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Sample records for carbohydrate binding modules

  1. Using structure to inform carbohydrate binding module function

    NARCIS (Netherlands)

    Abbott, D. Wade; Lammerts van Bueren, Alicia

    2014-01-01

    Generally, non-catalytic carbohydrate binding module (CBM) specificity has been shown to parallel the catalytic activity of the carbohydrate active enzyme (CAZyme) module it is appended to. With the rapid expansion in metagenomic sequence space for the potential discovery of new CBMs in addition to

  2. Modeling of Carbohydrate Binding Modules Complexed to Cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Nimlos, M. R.; Beckham, G. T.; Bu, L.; Himmel, M. E.; Crowley, M. F.; Bomble, Y. J.

    2012-01-01

    Modeling results are presented for the interaction of two carbohydrate binding modules (CBMs) with cellulose. The family 1 CBM from Trichoderma reesei's Cel7A cellulase was modeled using molecular dynamics to confirm that this protein selectively binds to the hydrophobic (100) surface of cellulose fibrils and to determine the energetics and mechanisms for locating this surface. Modeling was also conducted of binding of the family 4 CBM from the CbhA complex from Clostridium thermocellum. There is a cleft in this protein, which may accommodate a cellulose chain that is detached from crystalline cellulose. This possibility is explored using molecular dynamics.

  3. Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

    Science.gov (United States)

    Shoseyov, Oded; Shani, Ziv; Levy, Ilan

    2006-01-01

    Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular “Velcro” for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications. PMID:16760304

  4. Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-Binding Module

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, C. B.; Talib, M. F.; McCabe, C.; Bu, L.; Adney, W. S.; Himmel, M. E.; Crowley, M. F.; Beckham, G. T.

    2012-01-27

    Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3-6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function.

  5. A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module.

    Science.gov (United States)

    Matthews, Peter R; Schindler, Michael; Howles, Paul; Arioli, Tony; Williamson, Richard E

    2010-10-01

    Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5' and 3' rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed.

  6. Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

    Directory of Open Access Journals (Sweden)

    Cicortas Gunnarsson Lavinia

    2009-10-01

    Full Text Available Abstract Background Molecular evolution of carbohydrate binding modules (CBM is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

  7. The carbohydrate-binding module family 20-diversity, structure, and function

    DEFF Research Database (Denmark)

    Christiansen, Camilla; Abou Hachem, Maher; Janecek, S.

    2009-01-01

    Starch-active enzymes often possess starch-binding domains (SBDs) mediating attachment to starch granules and other high molecular weight substrates. SBDs are divided into nine carbohydrate-binding module (CBM) families, and CBM20 is the earliest-assigned and best characterized family. High...... diversity characterizes CBM20s, which occur in starch-active glycoside hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch-phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases......, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily...

  8. Binding Preferences, Surface Attachment, Diffusivity, and Orientation of a Family 1 Carbohydrate-Binding Module on Cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Nimlos, M. R.; Beckham, G. T.; Matthews, J. F.; Bu, L.; Himmel, M. E.; Crowley, M. F.

    2012-06-08

    Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 {mu}s of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.

  9. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  10. Engineering and Comparative Characteristics of Double Carbohydrate Binding Modules as a Strength Additive for Papermaking Applications

    Directory of Open Access Journals (Sweden)

    Xiaoran Shi

    2014-04-01

    Full Text Available In this study, four engineered proteins containing two family 1 and/or family 3 carbohydrate binding modules (CBMs were constructed and expressed as soluble forms in Escherichia coli. Their binding performances and effect on paper’s mechanical properties were comprehensively studied with the aim to design suitably engineered CBMs as novel biomaterials for use in the production of new cellulose materials. The recombinant engineered double CBMs exhibited obvious differences in their adsorption to different cellulosic substrates. The CBM3-GS-CBM3 was the most effective in enhancing paper mechanical properties in terms of folding endurance (27.4% and tensile strength (15.5% among the four engineered double CBMs, but it gave rise to only a slight increase in bursting strength (3.1%. On the other hand, CBM1-NL-CBM1 achieved a significant simultaneous increase in tensile strength (12.6% and burst strength (8.8%, as well as folding endurance (16.7%. Unexpectedly, CBM3-GS-CBM1 and CBM3-NL-CBM1 had the lowest effective paper property improvement. The differences in types of CBMs and linker peptides in engineered double CBMs may contribute to the considerable differences in their cellulose binding and paper property modification. Our data suggested that CBM1-NL-CBM1 may provide a better upgrade of the secondary pulp, which makes it very suitable for fiber recycling. Meanwhile, CBM3-GS-CBM3 may have particular potential for paper manufacture requiring high folding endurance.

  11. RETENTION AND PAPER-STRENGTH CHARACTERISTICS OF ANIONIC POLYACRYLAMIDES CONJUGATED WITH CARBOHYDRATE-BINDING MODULES

    Directory of Open Access Journals (Sweden)

    Shingo Yokota

    2009-02-01

    Full Text Available The retention behavior of polymers having the specific affinities of glyco-hydrolases for pulp fibers was investigated with regard to paper-strength enhancement in contaminated papermaking systems. Carbohydrate-binding modules (CBMs of cellulases derived from Trichoderma viride and T. reesei, and of xylanase from Thermomyces lanuginosus, were obtained by site-directed digestion with papain, then introduced into anionic polyacrylamide (A-PAM via a peptide condensation reaction. Three types of CBM-conjugated A-PAMs (CBM-A-PAMs displayed different retention behavior, depending on the kind of pulp substrates, i.e. hardwood and softwood fibers. The CBM-A-PAM from T. viride demonstrated good additive retention for hardwood pulp fibers, resulting in high tensile strength of paper sheets, even under contaminated conditions in the presence of Ca2+ ions and ligninsulfonate. The CBM-A-PAM from T. reesei showed better performance for softwood than for hardwood sheets. The xylanase CBM-A-PAM was preferentially retained on hardwood fibers in which hemicelluloses might be present. Such an additive retention system, with inherent affinities of enzymes for pulp fibers, is expected to expand the application range of CBM-polymers in practical wet-end processes.

  12. Specific tracking of xylan using fluorescent-tagged carbohydrate-binding module 15 as molecular probe

    OpenAIRE

    Khatri, Vinay; Hébert-Ouellet, Yannick; Meddeb-Mouelhi, Fatma; Beauregard,Marc

    2016-01-01

    Background Xylan has been identified as a physical barrier which limits cellulose accessibility by covering the outer surface of fibers and interfibrillar space. Therefore, tracking xylan is a prerequisite for understanding and optimizing lignocellulosic biomass processes. Results In this study, we developed a novel xylan tracking approach using a two-domain probe called OC15 which consists of a fusion of Cellvibrio japonicus carbohydrate-binding domain 15 with the fluorescent protein mOrange...

  13. Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7.

    Science.gov (United States)

    Mamo, Gashaw; Hatti-Kaul, Rajni; Mattiasson, Bo

    2007-01-01

    Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)(6) tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.

  14. Conformational Analysis of the Streptococcus pneumoniae Hyaluronate Lyase and Characterization of Its Hyaluronan-specific Carbohydrate-binding Module*

    Science.gov (United States)

    Suits, Michael D. L.; Pluvinage, Benjamin; Law, Adrienne; Liu, Yan; Palma, Angelina S.; Chai, Wengang; Feizi, Ten; Boraston, Alisdair B.

    2014-01-01

    For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl. PMID:25100731

  15. Expression of the C-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum in tobacco plant

    NARCIS (Netherlands)

    Olawole, O.

    2009-01-01

    Carbohydrate-binding modules have been shown to alter plant cell wall structural architecture. Hence, they have the potential application of being used to engineer the plant to produce tailor-made natural fibers in the cell wall. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that

  16. Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Binti Jamek, Shariza; Nyffenegger, Christian; Muschiol, Jan

    2017-01-01

    /α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type...... A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools...

  17. The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

    Directory of Open Access Journals (Sweden)

    Luke J Alderwick

    2011-02-01

    Full Text Available The D-arabinan-containing polymers arabinogalactan (AG and lipoarabinomannan (LAM are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM. Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985 at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.

  18. Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch; Seo, E.S.; Dilokpimol, Adiphol

    2008-01-01

    with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit...... that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley alpha-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends...

  19. Expression of the C-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum in tobacco plant

    OpenAIRE

    Olawole, O.

    2009-01-01

    Carbohydrate-binding modules have been shown to alter plant cell wall structural architecture. Hence, they have the potential application of being used to engineer the plant to produce tailor-made natural fibers in the cell wall. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The C-terminal CBM22-2 of the glycoside hydrolase (GH) 10 had been characterized to interact with xylan, a major hemicellulosic component in the secondary cell wall of ...

  20. A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

    Science.gov (United States)

    Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd

    2014-02-10

    Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.

  1. Carbohydrate Binding Module and Its Applications%碳水化合物结合结构域及其应用

    Institute of Scientific and Technical Information of China (English)

    周顺华

    2016-01-01

    碳水化合物结合结构域(Carbohydrate binding module),简称CBM.其是一种具有独立折叠结构的蛋白质.主要功能是特异性结合多糖(尤其不溶性多糖),并能通过4种机制来提高碳水化合物活性酶类的催化效率.其在生物工艺、环境的生物疗法、分析诊断工具及改善多糖水解酶酶特性上均具有广泛的应用.

  2. Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution.

    Science.gov (United States)

    Li, Shangyong; Yang, Xuemei; Bao, Mengmeng; Wu, Ying; Yu, Wengong; Han, Feng

    2015-05-01

    The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

  3. A starch-binding domain identified in α-amylase (AmyP) represents a new family of carbohydrate-binding modules that contribute to enzymatic hydrolysis of soluble starch.

    Science.gov (United States)

    Peng, Hui; Zheng, Yunyun; Chen, Maojiao; Wang, Ying; Xiao, Yazhong; Gao, Yi

    2014-04-02

    A novel starch-binding domain (SBD) that represents a new carbohydrate-binding module family (CBM69) was identified in the α-amylase (AmyP) of the recently established alpha-amylase subfamily GH13_37. The SBD and its homologues come mostly from marine bacteria, and phylogenetic analysis indicates that they are closely related to the CBM20 and CBM48 families. The SBD exhibited a binding preference toward raw rice starch, but the truncated mutant (AmyPΔSBD) still retained similar substrate preference. Kinetic analyses revealed that the SBD plays an important role in soluble starch hydrolysis because different catalytic efficiencies have been observed in AmyP and the AmyPΔSBD.

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

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...... for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon...... sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites...

  5. Treatment with Ginger Ameliorates Fructose-Induced Fatty Liver and Hypertriglyceridemia in Rats: Modulation of the Hepatic Carbohydrate Response Element-Binding Protein-Mediated Pathway

    Directory of Open Access Journals (Sweden)

    Huanqing Gao

    2012-01-01

    Full Text Available Ginger has been demonstrated to improve lipid derangements. However, its underlying triglyceride-lowering mechanisms remain unclear. Fructose overconsumption is associated with increase in hepatic de novo lipogenesis, thereby resulting in lipid derangements. Here we found that coadministration of the alcoholic extract of ginger (50 mg/kg/day, oral gavage, once daily over 5 weeks reversed liquid fructose-induced increase in plasma triglyceride and glucose concentrations and hepatic triglyceride content in rats. Plasma nonesterified fatty acid concentration was also decreased. Attenuation of the increased vacuolization and Oil Red O staining area was evident on histological examination of liver in ginger-treated rats. However, ginger treatment did not affect chow intake and body weight. Further, ginger treatment suppressed fructose-stimulated overexpression of carbohydrate response element-binding protein (ChREBP at the mRNA and protein levels in the liver. Consequently, hepatic expression of the ChREBP-targeted lipogenic genes responsible for fatty acid biosynthesis was also downregulated. In contrast, expression of neither peroxisome proliferator-activated receptor- (PPAR- alpha and its downstream genes, nor PPAR-gamma and sterol regulatory element-binding protein 1c was altered. Thus the present findings suggest that in rats, amelioration of fructose-induced fatty liver and hypertriglyceridemia by ginger treatment involves modulation of the hepatic ChREBP-mediated pathway.

  6. The structure of a Streptomyces avermitilis α-L-rhamnosidase reveals a novel carbohydrate-binding module CBM67 within the six-domain arrangement.

    Science.gov (United States)

    Fujimoto, Zui; Jackson, Adam; Michikawa, Mari; Maehara, Tomoko; Momma, Mitsuru; Henrissat, Bernard; Gilbert, Harry J; Kaneko, Satoshi

    2013-04-26

    α-L-rhamnosidases hydrolyze α-linked L-rhamnosides from oligosaccharides or polysaccharides. We determined the crystal structure of the glycoside hydrolase family 78 Streptomyces avermitilis α-L-rhamnosidase (SaRha78A) in its free and L-rhamnose complexed forms, which revealed the presence of six domains N, D, E, F, A, and C. In the ligand complex, L-rhamnose was bound in the proposed active site of the catalytic module, revealing the likely catalytic mechanism of SaRha78A. Glu(636) is predicted to donate protons to the glycosidic oxygen, and Glu(895) is the likely catalytic general base, activating the nucleophilic water, indicating that the enzyme operates through an inverting mechanism. Replacement of Glu(636) and Glu(895) resulted in significant loss of α-rhamnosidase activity. Domain D also bound L-rhamnose in a calcium-dependent manner, with a KD of 135 μm. Domain D is thus a non-catalytic carbohydrate binding module (designated SaCBM67). Mutagenesis and structural data identified the amino acids in SaCBM67 that target the features of L-rhamnose that distinguishes it from the other major sugars present in plant cell walls. Inactivation of SaCBM67 caused a substantial reduction in the activity of SaRha78A against the polysaccharide composite gum arabic, but not against aryl rhamnosides, indicating that SaCBM67 contributes to enzyme function against insoluble substrates.

  7. Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module.

    Science.gov (United States)

    Kurokawa, J; Hemjinda, E; Arai, T; Kimura, T; Sakka, K; Ohmiya, K

    2002-08-01

    The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548-554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTDeltadoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTDeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan, and low activity toward xylan. The V(max) and K(m) values were 137 micro mol min(-1) mg(-1) and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.

  8. Dynamics of starch granule biogenesis - the role of redox-regulated enzymes and low-affinity carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Blennow, A.; Svensson, Birte

    2010-01-01

    The deposition and degradation of starch in plants is subject to extensive post-translational regulation. To permit degradation of B-type crystallites present in tuberous and leaf starch these starch types are phosphorylated by glucan, water dikinase (GWD). At the level of post-translational redo...... families and can enable diurnal dynamics of starch-enzyme recognition. Such diurnal changes in starch binding have been indicated for the redox-regulated GWD and SEX4....

