Science.gov

Sample records for glial heparan sulfate

  1. Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation

    PubMed Central

    Higginson, Jennifer R; Thompson, Sophie M; Santos-Silva, Alessandra; Guimond, Scott E; Turnbull, Jeremy E; Barnett, Susan C

    2012-01-01

    Previously, it has been shown that rat Schwann cells (SCs), but not olfactory ensheathing cells (OECs), form a boundary with astrocytes, due to a SC-specific secreted factor. Here, we identify highly sulfated heparan sulfates (HS), and fibroblast growth factors (FGF) 1 and FGF9, as possible determinants of boundary formation induced by rat SCs. Disaccharide analysis of HS in SC and rat OEC conditioned medium showed that SCs secrete more highly sulfated HS than OECs. The dependence of the boundary-forming activity on high levels of sulfation was confirmed using a panel of semi-synthetic modified heparins with variable levels of sulfation. Furthermore, extracellular HS 6-O-endosulfatase enzymes, Sulf 1 and Sulf 2, were expressed at a significantly lower level by SCs compared to OECs and siRNA reduction of Sulfs in OECs was, in itself, sufficient to induce boundary formation. This demonstrates a key role for remodelling (reduction) of HS 6-O-sulfation by OECs to suppress boundary formation, in comparison to SCs. Furthermore, specific anti-FGF1 and FGF9 antibodies disrupted SC/astrocyte boundary formation, supporting a role for an HS sulfation-dependent FGF signalling mechanism via FGF receptors (FGFR) on astrocytes. We propose a model in which FGF1 and FGF9 signalling is differentially modulated by patterns of glial cell HS sulfation, dependent on Sulf 1 and Sulf 2 expression, to control FGFR3-IIIb mediated astrocytic responses. Moreover, these data suggest manipulation of HS sulfation after CNS injury as a potential novel approach for therapeutic intervention in CNS repair. PMID:23136428

  2. Differential sulfation remodelling of heparan sulfate by extracellular 6-O-sulfatases regulates fibroblast growth factor-induced boundary formation by glial cells: implications for glial cell transplantation.

    PubMed

    Higginson, Jennifer R; Thompson, Sophie M; Santos-Silva, Alessandra; Guimond, Scott E; Turnbull, Jeremy E; Barnett, Susan C

    2012-11-07

    Previously, it has been shown that rat Schwann cells (SCs), but not olfactory ensheathing cells (OECs), form a boundary with astrocytes, due to a SC-specific secreted factor. Here, we identify highly sulfated heparan sulfates (HSs) and fibroblast growth factors (FGFs) 1 and 9 as possible determinants of boundary formation induced by rat SCs. Disaccharide analysis of HS in SC-conditioned and rat OEC-conditioned media showed that SCs secrete more highly sulfated HS than OECs. The dependence of the boundary-forming activity on high levels of sulfation was confirmed using a panel of semisynthetic modified heparins with variable levels of sulfation. Furthermore, extracellular HS 6-O-endosulfatase enzymes, Sulf 1 and Sulf 2, were expressed at a significantly lower level by SCs compared with OECs, and siRNA reduction of Sulfs in OECs was, in itself, sufficient to induce boundary formation. This demonstrates a key role for remodelling (reduction) of HS 6-O-sulfation by OECs, compared with SCs, to suppress boundary formation. Furthermore, specific anti-FGF1 and anti-FGF9 antibodies disrupted SC-astrocyte boundary formation, supporting a role for an HS sulfation-dependent FGF signaling mechanism via FGF receptors on astrocytes. We propose a model in which FGF1 and FGF9 signaling is differentially modulated by patterns of glial cell HS sulfation, dependent on Sulf 1 and Sulf 2 expression, to control FGF receptor 3-IIIb-mediated astrocytic responses. Moreover, these data suggest manipulation of HS sulfation after CNS injury as a potential novel approach for therapeutic intervention in CNS repair.

  3. Heparan Sulfate Proteoglycans

    PubMed Central

    Sarrazin, Stephane; Lamanna, William C.; Esko, Jeffrey D.

    2011-01-01

    Heparan sulfate proteoglycans are found at the cell surface and in the extracellular matrix, where they interact with a plethora of ligands. Over the last decade, new insights have emerged regarding the mechanism and biological significance of these interactions. Here, we discuss changing views on the specificity of protein–heparan sulfate binding and the activity of HSPGs as receptors and coreceptors. Although few in number, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal level. PMID:21690215

  4. Bioengineered heparins and heparan sulfates.

    PubMed

    Fu, Li; Suflita, Matthew; Linhardt, Robert J

    2016-02-01

    Heparin and heparan sulfates are closely related linear anionic polysaccharides, called glycosaminoglycans, which exhibit a number of important biological and pharmacological activities. These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of animal cells. While heparan sulfate is a widely distributed membrane and extracellular glycosaminoglycan, heparin is found primarily intracellularly in the granules of mast cells. While heparin has historically received most of the scientific attention for its anticoagulant activity, interest has steadily grown in the multi-faceted role heparan sulfate plays in normal and pathophysiology. The chemical synthesis of these glycosaminoglycans is largely precluded by their structural complexity. Today, we depend on livestock animal tissues for the isolation and the annual commercial production of hundred ton quantities of heparin used in the manufacture of anticoagulant drugs and medical device coatings. The variability of animal-sourced heparin and heparan sulfates, their inherent impurities, the limited availability of source tissues, the poor control of these source materials and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans, driven by both therapeutic applications and as probes to study their natural functions. This review focuses on the complex biology of these glycosaminoglycans in human health and disease, and the use of recombinant technology in the chemoenzymatic synthesis and metabolic engineering of heparin and heparan sulfates.

  5. Divergent Synthesis of Heparan Sulfate Oligosaccharides

    PubMed Central

    2015-01-01

    Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis. PMID:26574650

  6. Artemin Crystal Structure Reveals Insights into Heparan Sulfate Binding

    SciTech Connect

    Silvian,L.; Jin, P.; Carmillo, P.; Boriack-Sjodin, P.; Pelletier, C.; Rushe, M.; Gong, B.; Sah, D.; Pepinsky, B.; Rossomando, A.

    2006-01-01

    Artemin (ART) promotes the growth of developing peripheral neurons by signaling through a multicomponent receptor complex comprised of a transmembrane tyrosine kinase receptor (cRET) and a specific glycosylphosphatidylinositol-linked co-receptor (GFR{alpha}3). Glial cell line-derived neurotrophic factor (GDNF) signals through a similar ternary complex but requires heparan sulfate proteoglycans (HSPGs) for full activity. HSPG has not been demonstrated as a requirement for ART signaling. We crystallized ART in the presence of sulfate and solved its structure by isomorphous replacement. The structure reveals ordered sulfate anions bound to arginine residues in the pre-helix and amino-terminal regions that were organized in a triad arrangement characteristic of heparan sulfate. Three residues in the pre-helix were singly or triply substituted with glutamic acid, and the resulting proteins were shown to have reduced heparin-binding affinity that is partly reflected in their ability to activate cRET. This study suggests that ART binds HSPGs and identifies residues that may be involved in HSPG binding.

  7. Heparan sulfate in skeletal muscle development

    SciTech Connect

    Noonan, D.M.

    1985-01-01

    In this study, chick breast skeletal muscle cells developing in vitro from myoblasts to myotubes were found to synthesize heparan sulfate (HS), chrondroitin-6-sulfate, chrondroitin-4-sulfate, dermatan sulfate, unsulfated chrondroitin and hyaluronic acid in both the substratum attached material (SAM) and the cellular fraction. SAM was found to contain predominantly chrondroitin-6-sulfate and relatively little HS whereas the cellular fraction contained relatively higher levels of HS and lower levels of chrondroitin-6-sulfate. Hyaluronic acid was also a major component in both fractions with the other glycosaminoglycan isomers present as minor components. Muscle derived fibroblast cultures had higher levels of dermatan sulfate in the cell layer and higher levels of HS in the SAM fraction than did muscle cultures. The structure of the proteoglycans were partially characterized in /sup 35/SO/sub 4//sup 2 -/ radio-labeled cultures which indicated an apparent increase in the hydrodynamic size of the cell fraction heparan sulfate proteoglycan (HS PG). Myotubes incorporated /sup 35/SO/sub 4//sup 2 -/ into HS PG at a rate 3 times higher than myoblasts. The turnover rate of HS in the cellular fraction was the same for myoblasts and myotubes, with a t/sub 1/2/ of approximately 5 hours. Fibroblasts in culture synthesized the smallest HS PG, and incorporated /sup 35/SO/sub 4//sup 2 -/ into HS PG at a rate lower than that of myotubes. Studies in which fusion was reversibly inhibited with decreased medium (Ca/sup + +/) closely linked the increased synthesis of cell fraction, but not SAM fraction, HS with myotube formation. However, decreasing medium calcium appeared to cause significant alterations in the metabolism of inorganic sulfate.

  8. Heparan sulfate structure: methods to study N-sulfation and NDST action.

    PubMed

    Dagälv, Anders; Lundequist, Anders; Filipek-Górniok, Beata; Dierker, Tabea; Eriksson, Inger; Kjellén, Lena

    2015-01-01

    Heparan sulfate proteoglycans are important modulators of cellular processes where the negatively charged polysaccharide chains interact with target proteins. The sulfation pattern of the heparan sulfate chains will determine the proteins that will bind and the affinity of the interactions. The N-deacetylase/N-sulfotransferase (NDST) enzymes are of key importance during heparan sulfate biosynthesis when the sulfation pattern is determined. In this chapter, metabolic labeling of heparan sulfate with [(35)S]sulfate or [(3)H]glucosamine in cell cultures is described, in addition to characterization of polysaccharide chain length and degree of N-sulfation. Methods to measure NDST enzyme activity are also presented.

  9. Heparan sulfate and heparin interactions with proteins

    PubMed Central

    Meneghetti, Maria C. Z.; Hughes, Ashley J.; Rudd, Timothy R.; Nader, Helena B.; Powell, Andrew K.; Yates, Edwin A.; Lima, Marcelo A.

    2015-01-01

    Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed. PMID:26289657

  10. Heparan sulfate and heparin interactions with proteins.

    PubMed

    Meneghetti, Maria C Z; Hughes, Ashley J; Rudd, Timothy R; Nader, Helena B; Powell, Andrew K; Yates, Edwin A; Lima, Marcelo A

    2015-09-06

    Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure-activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure-activity relationships and regulation will be discussed.

  11. Chemical Tumor Biology of Heparan Sulfate Proteoglycans

    PubMed Central

    Raman, Karthik; Kuberan, Balagurunathan

    2010-01-01

    Heparan sulfate proteoglycans (HSPGs) play vital roles in every step of tumor progression allowing cancer cells to proliferate, escape from immune response, invade neighboring tissues, and metastasize to distal sites away from the primary site. Several cancers including breast, lung, brain, pancreatic, skin, and colorectal cancers show aberrant modulation of several key HS biosynthetic enzymes such as 3-O Sulfotransferase and 6-O Sulfotransferase, and also catabolic enzymes such as HSulf-1, HSulf-2 and heparanase. The resulting tumor specific HS fine structures assist cancer cells to breakdown ECM to spread, misregulate signaling pathways to facilitate their proliferation, promote angiogenesis to receive nutrients, and protect themselves against natural killer cells. This review focuses on the changes in the expression of HS biosynthetic and catabolic enzymes in several cancers, the resulting changes in HS fine structures, and the effects of these tumor specific HS signatures on promoting invasion, proliferation, and metastasis. It is possible to retard tumor progression by modulating the deregulated biosynthetic and catabolic pathways of HS chains through novel chemical biology approaches. PMID:20596243

  12. Inhibition of synthesis of heparan sulfate by selenate: Possible dependence on sulfation for chain polymerization

    SciTech Connect

    Dietrich, C.P.; Nader, H.B. ); Buonassisi, V.; Colburn, P. )

    1988-01-01

    Selenate, a sulfation inhibitor, blocks the synthesis of heparan sulfate and chondroitin sulfate by cultured endothelial cells. In contrast, selenate does not affect the production of hyaluronic acid, a nonsulfated glycosaminoglycan. No differences in molecular weight, ({sup 3}H)glucosamine/({sup 35}S)sulfuric acid ratios, or disaccharide composition were observed when the heparan sulfate synthesized by selenate-treated cells was compared with that of control cells. The absence of undersulfated chains in preparations from cultures exposed to selenate supports the concept that, in the intact cell, the polymerization of heparan sulfate might be dependent on the sulfation of the saccharide units added to the growing glycosaminoglycan chain.

  13. Heparan Sulfate-Dependent Enhancement of Henipavirus Infection

    PubMed Central

    Mathieu, Cyrille; Dhondt, Kévin P.; Châlons, Marie; Mély, Stéphane; Raoul, Hervé; Negre, Didier; Cosset, François-Loïc; Gerlier, Denis; Vivès, Romain R.

    2015-01-01

    ABSTRACT Nipah virus and Hendra virus are emerging, highly pathogenic, zoonotic paramyxoviruses that belong to the genus Henipavirus. They infect humans as well as numerous mammalian species. Both viruses use ephrin-B2 and -B3 as cell entry receptors, and following initial entry into an organism, they are capable of rapid spread throughout the host. We have previously reported that Nipah virus can use another attachment receptor, different from its entry receptors, to bind to nonpermissive circulating leukocytes, thereby promoting viral dissemination within the host. Here, this attachment molecule was identified as heparan sulfate for both Nipah virus and Hendra virus. Cells devoid of heparan sulfate were not able to mediate henipavirus trans-infection and showed reduced permissivity to infection. Virus pseudotyped with Nipah virus glycoproteins bound heparan sulfate and heparin but no other glycosaminoglycans in a surface plasmon resonance assay. Furthermore, heparin was able to inhibit the interaction of the viruses with the heparan sulfate and to block cell-mediated trans-infection of henipaviruses. Moreover, heparin was shown to bind to ephrin-B3 and to restrain infection of permissive cells in vitro. Consequently, treatment with heparin devoid of anticoagulant activity improved the survival of Nipah virus-infected hamsters. Altogether, these results reveal heparan sulfate as a new attachment receptor for henipaviruses and as a potential therapeutic target for the development of novel approaches against these highly lethal infections. PMID:25759505

  14. Ca2+-mediated association of human serum amyloid P component with heparan sulfate and dermatan sulfate.

    PubMed

    Hamazaki, H

    1987-02-05

    The serum amyloid P component (SAP) is a precursor glycoprotein of amyloid P component found in all types of amyloid deposits. The binding of human SAP to heparan sulfate and dermatan sulfate was studied using Sepharose-immobilized SAP. The apparent dissociation constants of heparan sulfate and dermatan sulfate for immobilized-SAP were estimated to be approximately 2 X 10(-7) M in the presence of 2 mM CaCl2 at neutral pH and physiological ionic strength. Both the binding affinity of SAP for these glycosaminoglycans and the numbers of binding sites of SAP depended on calcium concentration. Cadmium partially substituted for calcium as an activator of glycosaminoglycan binding to SAP. No binding occurs in the absence of added metal, or in the presence of barium, copper, magnesium, manganese, and strontium. The calcium-dependent binding of [3H]heparan sulfate and [3H]dermatan sulfate to SAP was strongly inhibited by heparan sulfate, heparin, and dermatan sulfate. Chondroitin 6-sulfate was a moderate inhibitor, whereas hyaluronic acid, chondroitin 4-sulfate, and keratan sulfate were not potent inhibitors. The calcium-dependent binding of amyloid P component to heparan sulfate and/or dermatan sulfate may be a cause of the coexistence of the particular glycoprotein and these glycosaminoglycans in amyloid tissues.

  15. Heparan sulfate 3-O-sulfation: a rare modification in search of a function.

    PubMed

    Thacker, Bryan E; Xu, Ding; Lawrence, Roger; Esko, Jeffrey D

    2014-04-01

    Many protein ligands bind to heparan sulfate, which results in their presentation, protection, oligomerization or conformational activation. Binding depends on the pattern of sulfation and arrangement of uronic acid epimers along the chains. Sulfation at the C3 position of glucosamine is a relatively rare, yet biologically significant modification, initially described as a key determinant for binding and activation of antithrombin and later for infection by type I herpes simplex virus. In mammals, a family of seven heparan sulfate 3-O-sulfotransferases installs sulfate groups at this position and constitutes the largest group of sulfotransferases involved in heparan sulfate formation. However, to date very few proteins or biological systems have been described that are influenced by 3-O-sulfation. This review describes our current understanding of the prevalence and structure of 3-O-sulfation sites, expression and substrate specificity of the 3-O-sulfotransferase family and the emerging roles of 3-O-sulfation in biology.

  16. Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

    PubMed Central

    1984-01-01

    A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation. PMID

  17. Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability*

    PubMed Central

    Xu, Ding; Fuster, Mark M.; Lawrence, Roger; Esko, Jeffrey D.

    2011-01-01

    VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF165 and VEGF121. Because VEGF121 does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF165 and VEGF121. Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF165 and VEGF121, suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease. PMID:20974861

  18. Gucosamine-6-sulfamate Analogs of Heparan Sulfate as Inhibitors of Endosulfatases

    PubMed Central

    Schelwies, Mathias; Brinson, Diana; Otsuki, Shuhei; Hong, Young-Hoon; Lotz, Martin K.; Wong, Chi-Huey; Hanson, Sarah R.

    2011-01-01

    Keeping Sulfate. The extracellular endosulfatases, which modulate signalling pathways by removing sulfate groups from heparan, can be inhibited by replacing the 6-sulfate destined for cleavage with an inhibitory sulfamate motif, as demonstrated by simple glucosamine-6-sulfamate analogs of heparan sulfate. PMID:20973023

  19. A zinc complex of heparan sulfate destabilises lysozyme and alters its conformation

    SciTech Connect

    Hughes, Ashley J.; Hussain, Rohanah; Cosentino, Cesare; Guerrini, Marco; Siligardi, Giuliano; Yates, Edwin A.; Rudd, Timothy R.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Zinc-heparan sulfate complex destabilises lysozyme, a model amyloid protein. Black-Right-Pointing-Pointer Addition of zinc, without heparan sulfate, stabilises lysozyme. Black-Right-Pointing-Pointer Heparan sulfate cation complexes provide alternative protein folding routes. -- Abstract: The naturally occurring anionic cell surface polysaccharide heparan sulfate is involved in key biological activities and is implicated in amyloid formation. Following addition of Zn-heparan sulfate, hen lysozyme, a model amyloid forming protein, resembled {beta}-rich amyloid by far UV circular dichroism (increased {beta}-sheet: +25%), with a significantly reduced melting temperature (from 68 to 58 Degree-Sign C) by fluorescence shift assay. Secondary structure stability of the Zn-heparan sulfate complex with lysozyme was also distinct from that with heparan sulfate, under stronger denaturation conditions using synchrotron radiation circular dichroism. Changing the cation associated with heparan sulfate is sufficient to alter the conformation and stability of complexes formed between heparan sulfate and lysozyme, substantially reducing the stability of the protein. Complexes of heparan sulfate and cations, such as Zn, which are abundant in the brain, may provide alternative folding routes for proteins.

  20. Heparan sulfate proteoglycans: a sugar code for vertebrate development?

    PubMed Central

    Poulain, Fabienne E.; Yost, H. Joseph

    2015-01-01

    Heparan sulfate proteoglycans (HSPGs) have long been implicated in a wide range of cell-cell signaling and cell-matrix interactions, both in vitro and in vivo in invertebrate models. Although many of the genes that encode HSPG core proteins and the biosynthetic enzymes that generate and modify HSPG sugar chains have not yet been analyzed by genetics in vertebrates, recent studies have shown that HSPGs do indeed mediate a wide range of functions in early vertebrate development, for example during left-right patterning and in cardiovascular and neural development. Here, we provide a comprehensive overview of the various roles of HSPGs in these systems and explore the concept of an instructive heparan sulfate sugar code for modulating vertebrate development. PMID:26487777

  1. Heparan sulfate proteoglycans: a sugar code for vertebrate development?

    PubMed

    Poulain, Fabienne E; Yost, H Joseph

    2015-10-15

    Heparan sulfate proteoglycans (HSPGs) have long been implicated in a wide range of cell-cell signaling and cell-matrix interactions, both in vitro and in vivo in invertebrate models. Although many of the genes that encode HSPG core proteins and the biosynthetic enzymes that generate and modify HSPG sugar chains have not yet been analyzed by genetics in vertebrates, recent studies have shown that HSPGs do indeed mediate a wide range of functions in early vertebrate development, for example during left-right patterning and in cardiovascular and neural development. Here, we provide a comprehensive overview of the various roles of HSPGs in these systems and explore the concept of an instructive heparan sulfate sugar code for modulating vertebrate development.

  2. Heparan sulfate regulates ADAM12 through a molecular switch mechanism.

    PubMed

    Sørensen, Hans Peter; Vivès, Romain R; Manetopoulos, Christina; Albrechtsen, Reidar; Lydolph, Magnus C; Jacobsen, Jonas; Couchman, John R; Wewer, Ulla M

    2008-11-14

    The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.

  3. Heparan sulfate chains potentiate cadmium cytotoxicity in cultured vascular endothelial cells.

    PubMed

    Fujiwara, Yasuyuki; Yamamoto, Chika; Yoshida, Eiko; Kumagai, Yoshito; Kaji, Toshiyuki

    2016-02-01

    The monolayer of vascular endothelial cells, which is rich in heparan sulfate chains, is an important target of cadmium cytotoxicity. To investigate the effects of heparan sulfate chains on cadmium cytotoxicity, bovine aortic endothelial cells were cultured in the presence of cadmium, with or without exogenous heparan sulfate. The following results were obtained: (1) Heparan sulfate chains potentiated cadmium cytotoxicity. (2) Such a potentiation did not occur in bovine aortic smooth muscle cells. (3) Heparin chains as well as heparan sulfate chains potentiated cadmium cytotoxicity, while other glycosaminoglycan chains failed to exhibit such an activity. (4) The disaccharide units of heparan sulfate chains did not potentiate cadmium cytotoxicity in the endothelial cells. (5) Heparan sulfate chains did not potentiate mercury and arsenite cytotoxicity. (6) Fibroblast growth factor-2 (FGF-2) also potentiated cadmium cytotoxicity in the endothelial cells. (7) Heparan sulfate chains significantly increased intracellular cadmium accumulation by inducing the expression of metallothionein. Taken together, these results suggest that heparan sulfate chains activate FGF-2, which in turn elevates the expression and/or activity of metal transporter(s) that facilitate cadmium influx from the extracellular space into the cytoplasm.

  4. N-sulfation of heparan sulfate regulates early branching events in the developing mammary gland.

    PubMed

    Bush, Kevin T; Crawford, Brett E; Garner, Omai B; Nigam, Kabir B; Esko, Jeffrey D; Nigam, Sanjay K

    2012-12-07

    Branching morphogenesis, a fundamental process in the development of epithelial organs (e.g. breast, kidney, lung, salivary gland, prostate, pancreas), is in part dependent on sulfation of heparan sulfate proteoglycans. Proper sulfation is mediated by biosynthetic enzymes, including exostosin-2 (Ext2), N-deacetylase/N-sulfotransferases and heparan sulfate O-sulfotransferases. Recent conditional knockouts indicate that whereas primary branching is dependent on heparan sulfate, other stages are dependent upon selective addition of N-sulfate and/or 2-O sulfation (Crawford, B .E., Garner, O. B., Bishop, J. R., Zhang, D. Y., Bush, K. T., Nigam, S. K., and Esko, J. D. (2010) PLoS One 5, e10691; Garner, O .B., Bush, K. T., Nigam, S .K., Yamaguchi, Y., Xu, D., Esko, J. D., and Nigam, S. K. (2011) Dev. Biol. 355, 394-403). Here, we analyzed the effect of deleting both Ndst2 and Ndst1. Whereas deletion of Ndst1 has no major effect on primary or secondary branching, deletion of Ndst2 appears to result in a mild increase in branching. When both genes were deleted, ductal growth was variably diminished (likely due to variable Cre-recombinase activity), but an overabundance of branched structures was evident irrespective of the extent of gland growth or postnatal age. "Hyperbranching" is an unusual phenotype. The effects on N-sulfation and growth factor binding were confirmed biochemically. The results indicate that N-sulfation or a factor requiring N-sulfation regulates primary and secondary branching events in the developing mammary gland. Together with previous work, the data indicate that different stages of ductal branching and lobuloalveolar formation are regulated by distinct sets of heparan sulfate biosynthetic enzymes in an appropriate growth factor context.

  5. Renal localization of heparan sulfate proteoglycan by immunohistochemistry.

    PubMed Central

    Klein, D. J.; Oegema, T. R.; Eisenstein, R.; Furcht, L.; Michael, A. F.; Brown, D. M.

    1983-01-01

    Glomerular localization of heparan sulfate proteoglycan (HS-proteoglycan) has been studied immunohistochemically with a highly purified antiserum to bovine aorta HS-proteoglycan core protein. The specificity of the antiserum was enhanced by consecutive fibronectin and chondroitin sulfate-dermatan sulfate proteoglycan (CS-DS proteoglycan) affinity chromatography. The affinity-purified HS-proteoglycan antibody lacked cross-reactivity by enzyme-linked immunosorbent assays (ELISA) with CS-DS proteoglycan, fibronectin, laminin, and Type IV collagen. Reactivity of the antiserum with HS-proteoglycan antigen by ELISA was inhibited by HS core protein derived from CsCl density gradient centrifugation after heparinase treatment of the HS-proteoglycan. Immunofluorescent reactivity of the HS-proteoglycan antiserum was observed with bovine glomerular basement membrane, renal interstitium, Bowman's capsule, renal arterioles, and bovine aorta. No staining was seen with rat, mouse, or human glomeruli. Images Figure 4 Figure 5 PMID:6222657

  6. Stromal heparan sulfate differentiates neuroblasts to suppress neuroblastoma growth.

    PubMed

    Knelson, Erik H; Gaviglio, Angela L; Nee, Jasmine C; Starr, Mark D; Nixon, Andrew B; Marcus, Stephen G; Blobe, Gerard C

    2014-07-01

    Neuroblastoma prognosis is dependent on both the differentiation state and stromal content of the tumor. Neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here, we identified critical growth-limiting components of the differentiating stroma secretome and designed a potential therapeutic strategy based on their central mechanism of action. We demonstrated that expression of heparan sulfate proteoglycans (HSPGs), including TβRIII, GPC1, GPC3, SDC3, and SDC4, is low in neuroblasts and high in the Schwannian stroma. Evaluation of neuroblastoma patient microarray data revealed an association between TGFBR3, GPC1, and SDC3 expression and improved prognosis. Treatment of neuroblastoma cell lines with soluble HSPGs promoted neuroblast differentiation via FGFR1 and ERK phosphorylation, leading to upregulation of the transcription factor inhibitor of DNA binding 1 (ID1). HSPGs also enhanced FGF2-dependent differentiation, and the anticoagulant heparin had a similar effect, leading to decreased neuroblast proliferation. Dissection of individual sulfation sites identified 2-O, 3-O-desulfated heparin (ODSH) as a differentiating agent, and treatment of orthotopic xenograft models with ODSH suppressed tumor growth and metastasis without anticoagulation. These studies support heparan sulfate signaling intermediates as prognostic and therapeutic neuroblastoma biomarkers and demonstrate that tumor stroma biology can inform the design of targeted molecular therapeutics.

  7. PAPST1 regulates sulfation of heparan sulfate proteoglycans in epithelial MDCK II cells.

    PubMed

    Dick, Gunnar; Akslen-Hoel, Linn Kristin; Grøndahl, Frøy; Kjos, Ingrid; Maccarana, Marco; Prydz, Kristian

    2015-01-01

    Proteoglycan (PG) sulfation depends on activated nucleotide sulfate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Transporters in the Golgi membrane translocate PAPS from the cytoplasm into the organelle lumen where PG sulfation occurs. Silencing of PAPS transporter (PAPST) 1 in epithelial MDCK cells reduced PAPS uptake into Golgi vesicles. Surprisingly, at the same time sulfation of heparan sulfate (HS) was stimulated. The effect was pathway specific in polarized epithelial cells. Basolaterally secreted proteoglycans (PGs) displayed an altered HS sulfation pattern and increased growth factor binding capacity. In contrast, the sulfation pattern of apically secreted PGs was unchanged while the secretion was reduced. Regulation of PAPST1 allows epithelial cells to prioritize between PG sulfation in the apical and basolateral secretory routes at the level of the Golgi apparatus. This provides sulfation patterns that ensure PG functions at the extracellular level, such as growth factor binding.

  8. Heparin-like heparan sulfate from rabbit cartilage.

    PubMed

    Parra, Alessandro; Veraldi, Noemi; Locatelli, Manuela; Fini, Milena; Martini, Lucia; Torri, Giangiacomo; Sangiorgi, Luca; Bisio, Antonella

    2012-02-01

    Glycosaminoglycans were extracted from both young rabbit growth plate (GRP) and articular (ART) cartilage tissues and enzymatically treated to selectively eliminate chondroitin sulfates and hyaluronic acid. The procedure avoided any fractionation step that could enrich the extract with over- or under-sulfated species. Isolated heparan sulfate (HS) was characterized by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy to quantify their specific structural features and/or by mass spectrometry to establish the disaccharide composition. Both GRP and ART HSs, despite differing in their yield (GRP at least 100 times greater than ART), exhibited a surprisingly high degree of sulfation. Quantitative two-dimensional heteronuclear single-quantum coherence-NMR analysis of GRP HS revealed unusually high N-sulfated glucosamine and 2-O-sulfated iduronic acid contents, similar to heparin. The unique pentasaccharide sequence of the binding site for antithrombin was also detected in a significant amount. High-performance liquid chromatography mass spectrometry analysis of the enzymatic digests with a cocktail of heparin lyases of both cartilaginous HSs confirmed the NMR results. As well as the discovery of an unusual HS structure in the two different types of rabbit cartilage, the feasibility of the analytical method adopted here has been demonstrated within this study. Such a method can be used to isolate and analyze HS from both normal and pathologic tissues. Characterization of healthy and pathological HS structures will contribute to improve the understanding of diseases related to malfunctions of HS biosynthesis and/or metabolism.

  9. Is N-sulfation just a gateway modification during heparan sulfate biosynthesis?

    PubMed

    Raman, Karthik; Nguyen, Thao Kim Nu; Kuberan, Balagurunathan

    2011-11-04

    Several biologically important growth factor-heparan sulfate (HS) interactions are regulated by HS sulfation patterns. However, the biogenesis of these combinatorial sulfation patterns is largely unknown. N-Deacetylase/N-sulfotrasferase (NDST) converts N-acetyl-d-glucosamine residues to N-sulfo-d-glucosamine residues. This enzyme is suggested to be a gateway enzyme because N-sulfation dictates the final HS sulfation pattern. It is known that O-sulfation blocks C5-epimerase, which acts immediately after NDST action. However, it is still unknown whether O-sulfation inhibits NDST action in a similar manner. In this article we radically change conventional assumptions regarding HS biosynthesis by providing in vitro evidence that N-sulfation is not necessarily just a gateway modification during HS biosynthesis.

  10. Changes in glomerular heparan sulfate in puromycin aminonucleoside nephrosis.

    PubMed Central

    Groggel, G. C.; Hovingh, P.; Border, W. A.; Linker, A.

    1987-01-01

    Changes in glomerular anionic sites in puromycin aminonucleoside nephrosis (PAN) in the rat are controversial. The authors examined glomerular anionic sites in PAN by in vivo staining with polyethyleneimine (PEI). They also quantitated and characterized glomerular heparan sulfate (HS), which is known to be a major glomerular polyanion in PAN, using in vivo incorporation of 35S-sulfate. PAN rats had a mean protein excretion of 96 +/- 23 mg per 24 hours. Staining of anionic sites with PEI showed 15.3 +/- 2.8 sites per 1000-nm length of glomerular basement membrane in controls, 13.7 +/- 1.9 sites in PAN rats (P greater than 0.05), and 50% of rats with early PAN had absent staining. Total 35S-sulfate incorporation was similar in both the controls and established PAN rats (2900 +/- 150 dpm/mg dry wt of glomeruli versus 3005 +/- 260, P greater than 0.05) but decreased in early PAN rats (2025 +/- 148). The percentage of 35S-sulfate incorporated into chondroitin sulfate was similar in all three groups of animals. HS uronic acid was also similar (1.8 +/- 0.2 g/mg dry wt of glomeruli versus 1.7 +/- 0.3, P greater than 0.05) but decreased in early PAN (1.1 +/- 0.2). The distribution of 35S-sulfate activity within the HS subfractions was examined by ion-exchange chromatography and showed a shift in percent present from 1.0 M to 1.25 M fraction in established and early PAN animals (control 1.0 M 37% +/- 3.2% versus PAN 19% +/- 3.4%, P less than 0.01, and 1.25 M 36% +/- 2.9% versus 53% +/- 2.9%, P less than 0.01). These results demonstrate that glomerular heparan sulfate is unchanged in established PAN but decreased in early PAN. SO4 incorporation is unchanged in established PAN and diminished in early PAN. Thus, early in PAN HS synthesis is impaired, but in established PAN the HS is normal, and changes in glomerular HS cannot explain the increased permeability. Images Figure 1 PMID:2443012

  11. A role for heparan sulfate in viral surfing.

    PubMed

    Oh, Myung-Jin; Akhtar, Jihan; Desai, Prashant; Shukla, Deepak

    2010-01-01

    Heparan sulfate (HS) moieties on cell surfaces are known to provide attachment sites for many viruses including herpes simplex virus type-1 (HSV-1). Here, we demonstrate that cells respond to HSV-1 infection by enhancing filopodia formation. Filopodia express HS and are subsequently utilized for the transport of HSV-1 virions to cell bodies in a surfing-like phenomenon, which is facilitated by the underlying actin cytoskeleton and is regulated by transient activation of a small Rho GTPase, Cdc42. We also demonstrate that interaction between a highly conserved herpesvirus envelope glycoprotein B (gB) and HS is required for surfing. A HSV-1 mutant that lacks gB fails to surf and quantum dots conjugated with gB demonstrate surfing-like movements. Our data demonstrates a novel use of a common receptor, HS, which could also be exploited by multiple viruses and quite possibly, many additional ligands for transport along the plasma membrane.

  12. A role for heparan sulfate in viral surfing

    SciTech Connect

    Oh, Myung-Jin; Akhtar, Jihan; Desai, Prashant; Shukla, Deepak

    2010-01-01

    Heparan sulfate (HS) moieties on cell surfaces are known to provide attachment sites for many viruses including herpes simplex virus type-1 (HSV-1). Here, we demonstrate that cells respond to HSV-1 infection by enhancing filopodia formation. Filopodia express HS and are subsequently utilized for the transport of HSV-1 virions to cell bodies in a surfing-like phenomenon, which is facilitated by the underlying actin cytoskeleton and is regulated by transient activation of a small Rho GTPase, Cdc42. We also demonstrate that interaction between a highly conserved herpesvirus envelope glycoprotein B (gB) and HS is required for surfing. A HSV-1 mutant that lacks gB fails to surf and quantum dots conjugated with gB demonstrate surfing-like movements. Our data demonstrates a novel use of a common receptor, HS, which could also be exploited by multiple viruses and quite possibly, many additional ligands for transport along the plasma membrane.

  13. Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans

    PubMed Central

    1992-01-01

    The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV. PMID:1310996

  14. Heparan sulfation is essential for the prevention of cellular senescence

    PubMed Central

    Jung, S H; Lee, H C; Yu, D-M; Kim, B C; Park, S M; Lee, Y-S; Park, H J; Ko, Y-G; Lee, J-S

    2016-01-01

    Cellular senescence is considered as an important tumor-suppressive mechanism. Here, we demonstrated that heparan sulfate (HS) prevents cellular senescence by fine-tuning of the fibroblast growth factor receptor (FGFR) signaling pathway. We found that depletion of 3′-phosphoadenosine 5′-phosphosulfate synthetase 2 (PAPSS2), a synthetic enzyme of the sulfur donor PAPS, led to premature cell senescence in various cancer cells and in a xenograft tumor mouse model. Sodium chlorate, a metabolic inhibitor of HS sulfation also induced a cellular senescence phenotype. p53 and p21 accumulation was essential for PAPSS2-mediated cellular senescence. Such senescence phenotypes were closely correlated with cell surface HS levels in both cancer cells and human diploid fibroblasts. The determination of the activation of receptors such as FGFR1, Met, and insulin growth factor 1 receptor β indicated that the augmented FGFR1/AKT signaling was specifically involved in premature senescence in a HS-dependent manner. Thus, blockade of either FGFR1 or AKT prohibited p53 and p21 accumulation and cell fate switched from cellular senescence to apoptosis. In particular, desulfation at the 2-O position in the HS chain contributed to the premature senescence via the augmented FGFR1 signaling. Taken together, we reveal, for the first time, that the proper status of HS is essential for the prevention of cellular senescence. These observations allowed us to hypothesize that the FGF/FGFR signaling system could initiate novel tumor defenses through regulating premature senescence. PMID:26250908

  15. Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan

    SciTech Connect

    Nader, H.B.; Dietrich, C.P.; Buonassisi, V.; Colburn, P.

    1987-06-01

    The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. (/sup 35/S)sulfuric acid and/or (/sup 3/H) glucosamine ucre used in preparing heparan sulfate proteoglycan. This proteoglycan inhibits the thromboplastin-activated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I, an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, as assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase, which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.

  16. Adeno-associated virus type 2 binding study on model heparan sulfate surface

    NASA Astrophysics Data System (ADS)

    Negishi, Atsuko; Liu, Jian; McCarty, Douglas; Samulski, Jude; Superfine, Richard

    2003-11-01

    Understanding the mechanisms involved in virus infections is useful in its application in areas such as gene therapy, drug development and delivery, and biosensors. In collaboration with UNC Gene Therapy Center and School of Pharmacy, we are specifically looking at the interaction between human parvovirus adeno-associated virus type 2 (AAV2), a potential viral vector, and heparan sulfate proteoglycan (HSPG), a known cell surface receptor for AAV2. Recent development in glycobiology has shown that some protein-polysaccharide binding is sugar sequence dependent. Heparan sulfate (HS) is a polysaccharide chain of sulfated iduronic/glucuronic and sulfate glucosamine residues and can be differentiated into sequence specific structures by enzymes. These enzymatic modifications, known as heparan sulfate sulfotransferase modified modifications, have been shown to change the biological nature of heparan sulfate such as specific binding to proteins and viruses. For understanding HS-assisted viral infection mechanisms, we are interested in investigating the binding affinity and stability of AAV to different HS structures. We have developed a model heparan sulfate surface in which AAV adsorption studies are done and analyzed using the atomic force microscope (AFM). In addition, a miniArray assay has been created to facilitate to this study. Adsorption studies are done in 4 white LED wells with approximately 3 mm2 reaction areas which minimize sample use and waste.

  17. Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells.

    PubMed

    Salmivirta, M; Safaiyan, F; Prydz, K; Andresen, M S; Aryan, M; Kolset, S O

    1998-10-01

    Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O-sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.

  18. Developmental changes in heparan sulfate expression: in situ detection with mAbs

    PubMed Central

    1992-01-01

    Two mAbs that are specific for heparan sulfate-related epitopes have been raised and used to analyze the cellular and tissular distribution of this glycosaminoglycan during development. mAb 10E4 reacts with an epitope that occurs in native heparan sulfate chains and that is destroyed by N-desulfation of the glycosaminoglycan. The antibody does not react with hyaluronate, chondroitin sulfate, or DNA, and reacts only poorly with heparin. The reactivity of proteoglycan extracts or tissue sections with the 10E4 antibody is completely abolished by heparitinase, but is only partially affected by heparinase. mAb 3G10, in contrast, reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections. The 3G10 epitope is destroyed by treatment with mercuric acetate, which indicates that the desaturated uronate generated by the lyase is essential for the reactivity of the antibody. The 3G10 epitope is not generated by treating heparan sulfate proteoglycans with heparinase or chondroitin sulfate proteoglycans with chondroitin sulfate lyases, which indicates that the 3G10 antibody recognizes desaturated uronates that occur in specific structural contexts. The antibody 10E4 and, after heparitinase treatment, the antibody 3G10 decorate the surfaces of many cell types and the extracellular matrix in proximity of the cells, in particular, the basement membranes. The analysis of embryonic and adult tissues reveals important temporal and regional differences in the abundance of the 10E4 and 3G10 epitopes at these sites. Moreover, the staining pattern of the two antibodies is not always superimposable, which is indicative of regional differences in the exposure or structure of the tissular heparan sulfates. As a whole the results suggest that heparan sulfate abounds at sites of active morphogenesis and that the expression of this glycosaminoglycan is developmentally regulated. PMID:1385449

  19. Isolation of low-molecular-weight heparin/heparan sulfate from marine sources.

    PubMed

    Saravanan, Ramachandran

    2014-01-01

    The glycosaminoglycan (heparin and heparan sulfate) are polyanionic sulfated polysaccharides mostly recognized for its anticoagulant activity. In many countries, low-molecular-weight heparins have replaced the unfractionated heparin, owing to its high bioavailability, half-life, and less adverse effect. The low-molecular-weight heparins differ in mode of preparation (chemical or enzymatic synthesis and chromatography fractionations) and as a consequence in molecular weight distribution, chemical structure, and pharmacological activities. Bovine and porcine body parts are at present used for manufacturing of commercial heparins, and the appearance of mad cow disease and Creutzfeldt-Jakob disease in humans has limited the use of bovine heparin. Consequently, marine organisms come across the new resource for the production of low-molecular-weight heparin and heparan sulfate. The importance of this chapter suggests that the low-molecular-weight heparin and heparan sulfate from marine species could be alternative sources for commercial heparin.

  20. Heparan sulfate biosynthetic gene Ndst1 is required for FGF signaling in early lens development.

    PubMed

    Pan, Yi; Woodbury, Andrea; Esko, Jeffrey D; Grobe, Kay; Zhang, Xin

    2006-12-01

    Multiple signaling molecules, including bone morphogenic proteins (BMP) and fibroblast growth factors (FGF), play important roles in early lens development. However, how these morphogens are regulated is still largely unknown. Heparan sulfate participates in both morphogen transport and morphogen-receptor interaction. In this study, we demonstrate that inactivation of the heparan sulfate biosynthetic gene Ndst1 resulted in invagination defects of the early lens and in the disruption of lens-determination gene expression, leading to severe lens hypoplasia or anophthalmia. Ndst1 mutants exhibited reduced sulfation of heparan sulfate, but both BMP- and Wnt-signaling remained unchanged. Instead, these embryos showed diminished binding of a subset of FGF proteins to FGF receptors. Consistent with disruption of FGF signaling, expression of phospho-Erk and ERM were also downregulated in Ndst1-mutant lenses. Taken together, these results establish an important role of Ndst1 function in FGF signaling during lens development.

  1. Complex Cooperative Functions of Heparan Sulfate Proteoglycans Shape Nervous System Development in Caenorhabditis elegans

    PubMed Central

    Díaz-Balzac, Carlos A.; Lázaro-Peña, María I.; Tecle, Eillen; Gomez, Nathali; Bülow, Hannes E.

    2014-01-01

    The development of the nervous system is a complex process requiring the integration of numerous molecular cues to form functional circuits. Many cues are regulated by heparan sulfates, a class of linear glycosaminoglycan polysaccharides. These sugars contain distinct modification patterns that regulate protein–protein interactions. Misexpressing the homolog of KAL-1/anosmin-1, a neural cell adhesion molecule mutant in Kallmann syndrome, in Caenorhabditis elegans causes a highly penetrant, heparan sulfate–dependent axonal branching phenotype in AIY interneurons. In an extended forward genetic screen for modifiers of this phenotype, we identified alleles in new as well as previously identified genes involved in HS biosynthesis and modification, namely the xylosyltransferase sqv-6, the HS-6-O-sulfotransferase hst-6, and the HS-3-O-sulfotransferase hst-3.2. Cell-specific rescue experiments showed that different HS biosynthetic and modification enzymes can be provided cell-nonautonomously by different tissues to allow kal-1-dependent branching of AIY. In addition, we show that heparan sulfate proteoglycan core proteins that carry the heparan sulfate chains act genetically in a highly redundant fashion to mediate kal-1-dependent branching in AIY neurons. Specifically, lon-2/glypican and unc-52/perlecan act in parallel genetic pathways and display synergistic interactions with sdn-1/syndecan to mediate kal-1 function. Because all of these heparan sulfate core proteins have been shown to act in different tissues, these studies indicate that KAL-1/anosmin-1 requires heparan sulfate with distinct modification patterns of different cellular origin for function. Our results support a model in which a three-dimensional scaffold of heparan sulfate mediates KAL-1/anosmin-1 and intercellular communication through complex and cooperative interactions. In addition, the genes we have identified could contribute to the etiology of Kallmann syndrome in humans. PMID:25098771

  2. Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix.

    PubMed

    Kokenyesi, R

    Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.

  3. The heparanome--the enigma of encoding and decoding heparan sulfate sulfation.

    PubMed

    Lamanna, William C; Kalus, Ina; Padva, Michael; Baldwin, Rebecca J; Merry, Catherine L R; Dierks, Thomas

    2007-04-30

    Heparan sulfate (HS) is a cell surface carbohydrate polymer modified with sulfate moieties whose highly ordered composition is central to directing specific cell signaling events. The ability of the cell to generate these information rich glycans with such specificity has opened up a new field of "heparanomics" which seeks to understand the systems involved in generating these cell type and developmental stage specific HS sulfation patterns. Unlike other instances where biological information is encrypted as linear sequences in molecules such as DNA, HS sulfation patterns are generated through a non-template driven process. Thus, deciphering the sulfation code and the dynamic nature of its generation has posed a new challenge to system biologists. The recent discovery of two sulfatases, Sulf1 and Sulf2, with the unique ability to edit sulfation patterns at the cell surface, has opened up a new dimension as to how we understand the regulation of HS sulfation patterning and pattern-dependent cell signaling events. This review will focus on the functional relationship between HS sulfation patterning and biological processes. Special attention will be given to Sulf1 and Sulf2 and how these key editing enzymes might act in concert with the HS biosynthetic enzymes to generate and regulate specific HS sulfation patterns in vivo. We will further explore the use of knock out mice as biological models for understanding the dynamic systems involved in generating HS sulfation patterns and their biological relevance. A brief overview of new technologies and innovations summarizes advances in the systems biology field for understanding non-template molecular networks and their influence on the "heparanome".

  4. Cytokines and growth factors cross-link heparan sulfate

    PubMed Central

    Migliorini, Elisa; Thakar, Dhruv; Kühnle, Jens; Sadir, Rabia; Dyer, Douglas P.; Li, Yong; Sun, Changye; Volkman, Brian F.; Handel, Tracy M.; Coche-Guerente, Liliane; Fernig, David G.; Lortat-Jacob, Hugues; Richter, Ralf P.

    2015-01-01

    The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors. PMID:26269427

  5. Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans.

    PubMed

    Ortmann, Corinna; Pickhinke, Ute; Exner, Sebastian; Ohlig, Stefanie; Lawrence, Roger; Jboor, Hamodah; Dreier, Rita; Grobe, Kay

    2015-06-15

    All Hedgehog morphogens are released from producing cells, despite being synthesized as N- and C-terminally lipidated molecules, a modification that firmly tethers them to the cell membrane. We have previously shown that proteolytic removal of both lipidated peptides, called shedding, releases bioactive Sonic hedgehog (Shh) morphogens from the surface of transfected Bosc23 cells. Using in vivo knockdown together with in vitro cell culture studies, we now show that glypican heparan sulfate proteoglycans regulate this process, through their heparan sulfate chains, in a cell autonomous manner. Heparan sulfate specifically modifies Shh processing at the cell surface, and purified glycosaminoglycans enhance the proteolytic removal of N- and C-terminal Shh peptides under cell-free conditions. The most likely explanation for these observations is direct Shh processing in the extracellular compartment, suggesting that heparan sulfate acts as a scaffold or activator for Shh ligands and the factors required for their turnover. We also show that purified heparan sulfate isolated from specific cell types and tissues mediates the release of bioactive Shh from pancreatic cancer cells, revealing a previously unknown regulatory role for these versatile molecules in a pathological context.

  6. Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function

    PubMed Central

    Bode, Lars; Salvestrini, Camilla; Park, Pyong Woo; Li, Jin-Ping; Esko, Jeffrey D.; Yamaguchi, Yu; Murch, Simon; Freeze, Hudson H.

    2007-01-01

    Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-γ, TNF-α, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate– or syndecan-1–deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-γ, TNF-α, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients. PMID:18064305

  7. Discovery of a heparan sulfate 3-O-sulfation specific peeling reaction.

    PubMed

    Huang, Yu; Mao, Yang; Zong, Chengli; Lin, Cheng; Boons, Geert-Jan; Zaia, Joseph

    2015-01-06

    Heparan sulfate (HS) 3-O-sulfation determines the binding specificity of HS/heparin for antithrombin III and plays a key role in herpes simplex virus (HSV) infection. However, the low natural abundance of HS 3-O-sulfation poses a serious challenge for functional studies other than the two cases mentioned above. By contrast, multiple distinct isoforms of 3-O-sulfotranserases exist in mammals (up to seven isoenzymes). Here we describe a novel peeling reaction that specifically degrades HS chains with 3-O-sulfated glucosamine at the reducing-end. When HS/heparin is enzymatically depolymerized for compositional analysis, 3-O-sulfated glucosamine at the reducing ends appears to be susceptible to degradation under mildly basic conditions. We propose a 3-O-desulfation initiated peeling reaction mechanism based on the intermediate and side-reaction products observed. Our discovery calls for the re-evaluation of the natural abundance and functions of HS 3-O-sulfation by taking into consideration the negative impact of this novel peeling reaction.

  8. Enterovirus 71 Uses Cell Surface Heparan Sulfate Glycosaminoglycan as an Attachment Receptor

    PubMed Central

    Tan, Chee Wah; Poh, Chit Laa; Sam, I-Ching

    2013-01-01

    Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor. PMID:23097443

  9. Heparan Sulfate Degradation: Relation to Tumor Invasive and Metastatic Properties of Mouse B16 Melanoma Sublines

    NASA Astrophysics Data System (ADS)

    Nakajima, Motowo; Irimura, Tatsuro; di Ferrante, Daniela; di Ferrante, Nicola; Nicolson, Garth L.

    1983-05-01

    After transport in the blood and implantation in the microcirculation, metastatic tumor cells must invade the vascular endothelium and underlying basal lamina. Mouse B16 melanoma sublines were used to determine the relation between metastatic properties and the ability of the sublines to degrade enzymatically the sulfated glycosaminoglycans present in the extracellular matrix of cultured vascular endothelial cells. Highly invasive and metastatic B16 sublines degraded matrix glycosaminoglycans faster than did sublines of lower metastatic potential. The main products of this matrix degradation were heparan sulfate fragments. Intact B16 cells (or their cell-free homogenates) with a high potential for lung colonization degraded purified heparan sulfate from bovine lung at higher rates than did B16 cells with a poor potential for lung colonization. Analysis of the degradation fragments indicated that B16 cells have a heparan sulfate endoglycosidase. Thus the abilities of B16 melanoma cells to extravasate and successfully colonize the lung may be related to their capacities to degrade heparan sulfate in the walls of pulmonary blood vessels.

  10. Fractone-heparan sulfates mediate BMP-7 inhibition of cell proliferation in the adult subventricular zone.

    PubMed

    Douet, Vanessa; Arikawa-Hirasawa, Eri; Mercier, Frederic

    2012-10-24

    Bone morphogenetic protein-7 (BMP-7) is a heparin-binding growth factor that inhibits cell proliferation in the subventricular zone (SVZ) of the lateral ventricle, the primary neurogenic niche in the adult brain. However, the physiological mechanisms regulating the activity of BMP-7 in the SVZ are unknown. Here, we report the inhibitory effect of BMP-7 on cell proliferation through the anterior SVZ after intracerebroventricular injection in the adult mouse. To determine whether the inhibition of cell proliferation induced by BMP-7 is dependant on heparin-binding, heparitinase-1 was intracerebroventricularly injected to N-desulfate heparan sulfate proteoglycans before BMP-7 was injected. Heparatinase-1 drastically reduced the inhibitory effect of BMP-7 on cell proliferation in the SVZ. To determine where BMP-7 binds within the niche, we visualized biotinylated-BMP-7 after intracerebroventricular injection, using streptavidin Texas red on frozen brain sections. BMP-7 binding was seen as puncta in the SVZ at the location of fractones, the particulate specialized extracellular matrix of the SVZ, which have been identified primarily by N-sulfated heparan sulfate immunoreactivity (NS-HS+). BMP binding was also seen in NS-HS+ blood vessels of the SVZ. Injection of heparitinase-1 prior to biotinylated BMP-7 resulted in the absence of signal for biotinylated-BMP-7 in the fractones and blood vessels, indicating that the binding is heparan sulfate dependant. These results indicate that BMP-7 requires heparan sulfates to bind and inhibit cell proliferation in the SVZ neurogenic niche. Heparan sulfates concentrated in fractones and SVZ blood vessels emerge as a functional stem cell niche component involved in growth factor activity.

  11. Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia

    PubMed Central

    Zhang, Bing; Xiao, Wenyuan; Qiu, Hong; Zhang, Fuming; Moniz, Heather A.; Jaworski, Alexander; Condac, Eduard; Gutierrez-Sanchez, Gerardo; Heiss, Christian; Clugston, Robin D.; Azadi, Parastoo; Greer, John J.; Bergmann, Carl; Moremen, Kelley W.; Li, Dean; Linhardt, Robert J.; Esko, Jeffrey D.; Wang, Lianchun

    2013-01-01

    Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development. PMID:24355925

  12. Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides

    PubMed Central

    Hsieh, Po-Hung; Xu, Yongmei; Keire, David A; Liu, Jian

    2014-01-01

    Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. Nuclear magnetic resonance analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin (AT) binding affinities of a GlcA2S- and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to AT, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate. PMID:24770491

  13. Identification of the heparan sulfate binding sites in the cellular prion protein.

    PubMed

    Warner, Richard G; Hundt, Christoph; Weiss, Stefan; Turnbull, Jeremy E

    2002-05-24

    Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.

  14. Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans

    PubMed Central

    1985-01-01

    Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production. PMID:3160714

  15. Expanding the role of 3-O sulfated heparan sulfate in herpes simplex virus type-1 entry

    SciTech Connect

    O'Donnell, Christopher D.; Kovacs, Maria; Akhtar, Jihan; Valyi-Nagy, Tibor; Shukla, Deepak

    2010-02-20

    Heparan sulfate (HS) proteoglycans are commonly exploited by multiple viruses for initial attachment to host cells. Herpes simplex virus-1 (HSV-1) is unique because it can use HS for both attachment and penetration, provided specific binding sites for HSV-1 envelope glycoprotein gD are present. The interaction with gD is mediated by specific HS moieties or 3-O sulfated HS (3-OS HS), which are generated by all but one of the seven isoforms of 3-O sulfotransferases (3-OSTs). Here we demonstrate that several common experimental cell lines express unique sets of 3-OST isoforms. While the isoforms 3-OST-3, -5 and -6 were most commonly expressed, isoforms 3-OST-2 and -4 were undetectable in the cell lines examined. Since most cell lines expressed multiple 3-OST isoforms, we addressed the significance of 3-OS HS in HSV-1 entry by down-regulating 2-O-sulfation, a prerequisite for 3-OS HS formation, by knocking down 2-OST expression by RNA interference (RNAi). 2-OST knockdown was verified by reverse-transcriptase PCR and Western blot analysis, while 3-OS HS knockdown was verified by immunofluorescence. Cells showed a significant decrease in viral entry, suggesting an important role for 3-OS HS. Implicating 3-OS HS further, cells knocked down for 2-OST expression also demonstrated decreased cell-cell fusion when cocultivated with effector cells transfected with HSV-1 glycoproteins. Our findings suggest that 3-OS HS may play an important role in HSV-1 entry into many different cell lines.

  16. The "in and out" of glucosamine 6-O-sulfation: the 6th sense of heparan sulfate.

    PubMed

    El Masri, Rana; Seffouh, Amal; Lortat-Jacob, Hugues; Vivès, Romain R

    2016-11-03

    The biological properties of Heparan sulfate (HS) polysaccharides essentially rely on their ability to bind and modulate a multitude of protein ligands. These interactions involve internal oligosaccharide sequences defined by their sulfation patterns. Amongst these, the 6-O-sulfation of HS contributes significantly to the polysaccharide structural diversity and is critically involved in the binding of many proteins. HS 6-O-sulfation is catalyzed by 6-O-sulfotransferases (6OSTs) during biosynthesis, and it is further modified by the post-synthetic action of 6-O-endosulfatases (Sulfs), two enzyme families that remain poorly characterized. The aim of the present review is to summarize the contribution of 6-O-sulfates in HS structure/function relationships and to discuss the present knowledge on the complex mechanisms regulating HS 6-O-sulfation.

  17. Homocysteine, a thrombogenic agent, suppresses anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells.

    PubMed

    Nishinaga, M; Ozawa, T; Shimada, K

    1993-09-01

    Previous studies showed that homocysteine, a thrombo-atherogenic and atherogenic agent, inhibits an endothelial thrombomodulin-protein C anticoagulant pathway. We examined whether homocysteine might affect another endothelial anticoagulant mechanism; i.e., heparin-like glycosaminoglycan-antithrombin III interactions. Incubations of porcine aortic endothelial cell cultures with homocysteine reduced the amount of antithrombin III bound to the cell surface in a dose- and time-dependent fashion. The inhibitory effect was observed at a homocysteine concentration as low as 0.1 mM, and the maximal suppression occurred at 1 mM of homocysteine after 24 h. In contrast with a marked reduction in the maximal antithrombin III binding capacity (approximately 30% of control), the radioactivity of [35S]sulfate incorporated into heparan sulfate on the cell surface was minimally (< 15%) reduced. The cells remained viable after homocysteine treatment. Although neither net negative charge nor proportion in total glycosaminoglycans of cell surface heparan sulfate was altered by homocysteine treatment, a substantial reduction in antithrombin III binding capacity of heparan sulfate isolated from homocysteine-treated endothelial cells was found using both affinity chromatography and dot blot assay techniques. The antithrombin III binding activity of endothelial cells decreased after preincubation with 1 mM homocysteine, cysteine, or 2-mercaptoethanol; no reduction in binding activity was observed after preincubation with the same concentration of methionine, alanine, or valine. This sulfhydryl effect may be caused by generation of hydrogen peroxide, as incubation of catalase, but not superoxide dismutase, with homocysteine-treated endothelial cells prevented this reduction, whereas copper augmented the inhibitory effects of the metabolite. Thus, our data suggest that the inhibited expression of anticoagulant heparan sulfate may contribute to the thrombogenic property resulting from the

  18. Podocyte-specific deletion of NDST1, a key enzyme in the sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo.

    PubMed

    Sugar, Terrel; Wassenhove-McCarthy, Deborah J; Esko, Jeffrey D; van Kuppevelt, Toin H; Holzman, Lawrence; McCarthy, Kevin J

    2014-02-01

    Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to--and pedicel organization on--the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1(-/-)) was developed, having podocyte-specific deletion of Ndst1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1(-/-) mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

  19. Heparan sulfates in the lung: structure, diversity, and role in pulmonary emphysema.

    PubMed

    Smits, Nicole C; Shworak, Nicholas W; Dekhuijzen, P N Richard; van Kuppevelt, Toin H

    2010-06-01

    There is an emerging interest in the extracellular matrix (ECM) of the lung, especially in the role it plays in development and disease. There is a rapid change from the classical view of the ECM as a supporting structure towards a view of the ECM as a regulatory entity with profound effects on proliferation, migration, and differentiation of pulmonary cells. In the ECM, a variety of molecules is present in a highly organized pattern. Next to the abundant fiber-forming molecules such as collagens and elastin, a large number of less abundant molecules are part of the ECM, including proteoglycans. In this review, we will focus on one class of proteoglycans, the heparan sulfate proteoglycans. We will particularly address the structure, biosynthesis, and function of their saccharide moiety, the heparan sulfates, including their role in development and (patho)physiology.

  20. ELECTRON DETACHMENT DISSOCIATION OF SYNTHETIC HEPARAN SULFATE GLYCOSAMINOGLYCAN TETRASACCHARIDES VARYING IN DEGREE OF SULFATION AND HEXURONIC ACID STEREOCHEMISTRY.

    PubMed

    Leach, Franklin E; Arungundram, Sailaja; Al-Mafraji, Kanar; Venot, Andre; Boons, Geert-Jan; Amster, I Jonathan

    2012-12-15

    Glycosaminoglycan (GAG) carbohydrates provide a challenging analytical target for structural determination due to their polydisperse nature, non-template biosynthesis, and labile sulfate modifications. The resultant structures, although heterogeneous, contain domains which indicate a sulfation pattern or code that correlates to specific function. Mass spectrometry, in particular electron detachment dissociation Fourier transform ion cyclotron resonance (EDD FT-ICR MS), provides a highly sensitive platform for GAG structural analysis by providing cross-ring cleavages for sulfation location and product ions specific to hexuronic acid stereochemistry. To investigate the effect of sulfation pattern and variations in stereochemistry on EDD spectra, a series of synthetic heparan sulfate (HS) tetrasaccharides are examined. Whereas previous studies have focused on lowly sulfated compounds (0.5-1 sulfate groups per disaccharide), the current work extends the application of EDD to more highly sulfated tetrasaccharides (1-2 sulfate groups per disaccharide) and presents the first EDD of a tetrasaccharide containing a sulfated hexuronic acid. For these more highly sulfated HS oligomers, alternative strategies are shown to be effective for extracting full structural details. These strategies inlcude sodium cation replacement of protons, for determining the sites of sulfation, and desulfation of the oligosaccharides for the generation of product ions for assigning uronic acid stereochemistry.

  1. Incorporation of /sup 35/S-sulfate and /sup 3/H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells

    SciTech Connect

    Blair, O.C.; Sartorelli, A.C.

    1984-05-01

    Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of /sup 35/S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of /sup 35/S-sulfate and /sup 3/H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M.

  2. Fluorescent Tagged Heparan Sulfate Precursor Oligosaccharides to Probe the Enzymatic Action of Heparitinase I

    PubMed Central

    Babu, Ponnusamy; Kuberan, Balagurunathan

    2009-01-01

    Heparitinase I, a key lyase enzyme essential for structural analysis of heparan sulfate (HS), degrades HS domains which are undersulfated at glucuronyl residues through an elimination mechanism. Earlier studies employed viscosimetric measurements and electrophoresis to deduce the mechanism of action of heparitinase I and two other related lyases, heparitinase II and heparitinase III. However, these findings lack molecular evidence for the intermediates formed and could not distinguish whether the cleavage occurred from the reducing end or non-reducing end. In this report, 2-aminoacridone (2-AMAC) labeled HS precursor oligosaccharides of various sizes were prepared to investigate the mechanism of heparitinase I mediated depolymerization using sensitive and quantitative methodologies. Furthermore, fluorescence (2-AMAC) tagging of heparan sulfate precursor oligosaccharides allowed us to distinguish fragments that result from cleavage of the substrates at various time intervals and sites further away from the reducing and non-reducing ends of oligosaccharide substrates. This study provides first direct molecular evidence for a predominantly random endolytic mechanism of elimination of HS precursor oligosaccharides by heparitinase I. This robust strategy can be adapted to deduce the mechanism of action of other heparitinases and also to deduce structural information of complex heparan sulfate oligosaccharides of biological importance. PMID:19732739

  3. Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning

    PubMed Central

    Saez, Borja; Ferraro, Francesca; Yusuf, Rushdia Z.; Cook, Colleen M.; Yu, Vionnie W. C.; Pardo-Saganta, Ana; Sykes, Stephen M.; Palchaudhuri, Rahul; Schajnovitz, Amir; Lotinun, Sutada; Lymperi, Stefania; Mendez-Ferrer, Simon; del Toro, Raquel; Day, Robyn; Vasic, Radovan; Acharya, Sanket S.; Baron, Roland; Lin, Charles P.; Yamaguchi, Yu; Wagers, Amy J.

