Stanley; Jester, Benjamin Ward; Tinberg, Christine Elaine

    2017-09-19

    Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

  3. TWO LIGAND OXIDIO-VANADIUM(IV) COMPLEXES AS NOVEL ...

    African Journals Online (AJOL)

    TWO LIGAND OXIDIO-VANADIUM(IV) COMPLEXES AS NOVEL EFFICIENT. CATALYSTS IN MULTICOMPONENT REACTIONS FOR SYNTHESIS OF. TETRAHYDROBENZOPYRAN DERIVATIVES. Mehdi Abaszadeh1,2*, Mohammad Seifi3 and S. Yousef Ebrahimipour 4,5. 1Pharmaceutics Research Center, Institute of ...

  4. Biscalix[4]arene ligands for dinuclear lanthanide ion complexation

    NARCIS (Netherlands)

    Oude Wolbers, M.P.; van Veggel, F.C.J.M.; Heeringa, Remco H.M.; Hofstraat, Johannes W.; Geurts, Frank A.J.; van Hummel, G.J.; Harkema, Sybolt; Reinhoudt, David

    1997-01-01

    Three types of lower-lower rim linked biscalix[4]arenes that contain carboxylic ester (1) and/or amide functions (2 and 3) at their remaining phenolic oxygen atoms were synthesized. The homo- and heterodinuclear lanthanide ion complexes based on these ligands were used to study the energy transfer

  5. Synthesis and spectra characterization of mixed ligand complexes ...

    African Journals Online (AJOL)

    The synthesized ligand and its metal complexes have been characterized by elemental analysis and spectroscopic (i.r. and electronic) methods. An octahedral geometric structure is proposed for the metal complexes. The six coordinate environment of the metals is composed of N5X core with thr ee nitrogen atoms from the ...

  6. Design and synthesis of multidentate ligands via metal promoted C ...

    Indian Academy of Sciences (India)

    Unknown

    The coordination chemistry of the HL1 ligand involving some 3d-metal ions is described. Two unusual low-spin complexes of manganese(II) and iron(III) are reported. The ferric complex displayed a rhombic EPR while, the corresponding manganese compound showed a complex pattern due to hyperfine coupling. All the.

  7. REACTIVITY PROFILE OF CONFORMATIONALLY-FLEXIBLE RETINOID RECEPTOR LIGANDS

    Science.gov (United States)

    Retinoids and associated derivatives represent a class of endogenousr hormones that bind to and activate different families of retinoic acid receptors (RARs, RXRs), and control many aspects of normal vertebrate development. Identification of potential RAR and RXRs ligands is of i...

  8. Ligands that store and release electrons during catalysis

    NARCIS (Netherlands)

    Dzik, W.I.; van der Vlugt, J.I.; Reek, J.N.H.; de Bruin, B.

    2011-01-01

    First-row transition metals can be given a noble character by redox-active ligands, thus enabling two-electron oxidative addition and reductive elimination steps (see scheme). A recently reported cobalt-mediated Negishi-type cross-coupling reaction provides an illustrative example of this concept

  9. GluR2 ligand-binding core complexes

    DEFF Research Database (Denmark)

    Kasper, C; Lunn, M-L; Liljefors, T

    2002-01-01

    X-ray structures of the GluR2 ligand-binding core in complex with (S)-Des-Me-AMPA and in the presence and absence of zinc ions have been determined. (S)-Des-Me-AMPA, which is devoid of a substituent in the 5-position of the isoxazolol ring, only has limited interactions with the partly hydrophobic...

  10. Phenalenyl-based ligand for transition metal chemistry: Application ...

    Indian Academy of Sciences (India)

    mandal@iiserkol.ac.in. Abstract. We report the synthesis and characterization of the first transition metal complex of a phenalenyl- based ligand. The reaction of Cu(OAc)2.H2O with 9-N-methylamino-1-N -methylimino-phenalene (LH) in 1:1.

  11. Synthesis and characterization of mixed ligand complexes of bio ...

    Indian Academy of Sciences (India)

    In these complexes, the nucleoside (uridine) acts as a monodentate ligand coordinating through O(4) under the conditions of investigation, whereas the amino acids coordinate through the carboxylate oxygen and the amino nitrogen. Distorted octahedral geometry for Cu(II) and octahedral geometries for both Ni(II) and ...