  9. Dissecting the functional significance of non-catalytic carbohydrate binding modules in the deconstruction of plant cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, Michael G. [Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center

    2017-03-16

    The project seeks to investigate the mechanism by which CBMs potentiate the activity of glycoside hydrolases against complete plant cell walls. The project is based on the hypothesis that the wide range of CBMs present in bacterial enzymes maximize the potential target substrates by directing the cognate enzymes not only to different regions of a specific plant cell wall, but also increases the range of plant cell walls that can be degraded. In addition to maximizing substrate access, it was also proposed that CBMs can target specific subsets of hydrolases with complementary activities to the same region of the plant cell wall, thereby maximizing the synergistic interactions between these enzymes. This synergy is based on the premise that the hydrolysis of a specific polysaccharide will increase the access of closely associated polymers to enzyme attack. In addition, it is unclear whether the catalytic module and appended CBM of modular enzymes have evolved unique complementary activities.

  10. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    Carbohydrate active enzymes, particularly those that are active on polysaccharides, are often found associated with carbohydrate binding modules (CBMs), which can play several roles in supporting enzyme function, such as localizing the enzyme to the substrate. However, the presence of CBMs...

  11. Unique carbohydrate binding platforms employed by the glucan phosphatases.

    Science.gov (United States)

    Emanuelle, Shane; Brewer, M Kathryn; Meekins, David A; Gentry, Matthew S

    2016-07-01

    Glucan phosphatases are a family of enzymes that are functionally conserved at the enzymatic level in animals and plants. These enzymes bind and dephosphorylate glycogen in animals and starch in plants. While the enzymatic function is conserved, the glucan phosphatases employ distinct mechanisms to bind and dephosphorylate glycogen or starch. The founding member of the family is a bimodular human protein called laforin that is comprised of a carbohydrate binding module 20 (CBM20) followed by a dual specificity phosphatase domain. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a chloroplast targeting peptide, dual specificity phosphatase (DSP) domain, a CBM45, and a carboxy-terminal motif. LSF2 is comprised of simply a chloroplast targeting peptide, DSP domain, and carboxy-terminal motif. SEX4 employs an integrated DSP-CBM glucan-binding platform to engage and dephosphorylate starch. LSF2 lacks a CBM and instead utilizes two surface binding sites to bind and dephosphorylate starch. Laforin is a dimeric protein in solution and it utilizes a tetramodular architecture and cooperativity to bind and dephosphorylate glycogen. This chapter describes the biological role of glucan phosphatases in glycogen and starch metabolism and compares and contrasts their ability to bind and dephosphorylate glucans.

  12. Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Ruzanski, Christian

    2014-01-01

    Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half ...

  13. Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35 for gluco- and galacto- substituted mannans and its calcium induced stability.

    Directory of Open Access Journals (Sweden)

    Arabinda Ghosh

    Full Text Available This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35 in polysaccharide recognition. CtCBM35 was cloned into pET28a (+ vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3 cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC. Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×10(4 M(-1, carob galactomannan (Ka = 12.4×10(4 M(-1 and negligible association (Ka = 12 µM(-1 with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca(2+ ion with konjac glucomannan (Ka = 41×10(4 M(-1 and carob galactomannan (Ka = 30×10(4 M(-1. The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (K a with konjac glucomannan and carob galactomannan, 14.3×10(4 M(-1 and 11.4×10(4 M(-1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG -25 kJ mole(-1 as compared to carob galactomannan (ΔG -22 kJ mole(-1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH as analysed by dynamic light scattering (DLS study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca(2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was

  14. The Multiple Carbohydrate Binding Specificities of Helicobacter pylori

    Science.gov (United States)

    Teneberg, Susann

    Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of peptic ulcer disease and gastric cancer. Adhesion of microbes to the target tissue is an important determinant for successful initiation, establishment and maintenance of infection, and a variety of different candidate carbohydrate receptors for H. pylori have been identified. Here the different the binding specifities, and their potential role in adhesion to human gastric epithelium are described. Finally, recent findings on the roles of sialic acid binding SabA adhesin in interactions with human neutrophils and erythrocytes are discussed.

  15. PROCARB: A Database of Known and Modelled Carbohydrate-Binding Protein Structures with Sequence-Based Prediction Tools

    Directory of Open Access Journals (Sweden)

    Adeel Malik

    2010-01-01

    Full Text Available Understanding of the three-dimensional structures of proteins that interact with carbohydrates covalently (glycoproteins as well as noncovalently (protein-carbohydrate complexes is essential to many biological processes and plays a significant role in normal and disease-associated functions. It is important to have a central repository of knowledge available about these protein-carbohydrate complexes as well as preprocessed data of predicted structures. This can be significantly enhanced by tools de novo which can predict carbohydrate-binding sites for proteins in the absence of structure of experimentally known binding site. PROCARB is an open-access database comprising three independently working components, namely, (i Core PROCARB module, consisting of three-dimensional structures of protein-carbohydrate complexes taken from Protein Data Bank (PDB, (ii Homology Models module, consisting of manually developed three-dimensional models of N-linked and O-linked glycoproteins of unknown three-dimensional structure, and (iii CBS-Pred prediction module, consisting of web servers to predict carbohydrate-binding sites using single sequence or server-generated PSSM. Several precomputed structural and functional properties of complexes are also included in the database for quick analysis. In particular, information about function, secondary structure, solvent accessibility, hydrogen bonds and literature reference, and so forth, is included. In addition, each protein in the database is mapped to Uniprot, Pfam, PDB, and so forth.

  16. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  17. Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity

    DEFF Research Database (Denmark)

    Bindesbøll, Christian; Fan, Qiong; Nørgaard, Rikke C;

    2015-01-01

    Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription...... metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity....

  18. Carbohydrates

    Science.gov (United States)

    Carbohydrates are one of the main types of nutrients. They are the most important source of energy for your body. Your digestive system changes carbohydrates into glucose (blood sugar). Your body uses this ...

  19. Multiplicity of carbohydrate-binding sites in -prism fold lectins: occurrence and possible evolutionary implications

    Indian Academy of Sciences (India)

    Alok Sharma; Divya Chandran; Desh D Singh; M Vijayan

    2007-09-01

    The -prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, -prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the -prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a -prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of -prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the -prism II fold, is related to the role of plant lectins in defence.

  20. Hepatitis C Virus Resistance to Carbohydrate-Binding Agents.

    Directory of Open Access Journals (Sweden)

    Laure Izquierdo

    Full Text Available Carbohydrate binding agents (CBAs, including natural lectins, are more and more considered as broad-spectrum antivirals. These molecules are able to directly inhibit many viruses such as Human Immunodeficiency Virus (HIV, Hepatitis C Virus (HCV, Dengue Virus, Ebola Virus or Severe Acute Respiratory Syndrome Coronavirus through binding to envelope protein N-glycans. In the case of HIV, it has been shown that CBAs select for mutant viruses with N-glycosylation site deletions which are more sensitive to neutralizing antibodies. In this study we aimed at evaluating the HCV resistance to CBAs in vitro. HCV was cultivated in the presence of increasing Galanthus nivalis agglutinin (GNA, Cyanovirin-N, Concanavalin-A or Griffithsin concentrations, during more than eight weeks. At the end of lectin exposure, the genome of the isolated strains was sequenced and several potential resistance mutations in the E1E2 envelope glycoproteins were identified. The effect of these mutations on viral fitness as well as on sensitivity to inhibition by lectins, soluble CD81 or the 3/11 neutralizing antibody was assessed. Surprisingly, none of these mutations, alone or in combination, conferred resistance to CBAs. In contrast, we observed that some mutants were more sensitive to 3/11 or CD81-LEL inhibition. Additionally, several mutations were identified in the Core and the non-structural proteins. Thus, our results suggest that in contrast to HIV, HCV resistance to CBAs is not directly conferred by mutations in the envelope protein genes but could occur through an indirect mechanism involving mutations in other viral proteins. Further investigations are needed to completely elucidate the underlying mechanisms.

  1. Hepatitis C Virus Resistance to Carbohydrate-Binding Agents

    Science.gov (United States)

    Izquierdo, Laure; Oliveira, Catarina; Fournier, Carole; Descamps, Véronique; Morel, Virginie; Dubuisson, Jean; Brochot, Etienne; Francois, Catherine; Castelain, Sandrine; Duverlie, Gilles; Helle, Francois

    2016-01-01

    Carbohydrate binding agents (CBAs), including natural lectins, are more and more considered as broad-spectrum antivirals. These molecules are able to directly inhibit many viruses such as Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Dengue Virus, Ebola Virus or Severe Acute Respiratory Syndrome Coronavirus through binding to envelope protein N-glycans. In the case of HIV, it has been shown that CBAs select for mutant viruses with N-glycosylation site deletions which are more sensitive to neutralizing antibodies. In this study we aimed at evaluating the HCV resistance to CBAs in vitro. HCV was cultivated in the presence of increasing Galanthus nivalis agglutinin (GNA), Cyanovirin-N, Concanavalin-A or Griffithsin concentrations, during more than eight weeks. At the end of lectin exposure, the genome of the isolated strains was sequenced and several potential resistance mutations in the E1E2 envelope glycoproteins were identified. The effect of these mutations on viral fitness as well as on sensitivity to inhibition by lectins, soluble CD81 or the 3/11 neutralizing antibody was assessed. Surprisingly, none of these mutations, alone or in combination, conferred resistance to CBAs. In contrast, we observed that some mutants were more sensitive to 3/11 or CD81-LEL inhibition. Additionally, several mutations were identified in the Core and the non-structural proteins. Thus, our results suggest that in contrast to HIV, HCV resistance to CBAs is not directly conferred by mutations in the envelope protein genes but could occur through an indirect mechanism involving mutations in other viral proteins. Further investigations are needed to completely elucidate the underlying mechanisms. PMID:26871442

  2. Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

    Science.gov (United States)

    Apichela, Silvana A; Valz-Gianinet, Jorge N; Schuster, Stefanie; Jiménez-Díaz, María A; Roldán-Olarte, Eugenia M; Miceli, Dora C

    2010-04-01

    Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (pllama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.

  3. A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T.M. (VA); (UCLA); (Vanderbilt); (UCSF)

    2014-10-02

    GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIb{alpha}. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB{sub BR}), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB{sub BR} structure revealed that it is comprised of three independently folded subdomains or modules: (1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; (2) a second Ig-fold resembling the binding region of mammalian Siglecs; (3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIb{alpha}. Further examination of purified GspB{sub BR}-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

  4. A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.

    Directory of Open Access Journals (Sweden)

    Tasia M Pyburn

    2011-07-01

    Full Text Available GspB is a serine-rich repeat (SRR adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB(BR, both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB(BR structure revealed that it is comprised of three independently folded subdomains or modules: 1 an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2 a second Ig-fold resembling the binding region of mammalian Siglecs; 3 a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspB(BR-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspB(BR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

  5. Interleukin-2 carbohydrate recognition modulates CTLL-2 cell proliferation.

    Science.gov (United States)

    Fukushima, K; Yamashita, K

    2001-03-01

    Interleukin-2 (IL-2) specifically recognizes high-mannose type glycans with five or six mannosyl residues. To determine whether the carbohydrate recognition activity of IL-2 contributes to its physiological activity, the inhibitory effects of high-mannose type glycans on IL-2-dependent CTLL-2 cell proliferation were investigated. Man(5)GlcNAc(2)Asn added to CTLL-2 cell cultures inhibited not only phosphorylation of tyrosine kinases but also IL-2-dependent cell proliferation. We found that a complex of IL-2, IL-2 receptor alpha, beta, gamma subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor alpha subunit was stained with Galanthus nivalis agglutinin which specifically recognizes high-mannose type glycans. This staining was diminished after digestion of the glycans with endo-beta-N-acetylglucosaminidase H or D, suggesting that at least a N-glycan containing Man(5)GlcNAc(2) is linked to the extracellular portion of the IL-2 receptor alpha subunit. Our findings indicate that IL-2 binds the IL-2 receptor alpha subunit through Man(5)GlcNAc(2) and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2Ralpha subunit, this may trigger formation of the high affinity complex of IL-2-IL-2Ralpha, -beta, and -gamma subunits, leading to cellular signaling.

  6. A role for carbohydrate recognition in mammalian sperm-egg binding

    Energy Technology Data Exchange (ETDEWEB)

    Clark, Gary F., E-mail: clarkgf@health.missouri.edu

    2014-08-01

    Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the egg cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented.

  7. A method for identifying a proposed carbohydrate-binding motif of proteins.

    Science.gov (United States)

    Baumann, M A; Anderson, B E

    1991-11-01

    An examination of the binding sites of four carbohydrate binding proteins (Escherichia coli lactose repressor, E. coli arabinose-binding protein, yeast hexokinase A and Concanavalin A) revealed certain similarities of amino acid sequences and residues forming hydrogen bonds and hydrophobic interactions with the bound carbohydrate. These were: (i) Asx-Asx, hydrogen bonding to the pyranose ring oxygen and anomeric-OH group; (ii) Arg-X-X-X-(Ser/Thr), or the reverse sequence, with the Arg hydrogen bonding to the pyranose ring oxygen; (iii) Lys-(Ser/Thr)-X-X-Asp, or the reverse sequence and with interchange of the Lys-(Ser/Thr) positions, with hydrogen bonding of either or both the Lys and Asp residues to the -OH groups at carbons 2, 3, 4 or 6; (iv) a diaromatic sequence with possible hydrophobic interactions to the faces of the pyranose ring structure. An algorithm was devised to search the amino acid sequences of a large number of proteins, those known to bind carbohydrates as well as those without known carbohydrate-binding activities, for the four amino acid sequence criteria. The algorithm incorporated a weighted distance value (WDV) to assess the approximate distance between any two criteria, with the WDV being based on the predicted secondary structure of the protein amino acid sequence. When the algorithm using criteria 1 and 2 plus the WDV was applied to the sequences of 125 proteins, the method indicated the presence of the potential carbohydrate-binding site motif for 42% of proteins with known carbohydrate binding, only 8% of proteins were predicted as false positives, and the accuracy of the method was calculated to be 61.6%.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Lactobacillus rhamnosus GG SpaC pilin subunit binds to the carbohydrate moieties of intestinal glycoconjugates.