    2014-01-01

    The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions. PMID:25202142

  4. Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans

    PubMed Central

    Blanchette, Cassandra R.; Thackeray, Andrea; Perrat, Paola N.; Hekimi, Siegfried; Bénard, Claire Y.

    2017-01-01

    The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development. PMID:28068429

  5. The role of heparan sulfate and perlecan in bone-regenerative procedures.

    PubMed

    DeCarlo, A A; Whitelock, J M

    2006-02-01

    Tissue engineering, grafting procedures, regeneration, and tissue remodeling are developing therapeutic modalities with great potential medical value, but these regenerative modalities are not as effective or predictable as clinicians and patients would like. Greater understanding of growth factors, cytokines, extracellular matrix molecules, and their roles in cell-mediated healing processes have made these regenerative therapies more clinically viable and will continue advancing the fields of tissue engineering and grafting. However, millions of oral and non-oral bone-grafting procedures are performed annually, and only a small percentage yield the most desirable results. Here we review the heparan-sulfate-decorated extracellular biomolecule named perlecan and the research relating to its potential as an adjunct in bone-regenerative procedures. The review includes an overview of bone graft substitutes and biological adjuncts to bone-regenerative procedures in medicine as they apply to periodontal disease, alveolar ridge augmentation, and barrier membrane therapy. Perlecan is discussed as a potential biological adjunct in terms of growth factor sequestration and delivery, and promoting cell adhesion, proliferation, differentiation, and angiogenesis. Further, we propose delivery and application schemes for perlecan and/or its domains in bone-regenerative procedures, with particular emphasis on its heparan-sulfate-decorated domain I. The perlecan molecule, with its heparan sulfate glycosylation, may provide a multi-faceted approach for the delivery of a more comprehensive stimulus than other single potential adjuncts currently available for bone-regenerative procedures.

  6. Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate.

    PubMed

    Shah, Mita M; Sakurai, Hiroyuki; Gallegos, Thomas F; Sweeney, Derina E; Bush, Kevin T; Esko, Jeffrey D; Nigam, Sanjay K

    2011-08-01

    Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific

  7. Sulf loss influences N-, 2-O-, and 6-O-sulfation of multiple heparan sulfate proteoglycans and modulates fibroblast growth factor signaling.

    PubMed

    Lamanna, William C; Frese, Marc-André; Balleininger, Martina; Dierks, Thomas

    2008-10-10

    Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes.

  8. Investigating the mechanism of the assembly of FGF1-binding heparan sulfate motifs

    PubMed Central

    Nguyen, Thao Kim Nu; Raman, Karthik; Trana, Vy My; Kuberan, Balagurunathan

    2011-01-01

    Heparan sulfate (HS) chains play crucial biological roles by binding to various signaling molecules including fibroblast growth factors (FGFs). Distinct sulfation patterns of HS chains are required for their binding to FGFs/FGF receptors (FGFRs). These sulfation patterns are putatively regulated by biosynthetic enzyme complexes, called GAGOSOMES, in the Golgi. While the structural requirements of HS-FGF interactions have been described previously, it is still unclear how the FGF-binding motif is assembled in vivo. In this study, we generated HS structures using biosynthetic enzymes in a sequential or concurrent manner to elucidate the potential mechanism by which the FGF1-binding HS motif is assembled. Our results indicate that the HS chains form ternary complexes with FGF1/FGFR when enzymes carry out modifications in a specific manner. PMID:21803043

  9. Combining measurements to estimate properties and characterization extent of complex biochemical mixtures; applications to Heparan Sulfate

    NASA Astrophysics Data System (ADS)

    Pradines, Joël R.; Beccati, Daniela; Lech, Miroslaw; Ozug, Jennifer; Farutin, Victor; Huang, Yongqing; Gunay, Nur Sibel; Capila, Ishan

    2016-04-01

    Complex mixtures of molecular species, such as glycoproteins and glycosaminoglycans, have important biological and therapeutic functions. Characterization of these mixtures with analytical chemistry measurements is an important step when developing generic drugs such as biosimilars. Recent developments have focused on analytical methods and statistical approaches to test similarity between mixtures. The question of how much uncertainty on mixture composition is reduced by combining several measurements still remains mostly unexplored. Mathematical frameworks to combine measurements, estimate mixture properties, and quantify remaining uncertainty, i.e. a characterization extent, are introduced here. Constrained optimization and mathematical modeling are applied to a set of twenty-three experimental measurements on heparan sulfate, a mixture of linear chains of disaccharides having different levels of sulfation. While this mixture has potentially over two million molecular species, mathematical modeling and the small set of measurements establish the existence of nonhomogeneity of sulfate level along chains and the presence of abundant sulfate repeats. Constrained optimization yields not only estimations of sulfate repeats and sulfate level at each position in the chains but also bounds on these levels, thereby estimating the extent of characterization of the sulfation pattern which is achieved by the set of measurements.

  10. Heparan Sulfate Proteoglycans and Their Binding Proteins in Embryo Implantation and Placentation

    PubMed Central

    Kirn-Safran, Catherine; D’Souza, Sonia S.; Carson, Daniel D.

    2008-01-01

    Complex interactions occur among embryonic, placental and maternal tissues during embryo implantation. Many of these interactions are controlled by growth factors, extracellular matrix and cell surface components that share the ability to bind heparan sulfate (HS) polysaccharides. HS is carried by several classes of cell surface and secreted proteins called HS proteoglycan that are expressed in restricted patterns during implantation and placentation. This review will discuss the expression of HS proteoglycans and various HS binding growth factors as well as extracellular matrix components and HS-modifying enzymes that can release HS-bound proteins in the context of implantation and placentation. PMID:17766150

  11. Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

    PubMed Central

    Shimada, K; Ozawa, T

    1985-01-01

    It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycosaminoglycans such as heparan sulfate, which could serve as antithrombin cofactor on the endothelium. In the present study, we have directly evaluated the binding of 125I-labeled bovine thrombin to cultured porcine aortic endothelial cells. The thrombin binding to the cell surface was rapid, reversible, and displaced by enzymatically inactive diisopropylphosphoryl-thrombin. The concentration of thrombin at half-maximal binding was approximately 20 nM. Both specific and nonspecific binding of 125I-thrombin to the endothelial cell surface was partially inhibited in the presence of protamine sulfate, after the removal of cell surface heparan sulfate by the treatment of cells with crude Flavobacterium heparinum enzyme or purified heparitinase. The binding as a function of the concentration of thrombin revealed that the maximal amount of specific binding was reduced by approximately 50% with little alteration in binding affinity by these enzymatic treatments. The reversibility and active-site independence as well as the rate of the binding did not change after heparitinase treatment. Whereas removal of chondroitin sulfates by chondroitin ABC lyase treatment of cells did not affect the binding, identical enzymatic treatments of [35S]sulfate-labeled cells showed that either heparan sulfate or chondroitin sulfate was selectively and completely removed from the cell surface by heparitinase or chondroitin ABC lyase treatment, respectively. Furthermore, proteolysis of cell surface proteins by the purified glycosaminoglycan lyases was excluded by the identical enzymatic treatments of [3H]leucine-labeled or cell surface radioiodinated cells. Our results provide the first direct evidence that heparan sulfate on the cell surface is involved in the high-affinity, active site-independent thrombin binding by endothelial cells, and also suggest the presence of thrombin

  12. Influencing hematopoietic differentiation of mouse embryonic stem cells using soluble heparin and heparan sulfate saccharides.

    PubMed

    Holley, Rebecca J; Pickford, Claire E; Rushton, Graham; Lacaud, Georges; Gallagher, John T; Kouskoff, Valerie; Merry, Catherine L R

    2011-02-25

    Heparan sulfate proteoglycans (HSPG) encompass some of the most abundant macromolecules on the surface of almost every cell type. Heparan sulfate (HS) chains provide a key interaction surface for the binding of numerous proteins such as growth factors and morphogens, helping to define the ability of a cell to respond selectively to environmental cues. The specificity of HS-protein interactions are governed predominantly by the order and positioning of sulfate groups, with distinct cell types expressing unique sets of HS epitopes. Embryos deficient in HS-synthesis (Ext1(-/-)) exhibit pre-gastrulation lethality and lack recognizable organized mesoderm and extraembryonic tissues. Here we demonstrate that embryonic stem cells (ESCs) derived from Ext1(-/-) embryos are unable to differentiate into hematopoietic lineages, instead retaining ESC marker expression throughout embryoid body (EB) culture. However hematopoietic differentiation can be restored by the addition of soluble heparin. Consistent with specific size and composition requirements for HS:growth factor signaling, chains measuring at least 12 saccharides were required for partial rescue of hematopoiesis with longer chains (18 saccharides or more) required for complete rescue. Critically N- and 6-O-sulfate groups were essential for rescue. Heparin addition restored the activity of multiple signaling pathways including bone morphogenic protein (BMP) with activation of phospho-SMADs re-established by the addition of heparin. Heparin addition to wild-type cultures also altered the outcome of differentiation, promoting hematopoiesis at low concentrations, yet inhibiting blood formation at high concentrations. Thus altering the levels of HS and HS sulfation within differentiating ESC cultures provides an attractive and accessible mechanism for influencing cell fate.

  13. Liposomal delivery of doxorubicin to hepatocytes in vivo by targeting heparan sulfate

    PubMed Central

    Longmuir, Kenneth J.; Haynes, Sherry M.; Baratta, Janie L.; Kasabwalla, Natasha; Robertson, Richard T.

    2009-01-01

    Previous work demonstrated that liposomes, containing an amino acid sequence that binds to hepatic heparan sulfate glycosaminoglycan, show effective targeting to liver hepatocytes. These liposomes were tested to determine whether they can deliver doxorubicin selectively to liver and hepatocytes in vivo. Fluid-phase liposomes contained a lipid-anchored 19-amino acid glycosaminoglycan targeting peptide. Liposomes were loaded with doxorubicin and were non-leaky in the presence of serum. After intravenous administration to mice, organs were harvested and the doxorubicin content extracted and measured by fluorescence intensity and by fluorescence microscopy. The liposomal doxorubicin was recovered almost entirely from liver, with only trace amounts detectable in heart, lung, and kidney. Fluorescence microscopy demonstrated doxorubicin preferentially in hepatocytes, also in non-parenchymal cells of the liver, but not in cells of heart, lung or kidney. The doxorubicin was localized within liver cell nuclei within five minutes after intravenous injection. These studies demonstrated that liposomal doxorubicin can be effectively delivered to hepatocytes by targeting the heparan sulfate glycosaminoglycan of liver tissue. With the composition described here, the doxorubicin was rapidly released from the liposomes without the need for an externally supplied stimulus. PMID:19664697

  14. Interactions between heparan sulfate and proteins-design and functional implications.

    PubMed

    Lindahl, Ulf; Li, Jin-ping

    2009-01-01

    Heparan sulfate (HS) proteoglycans at cell surfaces and in the extracellular matrix of most animal tissues are essential in development and homeostasis, and variously implicated in disease processes. Functions of HS polysaccharide chains depend on ionic interactions with a variety of proteins including growth factors and their receptors. Negatively charged sulfate and carboxylate groups are arranged in various types of domains, generated through strictly regulated biosynthetic reactions and with enormous potential for structural variability. The level of specificity of HS-protein interactions is assessed through binding experiments in vitro using saccharides of defined composition, signaling assays in cell culture, and targeted disruption of genes for biosynthetic enzymes followed by phenotype analysis. While some protein ligands appear to require strictly defined HS structure, others bind to variable saccharide domains without any apparent dependence on distinct saccharide sequence. These findings raise intriguing questions concerning the functional significance of regulation in HS biosynthesis.

  15. The Solution Structure of Heparan Sulfate Differs from That of Heparin

    PubMed Central

    Khan, Sanaullah; Rodriguez, Elizabeth; Patel, Rima; Gor, Jayesh; Mulloy, Barbara; Perkins, Stephen J.

    2011-01-01

    The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments degree of polymerization 6–18 (dp6–dp18) and dp24, corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s20,w of HS dp6–dp24 showed a small rotor speed dependence, where similar s20,w values of 0.82–1.26 S (absorbance optics) and 1.05–1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6–dp24 gave radius of gyration (RG) values from 1.03 to 2.82 nm, cross-sectional radius of gyration (RXS) values from 0.31 to 0.65 nm, and maximum lengths (L) from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5000–8000 conformationally randomized HS structures gave best fit dp6–dp16 molecular structures that were longer and more bent than their equivalents in heparin. No fits were obtained for HS dp18 or dp24, indicating their higher flexibility. We conclude that HS displays an extended bent conformation that is significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin. PMID:21576246

  16. The Solution Structure of Heparan Sulfate Differs from That of Heparin

    PubMed Central

    Khan, Sanaullah; Fung, Ka Wai; Rodriguez, Elizabeth; Patel, Rima; Gor, Jayesh; Mulloy, Barbara; Perkins, Stephen J.

    2013-01-01

    The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments dp6–dp24 corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s20,w of HS dp6–dp24 showed a small rotor speed dependence, where similar s20,w values of 0.82–1.26 S (absorbance optics) and 1.05–1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6–dp24 gave radii of gyration RG values from 1.03 to 2.82 nm, cross-sectional radii of gyration RXS values from 0.31 to 0.65 nm, and maximum lengths L from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5,000 to 12,000 conformationally randomized HS structures gave best fit dp6–dp24 molecular structures that were longer and more bent than their equivalents in heparin. Alternative fits were obtained for HS dp18 and dp24, indicating their higher bending and flexibility. We conclude that HS displays bent conformations that are significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin. PMID:23921391

  17. Heparan sulfate inhibits hematopoietic stem and progenitor cell migration and engraftment in mucopolysaccharidosis I.

    PubMed

    Watson, H Angharad; Holley, Rebecca J; Langford-Smith, Kia J; Wilkinson, Fiona L; van Kuppevelt, Toin H; Wynn, Robert F; Wraith, J Edmond; Merry, Catherine L R; Bigger, Brian W

    2014-12-26

    Mucopolysaccharidosis I Hurler (MPSI-H) is a pediatric lysosomal storage disease caused by genetic deficiencies in IDUA, coding for α-l-iduronidase. Idua(-/-) mice share similar clinical pathology with patients, including the accumulation of the undegraded glycosaminoglycans (GAGs) heparan sulfate (HS), and dermatan sulfate (DS), progressive neurodegeneration, and dysostosis multiplex. Hematopoietic stem cell transplantation (HSCT) is the most effective treatment for Hurler patients, but reduced intensity conditioning is a risk factor in transplantation, suggesting an underlying defect in hematopoietic cell engraftment. HS is a co-receptor in the CXCL12/CXCR4 axis of hematopoietic stem and progenitor cell (HSPC) migration to the bone marrow (BM), but the effect of HS alterations on HSPC migration, or the functional role of HS in MPSI-H are unknown. We demonstrate defective WT HSPC engraftment and migration in Idua(-/-) recipient BM, particularly under reduced intensity conditioning. Both intra- but especially extracellular Idua(-/-) BM HS was significantly increased and abnormally sulfated. Soluble heparinase-sensitive GAGs from Idua(-/-) BM and specifically 2-O-sulfated HS, elevated in Idua(-/-) BM, both inhibited CXCL12-mediated WT HSPC transwell migration, while DS had no effect. Thus we have shown that excess overly sulfated extracellular HS binds, and sequesters CXCL12, limiting hematopoietic migration and providing a potential mechanism for the limited scope of HSCT in Hurler disease.

  18. Pectin of Prunus domestica L. alters sulfated structure of cell-surface heparan sulfate in differentiated Caco-2 cells through stimulation of heparan sulfate 6-O-endosulfatase-2.

    PubMed

    Nishida, Mitsutaka; Murata, Kazuma; Kanamaru, Yoshihiro; Yabe, Tomio

    2014-01-01

    Although previous reports have suggested that pectin induces morphological changes of the small intestine in vivo, the molecular mechanisms have not been elucidated. As heparan sulfate plays important roles in development of the small intestine, to verify the involvement of heparan sulfate (HS) in the pectin-induced morphological changes of the small intestine, the effects of pectin from Prunus domestica L. on cell-surface HS were investigated using differentiated Caco-2 cells. Disaccharide compositional analysis revealed that sulfated structures of HS were markedly changed by pectin administration. Real-time RT-PCR showed that pectin upregulated human HS 6-O-endosulfatase-2 (HSulf-2) expression and markedly inhibited HSulf-1 expression. Furthermore, inhibition analysis suggested that pretreatment with fibronectin III1C fragment, RGD peptide, and ERK1/2 inhibitor suppressed pectin-induced HSulf-2 expression. These observations indicate that pectin induced the expression of HSulf-2 through the interaction with fibronectin, α5β1 integrin, and ERK1/2, thereby regulating the sulfated structure of HS on differentiated Caco-2 cells.

  19. Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the fibroblast growth factor receptor complex.

    PubMed

    Kan, M; Wang, F; To, B; Gabriel, J L; McKeehan, W L

    1996-10-18

    Polypeptides of the fibroblast growth factor (FGF) family are ubiquitous bioregulators within tissues whose activity is controlled by heparan sulfates within the pericellular matrix. FGF and the ectodomain of their transmembrane tyrosine kinase receptors (FGFR) exhibit heparin-binding domains that when juxtaposed in a FGF middle dotFGFR complex can accommodate a single, potentially bivalent, decameric polysaccharide chain in a ternary complex. Here we show that the interaction of heparin with FGF ligands is not affected by divalent cations. In contrast, the high affinity interaction (apparent Kd = 10 nM) of heparin with FGFR requires Ca2+ or Mg2+ at physiological concentrations. Divalent cations maintain FGFR in a heparan sulfate-dependent state in respect to FGF binding and an FGF- and heparan sulfate-dependent state in respect to autophosphorylation. A model is proposed where divalent cations and heparan sulfate cooperate to maintain FGFR in a conformation that restricts trans-phosphorylation between intracellular kinase domains. The restriction is overcome by FGF or constitutively as a common consequence of diverse mutations in FGFR associated with skeletal and craniofacial abnormalities.

  20. Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells.

    PubMed

    Levings, Daniel C; Arashiro, Takeshi; Nakato, Hiroshi

    2016-03-15

    Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub-GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control.

  1. Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells

    PubMed Central

    Levings, Daniel C.; Arashiro, Takeshi; Nakato, Hiroshi

    2016-01-01

    Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub–GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control. PMID:26792837

  2. Synthetic heparan sulfate dodecasaccharides reveal single sulfation site interconverts CXCL8 and CXCL12 chemokine biology.

    PubMed

    Jayson, Gordon C; Hansen, Steen U; Miller, Gavin J; Cole, Claire L; Rushton, Graham; Avizienyte, Egle; Gardiner, John M

    2015-09-18

    The multigram-scale synthesis of a sulfation-site programmed heparin-like dodecasaccharide is described. Evaluation alongside dodecasaccharides lacking this single glucosamine O6-sulfation, or having per-O6-sulfation, shows that site-specific modification of the terminal glucosamine dramatically interconverts regulation of in vitro and in vivo biology mediated by the two important chemokines, CXCL12 (SDF1α) or CXCL8 (IL-8).

  3. Preparation and characterization of 15N-enriched, size-defined heparan sulfate precursor oligosaccharides

    PubMed Central

    Sigulinsky, Crystal; Babu, Ponnusamy; Victor, Xylophone V.; Kuberan, Balagurunathan

    2009-01-01

    We report the preparation of size-defined [15N]N-acetylheparosan oligosaccharides from Escherichia coli-derived 15N-enriched N-acetylheparosan. Optimized growth conditions of E. coli in minimal media containing 15NH4Cl yielded [15N]N-acetylheparosan on a preparative scale. Depolymerization of [15N]N-acetylheparosan by heparitinase I yielded resolvable, even-numbered oligosaccharides ranging from disaccharide to icosaccharide. Anion-exchange chromatography-assisted fractionation afforded size-defined [15N]N-acetylheparosan oligosaccharides identifiable by ESI-TOFMS. These isotopically labeled oligosaccharides will prove to be valuable research tools for the chemoenzymatic synthesis of heparin and heparan sulfate oligosaccharides and for the study of their structural biology. PMID:19945695

  4. Characterization of Heparan Sulfate Proteoglycan-positive Recycling Endosomes Isolated from Glioma Cells

    PubMed Central

    A. PODYMA-INOUE, KATARZYNA; MORIWAKI, TAKUYA; R. RAJAPAKSHE, ANUPAMA; TERASAWA, KAZUE; HARA-YOKOYAMA, MIKI

    2016-01-01

    Background: Heparan sulfate proteoglycans (HSPGs)-dependent endocytic events have been involved in glioma progression. Thus, comprehensive understanding of the intracellular trafficking complexes formed in presence of HSPGs would be important for development of glioma treatments. Materials and Methods: Subcellular fractionation was used to separate vesicles containing HSPGs from the rat C6 glioma cell line. Isolated HSPG-positive vesicles were further characterized with liquid chromatography-mass spectrometry. Results: The HSPG-positive vesicular fractions, distinct from plasma membrane-derived material, were enriched in endocytic marker, Rab11. Proteomic analysis identified more than two hundred proteins to be associated with vesicular membrane, among them, over eighty were related to endosomal uptake, recycling or vesicular transport. Conclusion: Part of HSPGs in glioma cells is internalized through clathrin-dependent endocytosis and undergo recycling. The development of compounds regulating HSPG-mediated trafficking will likely enable design of effective glioma treatment. PMID:27807067

  5. Important role of heparan sulfate in postnatal islet growth and insulin secretion

    SciTech Connect

    Takahashi, Iwao; Noguchi, Naoya; Nata, Koji; Yamada, Shuhei; Kaneiwa, Tomoyuki; Mizumoto, Shuji; Ikeda, Takayuki; Sugihara, Kazushi; Asano, Masahide; Yoshikawa, Takeo; Yamauchi, Akiyo; Shervani, Nausheen Jamal; Uruno, Akira; Kato, Ichiro; Unno, Michiaki; Sugahara, Kazuyuki; Takasawa, Shin; and others

    2009-05-22

    Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet {beta}-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in {beta}-cells. These mice exhibited abnormal islet morphology with reduced {beta}-cell proliferation after 1 week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.

  6. Enzyme overexpression – an exercise toward understanding regulation of heparan sulfate biosynthesis

    PubMed Central

    Fang, Jianping; Song, Tianyi; Lindahl, Ulf; Li, Jin-Ping

    2016-01-01

    Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process. PMID:27511124

  7. Functions of Heparan Sulfate Proteoglycans in Development: Insights From Drosophila Models.

    PubMed

    Nakato, H; Li, J-P

    2016-01-01

    Heparan sulfate proteoglycans (HSPGs) are a class of carbohydrate-modified proteins involved in key biological processes, including growth factor signaling, cell adhesion, and enzymatic catalysis. HSPGs serve as coreceptors for a number of ligand molecules to regulate their signaling and distribution. These HS-dependent factors include fibroblast growth factors, bone morphogenetic proteins, Wnt-related factors, hedgehog, and cytokines. Several classes of HSPGs are evolutionarily conserved from humans to the genetically tractable model organism Drosophila. Sophisticated molecular genetic tools available in Drosophila provide for a powerful system to address unanswered questions regarding in vivo functions of HSPGs. These studies have highlighted the functions of HSPGs in the regulation of significant developmental events, such as morphogen gradient formation, nervous system formation, and the stem cell niche. Drosophila genetics has also established HSPGs as key factors in feedback controls that ensure robustness in developmental systems.

  8. Single‐Molecule Fluorescence Detection of a Synthetic Heparan Sulfate Disaccharide

    PubMed Central

    Dalton, Charlotte E.; Quinn, Steven D.; Rafferty, Aidan; Morten, Michael J.

    2016-01-01

    Abstract The first single‐molecule fluorescence detection of a structurally‐defined synthetic carbohydrate is reported: a heparan sulfate (HS) disaccharide fragment labeled with Alexa488. Single molecules have been measured whilst freely diffusing in solution and controlled encapsulation in surface‐tethered lipid vesicles has allowed extended observations of carbohydrate molecules down to the single‐molecule level. The diverse and dynamic nature of HS–protein interactions means that new tools to investigate pure HS fragments at the molecular level would significantly enhance our understanding of HS. This work is a proof‐of‐principle demonstration of the feasibility of single‐molecule studies of synthetic carbohydrates which offers a new approach to the study of pure glycosaminoglycan (GAG) fragments. PMID:27538128

  9. Differential Interactions of FGFs with Heparan Sulfate Control Gradient Formation and Branching Morphogenesis

    PubMed Central

    Makarenkova, Helen P.; Hoffman, Matthew P.; Beenken, Andrew; Eliseenkova, Anna V.; Meech, Robyn; Tsau, Cindy; Patel, Vaishali N.; Lang, Richard A.; Mohammadi, Moosa

    2010-01-01

    The developmental activities of morphogens are controlled by the gradients that they form in the extracellular matrix (ECM). In this report, we show that differences in the binding of fibroblast growth factor 7 (FGF7) and FGF10 to heparan sulfate (HS) underlie the formation of different gradients that dictate distinct activities during branching morphogenesis. Reducing the binding affinity of FGF10 for HS by mutating a single residue in its HS-binding pocket converted FGF10 into a functional mimic of FGF7 with respect to gradient formation and regulation of branching morphogenesis; in particular by causing lacrimal and salivary gland epithelium to branch rather than elongate. In contrast, mutations that reduced the affinity of the FGF10 for its receptor affected the extent, but not the nature, of the response. Our data may provide a general model for understanding how binding to HS regulates other morphogenetic gradients. PMID:19755711

  10. Intermediate heparan sulfate binding during HPV-16 infection in HaCaTs.

    PubMed

    Kumar, Annandita; Jacob, Taylor; Abban, Cynthia Y; Meneses, Patricio I

    2014-01-01

    Human papillomavirus (HPV) is the most prevalent sexually transmitted disease in the United States and can cause cancer with persistent infection. The most common cancer caused by HPV is cervical carcinoma with an average of 12,000 cases reported every year in the United States. Worldwide, over 500,000 cases of cervical cancer are reported yearly with over 250,000 deaths attributed to the disease. Although much is known about the serious health risks associated with HPV infection, there is still much to be discovered about how HPV binds and enters target cells. Understanding is required on how HPV infections will lead to strategies and therapies for reducing the number of infections and HPV-related diseases, including cancers. The HPV viral particle is composed of 2 viral proteins, L1 and L2. Data suggest that binding of the viral capsid to cells is dependent on the L1 protein. We hypothesize that this initial binding to a heparan sulfate is composed of 2 independent events: the first results in a structural change that exposes a hidden portion of the L1 protein leading to a second binding event on the heparan sulfate. Our experiments tested if this "hidden" portion of L1 is necessary for infection and explored the nature of this binding. We generated a peptide with the sequence of the "hidden" portion of L1. Infection of HaCaT cells in the presence of this peptide is highly reduced. Our results suggest that the binding of the L1 C-terminal domain is dependent on amino acid sequence and is necessary for infection.

  11. Epitope mapping by a Wnt-blocking antibody: evidence of the Wnt binding domain in heparan sulfate

    PubMed Central

    Gao, Wei; Xu, Yongmei; Liu, Jian; Ho, Mitchell

    2016-01-01

    Heparan sulfate (HS) is a polysaccharide known to modulate many important biological processes, including Wnt signaling. However, the biochemical interaction between HS and Wnt molecules is not well characterized largely due to the lack of suitable methods. To determine the Wnt binding domain in HS, we used a Wnt signaling-inhibitory antibody (HS20) and a panel of synthetic HS oligosaccharides with distinct lengths and sulfation modifications. We found that the binding of HS20 to heparan sulfate required sulfation at both the C2 position (2-O-sulfation) and C6 position (6-O-sulfation). The oligosaccharides with the greatest competitive effect for HS20 binding were between six and eight saccharide residues in length. Additionally, a four residue-long oligosaccharide could also be recognized by HS20 if an additional 3-O-sulfation modification was present. Furthermore, similar oligosaccharides with 2-O, 6-O and 3-O-sulfations showed inhibition for Wnt activation. These results have revealed that HS20 and Wnt recognize a HS structure containing IdoA2S and GlcNS6S, and that the 3-O-sulfation in GlcNS6S3S significantly enhances the binding of both HS20 and Wnt. This study provides the evidence for identifying the Wnt binding domain in HS and suggests a therapeutic approach to target the interaction of Wnt and HS in cancer and other diseases. PMID:27185050

  12. Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche.

    PubMed

    Gupta, P; Oegema, T R; Brazil, J J; Dudek, A Z; Slungaard, A; Verfaillie, C M

    1998-12-15

    Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P <.005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34(+)/HLA-DR- cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34(+) cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective

  13. Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

    PubMed Central

    Zaiss, Anne K.; Foley, Erin M.; Lawrence, Roger; Schneider, Lina S.; Hoveida, Hamidreza; Secrest, Patrick; Catapang, Arthur B.; Yamaguchi, Yu; Alemany, Ramon; Shayakhmetov, Dmitry M.; Esko, Jeffrey D.

    2015-01-01

    ABSTRACT Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1HEP mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1HEP mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1HEP mice. FX remained essential for Ad5 transduction in vivo in Ext1HEP mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. IMPORTANCE Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These

  14. Heparan Sulfates and Coxsackievirus-Adenovirus Receptor: Each One Mediates Coxsackievirus B3 PD Infection

    PubMed Central

    Zautner, A. E.; Körner, U.; Henke, A.; Badorff, C.; Schmidtke, M.

    2003-01-01

    Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-O-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR. PMID:12941917

  15. Synthesis of 3-O-sulfonated heparan sulfate octasaccharides that inhibit the herpes simplex virus type 1 host-cell interaction

    NASA Astrophysics Data System (ADS)

    Hu, Yu-Peng; Lin, Shu-Yi; Huang, Cheng-Yen; Zulueta, Medel Manuel L.; Liu, Jing-Yuan; Chang, Wen; Hung, Shang-Cheng

    2011-07-01

    Cell surface carbohydrates play significant roles in a number of biologically important processes. Heparan sulfate, for instance, is a ubiquitously distributed polysulfated polysaccharide that is involved, among other things, in the initial step of herpes simplex virus type 1 (HSV-1) infection. The virus interacts with cell-surface heparan sulfate to facilitate host-cell attachment and entry. 3-O-Sulfonated heparan sulfate has been found to function as an HSV-1 entry receptor. Achieving a complete understanding of these interactions requires the chemical synthesis of such oligosaccharides, but this remains challenging. Here, we present a convenient approach for the synthesis of two irregular 3-O-sulfonated heparan sulfate octasaccharides, making use of a key disaccharide intermediate to acquire different building blocks for the oligosaccharide chain assembly. Despite substantial structural differences, the prepared 3-O-sulfonated sugars blocked viral infection in a dosage-dependent manner with remarkable similarity to one another.

  16. A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling.

    PubMed

    Dani, Neil; Nahm, Minyeop; Lee, Seungbok; Broadie, Kendal

    2012-01-01

    A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects.

  17. Modulatory effect of bitter gourd (Momordica charantia LINN.) on alterations in kidney heparan sulfate in streptozotocin-induced diabetic rats.

    PubMed

    Kumar, G Suresh; Shetty, A K; Salimath, P V

    2008-01-17

    Glycoconjugates in the kidney play an important role in the maintenance of glomerular filtration barrier. Thickening of the glomerular basement membrane (GBM) is well characterized in diabetic nephropathy. Changes in GBM mainly include reduction and undersulfation of heparan sulfate, and laminin with accumulation of type IV collagen leading to kidney dysfunction and there is a need to identify therapies that arrest disease progression to end-stage renal failure. In the present investigation, effect of bitter gourd on streptozotocin-induced diabetic rats with particular emphasis on kidney heparan sulfate (HS) was studied. Earlier, our study showed partial reversal of all the diabetes-induced effects by bitter gourd. Increase in the components of glycoconjugates during diabetes was significantly decreased by bitter gourd feeding. Diabetes associated elevation in the activities of enzymes involved in the synthesis and degradation of glycosaminoglycans (GAGs) were significantly lowered by bitter gourd supplementation. GAGs composition revealed decrease in amino sugar, and uronic acid contents during diabetes and bitter gourd feeding was effective in countering this reduction. Decrease in sulfate content in the GAGs during diabetes was ameliorated by bitter gourd feeding. HS decreased by 43% in diabetic rats while bitter gourd feeding to diabetic rats showed 28% reduction. These results clearly indicate beneficial role of bitter gourd in controlling glycoconjugate and heparan sulfate related kidney complications during diabetes thus prolonging late complications of diabetes.

  18. The involvement of heparan sulfate proteoglycans in stem cell differentiation and in malignant glioma

    NASA Astrophysics Data System (ADS)

    Kundu, Soumi; Xiong, Anqi; Forsberg-Nilsson, Karin

    2016-04-01

    Heparan sulfate (HS) proteoglycans (HSPG) are major components of the extracellular matrix. They interact with a plethora of macromolecules that are of physiological importance. The pattern of sulfation of the HS chain determines the specificity of these interactions. The enzymes that synthesize and degrade HS are thus key regulators of processes ranging from embryonic development to tissue homeostasis and tumor development. Formation of the nervous system is also critically dependent on appropriate HSPGs as shown by several studies on the role of HS in neural induction from embryonic stem cells. High-grade glioma is the most common primary malignant brain tumor among adults, and the prognosis is poor. Neural and glioma stem cells share several traits, including sustained proliferation and highly efficient migration in the brain. There are also similarities between the neurogenic niche where adult neural stem cells reside and the tumorigenic niche, including their interactions with components of the extracellular matrix (ECM). The levels of many of these components, for example HSPGs and enzymes involved in the biosynthesis and modification of HS are attenuated in gliomas. In this paper, HS regulation of pathways involved in neural differentiation and how these may be of importance for brain development are discussed. The literature suggesting that modifications of HS could regulate glioma growth and invasion is reviewed. Targeting the invasiveness of glioma cells by modulating HS may improve upon present therapeutic options, which only marginally enhance the survival of glioma patients.

  19. Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans

    PubMed Central

    Noborn, Fredrik; Gomez Toledo, Alejandro; Green, Anders; Nasir, Waqas; Sihlbom, Carina; Nilsson, Jonas; Larson, Göran

    2016-01-01

    Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans. PMID:27694851

  20. New Glucuronic Acid Donors for the Modular Synthesis of Heparan Sulfate Oligosaccharides**

    PubMed Central

    Dhamale, Omkar P.; Zong, Chengli; Al-Mafraji, Kanar

    2014-01-01

    Although hundreds of heparan sulfate (HS) binding proteins have been implicated in a myriad of physiological and pathological processes, very little information is known about ligand requirements for binding and mediating biological activities by these proteins. We report here a streamlined approach for the preparation of modular disaccharide building blocks that will facilitate the assembly of libraries of HS oligosaccharides for structure-activity relationship studies. In particular, we have found that glucuronic acid donors, which usually perform poorly in glycosylations, can give high yields of coupling product when the C-2 hydroxyl is protected with a permanent 4-acetoxy-2,2-dimethyl butanoyl- (PivOAc) or temporary levulinoyl (Lev) ester and the C-4 hydroxyl modified with a selectively removable 2-methylnaphthyl (Nap) ether. It has been shown that the PivOAc ester can be removed without affecting sulfate esters making it an ideal protecting group for HS oligosaccharide assembly. Iduronic acid donors exhibit more favorable glycosyl donating properties and a compound protected with a Lev ester at C-2 and an Fmoc function at the C-4 hydroxyl gave coupling products in high yield. The new donors avoid post-glycosylation oxidation and therefore allow the facile preparation of modular disaccharide building blocks. PMID:24549353

  1. A molecular mechanism for the heparan sulfate dependence of slit-robo signaling.

    PubMed

    Hussain, Sadaf-Ahmahni; Piper, Michael; Fukuhara, Noémi; Strochlic, Laure; Cho, Gian; Howitt, Jason A; Ahmed, Yassir; Powell, Andrew K; Turnbull, Jeremy E; Holt, Christine E; Hohenester, Erhard

    2006-12-22

    Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.

  2. Bone-specific heparan sulfates induce osteoblast growth arrest and downregulation of retinoblastoma protein.

    PubMed

    Manton, Kerry J; Sadasivam, Murali; Cool, Simon M; Nurcombe, Victor

    2006-10-01

    The heparan sulfate (HSs) sugars of the extracellular matrix (ECM) play a key role during both development and wound repair in regulating the flow of growth and adhesive factors across their cell surface receptors. The aim of this study was to assess the structural and functional differences of HS chains extracted from the conditioned media (soluble), cell surface, and ECM of primary human osteoblast cultures, and to analyze their effects on osteoblast cell growth. HS chains from these compartments were characterized through a combination of enzymatic degradation, anion exchange chromatography, and molecular sieving. Although the chains were all approximately the same size, they varied systematically in their sulfate content, suggesting differences in their protein-binding domains. When added to pre-confluent hFOB1.19 osteoblast cultures, HS doses exceeding 500 ng/ml inhibited proliferation, without affecting viability, irrespective of their origin. Furthermore, HS doses of 500 ng/ml also downregulated retinoblastoma, Cyclin A and CDK1 protein expression, indicating that high doses of osteoblast HS negatively regulate cell cycle, resulting in growth arrest; when high doses of HS were withdrawn after a prolonged period, linear cell growth was reestablished. Thus, despite differences in sulfation, HS from either the soluble, cell surface, or matrix compartments of primary human osteoblast cultures are functionally similar with respect to their effects on growth. Binding assays revealed that the HS chains bound TGFbeta1, a known inhibitor of osteoprogenitor growth, at higher affinity than a suite of other bone-related, heparin-binding growth factors. Overcoming such sugar-mediated inhibition may prove important for wound repair.

  3. Heparan sulfate proteoglycans including syndecan-3 modulate BMP activity during limb cartilage differentiation.

    PubMed

    Fisher, Melanie C; Li, Yingcui; Seghatoleslami, M Reza; Dealy, Caroline N; Kosher, Robert A

    2006-01-01

    Bone morphogenetic proteins (BMPs) are involved in multiple aspects of limb development including regulation of cartilage differentiation. Several BMPs bind strongly to heparin, and heparan sulfate proteoglycans (HSPGs) at the cell surface or in the extracellular matrix have recently been implicated as modulators of BMP signaling in some developing systems. Here we have explored the role of HSPGs in regulating BMP activity during limb chondrogenesis by evaluating the effects of exogenous heparan sulfate (HS), heparitinase treatment, and overexpression of the HSPG syndecan-3 on the ability of BMP2 to modulate the chondrogenic differentiation of limb mesenchymal cells in micromass culture. Exogenous HS dramatically enhances the ability of BMP2 to stimulate chondrogenesis and cartilage specific gene expression, and reduces the concentration of BMP2 needed to stimulate chondrogenesis. Furthermore, HS stimulates BMP2-mediated phosphorylation of Smad1, Smad5, and Smad8, transcriptional mediators of BMP2 signaling, indicating that HS enhances the interaction of BMP2 with its receptors. Pretreatment of micromass cultures with heparitinase to degrade endogenous HSPGs also enhances the chondrogenic activity of BMP2, and reduces the concentration of BMP2 needed to promote chondrogenesis. Taken together these results indicate that exogenous HS or heparitinase enhance the chondrogenic activity of BMP2 by interfering with its interaction with endogenous HSPGs that would normally restrict its interaction with its receptors. Consistent with the possibility that HSPGs are negative modulators of BMP signaling during chondrogenesis, we have found that overexpression of syndecan-3, which is one of the major HSPGs normally expressed during chondrogenesis, greatly impairs the ability of BMP2 to promote cartilage differentiation. Furthermore, retroviral overexpression of syndecan-3 inhibits BMP2-mediated Smad phosphorylation in the regions of the cultures in which chondrogenesis is

  4. Analysis of Drosophila glucuronyl C5-epimerase: implications for developmental roles of heparan sulfate sulfation compensation and 2-O-sulfated glucuronic acid.

    PubMed

    Dejima, Katsufumi; Takemura, Masahiko; Nakato, Eriko; Peterson, Jesse; Hayashi, Yoshiki; Kinoshita-Toyoda, Akiko; Toyoda, Hidenao; Nakato, Hiroshi

    2013-11-29

    During the biosynthesis of heparan sulfate (HS), glucuronyl C5-epimerase (Hsepi) catalyzes C5-epimerization of glucuronic acid (GlcA), converting it to iduronic acid (IdoA). Because HS 2-O-sulfotransferase (Hs2st) shows a strong substrate preference for IdoA over GlcA, C5-epimerization is required for normal HS sulfation. However, the physiological significance of C5-epimerization remains elusive. To understand the role of Hsepi in development, we isolated Drosophila Hsepi mutants. Homozygous mutants are viable and fertile with only minor morphological defects, including the formation of an ectopic crossvein in the wing, but they have a short lifespan. We propose that two mechanisms contribute to the mild phenotypes of Hsepi mutants: HS sulfation compensation and possible developmental roles of 2-O-sulfated GlcA (GlcA2S). HS disaccharide analysis showed that loss of Hsepi resulted in a significant impairment of 2-O-sulfation and induced compensatory increases in N- and 6-O-sulfation. Simultaneous block of Hsepi and HS 6-O-sulfotransferase (Hs6st) activity disrupted tracheoblast formation, a well established FGF-dependent process. This result suggests that the increase in 6-O-sulfation in Hsepi mutants is critical for the rescue of FGF signaling. We also found that the ectopic crossvein phenotype can be induced by expression of a mutant form of Hs2st with a strong substrate preference for GlcA-containing units, suggesting that this phenotype is associated with abnormal GlcA 2-O-sulfation. Finally, we show that Hsepi formed a complex with Hs2st and Hs6st in S2 cells, raising the possibility that this complex formation contributes to the close functional relationships between these enzymes.

  5. Glycoprotein C of equine herpesvirus 4 plays a role in viral binding to cell surface heparan sulfate.

    PubMed

    Azab, Walid; Tsujimura, Koji; Maeda, Ken; Kobayashi, Kyousuke; Mohamed, Yassir Mahgoub; Kato, Kentaro; Matsumura, Tomio; Akashi, Hiroomi

    2010-07-01

    Heparan sulfate moieties of cell surface proteoglycans serve as receptors for several herpesviruses. For herpes simplex virus 1, pseudorabies virus and equine herpesvirus 1, glycoprotein C (gC) homologues have been shown to mediate the binding to cell surface heparan sulfate. However, the role of gC in equine herpesvirus 4 (EHV-4) infection has not yet been analyzed. Using pull-down assay, we first determined that EHV-4 gC as well as gB are heparin-binding glycoproteins. To study the role of gC in EHV-4 infection, we constructed a gC-deletion mutant, WA79DeltagC, where the kanamycin resistant gene was inserted instead of the open reading frame encoding gC. We found that soluble heparin was capable of blocking both wild-type EHV-4 and WA79DeltagC infection of fetal horse kidney. Furthermore, pretreatment of cells with heparinase reduces considerably the ability of both viruses to adsorb to these cells and to form plaques. Similar results were obtained when cellular glycosaminoglycan synthesis was inhibited by chlorate treatment. In addition, we did find that gC protects EHV-4 from complement-mediated neutralization. These results suggest that, like other herpesviruses, EHV-4 gC plays a role in the interaction of the virus with cellular heparan sulfate. Moreover, gC can protect the virus from complement-mediated neutralization.

  6. Inhibition of Human Metapneumovirus Binding to Heparan Sulfate Blocks Infection in Human Lung Cells and Airway Tissues

    PubMed Central

    Klimyte, Edita M.; Smith, Stacy E.; Oreste, Pasqua; Lembo, David

    2016-01-01

    ABSTRACT Human metapneumovirus (HMPV), a recently discovered paramyxovirus, infects nearly 100% of the world population and causes severe respiratory disease in infants, the elderly, and immunocompromised patients. We previously showed that HMPV binds heparan sulfate proteoglycans (HSPGs) and that HMPV binding requires only the viral fusion (F) protein. To characterize the features of this interaction critical for HMPV binding and the role of this interaction in infection in relevant models, we utilized sulfated polysaccharides, heparan sulfate mimetics, and occluding compounds. Iota-carrageenan demonstrated potent anti-HMPV activity by inhibiting binding to lung cells mediated by the F protein. Furthermore, analysis of a minilibrary of variably sulfated derivatives of Escherichia coli K5 polysaccharide mimicking the HS structure revealed that the highly O-sulfated K5 polysaccharides inhibited HMPV infection, identifying a potential feature of HS critical for HMPV binding. The peptide dendrimer SB105-A10, which binds HS, reduced binding and infection in an F-dependent manner, suggesting that occlusion of HS at the target cell surface is sufficient to prevent infection. HMPV infection was also inhibited by these compounds during apical infection of polarized airway tissues, suggesting that these interactions take place during HMPV infection in a physiologically relevant model. These results reveal key features of the interaction between HMPV and HS, supporting the hypothesis that apical HS in the airway serves as a binding factor during infection, and HS modulating compounds may serve as a platform for potential antiviral development. IMPORTANCE Human metapneumovirus (HMPV) is a paramyxovirus that causes respiratory disease worldwide. It has been previously shown that HMPV requires binding to heparan sulfate on the surfaces of target cells for attachment and infection. In this study, we characterize the key features of this binding interaction using heparan sulfate

  7. Differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization

    PubMed Central

    Migliorini, Elisa; Salanga, Catherina L.; Thakar, Dhruv

    2017-01-01

    Chemokines control the migration of cells in normal physiological processes and in the context of disease such as inflammation, autoimmunity and cancer. Two major interactions are involved: (i) binding of chemokines to chemokine receptors, which activates the cellular machinery required for movement; and (ii) binding of chemokines to glycosaminoglycans (GAGs), which facilitates the organization of chemokines into haptotactic gradients that direct cell movement. Chemokines can bind and activate their receptors as monomers; however, the ability to oligomerize is critical for the function of many chemokines in vivo. Chemokine oligomerization is thought to enhance their affinity for GAGs, and here we show that it significantly affects the ability of chemokines to accumulate on and be retained by heparan sulfate (HS). We also demonstrate that several chemokines differentially rigidify and cross-link HS, thereby affecting HS rigidity and mobility, and that HS cross-linking is significantly enhanced by chemokine oligomerization. These findings suggest that chemokine–GAG interactions may play more diverse biological roles than the traditional paradigms of physical immobilization and establishment of chemokine gradients; we hypothesize that they may promote receptor-independent events such as physical re-organization of the endothelial glycocalyx and extracellular matrix, as well as signalling through proteoglycans to facilitate leukocyte adhesion and transmigration. PMID:28123055

  8. Heparan Sulfate Proteoglycan: An Arbovirus Attachment Factor Integral to Mosquito Salivary Gland Ducts

    PubMed Central

    Ciano, Kristen A.; Saredy, Jason J.; Bowers, Doria F.

    2014-01-01

    Variants of the prototype Alphavirus, Sindbis (SINV), were used in per os infections of adult female mosquitoes to investigate arbovirus interaction with the salivary gland (SG). Infection of Aedine mosquitoes with AR339, a heparan sulfate proteoglycan (HSPG)-dependent variant, resulted in gross pathology in the SG lateral lobes while infection with TR339, a HSPG-independent variant, resulted in minimal SG pathology. HSPG was detected in the internal ducts of the SG lateral lobes by immunolabeling but not in the median lobe, or beyond the triad structure and external ducts. Reports that human lactoferrin interacts with HSPG, suggested an interference with virus attachment to receptors on vertebrate cells. Pre-incubation of Aedes albopictus cultured C7-10 cells with bovine lactoferrin (bLF) followed by adsorption of SINV resulted in earlier and greater intensity of cytopathic response to TR339 compared with AR339. Following pre-treatment of C7-10 cells with bLF, plaques from tissue culture-adapted high-titer SINVTaV-GFP-TC were observed at 48 h post-infection (p.i.), while plaques from low-titer SINVTaV-GFP-TC were not observed until 120 h p.i. Confocal optics detected this reporter virus at 30 days p.i. in the SG proximal lateral lobe, a region of HSPG-immunolocalization. Altogether these data suggest an association between SINV and HSPG in the host mosquito. PMID:25533661

  9. Heparan sulfate proteoglycan: an arbovirus attachment factor integral to mosquito salivary gland ducts.

    PubMed

    Ciano, Kristen A; Saredy, Jason J; Bowers, Doria F

    2014-12-22

    Variants of the prototype Alphavirus, Sindbis (SINV), were used in per os infections of adult female mosquitoes to investigate arbovirus interaction with the salivary gland (SG). Infection of Aedine mosquitoes with AR339, a heparan sulfate proteoglycan (HSPG)-dependent variant, resulted in gross pathology in the SG lateral lobes while infection with TR339, a HSPG-independent variant, resulted in minimal SG pathology. HSPG was detected in the internal ducts of the SG lateral lobes by immunolabeling but not in the median lobe, or beyond the triad structure and external ducts. Reports that human lactoferrin interacts with HSPG, suggested an interference with virus attachment to receptors on vertebrate cells. Pre-incubation of Aedes albopictus cultured C7-10 cells with bovine lactoferrin (bLF) followed by adsorption of SINV resulted in earlier and greater intensity of cytopathic response to TR339 compared with AR339. Following pre-treatment of C7-10 cells with bLF, plaques from tissue culture-adapted high-titer SINVTaV-GFP-TC were observed at 48 h post-infection (p.i.), while plaques from low-titer SINVTaV-GFP-TC were not observed until 120 h p.i. Confocal optics detected this reporter virus at 30 days p.i. in the SG proximal lateral lobe, a region of HSPG-immunolocalization. Altogether these data suggest an association between SINV and HSPG in the host mosquito.

  10. A Heparan Sulfate-Binding Cell Penetrating Peptide for Tumor Targeting and Migration Inhibition

    PubMed Central

    Kuo, Ping-Hsueh; Chang, Pei-Lin; Wang, Wen-Ching; Chuang, Yung-Jen; Chang, Margaret Dah-Tsyr

    2015-01-01

    As heparan sulfate proteoglycans (HSPGs) are known as co-receptors to interact with numerous growth factors and then modulate downstream biological activities, overexpression of HS/HSPG on cell surface acts as an increasingly reliable prognostic factor in tumor progression. Cell penetrating peptides (CPPs) are short-chain peptides developed as functionalized vectors for delivery approaches of impermeable agents. On cell surface negatively charged HS provides the initial attachment of basic CPPs by electrostatic interaction, leading to multiple cellular effects. Here a functional peptide (CPPecp) has been identified from critical HS binding region in hRNase3, a unique RNase family member with in vitro antitumor activity. In this study we analyze a set of HS-binding CPPs derived from natural proteins including CPPecp. In addition to cellular binding and internalization, CPPecp demonstrated multiple functions including strong binding activity to tumor cell surface with higher HS expression, significant inhibitory effects on cancer cell migration, and suppression of angiogenesis in vitro and in vivo. Moreover, different from conventional highly basic CPPs, CPPecp facilitated magnetic nanoparticle to selectively target tumor site in vivo. Therefore, CPPecp could engage its capacity to be developed as biomaterials for diagnostic imaging agent, therapeutic supplement, or functionalized vector for drug delivery. PMID:26064887

  11. Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation

    PubMed Central

    Brennan, Todd V.; Lin, Liwen; Brandstadter, Joshua D.; Rendell, Victoria R.; Dredge, Keith; Huang, Xiaopei; Yang, Yiping

    2015-01-01

    Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell–based antitumor therapies. PMID:26649979

  12. Heparanase Facilitates Cell Adhesion and Spreading by Clustering of Cell Surface Heparan Sulfate Proteoglycans

    PubMed Central

    Levy-Adam, Flonia; Feld, Sari; Suss-Toby, Edith; Vlodavsky, Israel; Ilan, Neta

    2008-01-01

    Heparanase is a heparan sulfate (HS) degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys158-Asp171, termed KKDC) was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity. PMID:18545691

  13. Heparan sulfate proteoglycan mediates the selective attachment and internalization of serotype 3 human adenovirus dodecahedron.

    PubMed

    Vivès, Romain R; Lortat-Jacob, Hugues; Chroboczek, Jadwiga; Fender, Pascal

    2004-04-10

    During adenovirus type 3 (Ad3) infection cycle, the penton (Pt) of the viral capsid, a noncovalent complex of fiber and penton base proteins, is produced in large excess and self-assembles to form a highly organized dodecahedral structure, termed dodecahedron (Dd). The physiological role of these particles is poorly understood, but we have recently reported that they can penetrate cells with high efficiency and thus may constitute an attractive tool for gene or protein delivery approaches. Surprisingly, Dd displayed the ability to enter cells non-permissive to Ad3, suggesting the existence of additional internalization modes. In this study, we show that Ad3 Dd binds to cell surface heparan sulfate (HS) through high affinity interaction with the penton base. Furthermore, binding to HS was found to be the prerequisite for a novel and Dd specific entry pathway that could not be used by Ad3. Overall, these data provide new insights in the possible role of Dd during viral infection and potential therapeutic applications.

  14. Nucleolin is a nuclear target of heparan sulfate derived from glypican-1.

    PubMed

    Cheng, Fang; Belting, Mattias; Fransson, Lars-Åke; Mani, Katrin

    2017-03-12

    The recycling, S-nitrosylated heparan sulfate (HS) proteoglycan glypican-1 releases anhydromannose (anMan)-containing HS chains by a nitrosothiol-catalyzed cleavage in endosomes that can be constitutive or induced by ascorbate. The HS-anMan chains are then transported to the nucleus. A specific nuclear target for HS-anMan has not been identified. We have monitored endosome-to-nucleus trafficking of HS-anMan by deconvolution and confocal immunofluorescence microscopy using an anMan-specific monoclonal antibody in non-growing, ascorbate-treated, and growing, untreated, wild-type mouse embryonic fibroblasts and hypoxia-exposed Alzheimer mouse Tg2576 fibroblasts and human U87 glioblastoma cells. In all cells, nuclear HS-anMan targeted a limited number of sites of variable size where it colocalized with DNA and nucleolin, an established marker for nucleoli. HS-anMan also colocalized with ethynyl uridine-tagged nascent RNA and two acetylated forms of histone H3. Acute hypoxia increased the formation of HS-anMan in both Tg2576 and U87 cells. A portion of HS-anMan colocalized with nucleolin at small discrete sites, while most of the nucleolin and nascent RNA was dispersed. In U87 cells, HS-anMan, nucleolin and nascent RNA reassembled after prolonged hypoxia. Nucleolar HS may modulate synthesis and/or release of rRNA.

  15. A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils.

    PubMed

    Massena, Sara; Christoffersson, Gustaf; Hjertström, Elina; Zcharia, Eyal; Vlodavsky, Israel; Ausmees, Nora; Rolny, Charlotte; Li, Jin-Ping; Phillipson, Mia

    2010-09-16

    During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients toward the afflicted site. The aim of this in vivo study was to determine whether a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a macrophage inflammatory protein-2 (MIP-2)-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase-overexpressing transgenic mice (hpa-tg) with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared with homogeneous extravascular chemokine concentration (MIP-2 superfusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased the ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes toward transmigration loci close to the infection site.

  16. Characterization of chicken syndecan-3 as a heparan sulfate proteoglycan and its expression during embryogenesis.

    PubMed

    Gould, S E; Upholt, W B; Kosher, R A

    1995-04-01

    Syndecan-3 is one of four identified members of a family of transmembrane proteoglycans (the syndecans) that possess highly similar cytoplasmic and transmembrane domains and may function as extracellular matrix receptors and/or low affinity receptors for signaling molecules such as FGF. We previously reported the cloning of a partial cDNA for chicken syndecan-3. Here we report the isolation of a syndecan-3 cDNA containing additional 5' sequence which includes a potential methionine start codon and putative signal sequence. In vitro translation of syndecan-3 cDNA in the presence and absence of microsomes suggests that the putative signal sequence is functional, suggesting that the cDNA may encompass the full coding sequence. We also identify syndecan-3 as a heparan sulfate proteoglycan and report its expression pattern during chicken embryogenesis using polyclonal antibodies raised against a recombinant fusion protein. We detect abundant syndecan-3 expression in the developing brain and neural tube, including a striking expression in the floor plate of the neural tube. During limb development, syndecan-3 is expressed in the distal mesenchymal cells of the limb bud which are undergoing outgrowth in response to the apical ectodermal ridge. Syndecan-3 is also transiently expressed during the formation of the precartilage condensations of the skeletal elements of the limb and subsequently in association with the differentiating osteoblasts of the periosteum. Expression is also observed in several areas of tissue interactions including the developing lens, otic vesicle, genital ridge, sclerotome, and feather buds.

  17. Heterogeneous distribution of a basement membrane heparan sulfate proteoglycan in rat tissues

    PubMed Central

    1987-01-01

    A heparan sulfate proteoglycan (HSPG) synthesized by murine parietal yolk sac (PYS-2) cells has been characterized and purified from culture supernatants. A monospecific polyclonal antiserum was raised against it which showed activity against the HSPG core protein and basement membrane specificity in immunohistochemical studies on frozen tissue sections from many rat organs. However, there was no reactivity with some basement membranes, notably those of several smooth muscle types and cardiac muscle. In addition, it was found that pancreatic acinar basement membranes also lacked the HSPG type recognized by this antiserum. Those basement membranes that lacked the HSPG strongly stained with antisera against laminin and type IV collagen. The striking distribution pattern is possibly indicative of multiple species of basement membrane HSPGs of which one type is recognized by this antiserum. Further evidence for multiple HSPGs was derived from the finding that skeletal neuromuscular junction and liver epithelia also did not contain this type of HSPG, though previous reports have indicated the presence of HSPGs at these sites. The PYS-2 HSPG was shown to be antigenically related to the large, low buoyant density HSPG from the murine Engelbreth-Holm swarm tumor. It was, however, confirmed that only a single population of antibodies was present in the serum. Despite the presence of similar epitopes on these two proteoglycans of different hydrodynamic properties, it was apparent that the PYS-2 HSPG represents a basement membrane proteoglycan of distinct properties reflected in its restricted distribution in vivo. PMID:2959669

  18. Herpes simplex virus type 1-induced hemagglutination: glycoprotein C mediates virus binding to erythrocyte surface heparan sulfate.

    PubMed Central

    Trybala, E; Svennerholm, B; Bergström, T; Olofsson, S; Jeansson, S; Goodman, J L

    1993-01-01

    We recently reported that herpes simplex virus type 1 (HSV-1) can cause agglutination of murine erythrocytes (E. Trybala, Z. Larski, and J. Wisniewski, Arch. Virol. 113:89-94, 1990). We now demonstrate that the mechanism of this hemagglutination is glycoprotein C-mediated binding of virus to heparan sulfate moieties at the surface of erythrocytes. Hemagglutination was found to be a common property of all gC-expressing laboratory strains and clinical isolates of HSV-1 tested. Mutants of HSV-1 deficient in glycoprotein C caused no specific hemagglutination, whereas their derivatives transfected with a functional gC-1 gene, thus reconstituting gC expression, regained full hemagglutinating activity. Hemagglutination activity was inhibited by antibodies against gC-1 but not by antibodies with specificity for glycoproteins gB, gD, or gE or by murine antiserum raised against the MP strain of HSV-1, which is gC deficient. Finally, purified gC-1 protein, like whole HSV-1 virions, showed high hemagglutinating activity which was inhibited by heparan sulfate and/or heparin and was completely prevented by pretreatment of erythrocytes with heparitinase, providing evidence that gC-1 mediates hemagglutination by binding to heparan sulfate at the cell surface. Thus, HSV-1-induced hemagglutination is gC-1 dependent and resembles the recently proposed mechanism by which HSV-1 attaches to surface heparans on susceptible cells, providing a simple model for initial events in the virus-cell interaction. Images PMID:8382294

  19. Regulation of Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycan Biosynthesis by 4-Fluoro-glucosamine in Murine Airway Smooth Muscle Cells*

    PubMed Central

    Nigro, Julie; Wang, Aimin; Mukhopadhyay, Durba; Lauer, Mark; Midura, Ronald J.; Sackstein, Robert; Hascall, Vincent C.

    2009-01-01

    The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 μm) reduced the quantity (by 64.1–76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (Kav shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7–73.0%, and the size showed a Kav shift from 0.30 to 0.50. Treatment of ASM cells with 45 μm and 179 μm 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-β-d-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (Kav of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc. PMID:19346253

  20. Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries.

    PubMed

    Billings, Paul C; Pacifici, Maurizio

    2015-01-01

    Heparan sulfate (HS) is a component of cell surface and matrix-associated proteoglycans (HSPGs) that, collectively, play crucial roles in many physiologic processes including cell differentiation, organ morphogenesis and cancer. A key function of HS is to bind and interact with signaling proteins, growth factors, plasma proteins, immune-modulators and other factors. In doing so, the HS chains and HSPGs are able to regulate protein distribution, bio-availability and action on target cells and can also serve as cell surface co-receptors, facilitating ligand-receptor interactions. These proteins contain an HS/heparin-binding domain (HBD) that mediates their association and contacts with HS. HBDs are highly diverse in sequence and predicted structure, contain clusters of basic amino acids (Lys and Arg) and possess an overall net positive charge, most often within a consensus Cardin-Weintraub (CW) motif. Interestingly, other domains and residues are now known to influence protein-HS interactions, as well as interactions with other glycosaminoglycans, such as chondroitin sulfate. In this review, we provide a description and analysis of HBDs in proteins including amphiregulin, fibroblast growth factor family members, heparanase, sclerostin and hedgehog protein family members. We discuss HBD structural and functional features and important roles carried out by other protein domains, and also provide novel conformational insights into the diversity of CW motifs present in Sonic, Indian and Desert hedgehogs. Finally, we review progress in understanding the pathogenesis of a rare pediatric skeletal disorder, Hereditary Multiple Exostoses (HME), characterized by HS deficiency and cartilage tumor formation. Advances in understanding protein-HS interactions will have broad implications for basic biology and translational medicine as well as for the development of HS-based therapeutics.

  1. Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development.

    PubMed

    Solursh, M; Reiter, R S; Jensen, K L; Kato, M; Bernfield, M

    1990-07-01

    Syndecan is an integral membrane proteoglycan that contains both heparan sulfate and chondroitin sulfate chains and that links the cytoskeleton to interstitial extracellular matrix components, including collagen and fibronectin. Immunohistochemistry with a monoclonal antibody directed to the core protein of the syndecan ectodomain has been used to analyze the distribution of this proteoglycan in the developing mouse limb bud and in high-density cultures of limb mesenchyme cells. By Day 9 of gestation when the limb buds are just apparent, syndecan is detected on cells throughout the limb region, including both ectodermal and mesenchymal components. This distribution does not change as the limb bud elongates along its proximodistal axis, except for its reduction in the apical ectodermal ridge. By Day 11, the intensity of immunofluorescence in the central core decreases relative to other regions. By Day 13 immunostaining is lost in the regions destined for chondrogenesis and myogenesis but persists in the limb ectoderm and peripheral and distal mesenchyme. In the limb mesenchyme cell cultures, syndecan is initially undetected, but is found throughout the culture by 24 hr. With further culture the antigen becomes reduced in chondrogenic foci and in association with myogenic cells. When chick limb ectoderm is placed on the high-density cultures, immunoreactivity in the mouse mesenchyme is enhanced suggesting that epithelial-mesenchymal interactions modulate syndecan expression in the limb bud. Based on analysis of 35S-labeled syndecan from the cultures, syndecan from limb mesenchyme cells contains more glycosaminoglycan chains and is larger in size than the previously described polymorphic forms of syndecan from various epithelia. The high affinity of syndecan for components of the extracellular matrix and its distribution in the early limb bud are consistent with a role in maintaining the morphologic integrity of the limb bud during the period of initiation and rapid

  2. Heparan sulfation-dependent fibroblast growth factor signaling maintains embryonic stem cells primed for differentiation in a heterogeneous state.