  12. Water-soluble diphosphadiazacyclooctanes as ligands for aqueous organometallic catalysis

    KAUST Repository

    Boulanger, Jérôme

    2012-12-01

    Two new water-soluble diphosphacyclooctanes been synthesized and characterized by NMR and surface tension measurements. Both phosphanes proved to coordinate rhodium in a very selective way as well-defined bidentates were obtained. When used in Rh-catalyzed hydroformylation of terminal alkenes, both ligands positively impacted the reaction chemoselectivity. © 2012 Elsevier B.V.

  13. Heteromultivalent ligand-decoration for actively targeted nanomedicine.

    Science.gov (United States)

    Modery-Pawlowski, Christa L; Gupta, Anirban Sen

    2014-03-01

    Active targeting has become an important component of nanomedicine design where nanovehicles are surface-decorated with cell receptor-specific or disease matrix-specific ligands to enable site-selective binding, retention and delivery of theranostic cargo. In this context, there have been numerous reports regarding surface-modification of nanovehicles with antibodies, antibody fragments, carbohydrates, aptamers and peptides as targeting ligands. However, majority of these reports have focused on using a single type of targeting moiety on the vehicle surface. In any disease development and progression, multiple receptors and proteins are often spatio-temporally upregulated simultaneously and heterogeneously. Rationalizing from this, a significant advantage can be envisioned in targeting multiple entities simultaneously using vehicle co-decoration with multiple types of ligands, to enhance binding activity and targeting specificity. To this end, we present a comprehensive up-to-date review on research endeavors in heteromultivalent ligand-modification of nanovehicles and provide a mechanistic rationale as well as an insightful discussion of this promising area, including findings from our own research. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Topological estimation of proton–ligand formation constants of ...

    Indian Academy of Sciences (India)

    Proton-ligand formation constants of salicylhydroxamic acids (SHA) and their nuclear substituted derivatives have been estimated topologically using the normalized Wiener index, referred to as mean square Wiener index (Wms). Regression analysis of the data indicates that Wms can be used successfully for estimating ...

  15. Assessing ligand efficiencies using template-based molecular ...

    Indian Academy of Sciences (India)

    Statistical modelling using artificial neural network (ANN: 2 = 0.922) and multiple linear regression method (MLR: 2 = 0.851) showed good correlation between the biological activity, binding affinity, and different ligand efficiencies of the compounds, which suggest the robustness of the template-based binding ...

  16. Dependence of the cone angles of phosphorus-containing ligands ...

    Indian Academy of Sciences (India)

    Administrator

    Dependence of the cone angles of phosphorus-containing ligands in the chemistry of hydride and dihydrogen complexes of ruthenium. NISHA MATHEW and BALAJI R JAGIRDAR. Department of Inorganic and Physical Chemistry, Indian Institute of Science,. Bangalore 560 012, India. A series of new hydride complexes of ...

  17. NMR-based screening of membrane protein ligands

    NARCIS (Netherlands)

    Yanamala, Naveena; Dutta, Arpana; Beck, Barbara; Van Fleet, Bart; Hay, Kelly; Yazbak, Ahmad; Ishima, Rieko; Doemling, Alexander; Klein-Seetharaman, Judith

    2010-01-01

    Membrane proteins pose problems for the application of NMR-based ligand-screening methods because of the need to maintain the proteins in a membrane mimetic environment such as detergent micelles: they add to the molecular weight of the protein, increase the viscosity of the solution, interact with

  18. Evidence for clustered mannose as a new ligand for hyaluronan ...

    Indian Academy of Sciences (India)

    Here, we show the affinity of rHABP1 towards D-mannosylated albumin (DMA) by overlay assay and purification using a DMA affinity column. Our data suggests that DMA is another ligand for HABP1. Furthermore, we have observed that DMA inhibits the binding of HA in a concentration-dependent manner, suggesting its ...

  19. Modeling of metal interaction geometries for protein-ligand docking.

    Science.gov (United States)