    Science.gov (United States)

    Nishiyama, Keita; Ueno, Shintaro; Sugiyama, Makoto; Yamamoto, Yuji; Mukai, Takao

    2016-06-01

    Lactobacillus rhamnosus GG (LGG) is a well-established probiotic strain. The beneficial properties of this strain are partially dependent on its prolonged residence in the gastrointestinal tract, and are likely influenced by its adhesion to the intestinal mucosa. The pilin SpaC subunit, located within the Spa pili structure, is the most well studied LGG adhesion factor. However, the binding epitopes of SpaC remain largely unknown. The aim of this study was to evaluate the binding properties of SpaC to the carbohydrate moieties of intestinal glycoconjugates using a recombinant SpaC protein. In a competitive enzyme-linked immunosorbent assay, SpaC binding was markedly reduced by addition of purified mucin and the mucin oligosaccharide fraction. Histochemical staining revealed that the binding of SpaC was drastically reduced by periodic acid treatment. Moreover, in the surface plasmon resonance-based Biacore assay, SpaC bound strongly to the carbohydrate moieties containing β-galactoside at the non-reducing terminus of glycolipids. We here provide the first demonstration that SpaC binds to the oligosaccharide chains of mucins, and that the carbohydrate moieties containing β-galactoside at the non-reducing termini of glycoconjugates play a crucial role in this binding. Our results demonstrate the importance of carbohydrates of SpaC for mucus interactions.

  9. Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.

    Science.gov (United States)

    Yadav, Rupali; Verma, Kuldeep; Chandra, Mintu; Mukherjee, Madhumita; Datta, Sunando

    2016-09-01

    Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion.

  10. Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

    2016-01-01

    The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

  11. Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry.

    Science.gov (United States)

    Zhang, Jingjing; Kitova, Elena N; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S

    2016-01-01

    The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

  12. Carbohydrate Staple Food Modulates Gut Microbiota of Mongolians in China

    Science.gov (United States)

    Li, Jing; Hou, Qiangchuan; Zhang, Jiachao; Xu, Haiyan; Sun, Zhihong; Menghe, Bilige; Zhang, Heping

    2017-01-01

    Gut microbiota is a determining factor in human physiological functions and health. It is commonly accepted that diet has a major influence on the gut microbial community, however, the effects of diet is not fully understood. The typical Mongolian diet is characterized by high and frequent consumption of fermented dairy products and red meat, and low level of carbohydrates. In this study, the gut microbiota profile of 26 Mongolians whom consumed wheat, rice and oat as the sole carbohydrate staple food for a week each consecutively was determined. It was observed that changes in staple carbohydrate rapidly (within a week) altered gut microbial community structure and metabolic pathway of the subjects. Wheat and oat favored bifidobacteria (Bifidobacterium catenulatum, Bifodobacteriumbifidum, Bifidobacterium adolescentis); whereas rice suppressed bifidobacteria (Bifidobacterium longum, Bifidobacterium adolescentis) and wheat suppresses Lactobaciilus, Ruminococcus and Bacteroides. The study exhibited two gut microbial clustering patterns with the preference of fucosyllactose utilization linking to fucosidase genes (glycoside hydrolase family classifications: GH95 and GH29) encoded by Bifidobacterium, and xylan and arabinoxylan utilization linking to xylanase and arabinoxylanase genes encoded by Bacteroides. There was also a correlation between Lactobacillus ruminis and sialidase, as well as Butyrivibrio crossotus and xylanase/xylosidase. Meanwhile, a strong concordance was found between the gastrointestinal bacterial microbiome and the intestinal virome. Present research will contribute to understanding the impacts of the dietary carbohydrate on human gut microbiome, which will ultimately help understand relationships between dietary factor, microbial populations, and the health of global humans. PMID:28377764

  13. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.

    Science.gov (United States)

    Ferrara, Claudia; Grau, Sandra; Jäger, Christiane; Sondermann, Peter; Brünker, Peter; Waldhauer, Inja; Hennig, Michael; Ruf, Armin; Rufer, Arne Christian; Stihle, Martine; Umaña, Pablo; Benz, Jörg

    2011-08-02

    Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

  14. Prediction of Carbohydrate-Binding Proteins from Sequences Using Support Vector Machines

    Directory of Open Access Journals (Sweden)

    Seizi Someya

    2010-01-01

    Full Text Available Carbohydrate-binding proteins are proteins that can interact with sugar chains but do not modify them. They are involved in many physiological functions, and we have developed a method for predicting them from their amino acid sequences. Our method is based on support vector machines (SVMs. We first clarified the definition of carbohydrate-binding proteins and then constructed positive and negative datasets with which the SVMs were trained. By applying the leave-one-out test to these datasets, our method delivered 0.92 of the area under the receiver operating characteristic (ROC curve. We also examined two amino acid grouping methods that enable effective learning of sequence patterns and evaluated the performance of these methods. When we applied our method in combination with the homology-based prediction method to the annotated human genome database, H-invDB, we found that the true positive rate of prediction was improved.

  15. Carbohydrate Microarrays in Plant Science

    DEFF Research Database (Denmark)

    Fangel, Jonatan Ulrik; Pedersen, H.L.; Vidal-Melgosa, S.

    2012-01-01

    industrially and nutritionally. Understanding the biological roles of plant glycans and the effective exploitation of their useful properties requires a detailed understanding of their structures, occurrence, and molecular interactions. Microarray technology has revolutionized the massively high......-throughput analysis of nucleotides, proteins, and increasingly carbohydrates. Using microarrays, the abundance of and interactions between hundreds and thousands of molecules can be assessed simultaneously using very small amounts of analytes. Here we show that carbohydrate microarrays are multifunctional tools...... for plant research and can be used to map glycan populations across large numbers of samples to screen antibodies, carbohydrate binding proteins, and carbohydrate binding modules and to investigate enzyme activities....

  16. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C

    1998-01-01

    Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon...... resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site...

  17. The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling.

    Science.gov (United States)

    Ohtsuka, Isao; Sadakane, Yutaka; Higuchi, Mari; Hada, Noriyasu; Hada, Junko; Kakiuchi, Nobuko; Sakushima, Akiyo

    2011-01-15

    Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.

  18. Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

    Energy Technology Data Exchange (ETDEWEB)

    Kohnken, R.E.; Berger, E.A.

    1987-12-29

    N-(4-Azidosalicyl) galactosamine (GalNASA), a photoactivatable, radioiodinatable analog of N-acetylgalactosamine (GalNAc), has been prepared and characterized. The authors have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a K/sub i,app/ of 800 ..mu..M, comparable to that of GalNAc. The K/sub i,app/ of GalNASA decreased to 40 ..mu..m upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl) ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with /sup 125/I-GalNASA was entirely dependent upon ultraviolet light. A portion of labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethyl-enediaminetetraacetic acid. The carbohydrate-sensitive fraction of discoidin I photolabeling with /sup 125/I-GalNASA exhibited a K/sub d/ of 15-40 ..mu..M, in agreement with the K/sub i,app/ of prephotolyzed GalNASA observed in the carbohydrate binding assay. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc. This indicated that the location of carbohydrate-sensitive labeling within the structure of discoidin I was restricted. One particular tryptic fragment, Tr1, was examined in detail. These data suggest that Tr1 is derived from the carbohydrate binding site of discoidin I.

  19. Related lectins from snowdrop and maize differ in their carbohydrate-binding specificity.

    Science.gov (United States)

    Fouquaert, Elke; Smith, David F; Peumans, Willy J; Proost, Paul; Balzarini, Jan; Savvides, Savvas N; Damme, Els J M Van

    2009-03-01

    Searches in an EST database from maize revealed the expression of a protein related to the Galanthus nivalis (GNA) agglutinin, referred to as GNA(maize). Heterologous expression of GNA(maize) in Pichia pastoris allowed characterization of the first nucleocytoplasmic GNA homolog from plants. GNA(maize) is a tetrameric protein which shares 64% sequence similarity with GNA. Glycan microarray analyses revealed important differences in the specificity. Unlike GNA, which binds strongly to high-mannose N-glycans, the lectin from maize reacts almost exclusively with more complex glycans. Interestingly, GNA(maize) prefers complex glycans containing beta1-2 GlcNAc residues. The obvious difference in carbohydrate-binding properties is accompanied by a 100-fold reduced anti-HIV activity. Although the sequences of GNA and GNA(maize) are clearly related they show only 28% sequence identity. Our results indicate that gene divergence within the family of GNA-related lectins leads to changes in carbohydrate-binding specificity, as shown on N-glycan arrays.

  20. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    with identical N-terminal tags remote from the ligand-binding surface. Although rat and mouse showed similar affinities for saccharide competitors, both differed markedly from the human protein. The human neck+CRD preferentially recognized N-acetyl-mannosamine, whereas the rat and mouse proteins showed greater...... of the corresponding murine sequence (Asn324-Asn325) conferred a capacity to interact with immobilized maltose. Thus, ligand recognition by human SP-D involves a complex interplay between saccharide presentation, the valency of trimeric subunits, and species-specific residues that flank the primary carbohydrate...

  1. Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines.

    Science.gov (United States)

    Arndt, Nadia X; Tiralongo, Joe; Madge, Paul D; von Itzstein, Mark; Day, Christopher J

    2011-09-01

    Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan-binding lectins. The screening of breast cancer MDA-MB435 cells, cervical cancer HeLa cells and colon cancer Caco-2, HCT116 and HCT116-FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4-deoxy-4-guanidino-Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell-lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan-lectin interactions that are exhibited by immortalised cell lines.

  2. Amino Acid Change in the Carbohydrate Response Element Binding Protein is associated with lower triglycerides and myocardial infarction incidence depending on level of adherence to the Mediterranean diet in the PREDIMED trial

    Science.gov (United States)

    A variant (rs3812316, C771G, and Gln241His) in the MLXIPL (Max-like protein X interacting protein-like) gene encoding the carbohydrate response element binding protein has been associated with lower triglycerides. However, its association with cardiovascular diseases and gene-diet interactions modul...

  3. Molecular cloning and expression of chicken carbohydrate response element binding protein and Max-like protein X gene homologues

    Science.gov (United States)

    Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not ...

  4. Screening for carbohydrate-binding proteins in extracts of Uruguayan plants

    Directory of Open Access Journals (Sweden)

    Plá A.

    2003-01-01

    Full Text Available The presence of carbohydrate-binding proteins, namely lectins, ß-galactosidases and amylases, was determined in aqueous extracts of plants collected in Uruguay. Twenty-six extracts were prepared from 15 Uruguayan plants belonging to 12 Phanerogam families. Among them, 18 extracts caused hemagglutination (HAG that was inhibited by mono- and disaccharides in 13 cases, indicating the presence of lectins. The other 8 extracts did not cause any HAG with the four systems used to detect HAG activity (rabbit and mouse red cells, trypsin-treated rabbit and mouse red cells. For the extracts prepared from Solanum commersonii, HAG activity and HAG inhibition were similar for those prepared from tubers, leaves and fruits, with the chitocompounds being responsible for all the inhibitions. Purification of the S. commersonii tuber lectin was carried out by affinity chromatography on asialofetuin-Sepharose, and SDS-PAGE under reducing conditions gave a single band of Mr of approximately 80 kDa. The monomer N-acetylglucosamine did not inhibit HAG induced by the purified lectin, but chitobiose inhibited HAG at 24 mM and chitotriose inhibited it at 1 mM. ß-Galactosidase activity was detected in leaves and stems of Cayaponia martiana, and in seeds from Datura ferox. Only traces of amylase activity were detected in some of the extracts analyzed. The present screening increases knowledge about the occurrence of carbohydrate-binding proteins present in regional plants.

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

    DEFF Research Database (Denmark)

    Wong, Jaslyn E M M; Midtgaard, Søren Roi; Gysel, Kira

    2015-01-01

    LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multi......LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement...... of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering...... solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers...

  6. Functional dissection of the Clostridium botulinum type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites.

    Directory of Open Access Journals (Sweden)

    Yo Sugawara

    Full Text Available Botulinum neurotoxin (BoNT inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH, and some toxin complexes contain another non-toxic protein, hemagglutinin (HA, in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.

  7. Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

    Science.gov (United States)

    Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

    2004-01-01

    Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

  8. Potential of carbohydrate-binding agents as therapeutics against enveloped viruses.

    Science.gov (United States)

    François, K O; Balzarini, J

    2012-03-01

    Twenty-seven years after the discovery of HIV as the cause of AIDS more than 25 drugs directed against four different viral targets (i.e. reverse transcriptase, protease, integrase, envelope gp41) and one cellular target (i.e. CCR5 co-receptor) are available for treatment. However, the search for an efficient vaccine is still ongoing. One of the main problems is the presence of a continuously evolving dense carbohydrate shield, consisting of N-linked glycans that surrounds the virion and protects it against efficient recognition and persistent neutralization by the immune system. However, several lectins from the innate immune system specifically bind to these glycans in an attempt to process the virus antigens to provoke an immune response. Across a wide variety of different species in nature lectins can be found that can interact with the glycosylated envelope of HIV-1 and can block the infection of susceptible cells by the virus. In this review, we will give an overview of the lectins from non-mammalian origin that are endowed with antiviral properties and discuss the complex interactions between lectins of the innate immune system and HIV-1. Also, attention will be given to different carbohydrate-related modalities that can be exploited for antiviral chemotherapy.

  9. An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

    Directory of Open Access Journals (Sweden)

    Julius W Kim

    Full Text Available BACKGROUND: Vectors based on human adenovirus serotype 5 (HAdV-5 continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. METHODOLOGY/PRINCIPAL FINDINGS: As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4. This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. CONCLUSIONS/SIGNIFICANCE: These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

  10. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    Science.gov (United States)

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  11. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

    DEFF Research Database (Denmark)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig;

    2007-01-01

    to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has......-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number......Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity...

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-03-01

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

  14. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ming V. [Program of Cardiovascular Sciences, Houston, TX 77030 (United States); Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Chen, Weiqin [Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Harmancey, Romain N. [Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip [Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Taegtmeyer, Heinrich [Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Chan, Lawrence, E-mail: lchan@bcm.tmc.edu [Program of Cardiovascular Sciences, Houston, TX 77030 (United States); Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); St. Luke' s Episcopal Hospital, Houston, TX 77030 (United States)

    2010-05-07

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

  15. Carbohydrate/glycan-binding specificity of legume lectins in respect to their proposed biological functions

    Directory of Open Access Journals (Sweden)

    Márcio Viana Ramos

    2000-01-01

    Full Text Available The lectins, proteins which specifically recognize carbohydrate moieties, have been extensively studied in many biochemical and structural aspects in order to establish the molecular basis of this non-catalytic event. On the other hand, their clinical and agricultural potentials have been growing fast. Although lectins, mainly those from legume plants, had been investigated for biological properties, studies about the physiological functions of lectins are scarce in literature. Therefore, despite the accumulated data on lectins (as proteins, the role played by these signalizing molecules is poorly discussed. In the light of our accumulated results on legume lectins, specially those obtained from plants belonging to the Diocleinae sub-tribe and available data in literature, we discuss here the main hypothesis of their functions according to their carbohydrate/glycan-binding specificity.As lectinas, proteinas que especificamente reconhecem estruturas que contém carboidratos, têm sido extensivamente estudadas em muitos aspectos bioquímicos e estruturais, objetivando estabelecer as bases moleculares deste evento não-catalítico. Por outro lado, os potenciais clínicos e agriculturais destas proteínas têm crescido rapidamente. Embora as lectinas, principalmente aquelas de legumes tenham sido bastante investigadas em suas propriedades biológicas, estudos sobre as funcões fisiológicas de lectinas são escassos na literatura. Além disto, a despeito da quantidade de dados acumulados sobre lectinas (como proteínas, o papel desempenhado por estas moléculas de sinalização é pobremente discutido. Valendo-se de nossos estudos sobre lectinas de leguminosas, principalmente da sub-tribo Diocleinae, e outros dados presentes na literatura, discutimos aqui, as principais hipóteses de suas funções com base na especificidade por carboidratos e glicanos complexos.