    PubMed

    Lanner, Fredrik; Lee, Kian Leong; Sohl, Marcus; Holmborn, Katarina; Yang, Henry; Wilbertz, Johannes; Poellinger, Lorenz; Rossant, Janet; Farnebo, Filip

    2010-02-01

    Embryonic stem (ES) cells continuously decide whether to maintain pluripotency or differentiate. While exogenous leukemia inhibitory factor and BMP4 perpetuate a pluripotent state, less is known about the factors initiating differentiation. We show that heparan sulfate (HS) proteoglycans are critical coreceptors for signals inducing ES cell differentiation. Genetic targeting of NDST1 and NDST2, two enzymes required for N-sulfation of proteoglycans, blocked differentiation. This phenotype was rescued by HS presented in trans or by soluble heparin. NaClO(3) (-), which reduces sulfation of proteoglycans, potently blocked differentiation of wild-type cells. Mechanistically, N-sulfation was identified to be critical for functional autocrine fibroblast growth factor 4 (FGF4) signaling. Microarray analysis identified the pluripotency maintaining transcription factors Nanog, KLF2/4/8, Tbx3, and Tcf3 to be negatively regulated, whereas markers of differentiation such as Gbx2, Dnmt3b, FGF5, and Brachyury were induced by sulfation-dependent FGF receptor (FGFR) signaling. We show that several of these genes are heterogeneously expressed in ES cells, and that targeting of heparan sulfation or FGFR-signaling facilitated a homogenous Nanog/KLF4/Tbx3 positive ES cell state. This finding suggests that the recently discovered heterogeneous state of ES cells is regulated by HS-dependent FGFR signaling. Similarly, culturing blastocysts with NaClO(3) (-) eliminated GATA6-positive primitive endoderm progenitors generating a homogenous Nanog-positive inner cell mass. Functionally, reduction of sulfation robustly improved de novo ES cell derivation efficiency. We conclude that N-sulfated HS is required for FGF4 signaling to maintain ES cells primed for differentiation in a heterogeneous state. Inhibiting this pathway facilitates a more naïve ground state.

  3. Genome-Wide Screening Uncovers the Significance of N-sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection.

    PubMed

    Tanaka, Atsushi; Tumkosit, Uranan; Nakamura, Shota; Motooka, Daisuke; Kishishita, Natsuko; Priengprom, Thongkoon; Sa-Ngasang, Areerat; Kinoshita, Taroh; Takeda, Naokazu; Maeda, Yusuke

    2017-04-12

    The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3-E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of a clinically isolated CHIKV Thai #16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). Whereas glycosaminoglycans were commonly required for efficient infectivity of CHIKV, JEV and YFV as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NDST1-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at the later steps after the CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection, because it is involved in proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism and pathogenesis.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we

  4. A systems biology approach for the investigation of the heparin/heparan sulfate interactome.

    PubMed

    Ori, Alessandro; Wilkinson, Mark C; Fernig, David G

    2011-06-03

    A large body of evidence supports the involvement of heparan sulfate (HS) proteoglycans in physiological processes such as development and diseases including cancer and neurodegenerative disorders. The role of HS emerges from its ability to interact and regulate the activity of a vast number of extracellular proteins including growth factors and extracellular matrix components. A global view on how protein-HS interactions influence the extracellular proteome and, consequently, cell function is currently lacking. Here, we systematically investigate the functional and structural properties that characterize HS-interacting proteins and the network they form. We collected 435 human proteins interacting with HS or the structurally related heparin by integrating literature-derived and affinity proteomics data. We used this data set to identify the topological features that distinguish the heparin/HS-interacting network from the rest of the extracellular proteome and to analyze the enrichment of gene ontology terms, pathways, and domain families in heparin/HS-binding proteins. Our analysis revealed that heparin/HS-binding proteins form a highly interconnected network, which is functionally linked to physiological and pathological processes that are characteristic of higher organisms. Therefore, we then investigated the existence of a correlation between the expansion of domain families characteristic of the heparin/HS interactome and the increase in biological complexity in the metazoan lineage. A strong positive correlation between the expansion of the heparin/HS interactome and biosynthetic machinery and organism complexity emerged. The evolutionary role of HS was reinforced by the presence of a rudimentary HS biosynthetic machinery in a unicellular organism at the root of the metazoan lineage.

  5. Transendothelial migration of effector T cells across inflamed endothelial barriers does not require heparan sulfate proteoglycans.

    PubMed

    Stoler-Barak, Liat; Barzilai, Sagi; Zauberman, Ayelet; Alon, Ronen

    2014-06-01

    Leukocyte diapedesis is a chemotactic multistep process that requires optimal chemoattractant presentation by the endothelial barrier. Recent studies have described a critical role for heparan sulfate glycosaminoglycans (HSGAGs) in the presentation and functions of chemokines essential for lymphocyte interactions with the lymph node vasculature. We wished to test whether HS expression by a prototypic endothelial cell type, i.e. human umbilical vein endothelial cells (HUVECs), is critical for their ability to support neutrophil and lymphocyte adhesion and transendothelial migration (TEM) under shear flow. We found that HUVECs deposit HS GAGs mainly at their basolateral compartments in both their resting and inflamed states. We next inactivated the key enzyme involved in HS biosynthesis, exostosin-1 (Ext1). Silencing Ext1 resulted in a complete loss of HS biosynthesis; nonetheless, TNF-α and IL-1β stimulation of key adhesion molecules and inflammatory chemokines necessary for neutrophil or lymphocyte adhesion and TEM remained intact. Ext1 silencing reduced neutrophil arrest and markedly impaired TEM, consistent with a role of basolateral HS GAGs in directing neutrophil crossing of inflamed endothelial barriers. Strikingly, however, the TEM of effector T cells across identically Ext1-silenced HUVECs remained normal. Importantly, the biosynthesis of the main promigratory chemokines for effector T cells and neutrophils, respectively, CCL2 and CXCL1, and their vesicle distributions were also Ext1 independent. These results suggest that transmigrating neutrophils must respond to chemokines transiently presented by apical and basolateral endothelial HS GAGs. In contrast, effector T cells can integrate chemotactic TEM signals directly from intra-endothelial chemokine stores rather than from externally deposited chemokines.

  6. Inhibition of Human Respiratory Syncytial Virus Infectivity by a Dendrimeric Heparan Sulfate-Binding Peptide

    PubMed Central

    Donalisio, Manuela; Rusnati, Marco; Cagno, Valeria; Civra, Andrea; Bugatti, Antonella; Giuliani, Andrea; Pirri, Giovanna; Volante, Marco; Papotti, Mauro; Landolfo, Santo

    2012-01-01

    Respiratory syncytial virus (RSV) interacts with cell surface heparan sulfate proteoglycans (HSPGs) to initiate infection. The interaction of RSV with HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In the present study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was screened with the aim of identifying peptides able to bind HSPGs and thus block RSV attachment and infectivity. Of the compounds identified, the dendrimer SB105-A10 was the most potent inhibitor of RSV infectivity, with 50% inhibitory concentrations (IC50s) of 0.35 μM and 0.25 μM measured in Hep-2 and A549 cells, respectively. SB105-A10 was found to bind to both cell types via HSPGs, suggesting that its antiviral activity is indeed exerted by competing with RSV for binding to cell surface HSPGs. SB105-A10 prevented RSV infection when added before the viral inoculum, in line with its proposed HSPG-binding mechanism of action; moreover, antiviral activity was also exhibited when SB105-A10 was added postinfection, as it was able to reduce the cell-to-cell spread of the virus. The antiviral potential of SB105-A10 was further assessed using human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. SB105-A10 strongly reduced RSV infectivity in this model and exhibited no signs of cytotoxicity or proinflammatory effects. Together, these features render SB105-A10 an attractive candidate for further development as a RSV inhibitor to be administered by aerosol delivery. PMID:22850525

  7. Hydrogen/Deuterium Exchange-LC-MS Approach to Characterize the Action of Heparan Sulfate C5-Epimerase

    PubMed Central

    Babu, Ponnusamy; Victor, Xylophone V.; Nelsen, Emily; Nguyen, Thao Kim Nu; Raman, Karthik; Kuberan, Balagurunathan

    2011-01-01

    Heparan sulfate (HS) proteoglycans regulate a number of biological functions in many systems. Most of the functions of HS are attributed to its unique structure, consisting of sulfated and non-sulfated domains, arising from the differential presence of iduronyl and glucuronyl residues along the polysaccharide chain. A single glucuronyl C5-epimerase enzyme acts on heparan sulfate precursor, converts glucuronyl residues into iduronyl residues and modulates subsequent biosynthetic steps in vivo. The ratios of non-sulfated epimers within the polysaccharide chain have been calculated by resolving radiolabeled GlcA-AManR and IdoA-AManR disaccharides using a tedious paper chromatography technique. Radioactive assay, based on measuring either the release or incorporation of 3H at C5 carbon of uronyl residues of 3H-labeled HS precursor substrate, has been in use over three decades to characterize the action of HS C5-epimerase. We have developed a non-radioactive assay to estimate the epimerase activity through resolving GlcA-AManR and IdoA-AManR disaccharides on HPLC in conjunction with hydrogen/deuterium exchange upon epimerization protocol-liquid chromatography mass spectrometry (DEEP-LC-MS). Utilizing this new, non-radioactive based assay, DEEP-LC-MS, we were able to determine the extent of both forward and reverse reaction on the same substrate catalyzed by C5-epimerase. Results from this study also provide insights into the action of C5-epimerase and provide an opportunity to delineate snapshots of biosynthetic events that occur during the HSPG assembly in the Golgi. PMID:21573838

  8. Human Immunodeficiency Virus and Heparan Sulfate: From Attachment to Entry Inhibition

    PubMed Central

    Connell, Bridgette J.; Lortat-Jacob, Hugues

    2013-01-01

    By targeting cells that provide protection against infection, HIV-1 causes acquired immunodeficiency syndrome. Infection starts when gp120, the viral envelope glycoprotein, binds to CD4 and to a chemokine receptor usually CCR5 or CXCR4. As many microorganisms, HIV-1 also interacts with heparan sulfate (HS), a complex group of cell surface associated anionic polysaccharides. It has been thought that this binding, occurring at a step prior to CD4 recognition, increases infectivity by pre-concentrating the virion particles at the cell surface. Early work, dating from before the identification of CCR5 and CXCR4, showed that a variety of HS mimetics bind to the gp120 V3 loop through electrostatic interactions, compete with cell surface associated HS to bind the virus and consequently, neutralize the infectivity of a number of T-cell line-adapted HIV-1 strains. However, progress made to better understand HIV-1 attachment and entry, coupled with the recent identification of additional gp120 regions mediating HS recognition, have considerably modified this view. Firstly, the V3 loop from CXCR4-using viruses is much more positively charged compared to those using CCR5. HS inhibition of cell attachment is thus restricted to CXCR4-using viruses (such as T-cell line-adapted HIV-1). Secondly, studies aiming at characterizing the gp120/HS complex revealed that HS binding was far more complex than previously thought: in addition to the V3 loop of CXCR4 tropic gp120, HS interacts with several other cryptic areas of the protein, which can be induced upon CD4 binding, and are conserved amongst CCR5 and CXCR4 viruses. In view of these data, this review will detail the present knowledge on HS binding to HIV-1, with regards to attachment and entry processes. It will discuss the perspective of targeting the gp120 co-receptor binding site with HS mimetic compounds, a strategy that recently gave rise to entry inhibitors that work in the low nanomolar range, independently of co

  9. Interaction of E2 Glycoprotein with Heparan Sulfate Is Crucial for Cellular Infection of Sindbis Virus

    PubMed Central

    Yang, Yiliang; Jia, Juan; Fu, Shihong; Feng, Yun; He, Ying; Li, Jin-Ping; Liang, Guodong

    2010-01-01

    Cell culture-adapted strains of Sindbis virus (SINV) initially attach to cells by the ability to interact with heparan sulfate (HS) through selective mutation for positively charged amino acid (aa) scattered in E2 glycoprotein (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72: 7357–7366, 1998). Here we have further confirmed that interaction of E2 protein with HS is crucial for cellular infection of SINV based on the reverse genetic system of XJ-160 virus, a Sindbis-like virus (SINLV). Both SINV YN87448 and SINLV XJ-160 displayed similar infectivity on BHK-21, Vero, or C6/36 cells, but XJ-160 failed to infect mouse embryonic fibroblast (MEF) cells. The molecular mechanisms underlying the selective infectivity of XJ-160 were approached by substituting the E1, E2, or both genes of XJ-160 with that of YN87448, and the chimeric virus was denominated as XJ-160/E1, XJ-160/E2, or XJ-160/E1E2, respectively. In contrast to the parental XJ-160, all chimeric viruses became infectious to wild-type MEF cells (MEF-wt). While MEF-Ext−/− cells, producing shortened HS chains, were resistant not only to XJ-160, but also to YN87448 as well as the chimeric viruses, indicating that the inability of XJ-160 to infect MEF-wt cells likely due to its incompetent discrimination of cellular HS. Treatment with heparin or HS-degrading enzyme resulted in a substantial decrease in plaque formation by YN87448, XJ-160/E2, and XJ-160/E1E2, but had marginal effect on XJ-160 and XJ-160/E1, suggesting that E2 glycoprotein from YN87448 plays a more important role than does E1 in mediating cellular HS-related cell infection. In addition, the peptide containing 145–150 aa from E2 gene of YN87448 specifically bound to heparin, while the corresponding peptide from the E2 gene of XJ-160 essentially showed no binding to heparin. As a new dataset, these results clearly confirm an essential role of E2 glycoprotein, especially the domain of 145–150 aa, in SINV cellular infection through

  10. A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection

    PubMed Central

    Riblett, Amber M.; Blomen, Vincent A.; Jae, Lucas T.; Altamura, Louis A.; Doms, Robert W.; Brummelkamp, Thijn R.

    2015-01-01

    ABSTRACT Rift Valley fever virus (RVFV) causes recurrent insect-borne epizootics throughout the African continent, and infection of humans can lead to a lethal hemorrhagic fever syndrome. Deep mutagenesis of haploid human cells was used to identify host factors required for RVFV infection. This screen identified a suite of enzymes involved in glycosaminoglycan (GAG) biogenesis and transport, including several components of the cis-oligomeric Golgi (COG) complex, one of the central components of Golgi complex trafficking. In addition, disruption of PTAR1 led to RVFV resistance as well as reduced heparan sulfate surface levels, consistent with recent observations that PTAR1-deficient cells exhibit altered Golgi complex morphology and glycosylation defects. A variety of biochemical and genetic approaches were utilized to show that both pathogenic and attenuated RVFV strains require GAGs for efficient infection on some, but not all, cell types, with the block to infection being at the level of virion attachment. Examination of other members of the Bunyaviridae family for GAG-dependent infection suggested that the interaction with GAGs is not universal among bunyaviruses, indicating that these viruses, as well as RVFV on certain cell types, employ additional unidentified virion attachment factors and/or receptors. IMPORTANCE Rift Valley fever virus (RVFV) is an emerging pathogen that can cause severe disease in humans and animals. Epizootics among livestock populations lead to high mortality rates and can be economically devastating. Human epidemics of Rift Valley fever, often initiated by contact with infected animals, are characterized by a febrile disease that sometimes leads to encephalitis or hemorrhagic fever. The global burden of the pathogen is increasing because it has recently disseminated beyond Africa, which is of particular concern because the virus can be transmitted by widely distributed mosquito species. There are no FDA-licensed vaccines or antiviral

  11. Expression of the cell-surface heparan sulfate proteoglycan syndecan-2 in developing rat anterior pituitary gland.

    PubMed

    Horiguchi, Kotaro; Syaidah, Rahimi; Fujiwara, Ken; Tsukada, Takehiro; Ramadhani, Dini; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

    2013-09-01

    In the anterior pituitary gland, folliculo-stellate cells and five types of hormone-producing cells are surrounded by an extracellular matrix (ECM) essential for these cells to perform their respective roles. Syndecans-type I transmembrane cell-surface heparan sulfate proteoglycans act as major ECM coreceptors via their respective heparan sulfate chains and efficiently transduce intracellular signals through the convergent action of their transmembrane and cytoplasmic domains. The syndecans comprise four family members in vertebrates: syndecan-1, -2, -3 and -4. However, whether syndecans are produced in the pituitary gland or whether they have a role as a coreceptor is not known. We therefore used (1) reverse transcription plus the polymerase chain reaction to analyze the expression of syndecan genes and (2) immunohistochemical techniques to identify the cells that produce the syndecans in the anterior pituitary gland of adult rat. Syndecan-2 mRNA expression was clearly detected in the corticotropes of the anterior pituitary gland. Moreover, the expression of syndecan-2 in the developing pituitary gland had a distinct temporospatial pattern. To identify the cells expressing syndecan-2 in the developing pituitary gland, we used double-immunohistochemistry for syndecan-2 and the cell markers E-cadherin (immature cells) and Ki-67 (proliferating cells). Some E-cadherin- and Ki-67-immunopositive cells expressed syndecan-2. Therefore, syndecan-2 expression occurs in developmentally regulated patterns and syndecan-2 probably has different roles in adult and developing anterior pituitary glands.

  12. Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness.

    PubMed

    Dolez, Morgane; Nicolas, Jean-François; Hirsinger, Estelle

    2011-01-01

    In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues, such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analysed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmp-responding cells are excluded from the central domain and that this pattern is modulated by laminins, but not by Hedgehog signalling. Knockdown of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos. Last, extracellular matrix-associated heparan sulfate proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that laminins, via heparan sulfate proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.

  13. Crosstalk between 2 organelles: Lysosomal storage of heparan sulfate causes mitochondrial defects and neuronal death in mucopolysaccharidosis III type C

    PubMed Central

    Pshezhetsky, Alexey V

    2015-01-01

    More than 30% of all lysosomal diseases are mucopolysaccharidoses, disorders affecting the enzymes needed for the stepwise degradation of glycosaminoglycans (mucopolysaccharides). Mucopolysaccharidosis type IIIC (MPS IIIC) is a severe neurologic disease caused by genetic deficiency of heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT). Through our studies, we have cloned the gene, identified molecular defects in MPS IIIC patients and most recently completed phenotypic characterization of the first animal model of the disease, a mouse with a germline inactivation of the Hgsnat gene.1 The obtained data have led us to propose that Hgsnat deficiency and lysosomal accumulation of heparan sulfate in microglial cells followed by their activation and cytokine release result in mitochondrial dysfunction in the neurons causing their death which explains why MPS IIIC manifests primarily as a neurodegenerative disease. The goal of this addendum is to summarize data yielding new insights into the mechanism of MPS IIIC and promising novel therapeutic solutions for this and similar disorders. PMID:26459666

  14. Tandem Mass Spectrometry of Heparan Sulfate Negative Ions: Sulfate Loss Patterns and Chemical Modification Methods for Improvement of Product Ion Profiles

    NASA Astrophysics Data System (ADS)

    Shi, Xiaofeng; Huang, Yu; Mao, Yang; Naimy, Hicham; Zaia, Joseph

    2012-09-01

    Heparan sulfate (HS) is a polysaccharide modified with sulfation, acetylation, and epimerization that enable its binding with protein ligands and regulation of important biological processes. Tandem mass spectrometry has been employed to sequence linear biomolecules e.g., proteins and peptides. However, its application in structural characterization of HS is limited due to the neutral loss of sulfate (SO3) during collisional induced dissociation (CID). In this report, we studied the dissociation patterns of HS disaccharides and demonstrate that the N-sulfate (N-S) bond is especially facile during CID. We identified factors that influence the propensities of such losses from precursor ions and proposed a Free Proton Index (FPI) to help select ions that are able to produce meaningful backbone dissociations. We then investigated the thermodynamics and kinetics of SO3 loss from sulfates that are protonated, deprotonated, and metal-adducted using density functional theory computations. The calculations showed that sulfate loss from a protonated site was much more facile than that from a deprotonated or metal-adducted site. Further, the loss of SO3 from N-sulfate was energetically favored by 3-8 kcal/mol in transition states relative to O-sulfates, making it more prone to this process by a substantial factor. In order to reduce the FPI, representing the number of labile sulfates in HS native chains and oligosaccharides, we developed a series of chemical modifications to selectively replace the N-sulfates of the glucosamine with deuterated acetyl group. These modifications effectively reduced the sulfate density on the HS oligosaccharides and generated considerably more backbone dissociation using on-line LC/tandem MS.

  15. Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues.

    PubMed

    Nesterenko, Alexey M; Orlov, Eugeny E; Ermakova, Galina V; Ivanov, Igor A; Semenyuk, Pavel I; Orlov, Victor N; Martynova, Natalia Y; Zaraisky, Andrey G

    Heparin binding motifs were found in many secreted proteins and it was suggested that they are responsible for retardation of the protein diffusion within the intercellular space due to the binding to heparan sulfate proteoglycanes (HSPG). Here we used synthetic FITC labeled heparin binding motif (HBM peptide) of the Xenopus laevis secreted BMP inhibitor Noggin1 to study its diffusion along the surface of the heparin beads by FRAP method. As a result, we have found out that diffusivity of HBM-labeled FITC was indeed much lesser than those predicted by theoretical calculations even for whole protein of the Noggin size. We also compared by isothermal titration calorimetry the binding affinity of HBM and the control oligolysine peptide to several natural polyanions including heparan sulfate (HS), heparin, the bacterial dextran sulfate and salmon sperm DNA, and demonstrated that HBM significantly exceeds oligolysine peptide in the affinity to HS, heparin and DNA. By contrast, oligolysine peptide bound with higher affinity to dextran sulfate. We speculate that such a difference may ensure specificity of the morphogen binding to HSPG and could be explained by steric constrains imposed by different distribution of the negative charges along a given polymeric molecule. Finally, by using EGFP-HBM recombinant protein we have visualized the natural pattern of the Noggin1 binding sites within the X. laevis gastrula and demonstrated that these sites forms a dorsal-ventral concentration gradient, with a maximum in the dorsal blastopore lip. In sum, our data provide a quantitative basis for modeling the process of Noggin1 diffusion in embryonic tissues, considering its interaction with HSPG.

  16. Heparanase-1-induced shedding of heparan sulfate from syndecan-1 in hepatocarcinoma cell facilitates lymphatic endothelial cell proliferation via VEGF-C/ERK pathway.

    PubMed

    Yu, Shengjin; Lv, Huiming; Zhang, He; Jiang, Yu; Hong, Yu; Xia, Rongjun; Zhang, Qifang; Ju, Weiwei; Jiang, Lili; Ou, Geng; Zhang, Jinhui; Wang, Shujing; Zhang, Jianing

    2017-02-13

    Heparanase-1/syndecan-1 axis plays critical roles in tumorigenesis and development. The main mechanism includes heparanase-1 (HPA-1) degrades the heparan sulfate chain of syndecan-1 (SDC-1), and the following shedding of heparan sulfate from tumor cell releases and activates SDC-1 sequestered growth factors. However, the significance of Heparanase-1/syndecan-1 axis and its effects on the microenvironment of lymphatic metastasis in hepatocellular carcinogenesis (HCC) procession have not been reported. Herein, we found that HPA-1 could degrade the heparan sulfate on hepatocarcinoma cell surface. Importantly, HPA-1-induced shedding of heparan sulfate chain from SDC-1 facilitated the release of vascular endothelial growth factor C (VEGF-C) from SDC-1/VEGF-C complex into the medium of hepatocarcinoma cell. Further studies indicated that VEGF-C secretion from hepatocarcinoma cell promoted lymphatic endothelial cell growth through activating extracellular signal-regulated kinase (ERK) signaling. Taken together, this study reveals a novel existence of Heparanase-1/syndecan-1 axis in hepatocarcinoma cell and its roles in the cross-talking with the microenvironment of lymphatic metastasis.

  17. Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum).

    PubMed

    Phan, Anne Q; Lee, Jangwoo; Oei, Michelle; Flath, Craig; Hwe, Caitlyn; Mariano, Rachele; Vu, Tiffany; Shu, Cynthia; Dinh, Andrew; Simkin, Jennifer; Muneoka, Ken; Bryant, Susan V; Gardiner, David M

    2015-08-01

    Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain-of-function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneration. We discovered that salamander limb ECM has a position-specific ability to either inhibit regeneration or induce de novo limb structure, and that this difference is dependent on heparan sulfates that are associated with differential expression of heparan sulfate sulfotransferases. We also discovered that an artificial ECM containing only heparan sulfate was sufficient to induce de novo limb pattern in salamander limb regeneration. Finally, ECM from mouse limbs is capable of inducing limb pattern in axolotl blastemas in a position-specific, developmental-stage-specific, and heparan sulfate-dependent manner. This study demonstrates a mechanism for positional information in regeneration and establishes a crucial functional link between salamander regeneration and mammals.

  18. Border Patrol: Insights into the Unique Role of Perlecan/Heparan Sulfate Proteoglycan2 at Cell and Tissue Borders

    PubMed Central

    Farach-Carson, Mary C.; Warren, Curtis R.; Harrington, Daniel A.; Carson, Daniel D.

    2013-01-01

    The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550M years) extracellular matrix molecules. In vertebrates, perlecan’s five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously

  19. Border patrol: insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders.

    PubMed

    Farach-Carson, Mary C; Warren, Curtis R; Harrington, Daniel A; Carson, Daniel D

    2014-02-01

    The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550 M years) extracellular matrix molecules. In vertebrates, perlecan's five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously

  20. 2-O Heparan Sulfate Sulfation by Hs2st Is Required for Erk/Mapk Signalling Activation at the Mid-Gestational Mouse Telencephalic Midline.

    PubMed

    Chan, Wai Kit; Howe, Katherine; Clegg, James M; Guimond, Scott E; Price, David J; Turnbull, Jeremy E; Pratt, Thomas

    2015-01-01

    Heparan sulfate (HS) is a linear carbohydrate composed of polymerized uronate-glucosamine disaccharide units that decorates cell surface and secreted glycoproteins in the extracellular matrix. In mammals HS is subjected to differential sulfation by fifteen different heparan sulfotransferase (HST) enzymes of which Hs2st uniquely catalyzes the sulfation of the 2-O position of the uronate in HS. HS sulfation is postulated to be important for regulation of signaling pathways by facilitating the interaction of HS with signaling proteins including those of the Fibroblast Growth Factor (Fgf) family which signal through phosphorylation of extracellular signal-regulated kinases Erk1/2. In the developing mouse telencephalon Fgf2 signaling regulates proliferation and neurogenesis. Loss of Hs2st function phenocopies the thinned cerebral cortex of mutant mice in which Fgf2 or Erk1/2 function are abrogated, suggesting the hypothesis that 2-O-sulfated HS structures play a specific role in Fgf2/Erk signaling pathway in this context in vivo. This study investigated the molecular role of 2-O sulfation in Fgf2/Erk signaling in the developing telencephalic midline midway through mouse embryogenesis at E12.5. We examined the expression of Hs2st, Fgf2, and Erk1/2 activity in wild-type and Hs2st-/- mice. We found that Hs2st is expressed at high levels at the midline correlating with high levels of Erk1/2 activation and Erk1/2 activation was drastically reduced in the Hs2st-/- mutant at the rostral telencephalic midline. We also found that 2-O sulfation is specifically required for the binding of Fgf2 protein to Fgfr1, its major cell-surface receptor at the rostral telencephalic midline. We conclude that 2-O sulfated HS structures generated by Hs2st are needed to form productive signaling complexes between HS, Fgf2 and Fgfr1 that activate Erk1/2 at the midline. Overall, our data suggest the interesting possibility that differential expression of Hs2st targets the rostral telencephalic

  1. Localization of human serum amyloid P component and heparan sulfate proteoglycan in in vitro-formed Abeta fibrils.

    PubMed

    Holm Nielsen, E; Nybo, M; Junker, K; Toftedal Hansen, P; Rasmussen, I M; Svehag, S E

    2000-08-01

    Ultrastructural studies of the localization of serum amyloid P component (SAP) in amyloid fibrils have given divergent results. We here report for the first time that electron microscopy of SAP coincubated with Abeta1-42 peptides or with mature Abeta1-42 fibrils, revealed SAP molecules coating the surface of the mature fibrils and that protofibrils of Abeta1-42 did not bind SAP. Also when incubated with extracted amyloid light chain (AL)-fibrils the SAP molecules aligned on the fibril surface. Heparan sulfate proteoglycan bound to the surface of the Abeta fibrils with a spacing of about 50 nm. We conclude that SAP does not bind to protofibrils but to the surface of mature Abeta fibrils and that it may stabilize and protect the fibrils.

  2. Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias

    PubMed Central

    Mizumoto, Shuji; Yamada, Shuhei; Sugahara, Kazuyuki

    2015-01-01

    Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide. PMID:26582078

  3. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    SciTech Connect

    Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

    2009-12-18

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  4. Synovial joint formation requires local Ext1 expression and heparan sulfate production in developing mouse embryo limbs and spine

    PubMed Central

    Mundy, Christina; Yasuda, Tadashi; Kinumatsu, Takashi; Yamaguchi, Yu; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi; Koyama, Eiki; Pacifici, Maurizio

    2011-01-01

    Heparan sulfate proteoglycans (HSPGs) regulate a number of major developmental processes, but their roles in synovial joint formation remain unknown. Here we created conditional mouse embryo mutants lacking Ext1 in developing joints by mating Ext1f/f and Gdf5-Cre mice. Ext1 encodes a subunit of the Ext1/Ext2 Golgi-associated protein complex responsible for heparan sulfate (HS) synthesis. The proximal limb joints did form in the Gdf5-Cre;Ext1f/f mutants, but contained an uneven articulating superficial zone that expressed very low lubricin levels. The underlying cartilaginous epiphysis was deranged as well and displayed random patterns of cell proliferation and matrillin-1 and collagen IIA expression, indicative of an aberrant phenotypic definition of the epiphysis itself. Digit joints were even more affected, lacked a distinct mesenchymal interzone and were often fused likely as a result of local abnormal BMP and hedgehog activity and signaling. Interestingly, overall growth and lengthening of long bones were also delayed in the mutants. To test whether Ext1 function is needed for joint formation at other sites, we examined the spine. Indeed, entire intervertebral discs, normally composed by nucleus pulposus surrounded by the annulus fibrosus, were often missing in Gdf5-Cre;Ext1f/f mice. When disc remnants were present, they displayed aberrant organization and defective joint marker expression. Similar intervertebral joint defects and fusions occurred in Col2-Cre;β-cateninf/f mutants. The study provides novel evidence that local Ext1 expression and HS production are needed to maintain the phenotype and function of joint-forming cells and coordinate local signaling by BMP, hedgehog and Wnt/β-catenin pathways. The data indicate also that defects in joint formation reverberate on, and delay, overall long bone growth. PMID:21185280

  5. Microfluidic Screening Reveals Heparan Sulfate Enhances Human Mesenchymal Stem Cell Growth by Modulating Fibroblast Growth Factor-2 Transport.

    PubMed

    Titmarsh, Drew M; Tan, Clarissa L L; Glass, Nick R; Nurcombe, Victor; Cooper-White, Justin J; Cool, Simon M

    2017-04-01

    Cost-effective expansion of human mesenchymal stem/stromal cells (hMSCs) remains a key challenge for their widespread clinical deployment. Fibroblast growth factor-2 (FGF-2) is a key hMSC mitogen often supplemented to increase hMSC growth rates. However, hMSCs also produce endogenous FGF-2, which critically interacts with cell surface heparan sulfate (HS). We assessed the interplay of FGF-2 with a heparan sulfate variant (HS8) engineered to bind FGF-2 and potentiate its activity. Bone marrow-derived hMSCs were screened in perfused microbioreactor arrays (MBAs), showing that HS8 (50 μg/ml) increased hMSC proliferation and cell number after 3 days, with an effect equivalent to FGF-2 (50 ng/ml). In combination, the effects of HS8 and FGF-2 were additive. Differential cell responses, from upstream to downstream culture chambers under constant flow of media in the MBA, provided insights into modulation of FGF-2 transport by HS8. HS8 treatment induced proliferation mainly in the downstream chambers, suggesting a requirement for endogenous FGF-2 accumulation, whereas responses to FGF-2 occurred primarily in the upstream chambers. Adding HS8 along with FGF-2, however, maximized the range of FGF-2 effectiveness. Measurements of FGF-2 in static cultures then revealed that this was because HS8 caused increased endogenous FGF-2 production and liberated FGF-2 from the cell surface into the supernatant. HS8 also sustained levels of supplemented FGF-2 available over 3 days. These results suggest HS8 enhances hMSC proliferation and expansion by leveraging endogenous FGF-2 production and maximizing the effect of supplemented FGF-2. This is an exciting strategy for cost-effective expansion of hMSCs. Stem Cells Translational Medicine 2017;6:1178-1190.

  6. The Synthetic Antimicrobial Peptide 19-2.5 Interacts with Heparanase and Heparan Sulfate in Murine and Human Sepsis

    PubMed Central

    Martin, Lukas; De Santis, Rebecca; Koczera, Patrick; Simons, Nadine; Haase, Hajo; Heinbockel, Lena; Brandenburg, Klaus; Marx, Gernot; Schuerholz, Tobias

    2015-01-01

    Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains from their proteoglycans. Thereby, heparanase liberates highly potent circulating heparan sulfate-fragments (HS-fragments) and triggers the fatal and excessive inflammatory response in sepsis. As a potential anti-inflammatory agent for sepsis therapy, peptide 19–2.5 belongs to the class of synthetic anti-lipopolysaccharide peptides; however, its activity is not restricted to Gram-negative bacterial infection. We hypothesized that peptide 19–2.5 interacts with heparanase and/or HS, thereby reducing the levels of circulating HS-fragments in murine and human sepsis. Our data indicate that the treatment of septic mice with peptide 19–2.5 compared to untreated control animals lowers levels of plasma heparanase and circulating HS-fragments and reduces heparanase activity. Additionally, mRNA levels of heparanase in heart, liver, lung, kidney and spleen are downregulated in septic mice treated with peptide 19–2.5 compared to untreated control animals. In humans, plasma heparanase level and activity are elevated in septic shock. The ex vivo addition of peptide 19–2.5 to plasma of septic shock patients decreases heparanase activity but not heparanase level. Isothermal titration calorimetry revealed a strong exothermic reaction between peptide 19–2.5 and heparanase and HS-fragments. However, a saturation character has been identified only in the peptide 19–2.5 and HS interaction. In conclusion, the findings of our current study indicate that peptide 19–2.5 interacts with heparanase, which is elevated in murine and human sepsis and consecutively attenuates the generation of circulating HS-fragments in systemic inflammation. Thus, peptide 19–2.5 seems to be a potential anti-inflammatory agent in sepsis. PMID:26600070

  7. Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum)

    PubMed Central

    Phan, Anne Q.; Lee, Jangwoo; Oei, Michelle; Flath, Craig; Hwe, Caitlyn; Mariano, Rachele; Vu, Tiffany; Shu, Cynthia; Dinh, Andrew; Simkin, Jennifer; Muneoka, Ken; Bryant, Susan V.

    2015-01-01

    Abstract Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain‐of‐function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneration. We discovered that salamander limb ECM has a position‐specific ability to either inhibit regeneration or induce de novo limb structure, and that this difference is dependent on heparan sulfates that are associated with differential expression of heparan sulfate sulfotransferases. We also discovered that an artificial ECM containing only heparan sulfate was sufficient to induce de novo limb pattern in salamander limb regeneration. Finally, ECM from mouse limbs is capable of inducing limb pattern in axolotl blastemas in a position‐specific, developmental‐stage‐specific, and heparan sulfate‐dependent manner. This study demonstrates a mechanism for positional information in regeneration and establishes a crucial functional link between salamander regeneration and mammals. PMID:27499874

  8. Improved liquid chromatography-MS/MS of heparan sulfate oligosaccharides via chip-based pulsed makeup flow.

    PubMed

    Huang, Yu; Shi, Xiaofeng; Yu, Xiang; Leymarie, Nancy; Staples, Gregory O; Yin, Hongfeng; Killeen, Kevin; Zaia, Joseph

    2011-11-01

    Microfluidic chip-based hydrophilic interaction chromatography (HILIC) is a useful separation system for liquid chromatography-mass spectrometry (LC-MS) in compositional profiling of heparan sulfate (HS) oligosaccharides; however, ions observed using HILIC LC-MS are low in charge. Tandem MS of HS oligosaccharide ions with low charge results in undesirable losses of SO(3) from precursor ions during collision induced dissociation. One solution is to add metal cations to stabilize sulfate groups. Another is to add a nonvolatile, polar compound such as sulfolane, a molecule known to supercharge proteins, to produce a similar effect for oligosaccharides. We demonstrate use of a novel pulsed makeup flow (MUF) HPLC-chip. The chip enables controlled application of additives during specified chromatographic time windows and thus minimizes the extent to which nonvolatile additives build up in the ion source. The pulsed MUF system was applied to LC-MS/MS of HS oligosaccharides. Metal cations and sulfolane were tested as additives. The most promising results were obtained for sulfolane, for which supercharging of the oligosaccharide ions increased their signal strengths relative to controls. Tandem MS of these supercharged precursor ions showed decreased abundances of product ions from sulfate losses yet more abundant product ions from backbone cleavages.

  9. Profiling sulfation/epimerization pattern of full-length heparan sulfate by NMR following cell culture 13C-glucose metabolic labeling.

    PubMed

    Pegeot, Mathieu; Sadir, Rabia; Eriksson, Inger; Kjellen, Lena; Simorre, Jean-Pierre; Gans, Pierre; Lortat-Jacob, Hugues

    2015-02-01

    Through its ability to interact with proteins, heparan sulfate (HS) fulfills a large variety of functions. Protein binding depends on the level of HS sulfation and epimerization which are cell specific and dynamically regulated. Characterization of this molecule, however, has been restricted to oligosaccharide fragments available in large amount for structural investigation or to sulfate distribution through compositional analysis. Here we developed a (1)H-(13)C 2D NMR-based approach, directly performed on HS isolated from (13)C-labeled cells. By integrating the peak volumes measured at different chemical shifts, this non-destructive analysis allows us to determine both the sulfation and the iduronic/glucuronic profiles of the polysaccharide. Applied to wild-type and N-deacetylase/N-sulfotransferase-deficient fibroblasts as well as to epithelial cells differentiation, it also gives insights into the functional relationships existing between HS biosynthetic enzymes. This approach should be of significant interest to better understand HS changes that occur through physiologic regulations or during pathological development.

  10. Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88

    PubMed Central

    Hossain, Md Motarab; Hosono-Fukao, Tomomi; Tang, Renhong; Sugaya, Noriko; van Kuppevelt, Toin H; Jenniskens, Guido J; Kimata, Koji; Rosen, Steven D; Uchimura, Kenji

    2010-01-01

    Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O-sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293, and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O, and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes. PMID:19822709

  11. Degradation of endothelial cell matrix heparan sulfate proteoglycan by elastase and the myeloperoxidase-H2O2-chloride system.

    PubMed Central

    Klebanoff, S. J.; Kinsella, M. G.; Wight, T. N.

    1993-01-01

    The degradation of the heparan sulfate proteoglycans of subendothelial matrix by neutrophil elastase and the myeloperoxidase-H2O2-chloride system added separately, sequentially, or together at pH 4.5 to 7.5 was determined by the release of lower molecular weight 35S-labeled material. Elastase alone and the myeloperoxidase system alone caused degradation, and when 4-hour exposure to elastase was followed by 15 minutes of exposure to the myeloperoxidase system, the effect was greater than additive. A greater than additive effect was not observed when elastase followed the myeloperoxidase system or the two were added together. Chloride (or sulfate) alone increased the release of 35S-labeled material from elastase-treated matrix, although the effect of 0.1 M chloride was not as great as that observed when an equivalent concentration of chloride was combined with myeloperoxidase and H2O2. The release of these systems at sites of adherence of neutrophils to glomerular basement membrane may contribute to neutrophil-associated proteinuria. Images Figure 1 Figure 2 Figure 5 Figure 7 PMID:8395774

  12. A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread

    SciTech Connect

    O'Donnell, Christopher D.; Tiwari, Vaibhav; Oh, Myung-Jin; Shukla, Deepak . E-mail: dshukla@uic.edu

    2006-03-15

    Heparan sulfate (HS) 3-O-sulfotransferase isoform-2 (3-OST-2), which belongs to a family of enzymes capable of generating herpes simplex virus type-1 (HSV-1) entry and spread receptors, is predominantly expressed in human brain. Despite its unique expression pattern, the ability of 3-OST-2 to mediate HSV-1 entry and cell-to-cell fusion is not known. Our results demonstrate that expression of 3-OST-2 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and mutant strains of HSV-1. Evidence for generation of gD receptors by 3-OST-2 were suggested by gD-mediated interference assay and the ability of 3-OST-2-expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases II/III). In addition, 3-OST-2-expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins, a phenomenon that mimics a way of viral spread in vivo. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together, our results raise the possibility of a role of 3-OST-2 in the spread of HSV-1 infection in the brain.

  13. Distinct 3-O-Sulfated Heparan Sulfate Modification Patterns Are Required for kal-1−Dependent Neurite Branching in a Context-Dependent Manner in Caenorhabditis elegans

    PubMed Central

    Tecle, Eillen; Diaz-Balzac, Carlos A.; Bülow, Hannes E.

    2013-01-01

    Heparan sulfate (HS) is an unbranched glycosaminoglycan exhibiting substantial molecular diversity due to multiple, nonuniformly introduced modifications, including sulfations, epimerization, and acetylation. HS modifications serve specific and instructive roles in neuronal development, leading to the hypothesis of a HS code that regulates nervous system patterning. Although the in vivo roles of many of the HS modifications have been investigated, very little is known about the function of HS 3-O-sulfation in vivo. By examining patterning of the Caenorhabditis elegans nervous system in loss of function mutants of the two 3-O-sulfotransferases, hst-3.1 and hst-3.2, we found HS 3-O-sulfation to be largely dispensable for overall neural development. However, generation of stereotypical neurite branches in hermaphroditic-specific neurons required hst-3.1, hst-3.2, as well as an extracellular cell adhesion molecule encoded by kal-1, the homolog of Kallmann Syndrome associated gene 1/anosmin-1. In contrast, kal-1−dependent neurite branching in AIY neurons required catalytic activity of hst-3.2 but not hst-3.1. The context-dependent requirement for hst-3.2 and hst-3.1 indicates that both enzymes generate distinct types of HS modification patterns in different cell types, which regulate kal-1 to promote neurite branching. We conclude that HS 3-O-sulfation does not play a general role in establishing the HS code in C. elegans but rather plays a specialized role in a context-dependent manner to establish defined aspects of neuronal circuits. PMID:23451335

  14. Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity.

    PubMed

    Christianson, Helena C; Svensson, Katrin J; van Kuppevelt, Toin H; Li, Jin-Ping; Belting, Mattias

    2013-10-22

    Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosome-associated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

  15. Age-related impairment of endothelial progenitor cell migration correlates with structural alterations of heparan sulfate proteoglycans.

    PubMed

    Williamson, Kate A; Hamilton, Andrew; Reynolds, John A; Sipos, Peter; Crocker, Ian; Stringer, Sally E; Alexander, Yvonne M

    2013-02-01

    Aging poses one of the largest risk factors for the development of cardiovascular disease. The increased propensity toward vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells to contribute to vascular repair and regeneration. Although there is evidence to suggest that colony forming unit-Hill cells and circulating angiogenic cells are subject to age-associated changes that impair their function, the impact of aging on human outgrowth endothelial cell (OEC) function has been less studied. We demonstrate that OECs isolated from cord blood or peripheral blood samples from young and old individuals exhibit different characteristics in terms of their migratory capacity. In addition, age-related structural changes were discovered in OEC heparan sulfate (HS), a glycocalyx component that is essential in many signalling pathways. An age-associated decline in the migratory response of OECs toward a gradient of VEGF significantly correlated with a reduction in the relative percentage of the trisulfated disaccharide, 2-O-sulfated-uronic acid, N, 6-O-sulfated-glucosamine (UA[2S]-GlcNS[6S]), within OEC cell surface HS polysaccharide chains. Furthermore, disruption of cell surface HS reduced the migratory response of peripheral blood-derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Together these findings suggest that aging is associated with alterations in the fine structure of HS on the cell surface of OECs. Such changes may modulate the migration, homing, and engraftment capacity of these repair cells, thereby contributing to the progression of endothelial dysfunction and age-related vascular pathologies.

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

    SciTech Connect

    Dahms, Sven O.; Mayer, Magnus C.; Roeser, Dirk; Multhaup, Gerd; Than, Manuel E.

    2015-03-01

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

  17. Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide.

    PubMed

    Gonçalves, Elisabete; Kitas, Eric; Seelig, Joachim

    2005-02-22

    Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting

  18. Characterization of uniformly and atom-specifically 13C-labeled heparin and heparan sulfate polysaccharide precursors using 13C NMR spectroscopy and ESI mass spectrometry

    PubMed Central

    Nguyen, Thao K. N.; Tran, Vy M.; Victor, Xylophone V.; Skalicky, Jack J.; Kuberan, Balagurunathan

    2010-01-01

    The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin/heparan sulfate oligosaccharide or disaccharide fragments using simple 1D 1H NMR spectroscopy is often complicated by the extensive signal overlap. 13C NMR signals, on the other hand, overlap less and therefore, 13C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific 13C-labeled heparin/heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly 13C- and 15N-labeled precursors were also produced and characterized by 13C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of 13C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% 13C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome–proteome interactions. PMID:20832774

  19. Sanfilippo syndrome type C: mutation spectrum in the heparan sulfate acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT) gene.

    PubMed

    Feldhammer, Matthew; Durand, Stéphanie; Mrázová, Lenka; Boucher, Renée-Myriam; Laframboise, Rachel; Steinfeld, Robert; Wraith, James E; Michelakakis, Helen; van Diggelen, Otto P; Hrebícek, Martin; Kmoch, Stanislav; Pshezhetsky, Alexey V

    2009-06-01

    Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA (AcCoA): alpha-glucosaminide N-acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by alpha-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death, causing neurodegeneration and severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice-site mutations, 11 insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php?select_db=HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS IIIC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT.

  20. ScFv anti-heparan sulfate antibodies unexpectedly activate endothelial and cancer cells through p38 MAPK: implications for antibody-based targeting of heparan sulfate proteoglycans in cancer.

    PubMed

    Christianson, Helena C; van Kuppevelt, Toin H; Belting, Mattias

    2012-01-01

    Tumor development requires angiogenesis and anti-angiogenic therapies have been introduced in the treatment of cancer. In this context, heparan sulfate proteoglycans (HSPGs) emerge as interesting targets, owing to their function as co-receptors of major, pro-angiogenic factors. Accordingly, previous studies have suggested anti-tumor effects of heparin, i.e. over-sulfated HS, and various heparin mimetics; however, a significant drawback is their unspecific mechanism of action and potentially serious side-effects related to their anticoagulant properties. Here, we have explored the use of human ScFv anti-HS antibodies (αHS) as a more rational approach to target HSPG function in endothelial cells (ECs). αHS were initially selected for their recognition of HS epitopes localized preferentially to the vasculature of patient glioblastoma tumors, i.e. highly angiogenic brain tumors. Unexpectedly, we found that these αHS exhibited potent pro-angiogenic effects in primary human ECs. αHS were shown to stimulate EC differentiation, which was associated with increased EC tube formation and proliferation. Moreover, αHS supported EC survival under hypoxia and starvation, i.e. conditions typical of the tumor microenvironment. Importantly, αHS-mediated proliferation was efficiently counter-acted by heparin and was absent in HSPG-deficient mutant cells, confirming HS-specific effects. On a mechanistic level, binding of αHS to HSPGs of ECs as well as glioblastoma cells was found to trigger p38 MAPK-dependent signaling resulting in increased proliferation. We conclude that several αHS that recognize HS epitopes abundant in the tumor vasculature may elicit a pro-angiogenic response, which has implications for the development of antibody-based targeting of HSPGs in cancer.

  1. The Heparan and Heparin Metabolism Pathway is Involved in Regulation of Fatty Acid Composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Six genes involved in the heparan sulfate and heparin metabolism pathway, DSEL (dermatan sulfate epimerase-like), EXTL1 (exostoses (multiple)-like 1), HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), HS6ST3 (heparan sulfate 6-O-sulfotransferase 3), NDST3 (N-deacetylase/N-sulfotransferase (heparan gl...

  2. Sweet on Hedgehogs: regulatory roles of heparan sulfate proteoglycans in Hedgehog-dependent cell proliferation and differentiation.

    PubMed

    Bandari, Shyam; Exner, Sebastian; Ortmann, Corinna; Bachvarova, Velina; Vortkamp, Andrea; Grobe, Kay

    2015-01-01

    Morphogens exert their effects over long distances, typically by spreading from cell to cell to activate signal transduction in surrounding tissues in concentration-dependent manner. One example of a morphogen is the signaling molecule Hedgehog (Hh), which controls growth and patterning during development and has also been implicated in the progression of numerous cancers. To this end, accessory mechanisms that release, transport, and receive Hhs are required to elicit temporally and spatially specific responses in cells and tissues. The Hh spreading mechanism is especially intriguing, because all Hhs are released from the producing cells despite being synthesized as dually lipidated, membrane-tethered molecules. In addition to this cellular association, Hhs bind strongly to extracellular heparan sulfate proteoglycans (HSPGs), which is expected to further reduce their spreading. Paradoxically, several lines of evidence suggest that Hh gradient formation actually requires HSPG expression, and that HSPGs act as both positive and negative regulators of Hh function. This article reviews the multiple roles that HSPGs play in Hh morphogen function, and discusses their congruity with proposed mechanisms of Hh solubilization, transport, and signal reception in vertebrate and invertebrate tissues.

  3. Increased Serum Hyaluronic Acid and Heparan Sulfate in Dengue Fever: Association with Plasma Leakage and Disease Severity

    PubMed Central

    Tang, Tommy Hing-Cheung; Alonso, Sylvie; Ng, Lisa Fong-Poh; Thein, Tun-Linn; Pang, Vincent Jun-Xiong; Leo, Yee-Sin; Lye, David Chien-Boon; Yeo, Tsin-Wen

    2017-01-01

    Plasma leakage is a major pathogenic mechanism of severe dengue, but the etiology remains unclear. The association between endothelial glycocalyx integrity and vascular permeability in older adults with dengue has not been evaluated. A prospective cohort study of adults with undifferentiated fever screened for dengue by RT-PCR or NS1 antigen testing was performed. Patients were assessed daily while symptomatic and at convalescence. Serum hyaluronic acid (HA), heparan sulfate (HS) and selected cytokines (TNF-α, IL-6, IL-10) were measured on enrollment and convalescence. Patients were diagnosed as dengue fever (DF, n = 30), dengue hemorrhagic fever (DHF, n = 20) and non-dengue (ND) febrile illness (n = 11). Acute HA and HS levels were significantly higher in all dengue patients compared to ND (p = 0.0033 and p = 0.0441 respectively), but not different between DF and DHF (p = 0.3426 and p = 0.9180 respectively). Enrolment HA inversely correlated with serum albumin, protein and platelets in all dengue and DHF (p < 0.05). HA and HS in all dengue patients decreased significantly at convalescence. Serum IL-10 was significantly associated with HA in all dengue patients (p = 0.002). Serum HA and HS levels were increased in adult dengue and HA was associated with markers of disease severity. Endothelial glycocalyx damage may have a role in vascular leakage in dengue. PMID:28393899

  4. Interaction of low molecular weight group IIA phospholipase A2 with apoptotic human T cells: role of heparan sulfate proteoglycans.

    PubMed

    Boilard, Eric; Bourgoin, Sylvain G; Bernatchez, Chantale; Poubelle, Patrice E; Surette, Marc E

    2003-06-01

    Human group IIA phospholipase A2 (hIIA PLA2) is a 14 kDa secreted enzyme associated with inflammatory diseases. A newly discovered property of hIIA PLA2 is the binding affinity for the heparan sulfate proteoglycan (HSPG) glypican-1. In this study, the binding of hIIA PLA2 to apoptotic human T cells was investigated. Little or no exogenous hIIA PLA2 bound to CD3-activated T cells but significant binding was measured on activated T cells induced to undergo apoptosis by anti-CD95. Binding to early apoptotic T cells was greater than to late apoptotic cells. The addition of heparin and the hydrolysis of HSPG by heparinase III only partially inhibited hIIA PLA2 binding to apoptotic cells, suggesting an interaction with both HSPG and other binding protein(s). Two low molecular weight HSPG were coimmunoprecipitated with hIIA PLA2 from apoptotic T cells, but not from living cells. Treatment of CD95-stimulated T cells with hIIA PLA2 resulted in the release of arachidonic acid but not oleic acid from cells and this release was blocked by heparin and heparinase III. Altogether, these results suggest a role for hIIA PLA2 in the release of arachidonic acid from apoptotic cells through interactions with HSPG and its potential implication in the progression of inflammatory diseases.

  5. Heparan sulfate acts as a bone morphogenetic protein coreceptor by facilitating ligand-induced receptor hetero-oligomerization.

    PubMed

    Kuo, Wan-Jong; Digman, Michelle A; Lander, Arthur D

    2010-11-15

    Cell surface heparan sulfate (HS) not only binds several major classes of growth factors but also sometimes potentiates their activities--an effect usually termed "coreception." A view that coreception is due to the stabilization of growth factor-receptor interactions has emerged primarily from studies of the fibroblast growth factors (FGFs). Recent in vivo studies have strongly suggested that HS also plays an important role in regulating signaling by the bone morphogenetic proteins (BMPs). Here, we provide evidence that the mechanism of coreception for BMPs is markedly different from that established for FGFs. First, we demonstrate a direct, stimulatory role for cell surface HS in the immediate signaling activities of BMP2 and BMP4, and we provide evidence that HS-BMP interactions are required for this effect. Next, using several independent assays of ligand binding and receptor assembly, including coimmunoprecipitation, cross-linking, and fluorescence fluctuation microscopy, we show that HS does not affect BMP binding to type I receptor subunits but instead enhances the subsequent recruitment of type II receptor subunits to BMP-type I receptor complexes. This suggests a view of HS as a catalyst of the formation of signaling complexes, rather than as a stabilizer of growth factor binding.

  6. Adenovirus dodecahedron cell attachment and entry are mediated by heparan sulfate and integrins and vary along the cell cycle.

    PubMed

    Fender, Pascal; Schoehn, Guy; Perron-Sierra, Françoise; Tucker, Gordon C; Lortat-Jacob, Hugues

    2008-02-05

    The adenovirus penton base is a strategic protein involved in the virus internalisation pathway through interaction between its RGD sequences and integrin. In some human adenovirus serotypes, this pentameric protein features the ability of interacting together by twelve, leading to the formation of a symmetric nanoparticle called dodecahedron (Dd). This non-infectious adenovirus-like particle exhibiting sixty RGD sequences interacts with integrin but also with heparan sulfate proteoglycans (HSPGs) expressed at the cell surface. In this study, we discriminate the respective importance of HSPGs and integrin on human adenovirus serotype 3 dodecahedron attachment and entry. Using different cell lines and a specific integrin inhibitor, we have determined that HSPGs are mainly responsible for particle attachment to the cell surface, favouring a strictly required interaction with integrin that triggers internalisation. No other receptors are involved in Dd entry and integrins on their own can mediate the particle entry in HSPGs-deficient cells. Moreover, integrin recognition by Dd is highly susceptible to cations and particularly to manganese that enhances particle binding by 4- to 7-fold compared to calcium. Interestingly, investigations on Dd receptors along the cell cycle revealed an enhanced particle targeting to mitotic cells and a loss of internalisation at this stage. This phenomenon observed with both HeLa- and HSPGs-deficient cells, depends on integrin remodelling during mitosis. This provides new clues for the use of this adenovirus nanoparticle as a delivery vector and sheds light on the integrin and HSPGs relationship in both resting and dividing cells.

  7. N-sulfotestosteronan, a novel substrate for heparan sulfate 6-O-sulfotransferases and its analysis by oxidative degradation

    PubMed Central

    Li, Guoyun; Masuko, Sayaka; Green, Dixy E.; Xu, Yongmei; Li, Lingyun; Zhang, Fuming; Xue, Changhu; Liu, Jian; DeAngelis, Paul. L.; Linhardt, Robert J.

    2013-01-01

    Testosteronan, an unusual glycosaminoglycan first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on β-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the glycosaminoglycan, heparan sulfate. Studies using 35S-labeled 3′-phosphodenosine-5′-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from 34S-labeled 6-O-sulfo-N-sulfotestosteroran using 34S-labeled PAPS because testosteronan was resistant to all of the tested glycosaminoglycan-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of 34S into ~14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to ~20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O–sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to antithrombin III. PMID:23606289

  8. Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells.

    PubMed

    Jakobs, P; Schulz, P; Ortmann, C; Schürmann, S; Exner, S; Rebollido-Rios, R; Dreier, R; Seidler, D G; Grobe, K

    2016-05-20

    Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells.

  9. Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells

    PubMed Central

    Jakobs, P.; Schulz, P.; Ortmann, C.; Schürmann, S.; Exner, S.; Rebollido-Rios, R.; Dreier, R.; Seidler, D. G.; Grobe, K.

    2016-01-01

    Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells. PMID:27199253

  10. Pigment epithelium-derived factor (PEDF) shares binding sites in collagen with heparin/heparan sulfate proteoglycans.

    PubMed

    Sekiya, Atsushi; Okano-Kosugi, Hitomi; Yamazaki, Chisato M; Koide, Takaki

    2011-07-29

    Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues, including the eye. It exhibits various biological functions, such as anti-angiogenic, neurotrophic, and neuroprotective activities. PEDF also interacts with extracellular matrix components such as collagen, heparan sulfate proteoglycans (HSPGs), and hyaluronan. The collagen-binding property has been elucidated to be important for the anti-angiogenic activity in vivo (Hosomichi, J., Yasui, N., Koide, T., Soma, K., and Morita, I. (2005) Biochem. Biophys. Res. Commun. 335, 756-761). Here, we investigated the collagen recognition mechanism by PEDF. We first narrowed down candidate PEDF-binding sequences by taking advantage of previously reported structural requirements in collagen. Subsequent searches for PEDF-binding sequences employing synthetic collagen-like peptides resulted in the identification of one of the critical binding sites for PEDF, human α1(I)(929-938) (IKGHRGFSGL). Further analysis revealed that the collagen recognition by PEDF is sequence- and conformation-specific, and the high affinity binding motif is KGXRGFXGL in the triple helix. The PEDF-binding motif significantly overlapped with the heparin/HSPG-binding motif, KGHRG(F/Y). The interaction of PEDF with collagen I was specifically competed with by heparin but not by chondroitin sulfate-C or hyaluronan. The binding sequences for PEDF and heparin/HSPG also overlapped with the covalent cross-linking sites between collagen molecules. These findings imply a functional relationship between PEDF and HSPGs during angiogenesis, and the interaction of these molecules is regulated by collagen modifications.

  11. Age-related changes in rat myocardium involve altered capacities of glycosaminoglycans to potentiate growth factor functions and heparan sulfate-altered sulfation.

    PubMed

    Huynh, Minh Bao; Morin, Christophe; Carpentier, Gilles; Garcia-Filipe, Stephanie; Talhas-Perret, Sofia; Barbier-Chassefière, Véronique; van Kuppevelt, Toin H; Martelly, Isabelle; Albanese, Patricia; Papy-Garcia, Dulce

    2012-03-30

    Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF(165) was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.

  12. Heparan Sulfate Proteoglycans Are Required for Cellular Binding of the Hepatitis E Virus ORF2 Capsid Protein and for Viral Infection▿ †

    PubMed Central

    Kalia, Manjula; Chandra, Vivek; Rahman, Sheikh Abdul; Sehgal, Deepak; Jameel, Shahid

    2009-01-01

    The hepatitis E virus (HEV), a nonenveloped RNA virus, is the causative agent of hepatitis E. The mode by which HEV attaches to and enters into target cells for productive infection remains unidentified. Open reading frame 2 (ORF2) of HEV encodes its major capsid protein, pORF2, which is likely to have the determinants for virus attachment and entry. Using an ∼56-kDa recombinant pORF2 that can self-assemble as virus-like particles, we demonstrated that cell surface heparan sulfate proteoglycans (HSPGs), specifically syndecans, play a crucial role in the binding of pORF2 to Huh-7 liver cells. Removal of cell surface heparan sulfate by enzymatic (heparinase) or chemical (sodium chlorate) treatment of cells or competition with heparin, heparan sulfate, and their oversulfated derivatives caused a marked reduction in pORF2 binding to the cells. Syndecan-1 is the most abundant proteoglycan present on these cells and, hence, plays a key role in pORF2 binding. Specificity is likely to be dictated by well-defined sulfation patterns on syndecans. We show that pORF2 binds syndecans predominantly via 6-O sulfation, indicating that binding is not entirely due to random electrostatic interactions. Using an in vitro infection system, we also showed a marked reduction in HEV infection of heparinase-treated cells. Our results indicate that, analogous to some enveloped viruses, a nonenveloped virus like HEV may have also evolved to use HSPGs as cellular attachment receptors. PMID:19812150

  13. A synthetic heparan sulfate oligosaccharide library reveals the novel enzymatic action of d-glucosaminyl 3-O-sulfotransferase-3a†

    PubMed Central

    Nu Nguyen, Thao Kim; Arungundram, Sailaja; Tran, Vy My; Raman, Karthik; Al-Mafraji, Kanar; Venot, Andre; Boons, Geert-Jan; Kuberan, Balagurunathan

    2014-01-01

    Heparan sulfate (HS) glucosaminyl 3-O-sulfotranferases sulfate the C3-hydroxyl group of certain glucosamine residues on heparan sulfate. Six different 3-OST isoforms exist, each of which can sulfate very distinct glucosamine residues within the HS chain. Among these isoforms, 3-OST1 has been shown to play a role in generating ATIII-binding HS anticoagulants whereas 3-OST2, 3-OST3, 3-OST4 and 3OST-6 have been shown to play a vital role in generating gD-binding HS chains that permit the entry of herpes simplex virus type 1 into cells. 3-OST5 has been found to generate both ATIII- and gD-binding HS motifs. Previous studies have examined the substrate specificities of all the 3-OST isoforms using HS polysaccharides. However, very few studies have examined the contribution of the epimer configuration of neighboring uronic acid residues next to the target site to 3-OST action. In this study, we utilized a well-defined synthetic oligosaccharide library to examine the substrate specificity of 3-OST3a and compared it to 3-OST1. We found that both 3-OST1 and 3-OST3a preferentially sulfate the 6-O-sulfated, N-sulfoglucosamine when an adjacent iduronyl residue is located to its reducing side. On the other hand, 2-O-sulfation of this uronyl residue can inhibit the action of 3-OST3a on the target residue. The results reveal novel substrate sites for the enzyme actions of 3-OST3a. It is also evident that both these enzymes have promiscuous and overlapping actions that are differentially regulated by iduronyl 2-O-sulfation. PMID:22116385

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

    PubMed

    Ziegler, André; Seelig, Joachim

    2004-01-01

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

  15. N- and 6-O-Sulfated Heparan Sulfates Mediate Internalization of Coxsackievirus B3 Variant PD into CHO-K1 Cells

    PubMed Central

    Zautner, Andreas E.; Jahn, Birgit; Hammerschmidt, Elke; Wutzler, Peter; Schmidtke, Michaela

    2006-01-01

    Recently, it was demonstrated that the coxsackievirus B3 variant PD (CVB3 PD) is able to infect coxsackievirus-adenovirus receptor (CAR)-lacking cells by using heparan sulfates (HS) as additional receptors (A. E. Zautner, U. Korner, A. Henke, C. Badorff, and M. Schmidtke, J. Virol. 77:10071-10077, 2003). For this study, competition experiments with growth factors binding to known HS sequences as well as with specifically desulfated heparins were performed with Chinese hamster ovary cells (CHO-K1) to determine the structural requirements of HS for interaction with CVB3. Hepatocyte growth factor interacting with HS sequences containing [IdUA-GlcNSO3(6OSO3)]n, but not basic fibroblast growth factor binding to [HexUA-GlcNSO3-HexUA-GlcNSO3-IdUA(2OSO3)]n, was shown to compete effectively with CVB3 PD for cell surface HS. Whereas unmodified heparin and 2-O-desulfated heparin strongly inhibited the CVB3 PD-induced cytopathic effect, the antiviral activity was markedly reduced after N-, O- and 6-O-desulfation of heparin. Taken together, these results indicate that 6-O- and N-sulfation of GlcNAc of HS is crucial for HS interaction with CVB3 PD and that the disaccharide [IdUA-GlcNSO3(6OSO3)]n is involved in viral binding. Results from experiments with various inhibitors of endocytic pathways suggest that HS-mediated virus internalization is pH dependent. Despite the fact that CVB3 PD initiates infection about four times slower by making use of HS as a receptor than by using CAR, the time required for a complete viral life cycle in Chinese hamster ovary cells was independent of the utilized receptor. PMID:16775350

  16. Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface

    PubMed Central

    1984-01-01

    After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46- 1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell- associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm- long electron-dense filaments, respectively, on the surface of the tumor

  17. An affinity adsorption media that mimics heparan sulfate proteoglycans for the treatment of drug-resistant bacteremia

    NASA Astrophysics Data System (ADS)

    McCrea, Keith R.; Ward, Robert S.