    Seebeck, Birte; Reulecke, Ingo; Kämper, Andreas; Rarey, Matthias

    2008-05-15

    The accurate modeling of metal coordination geometries plays an important role for structure-based drug design applied to metalloenzymes. For the development of a new metal interaction model, we perform a statistical analysis of metal interaction geometries that are relevant to protein-ligand complexes. A total of 43,061 metal sites of the Protein Data Bank (PDB), containing amongst others magnesium, calcium, zinc, iron, manganese, copper, cadmium, cobalt, and nickel, were evaluated according to their metal coordination geometry. Based on statistical analysis, we derived a model for the automatic calculation and definition of metal interaction geometries for the purpose of molecular docking analyses. It includes the identification of the metal-coordinating ligands, the calculation of the coordination geometry and the superposition of ideal polyhedra to identify the optimal positions for free coordination sites. The new interaction model was integrated in the docking software FlexX and evaluated on a data set of 103 metalloprotein-ligand complexes, which were extracted from the PDB. In a first step, the quality of the automatic calculation of the metal coordination geometry was analyzed. In 74% of the cases, the correct prediction of the coordination geometry could be determined on the basis of the protein structure alone. Secondly, the new metal interaction model was tested in terms of predicting protein-ligand complexes. In the majority of test cases, the new interaction model resulted in an improved docking accuracy of the top ranking placements. 2007 Wiley-Liss, Inc.

  20. Effect of second ligand on the luminescence of Samarium (III ...

    Indian Academy of Sciences (India)

    Effect of second ligand on the luminescence of Samarium (III) dibenzoylmethane complexes: Syntheses, crystal structures, thermal analysis and luminescence study. MUHAMMAD IDIRIS SALEH, MIN YEE CHOO, TAI WEI CHAN and MOHD R RAZALI. ∗. School of Chemical Sciences, Universiti Sains Malaysia, Penang, ...

  1. Study of the spectroscopic characteristics of methyl (ligand ...

    Indian Academy of Sciences (India)

    ... Lecture Workshops · Refresher Courses · Symposia. Home; Journals; Journal of Chemical Sciences; Volume 119; Issue 1. Study of the spectroscopic characteristics of methyl (ligand) cobaloximes and their antibacterial activity. N Navaneetha P A Nagarjun S Satyanarayana. Volume 119 Issue 1 January 2007 pp 29-34 ...

  2. Chelating ligands: enhancers of quality and purity of biogas ...

    African Journals Online (AJOL)

    The quality of biogas depends largely on the percentage of methane and hydrogen sulphide gas present. High concentration of hydrogen sulphide results in low quality biogas. This work employed the use of chelating ligands in scrubbing hydrogen sulphide gas while improving the yield of methane gas. Experimental ...

  3. Synergistic Effects of PPARγ Ligands and Retinoids in Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Masahito Shimizu

    2008-01-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the nuclear receptor superfamily. The activation of PPARs by their specific ligands is regarded as one of the promising strategies to inhibit cancer cell growth. However, recent clinical trials targeting several common cancers showed no beneficial effect when PPAR ligands are used as a monotherapy. Retinoid X receptors (RXRs, which play a critical role in normal cell proliferation as a master regulator for nuclear receptors, preferentially form heterodimers with PPARs. A malfunction of RXRα due to phosphorylation by the Ras/MAPK signaling pathway is associated with the development of certain types of human malignancies. The activation of PPARγ/RXR heterodimer by their respective ligands synergistically inhibits cell growth, while inducing apoptosis in human colon cancer cells when the phosphorylation of RXRα was inhibited. We herein review the synergistic antitumor effects produced by the combination of the PPAR, especially PPARγ, ligands plus other agents, especially retinoids, in a variety of human cancers. We also focus on the phosphorylation of RXRα because the inhibition of RXRα phosphorylation and the restoration of its physiological function may activate PPAR/RXR heterodimer and, therefore, be a potentially effective and critical strategy for the inhibition of cancer cell growth.

  4. Synthesis and characterization of mixed ligand complexes of Zn (II ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 117; Issue 3. Synthesis and characterization of mixed ligand complexes of Zn(II) and Co(II) with amino acids: Relevance to zinc binding sites in zinc fingers. P Rabindra Reddy M Radhika P Manjula. Volume 117 Issue 3 May 2005 pp 239-246 ...

  5. Nickel (II) complexes containing ONS donor ligands: Synthesis ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 127; Issue 4. Nickel(II) complexes containing ONS donor ligands: Synthesis, characterization, crystal structure and catalytic application towards C-C cross-coupling reactions. Panneerselvam Anitha Rajendran Manikandan Paranthaman Vijayan Govindan Prakash ...

  6. Quantifying ligand effects in high-oxidation-state metal catalysis

    Science.gov (United States)

    Billow, Brennan S.; McDaniel, Tanner J.; Odom, Aaron L.