  16. Exploring the free-energy landscape of carbohydrate-protein complexes: development and validation of scoring functions considering the binding-site topology

    Science.gov (United States)

    Eid, Sameh; Saleh, Noureldin; Zalewski, Adam; Vedani, Angelo

    2014-12-01

    Carbohydrates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the structure-based design of carbohydrate-based ligands. We assembled a diverse data set comprising 273 carbohydrate-protein crystal structures with known binding affinity and evaluated the prediction accuracy of a large collection of well-established scoring and free-energy functions, as well as combinations thereof. Unfortunately, the tested functions were not capable of reproducing binding affinities in the studied complexes. To simplify the complex free-energy surface of carbohydrate-protein systems, we classified the studied proteins according to the topology and solvent exposure of the carbohydrate-binding site into five distinct categories. A free-energy model based on the proposed classification scheme reproduced binding affinities in the carbohydrate data set with an r 2 of 0.71 and root-mean-squared-error of 1.25 kcal/mol ( N = 236). The improvement in model performance underlines the significance of the differences in the local micro-environments of carbohydrate-binding sites and demonstrates the usefulness of calibrating free-energy functions individually according to binding-site topology and solvent exposure.

  17. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism

    Directory of Open Access Journals (Sweden)

    Katsumi Iizuka

    2017-02-01

    Full Text Available Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase, fructolysis (Glut5, ketohexokinase, and lipogenesis (acetyl CoA carboxylase, fatty acid synthase. ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp−/− mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome.

  18. Antiviral activity of carbohydrate-binding agents against Nidovirales in cell culture.

    Science.gov (United States)

    van der Meer, F J U M; de Haan, C A M; Schuurman, N M P; Haijema, B J; Peumans, W J; Van Damme, E J M; Delputte, P L; Balzarini, J; Egberink, H F

    2007-10-01

    Coronaviruses are important human and animal pathogens, the relevance of which increased due to the emergence of new human coronaviruses like SARS-CoV, HKU1 and NL63. Together with toroviruses, arteriviruses, and roniviruses the coronaviruses belong to the order Nidovirales. So far antivirals are hardly available to combat infections with viruses of this order. Therefore, various antiviral strategies to counter nidoviral infections are under evaluation. Lectins, which bind to N-linked oligosaccharide elements of enveloped viruses, can be considered as a conceptionally new class of virus inhibitors. These agents were recently evaluated for their antiviral activity towards a variety of enveloped viruses and were shown in most cases to inhibit virus infection at low concentrations. However, limited knowledge is available for their efficacy towards nidoviruses. In this article the application of the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), Cymbidium sp. agglutinin (CA) and Urtica dioica agglutinin (UDA) as well as non-plant derived pradimicin-A (PRM-A) and cyanovirin-N (CV-N) as potential antiviral agents was evaluated. Three antiviral tests were compared based on different evaluation principles: cell viability (MTT-based colorimetric assay), number of infected cells (immunoperoxidase assay) and amount of viral protein expression (luciferase-based assay). The presence of carbohydrate-binding agents strongly inhibited coronaviruses (transmissible gastroenteritis virus, infectious bronchitis virus, feline coronaviruses serotypes I and II, mouse hepatitis virus), arteriviruses (equine arteritis virus and porcine respiratory and reproductive syndrome virus) and torovirus (equine Berne virus). Remarkably, serotype II feline coronaviruses and arteriviruses were not inhibited by PRM-A, in contrast to the other viruses tested.

  19. Carbohydrate-binding specificities of potential probiotic Lactobacillus strains in porcine jejunal (IPEC-J2) cells and porcine mucin.

    Science.gov (United States)

    Valeriano, Valerie Diane; Bagon, Bernadette B; Balolong, Marilen P; Kang, Dae-Kyung

    2016-07-01

    Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen-probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.

  20. Modulation of antioxidant status, carbohydrate and lipid metabolism by melatonin on streptozotocin induced diabetic rats

    Directory of Open Access Journals (Sweden)

    Mirunalini Sankaran*

    2012-08-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Melatonin, “synchronizer of the biological clock” is major hormones secreted from the pineal gland have various therapeutic effects. The present study was designed to explore the modulatory effect of melatonin on antioxidant status, glucose and lipid metabolism in streptozotocin (STZ induced diabetic rats. Male Wistar rats weighing 180-200 g were made diabetic by administration of streptozotocin (STZ (40 mg/kg body weight intraperitoneally. Melatonin was administered intraperitoneally at a dose of 2 mg/kg body weight to STZ-induced diabetic rats for 30 days. Body weight, blood glucose, carbohydrate metabolic enzyme, lipid profile, antioxidant and lipid peroxidation status were assessed. The level of the blood glucose, carbohydrate metabolic enzymes (glucose-6-phosphatase and fructose-1,6-bisphosphatase and lipid peroxidative marker (TBARS were increased in STZ induced diabetic rats while the melatonin treatment revert back to the near normal condition. In contrast, administered melatonin resulted in an increased in body weight and insulin secretion in diabetic rats. The enzymatic antioxidants (SOD, CAT and GPX and non-enzymatic antioxidants (GSH, vitamin C and vitamin E were also increased by melatonin treatment. The cholesterol and phospholipids which were elevated in diabetic rats were normalized by the melatonin administration. Hence these findings indicate that melatonin protects against STZ induced oxidative stress and thus explain its use in treatment of diabetes by modulating lipid and glucose metabolism.

  1. Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in Vitro between Pigs and Cattle

    Directory of Open Access Journals (Sweden)

    Minoru Nakano

    2013-01-01

    Full Text Available Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm–oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle.

  2. Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in Vitro between Pigs and Cattle.

    Science.gov (United States)

    Takahashi, Kazuya; Kikuchi, Kazuhiro; Uchida, Yasuomi; Kanai-Kitayama, Saeko; Suzuki, Reiichiro; Sato, Reiko; Toma, Kazunori; Geshi, Masaya; Akagi, Satoshi; Nakano, Minoru; Yonezawa, Naoto

    2013-01-01

    Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm-oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm-oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle.

  3. Highly effective recognition of carbohydrates by phenanthroline-based receptors: alpha- versus beta-anomer binding preference.

    Science.gov (United States)

    Mazik, Monika; Hartmann, Andrè; Jones, Peter G

    2009-09-14

    (1)H NMR spectroscopic titrations in competitive and non-competitive media, as well as binding studies in two-phase systems, such as phase transfer of sugars from aqueous into organic solvents and dissolution of solid carbohydrates in apolar media revealed both highly effective recognition of neutral carbohydrates and interesting binding preferences of an acyclic phenanthroline-based receptor 1. Compared to the previously described acyclic receptors, compound 1 displays significantly higher binding affinities, the rare capability to extract sugars from water into non-polar organic solutions and alpha- versus beta-anomer binding preference in the recognition of glycosides, which differs from those observed for other receptor systems. X-ray crystallographic investigations revealed the presence of water molecules in the binding pocket of 1 that are engaged in the formation of hydrogen-bonding motifs similar to those suggested by molecular modelling for the sugar OH groups in the receptor-sugar complexes. The molecular modelling calculations, synthesis, crystal structure and binding properties of 1 are described and compared with those of the previously described receptors.

  4. Carbohydrate-Binding Non-Peptidic Pradimicins for the Treatment of Acute Sleeping Sickness in Murine Models

    Science.gov (United States)

    Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Reichardt, Niels C.; Igarashi, Yasuhiro; Liekens, Sandra; Balzarini, Jan

    2016-01-01

    Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis in vitro. Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT. PMID:27662652

  5. Carbohydrate microarrays in plant science.

    Science.gov (United States)

    Fangel, Jonatan U; Pedersen, Henriette L; Vidal-Melgosa, Silvia; Ahl, Louise I; Salmean, Armando Asuncion; Egelund, Jack; Rydahl, Maja Gro; Clausen, Mads H; Willats, William G T

    2012-01-01

    Almost all plant cells are surrounded by glycan-rich cell walls, which form much of the plant body and collectively are the largest source of biomass on earth. Plants use polysaccharides for support, defense, signaling, cell adhesion, and as energy storage, and many plant glycans are also important industrially and nutritionally. Understanding the biological roles of plant glycans and the effective exploitation of their useful properties requires a detailed understanding of their structures, occurrence, and molecular interactions. Microarray technology has revolutionized the massively high-throughput analysis of nucleotides, proteins, and increasingly carbohydrates. Using microarrays, the abundance of and interactions between hundreds and thousands of molecules can be assessed simultaneously using very small amounts of analytes. Here we show that carbohydrate microarrays are multifunctional tools for plant research and can be used to map glycan populations across large numbers of samples to screen antibodies, carbohydrate binding proteins, and carbohydrate binding modules and to investigate enzyme activities.

  6. Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters

    DEFF Research Database (Denmark)

    Seppälä, Susanna; Solomon, Kevin V; Gilmore, Sean P.;

    2016-01-01

    machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display...... a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce....

  7. Carbohydrate structure and differential binding of prostate specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy.

    Science.gov (United States)

    Ohyama, Chikara; Hosono, Masahiro; Nitta, Kazuo; Oh-eda, Masayoshi; Yoshikawa, Kazuyuki; Habuchi, Tomonori; Arai, Yoichi; Fukuda, Minoru

    2004-08-01

    Serum prostate-specific antigen (PSA) assay is widely used for detection of prostate cancer. Because PSA is also synthesized from normal prostate, false positive diagnosis cannot be avoided by the conventional serum PSA test. To apply the cancer-associated carbohydrate alteration to the improvement of PSA assay, we first elucidated the structures of PSA purified from human seminal fluid. The predominant core structure of N-glycans of seminal fluid PSA was a complex type biantennary oligosaccharide and was consistent with the structure reported previously. However, we found the sialic acid alpha2-3 galactose linkage as an additional terminal carbohydrate structure on seminal fluid PSA. We then analyzed the carbohydrate moiety of serum PSA from the patients with prostate cancer and benign prostate hypertrophy using lectin affinity chromatography. Lectin binding was assessed by lectin affinity column chromatography followed by determining the amount of total and free PSA. Concanavalin A, Lens culinaris, Aleuria aurantia, Sambucus nigra, and Maackia amurensis lectins were tested for their binding to the carbohydrates on PSA. Among the lectins examined, the M. amurensis agglutinin-bound fraction of free serum PSA is increased in prostate cancer patients compared to benign prostate hypertrophy patients. The binding of PSA to M. amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, was also confirmed by surface plasmon resonance analysis. These results suggest that the differential binding of free serum PSA to M. amurensis agglutinin lectin between prostate cancer and benign prostate hypertrophy could be a potential measure for diagnosis of prostate cancer.

  8. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.

    Science.gov (United States)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig; Bennett, Eric P; Mandel, Ulla; Takeuchi, Hideyuki; Kato, Kentaro; Irimura, Tatsuro; Suryanarayanan, Ganesh; Hollingsworth, Michael A; Clausen, Henrik

    2007-04-01

    Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

  9. Structures of parasite calreticulins provide insights into their flexibility and dual carbohydrate/peptide-binding properties

    Science.gov (United States)

    Moreau, Christophe; Cioci, Gianluca; Iannello, Marina; Laffly, Emmanuelle; Chouquet, Anne; Ferreira, Arturo; Thielens, Nicole M.; Gaboriaud, Christine

    2016-01-01

    Calreticulin (CRT) is a multifaceted protein, initially discovered as an endoplasmic reticulum (ER) chaperone protein, that is essential in calcium metabolism. Various implications in cancer, early development and immunology have been discovered more recently for CRT, as well as its role as a dominant ‘eat-me’ prophagocytic signal. Intriguingly, cell-surface exposure/secretion of CRT is among the infective strategies used by parasites such as Trypanosoma cruzi, Entamoeba histolytica, Taenia solium, Leishmania donovani and Schistosoma mansoni. Because of the inherent flexibility of CRTs, their analysis by X-ray crystallography requires the design of recombinant constructs suitable for crystallization, and thus only the structures of two very similar mammalian CRT lectin domains are known. With the X-ray structures of two distant parasite CRTs, insights into species structural determinants that might be harnessed to fight against the parasites without affecting the functions of the host CRT are now provided. Moreover, although the hypothesis that CRT can exhibit both open and closed conformations has been proposed in relation to its chaperone function, only the open conformation has so far been observed in crystal structures. The first evidence is now provided of a complex conformational transition with the junction reoriented towards P-domain closure. SAXS experiments also provided additional information about the flexibility of T. cruzi CRT in solution, thus complementing crystallographic data on the open conformation. Finally, regarding the conserved lectin-domain structure and chaperone function, evidence is provided of its dual carbohydrate/protein specificity and a new scheme is proposed to interpret such unusual substrate-binding properties. These fascinating features are fully consistent with previous experimental observations, as discussed considering the broad spectrum of CRT sequence conservations and differences. PMID:27840680

  10. Trimethoxybenzene- and trimethylbenzene-based compounds bearing imidazole, indole and pyrrole groups as recognition units: synthesis and evaluation of the binding properties towards carbohydrates.

    Science.gov (United States)

    Rosien, Jan-Ruven; Seichter, Wilhelm; Mazik, Monika

    2013-10-14

    The aim of the study was to evaluate the potential of trimethoxybenzene- and trimethylbenzene-based compounds bearing imidazole or indole groups as recognition sites in the complexation of carbohydrates. Representatives of these compounds were prepared and their binding properties toward selected carbohydrates evaluated. The results of the binding studies were compared with those obtained for the prepared pyrrole bearing analogues and for the previously described triethylbenzene-based receptors.

  11. Anti-tumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding

    Science.gov (United States)

    Dings, Ruud P.M.; Miller, Michelle C.; Nesmelova, Irina; Astorgues-Xerri, Lucile; Kumar, Nigam; Serova, Maria; Chen, Xuimei; Raymond, Eric; Hoye, Thomas R.; Mayo, Kevin H.