    2016-06-01

    Removal of several drug-resistant bacteria from blood by affinity adsorption onto a heparin-functional media is reported. Heparin is a chemical analogue of heparan sulfate (HS) proteoglycans, found on transmembrane proteins of endothelial cells. Many blood-borne human pathogens, including bacteria, viruses, parasites, and fungi have been reported to target HS as an initial step in their pathogenesis. Here, we demonstrate the binding and removal of Methicillin-resistant Staphylococcus aureus (MRSA), Extended-Spectrum Betalactamase Klebsiella pneumoniae (ESBL), and two Carbapenem-resistant Enterobacteriaceae (both CRE Escherichia coli and CRE K. pneumoniae) using 300 μm polyethylene beads surface modified with end-point-attached heparin. Depending on the specific bacteria, the amount removed ranged between 39% (ESBL) and 99.9% (CRE). The total amount of bacteria adsorbed ranged between 2.8 × 105 and 8.6 × 105 colony forming units (CFU) per gram of adsorption media. Based on a polymicrobial challenge which showed no competitive binding, MRSA and CRE apparently utilize different binding sequences on the immobilized heparin ligand. Since the total circulating bacterial load during bacteremia seldom exceeds 5 × 105 CFUs, it appears possible to significantly reduce bacterial concentration in infected patients by multi-pass recirculation of their blood through a small extracorporeal affinity filter containing the heparin-functional adsorption media. This 'dialysis-like therapy' is expected to improve patient outcomes and reduce the cost of care, particularly when there are no anti-infective drugs available to treat the infection.

  18. Abnormally High Content of Free Glucosamine Residues Identified in a Preparation of Commercially Available Porcine Intestinal Heparan Sulfate

    PubMed Central

    2016-01-01

    Heparan sulfate (HS) polysaccharides are ubiquitous in animal tissues as components of proteoglycans, and they participate in many important biological processes. HS carbohydrate chains are complex and can contain rare structural components such as N-unsubstituted glucosamine (GlcN). Commercially available HS preparations have been invaluable in many types of research activities. In the course of preparing microarrays to include probes derived from HS oligosaccharides, we found an unusually high content of GlcN residue in a recently purchased batch of porcine intestinal mucosal HS. Composition and sequence analysis by mass spectrometry of the oligosaccharides obtained after heparin lyase III digestion of the polysaccharide indicated two and three GlcN in the tetrasaccharide and hexasaccharide fractions, respectively. 1H NMR of the intact polysaccharide showed that this unusual batch differed strikingly from other HS preparations obtained from bovine kidney and porcine intestine. The very high content of GlcN (30%) and low content of GlcNAc (4.2%) determined by disaccharide composition analysis indicated that N-deacetylation and/or N-desulfation may have taken place. HS is widely used by the scientific community to investigate HS structures and activities. Great care has to be taken in drawing conclusions from investigations of structural features of HS and specificities of HS interaction with proteins when commercial HS is used without further analysis. Pending the availability of a validated commercial HS reference preparation, our data may be useful to members of the scientific community who have used the present preparation in their studies. PMID:27295282

  19. Vaginal Heparan Sulfate Linked to Neutrophil Dysfunction in the Acute Inflammatory Response Associated with Experimental Vulvovaginal Candidiasis

    PubMed Central

    Yano, Junko; Noverr, Mairi C.

    2017-01-01

    ABSTRACT Despite acute inflammation by polymorphonuclear neutrophils (PMNs) during vulvovaginal candidiasis (VVC), clearance of Candida fails to occur. The purpose of this study was to uncover the mechanism of vaginal PMN dysfunction. Designs included assessing PMN migration, proinflammatory mediators, and tissue damage (by analysis of the activity of lactate dehydrogenase [LDH]) in mice susceptible (C3H/HeN-C57BL/6) or resistant (CD-1) to chronic VVC (CVVC-S or CVVC-R) and testing morphology-specific Candida albicans strains under conditions of preinduced PMN migration (CVVC-S mice) or PMN depletion (CVVC-R mice). In vitro designs included evaluation of C. albicans killing by elicited vaginal or peritoneal PMNs in standard or vaginal conditioned medium (VCM). Results showed that despite significant migration of PMNs and high levels of vaginal beta interleukin-1 (IL-1β) and alarmin S100A8, CVVC-S mice failed to reduce vaginal fungal burden irrespective of morphology or whether PMNs were present pre- or postinoculation, and had high LDH levels. In contrast, CVVC-R mice had reduced fungal burden and low LDH levels following PMN recruitment and IL-1β/S100A8 production, but maintained colonization in the absence of PMNs. Elicited vaginal and peritoneal PMNs showed substantial killing activity in standard media or VCM from CVVC-R mice but not in VCM from CVVC-S mice. The inhibitory effect of VCM from CVVC-S mice was unaffected by endogenous or exogenous estrogen and was ablated following depletion/neutralization of Mac-1 ligands using Mac-1+/+ PMNs or recombinant Mac-1. Heparan sulfate (HS) was identified as the putative inhibitor as evidenced by the rescue of PMN killing following heparanase treatment of VCM, as well as by inhibition of killing by purified HS. These results suggest that vaginal HS is linked to PMN dysfunction in CVVC-S mice as a competitive ligand for Mac-1. PMID:28292981

  20. The Role of Heparanase and Sulfatases in the Modification of Heparan Sulfate Proteoglycans within the Tumor Microenvironment and Opportunities for Novel Cancer Therapeutics

    PubMed Central

    Hammond, Edward; Khurana, Ashwani; Shridhar, Viji; Dredge, Keith

    2014-01-01

    Heparan sulfate proteoglycans (HSPGs) are an integral and dynamic part of normal tissue architecture at the cell surface and within the extracellular matrix. The modification of HSPGs in the tumor microenvironment is known to result not just in structural but also functional consequences, which significantly impact cancer progression. As substrates for the key enzymes sulfatases and heparanase, the modification of HSPGs is typically characterized by the degradation of heparan sulfate (HS) chains/sulfation patterns via the endo-6-O-sulfatases (Sulf1 and Sulf2) or by heparanase, an endo-glycosidase that cleaves the HS polymers releasing smaller fragments from HSPG complexes. Numerous studies have demonstrated how these enzymes actively influence cancer cell proliferation, signaling, invasion, and metastasis. The activity or expression of these enzymes has been reported to be modified in a variety of cancers. Such observations are consistent with the degradation of normal architecture and basement membranes, which are typically compromised in metastatic disease. Moreover, recent studies elucidating the requirements for these proteins in tumor initiation and progression exemplify their importance in the development and progression of cancer. Thus, as the influence of the tumor microenvironment in cancer progression becomes more apparent, the focus on targeting enzymes that degrade HSPGs highlights one approach to maintain normal tissue architecture, inhibit tumor progression, and block metastasis. This review discusses the role of these enzymes in the context of the tumor microenvironment and their promise as therapeutic targets for the treatment of cancer. PMID:25105093

  1. Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates

    PubMed Central

    Merilahti, Pirjo; Karelehto, Eveliina; Susi, Petri

    2016-01-01

    Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections. PMID:26785353

  2. The Agmatine-Containing Poly(Amidoamine) Polymer AGMA1 Binds Cell Surface Heparan Sulfates and Prevents Attachment of Mucosal Human Papillomaviruses

    PubMed Central

    Cagno, Valeria; Donalisio, Manuela; Bugatti, Antonella; Civra, Andrea; Cavalli, Roberta; Ranucci, Elisabetta; Ferruti, Paolo; Rusnati, Marco

    2015-01-01

    The agmatine-containing poly(amidoamine) polymer AGMA1 was recently shown to inhibit the infectivity of several viruses, including human papillomavirus 16 (HPV-16), that exploit cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The aim of this work was to assess the antiviral activity of AGMA1 and its spectrum of activity against a panel of low-risk and high-risk HPVs and to elucidate its mechanism of action. AGMA1 was found to be a potent inhibitor of mucosal HPV types (i.e., types 16, 31, 45, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 0.34 μg/ml and 0.73 μg/ml, and no evidence of cytotoxicity was observed. AGMA1 interacted with immobilized heparin and with cellular heparan sulfates, exerting its antiviral action by preventing virus attachment to the cell surface. The findings from this study indicate that AGMA1 is a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections. PMID:26077258

  3. CXCL14 enhances proliferation and migration of NCI-H460 human lung cancer cells overexpressing the glycoproteins containing heparan sulfate or sialic acid.

    PubMed

    Park, Cho Rong; You, Dong-Joo; Kim, Dong-Kyu; Moon, Mi Jin; Lee, Cheolju; Oh, Seung-Hyun; Ahn, Curie; Seong, Jae Young; Hwang, Jong-Ik

    2013-05-01

    CXCL14 is a chemokine family member that is involved in various cellular responses in addition to immune cell activation. Although constitutive CXCL14 expression in normal epithelial cells may help protect against infection by activating immune systems, its expression in cancer cells has raised controversy regarding its possible role in tumorigenesis. However, the underlying mechanisms for this disparity remain unknown. Investigation of cellular CXCL14 binding properties might increase our understanding of the peptide's roles in tumorigenesis. In the present study, we found that CXCL14 binds to various cell types. Interestingly, binding to NCI-H460 cells was prevented by heparan sulfate and N-acetyl neuraminic acid. Next, we examined effect of CXCL14 binding in NCI-H460 and NCI-H23. CXCL14 enhanced proliferation and migration in NCI-H460 but had no effect on NCI-H23. A reporter gene assay with various transcription factor response elements revealed that only nuclear factor-κB (NF-κB) signaling was activated by CXCL14 in NCI-H460 cells, which was blocked by BAPTA-AM, TPCA-1, and brefeldin A. Exogenous expression of some glycoproteins such as syndecan-4, podoplanin, and CD43 in these cells enhanced CXCL14 binding and NF-κB activity. Collectively, these results demonstrate that CXCL14 binding to glycoproteins harboring heparan sulfate proteoglycans and sialic acids leads proliferation and migration of some cancer cells.

  4. Auto-associative heparin nanoassemblies: a biomimetic platform against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV.

    PubMed

    Lembo, David; Donalisio, Manuela; Laine, Claire; Cagno, Valeria; Civra, Andrea; Bianchini, Elsa P; Zeghbib, Narimane; Bouchemal, Kawthar

    2014-09-01

    A new, simple and green method was developed for the manufacturing of heparin nanoassemblies active against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV. These nanoassemblies were obtained by the auto-association of O-palmitoyl-heparin and α-cyclodextrin in water. The synthesized O-palmitoyl-heparin derivatives mixed with α-cyclodextrin resulted in the formation of crystalline hexagonal nanoassemblies as observed by transmission electron microscopy. The nanoassembly mean hydrodynamic diameters were modulated from 340 to 659 nm depending on the type and the initial concentration of O-palmitoyl-heparin or α-cyclodextrin. The antiviral activity of the nanoassemblies was not affected by the concentration of the components. However, the method of the synthesis of O-palmitoyl-heparin affected the antiviral activity of the formulations. We showed that reduced antiviral activity is correlated with lower sulfation degree and anticoagulant activity.

  5. Gas-Phase Analysis of the Complex of Fibroblast GrowthFactor 1 with Heparan Sulfate: A Traveling Wave Ion Mobility Spectrometry (TWIMS) and Molecular Modeling Study

    NASA Astrophysics Data System (ADS)

    Zhao, Yuejie; Singh, Arunima; Xu, Yongmei; Zong, Chengli; Zhang, Fuming; Boons, Geert-Jan; Liu, Jian; Linhardt, Robert J.; Woods, Robert J.; Amster, I. Jonathan

    2017-01-01

    Fibroblast growth factors (FGFs) regulate several cellular developmental processes by interacting with cell surface heparan proteoglycans and transmembrane cell surface receptors (FGFR). The interaction of FGF with heparan sulfate (HS) is known to induce protein oligomerization, increase the affinity of FGF towards its receptor FGFR, promoting the formation of the HS-FGF-FGFR signaling complex. Although the role of HS in the signaling pathways is well recognized, the details of FGF oligomerization and formation of the ternary signaling complex are still not clear, with several conflicting models proposed in literature. Here, we examine the effect of size and sulfation pattern of HS upon FGF1 oligomerization, binding stoichiometry and conformational stability, through a combination of ion mobility (IM) and theoretical modeling approaches. Ion mobility-mass spectrometry (IMMS) of FGF1 in the presence of several HS fragments ranging from tetrasaccharide (dp4) to dodecasaccharide (dp12) in length was performed. A comparison of the binding stoichiometry of variably sulfated dp4 HS to FGF1 confirmed the significance of the previously known high-affinity binding motif in FGF1 dimerization, and demonstrated that certain tetrasaccharide-length fragments are also capable of inducing dimerization of FGF1. The degree of oligomerization was found to increase in the presence of dp12 HS, and a general lack of specificity for longer HS was observed. Additionally, collision cross-sections (CCSs) of several FGF1-HS complexes were calculated, and were found to be in close agreement with experimental results. Based on the (CCSs) a number of plausible binding modes of 2:1 and 3:1 FGF1-HS are proposed.

  6. Synthesis of a targeted library of heparan sulfate hexa- to dodecasaccharides as inhibitors of β-secretase: potential therapeutics for Alzheimer's disease.

    PubMed

    Schwörer, Ralf; Zubkova, Olga V; Turnbull, Jeremy E; Tyler, Peter C

    2013-05-17

    Heparan sulfates (HS) are a class of sulfated polysaccharides that function as dynamic biological regulators of the functions of diverse proteins. The structural basis of these interactions, however, remains elusive, and chemical synthesis of defined structures represents a challenging but powerful approach for unravelling the structure-activity relationships of their complex sulfation patterns. HS has been shown to function as an inhibitor of the β-site cleaving enzyme β-secretase (BACE1), a protease responsible for generating the toxic Aβ peptides that accumulate in Alzheimer's disease (AD), with 6-O-sulfation identified as a key requirement. Here, we demonstrate a novel generic synthetic approach to HS oligosaccharides applied to production of a library of 16 hexa- to dodecasaccharides targeted at BACE1 inhibition. Screening of this library provided new insights into structure-activity relationships for optimal BACE1 inhibition, and yielded a number of potent non-anticoagulant BACE1 inhibitors with potential for development as leads for treatment of AD through lowering of Aβ peptide levels.

  7. The in vivo regulation of pioneer axon growth by FGF-2 and heparan sulfate proteoglycans in cultured embryos of the cockroach.

    PubMed

    Nyhus, J K; Denburg, J L

    1998-08-01

    Antibody perturbation experiments on cultured cockroach embryos demonstrated that a localized source of an FGF-2-like immunoreactive molecule in the head is required for the proper growth of pioneer axons in the leg. The study of axon growth in various fragments of cultured embryos and in the presence of various conditioned media showed that FGF-2 is needed to counteract the effects of an inhibitor of axon growth produced in the body trunk of the embryo. Endogenous heparan sulfate proteoglycans mediate these effects of FGF-2 on axon growth. The results of experiments with FGF-2 and/or body trunk axon growth inhibitor added to the culture medium indicate that more globally and uniformly distributed molecules may play as important a role in axon guidance as the more spatially restricted guidance cues. The results are interpreted in terms of a model that is consistent with a role for the FGF-2 receptor in axon growth.

  8. 1H and 15N NMR Analyses on Heparin, Heparan Sulfates and Related Monosaccharides Concerning the Chemical Exchange Regime of the N-Sulfo-Glucosamine Sulfamate Proton

    PubMed Central

    Pomin, Vitor H.

    2016-01-01

    Heparin and heparan sulfate are structurally related glycosaminoglycans (GAGs). Both GAGs present, although in different concentrations, N-sulfo-glucosamine (GlcNS) as one of their various composing units. The conditional fast exchange property of the GlcNS sulfamate proton in these GAGs has been pointed as the main barrier to its signal detection via NMR experiments, especially 1H-15N HSQC. Here, a series of NMR spectra is collected on heparin, heparan sulfate and related monosaccharides. The N-acetyl glucosamine-linked uronic acid types of these GAGs were properly assigned in the 1H-15N HSQC spectra. Dynamic nuclear polarization (DNP) was employed in order to facilitate 1D spectral acquisition of the sulfamate 15N signal of free GlcNS. Analyses on the multiplet pattern of scalar couplings of GlcNS 15N has helped to understand the chemical properties of the sulfamate proton in solution. The singlet peak observed for GlcNS happens due to fast chemical exchange of the GlcNS sulfamate proton in solution. Analyses on kinetics of alpha-beta anomeric mutarotation via 1H NMR spectra have been performed in GlcNS as well as other glucose-based monosaccharides. 1D 1H and 2D 1H-15N HSQC spectra recorded at low temperature for free GlcNS dissolved in a proton-rich solution showed signals from all exchangeable protons, including those belonging to the sulfamate group. This work suits well to the current grand celebration of one-century-anniversary of the discovery of heparin. PMID:27618066

  9. Natural variation in the heparan sulfate binding domain of the eastern equine encephalitis virus E2 glycoprotein alters interactions with cell surfaces and virulence in mice.

    PubMed

    Gardner, Christina L; Choi-Nurvitadhi, Jo; Sun, Chengqun; Bayer, Avraham; Hritz, Jozef; Ryman, Kate D; Klimstra, William B

    2013-08-01

    Recently, we compared amino acid sequences of the E2 glycoprotein of natural North American eastern equine encephalitis virus (NA-EEEV) isolates and demonstrated that naturally circulating viruses interact with heparan sulfate (HS) and that this interaction contributes to the extreme neurovirulence of EEEV (C. L. Gardner, G. D. Ebel, K. D. Ryman, and W. B. Klimstra, Proc. Natl. Acad. Sci. U. S. A., 108:16026-16031, 2011). In the current study, we have examined the contribution to HS binding of each of three lysine residues in the E2 71-to-77 region that comprise the primary HS binding site of wild-type (WT) NA-EEEV viruses. We also report that the original sequence comparison identified five virus isolates, each with one of three amino acid differences in the E2 71-to-77 region, including mutations in residues critical for HS binding by the WT virus. The natural variant viruses, which possessed either a mutation from lysine to glutamine at E2 71, a mutation from lysine to threonine at E2 71, or a mutation from threonine to lysine at E2 72, exhibited altered interactions with heparan sulfate and cell surfaces and altered virulence in a mouse model of EEEV disease. An electrostatic map of the EEEV E1/E2 heterotrimer based upon the recent Chikungunya virus crystal structure (J. E. Voss, M. C. Vaney, S. Duquerroy, C. Vonrhein, C. Girard-Blanc, E. Crublet, A. Thompson, G. Bricogne, and F. A. Rey, Nature, 468:709-712, 2010) showed the HS binding site to be at the apical surface of E2, with variants affecting the electrochemical nature of the binding site. Together, these results suggest that natural variation in the EEEV HS binding domain may arise during EEEV sylvatic cycles and that this variation may influence receptor interaction and the severity of EEEV disease.

  10. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    SciTech Connect

    Roehrig, John T.; Butrapet, Siritorn; Liss, Nathan M.; Bennett, Susan L.; Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E.; Blair, Carol D.; Huang, Claire Y.-H.

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  11. Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins.

    PubMed

    Gupta, P; Oegema, T R; Brazil, J J; Dudek, A Z; Slungaard, A; Verfaillie, C M

    2000-01-01

    We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell "niches" in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1alpha or PF4 (LTC-IC maintenance without GAGs, 32 +/- 2%; with GAGs, 95 +/- 7%; P <.001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1alpha, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 +/- 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1alpha. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1alpha, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)

  12. The binding of the bone morphogenetic protein antagonist gremlin to kidney heparan sulfate: Such binding is not essential for BMP antagonism.

    PubMed

    Tatsinkam, Arnold Junior; Rune, Naomi; Smith, Joy; Norman, Jill T; Mulloy, Barbara; Rider, Christopher C

    2017-02-01

    Gremlin-1, a bone morphogenetic protein (BMP) antagonist, has essential roles in kidney and limb bone development, and is important in chronic diseases including tissue fibrosis. It also functions as an activating ligand of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), and binds strongly to the sulfated polysaccharide, heparin. Here we investigated the extent to which gremlin binds to the related polysaccharide heparan sulfate (HS), which unlike heparin is widely distributed spread within tissues. We determined that both highly sulfated HS and kidney HS are able to partially compete for the binding of heparin to gremlin, whereas low sulfated HS is a poor competitor. In further investigations of the interaction between gremlin and HS, we found that wild-type gremlin is able to bind broadly across the various regions of kidney in an HS-dependent manner, with particularly intense binding to tubular structures in the renal cortex. In a model of chronic kidney disease, fibrotic changes in the kidney result in a loss of gremlin binding sites. Gremlin mutants with reduced affinity for heparin showed negligible binding under the same conditions. These mutants nonetheless remain functional as BMP antagonists on C2C12 myoblastic cells transfected with a Smad 1 reporter gene construct. Overall our findings indicate that on secretion, gremlin will bind to HS structures on the cell surface and in the extracellular matrix, thus providing for a localised reservoir which can modulate BMP activity in a temporospatially restricted manner. Although binding of heparin/HS to gremlin has been shown elsewhere to be necessary for gremlin activation of VEGFR2, this does not appear to be essential for BMP antagonism by gremlin. Thus these sulfated polysaccharides differentially regulate the activities of gremlin.

  13. Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

    PubMed

    Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

    2009-12-11

    Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

  14. [The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

    PubMed

    Snigireva, A V; Vrublevskaya, V V; Skarga, Y Y; Morenkov, O S

    2016-01-01

    Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90β, we showed that membrane-bound Hsp90α and Hsp90β play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90β to the plasma membrane.

  15. Heparan Sulfate Proteoglycans Containing a Glypican 5 Core and 2-O-Sulfo-iduronic Acid Function as Sonic Hedgehog Co-receptors to Promote Proliferation*♦

    PubMed Central

    Witt, Rochelle M.; Hecht, Marie-Lyn; Pazyra-Murphy, Maria F.; Cohen, Samuel M.; Noti, Christian; van Kuppevelt, Toin H.; Fuller, Maria; Chan, Jennifer A.; Hopwood, John J.; Seeberger, Peter H.; Segal, Rosalind A.

    2013-01-01

    Sonic Hedgehog (Shh) signaling is crucial for growth, cell fate determination, and axonal guidance in the developing nervous system. Although the receptors Patched (Ptch1) and Smoothened (Smo) are required for Shh signaling, a number of distinct co-receptors contribute to these critical responses to Shh. Several membrane-embedded proteins such as Boc, Cdo, and Gas1 bind Shh and promote signaling. In addition, heparan sulfate proteoglycans (HSPGs) have also been implicated in the initiation of Shh responses. However, the attributes of HSPGs that function as co-receptors for Shh have not yet been defined. Here, we identify HSPGs containing a glypican 5 core protein and 2-O-sulfo-iduronic acid residues at the nonreducing ends of the glycans as co-receptors for Shh. These HSPG co-receptors are expressed by cerebellar granule cell precursors and promote Shh binding and signaling. At the subcellular level, these HSPG co-receptors are located adjacent to the primary cilia that act as Shh signaling organelles. Thus, Shh binds to HSPG co-receptors containing a glypican 5 core and 2-O-sulfo-iduronic acid to promote neural precursor proliferation. PMID:23867465

  16. Heparan sulfate proteoglycans containing a glypican 5 core and 2-O-sulfo-iduronic acid function as Sonic Hedgehog co-receptors to promote proliferation.

    PubMed

    Witt, Rochelle M; Hecht, Marie-Lyn; Pazyra-Murphy, Maria F; Cohen, Samuel M; Noti, Christian; van Kuppevelt, Toin H; Fuller, Maria; Chan, Jennifer A; Hopwood, John J; Seeberger, Peter H; Segal, Rosalind A

    2013-09-06

    Sonic Hedgehog (Shh) signaling is crucial for growth, cell fate determination, and axonal guidance in the developing nervous system. Although the receptors Patched (Ptch1) and Smoothened (Smo) are required for Shh signaling, a number of distinct co-receptors contribute to these critical responses to Shh. Several membrane-embedded proteins such as Boc, Cdo, and Gas1 bind Shh and promote signaling. In addition, heparan sulfate proteoglycans (HSPGs) have also been implicated in the initiation of Shh responses. However, the attributes of HSPGs that function as co-receptors for Shh have not yet been defined. Here, we identify HSPGs containing a glypican 5 core protein and 2-O-sulfo-iduronic acid residues at the nonreducing ends of the glycans as co-receptors for Shh. These HSPG co-receptors are expressed by cerebellar granule cell precursors and promote Shh binding and signaling. At the subcellular level, these HSPG co-receptors are located adjacent to the primary cilia that act as Shh signaling organelles. Thus, Shh binds to HSPG co-receptors containing a glypican 5 core and 2-O-sulfo-iduronic acid to promote neural precursor proliferation.

  17. Human beta-defensins 2 and -3 cointernalize with human immunodeficiency virus via heparan sulfate proteoglycans and reduce infectivity of intracellular virions in tonsil epithelial cells

    PubMed Central

    Herrera, Rossana; Morris, Michael; Rosbe, Kristina; Feng, Zhimin; Weinberg, Aaron; Tugizov, Sharof

    2015-01-01

    We previously showed that expression of the anti-HIV innate proteins human beta-defensin 2 (hBD2) and hBD3 in adult oral epithelial cells reduces HIV transepithelial transmission by inactivation of virus. However, fetal/infant oral epithelia lack beta-defensin expression, leading to transmission of HIV. The mechanisms of hBD2- and hBD3-mediated HIV inactivation in adult oral epithelial cells are poorly understood. Here we found that heparan sulfate proteoglycans (HSPGs) on the apical surfaces of epithelial cells facilitate simultaneous binding of hBDs and HIV gp120 to the cell surface. HSPG-facilitated binding of hBDs and HIV gp120 to the cell surface did not affect viral attachment. HBD2 or -3 cointernalized with virions in endosomes, formed oligomers, and reduced infectivity of HIV. The anti-HIV effect of combining hBD2 and hBD3 was substantially higher than that of the individual peptides. These findings advance our understanding of the mechanisms of anti-HIV resistance in adult oral epithelium. PMID:26539799

  18. Assignment Of Hexuronic Acid Stereochemistry In Synthetic Heparan Sulfate Tetrasaccharides With 2-O-Sulfo Uronic Acids Using Electron Detachment Dissociation.

    PubMed

    Agyekum, Isaac; Patel, Anish B; Zong, Chengli; Boons, Geert-Jan; Amster, Jonathan

    2015-11-15

    The present work focuses on the assignment of uronic acid stereochemistry in heparan sulfate (HS) oligomers. The structural elucidation of HS glycosaminoglycans is the subject of considerable importance due to the biological and biomedical significance of this class of carbohydrates. They are highly heterogeneous due to their non-template biosynthesis. Advances in tandem mass spectrometry activation methods, particularly electron detachment dissociation (EDD), has led to the development of methods to assign sites of sulfo modification in glycosaminoglycan oligomers, but there are few reports of the assignment of uronic acid stereochemistry, necessary to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA). Whereas preceding studies focused on uronic acid epimers with no sulfo modification, the current work extends the assignment of the hexuronic acid stereochemistry to 2-O-sulfo uronic acid epimeric tetrasaccharides. The presence of a 2-O-sulfo group on the central uronic acid was found to greatly influence the formation of B3, C2, Z2, and Y1 ions in glucuronic acid and Y2 and (1,5)X2 for iduronic acid. The intensity of these peaks can be combined to yield a diagnostic ratios (DR), which can be used to confidently assign the uronic acid stereochemistry.

  19. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion.

    PubMed

    Roehrig, John T; Butrapet, Siritorn; Liss, Nathan M; Bennett, Susan L; Luy, Betty E; Childers, Thomas; Boroughs, Karen L; Stovall, Janae L; Calvert, Amanda E; Blair, Carol D; Huang, Claire Y-H

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion.

  20. Extracellular matrix of cultured glial cells: Selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

    SciTech Connect

    Gallo, V.; Bertolotto, A. )

    1990-04-01

    We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with (3H)glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various (3H)glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.

  1. Cobra CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate-dependent transcriptional regulation of endothelial cell adhesion molecules.

    PubMed

    Wang, Yu-Ling; Kuo, Je-Hung; Lee, Shao-Chen; Liu, Jai-Shin; Hsieh, Yin-Cheng; Shih, Yu-Tsung; Chen, Chun-Jung; Chiu, Jeng-Jiann; Wu, Wen-Guey

    2010-11-26

    Cysteine-rich secretory proteins (CRISPs) have been identified as a toxin family in most animal venoms with biological functions mainly associated with the ion channel activity of cysteine-rich domain (CRD). CRISPs also bind to Zn(2+) at their N-terminal pathogenesis-related (PR-1) domain, but their function remains unknown. Interestingly, similar the Zn(2+)-binding site exists in all CRISP family, including those identified in a wide range of organisms. Here, we report that the CRISP from Naja atra (natrin) could induce expression of vascular endothelial cell adhesion molecules, i.e. intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin, to promote monocytic cell adhesion in a heparan sulfate (HS)- and Zn(2+)-dependent manner. Using specific inhibitors and small interfering RNAs, the activation mechanisms are shown to involve both mitogen-activated protein kinases and nuclear factor-κB. Biophysical characterization of natrin by using fluorescence, circular dichroism, and x-ray crystallographic methods further reveals the presence of two Zn(2+)-binding sites for natrin. The strong binding site is located near the putative Ser-His-Glu catalytic triad of the N-terminal domain. The weak binding site remains to be characterized, but it may modulate HS binding by enhancing its interaction with long chain HS. Our results strongly suggest that natrin may serve as an inflammatory modulator that could perturb the wound-healing process of the bitten victim by regulating adhesion molecule expression in endothelial cells. Our finding uncovers a new aspect of the biological role of CRISP family in immune response and is expected to facilitate future development of new therapeutic strategy for the envenomed victims.

  2. Real time monitoring of the effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cell adhesion process using thickness shear mode (TSM) sensor.

    PubMed

    Ergezen, E; Hong, S; Barbee, K A; Lec, R

    2007-04-15

    The effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cellular interactions of the cell membrane with different substrates to determine the kinetics of cell adhesion was studied using thickness shear mode (TSM) sensor. The TSM sensor was operated at its first, third, fifth and seventh harmonics. Since the penetration depth of the shear wave decreases with increases in frequency, the multi-resonance operation of the TSM sensor was used to monitor the changes in the kinetics of the cell-substrate interaction at different distances from the sensor surface. During the sedimentation and the initial attachment of the cells on the sensor surface, the changes in the sensor resonant frequency and the magnitude response were monitored. First, HSPGs were partially digested with the enzyme Heparinase III to evaluate the effect of HSPG on the cell adhesion process. The results indicated that HSPG did not have any effect on the kinetics of the initial attachment, but it did reduce the strength of steady-state cell adhesion. Next, we investigated the effect of the electrostatic interactions of the cell membrane with the substrate on the cell adhesion. In this case, the sensor surface was coated with positively charged Poly-D-Lysine (PDL). It was observed that electrostatic interaction of the negatively charged cell membrane with the PDL surface promoted the initial cell adhesion but did not support long-term cell adhesion. The multi-resonant TSM technique was shown to be a very promising method for monitoring specific interfacial effects involving in cell adhesion process in real-time.

  3. The Heparan Sulfate Proteoglycan Glypican-6 Is Upregulated in the Failing Heart, and Regulates Cardiomyocyte Growth through ERK1/2 Signaling

    PubMed Central

    Melleby, Arne O.; Strand, Mari E.; Romaine, Andreas; Herum, Kate M.; Skrbic, Biljana; Dahl, Christen P.; Sjaastad, Ivar; Fiane, Arnt E.; Filmus, Jorge; Christensen, Geir; Lunde, Ida G.

    2016-01-01

    Pressure overload is a frequent cause of heart failure. Heart failure affects millions of patients worldwide and is a major cause of morbidity and mortality. Cell surface proteoglycans are emerging as molecular players in cardiac remodeling, and increased knowledge about their regulation and function is needed for improved understanding of cardiac pathogenesis. Here we investigated glypicans (GPC1-6), a family of evolutionary conserved heparan sulfate proteoglycans anchored to the extracellular leaflet of the cell membrane, in experimental and clinical heart failure, and explored the function of glypican-6 in cardiac cells in vitro. In mice subjected to pressure overload by aortic banding (AB), we observed elevated glypican-6 levels during hypertrophic remodeling and dilated, end-stage heart failure. Consistently, glypican-6 mRNA was elevated in left ventricular myocardium from explanted hearts of patients with end-stage, dilated heart failure with reduced ejection fraction. Glypican-6 levels correlated negatively with left ventricular ejection fraction in patients, and positively with lung weight after AB in mice. Glypican-6 mRNA was expressed in both cardiac fibroblasts and cardiomyocytes, and the corresponding protein displayed different sizes in the two cell types due to tissue-specific glycanation. Importantly, adenoviral overexpression of glypican-6 in cultured cardiomyocytes increased protein synthesis and induced mRNA levels of the pro-hypertrophic signature gene ACTA1 and the hypertrophy and heart failure signature genes encoding natriuretic peptides, NPPA and NPPB. Overexpression of GPC6 induced ERK1/2 phosphorylation, and co-treatment with the ERK inhibitor U0126 attenuated the GPC6-induced increase in NPPA, NPPB and protein synthesis. In conclusion, our data suggests that glypican-6 plays a role in clinical and experimental heart failure progression by regulating cardiomyocyte growth through ERK signaling. PMID:27768722

  4. Syndecan-1-Induced ECM Fiber Alignment Requires Integrin αvβ3 and Syndecan-1 Ectodomain and Heparan Sulfate Chains.

    PubMed

    Yang, Ning; Friedl, Andreas

    2016-01-01

    Expression of the cell surface proteoglycan syndecan-1 (Sdc1) is frequently induced in stromal fibroblasts of invasive breast carcinomas. We have recently identified a correlation between stromal Sdc1 expression and extracellular matrix (ECM) fiber alignment, both in vitro and in vivo. ECMs derived from Sdc1-positive human mammary fibroblasts (HMF) showed an aligned fiber architecture, which contrasted markedly with the more random fiber arrangement in the ECM produced by Sdc1-negative HMFs. We further demonstrated that aligned fiber architecture promotes the directional migration and invasion of breast carcinoma cells. To decipher the molecular mechanisms governing the formation of an aligned, invasion-permissive ECM, a series of Sdc1 mutants was introduced into HMF. We found that both the ectodomain and heparan sulfate chains of Sdc1 were required for full activity of Sdc1 in regulating ECM alignment, while transmembrane and cytoplasmic domains were dispensable. Sdc1 regulates the activities of several integrins via its ectodomain. Integrins are key players in the assembly of fibronectin-rich ECM. In addition, integrins are capable of regulating cell morphology and cell shape and orientation may affect ECM architecture. Therefore, we investigated the role of integrins in Sdc1-mediated ECM fiber alignment. Sdc1-overexpressing HMF gained an enhanced spindle-shaped morphology when cultured in an overconfluent state under conditions permissive for ECM production, which was partially reversed by siRNA-mediated silencing of β3 integrin expression. Moreover, suppression of αvβ3 integrin activity by a function-blocking antibody or β3 knockdown largely abolished the aligned ECM fiber architecture and consequently the invasion-permissive properties of the ECM induced by Sdc1. The results suggest that Sdc1 may modulate fibronectin fibrillogenesis and/or alter cell morphology during ECM production through αvβ3 integrin, thereby mediating ECM fiber alignment

  5. The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

    SciTech Connect

    Hirano, Kazumi; Van Kuppevelt, Toin H.; Nishihara, Shoko

    2013-01-18

    Highlights: ► Fas transcript increases during the transition from the naïve to the primed state. ► 3OST-5 transcript, the HS4C3 epitope synthesis gene, increases during the transition. ► Fas signaling regulates the transition from the naïve to the primed state. ► HS4C3-binding epitope regulates the transition from the naïve to the primed state. ► Fas signaling is regulated by the HS4C3 epitope during the transition. -- Abstract: The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope

  6. Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity.

    PubMed

    Ahmed, Yassir A; Yates, Edwin A; Moss, Diana J; Loeven, Markus A; Hussain, Sadaf-Ahmahni; Hohenester, Erhard; Turnbull, Jeremy E; Powell, Andrew K

    2016-10-20

    Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo.

  7. Pectin from Prunus domestica L. induces proliferation of IEC-6 cells through the alteration of cell-surface heparan sulfate on differentiated Caco-2 cells in co-culture.

    PubMed

    Nishida, Mitsutaka; Murata, Kazuma; Oshima, Kazuya; Itoh, Chihiro; Kitaguchi, Kohji; Kanamaru, Yoshihiro; Yabe, Tomio

    2015-05-01

    Dietary fiber intake provides various physiological and metabolic effects for human health. Pectin, a water-soluble dietary fiber, induces morphological changes of the small intestine in vivo. However, the molecular mechanisms underlying pectin-derived morphological alterations have not been elucidated. Previously, we found that pectin purified from Prunus domestica L. altered the sulfated structure of cell-surface heparan sulfate (HS) on differentiated Caco-2 cells via fibronectin and α5β1 integrin. In this study, we investigated the biological significance of the effect of pectin on HS in differentiated Caco-2 cells. An in vitro intestinal epithelium model was constructed by co-culture of differentiated Caco-2 cells and rat IEC-6 cells, which were used as models of intestinal epithelium and intestinal crypt cells, respectively. We found that pectin-treated differentiated Caco-2 cells promoted growth of IEC-6 cells. Real-time RT-PCR analysis and western blotting showed that relative mRNA and protein expression levels of Wnt3a were upregulated by pectin treatment in differentiated Caco-2 cells. Analysis by surface plasmon resonance spectroscopy demonstrated that pectin-induced structural alteration of HS markedly decreased the interaction with Wnt3a. However, depression in the secretion of Wnt3a from Caco-2 cells by anti-Wnt3a antibody did not affect the proliferation of IEC-6 cells in co-culture system. These observations indicated that pectin altered the sulfated structure of cell-surface HS to promote secretion of Wnt3a from differentiated Caco-2 cells and Wnt3a indirectly stimulated the proliferation of IEC-6 cells.

  8. The structural plasticity of heparan sulfate NA-domains and hence their role in mediating multivalent interactions is confirmed by high-accuracy 15N-NMR relaxation studies

    PubMed Central

    Mobli, Mehdi; Nilsson, Mathias

    2007-01-01

    Considering the biological importance of heparan sulfate (HS) and the significant activity of its highly-sulfated regions (S-domains), the paucity of known functions for the non-sulfated NA-domains is somewhat puzzling. It has been suggested that chain dynamics within the NA-domains are the key to their functional role in HS. In this study, we investigate this hypothesis using state-of-the-art nuclear magnetic resonance (NMR) experiments at multiple frequencies. To resolve the problem of severe overlap in 1H-NMR spectra of repetitive polysaccharides from proteoglycans, we have prepared oligosaccharides with the chemical structure of HS NA-domains containing the 15N nucleus, which has enough chemical shift dispersion to probe the central residues of octasaccharides at atomic resolution using 600 MHz NMR. By performing NMR relaxation experiments at three magnetic-field strengths, high quality data on internal dynamics and rotational diffusion was obtained. Furthermore, translational diffusion could also be measured by NMR using pulse field gradients. These experimental data were used, in concert with molecular dynamics simulations, to provide information on local molecular shape, greatly aiding our relaxation analyses. Our results, which are more accurate than those presented previously, confirm the higher flexibility of the NA-domains as compared with reported data on S-domains. It is proposed that this flexibility has two functional roles. First, it confers a greater area of interaction from the anchoring point on the core protein for the bioactive S-domains. Secondly, it allows multiple interactions along the same HS chain that are dynamically independent of each other. Electronic Supplementary Material The online version of this article (doi:10.1007/s10719-007-9081-9) contains supplementary material, which is available to authorized users. PMID:18080183

  9. Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin disaccharides derivatized with 2-aminoacridone.

    PubMed

    Galeotti, Fabio; Volpi, Nicola

    2011-09-01

    A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ~15-21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of the commercial products prior to enzymatic digestion or prepurification steps to eliminate any excess of AMAC reagent or interference from proteins, peptides, and other sample impurities before RP-HPLC-Fd-ESI-MS injection were necessary. This method has applicability for the rapid differentiation of pharmaceutical Heps and LMW-Heps prepared by means of different depolymerization processes and for compositional analysis of small amounts of samples derived from biological sources by using the highly sensitive fluorescence detector.

  10. Multivariate analysis of electron detachment dissociation and infrared multiphoton dissociation mass spectra of heparan sulfate tetrasaccharides differing only in hexuronic acid stereochemistry.

    PubMed

    Oh, Han Bin; Leach, Franklin E; Arungundram, Sailaja; Al-Mafraji, Kanar; Venot, Andre; Boons, Geert-Jan; Amster, I Jonathan

    2011-03-01

    The structural characterization of glycosaminoglycan (GAG) carbohydrates by mass spectrometry has been a long-standing analytical challenge due to the inherent heterogeneity of these biomolecules, specifically polydispersity, variability in sulfation, and hexuronic acid stereochemistry. Recent advances in tandem mass spectrometry methods employing threshold and electron-based ion activation have resulted in the ability to determine the location of the labile sulfate modification as well as assign the stereochemistry of hexuronic acid residues. To facilitate the analysis of complex electron detachment dissociation (EDD) spectra, principal component analysis (PCA) is employed to differentiate the hexuronic acid stereochemistry of four synthetic GAG epimers whose EDD spectra are nearly identical upon visual inspection. For comparison, PCA is also applied to infrared multiphoton dissociation spectra (IRMPD) of the examined epimers. To assess the applicability of multivariate methods in GAG mixture analysis, PCA is utilized to identify the relative content of two epimers in a binary mixture.

  11. Gene expression profiles identify both MyD88-independent and MyD88-dependent pathways involved in the maturation of dendritic cells mediated by heparan sulfate: A novel adjuvant

    PubMed Central

    Wu, Meini; Wang, Haixuan; Shi, Jiandong; Sun, Jing; Duan, Zhiqing; Li, Yanhan; Li, Jianfang; Hu, Ningzhu; Wei, Yiju; Chen, Yang; Hu, Yunzhang

    2015-01-01

    The traditional vaccine adjuvant research is mainly based on the trial and error method, and the mechanisms underlying the immune system stimulation remaining largely unknown. We previously demonstrated that heparan sulfate (HS), a TLR-4 ligand and endogenous danger signal, effectively enhanced humoral and cellular immune responses in mice immunized by HBsAg. This study aimed to evaluate whether HS induces better humoral immune responses against inactivated Hepatitis A or Rabies Vaccines, respectively, compared with traditional adjuvants (e.g. Alum and complete Freund's adjuvant). In order to investigate the molecular mechanisms of its adjuvanticity, the gene expression pattern of peripheral blood monocytes derived DCs (dendritic cells) stimulated with HS was analyzed at different times points. Total RNA was hybridized to Agilent SurePrint G3 Human Gene Expression 8 × 60 K one-color oligo-microarray. Through intersection analysis of the microarray results, we found that the Toll-like receptor signaling pathway was significantly activated, and NF-kB, TRAF3 and IRF7 were activated as early as 12 h, and MyD88 was activated at 48 h post-stimulation. Furthermore, the expression of the surface marker CD83 and the co-stimulatory molecules CD80 and CD86 was up-regulated as early as 24 h. Therefore, we speculated that HS-induced human monocyte-derived DC maturation may occur through both MyD88-independent and dependent pathways, but primarily through the former (TRIF pathway). These data provide an important basis for understanding the mechanisms underlying HS enhancement of the immune response. PMID:25668674

  12. The effect of nutritional status and myogenic satellite cell age on turkey satellite cell proliferation, differentiation, and expression of myogenic transcriptional regulatory factors and heparan sulfate proteoglycans syndecan-4 and glypican-1.

    PubMed

    Harthan, Laura B; McFarland, Douglas C; Velleman, Sandra G

    2014-01-01

    Posthatch satellite cell mitotic activity is a critical component of muscle development and growth. Satellite cells are myogenic stem cells that can be induced by nutrition to follow other cellular developmental pathways, and whose mitotic activity declines with age. The objective of the current study was to determine the effect of restricting protein synthesis on the proliferation and differentiation, expression of myogenic transcriptional regulatory factors myogenic determination factor 1, myogenin, and myogenic regulatory factor 4, and expression of the heparan sulfate proteoglycans syndecan-4 and glypican-1 in satellite cells isolated from 1-d-, 7-wk-, and 16-wk-old turkey pectoralis major muscle (1 d, 7 wk, and 16 wk cells, respectively) by using variable concentrations of Met and Cys. Four Met concentrations-30 (control), 7.5, 3, or 0 mg/L with 3.2 mg/L of Cys per 1 mg/L of Met-were used for culture of satellite cells to determine the effect of nutrition and age on satellite cell behavior during proliferation and differentiation. Proliferation was reduced by lower Met and Cys concentrations in all ages at 96 h of proliferation. Differentiation was increased in the 1 d Met-restricted cells, whereas the 7 wk cells treated with 3 mg/L of Met had decreased differentiation. Reduced Met and Cys levels from the control did not significantly affect the 16 wk cells at 72 h of differentiation. However, medium with no Met or Cys suppressed differentiation at all ages. The expression of myogenic determination factor 1, myogenin, myogenic regulatory factor 4, syndecan-4, and glypican-1 was differentially affected by age and Met or Cys treatment. These data demonstrate the age-specific manner in which turkey pectoralis major muscle satellite cells respond to nutritional availability and the importance of defining optimal nutrition to maximize satellite cell proliferation and differentiation for subsequent muscle mass accretion.

  13. The healing of alkali-injured cornea is stimulated by a novel matrix regenerating agent (RGTA, CACICOL20): a biopolymer mimicking heparan sulfates reducing proteolytic, oxidative and nitrosative damage.

    PubMed

    Cejkova, Jitka; Olmiere, Celine; Cejka, Cestmir; Trosan, Peter; Holan, Vladimir

    2014-04-01

    The efficacy of a chemically modified dextran - heparan sulfate mimicking regenerating agent (RGTA) on the healing of the rabbit cornea injured with alkali was examined. The eyes were injured with 0.15 N NaOH applied on the cornea or with 1.0 N NaOH using a 8 mm diameter filter paper disk. Then RGTA or placebo was applied on the cornea. In the last group of rabbits, corneas injured with the high alkali concentration were left without any treatment for four weeks; subsequently, the corneas were treated with RGTA or placebo. The central corneal thickness was measured using a pachymeter. The corneas were examined morphologically, immunohistochemically and for real time-PCR. Compared to control (unaffected) corneas, following the application of low alkali concentration the expression of urokinase-type plasminogen activator, metalloproteinase 9, nitric oxide synthase and xanthine oxidase was increased in the injured corneal epithelium of placebo-treated eyes, whereas the expression of antioxidant enzymes was reduced. Nitrotyrosine and malondialdehyde stainings appeared in the corneal epithelium. RGTA application suppressed the antioxidant/prooxidant imbalance and reduced the expression of the above-mentioned immunohistochemical markers. The corneal thickness increased after alkali injury, decreased during corneal healing after RGTA treatment faster than after placebo application. Following the injury with the high alkali concentration, corneal inflammation and neovascularization were highly pronounced in placebo-treated corneas, whereas in RGTA-treated corneas they were significantly supressed. When RGTA or placebo application was started later after alkali injury and corneas were ulcerated, subsequent RGTA treatment healed the majority of them. In conclusion, RGTA facilitates the healing of injured corneas via a reduction of proteolytic, oxidative and nitrosative damage.

  14. The role of heparan sulfate deficiency in autistic phenotype: potential involvement of Slit/Robo/srGAPs-mediated dendritic spine formation.

    PubMed

    Pérez, Christine; Sawmiller, Darrell; Tan, Jun

    2016-04-18

    Autism Spectrum Disorders (ASD) are the second most common developmental cause of disability in the United States. ASDs are accompanied with substantial economic and emotional cost. The brains of ASD patients have marked structural abnormalities, in the form of increased dendritic spines and decreased long distance connections. These structural differences may be due to deficiencies in Heparin Sulfate (HS), a proteoglycan involved in a variety of neurodevelopmental processes. Of particular interest is its role in the Slit/Robo pathway. The Slit/Robo pathway is known to be involved in the regulation of axonal guidance and dendritic spine formation. HS mediates the Slit/Robo interaction; without its presence Slit's repulsive activity is abrogated. Slit/Robo regulates dendritic spine formation through its interaction with srGAPs (slit-robo GTPase Activating Proteins), which leads to downstream signaling, actin cytoskeleton depolymerization and dendritic spine collapse. Through interference with this pathway, HS deficiency can lead to excess spine formation.

  15. The heparan and heparin metabolism pathway is involved in regulation of fatty acid composition.

    PubMed

    Jiang, Zhihua; Michal, Jennifer J; Wu, Xiao-Lin; Pan, Zengxiang; MacNeil, Michael D

    2011-01-01

    Six genes involved in the heparan sulfate and heparin metabolism pathway, DSEL (dermatan sulfate epimerase-like), EXTL1 (exostoses (multiple)-like 1), HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), HS6ST3 (heparan sulfate 6-O-sulfotransferase 3), NDST3 (N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3), and SULT1A1 (sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1), were investigated for their associations with muscle lipid composition using cattle as a model organism. Nineteen single nucleotide polymorphisms (SNPs)/multiple nucleotide length polymorphisms (MNLPs) were identified in five of these six genes. Six of these mutations were then genotyped on 246 Wagyu x Limousin F(2) animals, which were measured for 5 carcass, 6 eating quality and 8 fatty acid composition traits. Association analysis revealed that DSEL, EXTL1 and HS6ST1 significantly affected two stearoyl-CoA desaturase activity indices, the amount of conjugated linoleic acid (CLA), and the relative amount of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in skeletal muscle (P<0.05). In particular, HS6ST1 joined our previously reported SCD1 and UQCRC1 genes to form a three gene network for one of the stearoyl-CoA desaturase activity indices. These results provide evidence that genes involved in heparan sulfate and heparin metabolism are also involved in regulation of lipid metabolism in bovine muscle. Whether the SNPs affected heparan sulfate proteoglycan structure is unknown and warrants further investigation.

  16. Guanidinylated Neomycin Mediates Heparan Sulfate–dependent Transport of Active Enzymes to Lysosomes

    PubMed Central

    Sarrazin, Stéphane; Wilson, Beth; Sly, William S; Tor, Yitzhak; Esko, Jeffrey D

    2010-01-01

    Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, β--glucuronidase (GUS) and α--iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells. PMID:20442709

  17. A Cinnamon-Derived Procyanidin Compound Displays Anti-HIV-1 Activity by Blocking Heparan Sulfate- and Co-Receptor- Binding Sites on gp120 and Reverses T Cell Exhaustion via Impeding Tim-3 and PD-1 Upregulation

    PubMed Central

    Connell, Bridgette Janine; Chang, Sui-Yuan; Prakash, Ekambaranellore; Yousfi, Rahima; Mohan, Viswaraman; Posch, Wilfried; Wilflingseder, Doris; Moog, Christiane; Kodama, Eiichi N.; Clayette, Pascal; Lortat-Jacob, Hugues

    2016-01-01

    Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1–7 μM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS) as well as to an antibody (mAb 17b) directed against the gp120 co-receptor binding site with an IC50 in the low μM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1. PMID:27788205

  18. Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity† †Electronic supplementary information (ESI) available: NMR chemical shift characterisation of modified heparins, protein sequence alignment methodology and data, protein binding and activity assay dose-response curves. See DOI: 10.1039/c6mb00432f Click here for additional data file.

    PubMed Central

    Ahmed, Yassir A.; Yates, Edwin A.; Moss, Diana J.; Loeven, Markus A.; Hussain, Sadaf-Ahmahni; Hohenester, Erhard; Turnbull, Jeremy E.

    2016-01-01

    Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit–Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo. PMID:27502551

  19. Secondary Storage of Dermatan Sulfate in Sanfilippo Disease*

    PubMed Central

    Lamanna, William C.; Lawrence, Roger; Sarrazin, Stéphane; Esko, Jeffrey D.

    2011-01-01

    Mucopolysaccharidoses are a group of genetically inherited disorders that result from the defective activity of lysosomal enzymes involved in glycosaminoglycan catabolism, causing their intralysosomal accumulation. Sanfilippo disease describes a subset of mucopolysaccharidoses resulting from defects in heparan sulfate catabolism. Sanfilippo disorders cause severe neuropathology in affected children. The reason for such extensive central nervous system dysfunction is unresolved, but it may be associated with the secondary accumulation of metabolites such as gangliosides. In this article, we describe the accumulation of dermatan sulfate as a novel secondary metabolite in Sanfilippo. Based on chondroitinase ABC digestion, chondroitin/dermatan sulfate levels in fibroblasts from Sanfilippo patients were elevated 2–5-fold above wild-type dermal fibroblasts. Lysosomal turnover of chondroitin/dermatan sulfate in these cell lines was significantly impaired but could be normalized by reducing heparan sulfate storage using enzyme replacement therapy. Examination of chondroitin/dermatan sulfate catabolic enzymes showed that heparan sulfate and heparin can inhibit iduronate 2-sulfatase. Analysis of the chondroitin/dermatan sulfate fraction by chondroitinase ACII digestion showed dermatan sulfate storage, consistent with inhibition of iduronate 2-sulfatase. The discovery of a novel storage metabolite in Sanfilippo patients may have important implications for diagnosis and understanding disease pathology. PMID:21193389

  20. Secondary storage of dermatan sulfate in Sanfilippo disease.

    PubMed

    Lamanna, William C; Lawrence, Roger; Sarrazin, Stéphane; Esko, Jeffrey D

    2011-03-04

    Mucopolysaccharidoses are a group of genetically inherited disorders that result from the defective activity of lysosomal enzymes involved in glycosaminoglycan catabolism, causing their intralysosomal accumulation. Sanfilippo disease describes a subset of mucopolysaccharidoses resulting from defects in heparan sulfate catabolism. Sanfilippo disorders cause severe neuropathology in affected children. The reason for such extensive central nervous system dysfunction is unresolved, but it may be associated with the secondary accumulation of metabolites such as gangliosides. In this article, we describe the accumulation of dermatan sulfate as a novel secondary metabolite in Sanfilippo. Based on chondroitinase ABC digestion, chondroitin/dermatan sulfate levels in fibroblasts from Sanfilippo patients were elevated 2-5-fold above wild-type dermal fibroblasts. Lysosomal turnover of chondroitin/dermatan sulfate in these cell lines was significantly impaired but could be normalized by reducing heparan sulfate storage using enzyme replacement therapy. Examination of chondroitin/dermatan sulfate catabolic enzymes showed that heparan sulfate and heparin can inhibit iduronate 2-sulfatase. Analysis of the chondroitin/dermatan sulfate fraction by chondroitinase ACII digestion showed dermatan sulfate storage, consistent with inhibition of iduronate 2-sulfatase. The discovery of a novel storage metabolite in Sanfilippo patients may have important implications for diagnosis and understanding disease pathology.

  1. Chlorate: a reversible inhibitor of proteoglycan sulfation

    SciTech Connect

    Humphries, D.E.; Silbert, J.E.

    1988-07-15

    Bovine aorta endothelial cells were cultured in medium containing (/sup 3/H)glucosamine, (/sup 35/S)sulfate, and various concentrations of chlorate. Cell growth was not affected by 10 mM chlorate, while 30 mM chlorate had a slight inhibitory effect. Chlorate concentrations greater than 10 mM resulted in significant undersulfation of chondroitin. With 30 mM chlorate, sulfation of chondroitin was reduced to 10% and heparan to 35% of controls, but (/sup 3/H)glucosamine incorporation on a per cell basis did not appear to be inhibited. Removal of chlorate from the culture medium of cells resulted in the rapid resumption of sulfation.

  2. Glucosamine sulfate

    MedlinePlus

    ... Glucosamine Sulphate KCl, Glucosamine-6-Phosphate, GS, Mono-Sulfated Saccharide, Poly-(1->3)-N-Acetyl-2-Amino- ... Sulfate de Glucosamine, Sulfate de Glucosamine 2KCl, SG, Sulfated Monosaccharide, Sulfated Saccharide, Sulfato de Glucosamina. Glucosamine Hydrochloride ...

  3. Human Genetic Disorders Caused by Mutations in Genes Encoding Biosynthetic Enzymes for Sulfated Glycosaminoglycans*

    PubMed Central

    Mizumoto, Shuji; Ikegawa, Shiro; Sugahara, Kazuyuki

    2013-01-01

    A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs. PMID:23457301

  4. Structural characterization of a serendipitously discovered bioactive macromolecule, lignin sulfate.

    PubMed

    Raghuraman, Arjun; Tiwari, Vaibhav; Thakkar, Jay N; Gunnarsson, Gunnar T; Shukla, Deepak; Hindle, Michael; Desai, Umesh R

    2005-01-01

    The herpes simplex virus-1 (HSV-1) utilizes cell-surface glycosaminoglycan, heparan sulfate, to gain entry into cells and cause infection. In a search for synthetic mimics of heparan sulfate to prevent HSV infection, we discovered potent inhibitory activity arising from sulfation of a monomeric flavonoid. Yet, detailed screening indicated that the sulfated flavonoid was completely inactive and the potent inhibitory activity arose from a macromolecular substance present in the parent flavonoid. The active principle was identified through a battery of biophysical and chemical analyses as a sulfated form of lignin, a three-dimensional network polymer composed of substituted phenylpropanoid monomers. Mass spectral analysis of the parent lignin and its sulfated derivative indicates the presence of p-coumaryl monomers interconnected through uncondensed beta-O-4-linkages. Elemental analysis of lignin sulfate correlates primarily with a polymer of p-coumaryl alcohol containing one sulfate group. High-performance size exclusion chromatography shows a wide molecular weight distribution from 1.5 to 40 kDa suggesting significant polydispersity. Polyacrylamide gel electrophoresis (PAGE) analysis indicates a highly networked polymer that differs significantly from linear charged polymers with respect to its electrophoretic mobility. Overall, macromolecular lignin sulfate presents a multitude of substructures that can interact with biomolecules, including viral glycoproteins, using hydrophobic, hydrogen-bonding, and ionic forces. Thus, lignin sulfate represents a large number of interesting structures with potential medicinal benefits.

  5. Chondroitin sulfate

    MedlinePlus

    ... in combination with glucosamine sulfate, shark cartilage, and camphor. Some people also inject chondroitin sulfate into the ... in combination with glucosamine sulfate, shark cartilage, and camphor seems to reduce arthritis symptoms. However, any symptom ...

  6. Understanding the NG2 Glial Scar after Spinal Cord Injury

    PubMed Central

    Hackett, Amber R.; Lee, Jae K.

    2016-01-01

    NG2 cells, also known as oligodendrocyte progenitor cells, are located throughout the central nervous system and serve as a pool of progenitors to differentiate into oligodendrocytes. In response to spinal cord injury (SCI), NG2 cells increase their proliferation and differentiation into remyelinating oligodendrocytes. While astrocytes are typically associated with being the major cell type in the glial scar, many NG2 cells also accumulate within the glial scar but their function remains poorly understood. Similar to astrocytes, these cells hypertrophy, upregulate expression of chondroitin sulfate proteoglycans, inhibit axon regeneration, contribute to the glial-fibrotic scar border, and some even differentiate into astrocytes. Whether NG2 cells also have a role in other astrocyte functions, such as preventing the spread of infiltrating leukocytes and expression of inflammatory cytokines, is not yet known. Thus, NG2 cells are not only important for remyelination after SCI but are also a major component of the glial scar with functions that overlap with astrocytes in this region. In this review, we describe the signaling pathways important for the proliferation and differentiation of NG2 cells, as well as the role of NG2 cells in scar formation and tissue repair. PMID:27895617

  7. Insights into the role of sulfated glycans in cancer cell adhesion and migration through use of branched peptide probe

    PubMed Central

    Brunetti, Jlenia; Depau, Lorenzo; Falciani, Chiara; Gentile, Mariangela; Mandarini, Elisabetta; Riolo, Giulia; Lupetti, Pietro; Pini, Alessandro; Bracci, Luisa

    2016-01-01

    The tetra-branched peptide NT4 selectively binds to different human cancer cells and tissues. NT4 specifically binds to sulfated glycosaminoglycans on cancer cell membranes. Since sulfated glycosaminoglycans are involved in cancer cell interaction with the extracellular matrix, we evaluated the effect of NT4 on cancer cell adhesion and migration. We demonstrated here that the branched peptide NT4 binds sulfated glycosaminoglycans with high affinity and with preferential binding to heparan sulfate. NT4 inhibits cancer cell adhesion and migration on different proteins, without modifying cancer cell morphology or their ability to produce protrusions, but dramatically affecting the directionality and polarity of cell movement. Results obtained by taking advantage of the selective targeting of glycosaminoglycans chains by NT4, provide insights into the role of heparan sulfate proteoglycans in cancer cell adhesion and migration and suggest a determinant role of sulfated glycosaminoglycans in the control of cancer cell directional migration. PMID:27255651

  8. Purification and characterization of heparan sulphate proteoglycan from bovine brain.

    PubMed Central

    Park, Y; Yu, G; Gunay, N S; Linhardt, R J

    1999-01-01

    A heparan sulphate proteoglycan was purified from adult bovine brain tissues and its structure was characterized. The major heparan sulphate proteoglycan from whole bovine brain had a molecular mass of >200 kDa on denaturing SDS/PAGE and a core protein size of 66 kDa following the removal of glycosaminoglycan chains. Fractionation on DEAE-Sephacel showed that this proteoglycan consisted of three major forms having high, intermediate and low overall charge. All core proteins were identical in size and reacted with heparan sulphate proteoglycan-stub antibody and an antibody made to a synthetic peptide based on rat glypican. The three forms of proteoglycans had identical peptide maps and their amino acid compositional analysis did not match any of the known glypicans. The internal sequence of a major peptide showed only 37.5% sequence similarity with human glypican 5. The glycosaminoglycan chain sizes of the three forms of this proteoglycan, determined after beta-elimination by PAGE, were identical. The disaccharide compositional analysis on the heparan sulphate chains from the three forms of the proteoglycan, determined by treatment with a mixture of heparin lyases followed by high-resolution capillary electrophoresis, showed that they differed primarily by degree of sulphation. The most highly sulphated proteoglycan isolated had a disaccharide composition similar to heparan sulphate glycosaminoglycans found in brain tissue. Based on their sensitivity to low pH nitrous acid treatment, the N-sulphate groups in these proteoglycans were found to be primarily in the smaller glycosaminoglycan chains. The heparan sulphate proteoglycans were also heavily glycosylated with O-linked glycans and no glycosylphosphatidylinositol anchor could be detected. PMID:10585858

  9. Central neuron-glial and glial-glial interactions following axon injury.

    PubMed

    Aldskogius, H; Kozlova, E N

    1998-05-01

    Axon injury rapidly activates microglial and astroglial cells close to the axotomized neurons. Following motor axon injury, astrocytes upregulate within hour(s) the gap junction protein connexin-43, and within one day glial fibrillary acidic protein (GFAP). Concomitantly, microglial cells proliferate and migrate towards the axotomized neuron perikarya. Analogous responses occur in central termination territories of peripherally injured sensory ganglion cells. The activated microglia express a number of inflammatory and immune mediators. When neuron degeneration occurs, microglia act as phagocytes. This is uncommon after peripheral nerve injury in the adult mammal, however, and the functional implications of the glial cell responses in this situation are unclear. When central axons are injured, the glial cell responses around the affected neuron perikarya appears to be minimal or absent, unless neuron degeneration occurs. Microglia proliferate, and astrocytes upregulate GFAP along central axons undergoing anterograde, Wallerian, degeneration. Although microglia develop into phagocytes, they eliminate the disintegrating myelin very slowly, presumably because they fail to release molecules which facilitate phagocytosis. During later stages of Wallerian degeneration, oligodendrocytes express clusterin, a glycoprotein implicated in several conditions of cell degeneration. A hypothetical scheme for glial cell activation following axon injury is discussed, implying the injured neurons initially interact with adjacent astrocytes. Subsequently, neighbouring resting microglia are activated. These glial reactions are amplified by paracrine and autocrine mechanisms, in which cytokines appear to be important mediators. The specific functional properties of the activated glial cells will determine their influence on neuronal survival, axon regeneration, and synaptic plasticity. The control of the induction and progression of these responses are therefore likely to be critical

  10. [Glial activation and brain aging].