    2017-09-01

    Catalysis by high-valent metals such as titanium(IV) impacts our lives daily through reactions like olefin polymerization. In any catalysis, optimization involves a careful choice of not just the metal but also the ancillary ligands. Because these choices dramatically impact the electronic structure of the system and, in turn, catalyst performance, new tools for catalyst development are needed. Understanding ancillary ligand effects is arguably one of the most critical aspects of catalyst optimization and, while parameters for phosphines have been used for decades with low-valent systems, a comparable system does not exist for high-valent metals. A new electronic parameter for ligand donation, derived from experiments on a high-valent chromium species, is now available. Here, we show that the new parameters enable quantitative determination of ancillary ligand effects on catalysis rate and, in some cases, even provide mechanistic information. Analysing reactions in this way can be used to design better catalyst architectures and paves the way for the use of such parameters in a host of high-valent processes.

  7. Colloidal-quantum-dot photovoltaics using atomic-ligand passivation

    KAUST Repository

    Tang, Jiang

    2011-09-18

    Colloidal-quantum-dot (CQD) optoelectronics offer a compelling combination of solution processing and spectral tunability through quantum size effects. So far, CQD solar cells have relied on the use of organic ligands to passivate the surface of the semiconductor nanoparticles. Although inorganic metal chalcogenide ligands have led to record electronic transport parameters in CQD films, no photovoltaic device has been reported based on such compounds. Here we establish an atomic ligand strategy that makes use of monovalent halide anions to enhance electronic transport and successfully passivate surface defects in PbS CQD films. Both time-resolved infrared spectroscopy and transient device characterization indicate that the scheme leads to a shallower trap state distribution than the best organic ligands. Solar cells fabricated following this strategy show up to 6% solar AM1.5G power-conversion efficiency. The CQD films are deposited at room temperature and under ambient atmosphere, rendering the process amenable to low-cost, roll-by-roll fabrication. © 2011 Macmillan Publishers Limited. All rights reserved.

  8. Some new IIB group complexes of an imidazolidine ligand ...

    Indian Academy of Sciences (India)

    inosa, Staphylococcus aureus and Bacillus subtillis bac- teria and also for their inhibitory effects on the growth of Candid albicans using disc diffusion and MIC meth- ods. The antibacterial activities of the ligand and its complexes as inhibition zone (mm) of the bacterial and fungal growth have been classified in table 5 and.

  9. Kinetics and mechanism of interaction of some bioactive ligands ...

    Indian Academy of Sciences (India)

    The association equilibrium constant (KE) for the outer sphere complex formation has been evaluated together with rate constants for the two subsequent steps. The rate constants increase with increasing ligand concentration and the evaluated activation parameters for all reactions suggest an associative substitution ...

  10. synthesis and spectra characterization of mixed- ligand complexes ...

    African Journals Online (AJOL)

    BARTH EKWUEME

    “Let's Twist Again”-Double-Stranded,. Triple-Stranded, and Circular Helicates. Chemical Reviews 101(11): 3457-3498. Belfrekh, N., Dietrich-Buchecker, C. and Sauvage, J. P.. 2000. Synthesis and Characterization of Dinuclear Metal. Complexes Stabilized by Tetradentate Schiff base ligands.Inorganic Chemistry 39: 5169-.

  11. Structural basis for AMPA receptor activation and ligand selectivity

    DEFF Research Database (Denmark)

    Hogner, A; Kastrup, Jette Sandholm Jensen; Jin, R

    2002-01-01

    with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent...

  12. Selective Electrocatalytic Activity of Ligand Stabilized Copper Oxide Nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Kauffman, Douglas R; Ohodnicki, Paul R; Kail, Brian W; Matranga, Christopher

    2011-01-01

    Ligand stabilization can influence the surface chemistry of Cu oxide nanoparticles (NPs) and provide unique product distributions for electrocatalytic methanol (MeOH) oxidation and CO{sub 2} reduction reactions. Oleic acid (OA) stabilized Cu{sub 2}O and CuO NPs promote the MeOH oxidation reaction with 88% and 99.97% selective HCOH formation, respectively. Alternatively, CO{sub 2} is the only reaction product detected for bulk Cu oxides and Cu oxide NPs with no ligands or weakly interacting ligands. We also demonstrate that OA stabilized Cu oxide NPs can reduce CO{sub 2} into CO with a {approx}1.7-fold increase in CO/H{sub 2} production ratios compared to bulk Cu oxides. The OA stabilized Cu oxide NPs also show 7.6 and 9.1-fold increases in CO/H{sub 2} production ratios compared to weakly stabilized and non-stabilized Cu oxide NPs, respectively. Our data illustrates that the presence and type of surface ligand can substantially influence the catalytic product selectivity of Cu oxide NPs.