    2012-01-01

    Calix[4]arene compound 0118 is an angiostatic agent that inhibits tumor growth in mice. Although 0118 is a topomimetic of galectin-1-targeting angiostatic amphipathic peptide anginex, we had yet to prove that 0118 targets galectin-1. Galectin-1 is involved in pathological disorders like tumor endothelial cell adhesion and migration and therefore presents a relevant target for therapeutic intervention against cancer. Here, 15N-1H HSQC NMR spectroscopy demonstrates that 0118 indeed targets galectin-1 at a site away from the lectin’s carbohydrate binding site, and thereby attenuates lactose binding to the lectin. Flow cytometry and agglutination assays show that 0118 attenuates binding of galectin-1 to cell surface glycans, and the inhibition of cell proliferation by 0118 is found to be correlated with the cellular expression of the lectin. In general, our data indicate that 0118 targets galectin-1 as an allosteric inhibitor of glycan/carbohydrate binding. This work contributes to the clinical development of anti-tumor calixarene compound 0118. PMID:22575017

  12. Binding Sites for Acylated Trehalose Analogs of Glycolipid Ligands on an Extended Carbohydrate Recognition Domain of the Macrophage Receptor Mincle*

    Science.gov (United States)

    Feinberg, Hadar; Rambaruth, Neela D. S.; Jégouzo, Sabine A. F.; Jacobsen, Kristian M.; Djurhuus, Rasmus; Poulsen, Thomas B.; Weis, William I.; Taylor, Maureen E.; Drickamer, Kurt

    2016-01-01

    The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis. Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages. PMID:27542410

  13. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

    Directory of Open Access Journals (Sweden)

    Moffatt Barbara A

    2010-08-01

    Full Text Available Abstract Background Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB for coplanar aromatic motifs similar to those found in known glycan-binding proteins. Results The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192 in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Conclusions Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  14. Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity.

    Science.gov (United States)

    Moulaei, Tinoush; Shenoy, Shilpa R; Giomarelli, Barbara; Thomas, Cheryl; McMahon, James B; Dauter, Zbigniew; O'Keefe, Barry R; Wlodawer, Alexander

    2010-09-08

    Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviral activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.

  15. Mutational analysis of the pumpkin (Cucurbita maxima) phloem exudate lectin, PP2 reveals Ser-104 is crucial for carbohydrate binding.

    Science.gov (United States)

    Bobbili, Kishore Babu; Bandari, Shyam; Grobe, Kay; Swamy, Musti J

    2014-07-18

    The pumpkin phloem lectin (PP2) is an RNA-binding, defense-related, chitooligosaccharide-specific, homodimeric lectin of Mr 48 kDa expressed at high concentrations in the sieve elements and companion cells of pumpkin (Cucurbita maxima). In the present study, PP2 was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway as a prerequisite for unimpaired folding and posttranslational glycosylation of recombinant PP2. Previous computational modeling and ligand docking studies predicted a putative chitooligosaccharide-binding site on the PP2 surface, which was divided into three subsites, with two amino acid residues in each subsite identified as possible candidates for interaction with chitooligosaccharides (CHOs). In this work, mutational analysis and hemagglutination assays were employed to verify the role of the predicted residues in the carbohydrate binding activity of the protein. The results obtained revealed that mutation of Ser-104 to Ala (S104A) at subsite-2 resulted in about 90% loss of agglutination activity of the protein, indicating that Ser-104 is crucial for the binding of CHOs to PP2. Also, L100A (at subsite-1) and K200A (at subsite-3) independently decreased the lectin activity by about 40%, indicating that these two residues also contribute significantly to sugar binding by PP2. Together, these findings confirm that all the three subsites contribute to varying degrees toward PP2-carbohydrate interaction, and confirm the validity of the computational model, as proposed earlier.

  16. Eggs modulate the inflammatory response to carbohydrate restricted diets in overweight men

    Directory of Open Access Journals (Sweden)

    Volek Jeff S

    2008-02-01

    Full Text Available Abstract Background Carbohydrate restricted diets (CRD consistently lower glucose and insulin levels and improve atherogenic dyslipidemia [decreasing triglycerides and increasing HDL cholesterol (HDL-C]. We have previously shown that male subjects following a CRD experienced significant increases in HDL-C only if they were consuming a higher intake of cholesterol provided by eggs compared to those individuals who were taking lower concentrations of dietary cholesterol. Here, as a follow up of our previous study, we examined the effects of eggs (a source of both dietary cholesterol and lutein on adiponectin, a marker of insulin sensitivity, and on inflammatory markers in the context of a CRD. Methods Twenty eight overweight men [body mass index (BMI 26–37 kg/m2] aged 40–70 y consumed an ad libitum CRD (% energy from CHO:fat:protein = 17:57:26 for 12 wk. Subjects were matched by age and BMI and randomly assigned to consume eggs (EGG, n = 15 (640 mg additional cholesterol/day provided by eggs or placebo (SUB, n = 13 (no additional dietary cholesterol. Fasting blood samples were drawn before and after the intervention to assess plasma lipids, insulin, adiponectin and markers of inflammation including C-reactive protein (CRP, tumor necrosis factor-alpha (TNF-α, interleukin-8 (IL-8, monocyte chemoattractant protein-1 (MCP-1, intercellular adhesion molecule-1 (ICAM-1, and vascular cell adhesion molecule-1(VCAM-1. Results Body weight, percent total body fat and trunk fat were reduced for all subjects after 12 wk (P Conclusion A CRD with daily intake of eggs decreased plasma CRP and increased plasma adiponectin compared to a CRD without eggs. These findings indicate that eggs make a significant contribution to the anti-inflammatory effects of CRD, possibly due to the presence of cholesterol, which increases HDL-C and to the antioxidant lutein which modulates certain inflammatory responses.

  17. α-Mangostin Mediated Pharmacological Modulation of Hepatic Carbohydrate Metabolism in Diabetes Induced Wistar Rat

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    Vikas Kumar

    2016-09-01

    Full Text Available Garcinia mangostana L. (Fruit has been commonly used as folklore drug in the treatment of various types of diseases. The present experiment was designed to evaluate the potential effect of α-mangostin mediated pharmacological modulation of hepatic carbohydrate metabolism in streptozotocin (STZ induced diabetic rats. Oral glucose tolerance test (OGTT was performed in normoglycemic rats. Single intraperitoneal injection of STZ (60 mg/kg, body weight was used for induction the diabetes in Swiss albino (Wistar strain rats. The rats were divided into different groups. Blood glucose level, body weight, insulin, glycated hemoglobin and hemoglobin levels were recorded at regular intervals. Biochemical parameters, liver enzymes, lipid profile, antioxidant parameters and inflammatory cytokine mediators were also scrutinized. Histopathology study of kidney, pancreas and liver were performed. The result of OGTT study depicted the better utilization of glucose in experimental rats. STZ induced diabetic rats treated with α-mangostin (25, 50 and 100 mg/kg, p.o. and glibenclamide depicted the decline in the level of blood glucose; enhanced body weight and showed the better utilization of glucose by different organs. STZ induced diabetic rats treated with α-mangostin illustrated the increased level of plasma insulin, hemoglobin, hexokinase, HDL, total protein, SOD, CAT, GSH and declined level of glycated hemoglobin, fructose-1-6-biphosphatase, glucose-6-Phosphatase, TC, TG, LDL, VLDL, CRE, BUN, SGOT, SGPT, ALP and LPO at effective dose dependent manners. Histological study showed the inflamed blood vessels in diabetic kidney, which was less in α-mangostin treated rats; diabetic pancreatic showed the complete damage of β cells, islets, aciini and producing necrosis, but all damage was less obvious in α-mangostin treating group rats; diabetic liver showed the damage of hepatocytes as well as central vein but was less in treated groups. Considering the

  18. Simultaneous Determination of Binding Constants for Multiple Carbohydrate Hosts in Complex Mixtures

    DEFF Research Database (Denmark)

    Meier, Sebastian; Beeren, Sophie

    2014-01-01

    to determine binding constants for all other detectable and resolvable hosts. With the use of high-resolution 1H−13C HSQC experiments, complexes of amphiphiles with more than 10 different maltooligosaccharides can be resolved. Hereby, the binding capabilities of a set of structurally related hosts can...

  19. Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.

    Science.gov (United States)

    Chovanec, M; Smetana, K; Purkrábková, T; Holíková, Z; Dvoránková, B; André, S; Pytlík, R; Hozák, P; Plzák, J; Sedo, A; Vacík, J; Gabius, H

    2004-01-01

    The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.

  20. A new zinc binding fold underlines the versatility of zinc binding modules in protein evolution.

    Science.gov (United States)

    Sharpe, Belinda K; Matthews, Jacqueline M; Kwan, Ann H Y; Newton, Anthea; Gell, David A; Crossley, Merlin; Mackay, Joel P

    2002-05-01

    Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.

  1. A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

    Science.gov (United States)

    Chang, Tsung-Che; Adak, Avijit K; Lin, Ting-Wei; Li, Pei-Jhen; Chen, Yi-Ju; Lai, Chain-Hui; Liang, Chien-Fu; Chen, Yu-Ju; Lin, Chun-Cheng

    2016-03-15

    The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods.

  2. Modulation of RNase E activity by alternative RNA binding sites.

    Directory of Open Access Journals (Sweden)

    Daeyoung Kim

    Full Text Available Endoribonuclease E (RNase E affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37 of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne that was UV crosslinked with a 5'-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13 containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433, whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71. Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.

  3. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    Science.gov (United States)

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

  4. PPARγ regulates expression of carbohydrate sulfotransferase 11 (CHST11/C4ST1, a regulator of LPL cell surface binding.

    Directory of Open Access Journals (Sweden)

    Ismayil Tasdelen

    Full Text Available The transcription factor PPARγ is the key regulator of adipocyte differentiation, function and maintenance, and the cellular target of the insulin-sensitizing thiazolidinediones. Identification and functional characterization of genes regulated by PPARγ will therefore lead to a better understanding of adipocyte biology and may also contribute to the development of new anti-diabetic drugs. Here, we report carbohydrate sulfotransferase 11 (Chst11/C4st1 as a novel PPARγ target gene. Chst11 can sulphate chondroitin, a major glycosaminoglycan involved in development and disease. The Chst11 gene contains two functional intronic PPARγ binding sites, and is up-regulated at the mRNA and protein level during 3T3-L1 adipogenesis. Chst11 knockdown reduced intracellular lipid accumulation in mature adipocytes, which is due to a lowered activity of lipoprotein lipase, which may associate with the adipocyte cell surface through Chst11-mediated sulfation of chondroitin, rather than impaired adipogenesis. Besides directly inducing Lpl expression, PPARγ may therefore control lipid accumulation by elevating the levels of Chst11-mediated proteoglycan sulfation and thereby increasing the binding capacity for Lpl on the adipocyte cell surface.

  5. Targeting the Cryptococcus neoformans var. grubii Cell Wall Using Lectins: Study of the Carbohydrate-Binding Domain

    Directory of Open Access Journals (Sweden)

    Pamella de Brito Ximenes

    2015-02-01

    Full Text Available Cryptococcus neoformans var. grubii is considered to be the major cause of cryptococcosis in immunosuppressed patients. Understanding cell wall glycoproteins using lectins is of medical interest and can contribute to specific therapy. The aim of this study was to evaluate the carbohydrates on the cell wall of Cryptococcus neoformans var. grubii clinical isolates, using a fluorescein isothiocyanate-lectin binding protocol. Thirty yeast strains stocked in the culture collection were cultivated for 2 days at 30 °C with shaking. Cells were obtained by centrifugation, washed in phosphate-buffered saline, and a suspension of 107 cells/mL was obtained. To determine the binding profile of lectins, concanavalin A (Con A, wheat germ agglutinin (WGA, Ulex europaeus agglutinin I (UEA-I, and peanut agglutinin (PNA conjugated to fluorescein were used. All the tested clinical isolates of Cryptococcus neoformans var. grubii were intensely stained by WGA, moderately stained by Con A, and weakly stained by PNA and UEA-I. Thus, Cryptococcus can be detected in clinical specimens such as blood and cerebrospinal fluid using the fluorescent lectin WGA, which may be considered as an option for detection in cases of suspected cryptococcosis with low laboratory sensitivity. Future applications may be developed using this basic tool.

  6. Exploring the interactions between bacteriophage-encoded glycan binding proteins and carbohydrates.

    Science.gov (United States)

    Simpson, David J; Sacher, Jessica C; Szymanski, Christine M

    2015-10-01

    There is an unprecedented interest in glycobiology due to the increasing appreciation of its impact on all aspects of life. Likewise, bacteriophage biology is enjoying a new renaissance as the post-antibiotic era fuels the search for novel ways to control harmful bacteria. Phages have spent the last 3 billion years developing ways of recognizing and manipulating bacterial surface glycans. Therefore, phages comprise a massive reservoir of glycan-binding and -hydrolyzing proteins with the potential to be exploited for glycan analysis, bacterial diagnostics and therapeutics. We discuss phage tail proteins that recognize bacterial surface polysaccharides, endolysins that bind and cleave peptidoglycan, Ig-like proteins that attach to mucin glycans, and phage effector proteins that recognize both bacterial and eukaryotic oligosaccharides.

  7. Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals

    DEFF Research Database (Denmark)

    Janeček, Štefan; Svensson, Birte; MacGregor, E. Ann

    2011-01-01

    Starch-binding domains (SBDs) comprise distinct protein modules that bind starch, glycogen or related carbohydrates and have been classified into different families of carbohydrate-binding modules (CBMs). The present review focuses on SBDs of CBM20 and CBM48 found in amylolytic enzymes from sever...

  8. Serum carbohydrate-binding IgM are present in Vietnamese striped catfish (Pangasianodon hypophthalmus) but not in North African catfish (Clarias gariepinus).

    Science.gov (United States)

    Giang, Duong Thi Huong; Van Driessche, Edilbert; Beeckmans, Sonia

    2012-02-01

    Pangasianodon hypophthalmus serum was fractionated by affinity chromatography on 12 different Sepharose-carbohydrate columns and proteins eluted by the corresponding sugar. Binding to the affinity matrices is dependent on Ca(2+) ions. Upon gel filtration using Superose-12, essentially one fraction was obtained, eluting as a protein with a molecular mass of about 900 kDa. SDS-PAGE in reducing conditions revealed the presence of large (72 kDa) subunits (H-chains) and one up to three small (24, 26 and/or 28-29 kDa) subunits (L-chains). The isolated proteins were shown to be IgM since they bind monoclonal anti-P. hypophthalmus IgM antibodies. Rabbit polyclonal anti-galactose-binding IgM only cross-react with some sugar-binding IgM. The H-chains of the anti-carbohydrate IgM are glycosylated. Circular dichroism studies revealed that the IgMs have an "all-β" type of structure, and that Ca(2+) ions, though essential for carbohydrate-binding activity, are not required for the structural integrity of the molecules. In non-reducing SDS-PAGE, only monomers and halfmers were obtained, showing that there are no disulfide bonds linking the monomers, and that a disulfide bond connecting both H-chains within one monomer is only present in 45% of the molecules. Both the monomers and the halfmers display molecular mass heterogeneity which is indicative for redox forms at the level of the intradomain disulfide bonds. The native carbohydrate-binding IgMs agglutinate erythrocytes from different animals, as well as fish pathogenic bacteria. Similar proteins could not be isolated from another catfish, Clarias gariepinus.