    PubMed

    Sugaya, K

    2001-10-01

    While basal forebrain cholinergic neurons degenerate in aging and Alzheimer's disease, the cholinergic groups of the upper brainstem are preserved. Since the brainstem reticular-like cholinergic neurons differ from the rostral cholinergic phenotype by their high expression of nitric oxide synthase (NOS) mRNA, we hypothesized that they contain biochemical mechanisms to protect themselves against self-induced damage by nitric oxide (NO). Our initial question was a source of the NO during the aging process. We found a significant correlation between cognitive function and markers for glial activation and oxidative stress using aged rats. This result indicates that oxidative stress accompanied by glial activation may be occurred in the cognitively impaired animals. We also found mitochondrial DNA (mDNA) was significantly damaged in these animals, while accumulation of oxidative damage was not evident in other molecules. Therefore, oxidative damage to the mDNA by glial activation may occur in the cells having poor protection against oxidative stress during aging. Then the dysfunction of mitochondria, induced by the mDNA damage, may induce cell death as well as produce another oxidative stress to cause neuronal damage. The damaged neurons induce further glial activation and such self-accelerated immune-like response results in progressive neurodegeneration.

  11. Regulation of sulfated glycosaminoglycan production by prostaglandin E2 in cultured lung fibroblasts

    SciTech Connect

    Karlinsky, J.B.; Goldstein, R.H. )

    1989-08-01

    Prostaglandin E2 (PGE2) has been shown to increase the synthesis of hyaluronic acid in cultured fibroblasts by increasing the activity of hyaluronate synthetase, a group of plasma membrane-bound synthetic enzymes. We examined whether PGE2 also increased the activity of those enzyme systems involved in the synthesis of sulfated glycosaminoglycan in the human embryonic lung fibroblast. Exposure of cells to PGE2 resulted in dose-dependent increases in glucosamine incorporation into all sulfated glycosaminoglycan subtypes. PGE2 at 10(-7) mol/L increased total glycosaminoglycan per dish to 21.6 +/- 3.1 micrograms versus 12.0 +/- 2.5 micrograms in control untreated cultures. Stimulation of endogenous PGE2 production by bradykinin had a similar effect on glycosaminoglycan synthesis. To examine whether PGE2 affected sulfated glycosaminoglycan protein core production, cells were labeled with tritiated glucosamine in the presence of cycloheximide. Under these conditions, incorporation of radiolabel into all glycosaminoglycan subtypes was reduced. However, when exogenous sulfated glycosaminoglycan chain initiator (p-nitrophenyl beta-D-xyloside) was added, incorporation of tritiated glucosamine into sulfated glycosaminoglycan increased but not to levels found in control cultures. Application of PGE2 to cultures treated with cycloheximide alone, or to cultures treated with cycloheximide plus xyloside, increased tritiated glucosamine incorporation into chondroitin, dermatan sulfate, and to a lesser extent into heparan sulfate. We conclude that PGE2 stimulates synthesis of all sulfated glycosaminoglycan even in the absence of new protein core production, probably by increasing activities of sulfated glycosaminoglycan synthetase enzymes. PGE2 stimulation of heparan sulfate synthesis is partially dependent on the availability of heparan sulfate-specific protein core.

  12. Glial cells and energy balance.

    PubMed

    Argente-Arizón, Pilar; Guerra-Cantera, Santiago; Garcia-Segura, Luis Miguel; Argente, Jesús; Chowen, Julie A

    2017-01-01

    The search for new strategies and drugs to abate the current obesity epidemic has led to the intensification of research aimed at understanding the neuroendocrine control of appetite and energy expenditure. This intensified investigation of metabolic control has also included the study of how glial cells participate in this process. Glia, the most abundant cell type in the central nervous system, perform a wide spectrum of functions and are vital for the correct functioning of neurons and neuronal circuits. Current evidence indicates that hypothalamic glia, in particular astrocytes, tanycytes and microglia, are involved in both physiological and pathophysiological mechanisms of appetite and metabolic control, at least in part by regulating the signals reaching metabolic neuronal circuits. Glia transport nutrients, hormones and neurotransmitters; they secrete growth factors, hormones, cytokines and gliotransmitters and are a source of neuroprogenitor cells. These functions are regulated, as glia also respond to numerous hormones and nutrients, with the lack of specific hormonal signaling in hypothalamic astrocytes disrupting metabolic homeostasis. Here, we review some of the more recent advances in the role of glial cells in metabolic control, with a special emphasis on the differences between glial cell responses in males and females.

  13. Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling

    PubMed Central

    Ramachandra, Rashmi; Namburi, Ramesh B; Ortega-Martinez, Olga; Shi, Xiaofeng; Zaia, Joseph; Dupont, Sam T; Thorndyke, Michael C; Lindahl, Ulf; Spillmann, Dorothe

    2014-01-01

    Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates. PMID:24253764

  14. Heparin-like properties of sulfated alginates with defined sequences and sulfation degrees.

    PubMed

    Arlov, Øystein; Aachmann, Finn Lillelund; Sundan, Anders; Espevik, Terje; Skjåk-Bræk, Gudmund

    2014-07-14

    Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar molecular weights were chemically sulfated and structurally characterized by the use of NMR and elemental analysis. These sulfated alginates were shown to bind and displace HGF from the surface of myeloma cells in a manner similar to heparin. We observed dependence on the sulfation degree (DS) as well as variation in efficacy based on the alginate monosaccharide sequence, relating to relative flexibility and charge density in the polysaccharide chains. Co-incubation with human plasma showed complement compatibility of the alginates and lowering of soluble terminal complement complex levels by sulfated alginates. The sulfated polyalternating (poly-MG) alginate proved to be the most reproducible in terms of precise sulfation degrees and showed the greatest relative degree of complement inhibition and HGF interaction, maintaining high activity at low DS values.

  15. Murine T lymphocytes and T-lymphoma cells produce chondroitin sulphate and heparan sulphate proteoglycans and free heparan sulphate glycosaminoglycan.

    PubMed Central

    Wilson, A P; Rider, C C

    1991-01-01

    Normal murine splenic T lymphocytes and T-lymphoma cells were incubated with [35S]sulphate in low-sulphate medium for 4 hr. Gel filtration and SDS-PAGE revealed that the radiolabelled macromolecules secreted by these cells were almost exclusively chondroitin sulphate and heparan sulphate proteoglycans of relatively low molecular weight (MW), 100,000-200,000. Triton X-100 extracts of the cells contained similar proteoglycans. Under the conditions employed the incorporation of radiolabel by cells grown in vivo was equally distributed between cell-retained and secreted fractions, whereas cells grown in vitro retained some 75% of incorporated label. In general heparan sulphate predominated over chondroitin sulphate in both secreted and cell-retained fractions. Cell extracts also contained a minor proportion of free glycosaminoglycan, which is almost exclusively heparan sulphate. These chains, like those incorporated into the proteoglycan, were around 12,000 MW. The T-lymphoma cells RDM-4, whether grown in vitro or in vivo, also incorporated a substantial proportion of [35S]sulphate into a single, cell-retained protein, 100,000 MW. No such radiolabelled protein was detectable in T cells. Images Figure 3 Figure 4 Figure 5 PMID:1900056

  16. Glial dysfunction in abstinent methamphetamine abusers.

    PubMed

    Sailasuta, Napapon; Abulseoud, Osama; Harris, Kent C; Ross, Brian D

    2010-05-01

    Persistent neurochemical abnormalities in frontal brain structures are believed to result from methamphetamine use. We developed a localized (13)C magnetic resonance spectroscopy (MRS) assay on a conventional MR scanner, to quantify selectively glial metabolic flux rate in frontal brain of normal subjects and a cohort of recovering abstinent methamphetamine abusers. Steady-state bicarbonate concentrations were similar, between 11 and 15 mmol/L in mixed gray-white matter of frontal brain of normal volunteers and recovering methamphetamine-abusing subjects (P>0.1). However, glial (13)C-bicarbonate production rate from [1-(13)C]acetate, equating with glial tricarboxylic acid (TCA) cycle rate, was significantly reduced in frontal brain of abstinent methamphetamine-addicted women (methamphetamine 0.04 micromol/g per min (N=5) versus controls 0.11 micromol/g per min (N=5), P=0.001). This is equivalent to 36% of the normal glial TCA cycle rate. Severe reduction in glial TCA cycle rate that normally comprises 10% of total cerebral metabolic rate may impact operation of the neuronal glial glutamate cycle and result in accumulation of frontal brain glutamate, as observed in these recovering methamphetamine abusers. Although these are the first studies to define directly an abnormality in glial metabolism in human methamphetamine abuse, sequential studies using analogous (13)C MRS methods may determine 'cause and effect' between glial failure and neuronal injury.

  17. ((35)S)sulfate incorporation into glomerular basement membrane glycosaminoglycans is decreased in experimental diabetes

    SciTech Connect

    Cohen, M.P.; Surma, M.L.

    1981-11-01

    Isolated rat renal glomeruli incorporate radioactive sulfate into glycosaminoglycans, which are integral components of the glomerular basement membrane. Cellulose acetate electrophoresis and specific enzymatic sensitivities of glycosaminoglycans prepared after pronase digestion of purified glomerular basement membrane indicate the presence of heparan sulfate. We examined the effect of experimental diabetes on the incorporation of ((35)S)-sulfate into glycosaminoglycans deposited into newly synthesized glomerular basement membrane in vitro. Basement membranes were purified from glomeruli isolated from normal and streptozotocin-diabetic rats after incubation for 2 hr with radiolabeled sulfate and then were subjected to pronase digestion for isolation of the glycosaminoglycans. ((35)S) incorporation into basement membrane glycosaminoglycans was significantly decreased in glomeruli from diabetic animals. The addition of insulin (100 micron U/ml) in vitro did not affect ((35)S) incorporation into glycosaminoglycans of the glomerular basement membranes in normal or diabetic glomeruli. High glucose concentration (5 vs. 20 mM) was without effect in short-term incubations of glomeruli from normal animals. The results indicate that experimental diabetes influences ((35)S) sulfate incorporation into glomerular basement membrane glycosaminoglycans and suggest that decreased heparan sulfate production and/or sulfation may contribute to the increased permeability of the glomerular basement membrane in diabetes.

  18. Nitric oxide degradation of heparin and heparan sulphate.

    PubMed Central

    Vilar, R E; Ghael, D; Li, M; Bhagat, D D; Arrigo, L M; Cowman, M K; Dweck, H S; Rosenfeld, L

    1997-01-01

    NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines. PMID:9182706

  19. The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans

    PubMed Central

    Ludington, Jacob G.

    2016-01-01

    The apicomplexan parasite Cryptosporidium causes significant diarrheal disease worldwide. Effective anticryptosporidial agents are lacking, in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood. Previously, we identified and characterized a novel Cryptosporidium parvum C-type lectin domain-containing mucin-like glycoprotein, CpClec. In this study, we evaluated the mechanisms underlying interactions of CpClec with intestinal epithelial cells by using an Fc-tagged recombinant protein. CpClec-Fc displayed Ca2+-dependent, saturable binding to HCT-8 and Caco-2 cells and competitively inhibited C. parvum attachment to and infection of HCT-8 cells. Binding of CpClec-Fc was specifically inhibited by sulfated glycosaminoglycans, particularly heparin and heparan sulfate. Binding was reduced after the removal of heparan sulfate and following the inhibition of glycosaminoglycan synthesis or sulfation in HCT-8 cells. Like CpClec-Fc binding, C. parvum attachment to and infection of HCT-8 cells were inhibited by glycosaminoglycans and were reduced after heparan sulfate removal or inhibition of glycosaminoglycan synthesis or sulfation. Lastly, CpClec-Fc binding and C. parvum sporozoite attachment were significantly decreased in CHO cell mutants defective in glycosaminoglycan synthesis. Together, these results indicate that CpClec is a novel C-type lectin that mediates C. parvum attachment and infection via Ca2+-dependent binding to sulfated proteoglycans on intestinal epithelial cells. PMID:26975991

  20. Crystal packing in three related disaccharides: precursors to heparan sulfate oligosaccharides

    PubMed Central

    Gainsford, Graeme J.; Schwörer, Ralf; Tyler, Peter C.; Zubkova, Olga V.

    2015-01-01

    The three title compounds form part of a set of important precursor dissacharides which lead to novel therapeutics, in particular for Alzheimer’s disease. All three crystallize as poorly diffracting crystals with one independent mol­ecule in the asymmetric unit. Two of them are isostructural: 4-meth­oxy­phenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-de­oxy-4-O-(9-fluor­en­yl­methyl­oxycarbon­yl)-α-d-gluco­pyranos­yl]-2-O-benzoyl-3-O-benzyl-6-O-chloro­acetyl-α-l-ido­pyran­oside, C59H56ClN3O16, (I), the ido-relative of a reported gluco-disaccharide [Gainsford et al., 2013 ▸). Acta Cryst. C69, 679–682] and 4-meth­oxy­phenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-de­oxy-4-O-(9-fluorenyl­methyl­oxycarbon­yl)-α-d-gluco­pyranos­yl]-2-O-benzoyl-3-O-benzyl-6-O-meth­oxy­acetyl-α-l-ido­pyran­oside, C60H59N3O17, (II). Both exhibit similar conformational disorder of pendant groups. The third compound 4-meth­oxy­phenyl 4-O-[6-O-acetyl-2-azido-3,4-di-O-benzyl-2-de­oxy-α-d-gluco­pyranos­yl]-2-O-benzoyl-3-O-benzyl-6-O-meth­oxy­oacetyl-β-d-gluco­pyran­oside, C52H55N3O15, (III), illustrates that a slightly larger set of weak inter­molecular inter­actions can result in a less disordered mol­ecular arrangement. The mol­ecules are bound by weak C—H⋯O(ether) hydrogen bonds in (I) and (II), augmented by C—H⋯π inter­actions in (III). The absolute configurations were determined, although at varying levels of significance from the limited observed data. PMID:26090127

  1. The Role(s) of Heparan Sulfate Proteoglycan(s) in the wnt-1 Signaling Pathway

    DTIC Science & Technology

    1998-08-01

    and invertebrates , and are initiated by formation of a polysaccharide chain consisting of (D-glucuronic acido....N-acetyl-D- glucosamine...M.C. Ellis, T.B. Kornberg, and C.S. Goodman. 1989. Expression of Engrailed protein in arthropods, annelids and chordates . Cell 58: 955-968. Patel N.H

  2. Sulfation patterns determine cellular internalization of heparin-like polysaccharides

    PubMed Central

    Raman, Karthik; Mencio, Caitlin; Desai, Umesh R.; Kuberan, Balagurunathan

    2013-01-01

    Heparin is a highly sulfated polysaccharide which serves biologically relevant roles as an anticoagulant and anti-cancer agent. While it is well known that modification of heparin’s sulfation pattern can drastically influence its ability to bind growth factors and other extracellular molecules, very little is known about the cellular uptake of heparin and the role sulfation patterns serve in affecting its internalization. In this study, we chemically synthesized several fluorescently-labeled heparins consisting of a variety of sulfation patterns. These polysaccharides were thoroughly characterized using anion exchange chromatography and size exclusion chromatography. Subsequently, we utilized flow cytometry and confocal imaging to show that sulfation patterns differentially affect the amount of heparin uptake in multiple cell types. This study provides the first comprehensive analysis of the effect of sulfation pattern on the cellular internalization of heparin or heparan sulfate like polysaccharides. The results of this study expand current knowledge regarding heparin internalization and provide insights into developing more effective heparin-based drug conjugates for applications in intracellular drug delivery. PMID:23398560

  3. Sulfation patterns determine cellular internalization of heparin-like polysaccharides.

    PubMed

    Raman, Karthik; Mencio, Caitlin; Desai, Umesh R; Kuberan, Balagurunathan

    2013-04-01

    Heparin is a highly sulfated polysaccharide that serves biologically relevant roles as an anticoagulant and anticancer agent. While it is well-known that modification of heparin's sulfation pattern can drastically influence its ability to bind growth factors and other extracellular molecules, very little is known about the cellular uptake of heparin and the role sulfation patterns serve in affecting its internalization. In this study, we chemically synthesized several fluorescently labeled heparins consisting of a variety of sulfation patterns. These polysaccharides were thoroughly characterized using anion exchange chromatography and size exclusion chromatography. Subsequently, we utilized flow cytometry and confocal imaging to show that sulfation patterns differentially affect the amount of heparin uptake in multiple cell types. This study provides the first comprehensive analysis of the effect of sulfation pattern on the cellular internalization of heparin or heparan sulfate like polysaccharides. The results of this study expand current knowledge regarding heparin internalization and provide insights into developing more effective heparin-based drug conjugates for applications in intracellular drug delivery.

  4. Diethyl sulfate

    Integrated Risk Information System (IRIS)

    Diethyl sulfate ; CASRN 64 - 67 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  5. Barium Sulfate

    MedlinePlus

    ... uses a computer to put together x-ray images to create cross-sectional or three dimensional pictures of the inside of the body). Barium sulfate is in a class of medications called radiopaque contrast media. It works by coating the esophagus, stomach, or ...

  6. Dimethyl sulfate

    Integrated Risk Information System (IRIS)

    Dimethyl sulfate ; CASRN 77 - 78 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  7. The interface between glial progenitors and gliomas

    PubMed Central

    Canoll, Peter

    2009-01-01

    The mammalian brain and spinal cord contain heterogeneous populations of cycling, immature cells. These include cells with stem cell-like properties as well as progenitors in various stages of early glial differentiation. This latter population is distributed widely throughout gray and white matter and numerically represents an extremely large cell pool. In this review, we discuss the possibility that the glial progenitors that populate the adult CNS are one source of gliomas. Indeed, the marker phenotypes, morphologies, and migratory properties of cells in gliomas strongly resemble glial progenitors in many ways. We review briefly some salient features of normal glial development and then examine the similarities and differences between normal progenitors and cells in gliomas, focusing on the phenotypic plasticity of glial progenitors and the responses to growth factors in promoting proliferation and migration of normal and glioma cells, and discussing known mutational changes in gliomas in the context of how these might affect the proliferative and migratory behaviors of progenitors. Finally, we will discuss the “cancer stem cell” hypothesis in light of the possibility that glial progenitors can generate gliomas. PMID:18784926

  8. Chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) involved in chondroitin sulfate initiation: Impact of sulfation on activity and specificity.

    PubMed

    Gulberti, Sandrine; Jacquinet, Jean-Claude; Chabel, Matthieu; Ramalanjaona, Nick; Magdalou, Jacques; Netter, Patrick; Coughtrie, Michael W H; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie

    2012-04-01

    Glycosaminoglycan (GAG) assembly initiates through the formation of a linkage tetrasaccharide region serving as a primer for both chondroitin sulfate (CS) and heparan sulfate (HS) chain polymerization. A possible role for sulfation of the linkage structure and of the constitutive disaccharide unit of CS chains in the regulation of CS-GAG chain synthesis has been suggested. To investigate this, we determined whether sulfate substitution of galactose (Gal) residues of the linkage region or of N-acetylgalactosamine (GalNAc) of the disaccharide unit influences activity and specificity of chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1), a key glycosyltransferase of CS biosynthesis. We synthesized a series of sulfated and unsulfated analogs of the linkage oligosaccharide and of the constitutive unit of CS and tested these molecules as potential acceptor substrates for the recombinant human CSGalNAcT-1. We show here that sulfation at C4 or C6 of the Gal residues markedly influences CSGalNAcT-1 initiation activity and catalytic efficiency. Kinetic analysis indicates that CSGalNAcT-1 exhibited 3.6-, 1.6-, and 2.2-fold higher enzymatic efficiency due to lower K(m) values toward monosulfated trisaccharides substituted at C4 or C6 position of Gal1, and at C6 of Gal2, respectively, compared with the unsulfated oligosaccharide. This highlights the critical influence of Gal substitution on both CSGalNAcT-1 activity and specifity. No GalNAcT activity was detected toward sulfated and unsulfated analogs of the CS constitutive disaccharide (GlcA-β1,3-GalNAc), indicating that CSGalNAcT-1 was involved in initiation but not in elongation of CS chains. Our results strongly suggest that sulfation of the linkage region acts as a regulatory signal in CS chain initiation.

  9. Identification of keratan sulfate disaccharide at C-3 position of glucuronate of chondroitin sulfate from Mactra chinensis

    PubMed Central

    Higashi, Kyohei; Takeda, Keita; Mukuno, Ann; Okamoto, Yusuke; Masuko, Sayaka; Linhardt, Robert J.; Toida, Toshihiko

    2016-01-01

    Glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate, heparin, heparan sulfate and keratan sulfate (KS) are linear sulfated repeating disaccharide sequences containing hexosamine and uronic acid [or galactose (Gal) in the case of KS]. Among the GAGs, CS shows structural variations, such as sulfation patterns and fucosylation, which are responsible for their physiological functions through CS interaction with CS-binding proteins. Here, we solved the structure of KS-branched CS-E derived from a clam, Mactra chinensis. KS disaccharide [d-GlcNAc6S-(1→3)-β-d-Gal-(1→] was attached to the C-3 position of GlcA, and consecutive KS-branched disaccharide sequences were found in a CS chain. KS-branched polysaccharides clearly exhibited resistance to degradation by chondroitinase ABC or ACII (at low concentrations) compared with typical CS structures. Furthermore, KS-branched polysaccharides stimulated neurite outgrowth of hippocampal neurons. These results strongly suggest that M. chinensis is a rich source of KS-branched CS, and it has important biological activities. PMID:27647934

  10. Glial chain migration requires pioneer cells.

    PubMed

    Aigouy, Benoît; Lepelletier, Léa; Giangrande, Angela

    2008-11-05

    The migration of glial chains along the nerve entails directional and coordinated movement. Despite its importance in the formation of the nervous system, this process remains poorly understood, because of the difficulty of manipulating identified cells. Using confocal time-lapse and cell ablation in the whole animal, we provide direct evidence for a discrete number of Drosophila peripheral glial cells acting as pioneers and guiding the rest of the migratory chain. These cells are in direct contact with several follower cells through a very long and stable cytoplasmic extension. The presence of pioneer cells and homotypic interactions at the tip of the chain allows coordinated movement and the formation of a continuous sheath around the nerve. These in vivo data open novel perspectives for understanding the cellular bases of vertebrate glial migration in physiological and pathological conditions.

  11. Calcineurin and glial signaling: neuroinflammation and beyond.

    PubMed

    Furman, Jennifer L; Norris, Christopher M

    2014-09-10

    Similar to peripheral immune/inflammatory cells, neuroglial cells appear to rely on calcineurin (CN) signaling pathways to regulate cytokine production and cellular activation. Several studies suggest that harmful immune/inflammatory responses may be the most impactful consequence of aberrant CN activity in glial cells. However, newly identified roles for CN in glutamate uptake, gap junction regulation, Ca2+ dyshomeostasis, and amyloid production suggest that CN's influence in glia may extend well beyond neuroinflammation. The following review will discuss the various actions of CN in glial cells, with particular emphasis on astrocytes, and consider the implications for neurologic dysfunction arising with aging, injury, and/or neurodegenerative disease.

  12. Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system.

    PubMed

    von Hilchen, Christian M; Bustos, Alvaro E; Giangrande, Angela; Technau, Gerhard M; Altenhein, Benjamin

    2013-09-01

    One of the numerous functions of glial cells in Drosophila is the ensheathment of neurons to isolate them from the potassium-rich haemolymph, thereby establishing the blood-brain barrier. Peripheral nerves of flies are surrounded by three distinct glial cell types. Although all embryonic peripheral glia (ePG) have been identified on a single-cell level, their contribution to the three glial sheaths is not known. We used the Flybow system to label and identify each individual ePG in the living embryo and followed them into third instar larva. We demonstrate that all ePG persist until the end of larval development and some even to adulthood. We uncover the origin of all three glial sheaths and describe the larval differentiation of each peripheral glial cell in detail. Interestingly, just one ePG (ePG2) exhibits mitotic activity during larval stages, giving rise to up to 30 glial cells along a single peripheral nerve tract forming the outermost perineurial layer. The unique mitotic ability of ePG2 and the layer affiliation of additional cells were confirmed by in vivo ablation experiments and layer-specific block of cell cycle progression. The number of cells generated by this glial progenitor and hence the control of perineurial hyperplasia correlate with the length of the abdominal nerves. By contrast, the wrapping and subperineurial glia layers show enormous hypertrophy in response to larval growth. This characterisation of the embryonic origin and development of each glial sheath will facilitate functional studies, as they can now be addressed distinctively and genetically manipulated in the embryo.

  13. Negative regulation of glial engulfment activity by Draper terminates glial responses to axon injury

    PubMed Central

    Logan, Mary A.; Hackett, Rachel; Doherty, Johnna; Sheehan, Amy; Speese, Sean D.; Freeman, Marc R.

    2012-01-01

    Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function, and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial reponses to axon injury are molecularly encoded by unique isoforms of the Drosophila engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a novel immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II/Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. Our work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates a balance of opposing Draper-I/-II signaling events is essential to maintain glial sensitivity to brain injury. PMID:22426252

  14. Interplay between transglutaminases and heparan sulphate in progressive renal scarring

    PubMed Central

    Burhan, Izhar; Furini, Giulia; Lortat-Jacob, Hugues; Atobatele, Adeola G.; Scarpellini, Alessandra; Schroeder, Nina; Atkinson, John; Maamra, Mabrouka; Nutter, Faith H.; Watson, Philip; Vinciguerra, Manlio; Johnson, Timothy S.; Verderio, Elisabetta A. M.

    2016-01-01

    Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-β1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS. PMID:27694984

  15. Isolation and characterisation of putative adhesins from Helicobacter pylori with affinity for heparan sulphate proteoglycan.

    PubMed

    Ruiz-Bustos, E; Ochoa, J L; Wadström, T; Ascencio, F

    2001-03-01

    A pool of heparan sulphate-binding proteins (HSBPs) from Helicobacter pylori culture supernates was obtained by sequential ammonium sulphate precipitation and affinity chromatography on heparin-Sepharose. The chromatographic procedure yielded one major fraction that contained proteins with heparan sulphate affinity as revealed by inhibition studies of heparan sulphate binding to H. pylori cells. Preparative iso-electric focusing, SDS-PAGE and blotting experiments, with peroxidase(POD)-labelled heparan sulphate as a probe, indicated the presence of two major extracellular proteins with POD-heparan sulphate affinity. One protein had a molecular mass of 66.2 kDa and a pI of 5.4, whilst the second protein had a molecular mass of 71.5 kDa and a pI of 5.0. The N-terminal amino acid sequence of the 71.5-kDa HSBP did not show homology to any other heparin-binding protein, nor to known proteins of H. pylori, whereas the 66.2-kDa HSBP showed a high homology to an Escherichia coli chaperon protein and equine haemoglobin. A third HSBP was isolated from an outer-membrane protein (OMP) fraction of H. pylori cells with a molecular mass of 47.2 kDa. The amino acid sequence of an internal peptide of the OMP-HSBP did not show homology to the extracellular HSBP of H. pylori, or to another microbial HSBP.

  16. Intrathoracic glial implants in a child with gliomatosis peritonei.

    PubMed

    Lipskar, Aaron M; Rothstein, David H; Soffer, Samuel Z; Edelman, Morris; Glick, Richard D

    2009-09-01

    Glial peritoneal implants, commonly referred to as gliomatosis peritonei, are an occasional feature of ovarian teratomas. They are benign nodules of mature glial tissue and usually do not adversely affect outcome. We present the case of a 12-year-old girl who underwent excision of an immature ovarian teratoma, along with biopsies of multiple glial peritoneal implants. She also had a 2-cm right-sided pleural mass, which turned out to be normal glial tissue that was histologically indistinguishable from the peritoneal glial tissue. Pleural gliomatosis has not been described in the literature. The pathophysiology of gliomatosis peritonei was originally thought to be the direct extrusion or lymphatic spread of glial cells from the associated teratoma, although it has been postulated that the glial implants may instead be the result of pluripotent Mullerian stem cells that undergo metaplasia. This report provides evidence to bolster the metaplastic theory.

  17. Glial Cell Development and Function in Zebrafish

    PubMed Central

    Lyons, David A.; Talbot, William S.

    2015-01-01

    The zebrafish is a premier vertebrate model system that offers many experimental advantages for in vivo imaging and genetic studies. This review provides an overview of glial cell types in the central and peripheral nervous system of zebrafish. We highlight some recent work that exploited the strengths of the zebrafish system to increase the understanding of the role of Gpr126 in Schwann cell myelination and illuminate the mechanisms controlling oligodendrocyte development and myelination. We also summarize similarities and differences between zebrafish radial glia and mammalian astrocytes and consider the possibility that their distinct characteristics may represent extremes in a continuum of cell identity. Finally, we focus on the emergence of zebrafish as a model for elucidating the development and function of microglia. These recent studies have highlighted the power of the zebrafish system for analyzing important aspects of glial development and function. PMID:25395296

  18. White Matter Glial Pathology in Autism

    DTIC Science & Technology

    2014-09-01

    The majority of total nuclear-encoded non-ribosomal RNA in a human cell is “ dark matter ” un-annotated RNA. BMC Biol. 8, 149 (2010). 9. Ginsberg, S...DATE September 2014 2. REPORT TYPE Annual 3. DATES COVERED 1 Sep 2013 - 31 Aug 2014 4. TITLE AND SUBTITLE White Matter Glial Pathology in...stages of preparation due to poor sample quality factors such as low RIN or insufficient sequencing reads. Superficial white matter was laser

  19. Human brain glial cells synthesize thrombospondin.

    PubMed Central

    Asch, A S; Leung, L L; Shapiro, J; Nachman, R L

    1986-01-01

    Thrombospondin, a 450-kDa multinodular glycoprotein with lectin-type activity, is found in human platelets, endothelial cells, fibroblasts, smooth muscle cells, monocytes, and granular pneumocytes. Thrombospondin interacts with heparin, fibrinogen, fibronectin, collagen, histidine-rich glycoprotein, and plasminogen. Recently, thrombospondin synthesis by smooth muscle cells has been reported to be augmented by platelet-derived growth factor. We present evidence that thrombospondin is present within and synthesized by astrocytic neuroglial cells. Heparin-Sepharose affinity chromatography of material derived from a human brain homogenate yielded a protein that, when reduced, had an apparent size of 180 kDa and comigrated with reduced platelet thrombospondin on NaDodSO4/PAGE. Immunoblot analysis with monospecific anti-thrombospondin confirmed the presence of immunoreactive thrombospondin. Indirect immunofluorescence of cultured human glial cells indicated the presence of thrombospondin. Metabolic labeling of glial cell cultures with [35S]methionine followed by immunoprecipitation with monospecific anti-thrombospondin revealed synthesis of a 180-kDa polypeptide that comigrated with platelet thrombospondin on NaDodSO4/PAGE. Cultured human glial cells were incubated for 48 hr in serum-free medium with purified platelet-derived growth factor at concentrations up to 50 ng/ml. Aliquots taken at intervals were analyzed by a quantitative double-antibody ELISA. The growth factor stimulated the release of thrombospondin into the culture medium by as much as 10-fold over control cultures. The presence of thrombospondin within glial cells of the central nervous system and the augmentation of its synthesis by platelet-derived growth factor suggest that thrombospondin may play an important role in regulating cell-cell and cell-matrix interactions during periods of cell division and growth. Images PMID:2939460

  20. Physiological Functions of Glial Cell Hemichannels.

    PubMed

    Orellana, Juan A

    2016-01-01

    The brain performs exceptionally complex and dynamic tasks that depend on the coordinated interaction of neurons, glial cells, endothelial cells, pericytes, smooth muscle cells, ependymal cells, and circulating blood cells. Among these cells, glial cells have emerged as crucial protagonists in the regulation of synaptic transmission and neural function. Indeed, these cells express a wide range of receptors that enable them to sense changes in neuronal activity and the microenvironment by responding locally via the release of bioactive molecules known as gliotransmitters. In the central nervous system (CNS), a novel mechanism that allows gliotransmission via the opening of hemichannels has been proposed. These channels are composed of six protein subunits consisting of connexins or pannexins, which are two highly conserved protein families that are encoded by 21 and 3 genes, respectively, in humans. Typically, glial cell hemichannels exhibit low levels of activity, but this activity is sufficient to ensure the release of a broad spectrum of gliotransmitters, including ATP, D-serine, glutamate, adenosine, and glutathione. Here, we briefly review the current findings regarding the effects of the hemichannel-dependent release of gliotransmitters on the physiology of the CNS.

  1. Glial Cell Contributions to Auditory Brainstem Development

    PubMed Central

    Cramer, Karina S.; Rubel, Edwin W

    2016-01-01

    Glial cells, previously thought to have generally supporting roles in the central nervous system, are emerging as essential contributors to multiple aspects of neuronal circuit function and development. This review focuses on the contributions of glial cells to the development of auditory pathways in the brainstem. These pathways display specialized synapses and an unusually high degree of precision in circuitry that enables sound source localization. The development of these pathways thus requires highly coordinated molecular and cellular mechanisms. Several classes of glial cells, including astrocytes, oligodendrocytes and microglia, have now been explored in these circuits in both avian and mammalian brainstems. Distinct populations of astrocytes are found over the course of auditory brainstem maturation. Early appearing astrocytes are associated with spatial compartments in the avian auditory brainstem. Factors from late appearing astrocytes promote synaptogenesis and dendritic maturation, and astrocytes remain integral parts of specialized auditory synapses. Oligodendrocytes play a unique role in both birds and mammals in highly regulated myelination essential for proper timing to decipher interaural cues. Microglia arise early in brainstem development and may contribute to maturation of auditory pathways. Together these studies demonstrate the importance of non-neuronal cells in the assembly of specialized auditory brainstem circuits. PMID:27818624

  2. The role of the PI3K/Akt/mTOR pathway in glial scar formation following spinal cord injury.

    PubMed

    Chen, Chun-Hong; Sung, Chun-Sung; Huang, Shi-Ying; Feng, Chien-Wei; Hung, Han-Chun; Yang, San-Nan; Chen, Nan-Fu; Tai, Ming-Hong; Wen, Zhi-Hong; Chen, Wu-Fu

    2016-04-01

    Several studies suggest that glial scars pose as physical and chemical barriers that limit neurite regeneration after spinal cord injury (SCI). Evidences suggest that the activation of the PI3K/Akt/mTOR signaling pathway is involved in glial scar formation. Therefore, inhibition of the PI3K/Akt/mTOR pathway may beneficially attenuate glial scar formation after SCI. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulates the PI3K/Akt/mTOR pathway. Therefore, we hypothesized that the overexpression of PTEN in the spinal cord will have beneficial effects after SCI. In the present study, we intrathecally injected a recombinant adenovirus carrying the pten gene (Ad-PTEN) to cause overexpression of PTEN in rats with contusion injured spinal cords. The results suggest overexpression of PTEN in spinal cord attenuated glial scar formation and led to improved locomotor function after SCI. Overexpression of PTEN following SCI attenuated gliosis, affected chondroitin sulfate proteoglycan expression, and improved axon regeneration into the lesion site. Furthermore, we suggest that the activation of the PI3K/Akt/mTOR pathway in astrocytes at 3 days after SCI may be involved in glial scar formation. Because delayed treatment with Ad-PTEN enhanced motor function recovery more significantly than immediate treatment with Ad-PTEN after SCI, the results suggest that the best strategy to attenuate glial scar formation could be to introduce 3 days after SCI. This study's findings thus have positive implications for patients who are unable to receive immediate medical attention after SCI.

  3. Refractory sickle cell leg ulcer: is heparan sulphate a new hope?

    PubMed

    Hayek, Shady; Dibo, Saad; Baroud, Joe; Ibrahim, Amir; Barritault, Denis

    2016-02-01

    Patients with sickle cell disease are known to have recurrent lower extremity ulcers that have a high pain score and are resistant to conventional means of wound therapy. This study reports the successful use of synthetic heparan sulphate (Cacipliq20(®) , OTR3, Paris, France) in the treatment of a sickle cell ulcer that had failed to respond to several other means of treatment. Therapeutic success was assessed by complete wound coverage and vast improvement in pain score. This is the first study to report use of heparan sulphate in sickle cell ulcers.

  4. Chondroitin Sulfate is the Primary Receptor for a Peptide-Modified AAV That Targets Brain Vascular Endothelium In Vivo.

    PubMed

    Geoghegan, James C; Keiser, Nicholas W; Okulist, Anna; Martins, Inês; Wilson, Matthew S; Davidson, Beverly L

    2014-10-14

    Recently, we described a peptide-modified AAV2 vector (AAV-GMN) containing a capsid-displayed peptide that directs in vivo brain vascular targeting and transduction when delivered intravenously. In this study, we sought to identify the receptor that mediates transduction by AAV-GMN. We found that AAV-GMN, but not AAV2, readily transduces the murine brain endothelial cell line bEnd.3, a result that mirrors previously observed in vivo transduction profiles of brain vasculature. Studies in vitro revealed that the glycosaminoglycan, chondroitin sulfate C, acts as the primary receptor for AAV-GMN. Unlike AAV2, chondroitin sulfate expression is required for cell transduction by AAV-GMN, and soluble chondroitin sulfate C can robustly inhibit AAV-GMN transduction of brain endothelial cells. Interestingly, AAV-GMN retains heparin-binding properties, though in contrast to AAV2, it poorly transduces cells that express heparan sulfate but not chondroitin sulfate, indicating that the peptide insertion negatively impacts heparan-mediated transduction. Lastly, when delivered directly, this modified virus can transduce multiple brain regions, indicating that the potential of AAV-GMN as a therapeutic gene delivery vector for central nervous system disorders is not restricted to brain vascular endothelium.

  5. Glycosaminoglycan sulfation determines the biochemical properties of prion protein aggregates.

    PubMed

    Ellett, Laura J; Coleman, Bradley M; Shambrook, Mitch C; Johanssen, Vanessa A; Collins, Steven J; Masters, Colin L; Hill, Andrew F; Lawson, Victoria A

    2015-07-01

    Prion diseases are transmissible neurodegenerative disorders associated with the conversion of the cellular prion protein, PrP(C), to a misfolded isoform called PrP(Sc). Although PrP(Sc) is a necessary component of the infectious prion, additional factors, or cofactors, have been shown to contribute to the efficient formation of transmissible PrP(Sc). Glycosaminoglycans (GAGs) are attractive cofactor candidates as they can be found associated with PrP(Sc) deposits, have been shown to enhance PrP misfolding in vitro, are found in the same cellular compartments as PrP(C) and have been shown to be disease modifying in vivo. Here we investigated the effects of the sulfated GAGs, heparin and heparan sulfate (HS), on disease associated misfolding of full-length recombinant PrP. More specifically, the degree of sulfation of these molecules was investigated for its role in modulating the disease-associated characteristics of PrP. Both heparin and HS induced a β-sheet conformation in recombinant PrP that was associated with the formation of aggregated species; however, the biochemical properties of the aggregates formed in the presence of heparin or HS varied in solubility and protease resistance. Furthermore, these properties could be modified by changes in GAG sulfation, indicating that subtle changes in the properties of prion disease cofactors could initiate disease associated misfolding.

  6. Nematodes join the family of chondroitin sulfate-synthesizing organisms: Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans

    PubMed Central

    Dierker, Tabea; Shao, Chun; Haitina, Tatjana; Zaia, Joseph; Hinas, Andrea; Kjellén, Lena

    2016-01-01

    Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoans and Ecdysozoans. The general view that nematodes such as Caenorhabditis elegans, which belong to Ecdysozoa, produce HS but only chondroitin without sulfation has therefore been puzzling. We have analyzed GAGs in C. elegans using reversed-phase ion-pairing HPLC, mass spectrometry and immunohistochemistry. Our analyses included wild type C. elegans but also a mutant lacking two HS sulfotransferases (hst-6 hst-2), as we suspected that the altered HS structure could boost CS sulfation. We could indeed detect sulfated CS in both wild type and mutant nematodes. While 4-O-sulfation of galactosamine dominated, we also detected 6-O-sulfated galactosamine residues. Finally, we identified the product of the gene C41C4.1 as a C. elegans CS-sulfotransferase and renamed it chst-1 (CarboHydrate SulfoTransferase) based on loss of CS-4-O-sulfation in a C41C4.1 mutant and in vitro sulfotransferase activity of recombinant C41C4.1 protein. We conclude that C. elegans indeed manufactures CS, making this widely used nematode an interesting model for developmental studies involving CS. PMID:27703236

  7. Nematodes join the family of chondroitin sulfate-synthesizing organisms: Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans.

    PubMed

    Dierker, Tabea; Shao, Chun; Haitina, Tatjana; Zaia, Joseph; Hinas, Andrea; Kjellén, Lena

    2016-10-05

    Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoans and Ecdysozoans. The general view that nematodes such as Caenorhabditis elegans, which belong to Ecdysozoa, produce HS but only chondroitin without sulfation has therefore been puzzling. We have analyzed GAGs in C. elegans using reversed-phase ion-pairing HPLC, mass spectrometry and immunohistochemistry. Our analyses included wild type C. elegans but also a mutant lacking two HS sulfotransferases (hst-6 hst-2), as we suspected that the altered HS structure could boost CS sulfation. We could indeed detect sulfated CS in both wild type and mutant nematodes. While 4-O-sulfation of galactosamine dominated, we also detected 6-O-sulfated galactosamine residues. Finally, we identified the product of the gene C41C4.1 as a C. elegans CS-sulfotransferase and renamed it chst-1 (CarboHydrate SulfoTransferase) based on loss of CS-4-O-sulfation in a C41C4.1 mutant and in vitro sulfotransferase activity of recombinant C41C4.1 protein. We conclude that C. elegans indeed manufactures CS, making this widely used nematode an interesting model for developmental studies involving CS.

  8. Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

    PubMed

    de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

    2010-12-15

    Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology.

  9. Sex steroids inhibit osmotic swelling of retinal glial cells.

    PubMed

    Neumann, Florian; Wurm, Antje; Linnertz, Regina; Pannicke, Thomas; Iandiev, Ianors; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2010-04-01

    Osmotic swelling of glial cells may contribute to the development of retinal edema. We investigated whether sex steroids inhibit the swelling of glial somata in acutely isolated retinal slices and glial cells of the rat. Superfusion of retinal slices or cells from control animals with a hypoosmolar solution did not induce glial swelling, whereas glial swelling was observed in slices of postischemic and diabetic retinas. Progesterone, testosterone, estriol, and 17beta-estradiol prevented glial swelling with half-maximal effects at approximately 0.3, 0.6, 6, and 20 microM, respectively. The effect of progesterone was apparently mediated by transactivation of metabotropic glutamate receptors, P2Y1, and adenosine A1 receptors. The data suggest that sex steroids may inhibit cytotoxic edema in the retina.

  10. Biological function of unique sulfated glycosaminoglycans in primitive chordates.

    PubMed

    Karamanou, Konstantina; Espinosa, Diana Carolina Restrepo; Fortuna-Costa, Anneliese; Pavão, Mauro Sérgio Gonçalves

    2016-09-10

    Glycosaminoglycans with unique sulfation patterns have been identified in different species of ascidians (sea squirts), a group of marine invertebrates of the Phylum Chordata, sub-phylum Tunicata (or Urochordata). Oversulfated dermatan sulfate composed of [4-α-L-IdoA-(2-O-SO3)(-1) → 3-β-D-GalNAc(4-OSO3)(-1)]n repeating disaccharide units is found in the extracellular matrix of several organs, where it seems to interact with collagen fibers. This dermatan sulfate co-localizes with a decorin-like protein, as indicated by immunohistochemical analysis. Low sulfated heparin/heparan sulfate-like glycans composed mainly of [4-α-L-IdoA-(2-OSO3)(-1) → 4-α-D-GlcN(SO3)(-1) (6-O-SO3)(-1)]n and [4-α-L-IdoA-(2-O-SO3)(-1) → 4-α-D-GlcN(SO3)(-1)]n have also been described in ascidians. These heparin-like glycans occur in intracellular granules of oocyte assessory cells, named test cells, in circulating basophil-like cells in the hemolymph, and at the basement membrane of different ascidian organs. In this review, we present an overview of the structure, distribution, extracellular and intracellular localization of the sulfated glycosaminoglycans in different species and tissues of ascidians. Considering the phylogenetic position of the subphylum Tunicata in the phylum Chordata, a careful analysis of these data can reveal important information about how these glycans evolved from invertebrate to vertebrate animals.

  11. Ferrous Sulfate (Iron)

    MedlinePlus

    Ferrous sulfate provides the iron needed by the body to produce red blood cells. It is used to ... Ferrous sulfate comes as regular, coated, and extended-release (long-acting) tablets; regular and extended-release capsules; and ...

  12. Glial cells: Old cells with new twists

    PubMed Central

    Ndubaku, Ugo; de Bellard, Maria Elena

    2008-01-01

    Summary Based on their characteristics and function – migration, neural protection, proliferation, axonal guidance and trophic effects – glial cells may be regarded as probably the most versatile cells in our body. For many years, these cells were considered as simply support cells for neurons. Recently, it has been shown that they are more versatile than previously believed – as true stem cells in the nervous system – and are important players in neural function and development. There are several glial cell types in the nervous system: the two most abundant are oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Although both of these cells are responsible for myelination, their developmental origins are quite different. Oligodendrocytes originate from small niche populations from different regions of the central nervous system, while Schwann cells develop from a stem cell population (the neural crest) that gives rise to many cell derivatives besides glia and which is a highly migratory group of cells. PMID:18068219

  13. Glial Na(+) -dependent ion transporters in pathophysiological conditions.

    PubMed

    Boscia, Francesca; Begum, Gulnaz; Pignataro, Giuseppe; Sirabella, Rossana; Cuomo, Ornella; Casamassa, Antonella; Sun, Dandan; Annunziato, Lucio

    2016-10-01

    Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na(+) signaling influences and regulates important glial activities, and plays a role in neuron-glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na(+) pumps and Na(+) -dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na(+) homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na(+) -dependent ion transporters, including Na(+) /K(+) ATPase, Na(+) /Ca(2+) exchangers, Na(+) /H(+) exchangers, Na(+) -K(+) -Cl(-) cotransporters, and Na(+) - HCO3- cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na(+) -dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na(+) dynamics in different neurological disorders. GLIA 2016;64:1677-1697.

  14. Glial heterotopia in an adult: A rare orbital mass

    PubMed Central

    Sundaresh, Divya Dabir; Mangala Gouri, S R

    2016-01-01

    Heterotopic glial tissue is very rare in the orbit. Our case was an adult, which is unique since most cases reported in literature involve children. We describe a case of a 60-year-old man who presented with an orbital mass, which histopathologically revealed heterotopic glial tissue. PMID:27958209

  15. Mast Cells Produce a Unique Chondroitin Sulfate Epitope

    PubMed Central

    Farrugia, Brooke L.; Whitelock, John M.; O’Grady, Robert; Caterson, Bruce; Lord, Megan S.

    2015-01-01

    The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells. PMID:26586669

  16. Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

    PubMed

    Farrugia, Brooke L; Whitelock, John M; O'Grady, Robert; Caterson, Bruce; Lord, Megan S

    2016-02-01

    The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells.

  17. Glial heterotopia of the lip: A rare presentation

    PubMed Central

    Dadaci, Mehmet; Bayram, Fazli Cengiz; Ince, Bilsev; Bilgen, Fatma

    2016-01-01

    Glial heterotopia represents collections of normal glial tissue in an abnormal location distant to the central nervous system or spinal canal with no intracranial connectivity. Nasal gliomas are non-neoplastic midline tumours, with limited growth potential and no similarity to the central nervous system gliomas. The nose and the nasopharynx are the most common sites of location. Existence of glial heterotopia in the lip region is a rare developmental disorder. We report a case of large glial heterotopia in the upper lip region in a full-term female newborn which had intracranial extension with a fibrotic band. After the surgery, there was no recurrence in the follow-up period of 3 years. When glial heterotopia, which is a rare midline anomaly, is suspected, possible intracranial connection and properties of the mass should be evaluated by magnetic resonance imaging. By this way, lower complication rate and better aesthetic results can be achieved with early diagnosis and proper surgery. PMID:27274134

  18. Neuron-Glial Interactions in Blood-Brain Barrier Formation

    PubMed Central

    Banerjee, Swati; Bhat, Manzoor A.

    2010-01-01

    The blood brain barrier (BBB) evolved to preserve the microenvironment of the highly excitable neuronal cells to allow for action potential generation and propagation. Intricate molecular interactions between two main cell types, the neurons and the glial cells, form the underlying basis of the critical functioning of the nervous system across species. In invertebrates, interactions between neurons and glial cells are central in establishing a functional BBB. However, in vertebrates, the BBB formation and function is coordinated by interactions between neurons, glial cells, and endothelial cells. Here we review the neuron-glial interaction–based blood barriers in invertebrates and vertebrates and provide an evolutionary perspective as to how a glial-barrier system in invertebrates evolved into an endothelial barrier system. We also summarize the clinical relevance of the BBB as this protective barrier becomes disadvantageous in the pharmacological treatment of various neurological disorders. PMID:17506642

  19. N-acetylneuraminlactose sulfate in milk and its role in the synthesis of glycosaminoglycans during development

    SciTech Connect

    Kieras, F.J.; Kastin, S.; Rerecich, M.; Sturman, J.A.

    1986-05-01

    Mammals in early life are incapable of synthesizing inorganic sulfate, an important constituent of connective tissue glycosaminoglycans (GAGs). Studies have shown that (/sup 35/S)Na/sub 2/SO/sub 4/ injected into lactating rats is secreted into the milk as (/sup 35/S) N-acetylneuraminlactose sulfate (NLS); this compound accounts for greater than or equal to 95% of the radioactivity found in milk. They have studied the metabolic fate of the sulfate moiety of NLS in the newborn rat. Lactating rat dams were injected with (/sup 35/S) Na/sub 2/SO/sub 4/ at various times postpartum and pups were allowed to suckle for 48 hrs before sacrifice. Pups of varying ages (3 d. - 19 d.) were sacrificed and the GAGs were prepared from 9 different organs and tissues. Pups of all ages showed appreciable radiosulfate incorporated into all of the tissues and organs examined. In young pups (3 and 5 d old) a high of 75% of the total (/sup 35/S) sulfate in the homogenate was found in the GAGs of bone, and a low of 27% in the GAGs of liver. Analysis of the composition of the GAGs by enzymatic degradation showed that bone GAGs consisted entirely of chondroitin 4/6 sulfate (C4/6S) while liver GAGs contained 50% C4/6S, 10% dermatan sulfate (DS), and 40% chondroitinase resistant material (probably heparan sulfate). These data show that sulfate in the form of NLS is incorporated in substantial amounts into the sulfated GAGs of the developing rat and support the hypothesis that NLS is an important source of nutrient sulfate during rat development.

  20. Photodynamic damage of glial cells in crayfish ventral nerve cord

    NASA Astrophysics Data System (ADS)

    Kolosov, M. S.; Duz, E.; Uzdensky, A. B.

    2011-03-01

    Photodynamic therapy (PDT) is a promising method for treatment of brain tumors, the most of which are of glial origin. In the present work we studied PDT-mediated injury of glial cells in nerve tissue, specifically, in abdominal connectives in the crayfish ventral nerve cord. The preparation was photosensitized with alumophthalocyanine Photosens and irradiated 30 min with the diode laser (670 nm, 0.1 or 0.15 W/cm2). After following incubation in the darkness during 1- 10 hours it was fluorochromed with Hoechst 33342 and propidium iodide to reveal nuclei of living, necrotic and apoptotic cells. The chain-like location of the glial nuclei allowed visualization of those enveloping giant axons and blood vessels. The level of glial necrosis in control preparations was about 2-5 %. Apoptosis was not observed in control preparations. PDT significantly increased necrosis of glial cells to 52 or 67 % just after irradiation with 0.1 or 0.15 W/cm2, respectively. Apoptosis of glial cells was observed only at 10 hours after light exposure. Upper layers of the glial envelope of the connectives were injured stronger comparing to deep ones: the level of glial necrosis decreased from 100 to 30 % upon moving from the connective surface to the plane of the giant axon inside the connective. Survival of glial cells was also high in the vicinity of blood vessels. One can suggest that giant axons and blood vessels protect neighboring glial cells from photodynamic damage. The mechanism of such protective action remains to be elucidated.

  1. Photodynamic damage of glial cells in crayfish ventral nerve cord

    NASA Astrophysics Data System (ADS)

    Kolosov, M. S.; Duz, E.; Uzdensky, A. B.

    2010-10-01

    Photodynamic therapy (PDT) is a promising method for treatment of brain tumors, the most of which are of glial origin. In the present work we studied PDT-mediated injury of glial cells in nerve tissue, specifically, in abdominal connectives in the crayfish ventral nerve cord. The preparation was photosensitized with alumophthalocyanine Photosens and irradiated 30 min with the diode laser (670 nm, 0.1 or 0.15 W/cm2). After following incubation in the darkness during 1- 10 hours it was fluorochromed with Hoechst 33342 and propidium iodide to reveal nuclei of living, necrotic and apoptotic cells. The chain-like location of the glial nuclei allowed visualization of those enveloping giant axons and blood vessels. The level of glial necrosis in control preparations was about 2-5 %. Apoptosis was not observed in control preparations. PDT significantly increased necrosis of glial cells to 52 or 67 % just after irradiation with 0.1 or 0.15 W/cm2, respectively. Apoptosis of glial cells was observed only at 10 hours after light exposure. Upper layers of the glial envelope of the connectives were injured stronger comparing to deep ones: the level of glial necrosis decreased from 100 to 30 % upon moving from the connective surface to the plane of the giant axon inside the connective. Survival of glial cells was also high in the vicinity of blood vessels. One can suggest that giant axons and blood vessels protect neighboring glial cells from photodynamic damage. The mechanism of such protective action remains to be elucidated.

  2. Glial influence on the Blood Brain Barrier

    PubMed Central

    Alvarez, Jorge Ivan; Katayama, Takahiro; Prat, Alexandre

    2013-01-01

    The Blood Brain Barrier (BBB) is a specialized vascular structure tightly regulating central nervous system (CNS) homeostasis. Endothelial cells are the central component of the BBB and control of their barrier phenotype resides on astrocytes and pericytes. Interactions between these cells and the endothelium promote and maintain many of the physiological and metabolic characteristics that are unique to the BBB. In this review we describe recent findings related to the involvement of astroglial cells, including radial glial cells, in the induction of barrier properties during embryogenesis and adulthood. In addition, we describe changes that occur in astrocytes and endothelial cells during injury and inflammation with a particular emphasis on alterations of the BBB phenotype. GLIA 2013;61:1939–1958 PMID:24123158

  3. Glial Biology in Learning and Cognition

    PubMed Central

    Araque, Alfonso; Johansen-Berg, Heidi; Lim, Soo-Siang; Lynch, Gary; Nave, Klaus-Armin; Nedergaard, Maiken; Perez, Ray; Sejnowski, Terrence; Wake, Hiroaki

    2014-01-01

    Neurons are exquisitely specialized for rapid electrical transmission of signals, but some properties of glial cells, which do not communicate with electrical impulses, are well suited for participating in complex cognitive functions requiring broad spatial integration and long-term temporal regulation. Astrocytes, microglia, and oligodendrocytes all have biological properties that could influence learning and cognition. Myelination by oligodendrocytes increases conduction velocity, affecting spike timing and oscillations in neuronal activity. Astrocytes can modulate synaptic transmission and may couple multiple neurons and synapses into functional assemblies. Microglia can remove synapses in an activity-dependent manner altering neural networks. Incorporating glia into a bicellular mechanism of nervous system function may help answer long-standing questions concerning the cellular mechanisms of learning and cognition. PMID:24122821

  4. Ultraviolet irradiation induces the accumulation of chondroitin sulfate, but not other glycosaminoglycans, in human skin.

    PubMed

    Werth, Benjamin Boegel; Bashir, Muhammad; Chang, Laura; Werth, Victoria P

    2011-01-01

    Ultraviolet (UV) light alters cutaneous structure and function. Prior work has shown loss of dermal hyaluronan after UV-irradiation of human skin, yet UV exposure increases total glycosaminoglycan (GAG) content in mouse models. To more fully describe UV-induced alterations to cutaneous GAG content, we subjected human volunteers to intermediate-term (5 doses/week for 4 weeks) or single-dose UV exposure. Total dermal uronyl-containing GAGs increased substantially with each of these regimens. We found that UV exposure substantially increased dermal content of chondroitin sulfate (CS), but not hyaluronan, heparan sulfate, or dermatan sulfate. UV induced the accumulation of both the 4-sulfated (C4S) and 6-sulfated (C6S) isoforms of CS, but in distinct distributions. Next, we examined several CS proteoglycan core proteins and found a significant accumulation of dermal and endothelial serglycin, but not of decorin or versican, after UV exposure. To examine regulation in vitro, we found that UVB in combination with IL-1α, a cytokine upregulated by UV radiation, induced serglycin mRNA in cultured dermal fibroblasts, but did not induce the chondroitin sulfate synthases. Overall, our data indicate that intermediate-term and single-dose UVB exposure induces specific GAGs and proteoglycan core proteins in human skin in vivo. These molecules have important biologic functions and contribute to the cutaneous response to UV.

  5. Sulfated Glycosaminoglycans Control the Extracellular Trafficking and the Activity of the Metalloprotease Inhibitor TIMP-3

    PubMed Central

    Troeberg, Linda; Lazenbatt, Christopher; Anower-E-Khuda, Md. Ferdous; Freeman, Craig; Federov, Oleg; Habuchi, Hiroko; Habuchi, Osami; Kimata, Koji; Nagase, Hideaki

    2014-01-01

    Summary Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis. PMID:25176127

  6. GLIAL RESPONSES AFTER CHORDA TYMPANI NERVE INJURY

    PubMed Central

    Bartel, Dianna L.

    2013-01-01

    The chorda tympani (CT) nerve innervates lingual taste buds and is susceptible to damage during dental and inner ear procedures. Interruption of the CT results in a disappearance of taste buds, which can be accompanied by taste disturbances. Because the CT usually regenerates to reinnervate taste buds successfully in a few weeks, a persistence of taste disturbances may indicate alterations in central nervous function. Peripheral injury to other sensory nerves leads to glial responses at central terminals, which actively contribute to abnormal sensations arising from nerve damage. Therefore, the current study examined microglial and astrocytic responses in the first central gustatory relay -the nucleus of the solitary tract (nTS)- after transection of the CT. Damage to the CT resulted in significant microglial responses in terms of morphological reactivity and an increased density of microglial cells from 2-20 days after injury. This increased microglial population primarily resulted from microglial proliferation from 1.5-3 days, which was supplemented by microglial migration within sub-divisions of the nTS between days 2-3. Unlike other nerve injuries, CT injury did not result in recruitment of bone marrow-derived precursors. Astrocytes also reacted in the nTS with increased levels of GFAP by 3 days, although none showed evidence of cell division. GFAP levels remained increased at 30 days by which time microglial responses had resolved. These results show that nerve damage to the CT results in central glial responses, which may participate in long lasting taste alterations following CT lesion. PMID:22315167

  7. Culturing conditions determine neuronal and glial excitability.

    PubMed

    Stoppelkamp, Sandra; Riedel, Gernot; Platt, Bettina

    2010-12-15

    The cultivation of pure neuronal cultures is considered advantageous for the investigation of cell-type specific responses (such as transmitter release and also pharmacological agents), however, divergent results are a likely consequence of media modifications and culture composition. Using Fura-2 based imaging techniques, we here set out to compare calcium responses of rat hippocampal neurones and glia to excitatory stimulation with l-glutamate in different culture types and media. Neurones in neurone-enriched cultures had increased responses to 10 μM and 100 μM l-glutamate (+43 and 45%, respectively; p's< 0.001) and a slower recovery compared to mixed cultures, indicating heightened excitability. In matured (15-20 days in vitro) mixed cultures, neuronal responder rates were suppressed in a neurone-supportive medium (Neurobasal-A, NB: 65%) compared to a general-purpose medium (supplemented minimal essential medium, MEM: 96%). Glial response size in contrast did not differ greatly in isolated or mixed cultures maintained in MEM, but responder rates were suppressed in both culture types in NB (e.g. 10 μM l-glutamate responders in mixed cultures: 29% in NB, 71% in MEM). This indicates that medium composition is more important for glial excitability than the presence of neurones, whereas the presence of glia has an important impact on neuronal excitability. Therefore, careful consideration of culturing conditions is crucial for interpretation and comparison of experimental results. Especially for investigations of toxicity and neuroprotection mixed cultures may be more physiologically relevant over isolated cultures as they comprise aspects of mutual influences between glia and neurones.

  8. [Death of neurons and glial cells, induced by a photodynamic injury: signaling processes and neurone-glial interactions].

    PubMed

    Uzdenskiĭ, A B; Kolosov, M S; Lobanov, A V

    2007-01-01

    The mechanisms of photodynamic (PD) injury of neurons and glial cells are reviewed. Neuron responses: firing stimulation at high photosensitizer concentrations and inhibition at low concentrations (< 10(-7) M) that were followed by necrosis, are described. Glial cells died from both necrosis and apoptosis. Local laser inactivation of a neuron enhanced PD-induced apoptosis of glial cells, thus indicating that neuron maintained the survival of glia. Inter- and intracellular signaling mediated photodamage of these cells. Using inhibitors or activators of signaling proteins, the involvement of Ca(2+)-, adenylate cyclase- and tyrosine kinase-mediated signaling pathways in responses of neurons and glial cells to photosensitization was shown. Their pharmacological modulation can change selectivity of PD injury of neuronal and glial cells and efficiency of PD therapy.

  9. Inhibition of cultured bovine aortic endothelial cell proliferation by sodium spirulan, a new sulfated polysaccharide isolated from Spirulina platensis.