  13. Synthesis and evaluation of potential ligands for nuclear waste processing

    NARCIS (Netherlands)

    Iqbal, M.

    2012-01-01

    The research presented in this thesis deals with the synthesis and evaluation of new potential ligands for the complexation of actinide and lanthanide ions either for their extraction from bulk radioactive waste or their stripping from an extracted organic phase for final processing of the waste. In

  14. Mixed-ligand complexes of ruthenium (II) incorporating a diazo ...

    Indian Academy of Sciences (India)

    Binding of these complexes with calf thymus DNA (CT-DNA) has been investigated by absorption spectroscopy, steady-state emission spectroscopy and viscosity measurements. The experimental results indicate that the size and shape of the intercalating ligands have marked effect on the binding affinity of the complexes ...

  15. Designer Ligands. Part 13. Synthesis and Catalytic Activity of ...

    African Journals Online (AJOL)

    Copper(I), copper(II), cobalt(II) and zinc(II) complexes of a macrocyclic, multidentate Schiff-base ligand have been prepared and, with the exception of the zinc(II) complex, have been shown to exhibit biomimetic catecholase activity. Keywords: Copper(II);Cobalt(II); Zinc(II); Biomimetic complexes; Catecholase activity

  16. SLATE : a method for the superposition of flexible ligands

    NARCIS (Netherlands)

    Mills, J.E.; de Esch, I J; Perkins, T.D.; Dean, P.M.

    A novel program for the superposition of flexible molecules, SLATE, is presented. It uses simulated annealing to minimise the difference between the distance matrices calculated from the hydrogen-bonding and aromaticring properties of two ligands. A method for generating a molecular stack using

  17. Ligand.Info small-molecule Meta-Database.

    Science.gov (United States)

    von Grotthuss, Marcin; Koczyk, Grzegorz; Pas, Jakub; Wyrwicz, Lucjan S; Rychlewski, Leszek

    2004-12-01

    Ligand.Info is a compilation of various publicly available databases of small molecules. The total size of the Meta-Database is over 1 million entries. The compound records contain calculated three-dimensional coordinates and sometimes information about biological activity. Some molecules have information about FDA drug approving status or about anti-HIV activity. Meta-Database can be downloaded from the http://Ligand.Info web page. The database can also be screened using a Java-based tool. The tool can interactively cluster sets of molecules on the user side and automatically download similar molecules from the server. The application requires the Java Runtime Environment 1.4 or higher, which can be automatically downloaded from Sun Microsystems or Apple Computer and installed during the first use of Ligand.Info on desktop systems, which support Java (Ms Windows, Mac OS, Solaris, and Linux). The Ligand.Info Meta-Database can be used for virtual high-throughput screening of new potential drugs. Presented examples showed that using a known antiviral drug as query the system was able to find others antiviral drugs and inhibitors.

  18. Synthesis and Characterization of Metal Complexes with Schiff Base Ligands

    Science.gov (United States)

    Wilkinson, Shane M.; Sheedy, Timothy M.; New, Elizabeth J.

    2016-01-01

    In order for undergraduate laboratory experiments to reflect modern research practice, it is essential that they include a range of elements, and that synthetic tasks are accompanied by characterization and analysis. This intermediate general chemistry laboratory exercise runs over 2 weeks, and involves the preparation of a Schiff base ligand and…

  19. Ligand-targeted particulate nanomedicines undergoing clinical evaluations: current status

    NARCIS (Netherlands)

    van der Meel, Roy; Vehmeijer, Laurens J.C.; Kok, Robbert J.; Storm, Gerrit; van Gaal, Ethlinn V.B.

    2013-01-01

    Since the introduction of Doxil® on the market nearly 20 years ago, a number of nanomedicines have become part of treatment regimens in the clinic. With the exception of antibody–drug conjugates, these nanomedicines are all devoid of targeting ligands and rely solely on their physicochemical

  20. Synthesis and characterization of mixed ligand complexes of bio ...

    Indian Academy of Sciences (India)

    Unknown

    bio-metals with pyrimidine nucleoside (uridine) and amino acids. P RABINDRA REDDY* and A MOHAN REDDY. Department of Chemistry, Osmania University, Hyderabad 500 007, India. MS received 24 December 1999; revised 28 August 2000. Abstract. The mixed ligand complexes of Cu(II), Ni(II) and Co(II) with uridine ...