  9. Chromium picolinate modulates serotonergic properties and carbohydrate metabolism in a rat model of diabetes.

    Science.gov (United States)

    Komorowski, James R; Tuzcu, Mehmet; Sahin, Nurhan; Juturu, Vijaya; Orhan, Cemal; Ulas, Mustafa; Sahin, Kazim

    2012-10-01

    Chromium picolinate (CrPic) has shown both antidepressant and antidiabetic properties. In this study, the effects of CrPic on serotonergic properties and carbohydrate metabolism in diabetic rats were evaluated. Sixty male Sprague-Dawley rats were divided into four groups. (1) The control group received only standard diet (8 % fat). (2) The CrPic group was fed standard diet and CrPic (80 μg CrPic per kilogram body mass (b.m.)/day), for 10 weeks (microgram/kilogram b.m./day). (3) The HFD/STZ group fed a high-fat diet (HFD, 40 % fat) for 2 weeks and then received streptozotocin (STZ, 40 mg/kg, i.p.) (i.v.) HFD-STZ-CrPic group treated as the previous group and then were administered CrPic. CrPic administration to HFD/STZ-treated rats increased brain chromium levels and improved all measurements of carbohydrate metabolism and serotonergic properties (P<0.001). CrPic also significantly increased levels of insulin, tryptophan, and serotonin (P<0.001) in the serum and brain, and decreased cortisol levels in the serum (P<0.01). Except chromium levels, no significant effect of CrPic supplementation was detected on the overall measured parameters in the control group. CrPic administration was well tolerated without any adverse events. The results support the use of CrPic supplementation which improves serotonergic properties of brain in diabetes.

  10. Perilipin polymorphism interacts with dietary carbohydrates to modulate anthropometric traits in Hispanics of Caribbean origin

    Science.gov (United States)

    Perilipin (PLIN) is the major protein surrounding lipid droplets in adipocytes and regulates adipocyte metabolism by modulating the interaction between lipases and triacylglycerol stores. Associations between PLIN gene polymorphisms and obesity risk have been described, but interactions with die...

  11. In silico analysis of molecular mechanisms of Galanthus nivalis agglutinin-related lectin-induced cancer cell death from carbohydrate-binding motif evolution hypothesis.

    Science.gov (United States)

    Yu, Qi-Jia; Li, Zi-Yue; Yao, Shun; Ming, Miao; Wang, Shu-Ya; Liu, Bo; Bao, Jin-Ku

    2011-10-01

    Galanthus nivalis agglutinin-related lectins, a superfamily of strictly mannose-binding-specific lectins widespread amongst monotyledonous plants, have drawn a rising attention for their remarkable anti-proliferative and apoptosis-inducing activities toward various types of cancer cells; however, the precise molecular mechanisms by which they induce tumor cell apoptosis are still only rudimentarily understood. Herein, we found that the three conserved motifs "QXDXNXVXY," the mannose-specific binding sites, could mutate at one or more amino acid sites, which might be a driving force for the sequential evolution and thus ultimately leading to the complete disappearance of the three conserved motifs. In addition, we found that the motif evolution could result in the diversification of sugar-binding types that G. nivalis agglutinin-related lectins could bind from specific mannose receptors to more types of sugar-containing receptors in cancer cells. Subsequently, we indicated that some sugar-containing receptors such as TNFR1, EGFR, Hsp90, and Hsp70 could block downstream anti-apoptotic or survival signaling pathways, which, in turn, resulted in tumor cell apoptosis. Taken together, our hypothesis that carbohydrate-binding motif evolution may impact the G. nivalis agglutinin-related lectin-induced survival or anti-apoptotic pathways would provide a new perspective for further elucidating the intricate relationships between the carbohydrate-binding specificities and complex molecular mechanisms by which G. nivalis agglutinin-related lectins induce cancer cell death.

  12. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Lummis, S.C.R.; Johnston, G.A.R. (Univ. of Sydney, New South Wales (Australia)); Nicoletti, G. (Royal Melbourne Inst. of Tech. (Australia)); Holan, G. (CSIRO, Melbourne (Australia))

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.

  13. Two Secondary Carbohydrate Binding Sites on the Surface of Barley alpha-Amylase 1 Have Distinct Functions and Display Synergy in Hydrolysis of Starch Granules

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch; Bozonnet, Sophie; Seo, Eun-Seong

    2009-01-01

    Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)8-barrel and the noncatalytic C-terminal domain, respective...... to single alpha-glucan chains accessible for hydrolysis, is proposed. SBS1 and SBS2 also influence the kinetics of hydrolysis for amylose and maltooligosaccharides, the degree of multiple attack on amylose, and subsite binding energies....

  14. Carbohydrates as food allergens

    OpenAIRE

    SOH, Jian Yi; Huang, Chiung Hui; Lee, Bee Wah

    2015-01-01

    The literature supports the notion that carbohydrate epitopes, on their own, do not contribute significantly to the induction of allergic reactions. They bind weakly to IgE antibodies and have been termed as cross reactive carbohydrate determinants. These epitopes cause confusion in in vitro IgE testing through nonspecific cross-reactivity. Coincident with the rising trends in food allergy prevalence, there has recently been reports of anaphylaxis induced by carbohydrate epitopes. There are t...

  15. Modulation of carbohydrate metabolism and peptide hormones by soybean isoflavones and probiotics in obesity and diabetes.

    Science.gov (United States)

    Ali, Ali A; Velasquez, Manuel T; Hansen, Carl T; Mohamed, Ali I; Bhathena, Sam J

    2005-11-01

    Soybean and its isoflavones have been shown to have beneficial effects on carbohydrate and lipid metabolism and on renal function. Probiotics may potentiate the beneficial effects of isoflavones by converting the inactive isoflavone glycoside to aglycones, which are biologically active, thereby producing a synergistic effect. We therefore studied the effects of soybean isoflavones in the presence and absence of probiotics on glucose and triglyceride metabolism and the peptide hormones involved in their metabolism. Lean and obese SHR/N-cp rats were fed AIN-93 diets containing 0.1% soybean isoflavone mixture, 0.1% probiotics mixture or both. Plasma was analyzed for glucose, triglycerides, parameters of renal function and peptide hormones -- insulin, leptin, glucagon and ACTH -- that are involved in glucose and lipid metabolism. Isoflavones given alone lowered plasma glucose in both phenotypes while triglyceride was decreased only in lean animals. Isoflavones also lowered aspartate amino transferase and alanine amino transferase in both phenotypes. Isoflavones had significant effect on plasma insulin, leptin and glucagon in lean rats but not in obese rats. Thus, our data show that in lean animals, isoflavones have hypoglycemic and hypolipidemic effect, and the effect is mediated by changes in peptide hormones. When lipid levels are very high as in obese rats, isoflavones fail to lower plasma triglyceride levels. Probiotics do not appear to enhance the effect of isoflavones.

  16. Copper at synapse: Release, binding and modulation of neurotransmission.

    Science.gov (United States)

    D'Ambrosi, Nadia; Rossi, Luisa

    2015-11-01

    Over the last decade, a piece of the research studying copper role in biological systems was devoted to unravelling a still elusive, but extremely intriguing, aspect that is the involvement of copper in synaptic function. These studies were prompted to provide a rationale to the finding that copper is released in the synaptic cleft upon depolarization. The copper pump ATP7A, which mutations are responsible for diseases with a prominent neurodegenerative component, seems to play a pivotal role in the release of copper at synapses. Furthermore, it was found that, when in the synaptic cleft, copper can control, directly or indirectly, the activity of the neurotransmitter receptors (NMDA, AMPA, GABA, P2X receptors), thus affecting excitability. In turn, neurotransmission can affect copper trafficking and delivery in neuronal cells. Furthermore, it was reported that copper can also modulate synaptic vesicles trafficking and the interaction between proteins of the secretory pathways. Interestingly, proteins with a still unclear role in neuronal system though associated with the pathogenesis of neurodegenerative diseases (the amyloid precursor protein, APP, the prion protein, PrP, α-synuclein, α-syn) show copper-binding domains. They may act as copper buffer at synapses and participate in the interplay between copper and the neurotransmitters receptors. Given that copper dysmetabolism occurs in several diseases affecting central and peripheral nervous system, the findings on the contribution of copper in synaptic transmission, beside its more consolidate role as a neuronal enzymes cofactor, may open new insights for therapy interventions.

  17. The role of the glucose-sensing transcription factor carbohydrate-responsive element-binding protein pathway in termite queen fertility

    DEFF Research Database (Denmark)

    Sillam-Dussès, David; Hanus, Robert; Thomas-Poulsen, Michael

    2016-01-01

    Termites are among the few animals that themselves can digest the most abundant organic polymer, cellulose, into glucose. In mice and Drosophila, glucose can activate genes via the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to induce glucose utilization and de...... novo lipogenesis. Here, we identify a termite orthologue of ChREBP and its downstream lipogenic targets, including acetyl-CoA carboxylase and fatty acid synthase. We show that all of these genes, including ChREBP, are upregulated in mature queens compared with kings, sterile workers and soldiers...... in eight different termite species. ChREBP is expressed in several tissues, including ovaries and fat bodies, and increases in expression in totipotent workers during their differentiation into neotenic mature queens. We further show that ChREBP is regulated by a carbohydrate diet in termite queens...

  18. Counting carbohydrates

    Science.gov (United States)

    Carb counting; Carbohydrate-controlled diet; Diabetic diet; Diabetes-counting carbohydrates ... Many foods contain carbohydrates (carbs), including: Fruit and fruit juice Cereal, bread, pasta, and rice Milk and milk products, soy milk Beans, legumes, ...

  19. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

    DEFF Research Database (Denmark)

    Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska;

    2016-01-01

    γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently...

  20. Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3β protein.

    Science.gov (United States)

    Ge, Qiang; Huang, Nian; Wynn, R Max; Li, Yang; Du, Xinlin; Miller, Bonnie; Zhang, Hong; Uyeda, Kosaku

    2012-12-07

    Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex.

  1. Binding of Haemophilus ducreyi to carbohydrate receptors is mediated by the 58.5-kDa GroEL heat shock protein.

    Science.gov (United States)

    Pantzar, Martina; Teneberg, Susann; Lagergård, Teresa

    2006-08-01

    The bacterium Haemophilus ducreyi causes the sexually transmitted disease chancroid, which is characterized by the appearance of mucocutaneous, persistent ulcers on the external genitals. To identify carbohydrate receptors that mediate the attachment of this pathogen to host cells, we investigated the binding of 35S-methionine-labeled H. ducreyi strains to a panel of defined glycosphingolipids that were separated on thin layer chromatography plates. H. ducreyi bound to lactosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, neolactotetraosylceramide, the GM3 ganglioside, and sulfatide. To elucidate the role of the surface-located 58.5-kDa GroEL heat shock protein (HSP) of H. ducreyi in attachment, we investigated the binding of purified HSP to the same panel of glycosphingolipids. Our results suggest that the 58.5-kDa GroEL HSP of H. ducreyi is responsible for the attachment of this bacterium to the majority of the tested glycosphingolipids, and thus represents a potential bacterial adhesin.

  2. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    Energy Technology Data Exchange (ETDEWEB)

    Kraschnefski, Mark J.; Scott, Stacy A. [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia); Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von [Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010 (Australia); Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia)

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.

  3. The carboxy-terminal domain of Dictyostelium C-module-binding factor is an independent gene regulatory entity.

    Directory of Open Access Journals (Sweden)

    Jörg Lucas

    Full Text Available The C-module-binding factor (CbfA is a multidomain protein that belongs to the family of jumonji-type (JmjC transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD. An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

  4. Role of the carbohydrate-binding sites of griffithsin in the prevention of DC-SIGN-mediated capture and transmission of HIV-1.

    Directory of Open Access Journals (Sweden)

    Bart Hoorelbeke

    Full Text Available BACKGROUND: The glycan-targeting C-type DC-SIGN lectin receptor is implicated in the transmission of the human immunodeficiency virus (HIV by binding the virus and transferring the captured HIV-1 to CD4(+ T lymphocytes. Carbohydrate binding agents (CBAs have been reported to block HIV-1 infection. We have now investigated the potent mannose-specific anti-HIV CBA griffithsin (GRFT on its ability to inhibit the capture of HIV-1 to DC-SIGN, its DC-SIGN-directed transmission to CD4(+ T-lymphocytes and the role of the three carbohydrate-binding sites (CBS of GRFT in these processes. FINDINGS: GRFT inhibited HIV-1(IIIB infection of CEM and HIV-1(NL4.3 infection of C8166 CD4(+ T-lymphocytes at an EC50 of 0.059 and 0.444 nM, respectively. The single mutant CBS variants of GRFT (in which a key Asp in one of the CBS was mutated to Ala were about ∼20 to 60-fold less potent to prevent HIV-1 infection and ∼20 to 90-fold less potent to inhibit syncytia formation in co-cultures of persistently HIV-1 infected HuT-78 and uninfected C8166 CD4(+ T-lymphocytes. GRFT prevents DC-SIGN-mediated virus capture and HIV-1 transmission to CD4(+ T-lymphocytes at an EC50 of 1.5 nM and 0.012 nM, respectively. Surface plasmon resonance (SPR studies revealed that wild-type GRFT efficiently blocked the binding between DC-SIGN and immobilized gp120, whereas the point mutant CBS variants of GRFT were ∼10- to 15-fold less efficient. SPR-analysis also demonstrated that wild-type GRFT and its single mutant CBS variants have the capacity to expel bound gp120 from the gp120-DC-SIGN complex in a dose dependent manner, a property that was not observed for HHA, another mannose-specific potent anti-HIV-1 CBA. CONCLUSION: GRFT is inhibitory against HIV gp120 binding to DC-SIGN, efficiently prevents DC-SIGN-mediated transfer of HIV-1 to CD4(+ T-lymphocytes and is able to expel gp120 from the gp120-DC-SIGN complex. Functionally intact CBS of GRFT are important for the optimal action of