    PubMed

    Kaji, Toshiyuki; Fujiwara, Yasuyuki; Hamada, Chieko; Yamamoto, Chika; Shimada, Satomi; Lee, Jung-Bum; Hayashi, Toshimitsu

    2002-06-01

    Sodium spirulan (Na-SP) is a sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, which consists of two types of disaccharide repeating units, O-hexuronosyl-rhamnose (aldobiuronic acid) and O-rhamnosyl-3-O-methylrhamnose (acofriose) with sulfate groups, other minor saccharides and sodium ion. Vascular endothelial cells are present on the inner surface of blood vessels in a monolayer and have anticoagulant properties. To address the question whether Na-SP influences the maintenance of endothelial cell monolayers, we investigated the proliferation of cultured bovine aortic endothelial cells treated with Na-SP. It was found that Na-SP has an inhibitory activity on endothelial cell proliferation accompanied with suppression of whole protein synthesis but without non-specific cell damage. The inhibitory activity of Na-SP was the strongest when compared to that of heparan sulfate, heparin, dextran sulfate, dermatan sulfate, chondroitin sulfate A/C and hyaluronan. Furthermore, it was shown that the inhibitory activity of Na-SP disappeared by either desulfation or depolymerization. The present data suggest that Na-SP is a unique sulfated polysaccharide that strongly inhibits vascular endothelial cell proliferation, and the inhibitory activity requires polymerization of sulfated O-rhamnosyl-acofriose repeating units.

  10. Sulfate in fetal development.

    PubMed

    Dawson, Paul A

    2011-08-01

    Sulfate (SO(4)(2-)) is an important nutrient for human growth and development, and is obtained from the diet and the intra-cellular metabolism of sulfur-containing amino acids, including methionine and cysteine. During pregnancy, fetal tissues have a limited capacity to produce sulfate, and rely on sulfate obtained from the maternal circulation. Sulfate enters and exits placental and fetal cells via transporters on the plasma membrane, which maintain a sufficient intracellular supply of sulfate and its universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) for sulfate conjugation (sulfonation) reactions to function effectively. Sulfotransferases mediate sulfonation of numerous endogenous compounds, including proteins and steroids, which biotransforms their biological activities. In addition, sulfonation of proteoglycans is important for maintaining normal structure and development of tissues, as shown for reduced sulfonation of cartilage proteoglycans that leads to developmental dwarfism disorders and four different osteochondrodysplasias (diastrophic dysplasia, atelosteogenesis type II, achondrogenesis type IB and multiple epiphyseal dysplasia). The removal of sulfate via sulfatases is an important step in proteoglycan degradation, and defects in several sulfatases are linked to perturbed fetal bone development, including mesomelia-synostoses syndrome and chondrodysplasia punctata 1. In recent years, interest in sulfate and its role in developmental biology has expanded following the characterisation of sulfate transporters, sulfotransferases and sulfatases and their involvement in fetal growth. This review will focus on the physiological roles of sulfate in fetal development, with links to human and animal pathophysiologies.

  11. Heparan Sulfate-Binding Foot-and-Mouth Disease Virus Enters Cells Via Caveolae-Mediated Endocytosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV) utilizes different cell surface macromolecules to facilitate infection of cultured cells. Virus which is virulent for susceptible animals infects cells via four members of the alpha V subclass of cellular integrins. In contrast, tissue culture adaptation of some...

  12. Independent multimerization of Latent TGFβ Binding Protein-1 stabilized by cross-linking and enhanced by heparan sulfate

    PubMed Central

    Troilo, Helen; Steer, Ruth; Collins, Richard F.; Kielty, Cay M.; Baldock, Clair

    2016-01-01

    TGFβ plays key roles in fibrosis and cancer progression, and latency is conferred by covalent linkage to latent TGFβ binding proteins (LTBPs). LTBP1 is essential for TGFβ folding, secretion, matrix localization and activation but little is known about its structure due to its inherent size and flexibility. Here we show that LTBP1 adopts an extended conformation with stable matrix-binding N-terminus, extended central array of 11 calcium-binding EGF domains and flexible TGFβ-binding C-terminus. Moreover we demonstrate that LTBP1 forms short filament-like structures independent of other matrix components. The termini bind to each other to facilitate linear extension of the filament, while the N-terminal region can serve as a branch-point. Multimerization is enhanced in the presence of heparin and stabilized by the matrix cross-linking enzyme transglutaminase-2. These assemblies will extend the span of LTBP1 to potentially allow simultaneous N-terminal matrix and C-terminal fibrillin interactions providing tethering for TGFβ activation by mechanical force. PMID:27677855

  13. Syndecan-3: a cell-surface heparan sulfate proteoglycan important for chondrocyte proliferation and function during limb skeletogenesis.

    PubMed

    Pacifici, Maurizio; Shimo, Tsuyoshi; Gentili, Chiara; Kirsch, Thorsten; Freeman, Theresa A; Enomoto-Iwamoto, Motomi; Iwamoto, Masahiro; Koyama, Eiki

    2005-01-01

    Syndecans are single-pass integral membrane components that serve as co-receptors for growth factors and cytokines and can elicit signal transduction via their cytoplasmic tails. We review here previous studies from our groups on syndecan-3 biology and function in the growth plates of developing long bones in chick and mouse embryos. Gain- and loss-of-function data indicate that syndecan-3 has important roles in restricting mitotic activity to the proliferative zone of growth plate and may do so in close cooperation and interaction with the signaling molecule Indian hedgehog (IHH). Biochemical and protein-modeling data suggest a dimeric/oligomeric syndecan-3 configuration on the chondrocyte's cell surface. Analyses of embryos misexpressing syndecan-3 or lacking IHH provide further clues on syndecan-3/IHH interdependence and interrelationships. The data and the conclusions reached provide insights into mechanisms fine-tuning chondrocyte proliferation, maturation, and function in the developing and growing skeleton and into how abnormalities in these fundamental mechanisms may subtend human congenital pathologies, including osteochondromas in hereditary multiple exostoses syndrome.

  14. RGTA OTR4120, a heparan sulfate mimetic, is a possible long-term active agent to heal burned skin.

    PubMed

    Garcia-Filipe, S; Barbier-Chassefiere, V; Alexakis, C; Huet, E; Ledoux, D; Kerros, M E; Petit, E; Barritault, D; Caruelle, J P; Kern, P

    2007-01-01

    Burn-related skin fibrosis leads to loss of tissue function and hypertrophic scar formation with damaging consequences for the patient. There is therefore a great need for an efficient agent to treat burned skin. We report that ReGeneraTing Agent (RGTA) reduces burn-induced skin alteration. The tissue-regenerating effect of RGTA OTR4120 was evaluated after 1-6 days and after 10 months in a rat skin burn model. This effect was also examined in vitro using fibroblasts isolated from control and 6-day-old burned skins. We measured production of dermal collagen I, III, and V and activities of metalloproteinases 2 and 9 (MMP-2 and MMP-9). Ratio of collagen III over collagen I production increased 6 days after the burn, because of a decrease in collagen I production. After 10 months, ratio of collagen III over collagen I in burn sites was still increased compared with control skin, because of an increase in collagen III production. Both abnormalities were corrected by OTR4120. OTR4120 increased pro- and active MMP-2 and MMP-9, compared with healthy and burned controls and therefore accelerated remodeling. Similar data were obtained with cultured fibroblasts from healthy and burned skins. OTR4120 enhanced healing in short- and long-term after burns, reducing the formation of fibrotic tissue, and then represents a potential agent to improve burned skin healing.

  15. Sulfation pathways in plants.

    PubMed

    Koprivova, Anna; Kopriva, Stanislav

    2016-11-25

    Plants take up sulfur in the form of sulfate. Sulfate is activated to adenosine 5'-phosphosulfate (APS) and reduced to sulfite and then to sulfide when it is assimilated into amino acid cysteine. Alternatively, APS is phosphorylated to 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and sulfate from PAPS is transferred onto diverse metabolites in its oxidized form. Traditionally, these pathways are referred to as primary and secondary sulfate metabolism, respectively. However, the synthesis of PAPS is essential for plants and even its reduced provision leads to dwarfism. Here the current knowledge of enzymes involved in sulfation pathways of plants will be summarized, the similarities and differences between different kingdoms will be highlighted, and major open questions in the research of plant sulfation will be formulated.

  16. Isolation and partial characterization of heparan sulphate proteoglycans from human hepatic amyloid.

    PubMed Central

    Magnus, J H; Stenstad, T; Husby, G; Kolset, S O

    1992-01-01

    Proteoglycans were isolated from human amyloidotic liver by extraction with guanidine, followed by trichloroacetic acid precipitation, DEAE-Sephacel ion-exchange chromatography, and Sepharose CL-6B gel chromatography. A significant portion of the material was found to be free chondroitin/dermatan sulphate chains (30%), whereas the predominant part was heparan sulphate proteoglycan (HSPG) (70%). The approx. molecular mass of the HSPG was 200 kDa, as measured by gel electrophoresis and gel chromatography. The molecular mass of the core protein was shown to be 60 kDa by SDS/PAGE following de-aminative cleavage of the heparan sulphate chains. The heparan sulphate chains were liberated from the core protein by alkali treatment and found to have a molecular mass of approx. 35 kDa by Sepharose CL-6B gel chromatography. The core protein was shown, by immunoblotting, to react with a monoclonal antibody against bovine basement membrane HSPG. The presence of HSPG in amyloid deposits was further confirmed by immunohistochemistry on tissue sections from amyloidotic liver using the same antibody. Images Fig. 5 Fig. 6 PMID:1445267

  17. Chondroitin Sulfate Is Indispensable for Pluripotency and Differentiation of Mouse Embryonic Stem Cells

    PubMed Central

    Izumikawa, Tomomi; Sato, Ban; Kitagawa, Hiroshi

    2014-01-01

    Chondroitin sulfate (CS) proteoglycans are present on the surfaces of virtually all cells and in the extracellular matrix and are required for cytokinesis at early developmental stages. Studies have shown that heparan sulfate (HS) is essential for maintaining mouse embryonic stem cells (ESCs) that are primed for differentiation, whereas the function of CS has not yet been elucidated. To clarify the role of CS, we generated glucuronyltransferase-I-knockout ESCs lacking CS. We found that CS was required to maintain the pluripotency of ESCs and promoted initial ESC commitment to differentiation compared with HS. In addition, CS-A and CS-E polysaccharides, but not CS-C polysaccharides, bound to E-cadherin and enhanced ESC differentiation. Multiple-lineage differentiation was inhibited in chondroitinase ABC-digested wild-type ESCs. Collectively, these results suggest that CS is a novel determinant in controlling the functional integrity of ESCs via binding to E-cadherin. PMID:24424429

  18. Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction.

    PubMed

    Hoegg-Beiler, Maja B; Sirisi, Sònia; Orozco, Ian J; Ferrer, Isidre; Hohensee, Svea; Auberson, Muriel; Gödde, Kathrin; Vilches, Clara; de Heredia, Miguel López; Nunes, Virginia; Estévez, Raúl; Jentsch, Thomas J

    2014-03-19

    Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

  19. Globular glial tauopathies (GGT): consensus recommendations.

    PubMed

    Ahmed, Zeshan; Bigio, Eileen H; Budka, Herbert; Dickson, Dennis W; Ferrer, Isidro; Ghetti, Bernardino; Giaccone, Giorgio; Hatanpaa, Kimmo J; Holton, Janice L; Josephs, Keith A; Powers, James; Spina, Salvatore; Takahashi, Hitoshi; White, Charles L; Revesz, Tamas; Kovacs, Gabor G

    2013-10-01

    Recent studies have highlighted a group of 4-repeat (4R) tauopathies that are characterised neuropathologically by widespread, globular glial inclusions (GGIs). Tau immunohistochemistry reveals 4R immunoreactive globular oligodendroglial and astrocytic inclusions and the latter are predominantly negative for Gallyas silver staining. These cases are associated with a range of clinical presentations, which correlate with the severity and distribution of underlying tau pathology and neurodegeneration. Their heterogeneous clinicopathological features combined with their rarity and under-recognition have led to cases characterised by GGIs being described in the literature using various and redundant terminologies. In this report, a group of neuropathologists form a consensus on the terminology and classification of cases with GGIs. After studying microscopic images from previously reported cases with suspected GGIs (n = 22), this panel of neuropathologists with extensive experience in the diagnosis of neurodegenerative diseases and a documented record of previous experience with at least one case with GGIs, agreed that (1) GGIs were present in all the cases reviewed; (2) the morphology of globular astrocytic inclusions was different to tufted astrocytes and finally that (3) the cases represented a number of different neuropathological subtypes. They also agreed that the different morphological subtypes are likely to be part of a spectrum of a distinct disease entity, for which they recommend that the overarching term globular glial tauopathy (GGT) should be used. Type I cases typically present with frontotemporal dementia, which correlates with the fronto-temporal distribution of pathology. Type II cases are characterised by pyramidal features reflecting motor cortex involvement and corticospinal tract degeneration. Type III cases can present with a combination of frontotemporal dementia and motor neuron disease with fronto-temporal cortex, motor cortex and

  20. Globular glial tauopathies (GGT): consensus recommendations

    PubMed Central

    Bigio, Eileen H.; Budka, Herbert; Dickson, Dennis W.; Ferrer, Isidro; Ghetti, Bernardino; Giaccone, Giorgio; Hatanpaa, Kimmo J.; Holton, Janice L.; Josephs, Keith A.; Powers, James; Spina, Salvatore; Takahashi, Hitoshi; White, Charles L.; Revesz, Tamas

    2014-01-01

    Rrecent studies have highlighted a group of 4-repeat (4R) tauopathies that are characterised neuropathologically by widespread, globular glial inclusions (GGIs). Tau immunohistochemistry reveals 4R immunore-active globular oligodendroglial and astrocytic inclusions and the latter are predominantly negative for Gallyas silver staining. These cases are associated with a range of clinical presentations, which correlate with the severity and distribution of underlying tau pathology and neurodegeneration. Their heterogeneous clinicopathological features combined with their rarity and under-recognition have led to cases characterised by GGIs being described in the literature using various and redundant terminologies. In this report, a group of neuropathologists form a consensus on the terminology and classification of cases with GGIs. After studying microscopic images from previously reported cases with suspected GGIs (n = 22), this panel of neuropathologists with extensive experience in the diagnosis of neurodegenerative diseases and a documented record of previous experience with at least one case with GGIs, agreed that (1) GGIs were present in all the cases reviewed; (2) the morphology of globular astrocytic inclusions was different to tufted astrocytes and finally that (3) the cases represented a number of different neuropathological subtypes. They also agreed that the different morphological subtypes are likely to be part of a spectrum of a distinct disease entity, for which they recommend that the overarching term globular glial tauopathy (GGT) should be used. Type I cases typically present with frontotemporal dementia, which correlates with the fronto-temporal distribution of pathology. Type II cases are characterised by pyramidal features reflecting motor cortex involvement and corticospinal tract degeneration. Type III cases can present with a combination of frontotemporal dementia and motor neuron disease with fronto-temporal cortex, motor cortex and

  1. Extracellular environment contribution to astrogliosis—lessons learned from a tissue engineered 3D model of the glial scar

    PubMed Central

    Rocha, Daniela N.; Ferraz-Nogueira, José P.; Barrias, Cristina C.; Relvas, João B.; Pêgo, Ana P.

    2015-01-01

    Glial scars are widely seen as a (bio)mechanical barrier to central nervous system regeneration. Due to the lack of a screening platform, which could allow in-vitro testing of several variables simultaneously, up to now no comprehensive study has addressed and clarified how different lesion microenvironment properties affect astrogliosis. Using astrocytes cultured in alginate gels and meningeal fibroblast conditioned medium, we have built a simple and reproducible 3D culture system of astrogliosis mimicking many features of the glial scar. Cells in this 3D culture model behave similarly to scar astrocytes, showing changes in gene expression (e.g., GFAP) and increased extra-cellular matrix production (chondroitin 4 sulfate and collagen), inhibiting neuronal outgrowth. This behavior being influenced by the hydrogel network properties. Astrocytic reactivity was found to be dependent on RhoA activity, and targeting RhoA using shRNA-mediated lentivirus reduced astrocytic reactivity. Further, we have shown that chemical inhibition of RhoA with ibuprofen or indirectly targeting RhoA by the induction of extracellular matrix composition modification with chondroitinase ABC, can diminish astrogliosis. Besides presenting the extracellular matrix as a key modulator of astrogliosis, this simple, controlled and reproducible 3D culture system constitutes a good scar-like system and offers great potential in future neurodegenerative mechanism studies, as well as in drug screenings envisaging the development of new therapeutic approaches to minimize the effects of the glial scar in the context of central nervous system disease. PMID:26483632

  2. Structural differences and the presence of unsubstituted amino groups in heparan sulphates from different tissues and species.

    PubMed Central

    Toida, T; Yoshida, H; Toyoda, H; Koshiishi, I; Imanari, T; Hileman, R E; Fromm, J R; Linhardt, R J

    1997-01-01

    This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural and compositional studies on intact heparan sulphate chains. After enzymic digestion of heparan sulphate using heparin lyase I (EC 4.2.2.7) II and III (EC 4.2.2.8), the compositions of unsaturated disaccharides obtained were determined by analytical capillary electrophoresis. Correlations between the N-sulphated glucosamine residues and O-sulphation and between iduronic acid content and total sulphation were discovered using the data obtained by NMR and disaccharide analysis. Heparan sulphate chains could be classified into two groups based on the sulphation degree and the iduronic acid content. Heparan sulphate chains with a high degree of sulphation possessed also a significant number of iduronic acid residues and were isolated exclusively from porcine brain, liver and kidney medulla. The presence and amount of N-unsubstituted glucosamine residues (GlcNp) was established in all of the heparan sulphates examined. The structural context in which this residue occurs was demonstrated to be: high sulphation domain --> 4)-beta-D-GlcAp-(1 --> 4)-alpha-D-GlcNp-(1 --> 4)-beta-D-GlcAp-(1 --> low sulphation domain (where GlcNp is 2-amino-2-deoxyglucopyranose, and GlcAp is glucopyranosyluronic acid), based on the isolation and characterization of a novel, heparin lyase III-derived, GlcNp containing tetrasaccharide and hexasaccharide. The results presented suggest that structural differences may play a role in important biological events controlled by heparan sulphate in different tissues. PMID:9065769

  3. Serum albumin induces osmotic swelling of rat retinal glial cells.

    PubMed

    Löffler, Silvana; Wurm, Antje; Kutzera, Franziska; Pannicke, Thomas; Krügel, Katja; Linnertz, Regina; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2010-03-04

    Edema in the ischemic neural tissue develops by increased vascular permeability associated with extravasation of albumin, and by glial swelling. Here, we show that bovine serum albumin acutely administered to slices of the rat retina causes swelling of glial somata under hypoosmotic conditions. The effect of albumin was dose-dependent, with half-maximal and maximal effects at 10 nM and 1 microM, respectively, and was mediated by activation of transforming growth factor-beta receptor type II, oxidative stress, and the production of arachidonic acid and prostaglandins. Albumin-induced glial swelling was prevented by glutamate and purinergic receptor agonists. The data suggest that serum albumin may induce glial swelling in the presence of osmotic gradients.

  4. Nitric oxide mediates glial-induced neurodegeneration in Alexander disease.

    PubMed

    Wang, Liqun; Hagemann, Tracy L; Kalwa, Hermann; Michel, Thomas; Messing, Albee; Feany, Mel B

    2015-11-26

    Glia play critical roles in maintaining the structure and function of the nervous system; however, the specific contribution that astroglia make to neurodegeneration in human disease states remains largely undefined. Here we use Alexander disease, a serious degenerative neurological disorder caused by astrocyte dysfunction, to identify glial-derived NO as a signalling molecule triggering astrocyte-mediated neuronal degeneration. We further find that NO acts through cGMP signalling in neurons to promote cell death. Glial cells themselves also degenerate, via the DNA damage response and p53. Our findings thus define a specific mechanism for glial-induced non-cell autonomous neuronal cell death, and identify a potential therapeutic target for reducing cellular toxicity in Alexander disease, and possibly other neurodegenerative disorders with glial dysfunction.

  5. Complex and differential glial responses in Alzheimer's disease and ageing.

    PubMed

    Rodríguez, José J; Butt, Arthur M; Gardenal, Emanuela; Parpura, Vladimir; Verkhratsky, Alexei

    2016-01-01

    Glial cells and their association with neurones are fundamental for brain function. The emergence of complex neurone-glial networks assures rapid information transfer, creating a sophisticated circuitry where both types of neural cells work in concert, serving different activities. All glial cells, represented by astrocytes, oligodendrocytes, microglia and NG2-glia, are essential for brain homeostasis and defence. Thus, glia are key not only for normal central nervous system (CNS) function, but also to its dysfunction, being directly associated with all forms of neuropathological processes. Therefore, the progression and outcome of neurological and neurodegenerative diseases depend on glial reactions. In this review, we provide a concise account of recent data obtained from both human material and animal models demonstrating the pathological involvement of glia in neurodegenerative processes, including Alzheimer's disease (AD), as well as physiological ageing.

  6. Glial cell biology in the Great Lakes region.

    PubMed

    Feinstein, Douglas L; Skoff, Robert P

    2016-03-31

    We report on the tenth bi-annual Great Lakes Glial meeting, held in Traverse City, Michigan, USA, September 27-29 2015. The GLG meeting is a small conference that focuses on current research in glial cell biology. The array of functions that glial cells (astrocytes, microglia, oligodendrocytes, Schwann cells) play in health and disease is constantly increasing. Despite this diversity, GLG meetings bring together scientists with common interests, leading to a better understanding of these cells. This year's meeting included two keynote speakers who presented talks on the regulation of CNS myelination and the consequences of stress on Schwann cell biology. Twenty-two other talks were presented along with two poster sessions. Sessions covered recent findings in the areas of microglial and astrocyte activation; age-dependent changes to glial cells, Schwann cell development and pathology, and the role of stem cells in glioma and neural regeneration.

  7. Glial cells in (patho)physiology

    PubMed Central

    Parpura, Vladimir; Heneka, Michael T.; Montana, Vedrana; Oliet, Stéphane H.R.; Schousboe, Arne; Haydon, Philip. G.; Stout, Randy F.; Spray, David C.; Reichenbach, Andreas; Pannicke, Thomas; Pekny, Milos; Pekna, Marcela; Zorec, Robert; Verkhratsky, Alexei

    2012-01-01

    Neuroglial cells define brain homeostasis and mount defense against pathological insults. Astroglia regulate neurogenesis and development of brain circuits. In the adult brain, astrocytes enter into intimate dynamic relationship with neurons, especially at synaptic sites where they functionally form the tripartite synapse. At these sites astrocytes regulate ion and neurotransmitter homeostasis, metabolically support neurons and monitor synaptic activity; one of the readouts of the latter manifests in astrocytic intracellular Ca2+ signals. This form of astrocytic excitability can lead to release of chemical transmitters via Ca2+-dependent exocytosis. Once in the extracellular space, gliotransmitters can modulate synaptic plasticity and cause changes in behavior. Besides these physiological tasks, astrocytes are fundamental for progression and outcome of neurological diseases. In Alzheimer’s disease, for example, astrocytes may contribute to the etiology of this disorder. Highly lethal glial-derived tumors use signaling trickery to coerce normal brain cells to assist tumor invasiveness. This review sheds new light on the brain operation in health and disease, but also points to many unknowns. PMID:22251135

  8. Corticobasal syndrome with novel argyrophilic glial inclusions.

    PubMed

    Rippon, Gregory A; Staugaitis, S M; Chin, Steven S M; Goldman, James E; Marder, K

    2005-05-01

    A 42-year-old, left-handed woman first noted impaired dexterity of the dominant hand, soon followed by dysarthria and cognitive decline. Over a 4-year period, she developed severe left-sided apraxia with eventual neglect of the left arm and progressive extrapyramidal signs. Cognitive testing showed progressive executive, visuospatial, fluency, and naming impairment with relative preservation of memory. Single-photon emission computed tomography demonstrated asymmetric right posterior frontal and superior parietal hypoperfusion. The clinical impression was corticobasal degeneration. At autopsy, severe atrophy was seen in the perirolandic and frontal regions. There was marked neuronal loss and gliosis in the posterior frontal and precentral regions and less severe pathology in prefrontal, temporal, and parietal areas. Mild to moderate gliosis and neuronal loss were also seen in the putamen, globus pallidus, subthalamic, and dentate nuclei. Gallyas silver stain revealed numerous inclusions adjacent to oligodendrocyte nuclei in white and gray matter of affected cortical and subcortical regions. The gracile inclusions were wavy, slender, and stained positively with antibodies to ubiquitin and alphaB-crystallin but not to microtubule-associated proteins (tau, MAP1B, MAP2), tubulin, neurofilaments, glial fibrillary acidic protein, or alpha-synuclein. The argyrophilic inclusions identified in this case are distinct from those previously described in neurodegenerative diseases.

  9. Modeling cognition and disease using human glial chimeric mice.

    PubMed

    Goldman, Steven A; Nedergaard, Maiken; Windrem, Martha S

    2015-08-01

    As new methods for producing and isolating human glial progenitor cells (hGPCs) have been developed, the disorders of myelin have become especially compelling targets for cell-based therapy. Yet as animal modeling of glial progenitor cell-based therapies has progressed, it has become clear that transplanted hGPCs not only engraft and expand within murine hosts, but dynamically outcompete the resident progenitors so as to ultimately dominate the host brain. The engrafted human progenitor cells proceed to generate parenchymal astrocytes, and when faced with a hypomyelinated environment, oligodendrocytes as well. As a result, the recipient brains may become inexorably humanized with regards to their resident glial populations, yielding human glial chimeric mouse brains. These brains provide us a fundamentally new tool by which to assess the species-specific attributes of glia in modulating human cognition and information processing. In addition, the cellular humanization of these brains permits their use in studying glial infectious and inflammatory disorders unique to humans, and the effects of those disorders on the glial contributions to cognition. Perhaps most intriguingly, by pairing our ability to construct human glial chimeras with the production of patient-specific hGPCs derived from pluripotential stem cells, we may now establish mice in which a substantial proportion of resident glia are both human and disease-derived. These mice in particular may provide us new opportunities for studying the human-specific contributions of glia to psychopathology, as well as to higher cognition. As such, the assessment of human glial chimeric mice may provide us new insight into the species-specific contributions of glia to human cognitive evolution, as well as to the pathogenesis of human neurological and neuropsychiatric disease.

  10. Glial hemichannels and their involvement in aging and neurodegenerative diseases.

    PubMed

    Orellana, Juan A; von Bernhardi, Rommy; Giaume, Christian; Sáez, Juan C

    2012-01-26

    During the last two decades, it became increasingly evident that glial cells accomplish a more important role in brain function than previously thought. Glial cells express pannexins and connexins, which are member subunits of two protein families that form membrane channels termed hemichannels. These channels communicate intra- and extracellular compartments and allow the release of autocrine/paracrine signaling molecules [e.g., adenosine triphosphate (ATP), glutamate, nicotinamide adenine dinucleotide, and prostaglandin E2] to the extracellular milieu, as well as the uptake of small molecules (e.g., glucose). An increasing body of evidence has situated glial hemichannels as potential regulators of the beginning and maintenance of homeostatic imbalances observed in diverse brain diseases. Here, we review and discuss the current evidence about the possible role of glial hemichannels on neurodegenerative diseases. A subthreshold pathological threatening condition leads to microglial activation, which keeps active defense and restores the normal function of the central nervous system. However, if the stimulus is deleterious, microglial cells and the endothelium become overactivated, both releasing bioactive molecules (e.g., glutamate, cytokines, prostaglandins, and ATP), which increase the activity of glial hemichannels, reducing the astroglial neuroprotective functions, and further reducing neuronal viability. Because ATP and glutamate are released via glial hemichannels in neurodegenerative conditions, it is expected that they contribute to neurotoxicity. More importantly, toxic molecules released via glial hemichannels could increase the Ca2+ entry in neurons also via neuronal hemichannels, leading to neuronal death. Therefore, blockade of hemichannels expressed by glial cells and/or neurons during neuroinflammation might prevent neurodegeneration.

  11. A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation

    PubMed Central

    Frederick, Joshua P.; Tafari, A. Tsahai; Wu, Sheue-Mei; Megosh, Louis C.; Chiou, Shean-Tai; Irving, Ryan P.; York, John D.

    2008-01-01

    Sulfation is an important biological process that modulates the function of numerous molecules. It is directly mediated by cytosolic and Golgi sulfotransferases, which use 3′-phosphoadenosine 5′-phosphosulfate to produce sulfated acceptors and 3′-phosphoadenosine 5′-phosphate (PAP). Here, we identify a Golgi-resident PAP 3′-phosphatase (gPAPP) and demonstrate that its activity is potently inhibited by lithium in vitro. The inactivation of gPAPP in mice led to neonatal lethality, lung abnormalities resembling atelectasis, and dwarfism characterized by aberrant cartilage morphology. The phenotypic similarities of gPAPP mutant mice to chondrodysplastic models harboring mutations within components of the sulfation pathway lead to the discovery of undersulfated chondroitin in the absence of functional enzyme. Additionally, we observed loss of gPAPP leads to perturbations in the levels of heparan sulfate species in lung tissue and whole embryos. Our data are consistent with a model that clearance of the nucleotide product of sulfotransferases within the Golgi plays an important role in glycosaminoglycan sulfation, provide a unique genetic basis for chondrodysplasia, and define a function for gPAPP in the formation of skeletal elements derived through endochondral ossification. PMID:18695242

  12. De novo cellular synthesis of sulfated proteoglycans of the developing renal glomerulus in vivo.

    PubMed Central

    Kanwar, Y S; Jakubowski, M L; Rosenzweig, L J; Gibbons, J T

    1984-01-01

    The site of cellular synthesis of glomerular proteoglycans was investigated in developing glomeruli of 4- to 5-day-old rats. [35S]Sulfate was administered intravenously and animals were sacrificed 15 min to 12 hr later. The outermost layers of the kidney cortices were utilized for characterization of proteoglycans and electron microscopic autoradiography. Sepharose CL-6B chromatography and cellulose acetate electrophoresis revealed that most (approximately equal to 96%) of the radioactivity was associated with heparan sulfate-proteoglycan synthesized during maturation of glomerular capillaries. Tissue autoradiography revealed the following: (i) during the S-shaped body stage, there is rapid incorporation of [35S]sulfate by mesenchymal cells into the cleft region (site for development of future glomerular extracellular matrices); (ii) during the precapillary stage, mesenchyme-derived cells showed higher incorporation of radioisotope than did epithelial cells; and (iii) during the mature capillary stage, all glomerular cell types (mesangial, endothelial, and epithelial) incorporated [35S]sulfate, incorporation by mesangial cells being the greatest. Radiolabeling was also higher in the mesangial matrix than in the glomerular basement membrane of peripheral capillary loops. Synthesis of a single major species of sulfated glycosaminoglycan by cells of different embryologic origin may be unique to glomerular capillaries. Images PMID:6239287

  13. Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

    PubMed

    Takechi-Haraya, Yuki; Nadai, Ryo; Kimura, Hitoshi; Nishitsuji, Kazuchika; Uchimura, Kenji; Sakai-Kato, Kumiko; Kawakami, Kohsaku; Shigenaga, Akira; Kawakami, Toru; Otaka, Akira; Hojo, Hironobu; Sakashita, Naomi; Saito, Hiroyuki

    2016-06-01

    The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

  14. Localization and characterization of sulfated glycosaminoglycans in the body of the earthworm Eisenia andrei (Oligochaeta, Annelida).

    PubMed

    Amaral, Hanna B F; Mateus, Samuel H; Ferreira, Laina C; Ribeiro, Cristiane C; Palumbo-Junior, Antonio; Domingos, Maria-Aparecida O; Cinelli, Leonardo P; Costa-Filho, Adilson; Nasciutti, Luiz E; Silva, Luiz-Claudio F

    2011-07-01

    The aim of this study was to characterize the compartmental distribution of sulfated glycosaminoglycans (S-GAGs) in adults and their occurrence during the development of the earthworm Eisenia andrei. S-GAGs were extracted from the body of earthworms to identify their composition and the time of their appearance and disappearance in embryonic, newborn, juvenile, and adult earthworms. S-GAGs were also analyzed in earthworm tissue using histochemical metachromatic staining. Purified S-GAGs obtained from the whole body of adult earthworms were composed of chondroitin sulfate (CS) and heparan sulfate (HS). In addition, an unknown, highly sulfated polysaccharide (HSP) was detected. In order to characterize specifically the S-GAG composition in the integument, earthworms were dissected and as much as possible of their viscera was removed. HS and CS were the predominant sulfated polysaccharides in the dissected integument, whereas in viscera, CS, HS and the HSP were found in proportions similar to those identified in the body. The qualitative S-GAG composition in juveniles was similar to that obtained from adult earthworms. CS was the predominant S-GAG in newborn earthworms, accompanied by lesser amounts of HS and by tiny amounts of the HSP. This study provides a detailed descriptive account of the pattern of S-GAG synthesis during development, and also the characterization of the tissue distribution of these compounds in the body of earthworms.

  15. Asymptomatic and symptomatic glial cysts of the pineal gland.

    PubMed

    Taraszewska, Anna; Matyja, Ewa; Koszewski, Waldemar; Zaczyński, Artur; Bardadin, Krzysztof; Czernicki, Zbigniew

    2008-01-01

    Glial cysts of the pineal gland are benign and mostly asymptomatic incidental lesions found in the brain MRI or at autopsy examinations. In rare cases pineal cysts become symptomatic and require surgical intervention. Symptomatic glial cysts may be clinically and radiologically indistinguishable from cystic neoplasms of the pineal region; therefore, histopathological diagnosis is critical for further prognosis and therapy in operated patients. In this paper we present detailed histopathological characteristics of symptomatic glial cysts in 2 surgical cases and of asymptomatic cysts of the pineal gland found at random in 3 autopsy cases. Both surgical patients, a 19-year-old girl and a 17-year-old boy, presented with severe headaches, associated with syncope in one case and insomnia in the second one. Preoperative MR imaging suggested tumour of the pineal gland in case no. 2. Histopathological and immunohistochemical examination of the specimens from both surgical and all autopsy cases revealed a characteristic pattern of cystic structures within the pineal gland, surrounded by layers of a dense fibrillar glial tissue and pineal parenchyma, consistent with non-neoplastic glial cysts. Although histopathological findings in asymptomatic and symptomatic cysts are essentially the same, the cyst in surgical case 1 was unilocular and partly lined with ependymal cells, whereas the cysts in other cases were multilocular, comprising cavities of various size, formed in the central part of gliotic tissue or directly within the pineal parenchyma, and lacked ependymal lining. Possible pathophysiological and clinicopathological significance of some morphological variants of pineal glial cysts is discussed.

  16. Glial cell dysregulation: a new perspective on Alzheimer disease.

    PubMed

    von Bernhardi, Rommy

    2007-12-01

    Alzheimer disease (AD) is a major cause of dementia. Several mechanisms have been postulated to explain its pathogenesis, beta-amyloid (A beta toxicity, cholinergic dysfunction, Tau hyper-phosphorylation, oxidative damage, synaptic dysfunction and inflammation secondary to senile plaques, among others. Glial cells are the major producers of inflammatory mediators, and cytotoxic activation of glial cells is linked to several neurodegenerative diseases; however, whether inflammation is a consequence or the cause of neurodegeneration is still unclear. I propose that inflammation and cellular stress associated with aging are key events in the development of AD through the induction of glial dysfunction. Dysregulated inflammatory response can elicit glial cell activation by compounds which are normally poorly reactive. Inflammation can also be the major cause of defective handling of A beta and the amyloid precursor protein (APP). Here I review evidence that support the proposal that dysfunctional glia and the resulting neuroinflammation can explain many features of AD. Evidence supports the notion that damage caused by inflammation is not only a primary cause of neurodegeneration but also an inducer for the accumulation of A beta in AD. Dysfunctional glia can result in impaired neuronal function in AD, as well as in many progressive neurodegenerative disorders. We show that microglial cell activation is enhanced under pro-inflammatory conditions, indicating that glial cell responses to A beta related proteins can be critically dependent on the priming of glial cells by pro-inflammatory factors.

  17. Glycosaminoglycan in cerebrum, cerebellum and brainstem of young sheep brain with particular reference to compositional and structural variations of chondroitin-dermatan sulfate and hyaluronan.

    PubMed

    Kilia, Virginia; Skandalis, Spyros S; Theocharis, Achilleas D; Theocharis, Dimitrios A; Karamanos, Nikos K; Papageorgakopoulou, Nickoletta

    2008-09-01

    Recent advances in the structural biology of chondroitin sulfate chains have suggested important biological functions in the development of the brain. Several studies have demonstrated that the composition of chondroitin sulfate chains changes with aging and normal brain maturation. In this study, we determined the concentration of all glycosaminoglycan types, i.e. chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, hyaluronan and chondroitin in cerebrum, cerebellum and brainstem of young sheep brain. In all cases, chondroitin sulfate was the predominant glycosaminoglycan type, comprising about 54-58% of total glycosaminoglycans, with hyaluronan being present also in significant amounts of about 19-28%. Of particular interest was the increased presence of the disulfated disaccharides and dermatan sulfate in cerebellum and brainstem, respectively, as well as the detectable and measurable occurrence of chondroitin in young sheep brain. Among the three brain areas, cerebrum was found to be significantly richer in chondroitin sulfate and hyaluronan, two major extracellular matrix components. These findings imply that the extracellular matrix of the cerebrum is different from those of cerebellum and brainstem, and probably this fact is related to the particular histological and functional characteristics of each anatomic area of the brain.

  18. Analysis of crude heparin by (1)H NMR, capillary electrophoresis, and strong-anion-exchange-HPLC for contamination by over sulfated chondroitin sulfate.

    PubMed

    Keire, David A; Trehy, Michael L; Reepmeyer, John C; Kolinski, Richard E; Ye, Wei; Dunn, Jamie; Westenberger, Benjamin J; Buhse, Lucinda F

    2010-03-11

    We previously published a strong-anion-exchange-high performance liquid chromatography (SAX-HPLC) method for the detection of the contaminant over sulfated chondroitin sulfate (OSCS) in heparin sodium active pharmaceutical ingredient (API). While APIs have been processed to remove impurities, crude heparins contain insoluble material, chondroitin sulfates, heparan sulfate, and proteins that may interfere with the recovery and measurement of OSCS. We examined 500MHz (1)H NMR, capillary electrophoresis (CE), and SAX-HPLC to quantify OSCS in crude heparin. Using our standard API protocol on OSCS spiked crude heparin samples; we observed a weight percent LOD and LOQ for the NMR approach of 0.1% and 0.3%, respectively, while the SAX-HPLC method gave values of 0.03% and 0.09%, respectively. CE data was not amenable to quantitative measurement of OSCS in crude heparin. We developed a modified HPLC sample preparation protocol using crude dissolved at the 100mg/mL level with a 2.5M NaCl solution. This SAX-HPLC approach gave a weight percent LOD of 0.02% and a LOQ of 0.07% and had better performance characteristics than that of the protocol used for APIs.

  19. Modification of glial response in hibernation: a patch-clamp study on glial cells acutely isolated from hibernating land snail.

    PubMed

    Nikolic, Ljiljana; Bataveljic, Danijela; Andjus, Pavle R; Moldovan, Ivana; Nedeljkovic, Miodrag; Petkovic, Branka

    2014-12-01

    Hibernation is a dormant state of some animal species that enables them to survive harsh environmental conditions during the winter seasons. In the hibernating state, preservation of neuronal rhythmic activity at a low level is necessary for maintenance of suspended forms of behavior. As glial cells support rhythmic activity of neurons, preservation of brain function in the hibernating state implies accompanying modification of glial activity. A supportive role of glia in regulating neuronal activity is reflected through the activity of inwardly rectifying K+ channels (Kir). Therefore, we examined electrophysiological response, particularly Kir current response, of glial cells in mixture with neurons acutely isolated from active and hibernating land snail Helix pomatia. Our data show that hibernated glia have significantly lower inward current density, specific membrane conductance, and conductance density compared with active glia. The observed reduction could be attributed to the Kir currents, since the Ba2+-sensitive Kir current density was significantly lower in hibernated glia. Accordingly, a significant positive shift of the current reversal potential indicated a more depolarized state of hibernated glia. Data obtained show that modification of glial current response could be regulated by serotonin (5-HT) through an increase of cGMP as a secondary messenger, since extracellular addition of 5-HT or intracellular administration of cGMP to active glia induced a significant reduction of inward current density and thus mimicked the reduced response of hibernated glia. Lower Kir current density of hibernated glia accompanied the lower electrical activity of hibernated neurons, as revealed by a decrease in neuronal fast inward Na+ current density. Our findings reveal that glial response is reduced in the hibernating state and suggest seasonal modulation of glial activity. Maintenance of low glial activity in hibernation could be important for preservation of brain

  20. Distinctive glial and neuronal interfacing on nanocrystalline diamond.

    PubMed

    Bendali, Amel; Agnès, Charles; Meffert, Simone; Forster, Valérie; Bongrain, Alexandre; Arnault, Jean-Charles; Sahel, José-Alain; Offenhäusser, Andreas; Bergonzo, Philippe; Picaud, Serge

    2014-01-01

    Direct electrode/neuron interfacing is a key challenge to achieve high resolution of neuronal stimulation required for visual prostheses. Neuronal interfacing on biomaterials commonly requires the presence of glial cells and/or protein coating. Nanocrystalline diamond is a highly mechanically stable biomaterial with a remarkably large potential window for the electrical stimulation of tissues. Using adult retinal cell cultures from rats, we found that glial cells and retinal neurons grew equally well on glass and nanocrystalline diamond. The use of a protein coating increased cell survival, particularly for glial cells. However, bipolar neurons appeared to grow even in direct contact with bare diamond. We investigated whether the presence of glial cells contributed to this direct neuron/diamond interface, by using purified adult retinal ganglion cells to seed diamond and glass surfaces with and without protein coatings. Surprisingly, these fully differentiated spiking neurons survived better on nanocrystalline diamond without any protein coating. This greater survival was indicated by larger cell numbers and the presence of longer neurites. When a protein pattern was drawn on diamond, neurons did not grow preferentially on the coated area, by contrast to their behavior on a patterned glass. This study highlights the interesting biocompatibility properties of nanocrystalline diamond, allowing direct neuronal interfacing, whereas a protein coating was required for glial cell growth.

  1. Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders.

    PubMed

    Kaminsky, Natalie; Bihari, Ofer; Kanner, Sivan; Barzilai, Ari

    2016-06-01

    The DNA damage response (DDR) is a complex biological system activated by different types of DNA damage. Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration, premature aging, and various types of cancers. Intriguingly, malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases. For many years, brain degenerative disorders were thought to result from aberrant neural death. Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells (astrocytes, microglia, and oligodendrocytes). Impairment in the functionality of glial cells results in pathological neuro-glial interactions that, in turn, generate a "hostile" environment that impairs the functionality of neuronal cells. These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit.

  2. Glial dysfunction causes age-related memory impairment in Drosophila.

    PubMed

    Yamazaki, Daisuke; Horiuchi, Junjiro; Ueno, Kohei; Ueno, Taro; Saeki, Shinjiro; Matsuno, Motomi; Naganos, Shintaro; Miyashita, Tomoyuki; Hirano, Yukinori; Nishikawa, Hiroyuki; Taoka, Masato; Yamauchi, Yoshio; Isobe, Toshiaki; Honda, Yoshiko; Kodama, Tohru; Masuda, Tomoko; Saitoe, Minoru

    2014-11-19

    Several aging phenotypes, including age-related memory impairment (AMI), are thought to be caused by cumulative oxidative damage. In Drosophila, age-related impairments in 1 hr memory can be suppressed by reducing activity of protein kinase A (PKA). However, the mechanism for this effect has been unclear. Here we show that decreasing PKA suppresses AMI by reducing activity of pyruvate carboxylase (PC), a glial metabolic enzyme whose amounts increase upon aging. Increased PC activity causes AMI through a mechanism independent of oxidative damage. Instead, increased PC activity is associated with decreases in D-serine, a glia-derived neuromodulator that regulates NMDA receptor activity. D-serine feeding suppresses both AMI and memory impairment caused by glial overexpression of dPC, indicating that an oxidative stress-independent dysregulation of glial modulation of neuronal activity contributes to AMI in Drosophila.

  3. Abrogation of apoptosis through PDGF-BB-induced sulfated glycosaminoglycan synthesis and secretion.

    PubMed

    Cartel, Nicholas J; Post, Martin

    2005-02-01

    Platelet-derived growth factor (PDGF)-BB-stimulated glycosaminoglycan (GAG) synthesis/secretion in fetal lung fibroblasts is dependent on sequential activation of the PDGF beta-receptor, phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt-1,2, and the GTPase Rab3D. Because the Akt pathway has been implicated in cell survival mechanisms, we investigated whether the pathway regulating GAG synthesis/secretion was antiapoptotic. PDGF-BB treatment protected fetal lung fibroblasts against serum starvation-induced apoptosis, whereas wortmannin, an inhibitor of PI3K, abrogated this protective effect. Transfection of constitutively active Akt into fetal lung fibroblasts also safeguarded the cells from apoptosis induced by serum starvation. To determine whether the antiapoptotic response was due, at least in part, to GAGs, we treated lung fibroblasts with beta-D-xyloside as well as with topically applied GAGs, specifically those produced by fetal lung fibroblasts. beta-D-xyloside increased GAG synthesis/secretion and diminished apoptosis. Application of sulfated GAGs, chondroitin sulfate, and heparan sulfate, but not nonsulfated hyaluronan, also resulted in diminished apoptosis. Moreover, topically applied sulfated GAGs increased Bcl-associated death promoter phosphorylation and diminished caspase-3 and -7 cleavage, indicating an antiapototic response. These data are compatible with the PDGF-BB-GAG signaling pathway regulating programmed fibroblast death in the fetal lung.

  4. Uncovering the Relationship between Sulphation Patterns and Conformation of Iduronic Acid in Heparan Sulphate.

    PubMed

    Hsieh, Po-Hung; Thieker, David F; Guerrini, Marco; Woods, Robert J; Liu, Jian

    2016-07-14

    The L-iduronic acid (IdoA) residue is a critically important structural component in heparan sulphate polysaccharide for the biological functions. The pyranose ring of IdoA is present in (1)C4-chair, (2)SO-skew boat, and less frequently, in (4)C1-chair conformations. Here, we analyzed the conformation of IdoA residue in eight hexasaccharides by NMR. The data demonstrate a correlation between the conformation of IdoA and sulphations in the surrounding saccharide residues. For the 2-O-sulpho IdoA residue, a high degree of sulphation on neighboring residues drives ring dynamics towards the (2)SO-skew boat conformer. In contrast, the nonsulphated IdoA residue is pushed towards the (1)C4-chair conformer when the neighboring residues are highly sulphated. Our data suggest that the conformation of IdoA is regulated by the sulphation pattern of nearby saccharides that is genetically controlled by the heparan sulphate biosynthetic pathway.

  5. Uncovering the Relationship between Sulphation Patterns and Conformation of Iduronic Acid in Heparan Sulphate

    PubMed Central

    Hsieh, Po-Hung; Thieker, David F.; Guerrini, Marco; Woods, Robert J.; Liu, Jian

    2016-01-01

    The L-iduronic acid (IdoA) residue is a critically important structural component in heparan sulphate polysaccharide for the biological functions. The pyranose ring of IdoA is present in 1C4-chair, 2SO-skew boat, and less frequently, in 4C1-chair conformations. Here, we analyzed the conformation of IdoA residue in eight hexasaccharides by NMR. The data demonstrate a correlation between the conformation of IdoA and sulphations in the surrounding saccharide residues. For the 2-O-sulpho IdoA residue, a high degree of sulphation on neighboring residues drives ring dynamics towards the 2SO-skew boat conformer. In contrast, the nonsulphated IdoA residue is pushed towards the 1C4-chair conformer when the neighboring residues are highly sulphated. Our data suggest that the conformation of IdoA is regulated by the sulphation pattern of nearby saccharides that is genetically controlled by the heparan sulphate biosynthetic pathway. PMID:27412370

  6. Uncovering the Relationship between Sulphation Patterns and Conformation of Iduronic Acid in Heparan Sulphate

    NASA Astrophysics Data System (ADS)

    Hsieh, Po-Hung; Thieker, David F.; Guerrini, Marco; Woods, Robert J.; Liu, Jian

    2016-07-01

    The L-iduronic acid (IdoA) residue is a critically important structural component in heparan sulphate polysaccharide for the biological functions. The pyranose ring of IdoA is present in 1C4-chair, 2SO-skew boat, and less frequently, in 4C1-chair conformations. Here, we analyzed the conformation of IdoA residue in eight hexasaccharides by NMR. The data demonstrate a correlation between the conformation of IdoA and sulphations in the surrounding saccharide residues. For the 2-O-sulpho IdoA residue, a high degree of sulphation on neighboring residues drives ring dynamics towards the 2SO-skew boat conformer. In contrast, the nonsulphated IdoA residue is pushed towards the 1C4-chair conformer when the neighboring residues are highly sulphated. Our data suggest that the conformation of IdoA is regulated by the sulphation pattern of nearby saccharides that is genetically controlled by the heparan sulphate biosynthetic pathway.

  7. The accumulation of circulating histones on heparan sulphate in the capillary glycocalyx of the lungs.

    PubMed

    Freeman, Craig G; Parish, Christopher R; Knox, Karen J; Blackmore, Jessica L; Lobov, Sergei A; King, David W; Senden, Tim J; Stephens, Ross W

    2013-07-01

    Recent findings on the role of circulating histone proteins in mediating acute lung injury prompted us to investigate whether there is a specific mechanism for accumulation of histones in the lungs. Binding sites for polycations are already known in the vasculature of the lungs, and we postulated that these could also be involved in histone accumulation, since histones have a high content of positively charged amino acids. Using a histone-coated colloid of a radiolabelled nanocomposite to track histone biodistribution with imaging techniques, it was found that histones bind avidly in the lungs of rabbits after intravenous injection. Blocking experiments with competing polycations in vivo characterised histone lung binding as dependent on a charge interaction with microvessel polyanions. Pretreatment of rabbits with a specific heparinase confirmed that the lung binding sites consist of heparan sulphate in the endothelial glycocalyx. A range of heparan sulphate analogues was accordingly shown to prevent histone accumulation in the lungs by neutralising histones in blood. These findings provide a rational basis for the design of polyanions that can prevent accumulation of cytotoxic histones in the lungs and thereby intervene at an early key step in the development of acute lung injury.

  8. Human immunodeficiency virus can productively infect cultured human glial cells.

    PubMed

    Cheng-Mayer, C; Rutka, J T; Rosenblum, M L; McHugh, T; Stites, D P; Levy, J A

    1987-05-01

    Six isolates of the human immunodeficiency virus (HIV) showed differences in their ability to productively infect glioma-derived cell lines and early-passage human brain cell cultures. Susceptibility to HIV infection correlated well with the expression of the astrocyte marker glial fibrillary acidic protein. The CD4 molecule was expressed on some, but not all, of the brain-derived cells; however, no correlation was observed between CD4 protein expression and susceptibility to virus infection. The results show that HIV can productively infect human brain cells, particularly those of glial origin, and suggest that these cell types in the brain can harbor the virus.

  9. Sleep and immune function: glial contributions and consequences of aging.

    PubMed

    Ingiosi, Ashley M; Opp, Mark R; Krueger, James M

    2013-10-01

    The reciprocal interactions between sleep and immune function are well-studied. Insufficient sleep induces innate immune responses as evidenced by increased expression of pro-inflammatory mediators in the brain and periphery. Conversely, immune challenges upregulate immunomodulator expression, which alters central nervous system-mediated processes and behaviors, including sleep. Recent studies indicate that glial cells, namely microglia and astrocytes, are active contributors to sleep and immune system interactions. Evidence suggests glial regulation of these interactions is mediated, in part, by adenosine and adenosine 5'-triphosphate actions at purinergic type 1 and type 2 receptors. Furthermore, microglia and astrocytes may modulate declines in sleep-wake behavior and immunity observed in aging.

  10. Sulfate attack expansion mechanisms

    SciTech Connect

    Müllauer, Wolfram Beddoe, Robin E.; Heinz, Detlef

    2013-10-15

    A specially constructed stress cell was used to measure the stress generated in thin-walled Portland cement mortar cylinders caused by external sulfate attack. The effects of sulfate concentration of the storage solution and C{sub 3}A content of the cement were studied. Changes in mineralogical composition and pore size distribution were investigated by X-ray diffraction and mercury intrusion porosimetry, respectively. Damage is due to the formation of ettringite in small pores (10–50 nm) which generates stresses up to 8 MPa exceeding the tensile strength of the binder matrix. Higher sulfate concentrations and C{sub 3}A contents result in higher stresses. The results can be understood in terms of the effect of crystal surface energy and size on supersaturation and crystal growth pressure.

  11. Identification of an extended N-acetylated sequence adjacent to the protein-linkage region of fibroblast heparan sulphate.

    PubMed Central

    Lyon, M; Steward, W P; Hampson, I N; Gallagher, J T

    1987-01-01

    The distribution of N-sulphate groups within fibroblast heparan sulphate chains was investigated. The detergent-extractable heparan sulphate proteoglycan from adult human skin fibroblasts, radiolabelled with [3H]glucosamine and [35S]sulphate, was coupled to CNBr-activated Sepharose 4B. After partial depolymerization of the heparan sulphate with nitrous acid, the remaining Sepharose-bound fragments were removed by treatment with alkali. These fragments, of various sizes, but all containing an intact reducing xylose residue, were fractionated on Sephacryl S-300 and the distribution of the 3H and 35S radiolabels was analysed. A decreased degree of sulphation was observed towards the reducing termini of the chains. After complete nitrous acid hydrolysis of the Sepharose-bound proteoglycan, analysis of the proximity of N-sulphation to the reducing end revealed the existence of an extended N-acetylated sequence directly adjacent to the protein-linkage sequence. The size of this N-acetylated domain was estimated by gel filtration to be approximately eight disaccharide units. This domain appears to be highly conserved, being present in virtually all the chains derived from this proteoglycan, implying the existence of a mechanism capable of generating such a non-random sequence during the post-polymeric modification of heparan sulphate. Comparison with the corresponding situation in heparin suggests that different mechanisms regulate polymer N-sulphation in the vicinity of the protein-linkage region of these chemically related glycosaminoglycans. PMID:2954540

  12. Sulfated polysaccharides extracted from sea algae as potential antiviral drugs.

    PubMed

    Witvrouw, M; De Clercq, E

    1997-10-01

    also play an important role. Sulfated polysaccharides may act synergistically with other anti-HIV drugs (e.g., azidothymidine [AZT]). They are known to lead very slowly to virus-drug resistance development and they show activity against HIV mutants that have become resistant to reverse transcriptase inhibitors, such as AZT, tetrahydro-imidazo [4,5,l-jk] [1,4]-benzodiazepin-2(1H)-thione (TIBO) and others. From studies on their mechanism of action we concluded that polysulfates exert their anti-HIV activity by shielding off the positively charged sites in the V3 loop of the viral envelope glycoprotein (gp120). The V3 loop is necessary for virus attachment to cell surface heparan sulfate, a primary binding site, before more specific binding occurs to the CD4 receptor of CD4+ cells. This general mechanism also explains the broad antiviral activity of polysulfates against enveloped viruses. Variations in the viral envelope glycoprotein region may result in differences in the susceptibility of different enveloped viruses to compounds that interact with their envelope glycoproteins. The efficacy of polysulfates in the therapy and/or prophylaxis of retroviral infections and opportunistic infections remains to be demonstrated both in animal models and humans. It is important to consider not only treatment of patients who are already infected with HIV, but also prophylaxis and protection from HIV and/or other virus infections. Because (i) sexual transmission is responsible for the large majority of HIV infections worldwide; (ii) this transmission is mostly mediated via mononuclear cells that infect epithelial cells of the genital tract; and because (iii) polysulfates effectively inhibit cell-cell adhesion, polysulfates may be considered as potentially effective in a vaginal formulation to protect against HIV infection.

  13. Mechanisms of Aβ Clearance and Degradation by Glial Cells

    PubMed Central

    Ries, Miriam; Sastre, Magdalena

    2016-01-01

    Glial cells have a variety of functions in the brain, ranging from immune defense against external and endogenous hazardous stimuli, regulation of synaptic formation, calcium homeostasis, and metabolic support for neurons. Their dysregulation can contribute to the development of neurodegenerative disorders, including Alzheimer’s disease (AD). One of the most important functions of glial cells in AD is the regulation of Amyloid-β (Aβ) levels in the brain. Microglia and astrocytes have been reported to play a central role as moderators of Aβ clearance and degradation. The mechanisms of Aβ degradation by glial cells include the production of proteases, including neprilysin, the insulin degrading enzyme, and the endothelin-converting enzymes, able to hydrolyse Aβ at different cleavage sites. Besides these enzymes, other proteases have been described to have some role in Aβ elimination, such as plasminogen activators, angiotensin-converting enzyme, and matrix metalloproteinases. Other relevant mediators that are released by glial cells are extracellular chaperones, involved in the clearance of Aβ alone or in association with receptors/transporters that facilitate their exit to the blood circulation. These include apolipoproteins, α2macroglobulin, and α1-antichymotrypsin. Finally, astrocytes and microglia have an essential role in phagocytosing Aβ, in many cases via a number of receptors that are expressed on their surface. In this review, we examine all of these mechanisms, providing an update on the latest research in this field. PMID:27458370

  14. Methylphenidate Increases Glutamate Uptake in Bergmann Glial Cells.

    PubMed

    Guillem, Alain M; Martínez-Lozada, Zila; Hernández-Kelly, Luisa C; López-Bayghen, Esther; López-Bayghen, Bruno; Calleros, Oscar A; Campuzano, Marco R; Ortega, Arturo

    2015-11-01

    Glutamate, the main excitatory transmitter in the vertebrate brain, exerts its actions through the activation of specific membrane receptors present in neurons and glial cells. Over-stimulation of glutamate receptors results in neuronal death, phenomena known as excitotoxicity. A family of glutamate uptake systems, mainly expressed in glial cells, removes the amino acid from the synaptic cleft preventing an excessive glutamatergic stimulation and thus neuronal damage. Autism spectrum disorders comprise a group of syndromes characterized by impaired social interactions and anxiety. One or the most common drugs prescribed to treat these disorders is Methylphenidate, known to increase dopamine extracellular levels, although it is not clear if its sedative effects are related to a plausible regulation of the glutamatergic tone via the regulation of the glial glutamate uptake systems. To gain insight into this possibility, we used the well-established model system of cultured chick cerebellum Bergmann glia cells. A time and dose-dependent increase in the activity and protein levels of glutamate transporters was detected upon Methylphenidate exposure. Interestingly, this increase is the result of an augmentation of both the synthesis as well as the insertion of these protein complexes in the plasma membrane. These results favour the notion that glial cells are Methylphenidate targets, and that by these means could regulate dopamine turnover.

  15. Glial progenitor cell-based treatment of the childhood leukodystrophies

    PubMed Central

    Osorio, M. Joana; Goldman, Steven A.

    2017-01-01

    The childhood leukodystrophies comprise a group of hereditary disorders characterized by the absence, malformation or destruction of myelin. These disorders share common clinical, radiological and pathological features, despite their diverse molecular and genetic etiologies. Oligodendrocytes and astrocytes are the major affected cell populations, and are either structurally impaired or metabolically compromised through cell-intrinsic pathology, or are the victims of mis-accumulated toxic byproducts of metabolic derangement. In either case, glial cell replacement using implanted tissue or pluripotent stem cell-derived human neural or glial progenitor cells may comprise a promising strategy for both structural remyelination and metabolic rescue. A broad variety of pediatric white matter disorders, including the primary hypomyelinating disorders, the lysosomal storage disorders, and the broader group of non-lysosomal metabolic leukodystrophies, may all be appropriate candidates for glial progenitor cell-based treatment. Nonetheless, a variety of specific challenges remain before this therapeutic strategy can be applied to children. These include timely diagnosis, before irreparable neuronal injury has ensued; understanding the natural history of the targeted disease; defining the optimal cell phenotype for each disorder; achieving safe and scalable cellular compositions, designing age-appropriate controlled clinical trials; and for autologous therapy of genetic disorders, achieving the safe genetic editing of pluripotent stem cells. Yet these challenges notwithstanding, the promise of glial progenitor cell-based treatment of the childhood myelin disorders offers hope to the many victims of this otherwise largely untreatable class of disease. PMID:27170209

  16. Aluminum Sulfate 18 Hydrate

    ERIC Educational Resources Information Center

    Young, Jay A.

    2004-01-01

    A chemical laboratory information profile (CLIP) of the chemical, aluminum sulfate 18 hydrate, is presented. The profile lists physical and harmful properties, exposure limits, reactivity risks, and symptoms of major exposure for the benefit of teachers and students using the chemical in the laboratory.

  17. Hydrazine/Hydrazine sulfate

    Integrated Risk Information System (IRIS)

    Hydrazine / Hydrazine sulfate ; CASRN 302 - 01 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Non

  18. Effects of Cadmium on the Glial Architecture in Lizard Brain

    PubMed Central

    Favorito, Rossana; Monaco, Antonio; Grimaldi, Maria C.; Ferrandino, Ida

    2017-01-01

    The glial cells are positioned to be the first cells of the brain parenchyma to face molecules crossing the blood-brain barrier with a relevant neuroprotective role from cytotoxic action of heavy metals on the nervous system. Cadmium is a highly toxic metal and its levels in the environment are increasing due to industrial activities. This element can pass the blood-brain barrier and have neurotoxic activity. For this reason we have studied the effects of cadmium on the glial architecture in the lizard Podarcis siculus, a significant bioindicator of chemical exposure due to its persistence in a variety of habitats. The study was performed on two groups of lizards. The first group of P. siculus was exposed to an acute treatment by a single i.p. injection (2 mg/kg-BW) of CdCl2 and sacrificed after 2, 7 and 16 days. The second one was used as control. The histology of the brain was studied by Hematoxylin/Eosin and Cresyl/Violet stains while the glial structures were analyzed by immunodetection of the glial fibrillary acidic protein (GFAP), the most widely accepted marker for astroglial cells. Evident morphological alterations of the brain were observed at 7 and 16 days from the injection, when we revealed also a decrease of the GFAP-immunopositive structures in particular in the rhombencephalic ventricle, telencephalon and optic tectum. These results show that in the lizards an acute exposure to cadmium provokes morphological cellular alterations in the brain but also a decrement of the expression of GFAP marker with possible consequent damage of glial cells functions. PMID:28348417

  19. An unusual antithrombin-binding heparin octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence.

    PubMed

    Guerrini, Marco; Elli, Stefano; Mourier, Pierre; Rudd, Timothy R; Gaudesi, Davide; Casu, Benito; Boudier, Christian; Torri, Giangiacomo; Viskov, Christian

    2013-01-15

    The 3-O-sulfation of N-sulfated glucosamine is the last event in the biosynthesis of heparin/heparan sulfate, giving rise to the antithrombin-binding pentasaccharide sequence AGA*IA, which is largely associated with the antithrombotic activity of these molecules. The aim of the present study was the structural and biochemical characterization of a previously unreported AGA*IA*-containing octasaccharide isolated from the very-low-molecular-mass heparin semuloparin, in which both glucosamine residues of the pentasaccharide moiety located at the non-reducing end bear 3-O-sulfate groups. Two-dimensional and STD (saturation transfer difference) NMR experiments clearly confirmed its structure and identified its ligand epitope binding to antithrombin. The molecular conformation of the octasaccharide-antithrombin complex has been determined by NMR experiments and docking/energy minimization. The presence of the second 3-O-sulfated glucosamine in the octasaccharide induced more than one order of magnitude increase in affinity to antithrombin compared to the pentasaccharide AGA*IA.