  5. The role of the glucose-sensing transcription factor carbohydrate-responsive element-binding protein pathway in termite queen fertility

    Science.gov (United States)

    Sillam-Dussès, David; Hanus, Robert; Poulsen, Michael; Roy, Virginie; Favier, Maryline

    2016-01-01

    Termites are among the few animals that themselves can digest the most abundant organic polymer, cellulose, into glucose. In mice and Drosophila, glucose can activate genes via the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to induce glucose utilization and de novo lipogenesis. Here, we identify a termite orthologue of ChREBP and its downstream lipogenic targets, including acetyl-CoA carboxylase and fatty acid synthase. We show that all of these genes, including ChREBP, are upregulated in mature queens compared with kings, sterile workers and soldiers in eight different termite species. ChREBP is expressed in several tissues, including ovaries and fat bodies, and increases in expression in totipotent workers during their differentiation into neotenic mature queens. We further show that ChREBP is regulated by a carbohydrate diet in termite queens. Suppression of the lipogenic pathway by a pharmacological agent in queens elicits the same behavioural alterations in sterile workers as observed in queenless colonies, supporting that the ChREBP pathway partakes in the biosynthesis of semiochemicals that convey the signal of the presence of a fertile queen. Our results highlight ChREBP as a likely key factor for the regulation and signalling of queen fertility. PMID:27249798

  6. 碳水化合物反应元件结合蛋白的研究进展%Research Progress on Carbohydrate Response Element Binding Protein

    Institute of Scientific and Technical Information of China (English)

    唐慧; 付茂忠; 潘志雄; 王继文; 易军; 王淮; 易礼胜; 邓由飞; 甘佳; 俄木曲者

    2013-01-01

    Glycolipid metabolism is the foundation of the energy sources of normal growth and development in human and animals,directly affects the animal lipid traits,therefore it has become the breakthrough point of cultivating high quality livestock and poultry breed. Carbohydrate response element binding protein(ChREBP) plays an important role in regulation of animal glycolipid metabolism in recent years,especially in major site of glycolipid metabolism-liver. In this review,the author summarised the structure characteristics,tissue distributions,mechanism of glucose-responsive,biological functions,gene expression regulation of ChREBP,and it might provide theoretical basis for following research.%糖脂代谢是人和动物正常生长发育的能源基础,这将直接影响着动物的脂质性状,因此糖脂代谢目前已经成为培育优质畜禽品种的切入点.近年来,发现碳水化合物反应元件结合蛋白(carbohydrate response element binding protein,ChREBP)在调控动物糖脂代谢中具有重要的作用,特别在糖脂代谢的主要场所——肝脏中对糖脂代谢发挥着关键作用.作者就ChREBP的结构特征、组织分布、葡萄糖应答机制、生物学功能、表达调控等方面作一综述,以期为今后的研究提供理论依据.

  7. Two DNA-binding and Nick Recognition Modules in Human DNA Ligase III*

    OpenAIRE

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Tomkinson, Alan E.; Ellenberger, Tom

    2008-01-01

    Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bin...

  8. Carbohydrate Analysis

    Science.gov (United States)

    Bemiller, James N.

    Carbohydrates are important in foods as a major source of energy, to impart crucial textural properties, and as dietary fiber which influences physiological processes. Digestible carbohydrates, which are converted into monosaccharides, which are absorbed, provide metabolic energy. Worldwide, carbohydrates account for more than 70% of the caloric value of the human diet. It is recommended that all persons should limit calories from fat (the other significant source) to not more than 30% and that most of the carbohydrate calories should come from starch. Nondigestible polysaccharides (all those other than starch) comprise the major portion of dietary fiber (Sect. 10.5). Carbohydrates also contribute other attributes, including bulk, body, viscosity, stability to emulsions and foams, water-holding capacity, freeze-thaw stability, browning, flavors, aromas, and a range of desirable textures (from crispness to smooth, soft gels). They also provide satiety. Basic carbohydrate structures, chemistry, and terminology can be found in references (1, 2).

  9. Study on Binding Specificity of Tt APuX21 Module for Polysaccharides%结构域Tt APuX21与碳水化合物的结合特性分析

    Institute of Scientific and Technical Information of China (English)

    谢晚彬

    2009-01-01

    This study aimed to investigate whether Tt APuX21 was a functional carbohydrate-binding module (CBM). In order to test whether Tt APuX21 possessed affinity for polysaccharides, non-denaturing affinity electrophoresis (NDAE) was carried out using different carbohydrates as ligands. NDAE displayed that Tt APuX21 had affinity for soluble starch, dextrin and pullulan. The results showed that Tt APuX21 was a functional carbohydrate-binding module.%以多种碳水化合物为底物,通过非变性亲和电泳(NDAE)来检验Tt APuX21是否能与碳水化合物结合,以证明Tt APuX21是否确为有功能的碳水化合物结合结构域.NDAE的分析结果表明,Tt A-PuX21能与水溶性淀粉、糊精和普鲁兰糖结合,Tt APuX21是一个有功能的碳水化合物结合结构域.

  10. Carbohydrate-active enzymes from pigmented Bacilli: a genomic approach to assess carbohydrate utilization and degradation

    Directory of Open Access Journals (Sweden)

    Henrissat Bernard

    2011-09-01

    Full Text Available Abstract Background Spore-forming Bacilli are Gram-positive bacteria commonly found in a variety of natural habitats, including soil, water and the gastro-intestinal (GI-tract of animals. Isolates of various Bacillus species produce pigments, mostly carotenoids, with a putative protective role against UV irradiation and oxygen-reactive forms. Results We report the annotation of carbohydrate active enzymes (CAZymes of two pigmented Bacilli isolated from the human GI-tract and belonging to the Bacillus indicus and B. firmus species. A high number of glycoside hydrolases (GHs and carbohydrate binding modules (CBMs were found in both isolates. A detailed analysis of CAZyme families, was performed and supported by growth data. Carbohydrates able to support growth as the sole carbon source negatively effected carotenoid formation in rich medium, suggesting that a catabolite repression-like mechanism controls carotenoid biosynthesis in both Bacilli. Experimental results on biofilm formation confirmed genomic data on the potentials of B. indicus HU36 to produce a levan-based biofilm, while mucin-binding and -degradation experiments supported genomic data suggesting the ability of both Bacilli to degrade mammalian glycans. Conclusions CAZy analyses of the genomes of the two pigmented Bacilli, compared to other Bacillus species and validated by experimental data on carbohydrate utilization, biofilm formation and mucin degradation, suggests that the two pigmented Bacilli are adapted to the intestinal environment and are suited to grow in and colonize the human gut.

  11. Characterization of an endo-processive-type xyloglucanase having a β-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.

    Science.gov (United States)

    Ichinose, Hitomi; Araki, Yuko; Michikawa, Mari; Harazono, Koichi; Yaoi, Katsuro; Karita, Shuichi; Kaneko, Satoshi

    2012-11-01

    We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to β-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.

  12. Sulindac-derived RXRα modulators inhibit cancer cell growth by binding to a novel site.

    Science.gov (United States)

    Chen, Liqun; Wang, Zhi-Gang; Aleshin, Alexander E; Chen, Fan; Chen, Jiebo; Jiang, Fuquan; Alitongbieke, Gulimiran; Zeng, Zhiping; Ma, Yue; Huang, Mingfeng; Zhou, Hu; Cadwell, Gregory; Zheng, Jian-Feng; Huang, Pei-Qiang; Liddington, Robert C; Zhang, Xiao-kun; Su, Ying

    2014-05-22

    Retinoid X receptor-alpha (RXRα), an intriguing and unique drug target, can serve as an intracellular target mediating the anticancer effects of certain nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac. We report the synthesis and characterization of two sulindac analogs, K-8008 and K-8012, which exert improved anticancer activities over sulindac in a RXRα-dependent manner. The analogs inhibit the interaction of the N-terminally truncated RXRα (tRXRα) with the p85α subunit of PI3K, leading to suppression of AKT activation and induction of apoptosis. Crystal structures of the RXRα ligand-binding domain (LBD) with K-8008 or K-8012 reveal that both compounds bind to tetrameric RXRα LBD at a site different from the classical ligand-binding pocket. Thus, these results identify K-8008 and K-8012 as tRXRα modulators and define a binding mechanism for regulating the nongenomic action of tRXRα.

  13. Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

    Science.gov (United States)

    Mo, H; Winter, H C; Van Damme, E J; Peumans, W J; Misaki, A; Goldstein, I J

    2001-05-01

    Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.

  14. Carbohydrate Loading.

    Science.gov (United States)

    Csernus, Marilyn

    Carbohydrate loading is a frequently used technique to improve performance by altering an athlete's diet. The objective is to increase glycogen stored in muscles for use in prolonged strenuous exercise. For two to three days, the athlete consumes a diet that is low in carbohydrates and high in fat and protein while continuing to exercise and…

  15. Carbohydrate microarrays

    DEFF Research Database (Denmark)

    Park, Sungjin; Gildersleeve, Jeffrey C; Blixt, Klas Ola;

    2012-01-01

    In the last decade, carbohydrate microarrays have been core technologies for analyzing carbohydrate-mediated recognition events in a high-throughput fashion. A number of methods have been exploited for immobilizing glycans on the solid surface in a microarray format. This microarray-based technol...

  16. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    Science.gov (United States)

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  17. The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

    DEFF Research Database (Denmark)

    Morrill, Johan; Kulcinskaja, Evelina; Sulewska, Anna Maria

    2015-01-01

    β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here......, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8). BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan...

  18. The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

    DEFF Research Database (Denmark)

    Morrill, Johan; Kulcinskaja, Evelina; Sulewska, Anna Maria;

    2015-01-01

    β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here......, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8). BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan...

  19. Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB

    Energy Technology Data Exchange (ETDEWEB)

    Pyburn, Tasia M.; Yankovskaya, Victoria; Bensing, Barbara A.; Cecchini, Gary; Sullam, Paul M.; Iverson, T.M. (VA); (Vanderbilt); (UCSF)

    2012-07-11

    The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB{sub BR}) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB{sub BR} buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB{sub BR} in each buffer. While both sets of conditions supported crystal growth in space group P2{sub 1}2{sub 1}2{sub 1}, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 {angstrom} for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 {angstrom} for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 {angstrom} resolution. A complete data set has been collected to 1.3 {angstrom} resolution with an overall R{sub merge} value of 0.04 and an R{sub merge} value of 0.33 in the highest resolution shell.

  20. Sensitivity of transmitted and founder human immunodeficiency virus type 1 envelopes to carbohydrate-binding agents griffithsin, cyanovirin-N and Galanthus nivalis agglutinin.

    Science.gov (United States)

    Hu, Bodan; Du, Tao; Li, Chang; Luo, Sukun; Liu, Yalan; Huang, Xin; Hu, Qinxue

    2015-12-01

    Human immunodeficiency virus type 1 (HIV-1) transmission often results from infection by a single transmitted/founder (T/F) virus. Here, we investigated the sensitivity of T/F HIV-1 envelope glycoproteins (Envs) to microbicide candidate carbohydrate-binding agents (CBAs) griffithsin (GRFT), cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA), showing that T/F Envs demonstrated different sensitivity to CBAs, with IC50 values ranging from 0.006 ± 0.0003 to >10 nM for GRFT, from 0.6 ± 0.2 to 28.9 ± 2.9 nM for CV-N and from 1.3 ± 0.2 to >500 nM for GNA. We further revealed that deglycosylation at position 295 or 448 decreased the sensitivity of T/F Env to GRFT, and at 339 to both CV-N and GNA. Mutation of all the three glcyans rendered a CBA-sensitive T/F Env largely resistant to GRFT, indicating that the sensitivity of T/F Env to GRFT is mainly determined by glycans at 295, 339 and 448. Our study identified specific T/F Env residues associated with CBA sensitivity.

  1. Broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Marijke M F Alen

    Full Text Available BACKGROUND: Dendritic cells (DC, present in the skin, are the first target cells of dengue virus (DENV. Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN is present on DC and recognizes N-glycosylation sites on the E-glycoprotein of DENV. Thus, the DC-SIGN/E-glycoprotein interaction can be considered as an important target for inhibitors of viral replication. We evaluated various carbohydrate-binding agents (CBAs against all four described serotypes of DENV replication in Raji/DC-SIGN(+ cells and in monocyte-derived DC (MDDC. METHODOLOGY/PRINCIPAL FINDINGS: A dose-dependent anti-DENV activity of the CBAs Hippeastrum hybrid (HHA, Galanthus nivalis (GNA and Urtica dioica (UDA, but not actinohivin (AH was observed against all four DENV serotypes as analyzed by flow cytometry making use of anti-DENV antibodies. Remarkably, the potency of the CBAs against DENV in MDDC cultures was significantly higher (up to 100-fold than in Raji/DC-SIGN(+ cells. Pradimicin-S (PRM-S, a small-size non-peptidic CBA, exerted antiviral activity in MDDC but not in Raji/DC-SIGN(+ cells. The CBAs act at an early step of DENV infection as they bind to the viral envelope of DENV and subsequently prevent virus attachment. Only weak antiviral activity of the CBAs was detected when administered after the virus attachment step. The CBAs were also able to completely prevent the cellular activation and differentiation process of MDDC induced upon DENV infection. CONCLUSIONS/SIGNIFICANCE: The CBAs exerted broad spectrum antiviral activity against the four DENV serotypes, laboratory-adapted viruses and low passage clinical isolates, evaluated in Raji/DC-SIGN(+ cells and in primary MDDC.

  2. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

    DEFF Research Database (Denmark)

    Eghorn, Laura Friis; Høstgaard-Jensen, Kirsten; Kongstad, Kenneth Thermann

    2014-01-01

    conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed......γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate...... whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive...

  3. Calcium modulates promoter occupancy by the Entamoeba histolytica Ca2+-binding transcription factor URE3-BP.

    Science.gov (United States)

    Gilchrist, Carol A; Leo, Megan; Line, C Genghis; Mann, Barbara J; Petri, William A

    2003-02-14

    The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) binds to the URE3 sequence of the Gal/GalNAc-inhibitable lectin hgl5 and ferredoxin 1 (fdx) gene promoters. This binding can be inhibited in vitro by addition of calcium. Two EF-hand motifs, which are associated with the ability to bind calcium, are present in the amino acid sequence of URE3-BP. Mutation of the second EF-hand motif in URE3-BP resulted in the loss of calcium inhibition of DNA binding as monitored by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays revealed that URE3-BP was physically bound to the hgl5 and fdx promoters in vivo. Parasite intracellular calcium concentrations were altered by changes in extracellular calcium. Promoter occupancy was lost when intracellular calcium levels were increased by coordinate increases in extracellular calcium. Increased intracellular calcium also resulted in decreased levels of URE3-BP mRNA. Together these results demonstrate that changes in extracellular calcium result in changes in URE3-BP mRNA and in the ability of URE3-BP to bind to URE3-containing promoters. Modulation of URE3-BP by calcium may represent an important mechanism of control of gene expression in E. histolytica.