  20. Development of a glial network in the olfactory nerve: role of calcium and neuronal activity.

    PubMed

    Koussa, Mounir A; Tolbert, Leslie P; Oland, Lynne A

    2010-11-01

    In adult olfactory nerves of mammals and moths, a network of glial cells ensheathes small bundles of olfactory receptor axons. In the developing antennal nerve (AN) of the moth Manduca sexta, the axons of olfactory receptor neurons (ORNs) migrate from the olfactory sensory epithelium toward the antennal lobe. Here we explore developmental interactions between ORN axons and AN glial cells. During early stages in AN glial-cell migration, glial cells are highly dye coupled, dividing glia are readily found in the nerve and AN glial cells label strongly for glutamine synthetase. By the end of this period, dye-coupling is rare, glial proliferation has ceased, glutamine synthetase labeling is absent, and glial processes have begun to extend to enwrap bundles of axons, a process that continues throughout the remainder of metamorphic development. Whole-cell and perforated-patch recordings in vivo from AN glia at different stages of network formation revealed two potassium currents and an R-like calcium current. Chronic in vivo exposure to the R-type channel blocker SNX-482 halted or greatly reduced AN glial migration. Chronically blocking spontaneous Na-dependent activity by injection of tetrodotoxin reduced the glial calcium current implicating an activity-dependent interaction between ORNs and glial cells in the development of glial calcium currents.

  1. Guiding migration of transplanted glial progenitor cells in the injured spinal cord

    PubMed Central

    Yuan, Xiao-bing; Jin, Ying; Haas, Christopher; Yao, Lihua; Hayakawa, Kazuo; Wang, Yue; Wang, Chunlei; Fischer, Itzhak

    2016-01-01

    Transplantation of glial-restricted progenitors (GRPs) is a promising strategy for generating a supportive environment for axon growth in the injured spinal cord. Here we explored the possibility of producing a migratory stream of GRPs via directional cues to create a supportive pathway for axon regeneration. We found that the axon growth inhibitor chondroitin sulfate proteoglycan (CSPG) strongly inhibited the adhesion and migration of GRPs, an effect that could be modulated by the adhesion molecule laminin. Digesting glycosaminoglycan side chains of CSPG with chondroitinase improved GRP migration on stripes of CSPG printed on cover glass, although GRPs were still responsive to the remaining repulsive signals of CSPG. Of all factors tested, the basic fibroblast growth factor (bFGF) had the most significant effect in promoting the migration of cultured GRPs. When GRPs were transplanted into either normal spinal cord of adult rats or the injury site in a dorsal column hemisection model of spinal cord injury, a population of transplanted cells migrated toward the region that was injected with the lentivirus expressing chondroitinase or bFGF. These findings suggest that removing CSPG-mediated inhibition, in combination with guidance by attractive factors, can be a promising strategy to produce a migratory stream of supportive GRPs. PMID:26971438

  2. Macrophage polarization alters the expression and sulfation pattern of glycosaminoglycans.

    PubMed

    Martinez, Pierre; Denys, Agnès; Delos, Maxime; Sikora, Anne-Sophie; Carpentier, Mathieu; Julien, Sylvain; Pestel, Joël; Allain, Fabrice

    2015-05-01

    Macrophages are major cells of inflammatory process and take part in a large number of physiological and pathological processes. According to tissue environment, they can polarize into pro-inflammatory (M1) or alternative (M2) cells. Although many evidences have hinted to a potential role of cell-surface glycosaminoglycans (GAGs) in the functions of macrophages, the effect of M1 or M2 polarization on the biosynthesis of these polysaccharides has not been investigated so far. GAGs are composed of repeat sulfated disaccharide units. Heparan (HS) and chondroitin/dermatan sulfates (CS/DS) are the major GAGs expressed at the cell membrane. They are involved in numerous biological processes, which rely on their ability to selectively interact with a large panel of proteins. More than 20 genes encoding sulfotransferases have been implicated in HS and CS/DS biosynthesis, and the functional repertoire of HS and CS/DS has been related to the expression of these isoenzymes. In this study, we analyzed the expression of sulfotransferases as a response to macrophage polarization. We found that M1 and M2 activation drastically modified the profiles of expression of numerous HS and CS/DS sulfotransferases. This was accompanied by the expression of GAGs with distinct structural features. We then demonstrated that GAGs of M2 macrophages were efficient to present fibroblast growth factor-2 in an assay of tumor cell proliferation, thus indicating that changes in GAG structure may contribute to the functions of polarized macrophages. Altogether, our findings suggest a regulatory mechanism in which fine modifications in GAG biosynthesis may participate to the plasticity of macrophage functions.

  3. Hexuronic acid stereochemistry determination in chondroitin sulfate glycosaminoglycan oligosaccharides by electron detachment dissociation.

    PubMed

    Leach, Franklin E; Ly, Mellisa; Laremore, Tatiana N; Wolff, Jeremy J; Perlow, Jacob; Linhardt, Robert J; Amster, I Jonathan

    2012-09-01

    Electron detachment dissociation (EDD) has previously provided stereo-specific product ions that allow for the assignment of the acidic C-5stereochemistry in heparan sulfate glycosaminoglycans (GAGs), but application of the same methodology to an epimer pair in the chondroitin sulfate glycoform class does not provide the same result. A series of experiments have been conducted in which glycosaminoglycan precursor ions are independently activated by electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD) to assign the stereochemistry in chondroitin sulfate (CS) epimers and investigate the mechanisms for product ion formation during EDD in CS glycoforms. This approach allows for the assignment of electronic excitation products formed by EID and detachment products to radical pathways in NETD, both of which occur simultaneously during EDD. The uronic acid stereochemistry in electron detachment spectra produces intensity differences when assigned glycosidic and cross-ring cleavages are compared. The variations in the intensities of the doubly deprotonated (0,2)X(3) and Y(3) ions have been shown to be indicative of CS-A/DS composition during the CID of binary mixtures. These ions can provide insight into the uronic acid composition of binary mixtures in EDD, but the relative abundances, although reproducible, are low compared with those in a CID spectrum acquired on an ion trap. The application of principal component analysis (PCA) presents a multivariate approach to determining the uronic acid stereochemistry spectra of these GAGs by taking advantage of the reproducible peak distributions produced by electron detachment.

  4. GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system

    PubMed Central

    Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D.; Baba, Hiroko; Ikenaka, Kazuhiro

    2017-01-01

    Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy. PMID:28186137

  5. Off limits: sulfate below the sulfate-methane transition

    NASA Astrophysics Data System (ADS)

    Brunner, Benjamin; Arnold, Gail; Røy, Hans; Müller, Inigo; Jørgensen, Bo

    2016-07-01

    One of the most intriguing recent discoveries in biogeochemistry is the ubiquity of cryptic sulfur cycling. From subglacial lakes to marine oxygen minimum zones, and in marine sediments, cryptic sulfur cycling - the simultaneous sulfate consumption and production - has been observed. Though this process does not leave an imprint in the sulfur budget of the ambient environment - thus the term cryptic - it may have a massive impact on other element cycles and fundamentally change our understanding of biogeochemical processes in the subsurface. Classically, the sulfate-methane transition (SMT) in marine sediments is considered to be the boundary that delimits sulfate reduction from methanogenesis as the predominant terminal pathway of organic matter mineralization. Two sediment cores from Aarhus Bay, Denmark reveal the constant presence of sulfate (generally 0.1 to 0.2 mM) below the SMT. The sulfur and oxygen isotope signature of this deep sulfate (34S = 18.9‰, 18O = 7.7‰) was close to the isotope signature of bottom-seawater collected from the sampling site (34S = 19.8‰, 18O = 7.3‰). In one of the cores, oxygen isotope values of sulfate at the transition from the base of the SMT to the deep sulfate pool (18O = 4.5‰ to 6.8‰) were distinctly lighter than the deep sulfate pool. Our findings are consistent with a scenario where sulfate enriched in 34S and 18O is removed at the base of the SMT and replaced with isotopically light sulfate below. Here, we explore scenarios that explain this observation, ranging from sampling artifacts, such as contamination with seawater or auto-oxidation of sulfide - to the potential of sulfate generation in a section of the sediment column where sulfate is expected to be absent which enables reductive sulfur cycling, creating the conditions under which sulfate respiration can persist in the methanic zone.

  6. Satellite glial cells in sensory ganglia: from form to function.

    PubMed

    Hanani, Menachem

    2005-06-01

    Current information indicates that glial cells participate in all the normal and pathological processes of the central nervous system. Although much less is known about satellite glial cells (SGCs) in sensory ganglia, it appears that these cells share many characteristics with their central counterparts. This review presents information that has been accumulated recently on the physiology and pharmacology of SGCs. It appears that SGCs carry receptors for numerous neuroactive agents (e.g., ATP, bradykinin) and can therefore receive signals from other cells and respond to changes in their environment. Activation of SGCs might in turn influence neighboring neurons. Thus SGCs are likely to participate in signal processing and transmission in sensory ganglia. Damage to the axons of sensory ganglia is known to contribute to neuropathic pain. Such damage also affects SGCs, and it can be proposed that these cells have a role in pathological changes in the ganglia.

  7. Temporal Variant Frontotemporal Dementia is Associated with Globular Glial Tauopathy.

    PubMed

    Clark, Camilla N; Lashley, Tammaryn; Mahoney, Colin J; Warren, Jason D; Revesz, Tamas; Rohrer, Jonathan D

    2015-06-01

    Frontotemporal dementia (FTD) is a clinically and pathologically heterogeneous neurodegenerative disorder associated with atrophy of the frontal and temporal lobes. Most patients with focal temporal lobe atrophy present with either the semantic dementia subtype of FTD or the behavioral variant subtype. For patients with temporal variant FTD, the most common cause found on post-mortem examination has been a TDP-43 (transactive response DNA-binding protein 43 kDa) proteinopathy, but tauopathies have also been described, including Pick's disease and mutations in the microtubule-associated protein tau (MAPT) gene. We report the clinical and imaging features of 2 patients with temporal variant FTD associated with a rare frontotemporal lobar degeneration pathology known as globular glial tauopathy. The pathologic diagnosis of globular glial tauopathy should be considered in patients with temporal variant FTD, particularly those who have atypical semantic dementia or an atypical parkinsonian syndrome in association with the right temporal variant.

  8. Glial β-oxidation regulates Drosophila energy metabolism.

    PubMed

    Schulz, Joachim G; Laranjeira, Antonio; Van Huffel, Leen; Gärtner, Annette; Vilain, Sven; Bastianen, Jarl; Van Veldhoven, Paul P; Dotti, Carlos G

    2015-01-15

    The brain's impotence to utilize long-chain fatty acids as fuel, one of the dogmas in neuroscience, is surprising, since the nervous system is the tissue most energy consuming and most vulnerable to a lack of energy. Challenging this view, we here show in vivo that loss of the Drosophila carnitine palmitoyltransferase 2 (CPT2), an enzyme required for mitochondrial β-oxidation of long-chain fatty acids as substrates for energy production, results in the accumulation of triacylglyceride-filled lipid droplets in adult Drosophila brain but not in obesity. CPT2 rescue in glial cells alone is sufficient to restore triacylglyceride homeostasis, and we suggest that this is mediated by the release of ketone bodies from the rescued glial cells. These results demonstrate that the adult brain is able to catabolize fatty acids for cellular energy production.

  9. Glial β-Oxidation regulates Drosophila Energy Metabolism

    PubMed Central

    Schulz, Joachim G.; Laranjeira, Antonio; Van Huffel, Leen; Gärtner, Annette; Vilain, Sven; Bastianen, Jarl; Van Veldhoven, Paul P.; Dotti, Carlos G.

    2015-01-01

    The brain's impotence to utilize long-chain fatty acids as fuel, one of the dogmas in neuroscience, is surprising, since the nervous system is the tissue most energy consuming and most vulnerable to a lack of energy. Challenging this view, we here show in vivo that loss of the Drosophila carnitine palmitoyltransferase 2 (CPT2), an enzyme required for mitochondrial β-oxidation of long-chain fatty acids as substrates for energy production, results in the accumulation of triacylglyceride-filled lipid droplets in adult Drosophila brain but not in obesity. CPT2 rescue in glial cells alone is sufficient to restore triacylglyceride homeostasis, and we suggest that this is mediated by the release of ketone bodies from the rescued glial cells. These results demonstrate that the adult brain is able to catabolize fatty acids for cellular energy production. PMID:25588812

  10. Glial activation colocalizes with structural abnormalities in amyotrophic lateral sclerosis

    PubMed Central

    Alshikho, Mohamad J.; Zürcher, Nicole R.; Loggia, Marco L.; Cernasov, Paul; Chonde, Daniel B.; Izquierdo Garcia, David; Yasek, Julia E.; Akeju, Oluwaseun; Catana, Ciprian; Rosen, Bruce R.; Cudkowicz, Merit E.

    2016-01-01

    Objective: In this cross-sectional study, we aimed to evaluate brain structural abnormalities in relation to glial activation in the same cohort of participants. Methods: Ten individuals with amyotrophic lateral sclerosis (ALS) and 10 matched healthy controls underwent brain imaging using integrated MR/PET and the radioligand [11C]-PBR28. Diagnosis history and clinical assessments including Upper Motor Neuron Burden Scale (UMNB) were obtained from patients with ALS. Diffusion tensor imaging (DTI) analyses including tract-based spatial statistics and tractography were applied. DTI metrics including fractional anisotropy (FA) and diffusivities (mean, axial, and radial) were measured in regions of interest. Cortical thickness was assessed using surface-based analysis. The locations of structural changes, measured by DTI and the areas of cortical thinning, were compared to regional glial activation measured by relative [11C]-PBR28 uptake. Results: In this cohort of individuals with ALS, reduced FA and cortical thinning colocalized with regions demonstrating higher radioligand binding. [11C]-PBR28 binding in the left motor cortex was correlated with FA (r = −0.68, p < 0.05) and cortical thickness (r = −0.75, p < 0.05). UMNB was correlated with glial activation (r = +0.75, p < 0.05), FA (r = −0.77, p < 0.05), and cortical thickness (r = −0.75, p < 0.05) in the motor cortex. Conclusions: Increased uptake of the glial marker [11C]-PBR28 colocalizes with changes in FA and cortical thinning. This suggests a link between disease mechanisms (gliosis and inflammation) and structural changes (cortical thinning and white and gray matter changes). In this multimodal neuroimaging work, we provide an in vivo model to investigate the pathogenesis of ALS. PMID:27837005

  11. Glial Modulators as Potential Treatments of Psychostimulant Abuse

    PubMed Central

    Beardsley, Patrick M.; Hauser, Kurt F.

    2014-01-01

    Glia (including astrocytes, microglia and oligodendrocytes), which constitute the majority of cells in the brain. have many of the same receptors as neurons, secrete neurotransmitters and neurotrophic and neuroinflammatory factors, control clearance of neurotransmitters from synaptic clefts, and are intimately involved in synaptic plasticity. Despite their prevalence and spectrum of functions, appreciation of their potential general importance has been elusive since their identification in the mid-1800s, and only relatively recently have they been gaining their due respect. This development of appreciation has been nurtured by the growing awareness that drugs of abuse, including the psychostimulants, affect glial activity, and glial activity, in turn, has been found to modulate the effects of the psychostimulants. This developing awareness has begun to illuminate novel pharmacotherapeutic targets for treating psychostimulant abuse, for which targeting more conventional neuronal targets has not yet resulted in a single, approved medication. In this chapter, we discuss the molecular pharmacology, physiology and functional relationships that the glia have especially in the light in which they present themselves as targets for pharmacotherapeutics intended to treat psychostimulant abuse disorders. We then review a cross section of preclinical studies that have manipulated glial processes whose behavioral effects have been supportive of considering the glia as drug targets for psychostimulant-abuse medications. We then close with comments regarding the current clinical evaluation of relevant compounds for treating psychostimulant abuse, as well as the likelihood of future prospects. PMID:24484974

  12. Homocysteine Induces Glial Reactivity in Adult Rat Astrocyte Cultures.

    PubMed

    Longoni, Aline; Bellaver, Bruna; Bobermin, Larissa Daniele; Santos, Camila Leite; Nonose, Yasmine; Kolling, Janaina; Dos Santos, Tiago M; de Assis, Adriano M; Quincozes-Santos, André; Wyse, Angela T S

    2017-03-02

    Astrocytes are dynamic glial cells associated to neurotransmitter systems, metabolic functions, antioxidant defense, and inflammatory response, maintaining the brain homeostasis. Elevated concentrations of homocysteine (Hcy) are involved in the pathogenesis of age-related neurodegenerative disorders, such as Parkinson and Alzheimer diseases. In line with this, our hypothesis was that Hcy could promote glial reactivity in a model of cortical primary astrocyte cultures from adult Wistar rats. Thus, cortical astrocytes were incubated with different concentrations of Hcy (10, 30, and 100 μM) during 24 h. After the treatment, we analyzed cell viability, morphological parameters, antioxidant defenses, and inflammatory response. Hcy did not induce any alteration in cell viability; however, it was able to induce cytoskeleton rearrangement. The treatment with Hcy also promoted a significant decrease in the activities of Na(+), K(+) ATPase, superoxide dismutase (SOD), and glutathione peroxidase (GPx), as well as in the glutathione (GSH) content. Additionally, Hcy induced an increase in the pro-inflammatory cytokine release. In an attempt to elucidate the putative mechanisms involved in the Hcy-induced glial reactivity, we measured the nuclear factor kappa B (NFκB) transcriptional activity and heme oxygenase 1 (HO-1) expression, which were activated and inhibited by Hcy, respectively. In summary, our findings provide important evidences that Hcy modulates critical astrocyte parameters from adult rats, which might be associated to the aging process.

  13. Phenotypic changes in satellite glial cells in cultured trigeminal ganglia.

    PubMed

    Belzer, Vitali; Shraer, Nathanael; Hanani, Menachem

    2010-11-01

    Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24 h after culturing, but undergo major phenotypic changes at later times.

  14. Glial modulators as potential treatments of psychostimulant abuse.

    PubMed

    Beardsley, Patrick M; Hauser, Kurt F

    2014-01-01

    Glia (including astrocytes, microglia, and oligodendrocytes), which constitute the majority of cells in the brain, have many of the same receptors as neurons, secrete neurotransmitters and neurotrophic and neuroinflammatory factors, control clearance of neurotransmitters from synaptic clefts, and are intimately involved in synaptic plasticity. Despite their prevalence and spectrum of functions, appreciation of their potential general importance has been elusive since their identification in the mid-1800s, and only relatively recently have they been gaining their due respect. This development of appreciation has been nurtured by the growing awareness that drugs of abuse, including the psychostimulants, affect glial activity, and glial activity, in turn, has been found to modulate the effects of the psychostimulants. This developing awareness has begun to illuminate novel pharmacotherapeutic targets for treating psychostimulant abuse, for which targeting more conventional neuronal targets has not yet resulted in a single, approved medication. In this chapter, we discuss the molecular pharmacology, physiology, and functional relationships that the glia have especially in the light in which they present themselves as targets for pharmacotherapeutics intended to treat psychostimulant abuse disorders. We then review a cross section of preclinical studies that have manipulated glial processes whose behavioral effects have been supportive of considering the glia as drug targets for psychostimulant-abuse medications. We then close with comments regarding the current clinical evaluation of relevant compounds for treating psychostimulant abuse, as well as the likelihood of future prospects.

  15. Transcriptional regulation of the Drosophila glial gene repo.

    PubMed

    Lee, Bruce P; Jones, Bradley W

    2005-06-01

    reversed polarity (repo) is a putative target gene of glial cells missing (gcm), the primary regulator of glial cell fate in Drosophila. Transient expression of Gcm is followed by maintained expression of repo. Multiple Gcm binding sites are found in repo upstream DNA. However, while repo is expressed in Gcm positive glia, it is not expressed in Gcm positive hemocytes. These observations suggest factors in addition to Gcm are required for repo expression. Here we have undertaken an analysis of the cis-regulatory DNA elements of repo using lacZ reporter activity in transgenic embryos. We have found that a 4.2 kb DNA region upstream of the repo start site drives the wild-type repo expression pattern. We show that expression is dependent on multiple Gcm binding sites. By ectopically expressing Repo, we show that Repo can regulate its own enhancer. Finally, by systematically analyzing fragments of repo upstream DNA, we show that expression is dependent on multiple elements that are responsible for activity in subsets of glia, as well as repressing inappropriate expression in the epidermis. Our results suggest that Gcm acts synergistically with other factors to control repo transcription in glial cells.

  16. Chondroitin 6-Sulfate as a Novel Biomarker for Mucopolysaccharidosis IVA and VII.

    PubMed

    Shimada, Tsutomu; Tomatsu, Shunji; Yasuda, Eriko; Mason, Robert W; Mackenzie, William G; Shibata, Yuniko; Kubaski, Francyne; Giugliani, Roberto; Yamaguchi, Seiji; Suzuki, Yasuyuki; Orii, Kenji; Orii, Tadao

    2014-01-01

    Chondroitin 6-sulfate (C6S), a glycosaminoglycan (GAG), is distributed mainly in the growth plates, aorta, and cornea; however, the physiological function of C6S is not fully understood. One of the limitations is that no rapid, accurate quantitative method to measure C6S has been established. Mucopolysaccharidosis IVA and VII (MPS IVA and VII) are caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase and β-D-glucuronidase, respectively, resulting in accumulation of C6S and other GAG(s). While levels of keratan sulfate (KS), heparan sulfate, and dermatan sulfate in samples from MPS patients are well described, this is the first report of quantitative analysis of C6S levels in samples from MPS IVA and VII patients.We developed a method to digest polymeric C6S and measure resultant disaccharides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). C6S levels were measured in the blood from control subjects and patients with MPS IVA and VII aged from 0 to 58 years of age. We also assayed KS levels in the same samples for comparison with C6S.Levels of C6S in the blood decreased with age and were significantly elevated in patients with MPS IVA and VII, compared with age-matched controls. Levels of KS in patients with MPS IVA were also higher than those in age-matched controls, although differences were less pronounced than with C6S. Combining KS and C6S data, discriminated patients with MPS IVA from age-matched control subjects were better than either C6S or KS levels alone.In conclusion, this first report showing that blood levels of C6S are quantitatively evaluated in patients with MPS IVA and VII indicates that C6S could be a useful biomarker for these metabolic disorders.

  17. Role of satellite glial cells in gastrointestinal pain.

    PubMed

    Hanani, Menachem

    2015-01-01

    Gastrointestinal (GI) pain is a common clinical problem, for which effective therapy is quite limited. Sensations from the GI tract, including pain, are mediated largely by neurons in the dorsal root ganglia (DRG), and to a smaller extent by vagal afferents emerging from neurons in the nodose/jugular ganglia. Neurons in rodent DRG become hyperexcitable in models of GI pain (e.g., gastric or colonic inflammation), and can serve as a source for chronic pain. Glial cells are another element in the pain signaling pathways, and there is evidence that spinal glial cells (microglia and astrocytes) undergo activation (gliosis) in various pain models and contribute to pain. Recently it was found that satellite glial cells (SGCs), the main type of glial cells in sensory ganglia, might also contribute to chronic pain in rodent models. Most of that work focused on somatic pain, but in several studies GI pain was also investigated, and these are discussed in the present review. We have shown that colonic inflammation induced by dinitrobenzene sulfonic acid (DNBS) in mice leads to the activation of SGCs in DRG and increases gap junction-mediated coupling among these cells. This coupling appears to contribute to the hyperexcitability of DRG neurons that innervate the colon. Blocking gap junctions (GJ) in vitro reduced neuronal hyperexcitability induced by inflammation, suggesting that glial GJ participate in SGC-neuron interactions. Moreover, blocking GJ by carbenoxolone and other agents reduces pain behavior. Similar changes in SGCs were also found in the mouse nodose ganglia (NG), which provide sensory innervation to most of the GI tract. Following systemic inflammation, SGCs in these ganglia were activated, and displayed augmented coupling and greater sensitivity to the pain mediator ATP. The contribution of these changes to visceral pain remains to be determined. These results indicate that although visceral pain is unique, it shares basic mechanisms with somatic pain

  18. Role of satellite glial cells in gastrointestinal pain

    PubMed Central

    Hanani, Menachem

    2015-01-01

    Gastrointestinal (GI) pain is a common clinical problem, for which effective therapy is quite limited. Sensations from the GI tract, including pain, are mediated largely by neurons in the dorsal root ganglia (DRG), and to a smaller extent by vagal afferents emerging from neurons in the nodose/jugular ganglia. Neurons in rodent DRG become hyperexcitable in models of GI pain (e.g., gastric or colonic inflammation), and can serve as a source for chronic pain. Glial cells are another element in the pain signaling pathways, and there is evidence that spinal glial cells (microglia and astrocytes) undergo activation (gliosis) in various pain models and contribute to pain. Recently it was found that satellite glial cells (SGCs), the main type of glial cells in sensory ganglia, might also contribute to chronic pain in rodent models. Most of that work focused on somatic pain, but in several studies GI pain was also investigated, and these are discussed in the present review. We have shown that colonic inflammation induced by dinitrobenzene sulfonic acid (DNBS) in mice leads to the activation of SGCs in DRG and increases gap junction-mediated coupling among these cells. This coupling appears to contribute to the hyperexcitability of DRG neurons that innervate the colon. Blocking gap junctions (GJ) in vitro reduced neuronal hyperexcitability induced by inflammation, suggesting that glial GJ participate in SGC-neuron interactions. Moreover, blocking GJ by carbenoxolone and other agents reduces pain behavior. Similar changes in SGCs were also found in the mouse nodose ganglia (NG), which provide sensory innervation to most of the GI tract. Following systemic inflammation, SGCs in these ganglia were activated, and displayed augmented coupling and greater sensitivity to the pain mediator ATP. The contribution of these changes to visceral pain remains to be determined. These results indicate that although visceral pain is unique, it shares basic mechanisms with somatic pain

  19. Relationship between glial potassium regulation and axon excitability: a role for glial Kir4.1 channels.

    PubMed

    Bay, Virginia; Butt, Arthur M

    2012-04-01

    Uptake of K(+) released by axons during action potential propagation is a major function of astrocytes. Here, we demonstrate the importance of glial inward rectifying potassium channels (Kir) in regulating extracellular K(+) ([K(+)](o)) and axonal electrical activity in CNS white matter of the mouse optic nerve. Increasing optic nerve stimulation frequency from 1 Hz to 10-35 Hz for 120 s resulted in a rise in [K(+)](o) and consequent decay in the compound action potential (CAP), a measure of reduced axonal activity. On cessation of high frequency stimulation, rapid K(+) clearance resulted in a poststimulus [K(+)](o) undershoot, followed by a slow recovery of [K(+)](o) and the CAP, which were more protracted with increasing stimulation frequency. Blockade of Kir (100 μM BaCl(2)) slowed poststimulus recovery of [K(+)](o) and the CAP at all stimulation frequencies, indicating a primary function of glial Kir was redistributing K(+) to the extracellular space to offset active removal by Na(+)-K(+) pumps. At higher levels of axonal activity, Kir blockade also increased [K(+)](o) accumulation, exacerbating the decline in the CAP and impeding its subsequent recovery. In the Kir4.1-/- mouse, astrocytes displayed a marked reduction of inward currents and were severely depolarized, resulting in retarded [K(+)](o) regulation and reduced CAP. The results demonstrate the importance of glial Kir in K(+) spatial buffering and sustaining axonal activity in the optic nerve. Glial Kir have increasing importance in K(+) clearance at higher levels of axonal activity, helping to maintain the physiological [K(+)](o) ceiling and ensure the fidelity of signaling between the retina and brain.

  20. Crystal structure of tris-(piperidinium) hydrogen sulfate sulfate.

    PubMed

    Lukianova, Tamara J; Kinzhybalo, Vasyl; Pietraszko, Adam

    2015-12-01

    In the title molecular salt, 3C5H12N(+)·HSO4 (-)·SO4 (2-), each cation adopts a chair conformation. In the crystal, the hydrogen sulfate ion is connected to the sulfate ion by a strong O-H⋯O hydrogen bond. The packing also features a number of N-H⋯O hydrogen bonds, which lead to a three-dimensional network structure. The hydrogen sulfate anion accepts four hydrogen bonds from two cations, whereas the sulfate ion, as an acceptor, binds to five separate piperidinium cations, forming seven hydrogen bonds.

  1. Sulfate scale dissolution

    SciTech Connect

    Morris, R.L.; Paul, J.M.

    1992-01-28

    This patent describes a method for removing barium sulfate scale. It comprises contacting the scale with an aqueous solution having a pH of about 8 to about 14 and consisting essentially of a chelating agent comprising a polyaminopolycarboxylic acid or salt of such an acid in a concentration of 0.1 to 1.0 M, and anions of a monocarboxylic acid selected form mercaptoacetic acid, hydroxyacetic acid, aminoacetic acid, or salicyclic acid in a concentration of 0.1 to 1.0 M and which is soluble in the solution under the selected pH conditions, to dissolve the scale.

  2. Glucosamine and chondroitin sulfate.

    PubMed

    Miller, Karla L; Clegg, Daniel O

    2011-02-01

    Glucosamine and chondroitin sulfate, components of normal cartilage that are marketed as dietary supplements in the United States, have been evaluated for their potential role in the treatment of osteoarthritis. Due to claims of efficacy, increased prevalence of osteoarthritis, and a lack of other effective therapies, there has been substantial interest in using these dietary supplements as therapeutic agents for osteoarthritis. Though pharmacokinetic and bioavailability data are limited, use of these supplements has been evaluated for management of osteoarthritis symptoms and modification of disease progression. Relevant clinical trial efficacy and safety data are reviewed and summarized.

  3. Ferric sulfates on Mars

    NASA Technical Reports Server (NTRS)

    Burns, Roger G.

    1987-01-01

    Evidence is presented for the possible existence of ferric sulfato complexes and hydroxo ferric sulfate minerals in the permafrost of Mars. A sequential combination of ten unique conditions during the cooling history of Mars is suggested which is believed to have generated an environment within Martian permafrost that has stabilized Fe(3+)-SO4(2-)-bearing species. It is argued that minerals belonging to the jarosite and copiapite groups could be present in Martian regolith analyzed in the Viking XRF measurements at Chryse and Utopia, and that maghemite suspected to be coating the Viking magnet arrays is a hydrolysate of dissolved ferric sulfato complexes from exposed Martian permafrost.

  4. Arylsulfatase B modulates neurite outgrowth via astrocyte chondroitin-4-sulfate: dysregulation by ethanol.

    PubMed

    Zhang, Xiaolu; Bhattacharyya, Sumit; Kusumo, Handojo; Goodlett, Charles R; Tobacman, Joanne K; Guizzetti, Marina

    2014-02-01

    In utero ethanol exposure causes fetal alcohol spectrum disorders, associated with reduced brain plasticity; the mechanisms of these effects are not well understood, particularly with respect to glial involvement. Astrocytes release factors that modulate neurite outgrowth. We explored the hypothesis that ethanol inhibits neurite outgrowth by increasing the levels of inhibitory chondroitin sulfate proteoglycans (CSPGs) in astrocytes. Astrocyte treatment with ethanol inhibited the activity of arylsulfatase B (ARSB), the enzyme that removes sulfate groups from chondroitin-4-sulfate (C4S) and triggers the degradation of C4S, increased total sulfated glycosaminoglycans (GAGs), C4S, and neurocan core-protein content and inhibited neurite outgrowth in neurons cocultured with ethanol-treated astrocytes in vitro, effects reversed by treatment with recombinant ARSB. Ethanol also inhibited ARSB activity and increased sulfate GAG and neurocan levels in the developing hippocampus after in vivo ethanol exposure. ARSB silencing increased the levels of sulfated GAGs, C4S, and neurocan in astrocytes and inhibited neurite outgrowth in cocultured neurons, indicating that ARSB activity directly regulates C4S and affects neurocan expression. In summary, this study reports two major findings: ARSB modulates sulfated GAG and neurocan levels in astrocytes and astrocyte-mediated neurite outgrowth in cocultured neurons; and ethanol inhibits the activity of ARSB, increases sulfated GAG, C4S, and neurocan levels, and thereby inhibits astrocyte-mediated neurite outgrowth. An unscheduled increase in CSPGs in the developing brain may lead to altered brain connectivity and to premature decrease in neuronal plasticity and therefore represents a novel mechanism by which ethanol can exert its neurodevelopmental effects.

  5. Aggrecan and chondroitin-6-sulfate abnormalities in schizophrenia and bipolar disorder: a postmortem study on the amygdala

    PubMed Central

    Pantazopoulos, H; Markota, M; Jaquet, F; Ghosh, D; Wallin, A; Santos, A; Caterson, B; Berretta, S

    2015-01-01

    Perineuronal nets (PNNs) are specialized extracellular matrix aggregates surrounding distinct neuronal populations and regulating synaptic functions and plasticity. Previous findings showed robust PNN decreases in amygdala, entorhinal cortex and prefrontal cortex of subjects with schizophrenia (SZ), but not bipolar disorder (BD). These studies were carried out using a chondroitin sulfate proteoglycan (CSPG) lectin marker. Here, we tested the hypothesis that the CSPG aggrecan, and 6-sulfated chondroitin sulfate (CS-6) chains highly represented in aggrecan, may contribute to these abnormalities. Antibodies against aggrecan and CS-6 (3B3 and CS56) were used in the amygdala of healthy control, SZ and BD subjects. In controls, aggrecan immunoreactivity (IR) was observed in PNNs and glial cells. Antibody 3B3, but not CS56, also labeled PNNs in the amygdala. In addition, dense clusters of CS56 and 3B3 IR encompassed CS56- and 3B3-IR glia, respectively. In SZ, numbers of aggrecan- and 3B3-IR PNNs were decreased, together with marked reductions of aggrecan-IR glial cells and CS-6 (3B3 and CS56)-IR ‘clusters'. In BD, numbers of 3B3-IR PNNs and CS56-IR clusters were reduced. Our findings show disruption of multiple PNN populations in the amygdala of SZ and, more modestly, BD. Decreases of aggrecan-IR glia and CS-6-IR glial ‘clusters', in sharp contrast to increases of CSPG/lectin-positive glia previously observed, indicate that CSPG abnormalities may affect distinct glial cell populations and suggest a potential mechanism for PNN decreases. Together, these abnormalities may contribute to a destabilization of synaptic connectivity and regulation of neuronal functions in the amygdala of subjects with major psychoses. PMID:25603412

  6. 21 CFR 184.1315 - Ferrous sulfate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Ferrous sulfate. 184.1315 Section 184.1315 Food... GRAS § 184.1315 Ferrous sulfate. (a) Ferrous sulfate heptahydrate (iron (II) sulfate heptahydrate, Fe... pale, bluish-green crystals or granules. Progressive heating of ferrous sulfate heptahydrate...

  7. 21 CFR 184.1315 - Ferrous sulfate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ferrous sulfate. 184.1315 Section 184.1315 Food and... Substances Affirmed as GRAS § 184.1315 Ferrous sulfate. (a) Ferrous sulfate heptahydrate (iron (II) sulfate... as pale, bluish-green crystals or granules. Progressive heating of ferrous sulfate...

  8. Subretinal Glial Membranes in Eyes With Geographic Atrophy

    PubMed Central

    Edwards, Malia M.; McLeod, D. Scott; Bhutto, Imran A.; Grebe, Rhonda; Duffy, Maeve; Lutty, Gerard A.

    2017-01-01

    Purpose Müller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study evaluated the relationship between focal photoreceptors and RPE loss in geographic atrophy (GA) and Müller cell extension into the subretinal space. Methods Human donor eyes with no retinal disease or geographic atrophy (GA) were fixed and the eye cups imaged. The retinal posterior pole was stained for glial fibrillary acidic protein (GFAP; astrocytes and activated Müller cells) and vimentin (Müller cells) while the submacular choroids were labeled with Ulex Europaeus Agglutinin lectin (blood vessels). Choroids and retinas were imaged using a Zeiss 710 confocal microscope. Additional eyes were cryopreserved or processed for transmission electron microscopy (TEM) to better visualize the Müller cells. Results Vimentin staining of aged control retinas (n = 4) revealed a panretinal cobblestone-like ELM. While this pattern was also observed in the GA retinas (n = 7), each also had a distinct area in which vimentin+ and vimentin+/GFAP+ processes created a subretinal membrane. Subretinal glial membranes closely matched areas of RPE atrophy in the gross photos. Choroidal vascular loss was also evident in these atrophic areas. Smaller glial projections were noted, which correlated with drusen in gross photos. The presence of glia in the subretinal space was confirmed by TEM and cross cross-section immunohistochemistry. Conclusions In eyes with GA, subretinal Müller cell membranes present in areas of RPE atrophy may be a Müller cell attempt to replace the ELM. These membranes could interfere with treatments such as stem cell therapy. PMID:28249091

  9. Orbitrap mass spectrometry characterization of hybrid chondroitin/dermatan sulfate hexasaccharide domains expressed in brain.

    PubMed

    Robu, Adrian C; Popescu, Laurentiu; Munteanu, Cristian V A; Seidler, Daniela G; Zamfir, Alina D

    2015-09-15

    In the central nervous system, chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) modulate neurotrophic effects and glial cell maturation during brain development. Previous reports revealed that GAG composition could be responsible for CS/DS activities in brain. In this work, for the structural characterization of DS- and CS-rich domains in hybrid GAG chains extracted from neural tissue, we have developed an advanced approach based on high-resolution mass spectrometry (MS) using nanoelectrospray ionization Orbitrap in the negative ion mode. Our high-resolution MS and multistage MS approach was developed and applied to hexasaccharides obtained from 4- and 14-week-old mouse brains by GAG digestion with chondroitin B and in parallel with AC I lyase. The expression of DS- and CS-rich domains in the two tissues was assessed comparatively. The analyses indicated an age-related structural variability of the CS/DS motifs. The older brain was found to contain more structures and a higher sulfation of DS-rich regions, whereas the younger brain was found to be characterized by a higher sulfation of CS-rich regions. By multistage MS using collision-induced dissociation, we also demonstrated the incidence in mouse brain of an atypical [4,5-Δ-GlcAGalNAc(IdoAGalNAc)2], presenting a bisulfated CS disaccharide formed by 3-O-sulfate-4,5-Δ-GlcA and 6-O-sulfate-GalNAc moieties.

  10. The scoop on the fly brain: glial engulfment functions in Drosophila.

    PubMed

    Logan, Mary A; Freeman, Marc R

    2007-02-01

    Glial cells provide support and protection for neurons in the embryonic and adult brain, mediated in part through the phagocytic activity of glia. Glial cells engulf apoptotic cells and pruned neurites from the developing nervous system, and also clear degenerating neuronal debris from the adult brain after neural trauma. Studies indicate that Drosophila melanogaster is an ideal model system to elucidate the mechanisms of engulfment by glia. The recent studies reviewed here show that many features of glial engulfment are conserved across species and argue that work in Drosophila will provide valuable cellular and molecular insight into glial engulfment activity in mammals.

  11. Induction of oxidative stress and oxidative damage in rat glial cells by acrylonitrile.

    PubMed

    Kamendulis, L M; Jiang, J; Xu, Y; Klaunig, J E

    1999-08-01

    Chronic treatment of rats with acrylonitrile (ACN) resulted in a dose-related increase in glial cell tumors (astrocytomas). While the exact mechanism(s) for ACN-induced carcinogenicity remains unresolved, non-genotoxic and possibly tumor promotion modes of action appear to be involved in the induction of glial tumors. Recent studies have shown that ACN induced oxidative stress selectively in rat brain in a dose-responsive manner. The present study examined the ability of ACN to induce oxidative stress in a rat glial cell line, a target tissue, and in cultured rat hepatocytes, a non-target tissue of ACN carcinogenicity. Glial cells and hepatocytes were treated for 1, 4 and 24 h with sublethal concentrations of ACN. ACN induced an increase in oxidative DNA damage, as evidenced by increased production of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in glial cells but not in rat hepatocytes. Hydroxyl radical formation following ACN treatment was also selectively increased in glial cells. Following 1 and 4 h of ACN exposure, the levels of the non-enzymatic antioxidant glutathione, as well as the activities of the enzymatic antioxidants catalase and superoxide dismutase were significantly decreased in the rat glial cells. Lipid peroxidation and the activity of glutathione peroxidase were not affected by ACN treatment in rat glial cells. No changes in any of these biomarkers of oxidative stress were observed in hepatocytes treated with ACN. These data indicate that ACN selectively induced oxidative stress in rat glial cells.

  12. The glial fascicle: an ontogenic and phylogenic unit guiding, supplying and distributing mammalian cortical neurons.

    PubMed

    Gressens, P; Evrard, P

    1993-12-17

    Neurons destined for the mammalian neocortex migrate along radial glial cells (RGCs). This paper describes a comparative study of RGCs in the mouse, rat, hamster, cat and human fetus, selected as representative species of mammalian evolution. The glial fascicles display constant features throughout these species: they consist of 4-10 RGCs filled with glycogen. The glial fascicle guides and probably supplies metabolites to the migrating neurons and organizes the vertical lamination of the developing neocortical plate. The neuronal-glial unit which consists of the RGCs and their affiliated migrating neurons is conserved throughout the species studied, suggesting a designation as a phylogenic unit.

  13. Investigation of terahertz radiation influence on rat glial cells

    PubMed Central

    Borovkova, Mariia; Serebriakova, Maria; Fedorov, Viacheslav; Sedykh, Egor; Vaks, Vladimir; Lichutin, Alexander; Salnikova, Alina; Khodzitsky, Mikhail

    2016-01-01

    We studied an influence of continuous terahertz (THz) radiation (0.12 – 0.18 THz, average power density of 3.2 mW/cm2) on a rat glial cell line. A dose-dependent cytotoxic effect of THz radiation is demonstrated. After 1 minute of THz radiation exposure a relative number of apoptotic cells increased in 1.5 times, after 3 minutes it doubled. This result confirms the concept of biological hazard of intense THz radiation. Diagnostic applications of THz radiation can be restricted by the radiation power density and exposure time. PMID:28101417

  14. Axon-glial interactions at the Drosophila CNS midline.

    PubMed

    Crews, Stephen T

    2010-01-01

    The glia that reside at the midline of the Drosophila CNS are an important embryonic signaling center and also wrap the axons that cross the CNS. The development of the midline glia (MG) is characterized by migration, ensheathment, subdivision of axon commissures, apoptosis, and the extension of glial processes. All of these events are characterized by cell-cell contact between MG and adjacent neurons. Cell adhesion and signaling proteins that mediate different aspects of MG development and MG-neuron interactions have been identified. This provides a foundation for ultimately obtaining an integrated picture of how the MG assemble into a characteristic axonal support structure in the CNS.

  15. Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

    PubMed Central

    1989-01-01

    Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component. PMID:2592422

  16. 21 CFR 184.1307 - Ferric sulfate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Substances Affirmed as GRAS § 184.1307 Ferric sulfate. (a) Ferric sulfate (iron (III) sulfate, Fe2(SO4)3 CAS... treating ferric oxide or ferric hydroxide with sulfuric acid. (b) The ingredient must be of a...

  17. 21 CFR 184.1307 - Ferric sulfate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Substances Affirmed as GRAS § 184.1307 Ferric sulfate. (a) Ferric sulfate (iron (III) sulfate, Fe2(SO4)3 CAS... treating ferric oxide or ferric hydroxide with sulfuric acid. (b) The ingredient must be of a...

  18. Sulfation of von Willebrand factor

    SciTech Connect

    Carew, J.A.; Browning, P.J.; Lynch, D.C. )

    1990-12-15

    von Willebrand factor (vWF) is a multimeric adhesive glycoprotein essential for normal hemostasis. We have discovered that cultured human umbilical vein endothelial cells incorporate inorganic sulfate into vWF. Following immunoisolation and analysis by polyacrylamide or agarose gel electrophoresis, metabolically labeled vWF was found to have incorporated (35S)-sulfate into all secreted multimer species. The time course of incorporation shows that sulfation occurs late in the biosynthesis of vWF, near the point at which multimerization occurs. Quantitative analysis suggests the presence, on average, of one molecule of sulfate per mature vWF subunit. Virtually all the detectable sulfate is released from the mature vWF subunit by treatment with endoglycosidases that remove asparagine-linked carbohydrates. Sulfated carbohydrate was localized first to the N-terminal half of the mature subunit (amino acids 1 through 1,365) by partial proteolytic digestion with protease V8; and subsequently to a smaller fragment within this region (amino acids 273 through 511) by sequential digestions with protease V8 and trypsin. Thus, the carbohydrate at asparagine 384 and/or 468 appears to be the site of sulfate modification. Sodium chlorate, an inhibitor of adenosine triphosphate-sulfurylase, blocks sulfation of vWF without affecting either the ability of vWF to assemble into high molecular weight multimers or the ability of vWF multimers to enter Weible-Palade bodies. The stability of vWF multimers in the presence of an endothelial cell monolayer also was unaffected by the sulfation state. Additionally, we have found that the cleaved propeptide of vWF is sulfated on asparagine-linked carbohydrate.

  19. Glial cell plasticity in sensory ganglia induced by nerve damage.

    PubMed

    Hanani, M; Huang, T Y; Cherkas, P S; Ledda, M; Pannese, E

    2002-01-01

    Numerous studies have been done on the effect of nerve injury on neurons of sensory ganglia but little is known about the contribution of satellite glial cells (SCs) in these ganglia to post-injury events. We investigated cell-to-cell coupling and ultrastructure of SCs in mouse dorsal root ganglia after nerve injury (axotomy). Under control conditions SCs were mutually coupled, but mainly to other SCs around a given neuron. After axotomy SCs became extensively coupled to SCs that enveloped other neurons, apparently by gap junctions. Serial section electron microscopy showed that after axotomy SC sheaths enveloping neighboring neurons formed connections with each other. Such connections were absent in control ganglia. The number of gap junctions between SCs increased 6.5-fold after axotomy. We propose that axotomy induces growth of perineuronal SC sheaths, leading to contacts between SCs enveloping adjacent neurons and to formation of new gap junctions between SCs. These changes may be an important mode of glial plasticity and can contribute to neuropathic pain.

  20. DNA damage, neuronal and glial cell death and neurodegeneration.

    PubMed

    Barzilai, Ari

    2010-11-01

    The DNA damage response (DDR) is a key factor in the maintenance of genome stability. As such, it is a central axis in sustaining cellular homeostasis in a variety of contexts: development, growth, differentiation, and maintenance of the normal life cycle of the cell. It is now clear that diverse mechanisms encompassing cell cycle regulation, repair pathways, many aspects of cellular metabolism, and cell death are inter-linked and act in concert in response to DNA damage. Defects in the DDR in proliferating cells can lead to cancer, while DDR defects in neurons may result in neurodegeneration. Mature neurons are highly differentiated, post-mitotic cells that cannot be replenished after disease or trauma. Their high metabolic activity generates large amounts of reactive oxygen species with DNA damaging capacity. Moreover, their intense transcriptional activity increases the potential for genomic DNA damage. Respectively, neurons have elaborate mechanisms to defend the integrity of their genome, thus ensuring their longevity and functionality in the face of these threats. Over the course of the past two decades, there has been a substantial increase in our understanding of the role of glial cells in supporting the neuronal cell DDR and longevity. This review article focuses on the potential role of the DDR in the etiology and pathogenesis of neurodegenerative diseases, and in addition, it describes various aspects of glial cell functionality in two genomic instability disorders: ataxia telangiectasia (A-T) and Nijmegen breakage syndrome.

  1. Glutamate dehydrogenase 1 and SIRT4 regulate glial development.

    PubMed

    Komlos, Daniel; Mann, Kara D; Zhuo, Yue; Ricupero, Christopher L; Hart, Ronald P; Liu, Alice Y-C; Firestein, Bonnie L

    2013-03-01

    Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by an activation mutation of glutamate dehydrogenase 1 (GDH1), a mitochondrial enzyme responsible for the reversible interconversion between glutamate and α-ketoglutarate. The syndrome presents clinically with hyperammonemia, significant episodic hypoglycemia, seizures, and frequent incidences of developmental and learning defects. Clinical research has implicated that although some of the developmental and neurological defects may be attributed to hypoglycemia, some characteristics cannot be ascribed to low glucose and as hyperammonemia is generally mild and asymptomatic, there exists the possibility that altered GDH1 activity within the brain leads to some clinical changes. GDH1 is allosterically regulated by many factors, and has been shown to be inhibited by the ADP-ribosyltransferase sirtuin 4 (SIRT4), a mitochondrially localized sirtuin. Here we show that SIRT4 is localized to mitochondria within the brain. SIRT4 is highly expressed in glial cells, specifically astrocytes, in the postnatal brain and in radial glia during embryogenesis. Furthermore, SIRT4 protein decreases in expression during development. We show that factors known to allosterically regulate GDH1 alter gliogenesis in CTX8 cells, a novel radial glial cell line. We find that SIRT4 and GDH1 overexpression play antagonistic roles in regulating gliogenesis and that a mutant variant of GDH1 found in HI/HA patients accelerates the development of glia from cultured radial glia cells.

  2. Satellite glial cell responses to neuronal firing in the nervous system of Helix pomatia.

    PubMed

    Gommerat, I; Gola, M

    1994-03-01

    Patch clamp experiments were conducted on satellite glial cells attached to the cell body of neurons in place within the nervous system of the snail Helix pomatia. The glial cells were studied using cell-attached and whole-cell patch clamp configurations while the underlying neurons were under current or voltage clamp control. The resting potential of the glial cells (-69 mV) was more negative than that of the underlying neurons (-53 mV), due to their high K+ selectivity. Densely packed K+ channels were present, some of which were active at the cell resting potential. Neuronal firing elicited a cumulative depolarization of the glial cells. Large K+ currents flowing from V-clamped neurons depolarized the glial layer by up to 30 mV. The glial depolarization was directly correlated with the size of the neuronal K+ current. The glial cells recovered their resting potential within 2-5 sec. The neuronal depolarization induced a delayed (20-30 sec) and persistent (3-4 min) increase in the glial K+ channel opening probability. Likewise, pulses of K+ (20-50 mM)-rich saline activated the glial channels, unless the underlying neuron was held hyperpolarized. In low Ca(2+)-high Mg2+ saline, neuron depolarization and K(+)-rich saline did not activate the glial K+ channels. These data indicate that a calcium-dependent signal released from the neuronal cell body was involved in glial channel regulation. Neuron-induced channel opening may help eliminate the K+ ions flowing from active neurons.

  3. 2- and 6-O-sulfated proteoglycans have distinct and complementary roles in cranial axon guidance and motor neuron migration

    PubMed Central

    Maden, Charlotte H.; Davidson, Kathryn; Fantin, Alessandro

    2016-01-01

    The correct migration and axon extension of neurons in the developing nervous system is essential for the appropriate wiring and function of neural networks. Here, we report that O-sulfotransferases, a class of enzymes that modify heparan sulfate proteoglycans (HSPGs), are essential to regulate neuronal migration and axon development. We show that the 6-O-sulfotransferases HS6ST1 and HS6ST2 are essential for cranial axon patterning, whilst the 2-O-sulfotransferase HS2ST (also known as HS2ST1) is important to regulate the migration of facial branchiomotor (FBM) neurons in the hindbrain. We have also investigated how HS2ST interacts with other signals in the hindbrain and show that fibroblast growth factor (FGF) signalling regulates FBM neuron migration in an HS2ST-dependent manner. PMID:27048738

  4. Vascular dermatan sulfate regulates the antithrombotic activity of heparin cofactor II

    PubMed Central

    He, Li; Giri, Tusar K.; Vicente, Cristina P.

    2008-01-01

    Heparin cofactor II (HCII)–deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo. PMID:18281504

  5. A Model of Glial Scarring Analogous to the Environment of a Traumatically Injured Spinal Cord Using Kainate

    PubMed Central

    2016-01-01

    Objective To develop an in vitro model analogous to the environment of traumatic spinal cord injury (SCI), the authors evaluated change of astrogliosis following treatments with kainate and/or scratch, and degree of neurite outgrowth after treatment with a kainate inhibitor. Methods Astrocytes were obtained from the rat spinal cord. Then, 99% of the cells were confirmed to be GFAP-positive astrocytes. For chemical injury, the cells were treated with kainate at different concentrations (10, 50 or 100 µM). For mechanical injury, two kinds of uniform scratches were made using a plastic pipette tip by removing strips of cells. For combined injury (S/K), scratch and kainate were provided. Cord neurons from rat embryos were plated onto culture plates immediately after the three kinds of injuries and some cultures were treated with a kainate inhibitor. Results Astro-gliosis (glial fibrillary acidic protein [GFAP], vimentin, chondroitin sulfate proteoglycan [CSPG], rho-associated protein kinase [ROCK], and ephrin type-A receptor 4 [EphA4]) was most prominent after treatment with 50 µM kainate and extensive scratch injury in terms of single arm (p<0.001) and in the S/K-induced injury model in view of single or combination (p<0.001). Neurite outgrowth in the seeded spinal cord (β-III tubulin) was the least in the S/K-induced injury model (p<0.001) and this inhibition was reversed by the kainate inhibitor (p<0.001). Conclusion The current in vitro model combining scratch and kainate induced glial scarring and inhibitory molecules and restricted neurite outgrowth very strongly than either the mechanically or chemically-induced injury model; hence, it may be a useful tool for research on SCI. PMID:27847705

  6. Activation of paracrine growth factors by heparan sulphate induced by glucocorticoid in A549 lung carcinoma cells.

    PubMed

    Yevdokimova, N; Freshney, R I

    1997-01-01

    Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation.

  7. Activation of paracrine growth factors by heparan sulphate induced by glucocorticoid in A549 lung carcinoma cells.

    PubMed Central

    Yevdokimova, N.; Freshney, R. I.

    1997-01-01

    Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation. PMID:9252193

  8. Involvement of a membrane potassium channel in heparan sulphate-induced activation of macrophages.

    PubMed

    Ren, Jian-Dong; Fan, Li; Tian, Fu-Zhou; Fan, Kai-Hua; Yu, Bo-Tao; Jin, Wei-Hua; Tan, Yong-Hong; Cheng, Long

    2014-03-01

    Increasing evidence has demonstrated that Toll-like receptor 4 (TLR4) -mediated systemic inflammatory response syndrome accompanied by multiple organ failure, is one of the most common causes of death in patients with severe acute pancreatitis. Recent reports have revealed that heparan sulphate (HS) proteoglycan, a component of extracellular matrices, potentiates the activation of intracellular pro-inflammatory responses via TLR4, contributing to the aggravation of acute pancreatitis. However, little is known about the participants in the HS/TLR4-mediated inflammatory cascades. Our previous work provided a clue that a membrane potassium channel (MaxiK) is responsible for HS-induced production of inflammatory cytokines. Therefore, in this report we attempted to reveal the roles of MaxiK in the activation of macrophages stimulated by HS. Our results showed that incubation of RAW264.7 cells with HS up-regulated MaxiK and TLR4 expression levels. HS could also activate MaxiK channels to promote the efflux of potassium ions from cells, as measured by the elevated activity of caspase-1, whereas this was significantly abolished by treatment with paxilline, a specific blocker of the MaxiK channel. Moreover, it was found that paxilline substantially inhibited HS-induced activation of several different transcription factors in macrophages, including nuclear factor-κB, p38 and interferon regulatory factor-3, followed by decreased production of tumour necrosis factor-α and interferon-β. Taken together, our investigation provides evidence that the HS/TLR4-mediated intracellular inflammatory cascade depends on the activation of MaxiK, which may offer an important opportunity for a new approach in therapeutic strategies of severe acute pancreatitis.

  9. A Mathematical Model of Regenerative Axon Growing along Glial Scar after Spinal Cord Injury

    PubMed Central

    Chen, Xuning; Zhu, Weiping

    2016-01-01

    A major factor in the failure of central nervous system (CNS) axon regeneration is the formation of glial scar after the injury of CNS. Glial scar generates a dense barrier which the regenerative axons cannot easily pass through or by. In this paper, a mathematical model was established to explore how the regenerative axons grow along the surface of glial scar or bypass the glial scar. This mathematical model was constructed based on the spinal cord injury (SCI) repair experiments by transplanting Schwann cells as bridge over the glial scar. The Lattice Boltzmann Method (LBM) was used in this model for three-dimensional numerical simulation. The advantage of this model is that it provides a parallel and easily implemented algorithm and has the capability of handling complicated boundaries. Using the simulated data, two significant conclusions were made in this study: (1) the levels of inhibitory factors on the surface of the glial scar are the main factors affecting axon elongation and (2) when the inhibitory factor levels on the surface of the glial scar remain constant, the longitudinal size of the glial scar has greater influence on the average rate of axon growth than the transverse size. These results will provide theoretical guidance and reference for researchers to design efficient experiments. PMID:27274762

  10. p-Cresyl Sulfate

    PubMed Central

    Gryp, Tessa; Vanholder, Raymond; Vaneechoutte, Mario; Glorieux, Griet

    2017-01-01

    If chronic kidney disease (CKD) is associated with an impairment of kidney function, several uremic solutes are retained. Some of these exert toxic effects, which are called uremic toxins. p-Cresyl sulfate (pCS) is a prototype protein-bound uremic toxin to which many biological and biochemical (toxic) effects have been attributed. In addition, increased levels of pCS have been associated with worsening outcomes in CKD patients. pCS finds its origin in the intestine where gut bacteria metabolize aromatic amino acids, such as tyrosine and phenylalanine, leading to phenolic end products, of which pCS is one of the components. In this review we summarize the biological effects of pCS and its metabolic origin in the intestine. It appears that, according to in vitro studies, the intestinal bacteria generating phenolic compounds mainly belong to the families Bacteroidaceae, Bifidobacteriaceae, Clostridiaceae, Enterobacteriaceae, Enterococcaceae, Eubacteriaceae, Fusobacteriaceae, Lachnospiraceae, Lactobacillaceae, Porphyromonadaceae, Staphylococcaceae, Ruminococcaceae, and Veillonellaceae. Since pCS remains difficult to remove by dialysis, the gut microbiota could be a future target to decrease pCS levels and its toxicity, even at earlier stages of CKD, aiming at slowing down the progression of the disease and decreasing the cardiovascular burden. PMID:28146081

  11. Residual keratan sulfate in chondroitin sulfate formulations for oral administration.

    PubMed

    Pomin, Vitor H; Piquet, Adriana A; Pereira, Mariana S; Mourão, Paulo A S

    2012-10-01

    Chondroitin sulfate is a biomedical glycosaminoglycan (GAG) mostly used as a dietary supplement. We undertook analysis on some formulations of chondroitin sulfates available for oral administration. The analysis was based on agarose-gel electrophoresis, strong anion-exchange chromatography, digestibility with specific GAG lyases, uronic acid content, NMR spectroscopy, and size-exclusion chromatography. Keratan sulfate was detected in batches from shark cartilage, averaging ∼16% of the total GAG. Keratan sulfate is an inert material, and hazardous effects due to its presence in these formulations are unlikely to occur. However, its unexpected high percentage compromises the desired amounts of the real ingredient specified on the label claims, and forewarns the pharmacopeias to update their monographs. The techniques they recommended, especially cellulose acetate electrophoresis, are inefficient in detecting keratan sulfate in chondroitin sulfate formulations. In addition, this finding also alerts the manufacturers for improved isolation procedures as well as the supervisory agencies for better audits. Analysis based on strong anion-exchange chromatography is shown to be more reliable than the methods presently suggested by standard pharmacopeias.

  12. Protein misfolding and oxidative stress promote glial-mediated neurodegeneration in an Alexander disease model

    PubMed Central

    Wang, Liqun; Colodner, Kenneth J.; Feany, Mel B.

    2011-01-01

    Although alterations in glial structure and function commonly accompany death of neurons in neurodegenerative diseases, the role glia play in modulating neuronal loss is poorly understood. We have created a model of Alexander disease in Drosophila by expressing disease-linked mutant versions of glial fibrillary acidic protein (GFAP) in fly glia. We find aggregation of mutant human GFAP into inclusions bearing the hallmarks of authentic Rosenthal fibers. We also observe significant toxicity of mutant human GFAP to glia, which is mediated by protein aggregation and oxidative stress. Both protein aggregation and oxidative stress contribute to activation of a robust autophagic response in glia. Toxicity of mutant GFAP to glial cells induces a non-cell autonomous stress response and subsequent apoptosis in neurons, which is dependent on glial glutamate transport. Our findings thus establish a simple genetic model of Alexander disease and further identify cellular pathways critical for glial-induced neurodegeneration. PMID:21414908

  13. Honeybee retinal glial cells transform glucose and supply the neurons with metabolic substrate.

    PubMed Central

    Tsacopoulos, M; Evêquoz-Mercier, V; Perrottet, P; Buchner, E

    1988-01-01

    The retina of the honeybee drone is a nervous tissue in which glial cells and photoreceptor cells (sensory neurons) constitute two distinct metabolic compartments. Retinal slices incubated with 2-deoxy[3H]glucose convert this glucose analogue to 2-deoxy[3H]glucose 6-phosphate, but this conversion is made only in the glial cells. Hence, glycolysis occurs only in glial cells. In contrast, the neurons consume O2 and this consumption is sustained by the hydrolysis of glycogen, which is contained in large amounts in the glia. During photostimulation the increased oxidative metabolism of the neurons is sustained by a higher supply of carbohydrates from the glia. This clear case of metabolic interaction between neurons and glial cells supports Golgi's original hypothesis, proposed nearly 100 years ago, about the nutritive function of glial cells in the nervous system. Images PMID:3186756

  14. Honeybee retinal glial cells transform glucose and supply the neurons with metabolic substrate

    SciTech Connect

    Tsacopoulos, M.; Evequoz-Mercier, V.; Perrottet, P.; Buchner, E.

    1988-11-01

    The retina of the honeybee drone is a nervous tissue in which glial cells and photoreceptor cells (sensory neurons) constitute two distinct metabolic compartments. Retinal slices incubated with 2-deoxy(/sup 3/H)glucose convert this glucose analogue to 2-deoxy(/sup 3/H)glucose 6-phosphate, but this conversion is made only in the glial cells. Hence, glycolysis occurs only in glial cells. In contrast, the neurons consume O/sub 2/ and this consumption is sustained by the hydrolysis of glycogen, which is contained in large amounts in the glia. During photostimulation the increased oxidative metabolism of the neurons is sustained by a higher supply of carbohydrates from the glia. This clear case of metabolic interaction between neurons and glial cells supports Golgi's original hypothesis, proposed nearly 100 years ago, about the nutritive function of glial cells in the nervous system.

  15. Friend or foe? Resolving the impact of glial responses in glaucoma.

    PubMed

    Johnson, Elaine C; Morrison, John C

    2009-01-01

    Glaucomatous vision loss results from the progressive degeneration of optic nerve axons and the death of retinal ganglion cells. This process is accompanied by dramatic alterations in the functional properties and distribution of glial cells in both the retina and the optic nerve head in a reaction commonly referred to as glial activation. The recent availability of rodent and cell culture glaucoma models has substantially contributed to our knowledge of glial activation under glaucomatous conditions. Conclusions drawn from these studies have led to the refinement of existing hypotheses and the generation of new ones. Because these hypotheses encompass both protective and injurious roles for glia, the impact of specific aspects of glial activation are current topics of intensive research, speculation, and debate in the field. With these unresolved issues in mind, this review will summarize recent progress in our understanding of the process of glial activation in the glaucomatous optic nerve head and retina.

  16. Final report on the safety assessment of sodium cetearyl sulfate and related alkyl sulfates as used in cosmetics.

    PubMed

    Fiume, Monice; Bergfeld, Wilma F; Belsito, Donald V; Klaassen, Curtis D; Marks, James G; Shank, Ronald C; Slaga, Thomas J; Snyder, Paul W; Alan Andersen, F

    2010-05-01

    Sodium cetearyl sulfate is the sodium salt of a mixture of cetyl and stearyl sulfate. The other ingredients in this safety assessment are also alkyl salts, including ammonium coco-sulfate, ammonium myristyl sulfate, magnesium coco-sulfate, sodium cetyl sulfate, sodium coco/hydrogenated tallow sulfate, sodium coco-sulfate, sodium decyl sulfate, sodium ethylhexyl sulfate, sodium myristyl sulfate, sodium oleyl sulfate, sodium stearyl sulfate, sodium tallow sulfate, sodium tridecyl sulfate, and zinc coco-sulfate. These ingredients are surfactants used at concentrations from 0.1% to 29%, primarily in soaps and shampoos. Many of these ingredients are not in current use. The Cosmetic Ingredient Review (CIR) Expert Panel previously completed a safety assessment of sodium and ammonium lauryl sulfate. The data available for sodium lauryl sulfate and ammonium lauryl sulfate provide sufficient basis for concluding that sodium cetearyl sulfate and related alkyl sulfates are safe in the practices of use and concentration described in the safety assessment.

  17. Involvement of nucleotides in glial growth following scratch injury in avian retinal cell monolayer cultures.

    PubMed

    Silva, Thayane Martins; França, Guilherme Rapozeiro; Ornelas, Isis Moraes; Loiola, Erick Correia; Ulrich, Henning; Ventura, Ana Lucia Marques

    2015-06-01

    When retinal cell cultures were mechanically scratched, cell growth over the empty area was observed. Only dividing and migrating, 2 M6-positive glial cells were detected. Incubation of cultures with apyrase (APY), suramin, or Reactive Blue 2 (RB-2), but not MRS 2179, significantly attenuated the growth of glial cells, suggesting that nucleotide receptors other than P2Y1 are involved in the growth of glial cells. UTPγS but not ADPβS antagonized apyrase-induced growth inhibition in scratched cultures, suggesting the participation of UTP-sensitive receptors. No decrease in proliferating cell nuclear antigen (PCNA(+)) cells was observed at the border of the scratch in apyrase-treated cultures, suggesting that glial proliferation was not affected. In apyrase-treated cultures, glial cytoplasm protrusions were smaller and unstable. Actin filaments were less organized and alfa-tubulin-labeled microtubules were mainly parallel to scratch. In contrast to control cultures, very few vinculin-labeled adhesion sites could be noticed in these cultures. Increased Akt and ERK phosphorylation was observed in UTP-treated cultures, effect that was inhibited by SRC inhibitor 1 and PI3K blocker LY294002. These inhibitors and the FAK inhibitor PF573228 also decreased glial growth over the scratch, suggesting participation of SRC, PI3K, and FAK in UTP-induced growth of glial cells in scratched cultures. RB-2 decreased dissociated glial cell attachment to fibronectin-coated dishes and migration through transwell membranes, suggesting that nucleotides regulated adhesion and migration of glial cells. In conclusion, mechanical scratch of retinal cell cultures induces growth of glial cells over the empty area through a mechanism that is dependent on activation of UTP-sensitive receptors, SRC, PI3K, and FAK.

  18. Sox2 promotes survival of satellite glial cells in vitro

    SciTech Connect

    Koike, Taro Wakabayashi, Taketoshi; Mori, Tetsuji; Hirahara, Yukie; Yamada, Hisao

    2015-08-14

    Sox2 is a transcriptional factor expressed in neural stem cells. It is known that Sox2 regulates cell differentiation, proliferation and survival of the neural stem cells. Our previous study showed that Sox2 is expressed in all satellite glial cells of the adult rat dorsal root ganglion. In this study, to examine the role of Sox2 in satellite glial cells, we establish a satellite glial cell-enriched culture system. Our culture method succeeded in harvesting satellite glial cells with the somata of neurons in the dorsal root ganglion. Using this culture system, Sox2 was downregulated by siRNA against Sox2. The knockdown of Sox2 downregulated ErbB2 and ErbB3 mRNA at 2 and 4 days after siRNA treatment. MAPK phosphorylation, downstream of ErbB, was also inhibited by Sox2 knockdown. Because ErbB2 and ErbB3 are receptors that support the survival of glial cells in the peripheral nervous system, apoptotic cells were also counted. TUNEL-positive cells increased at 5 days after siRNA treatment. These results suggest that Sox2 promotes satellite glial cell survival through the MAPK pathway via ErbB receptors. - Highlights: • We established satellite glial cell culture system. • Function of Sox2 in satellite glial cell was examined using siRNA. • Sox2 knockdown downregulated expression level of ErbB2 and ErbB3 mRNA. • Sox2 knockdown increased apoptotic satellite glial cell. • Sox2 promotes satellite glial cell survival through ErbB signaling.

  19. Glial Cell Calcium Signaling Mediates Capillary Regulation of Blood Flow in the Retina

    PubMed Central

    Biesecker, Kyle R.; Srienc, Anja I.; Shimoda, Angela M.; Agarwal, Amit; Bergles, Dwight E.; Kofuji, Paulo

    2016-01-01

    The brain is critically dependent on the regulation of blood flow to nourish active neurons. One widely held hypothesis of blood flow regulation holds that active neurons stimulate Ca2+ increases in glial cells, triggering glial release of vasodilating agents. This hypothesis has been challenged, as arteriole dilation can occur in the absence of glial Ca2+ signaling. We address this controversy by imaging glial Ca2+ signaling and vessel dilation in the mouse retina. We find that sensory stimulation results in Ca2+ increases in the glial endfeet contacting capillaries, but not arterioles, and that capillary dilations often follow spontaneous Ca2+ signaling. In IP3R2−/− mice, where glial Ca2+ signaling is reduced, light-evoked capillary, but not arteriole, dilation is abolished. The results show that, independent of arterioles, capillaries actively dilate and regulate blood flow. Furthermore, the results demonstrate that glial Ca2+ signaling regulates capillary but not arteriole blood flow. SIGNIFICANCE STATEMENT We show that a Ca2+-dependent glial cell signaling mechanism is responsible for regulating capillary but not arteriole diameter. This finding resolves a long-standing controversy regarding the role of glial cells in regulating blood flow, demonstrating that glial Ca2+ signaling is both necessary and sufficient to dilate capillaries. While the relative contributions of capillaries and arterioles to blood flow regulation remain unclear, elucidating the mechanisms that regulate capillary blood flow may ultimately lead to the development of therapies for treating diseases where blood flow regulation is disrupted, including Alzheimer's disease, stroke, and diabetic retinopathy. This finding may also aid in revealing the underlying neuronal activity that generates BOLD fMRI signals. PMID:27605617

  20. Cement composition and sulfate attack

    SciTech Connect

    Shanahan, Natalya; Zayed, Abla . E-mail: zayed@eng.usf.edu

    2007-04-15

    Four cements were used to address the effect of tricalcium silicate content of cement on external sulfate attack in sodium sulfate solution. The selected cements had similar fineness and Bogue-calculated tricalcium aluminate content but variable tricalcium silicates. Durability was assessed using linear expansion and compressive strength. Phases associated with deterioration were examined using scanning electron microscopy and X-ray diffraction. Mineralogical phase content of the as-received cements was studied by X-ray diffraction using two methods: internal standard and Rietveld analysis. The results indicate that phase content of cements determined by X-ray mineralogical analysis correlates better with the mortar performance in sulfate environment than Bogue content. Additionally, it was found that in cements containing triclacium aluminate only in the cubic form, the observed deterioration is affected by tricalcium silicate content. Morphological similarities between hydration products of high tricalcium aluminate and high tricalcium silicate cements exposed to sodium sulfate environment were also observed.

  1. Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of human immunodeficiency virus-1 infectivity in vitro.

    PubMed

    Stone, A L; Melton, D J; Lewis, M S

    1998-07-01

    Heparins/heparan sulfates modulate the function of proteins and cell membranes in numerous biological systems including normal and disease processes in humans. Heparin has been used for many years as an anticoagulant, and anticoagulant heparin-mimetics were developed several decades ago by chemical sulfation of non-mammalian polysaccharides, e.g., an antithrombotic sulfated xylan. This pharmaceutical, which comprises a mixture of sulfated oligoxylans, also mimics most other biological actions of natural heparins in vitro, including inhibition of the human immunodeficiency virus, but the molecular basis for these actions has been unclear. Here, numerous Components of the sulfated oligoxylan mixture were isolated and when bioassayed in the case of anti-HIV-1 infectivity revealed that a structural specificity underlines the capacity of sulfated xylan to inhibit HIV-1, rather than a non-specific mechanism. Components were isolated by chromatographic fractionation through Bio-Gel P10 in 0.5 M ammonium bicarbonate. This fractionation revealed an elution range associated with apparent molecular weights of approximately 22000 to <1500 relative to standard heparin and heparan sulfates and newly prepared sulfated oligosaccharide standards. Components were characterized by metachromatic absorption spectroscopy, ultracentrifugation, GlcA analysis, and potency against HIV-1 infectivity, both in the tetrazolium cytotoxicity assay and in syncytium-forming assays, in CD4-lymphocytes. Structural specificity was indicated by the differential potencies exhibited by the Components: Highest activity (cytotoxicity) was exhibited by Components in the chromatographic region > or = approximately 5500 in mass (50% effective (inhibitory) concentration = 0.5-0.7 microg ml(-1) in the first fractionation series, and 0.1-0.5 microg ml(-1) in a second series). The potency declined sharply below approximately 5400 in mass, but with an exception; a second structure exhibiting relatively high

  2. In defense of magnesium sulfate.

    PubMed

    Elliott, John P; Lewis, David F; Morrison, John C; Garite, Thomas J

    2009-06-01

    Magnesium sulfate has been used by obstetricians for more than 25 years to treat preterm labor. Magnesium sulfate is effective in delaying delivery for at least 48 hours in patients with preterm labor when used in higher dosages. There do not seem to be any harmful effects of the drug on the fetus, and indeed there is a neuroprotective effect in reducing the incidence of cerebral palsy in premature newborns weighing less than 1,500 g.

  3. Gliopathic pain: when satellite glial cells go bad.

    PubMed

    Ohara, Peter T; Vit, Jean-Philippe; Bhargava, Aditi; Romero, Marcela; Sundberg, Christopher; Charles, Andrew C; Jasmin, Luc

    2009-10-01

    Neurons in sensory ganglia are surrounded by satellite glial cells (SGCs) that perform similar functions to the glia found in the CNS. When primary sensory neurons are injured, the surrounding SGCs undergo characteristic changes. There is good evidence that the SGCs are not just bystanders to the injury but play an active role in the initiation and maintenance of neuronal changes that underlie neuropathic pain. In this article the authors review the literature on the relationship between SGCs and nociception and present evidence that changes in SGC potassium ion buffering capacity and glutamate recycling can lead to neuropathic pain-like behavior in animal models. The role that SGCs play in the immune responses to injury is also considered. We propose the term gliopathic pain to describe those conditions in which central or peripheral glia are thought to be the principal generators of principal pain generators.

  4. Neuronal-glial networks as substrate for CNS integration

    PubMed Central

    Verkhratsky, A; Toescu, E C

    2006-01-01

    Astrocytes have been considered, for a long time, as the support and house-keeping cells of the nervous system. Indeed, the astrocytes play very important metabolic roles in the brain, but the catalogue of nervous system functions or activities that involve directly glial participation has extended dramatically in the last decade. In addition to the further refining of the signalling capacity of the neuroglial networks and the detailed reassessment of the interactions between glia and vascular bed in the brain, one of the important salient features of the increased glioscience activity in the last few years was the morphological and functional demonstration that protoplasmic astrocytes occupy well defined spatial territories, with only limited areas of morphological overlapping, but still able to communicate with adjacent neighbours through intercellular junctions. All these features form the basis for a possible reassessment of the nature of integration of activity in the central nervous system that could raise glia to a role of central integrator.

  5. Review: Glial lineages and myelination in the central nervous system

    PubMed Central

    COMPSTON, ALASTAIR; ZAJICEK, JOHN; SUSSMAN, JON; WEBB, ANNA; HALL, GILLIAN; MUIR, DAVID; SHAW, CHRISTOPHER; WOOD, ANDREW; SCOLDING, NEIL

    1997-01-01

    Oligodendrocytes, derived from stem cell precursors which arise in subventricular zones of the developing central nervous system, have as their specialist role the synthesis and maintenance of myelin. Astrocytes contribute to the cellular architecture of the central nervous system and act as a source of growth factors and cytokines; microglia are bone-marrow derived macrophages which function as primary immunocompetent cells in the central nervous system. Myelination depends on the establishment of stable relationships between each differentiated oligodendrocyte and short segments of several neighbouring axons. There is growing evidence, especially from studies of glial cell implantation, that oligodendrocyte precursors persist in the adult nervous system and provide a limited capacity for the restoration of structure and function in myelinated pathways damaged by injury or disease. PMID:9061442

  6. Glial Progenitors as Targets for Transformation in Glioma

    PubMed Central

    Ilkanizadeh, Shirin; Lau, Jasmine; Huang, Miller; Foster, Daniel J.; Wong, Robyn; Frantz, Aaron; Wang, Susan; Weiss, William A.; Persson, Anders I.

    2014-01-01

    Glioma is the most common primary malignant brain tumor and arises throughout the central nervous system (CNS). Recent focus on stem-like glioma cells has implicated neural stem cells (NSCs), a minor precursor population restricted to germinal zones, as a potential source of gliomas. In this review, we will focus on the relationship between oligodendrocyte progenitor cells (OPCs), the largest population of cycling glial progenitors in the postnatal brain, and gliomas. Recent studies suggest that OPCs can give rise to gliomas. Furthermore, signaling pathways often associated with NSCs also play key roles during OPC lineage development. Recent advances suggesting that gliomas can undergo a switch from progenitor- to stem-like phenotype after therapy, implicating that an OPC-origin is more likely than previously recognized. Future in-depth studies of OPC biology may shed light on the etiology of OPC-derived gliomas and reveal new therapeutic avenues. PMID:24889528

  7. Evidence for brain glial activation in chronic pain patients.

    PubMed

    Loggia, Marco L; Chonde, Daniel B; Akeju, Oluwaseun; Arabasz, Grae; Catana, Ciprian; Edwards, Robert R; Hill, Elena; Hsu, Shirley; Izquierdo-Garcia, David; Ji, Ru-Rong; Riley, Misha; Wasan, Ajay D; Zürcher, Nicole R; Albrecht, Daniel S; Vangel, Mark G; Rosen, Bruce R; Napadow, Vitaly; Hooker, Jacob M

    2015-03-01

    Although substantial evidence has established that microglia and astrocytes play a key role in the establishment and maintenance of persistent pain in animal models, the role of glial cells in human pain disorders remains unknown. Here, using the novel technology of integrated positron emission tomography-magnetic resonance imaging and the recently developed radioligand (11)C-PBR28, we show increased brain levels of the translocator protein (TSPO), a marker of glial activation, in patients with chronic low back pain. As the Ala147Thr polymorphism in the TSPO gene affects binding affinity for (11)C-PBR28, nine patient-control pairs were identified from a larger sample of subjects screened and genotyped, and compared in a matched-pairs design, in which each patient was matched to a TSPO polymorphism-, age- and sex-matched control subject (seven Ala/Ala and two Ala/Thr, five males and four females in each group; median age difference: 1 year; age range: 29-63 for patients and 28-65 for controls). Standardized uptake values normalized to whole brain were significantly higher in patients than controls in multiple brain regions, including thalamus and the putative somatosensory representations of the lumbar spine and leg. The thalamic levels of TSPO were negatively correlated with clinical pain and circulating levels of the proinflammatory citokine interleukin-6, suggesting that TSPO expression exerts pain-protective/anti-inflammatory effects in humans, as predicted by animal studies. Given the putative role of activated glia in the establishment and or maintenance of persistent pain, the present findings offer clinical implications that may serve to guide future studies of the pathophysiology and management of a variety of persistent pain conditions.

  8. Depression as a Glial-Based Synaptic Dysfunction

    PubMed Central

    Rial, Daniel; Lemos, Cristina; Pinheiro, Helena; Duarte, Joana M.; Gonçalves, Francisco Q.; Real, Joana I.; Prediger, Rui D.; Gonçalves, Nélio; Gomes, Catarina A.; Canas, Paula M.; Agostinho, Paula; Cunha, Rodrigo A.

    2016-01-01

    Recent studies combining pharmacological, behavioral, electrophysiological and molecular approaches indicate that depression results from maladaptive neuroplastic processes occurring in defined frontolimbic circuits responsible for emotional processing such as the prefrontal cortex, hippocampus, amygdala and ventral striatum. However, the exact mechanisms controlling synaptic plasticity that are disrupted to trigger depressive conditions have not been elucidated. Since glial cells (astrocytes and microglia) tightly and dynamically interact with synapses, engaging a bi-directional communication critical for the processing of synaptic information, we now revisit the role of glial cells in the etiology of depression focusing on a dysfunction of the “quad-partite” synapse. This interest is supported by the observations that depressive-like conditions are associated with a decreased density and hypofunction of astrocytes and with an increased microglia “activation” in frontolimbic regions, which is expected to contribute for the synaptic dysfunction present in depression. Furthermore, the traditional culprits of depression (glucocorticoids, biogenic amines, brain-derived neurotrophic factor, BDNF) affect glia functioning, whereas antidepressant treatments (serotonin-selective reuptake inhibitors, SSRIs, electroshocks, deep brain stimulation) recover glia functioning. In this context of a quad-partite synapse, systems modulating glia-synapse bidirectional communication—such as the purinergic neuromodulation system operated by adenosine 5′-triphosphate (ATP) and adenosine—emerge as promising candidates to “re-normalize” synaptic function by combining direct synaptic effects with an ability to also control astrocyte and microglia function. This proposed triple action of purines to control aberrant synaptic function illustrates the rationale to consider the interference with glia dysfunction as a mechanism of action driving the design of future

  9. Glial dysfunction in parkin null mice: effects of aging.

    PubMed

    Solano, Rosa M; Casarejos, Maria J; Menéndez-Cuervo, Jamie; Rodriguez-Navarro, Jose A; García de Yébenes, Justo; Mena, Maria A

    2008-01-16

    Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals, and abnormal neurotransmitter release. The role of glia in parkin deficiency is little known. We cultured midbrain glia from wild-type (WT) and parkin knock-out (PK-KO) mice. After 18-20 d in vitro, PK-KO glial cultures had less astrocytes, more microglia, reduced proliferation, and increased proapoptotic protein expression. PK-KO glia had greater levels of intracellular glutathione (GSH), increased mRNA expression of the GSH-synthesizing enzyme gamma-glutamylcysteine synthetase, and greater glutathione S-transferase and lower glutathione peroxidase activities than WT. The reverse happened in glia cultured in serum-free defined medium (EF12) or in old cultures. PK-KO glia was more susceptible than WT to transference to EF12 or neurotoxins (1-methyl-4-phenylpyridinium, blockers of GSH synthesis or catalase, inhibitors of extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3 kinases), aging of the culture, or combination of these insults. PK-KO glia was less susceptible than WT to Fe2+ plus H2O2 and less responsive to protection by deferoxamine. Old WT glia increased the expression of heat shock protein 70, but PK-KO did not. Glia conditioned medium (GCM) from PK-KO was less neuroprotective and had lower levels of GSH than WT. GCM from WT increased the levels of dopamine markers in midbrain neuronal cultures transferred to EF12 more efficiently than GCM from PK-KO, and the difference was corrected by supplementation with GSH. PK-KO-GCM was a less powerful suppressor of apoptosis and microglia in neuronal cultures. Our data prove that abnormal glial function is critical in parkin mutations, and its role increases with aging.

  10. Minocycline blocks glial cell activation and ventilatory acclimatization to hypoxia.

    PubMed

    Stokes, Jennifer Ann; Arbogast, Tara Elizabeth; Moya, Esteban A; Fu, Zhenxing; Powell, Frank L

    2017-01-18

    Ventilatory acclimatization to hypoxia (VAH) is the time-dependent increase in ventilation, which persists upon return to normoxia, and involves plasticity in both central nervous system respiratory centers and peripheral chemoreceptors. We investigated the role of glial cells in VAH in male Sprague Dawley rats using minocycline, an antibiotic that inhibits microglia activation and has anti-inflammatory properties, and barometric pressure plethysmography to measure ventilation. Rats received either minocycline (45mg/kg, i.p. daily) or saline beginning one day before and during 7 days of chronic hypoxia (CH, PIO2 = 70 Torr). Minocycline had no effect on normoxic control rats or the hypercapnic ventilatory response in CH rats, but minocycline significantly (p < 0.001) decreased ventilation during acute hypoxia in CH rats. However, minocycline administration during only the last 3 days of CH did not reverse VAH. Microglia and astrocyte activation in the nucleus tractus solitarius was quantified from 30 minutes to 7 days of CH. Microglia showed an active morphology (shorter and fewer branches) after 1 hour of hypoxia and returned to the control state (longer filaments and extensive branching) after 4 hours of CH. Astrocytes increased GFAP antibody immunofluorescent intensity, indicating activation, at both 4 and 24 hours of CH. Minocycline had no effect on glia in normoxia, but significantly decreased microglia activation at 1 hour of CH and astrocyte activation at 24 hours of CH. These results support a role for glial cells providing an early signal for the induction but not maintenance of neural plasticity underlying ventilatory acclimatization to hypoxia.

  11. The Caenorhabditis elegans Ephrin EFN-4 Functions Non-cell Autonomously with Heparan Sulfate Proteoglycans to Promote Axon Outgrowth and Branching

    PubMed Central

    Schwieterman, Alicia A.; Steves, Alyse N.; Yee, Vivian; Donelson, Cory J.; Bentley, Melissa R.; Santorella, Elise M.; Mehlenbacher, Taylor V.; Pital, Aaron; Howard, Austin M.; Wilson, Melissa R.; Ereddia, Danielle E.; Effrein, Kelsie S.; McMurry, Jonathan L.; Ackley, Brian D.; Chisholm, Andrew D.; Hudson, Martin L.

    2016-01-01

    The Eph receptors and their cognate ephrin ligands play key roles in many aspects of nervous system development. These interactions typically occur within an individual tissue type, serving either to guide axons to their terminal targets or to define boundaries between the rhombomeres of the hindbrain. We have identified a novel role for the Caenorhabditis elegans ephrin EFN-4 in promoting primary neurite outgrowth in AIY interneurons and D-class motor neurons. Rescue experiments reveal that EFN-4 functions non-cell autonomously in the epidermis to promote primary neurite outgrowth. We also find that EFN-4 plays a role in promoting ectopic axon branching in a C. elegans model of X-linked Kallmann syndrome. In this context, EFN-4 functions non-cell autonomously in the body-wall muscle and in parallel with HS modification genes and HSPG core proteins. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system. PMID:26645816

  12. RGTA OTR 4120, a heparan sulfate proteoglycan mimetic, increases wound breaking strength and vasodilatory capability in healing rat full-thickness excisional wounds.

    PubMed

    Tong, Miao; Zbinden, Mariken M; Hekking, Ineke J M; Vermeij, Marcel; Barritault, Denis; van Neck, Johan W

    2008-01-01

    ReGeneraTing Agents (RGTAs), a family of polymers engineered to protect and stabilize heparin-binding growth factors, have been shown to promote tissue repair and regeneration. In this study, the effects of one of these polymers, RGTA OTR4120, on healing of full-thickness excisional wounds in rats were investigated. Two 1.5 cm diameter circular full-thickness excisional wounds were created on the dorsum of a rat. After creation of the wounds, RGTA OTR4120 was applied. The progress of healing was assessed quantitatively by evaluating the wound closure rate, vasodilatory capability, and wound breaking strength. The results showed a triple increase of the local vascular response to heat provocation in the RGTA OTR4120-treated wounds as compared with vehicle-treated wounds. On days 14 and 79 after surgery, the wounds treated with RGTA OTR4120 gained skin strength 12% and 48% of the unwounded skin, respectively, and displayed a significantly increased gain in skin strength when compared with control animals. These results raise the possibility of efficacy of RGTA OTR4120 in accelerating surgically cutaneous wound healing by enhancing the wound breaking strength and improving the microcirculation.

  13. NG2 glial cells regulate neuroimmunological responses to maintain neuronal function and survival

    PubMed Central

    Nakano, Masayuki; Tamura, Yasuhisa; Yamato, Masanori; Kume, Satoshi; Eguchi, Asami; Takata, Kumi; Watanabe, Yasuyoshi; Kataoka, Yosky

    2017-01-01

    NG2-expressing neural progenitor cells (i.e., NG2 glial cells) maintain their proliferative and migratory activities even in the adult mammalian central nervous system (CNS) and produce myelinating oligodendrocytes and astrocytes. Although NG2 glial cells have been observed in close proximity to neuronal cell bodies in order to receive synaptic inputs, substantive non-proliferative roles of NG2 glial cells in the adult CNS remain unclear. In the present study, we generated NG2-HSVtk transgenic rats and selectively ablated NG2 glial cells in the adult CNS. Ablation of NG2 glial cells produced defects in hippocampal neurons due to excessive neuroinflammation via activation of the interleukin-1 beta (IL-1β) pro-inflammatory pathway, resulting in hippocampal atrophy. Furthermore, we revealed that the loss of NG2 glial cell-derived hepatocyte growth factor (HGF) exacerbated these abnormalities. Our findings suggest that NG2 glial cells maintain neuronal function and survival via the control of neuroimmunological function. PMID:28195192

  14. The role of Ca 2+-related signaling in photodynamic injury of nerve and glial cells

    NASA Astrophysics Data System (ADS)

    Lobanov, A. V.; Petin, Y. O.; Uzdensky, A. B.

    2007-05-01

    Photodynamic therapy (PDT) inhibited and irreversibly abolished firing, caused necrosis of neurons, necrosis, apoptosis and proliferation of glial cells in the isolated crayfish stretch receptor. The role in these processes of the central components of Ca 2+-mediated signaling pathway: phospholipase C, calmodulin, calmodulin-dependent kinase II, and protein kinase C was studied using their inhibitors: ET-18, fluphenazine, KN-93, or staurosporine, respectively. ET-18 reduced functional inactivation of neurons, necrosis and apoptosis of glial cells. Fluphenazine and KN-93 reduced PDT-induced necrosis of neurons and glial cells. Staurosporine enhanced PDT-induced glial apoptosis. PDTinduced gliosis was prevented by KN-93 and staurosporine. Therefore, phospholipase C participated in neuron inactivation and glial necrosis and apoptosis. Calmodulin and calmodulin-dependent kinase II were involved in PDT-induced necrosis of neurons and glial cells but not in glial apoptosis. Protein kinase C protected glia from apoptosis and participated in PDT-induced gliosis and loss of neuronal activity. These data may be used for modulation of PDT of brain tumors.

  15. Glutamate-mediated protection of crayfish glial cells from PDT-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Rudkovskii, M. V.; Romanenko, N. P.; Berezhnaya, E. V.; Kovaleva, V. D.; Uzdensky, A. B.

    2011-03-01

    Photodynamic treatment that causes intense oxidative stress and kills cells is currently used in neurooncology. However, along with tumor it damages surrounding healthy neurons and glial cells. In order to study the possible role of glutamate-related signaling pathways in photodynamic injury of neurons and glia, we investigated photodynamic effect of alumophthalocyanine Photosens on isolated crayfish stretch receptor that consists of a single neuron surrounded by glial cells. The laser diode (670 nm, 0.4 W/cm2) was used for dye photoexcitation. Application of glutamate increased photodynamically induced necrosis of neurons and glial cells but significantly decreased glial apoptosis. The natural neuroglial mediator N-acetylaspartylglutamate, which releases glutamate after cleavage in the extracellular space by glutamate carboxypeptidase II, also inhibited photoinduced apoptosis. Inhibition of glutamate carboxypeptidase II, oppositely, enhanced apoptosis of glial cells. These data confirm the anti-apoptotic activity of glutamate. Application of NMDA or inhibition of NMDA receptors by MK801 did not influence photodynamic death of neurons and glial cells that indicated nonparticipation of NMDA receptors in these processes. Inhibition of metabotropic glutamate receptors by AP-3 decreased PDT-induced apoptosis. One can suggest that crayfish neurons naturally secrete NAAG, which being cleaved by GCOP produces glutamate. Glutamate prevents photoinduced apoptosis of glial cells possibly through metabotropic but not ionotropic glutamate receptors.

  16. Glutamate-mediated protection of crayfish glial cells from PDT-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Rudkovskii, M. V.; Romanenko, N. P.; Berezhnaya, E. V.; Kovaleva, V. D.; Uzdensky, A. B.

    2010-10-01

    Photodynamic treatment that causes intense oxidative stress and kills cells is currently used in neurooncology. However, along with tumor it damages surrounding healthy neurons and glial cells. In order to study the possible role of glutamate-related signaling pathways in photodynamic injury of neurons and glia, we investigated photodynamic effect of alumophthalocyanine Photosens on isolated crayfish stretch receptor that consists of a single neuron surrounded by glial cells. The laser diode (670 nm, 0.4 W/cm2) was used for dye photoexcitation. Application of glutamate increased photodynamically induced necrosis of neurons and glial cells but significantly decreased glial apoptosis. The natural neuroglial mediator N-acetylaspartylglutamate, which releases glutamate after cleavage in the extracellular space by glutamate carboxypeptidase II, also inhibited photoinduced apoptosis. Inhibition of glutamate carboxypeptidase II, oppositely, enhanced apoptosis of glial cells. These data confirm the anti-apoptotic activity of glutamate. Application of NMDA or inhibition of NMDA receptors by MK801 did not influence photodynamic death of neurons and glial cells that indicated nonparticipation of NMDA receptors in these processes. Inhibition of metabotropic glutamate receptors by AP-3 decreased PDT-induced apoptosis. One can suggest that crayfish neurons naturally secrete NAAG, which being cleaved by GCOP produces glutamate. Glutamate prevents photoinduced apoptosis of glial cells possibly through metabotropic but not ionotropic glutamate receptors.

  17. Mutation of a NCKX Eliminates Glial Microdomain Calcium Oscillations and Enhances Seizure Susceptibility

    PubMed Central

    Melom, Jan E.; Littleton, J. Troy

    2013-01-01

    Glia exhibit spontaneous and activity-dependent fluctuations in intracellular Ca2+, yet it is unclear whether glial Ca2+ oscillations are required during neuronal signaling. Somatic glial Ca2+ waves are primarily mediated by the release of intracellular Ca2+ stores, and their relative importance in normal brain physiology has been disputed. Recently, near-membrane microdomain Ca2+ transients were identified in fine astrocytic processes and found to arise via an intracellular store-independent process. Here, we describe the identification of rapid, near-membrane Ca2+ oscillations in Drosophila cortex glia of the CNS. In a screen for temperature-sensitive conditional seizure mutants, we identified a glial-specific Na+/Ca2+, K+ exchanger (zydeco) that is required for microdomain Ca2+ oscillatory activity. We found that zydeco mutant animals exhibit increased susceptibility to seizures in response to a variety of environmental stimuli, and that zydeco is required acutely in cortex glia to regulate seizure susceptibility. We also found that glial expression of calmodulin is required for stress-induced seizures in zydeco mutants, suggesting a Ca2+/calmodulin-dependent glial signaling pathway underlies glial–neuronal communication. These studies demonstrate that microdomain glial Ca2+ oscillations require NCKX-mediated plasma membrane Ca2+ flux, and that acute dysregulation of glial Ca2+ signaling triggers seizures. PMID:23325253

  18. The glial investment of the adult and developing antennal lobe of Drosophila

    PubMed Central

    Oland, Lynne A.; Biebelhausen, John P.; Tolbert, Leslie P.

    2009-01-01

    In recent years, the Drosophila olfactory system, with its unparalleled opportunities for genetic dissection of development and functional organization, has been used to study the development of central olfactory neurons and the molecular basis of olfactory coding. The results of these studies have been interpreted in the absence of a detailed understanding of the steps in maturation of glial cells in the antennal lobe. Here, we present a high-resolution study of the glia associated with olfactory glomeruli in adult and developing antennal lobes. The study provides a basis for comparison of findings in Drosophila with those in the moth Manduca sexta that indicate a critical role for glia in antennal lobe development. Using flies expressing GFP under a Nervana2 driver to visualize glia for confocal microscopy, and probing at higher resolution with the electron microscope, we find that glial development in Drosophila differs markedly from that in moths: glial cell bodies remain in a rind around the glomerular neuropil; glial processes ensheathe axon bundles in the nerve layer but likely contribute little to axonal sorting; their processes insinuate between glomeruli only very late and then form only a sparse, open network around each glomerulus; and glial processes invade the synaptic neuropil. Taking our results in the context of previous studies, we conclude that glial cells in the developing Drosophila antennal lobe are unlikely to play a strong role in either axonal sorting or glomerulus stabilization and that in the adult, glial processes do not electrically isolate glomeruli from their neighbors. PMID:18537134

  19. Radial glial cells play a key role in echinoderm neural regeneration

    PubMed Central

    2013-01-01

    Background Unlike the mammalian central nervous system (CNS), the CNS of echinoderms is capable of fast and efficient regeneration following injury and constitutes one of the most promising model systems that can provide important insights into evolution of the cellular and molecular events involved in neural repair in deuterostomes. So far, the cellular mechanisms of neural regeneration in echinoderm remained obscure. In this study we show that radial glial cells are the main source of new cells in the regenerating radial nerve cord in these animals. Results We demonstrate that radial glial cells of the sea cucumber Holothuria glaberrima react to injury by dedifferentiation. Both glia and neurons undergo programmed cell death in the lesioned CNS, but it is the dedifferentiated glial subpopulation in the vicinity of the injury that accounts for the vast majority of cell divisions. Glial outgrowth leads to formation of a tubular scaffold at the growing tip, which is later populated by neural elements. Most importantly, radial glial cells themselves give rise to new neurons. At least some of the newly produced neurons survive for more than 4 months and express neuronal markers typical of the mature echinoderm CNS. Conclusions A hypothesis is formulated that CNS regeneration via activation of radial glial cells may represent a common capacity of the Deuterostomia, which is not invoked spontaneously in higher vertebrates, whose adult CNS does not retain radial glial cells. Potential implications for biomedical research aimed at finding the cure for human CNS injuries are discussed. PMID:23597108

  20. The Drosophila blood-brain barrier: development and function of a glial endothelium

    PubMed Central

    Limmer, Stefanie; Weiler, Astrid; Volkenhoff, Anne; Babatz, Felix; Klämbt, Christian

    2014-01-01

    The efficacy of neuronal function requires a well-balanced extracellular ion homeostasis and a steady supply with nutrients and metabolites. Therefore, all organisms equipped with a complex nervous system developed a so-called blood-brain barrier, protecting it from an uncontrolled entry of solutes, metabolites or pathogens. In higher vertebrates, this diffusion barrier is established by polarized endothelial cells that form extensive tight junctions, whereas in lower vertebrates and invertebrates the blood-brain barrier is exclusively formed by glial cells. Here, we review the development and function of the glial blood-brain barrier of Drosophila melanogaster. In the Drosophila nervous system, at least seven morphologically distinct glial cell classes can be distinguished. Two of these glial classes form the blood-brain barrier. Perineurial glial cells participate in nutrient uptake and establish a first diffusion barrier. The subperineurial glial (SPG) cells form septate junctions, which block paracellular diffusion and thus seal the nervous system from the hemolymph. We summarize the molecular basis of septate junction formation and address the different transport systems expressed by the blood-brain barrier forming glial cells. PMID:25452710

  1. Photodynamic therapy-induced nitric oxide production in neuronal and glial cells

    NASA Astrophysics Data System (ADS)

    Kovaleva, Vera D.; Uzdensky, Anatoly B.

    2016-10-01

    Nitric oxide (NO) has been recently demonstrated to enhance apoptosis of glial cells induced by photodynamic therapy (PDT), but to protect glial cells from PDT-induced necrosis in the crayfish stretch receptor, a simple neuroglial preparation that consists of a single mechanosensory neuron enveloped by satellite glial cells. We used the NO-sensitive fluorescent probe 4,5-diaminofluorescein diacetate to study the distribution and dynamics of PDT-induced NO production in the mechanosensory neuron and surrounding glial cells. The NO production in the glial envelope was higher than in the neuronal soma axon and dendrites both in control and in experimental conditions. In dark NO generator, DEA NONOate or NO synthase substrate L-arginine hydrochloride significantly increased the NO level in glial cells, whereas NO scavenger 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) or inhibitors of NO synthase L-NG-nitro arginine methyl ester and Nω-nitro-L-arginine decreased it. PDT induced the transient increase in NO production with a maximum at 4 to 7 min after the irradiation start followed by its inhibition at 10 to 40 min. We suggested that PDT stimulated neuronal rather than inducible NO synthase isoform in glial cells, and the produced NO could mediate PDT-induced apoptosis.

  2. Anticoagulant properties and cytotoxic effect against HCT116 human colon cell line of sulfated glycosaminoglycans isolated from the Norway lobster (Nephrops norvegicus) shell.

    PubMed

    Sayari, Nadhem; Balti, Rafik; Ben Mansour, Mohamed; Ben Amor, Ikram; Graiet, Imen; Gargouri, Jalel; Bougatef, Ali

    2016-05-01

    Sulfated glycosaminoglycans (SGNL) were extracted for the first time from Norway lobster (Nephrops norvegicus) shell. The monosaccharide composition analysed by GC/MS revealed the presence of galacturonic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine. The analysis of SGNL with acetate cellulose electrophoresis in Zn-acetate revealed the presence of heparan sulfate (HS) and dermatan sulfate (DS). SGNL were evaluated for their anticoagulant activities using activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. After 21h incubation, HCT116 cell proliferation was inhibited (p<0.05) between 39.7 and 54.8% at 1.5-7.5mg/mL of SGNL. SGNL don't show hemolytic activity towards bovine erythrocytes and no cytotoxicity against the normal lymphocytes. The antiproliferative efficacy of these lobster glycosaminoglycans were probably related with the higher sulfate content. SGNL demonstrated promising antiproliferative and anticoagulant potential, which may be used as a novel, effective and promising antithrombotic agent.

  3. DOPAL is Transmissible to and Oligomerizes Alpha-Synuclein in Human Glial Cells

    PubMed Central

    Jinsmaa, Yunden; Sullivan, Patricia; Sharabi, Yehonatan; Goldstein, David S.

    2016-01-01

    Introduction Glial cytoplasmic inclusions (GCIs) containing alpha-synuclein (AS) are a neuropathologic hallmark of multiple system atrophy (MSA). Oligomerized AS is thought to be the pathogenic form of the protein. Glial cells normally express little AS, but they can take up AS from the extracellular fluid. 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate in the intra-neuronal metabolism of dopamine (DA), potently oligomerizes AS. In this study we tested whether DOPAL is taken up by human glial cells and augments intracellular oligomerization of AS. Methods DOPAL (exogenous or endogenous from co-incubation with PC12 cells) and AS (native or A53T mutant form) were added to the incubation medium of glial cells (glioblastoma or MO3.13 oligodendrocytes). Glial cellular contents of DOPAL and its intracellular metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at up to 180 minutes of incubation. Glial cellular AS oligomers were quantified by Western blotting. Results Neither glioblastoma nor MO3.13 cells contained endogenous catecholamines or AS. Co-incubation of the cells with DA-producing PC12 cells produced time-related increases in DOPAL and DOPAC contents. Similarly, glial cellular DOPAL and DOPAC contents increased rapidly after addition of DOPAL to the medium. After addition of native or A53T-AS, intracellular AS also increased. Incubation of glial cells with both DOPAL and AS enhanced the intracellular oligomerization of native and A53T-AS. Conclusions DOPAL is transmissible to glial cells and enhances intracellular oligomerization of AS. An interaction of DOPAL with AS might help explain the formation of CGIs in MSA. PMID:26777075

  4. Chondroitin sulfate/dermatan sulfate sulfatases from mammals and bacteria.

    PubMed

    Wang, Shumin; Sugahara, Kazuyuki; Li, Fuchuan

    2016-12-01

    Sulfatases that specifically catalyze the hydrolysis of the sulfate groups on chondroitin sulfate (CS)/dermatan sulfate (DS) poly- and oligosaccharides belong to the formylglycine-dependent family of sulfatases and have been widely found in various mammalian and bacterial organisms. However, only a few types of CS/DS sulfatase have been identified so far. Recently, several novel CS/DS sulfatases have been cloned and characterized. Advanced studies have provided significant insight into the biological function and mechanism of action of CS/DS sulfatases. Moreover, further studies will provide powerful tools for structural and functional studies of CS/DS as well as related applications. This article reviews the recent progress in CS/DS sulfatase research and is expected to initiate further research in this field.

  5. Sulfate-rich Archean Oceans

    NASA Astrophysics Data System (ADS)

    Brainard, J. L.; Choney, A. P.; Ohmoto, H.

    2012-12-01

    There is a widely held belief that prior to 2.4 Ga, the Archean oceans and atmosphere were reducing, and therefore sulfate poor (concentrations <0.1 mmol). However, there is mounting evidence from diverse rock types of Archean ages that sulfate concentrations were likely similar to those in the modern ocean (~28 mmol). In this study we demonstrate that in different lithologies, representing a wide range of marine environments, there is ubiquitous evidence for abundant seawater sulfate. One of the more apparent lines of evidence for sulfate rich Archean waters are bedded barite (BaSO4) deposits, such as those in the ~3.4 Ga Fig Tree Group, South Africa and ~3.5 Ga Dresser Formation, Western Australia (WA). These deposits are thick (>100 m), widely distributed (> km2), and contain only minor amounts of sulfides. These barite beds may have developed from reactions between Ba-rich hydrothermal fluids and evaporate bodies. Simple mass balance calculations suggest that the sulfate contents of the pre-evaporitic seawater must have been greater than ~1 mM. Some researchers have suggested that the SO4 for these beds was derived from the hydrolysis of SO2-rich magmatic fluids. However, this was unlikely as the reaction, 4SO2 + 4H2O → 3H2SO4 + H2S would have produced large amounts of sulfide, as well as sulfate minerals. Many Archean-aged volcanogenic massive sulfide (VMS) deposits, much like those of the younger ages, record evidence for abundant seawater sulfate. As VMS deposits are most likely formed by submarine hydrothermal fluids that developed from seawater circulating through the seafloor rock, much of the seawater sulfate is reduced to from sulfides at depths. However, some residual sulfate in the hydrothermal fluids, with or without the addition of sulfate from the local seawater, can form sulfate minerals such as barite at near the seafloor. The d34S relationships between barites and pyrites in the Archean VMS deposits are similar to those of the younger VMS

  6. Methods of producing sulfate salts of cations from heteroatomic compounds and dialkyl sulfates and uses thereof

    DOEpatents

    Friesen, Cody A.; Wolfe, Derek; Johnson, Paul Bryan

    2015-09-29

    Methods of preparing sulfate salts of heteroatomic compounds using dialkyl sulfates as a primary reactant are disclosed. Also disclosed are methods of making ionic liquids from the sulfate salts of the heteroatomic compound, and electrochemical cells comprising the ionic liquids.

  7. [Fine structure of glial cells in the central nervous system of the tapeworm Grillotia erinaceus (Cestoda: Trypanorhyncha)].

    PubMed

    Biserova, N M

    2008-01-01

    The problem of glial cells existing in parasitic and free living flatworms is correlated with organization of parenchyma in platyhelmintes. In the contrary to the widespread opinion that myelin-like envelopes and glial cells do not exist in the nervous system of parasitic flatworms, it has been shown by ultrastructural researches that Amphilina foliacea (Cestoda, Amphilinidea) has well developed glial cells and myelin-like envelopes in the ganglia and main cords, which include both glial cells and intercellular components. The aim of our research was to reveal and investigate in details structural components corresponding to the concept of the glial cell in the CNS of Grillotia erinaceus (Cestoda: Trypanorhyncha). Three types of glial cells have been found. The first type is the fibroblast-like glial cells; cells locate in the cerebral ganglion, contain in cytoplasm and extract out fibrillar matrix, form desmosomes and have supporting function. The glial cells of the second type form myeline-like envelope of the giant axons and bulbar nerves in scolex and have laminar cytoplasm. These cells are numerous and exceed in number the neurons bodies into the nerve. The glial cells of the third type form multilayer envelopes in the main nerve cords; extra cellular fibers and gap-junctions take place between the layers. There are contacts between the glial cells of the third type and excretory epithelium but specialized contacts with neurons have been not found. The existing of glial cells in free living and parasitic flatworms is discussed.

  8. Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

    PubMed Central

    Freitas, Hercules R.; Ferraz, Gabriel; Ferreira, Gustavo C.; Ribeiro-Resende, Victor T.; Chiarini, Luciana B.; do Nascimento, José Luiz M.; Matos Oliveira, Karen Renata H.; Pereira, Tiago de Lima; Ferreira, Leonardo G. B.; Kubrusly, Regina C.; Faria, Robson X.

    2016-01-01

    Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit. PMID:27078878

  9. Early Triassic seawater sulfate drawdown

    NASA Astrophysics Data System (ADS)

    Song, Huyue; Tong, Jinnan; Algeo, Thomas J.; Song, Haijun; Qiu, Haiou; Zhu, Yuanyuan; Tian, Li; Bates, Steven; Lyons, Timothy W.; Luo, Genming; Kump, Lee R.

    2014-03-01

    The marine sulfur cycle is intimately linked to global carbon fluxes, atmospheric composition, and climate, yet relatively little is known about how it responded to the end-Permian biocrisis, the largest mass extinction of the Phanerozoic. Here, we analyze carbonate-associated-sulfate (CAS) from three Permo-Triassic sections in South China in order to document the behavior of the C-S cycle and its relationship to marine environmental changes during the mass extinction and its aftermath. We find that δ34SCAS varied from +9‰ to +44‰ at rates up to 100‰ Myr-1 during the Griesbachian-Smithian substages of the Early Triassic. We model the marine sulfur cycle to demonstrate that such rapid variation required drawdown of seawater sulfate concentrations to ⩽4 mM and a reduction in its residence time to ⩽200 kyr. This shorter residence time resulted in positive covariation with δ13Ccarb due to strong coupling of the organic carbon and pyrite burial fluxes. Carbon and sulfur isotopic shifts were associated with contemporaneous changes in climate, marine productivity, and microbial sulfate reduction rates, with negative shifts in δ13Ccarb and δ34SCAS linked to warming, decreased productivity, and reduced sulfate reduction. Sustained cooling during the Spathian re-invigorated oceanic overturning circulation, reduced marine anoxia, and limited pyrite burial. As seawater sulfate built to higher concentrations during the Spathian, the coupling of the marine C and S cycles came to an end and a general amelioration of marine environmental conditions set the stage for a recovery of invertebrate faunas. Variation in seawater sulfate during the Early Triassic was probably controlled by climate change, possibly linked to major eruptive phases of the Siberian Traps.

  10. Wastewater treatment using ferrous sulfate

    SciTech Connect

    Boetskaya, K.P.; Ioffe, E.M.

    1980-01-01

    Treatment of industrial wastewater with coagulants is used extensively in the thorough removal of emulsified tars and oils. The central plant laboratory at the Zhdanov Coke Works conducted investigations of the treatment of wastewater, subsequently used for quenching coke, with ferrous sulfate. Laboratory tests and subsequent industrial tests demonstrated the efficiency of the method. In order to further intensify the wastewater treatment process we conducted laboratory tests with the addition of certain quantities of other coagulation reagents, for example polyacrylamide (PAA) and caustic soda, in addition to the ferrous sulfate. The combined use of polyacrylamide and ferrous sulfate permits instant coagulation of the sludge and very rapid (5 to 10 min) clarification of the water. In addition, in this case the degree of purification of the water is less dependent on the initial concentration of impurities. The purification is also improved when caustic soda is added, raising the pH. From the data it is apparent that an identical degree of purification of the water may be achieved either by increasing the consumption of ferrous sulfate, or by adding PAA or NaOH. During industrial tests of the purification of wastewater with ferrous sulfate, we also investigated the resulting sludge. The use of ferrous sulfate causes a significant increase in its quantity (by a factor of 1.5 to 1.8) and in its oil content (by a factor of 2 to 2.5). The water content in the sludge decreases. The sludge (in the quantity of 0.6% of the charge) may be added to the coking charge.

  11. Acid Sulfate Alteration on Mars

    NASA Technical Reports Server (NTRS)

    Ming, D. W.; Morris, R. V.

    2016-01-01

    A variety of mineralogical and geochemical indicators for aqueous alteration on Mars have been identified by a combination of surface and orbital robotic missions, telescopic observations, characterization of Martian meteorites, and laboratory and terrestrial analog studies. Acid sulfate alteration has been identified at all three landing sites visited by NASA rover missions (Spirit, Opportunity, and Curiosity). Spirit landed in Gusev crater in 2004 and discovered Fe-sulfates and materials that have been extensively leached by acid sulfate solutions. Opportunity landing on the plains of Meridiani Planum also in 2004 where the rover encountered large abundances of jarosite and hematite in sedimentary rocks. Curiosity landed in Gale crater in 2012 and has characterized fluvial, deltaic, and lacustrine sediments. Jarosite and hematite were discovered in some of the lacustrine sediments. The high elemental abundance of sulfur in surface materials is obvious evidence that sulfate has played a major role in aqueous processes at all landing sites on Mars. The sulfate-rich outcrop at Meridiani Planum has an SO3 content of up to 25 wt.%. The interiors of rocks and outcrops on the Columbia Hills within Gusev crater have up to 8 wt.% SO3. Soils at both sites generally have between 5 to 14 wt.% SO3, and several soils in Gusev crater contain around 30 wt.% SO3. After normalization of major element compositions to a SO3-free basis, the bulk compositions of these materials are basaltic, with a few exceptions in Gusev crater and in lacustrine mudstones in Gale crater. These observations suggest that materials encountered by the rovers were derived from basaltic precursors by acid sulfate alteration under nearly isochemical conditions (i.e., minimal leaching). There are several cases, however, where acid sulfate alteration minerals (jarosite and hematite) formed in open hydrologic systems, e.g., in Gale crater lacustrine mudstones. Several hypotheses have been suggested for the

  12. Modulating the Delicate Glial-Neuronal Interactions in Neuropathic Pain: Promises and Potential Caveats

    PubMed Central

    Tiwari, Vinod; Guan, Yun; Raja, Srinivasa N.

    2014-01-01

    During neuropathic pain, glial cells (mainly astrocytes and microglia) become activated and initiate a series of signaling cascades that modulate pain processing at both spinal and supraspinal levels. It has been generally accepted that glial cell activation contributes to neuropathic pain because glia release proinflammatory cytokines, chemokines, and factors such as calcitonin gene-related peptide, substance P, and glutamate, which are known to facilitate pain signaling. However, recent research has shown that activation of glia also leads to some beneficial outcomes. Glia release anti-inflammatory factors that protect against neurotoxicity and restore normal pain. Accordingly, use of glial inhibitors might compromise the protective functions of glia in addition to suppressing their detrimental effects. With a better understanding of how different conditions affect glial cell activation, we may be able to promote the protective function of glia and pave the way for future development of novel, safe, and effective treatments of neuropathic pain. PMID:24820245

  13. An electrically resistive sheet of glial cells for amplifying signals of neuronal extracellular recordings.

    PubMed

    Matsumura, R; Yamamoto, H; Niwano, M; Hirano-Iwata, A

    2016-01-11

    Electrical signals of neuronal cells can be recorded non-invasively and with a high degree of temporal resolution using multielectrode arrays (MEAs). However, signals that are recorded with these devices are small, usually 0.01%-0.1% of intracellular recordings. Here, we show that the amplitude of neuronal signals recorded with MEA devices can be amplified by covering neuronal networks with an electrically resistive sheet. The resistive sheet used in this study is a monolayer of glial cells, supportive cells in the brain. The glial cells were grown on a collagen-gel film that is permeable to oxygen and other nutrients. The impedance of the glial sheet was measured by electrochemical impedance spectroscopy, and equivalent circuit simulations were performed to theoretically investigate the effect of covering the neurons with such a resistive sheet. Finally, the effect of the resistive glial sheet was confirmed experimentally, showing a 6-fold increase in neuronal signals. This technique feasibly amplifies signals of MEA recordings.

  14. A New Outlook on Mental Illnesses: Glial Involvement Beyond the Glue

    PubMed Central

    Elsayed, Maha; Magistretti, Pierre J.

    2015-01-01

    Mental illnesses have long been perceived as the exclusive consequence of abnormalities in neuronal functioning. Until recently, the role of glial cells in the pathophysiology of mental diseases has largely been overlooked. However recently, multiple lines of evidence suggest more diverse and significant functions of glia with behavior-altering effects. The newly ascribed roles of astrocytes, oligodendrocytes and microglia have led to their examination in brain pathology and mental illnesses. Indeed, abnormalities in glial function, structure and density have been observed in postmortem brain studies of subjects diagnosed with mental illnesses. In this review, we discuss the newly identified functions of glia and highlight the findings of glial abnormalities in psychiatric disorders. We discuss these preclinical and clinical findings implicating the involvement of glial cells in mental illnesses with the perspective that these cells may represent a new target for treatment. PMID:26733803

  15. Embryonic development of glial cells and myelin in the shark, Chiloscyllium punctatum

    PubMed Central

    Rotenstein, Lisa; Milanes, Anthony; Juarez, Marilyn; Reyes, Michelle; de Bellard, Maria Elena

    2009-01-01

    Glial cells are responsible for a wide range of functions in the nervous system of vertebrates. The myelinated nervous systems of extant elasmobranchs have the longest independent history of all gnathostomes. Much is known about the development of glia in other jawed vertebrates, but research in elasmobranchs is just beginning to reveal the mechanisms guiding neurodevelopment. This study examines the development of glial cells in the bamboo shark, Chiloscyllium punctatum, by identifying the expression pattern of several classic glial and myelin proteins. We show for the first time that glial development in the bamboo shark (Ch. punctamum) embryo follows closely the one observed in other vertebrates and that neural development seems to proceed at a faster rate in the PNS than in the CNS. In addition, we observed more myelinated tracts in the PNS than in the CNS, and as early as stage 32, suggesting that the ontogeny of myelin in sharks is closer to osteichthyans than agnathans. PMID:19733690

  16. An electrically resistive sheet of glial cells for amplifying signals of neuronal extracellular recordings

    NASA Astrophysics Data System (ADS)

    Matsumura, R.; Yamamoto, H.; Niwano, M.; Hirano-Iwata, A.

    2016-01-01

    Electrical signals of neuronal cells can be recorded non-invasively and with a high degree of temporal resolution using multielectrode arrays (MEAs). However, signals that are recorded with these devices are small, usually 0.01%-0.1% of intracellular recordings. Here, we show that the amplitude of neuronal signals recorded with MEA devices can be amplified by covering neuronal networks with an electrically resistive sheet. The resistive sheet used in this study is a monolayer of glial cells, supportive cells in the brain. The glial cells were grown on a collagen-gel film that is permeable to oxygen and other nutrients. The impedance of the glial sheet was measured by electrochemical impedance spectroscopy, and equivalent circuit simulations were performed to theoretically investigate the effect of covering the neurons with such a resistive sheet. Finally, the effect of the resistive glial sheet was confirmed experimentally, showing a 6-fold increase in neuronal signals. This technique feasibly amplifies signals of MEA recordings.

  17. Inflammation after Ischemic Stroke: The Role of Leukocytes and Glial Cells

    PubMed Central

    Kim, Jong Youl; Park, Joohyun; Chang, Ji Young; Kim, Sa-Hyun

    2016-01-01

    The immune response after stroke is known to play a major role in ischemic brain pathobiology. The inflammatory signals released by immune mediators activated by brain injury sets off a complex series of biochemical and molecular events which have been increasingly recognized as a key contributor to neuronal cell death. The primary immune mediators involved are glial cells and infiltrating leukocytes, including neutrophils, monocytes and lymphocyte. After ischemic stroke, activation of glial cells and subsequent release of pro- and anti-inflammatory signals are important for modulating both neuronal cell damage and wound healing. Infiltrated leukocytes release inflammatory mediators into the site of the lesion, thereby exacerbating brain injury. This review describes how the roles of glial cells and circulating leukocytes are a double-edged sword for neuroinflammation by focusing on their detrimental and protective effects in ischemic stroke. Here, we will focus on underlying characterize of glial cells and leukocytes under inflammation after ischemic stroke. PMID:27790058

  18. Implications of glial nitric oxide in neurodegenerative diseases

    PubMed Central

    Yuste, Jose Enrique; Tarragon, Ernesto; Campuzano, Carmen María; Ros-Bernal, Francisco

    2015-01-01

    Nitric oxide (NO) is a pleiotropic janus-faced molecule synthesized by nitric oxide synthases (NOS) which plays a critical role in a number of physiological and pathological processes in humans. The physiological roles of NO depend on its local concentrations, as well as its availability and the nature of downstream target molecules. Its double-edged sword action has been linked to neurodegenerative disorders. Excessive NO production, as the evoked by inflammatory signals, has been identified as one of the major causative reasons for the pathogenesis of several neurodegenerative diseases. Moreover, excessive NO synthesis under neuroinflammation leads to the formation of reactive nitrogen species and neuronal cell death. There is an intimate relation between microglial activation, NO and neuroinflammation in the human brain. The role of NO in neuroinflammation has been defined in animal models where this neurotransmitter can modulate the inflammatory process acting on key regulatory pathways, such as those associated with excitotoxicity processes induced by glutamate accumulation and microglial activation. Activated glia express inducible NOS and produce NO that triggers calcium mobilization from the endoplasmic reticulum, activating the release of vesicular glutamate from astroglial cells resulting in neuronal death. This change in microglia potentially contributes to the increased age-associated susceptibility and neurodegeneration. In the current review, information is provided about the role of NO, glial activation and age-related processes in the central nervous system (CNS) that may be helpful in the isolation of new therapeutic targets for aging and neurodegenerative diseases. PMID:26347610

  19. Glial Reactivity in Resistance to Methamphetamine-Induced Neurotoxicity

    PubMed Central

    Friend, Danielle M.; Keefe, Kristen A.

    2013-01-01

    Neurotoxic regimens of methamphetamine (METH) result in reactive microglia and astrocytes in striatum. Prior data indicate that rats with partial dopamine (DA) loss resulting from prior exposure to METH are resistant to further decreases in striatal DA when re-exposed to METH 30 days later. Such resistant animals also do not show an activated microglia phenotype, suggesting a relation between microglial activation and METH-induced neurotoxicity. To date, the astrocyte response in such resistance has not been examined. Thus, this study examined glial-fibrillary acidic protein (GFAP) and CD11b protein expression in striata of animals administered saline or a neurotoxic regimen of METH on postnatal days 60 and/or 90 (Saline:Saline, Saline:METH, METH:Saline, METH:METH). Consistent with previous work, animals experiencing acute toxicity (Saline:METH) showed both activated microglia and astocytes, whereas those resistant to the acute toxicity (METH:METH) did not show activated microglia. Interestingly, GFAP expression remained elevated in rats exposed to METH at PND60 (METH:Saline), and was not elevated further in resistant rats treated for the second time with METH (METH:METH). These data suggest that astrocytes remain reactive up to 30 days post-METH exposure. Additionally, these data indicate that astrocyte reactivity does not reflect acute, METH-induced DA terminal toxicity, whereas microglial reactivity does. PMID:23414433

  20. Anti-aging effects of guanosine in glial cells.

    PubMed

    Souza, Débora Guerini; Bellaver, Bruna; Bobermin, Larissa Daniele; Souza, Diogo Onofre; Quincozes-Santos, André

    2016-12-01

    Guanosine, a guanine-based purine, has been shown to exert beneficial roles in in vitro and in vivo injury models of neural cells. Guanosine is released from astrocytes and modulates important astroglial functions, including glutamatergic metabolism, antioxidant, and anti-inflammatory activities. Astrocytes are crucial for regulating the neurotransmitter system and synaptic information processes, ionic homeostasis, energy metabolism, antioxidant defenses, and the inflammatory response. Aging is a natural process that induces numerous changes in the astrocyte functionality. Thus, the search for molecules able to reduce the glial dysfunction associated with aging may represent an approach for avoiding the onset of age-related neurological diseases. Hence, the aim of this study was to evaluate the anti-aging effects of guanosine, using primary astrocyte cultures from newborn, adult, and aged Wistar rats. Concomitantly, we evaluated the role of heme oxygenase 1 (HO-1) in guanosine-mediated glioprotection. We observed age-dependent changes in glutamate uptake, glutamine synthetase (GS) activity, the glutathione (GSH) system, pro-inflammatory cytokine (tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β)) release, and the transcriptional activity of nuclear factor kB (NFkB), which were prevented by guanosine in an HO-1-dependent manner. Our findings suggest guanosine to be a promising therapeutic agent able to provide glioprotection during the aging process. Thus, this study contributes to the understanding of the cellular and molecular mechanisms of guanosine in the aging process.

  1. Glial activation in the collagenase model of nociception associated with osteoarthritis

    PubMed Central

    Almeida, Lígia; Potes, Catarina S; Ferreira, Ana Rita; Castro-Lopes, José M; Ferreira-Gomes, Joana; Neto, Fani L

    2017-01-01

    Background Experimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase. Methods Osteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3–L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection. Results GFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception. Conclusion Collagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord

  2. Glial activation in the collagenase model of nociception associated with osteoarthritis.

    PubMed

    Adães, Sara; Almeida, Lígia; Potes, Catarina S; Ferreira, Ana Rita; Castro-Lopes, José M; Ferreira-Gomes, Joana; Neto, Fani L

    2017-01-01

    Background Experimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase. Methods Osteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3-L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection. Results GFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception. Conclusion Collagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord

  3. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed

    Gommerat, I; Gola, M

    1996-09-15

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  4. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed Central

    Gommerat, I; Gola, M

    1996-01-01

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  5. Changes in composition and sulfation patterns of glycoaminoglycans in renal cell carcinoma.

    PubMed

    Ucakturk, Ebru; Akman, Orkun; Sun, Xiaojun; Baydar, Dilek Ertoy; Dolgun, Anil; Zhang, Fuming; Linhardt, Robert J

    2016-02-01

    Glycosaminoglycans (GAGs) are heterogeneous, linear, highly charged, anionic polysaccharides consisting of repeating disaccharides units. GAGs have some biological significance in cancer progression (invasion and metastasis) and cell signaling. In different cancer types, GAGs undergo specific structural changes. In the present study, in depth investigation of changes in sulfation pattern and composition of GAGs, heparan sulfate (HS)/heparin (HP), chondroitin sulfate (CS)/dermatan sulfate and hyaluronan (HA) in normal renal tissue (NRT) and renal cell carcinoma tissue (RCCT) were evaluated. The statistical evaluation showed that alteration of the HS (HSNRT = 415.1 ± 115.3; HSRCCT = 277.5 ± 134.3), and CS (CSNRT = 35.3 ± 12.3; CSRCCT = 166.7 ± 108.8) amounts (in ng/mg dry tissue) were statistically significant (p < 0.05). Sulfation pattern in NRT and RCCT was evaluated to reveal disaccharide profiles. Statistical analyses showed that RCCT samples contain significantly increased amounts (in units of ng/mg dry tissue) of 4SCS (NRT = 25.7 ± 9.4; RCCT = 117.1 ± 73.9), SECS (NRT = 0.7 ± 0.3; RCCT = 4.7 ± 4.5), 6SCS (NRT = 6.1 ± 2.7; RCCT = 39.4 ± 34.7) and significantly decreased amounts (in units of ng/mg dry tissue) of NS6SHS (RCCT = 28.6 ± 6.5, RCCT = 10.2 ± 8.0), NS2SHS (RCCT = 44.2 ± 13.8; RCCT = 27.2 ± 15.0), NSHS (NRT = 68.4 ± 15.8; RCCT = 50.4 ± 21.2), 2S6SHS (NRT = 1.0 ± 0.4; RCCT = 0.4 ± 0.3), and 6SHS (NRT = 60.6 ± 17.5; RCCT = 24.9 ± 12.3). If these changes in GAGs are proven to be specific and sensitive, they may serve as potential biomarkers in RCC. Our findings are likely to help us to show the direction for further investigations to be able to bring different diagnostic and prognostic approaches in renal tumors.

  6. Observations on glial inclusion bodies in a case of acute disseminated sclerosis

    PubMed Central

    Field, E. J.; Miller, Henry; Russell, Dorothy S.

    1962-01-01

    An unusual rod-like structure enclosed within a vacuole is described as occurring in enlarged glial cells associated with the lesions encountered in an uncommonly acute case of multiple sclerosis apparently heralded by an attack of `viral encephalitis'. Similar bodies were not found in a variety of other e