  4. Disabled-2 modulates homotypic and heterotypic platelet interactions by binding to sulfatides.

    Science.gov (United States)

    Welsh, John D; Charonko, John J; Salmanzadeh, Alireza; Drahos, Karen E; Shafiee, Hadi; Stremler, Mark A; Davalos, Rafael V; Capelluto, Daniel G S; Vlachos, Pavlos P; Finkielstein, Carla V

    2011-07-01

    Disabled-2 (Dab2) inhibits platelet aggregation by competing with fibrinogen for binding to the α(IIb) β(3) integrin receptor, an interaction that is modulated by Dab2 binding to sulfatides at the outer leaflet of the platelet plasma membrane. The disaggregatory function of Dab2 has been mapped to its N-terminus phosphotyrosine-binding (N-PTB) domain. Our data show that the surface levels of P-selectin, a platelet transmembrane protein known to bind sulfatides and promote cell-cell interactions, are reduced by Dab2 N-PTB, an event that is reversed in the presence of a mutant form of the protein that is deficient in sulfatide but not in integrin binding. Importantly, Dab2 N-PTB, but not its sulfatide binding-deficient form, was able to prevent sulfatide-induced platelet aggregation when tested under haemodynamic conditions in microfluidic devices at flow rates with shear stress levels corresponding to those found in vein microcirculation. Moreover, the regulatory role of Dab2 N-PTB extends to platelet-leucocyte adhesion and aggregation events, suggesting a multi-target role for Dab2 in haemostasis.

  5. Characterization of a modular enzyme of exo-1,5-alpha-L-arabinofuranosidase and arabinan binding module from Streptomyces avermitilis NBRC14893.

    Science.gov (United States)

    Ichinose, Hitomi; Yoshida, Makoto; Fujimoto, Zui; Kaneko, Satoshi

    2008-09-01

    A gene encoding an alpha-L-arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45 degrees C and was stable over the pH range of 5.0-6.5 at 30 degrees C. The enzyme hydrolyzed p-nitrophenol (PNP)-alpha-L-arabinofuranoside but did not hydrolyze PNP-alpha-L-arabinopyranoside, PNP-beta-D-xylopyranoside, or PNP-beta-D-galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside and methyl 3-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside were not. These data suggested that the enzyme only cleaves alpha-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-alpha-L-arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.

  6. Confined housing system increased abdominal and subcutaneous fat deposition and gene expressions of carbohydrate response element-binding protein and sterol regulatory element-binding protein 1 in chicken.

    Science.gov (United States)

    Li, Q; Zhao, X L; Gilbert, E R; Liu, Y P; Wang, Y; Qiu, M H; Zhu, Q

    2015-02-06

    Free-range production system is increasingly being used in poultry breeding and feed production in many countries. The objective of the current experiment was to evaluate the effects of different raising systems on fat-related traits and mRNA levels of liver lipogenesis genes in Erlang Mountainous chicken. Each of 10 birds (91 day old) from caged, indoor-floor housed, and free-range housing systems was slaughtered, and fat-related traits, live body weight (BW), subcutaneous fat thickness (SFT), abdominal fat weight (AFW), abdominal fat percentage (AFP), and intramuscular fat content were determined. The mRNA levels of liver X receptor α, carbohydrate response element-binding protein (ChREBP), sterol regulatory element-binding protein-1 (SREBP1), and fatty acid synthase were detected. The caged chicken exhibited significantly higher BW, SFT, and AFW than those of free-ranged chicken (P caged chicken, while the lowest level was found in free-ranged chicken. Association analysis indicated that there were significant (P caged chicken was probably induced by increased gene expression of ChREBP and SREBP1 in the liver.

  7. Carbohydrate malabsorption

    DEFF Research Database (Denmark)

    Rumessen, J J; Nordgaard-Andersen, I; Gudmand-Høyer, E

    1994-01-01

    Previous studies in small series of healthy adults have suggested that parallel measurement of hydrogen and methane resulting from gut fermentation may improve the precision of quantitative estimates of carbohydrate malabsorption. Systematic, controlled studies of the role of simultaneous hydrogen...

  8. Glutamate-containing dipeptides do not modulate ligand binding at excitatory amino acid receptors.

    Science.gov (United States)

    Baud, J; Fagg, G E

    1986-10-08

    Dipeptides of the structure X-Glu (e.g. X = Phe, Leu) have been proposed as allosteric modulators of excitatory amino acid receptors in rat brain membranes. Here we report that these dipeptides reduce the binding of L-[3H]Glu (predominantly N-methyl-D-aspartate-sensitive sites) and of [3H]kainate to postsynaptic density preparations isolated from rat brain. However, several observations indicate that the effects of these dipeptides are mediated not by allosteric modulation, but by free L-Glu liberated by the actions of a membrane-associated aminopeptidase. The absolute and relative potencies of the dipeptides are similar at all acidic amino acid binding sites examined to date, suggesting the involvement of a factor with similar activity at each site (e.g. L-Glu). N-Acetyl-Met-Glu is a weak inhibitor of L-Glu and kainate binding, and N-blocked peptides are known to be poor substrates of aminopeptidases. Bestatin, an inhibitor of aminopeptidases, decreases or abolishes the effects of substrate dipeptides on L-Glu and kainate receptor binding, while having no effect itself.

  9. IQGAP1 Binds to Yes-associated Protein (YAP) and Modulates Its Transcriptional Activity.

    Science.gov (United States)

    Sayedyahossein, Samar; Li, Zhigang; Hedman, Andrew C; Morgan, Chase J; Sacks, David B

    2016-09-09

    During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knock-out of endogenous IQGAP1 significantly increased the formation of nuclear YAP-TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP-TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling.

  10. Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

    Directory of Open Access Journals (Sweden)

    Corleta H.

    2000-01-01

    Full Text Available Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1 interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5% followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

  11. Reciprocal allosteric modulation of carbon monoxide and warfarin binding to ferrous human serum heme-albumin.

    Directory of Open Access Journals (Sweden)

    Alessio Bocedi

    Full Text Available Human serum albumin (HSA, the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s. As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i of carbon monoxide (CO binding to ferrous human serum heme-albumin (HSA-heme-Fe(II by warfarin (WF, and (ii of WF binding to HSA-heme-Fe(II by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II, respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands. This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II. The HSA-heme-Fe(II populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i upon CO binding a conformational change of HSA-heme-Fe(II takes place (likely reflecting the displacement of an endogenous ligand by CO, and (ii CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II.

  12. Does protein binding modulate the effect of angiotensin II receptor antagonists?

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  13. Insights into the modulation of dopamine transporter function by amphetamine, orphenadrine and cocaine binding

    Directory of Open Access Journals (Sweden)

    Mary Hongying Cheng

    2015-06-01

    Full Text Available Human dopamine transporter (hDAT regulates dopaminergic signaling in the central nervous system by maintaining the synaptic concentration of dopamine (DA at physiological levels, upon reuptake of DA into presynaptic terminals. DA translocation involves the co-transport of two sodium ions and the channeling of a chloride ion, and it is achieved via alternating access between outward-facing and inward-facing states of DAT. hDAT is a target for addictive drugs such as cocaine, amphetamine (AMPH and therapeutic antidepressants. Our recent quantitative systems pharmacology study suggested that orphenadrine (ORPH, an anticholinergic agent and anti-PD drug, might be repurposable as a DAT drug. Previous studies have shown that DAT-substrates like AMPH or -blockers like cocaine modulate the function of DAT in different ways. However, the molecular mechanisms of modulation remained elusive due to the lack of structural data on DAT. The newly resolved DAT structure from Drosophila melanogaster opens the way to a deeper understanding of the mechanism and time evolution of DAT-drug/ligand interactions. Using a combination of homology modeling, docking analysis, molecular dynamics simulations and molecular biology experiments, we performed a comparative study of the binding properties of DA, AMPH, ORPH and cocaine, and their modulation of hDAT function. Simulations demonstrate that binding DA or AMPH drives a structural transition towards a functional form predisposed to translocate the ligand. In contrast, ORPH appears to inhibit DAT function by arresting it in the outward-facing open conformation. The analysis shows that cocaine and ORPH competitively bind DAT, with the binding pose and affinity dependent on the conformational state of DAT. Further assays show that the effect of ORPH on DAT uptake and endocytosis is comparable to that of cocaine.

  14. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  15. Defining Starch Binding by Glucan Phosphatases

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2015-01-01

    phosphatases. The main objective of this study was to quantify the binding affinity of different enzymes that are involved in this cyclic process. We established a protocol to quickly, reproducibly, and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE...... glucan phosphatases showed similar affinities for the short oligosaccharide β-cyclodextrin. We performed structure-guided mutagenesis to define the mechanism of these differences. We found that the carbohydrate binding module (CBM) domain provided a stronger binding affinity compared to surface binding...

  16. Binding of histone H1 to DNA is differentially modulated by redox state of HMGB1.

    Directory of Open Access Journals (Sweden)

    Eva Polanská

    Full Text Available HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.

  17. Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2

    DEFF Research Database (Denmark)

    Krintel, Christian; Francotte, Pierre; Pickering, Darryl S;

    2016-01-01

    The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer’s disease. The modulators bind in a cleft formed by the interface of two neighboring...

  18. Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu

    2012-01-01

    CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism...

  19. Stereoselective synthesis of light-activatable perfluorophenylazide-conjugated carbohydrates for glycoarray fabrication and evaluation of structural effects on protein binding by SPR imaging.

    Science.gov (United States)

    Deng, Lingquan; Norberg, Oscar; Uppalapati, Suji; Yan, Mingdi; Ramström, Olof

    2011-05-07

    A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S- and O-linked structures with intermediate linker length.

  20. Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

    Science.gov (United States)

    Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

    2016-09-01

    In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

  1. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    Science.gov (United States)

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-05

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  2. Various Terpenoids Derived from Herbal and Dietary Plants Function as PPAR Modulators and Regulate Carbohydrate and Lipid Metabolism

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Goto

    2010-01-01

    Full Text Available Several herbal plants improve medical conditions. Such plants contain many bioactive phytochemicals. Terpenoids (also called “isoprenoids” constitute one of the largest families of natural products accounting for more than 40,000 individual compounds of both primary and secondary metabolisms. In particular, terpenoids are contained in many herbal plants, and several terpenoids have been shown to be available for pharmaceutical applications, for example, artemisinin and taxol as malaria and cancer medicines, respectively. Various terpenoids are contained in many plants for not only herbal use but also dietary use. In this paper, we describe several bioactive terpenoids contained in herbal or dietary plants, which can modulate the activities of ligand-dependent transcription factors, namely, peroxisome proliferator-activated receptors (PPARs. Because PPARs are dietary lipid sensors that control energy homeostasis, daily eating of these terpenoids might be useful for the management for obesity-induced metabolic disorders, such as type 2 diabetes, hyperlipidemia, insulin resistance, and cardiovascular diseases.

  3. It's not my fault: postdictive modulation of intentional binding by monetary gains and losses.

    Directory of Open Access Journals (Sweden)

    Keisuke Takahata

    Full Text Available Sense of agency refers to the feeling that one's voluntary actions caused external events. Past studies have shown that compression of the subjective temporal interval between actions and external events, called intentional binding, is closely linked to the experience of agency. Current theories postulate that the experience of agency is constructed via predictive and postdictive pathways. One remaining problem is the source of human causality bias; people often make misjudgments on the causality of voluntary actions and external events depending on their rewarding or punishing outcomes. Although human causality bias implies that sense of agency can be modified by post-action information, convincing empirical findings for this issue are lacking. Here, we hypothesized that sense of agency would be modified by affective valences of action outcomes. To examine this issue, we investigated how rewarding and punishing outcomes following voluntary action modulate behavioral measures of agency using intentional binding paradigm and classical conditioning procedures. In the acquisition phase, auditory stimuli were paired with positive, neutral or negative monetary outcomes. Tone-reward associations were evaluated using reaction times and preference ratings. In the experimental session, participants performed a variant of intentional binding task, where participants made timing judgments for onsets of actions and sensory outcomes while playing simple slot games. Our results showed that temporal binding was modified by affective valences of action outcomes. Specifically, intentional binding was attenuated when negative outcome occurred, consistent with self-serving bias. Our study not only provides evidence for postdictive modification of agency, but also proposes a possible mechanism of human causality bias.

  4. Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain.

    Science.gov (United States)

    Russo, C; Gao, Y; Mancini, P; Vanni, C; Porotto, M; Falasca, M; Torrisi, M R; Zheng, Y; Eva, A

    2001-06-01

    The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.

  5. Learning about Carbohydrates

    Science.gov (United States)

    ... What Happens in the Operating Room? Learning About Carbohydrates KidsHealth > For Kids > Learning About Carbohydrates A A ... of energy for the body. Two Types of Carbohydrates There are two major types of carbohydrates (or ...

  6. Modulating supramolecular binding of carbon dioxide in a redox-active porous metal-organic framework

    Science.gov (United States)

    Lu, Zhenzhong; Godfrey, Harry G. W.; da Silva, Ivan; Cheng, Yongqiang; Savage, Mathew; Tuna, Floriana; McInnes, Eric J. L.; Teat, Simon J.; Gagnon, Kevin J.; Frogley, Mark D.; Manuel, Pascal; Rudić, Svemir; Ramirez-Cuesta, Anibal J.; Easun, Timothy L.; Yang, Sihai; Schröder, Martin

    2017-01-01

    Hydrogen bonds dominate many chemical and biological processes, and chemical modification enables control and modulation of host–guest systems. Here we report a targeted modification of hydrogen bonding and its effect on guest binding in redox-active materials. MFM-300(VIII) {[VIII2(OH)2(L)], LH4=biphenyl-3,3′,5,5′-tetracarboxylic acid} can be oxidized to isostructural MFM-300(VIV), [VIV2O2(L)], in which deprotonation of the bridging hydroxyl groups occurs. MFM-300(VIII) shows the second highest CO2 uptake capacity in metal-organic framework materials at 298 K and 1 bar (6.0 mmol g−1) and involves hydrogen bonding between the OH group of the host and the O-donor of CO2, which binds in an end-on manner, =1.863(1) Å. In contrast, CO2-loaded MFM-300(VIV) shows CO2 bound side-on to the oxy group and sandwiched between two phenyl groups involving a unique ···c.g.phenyl interaction [3.069(2), 3.146(3) Å]. The macroscopic packing of CO2 in the pores is directly influenced by these primary binding sites. PMID:28194014

  7. Modulating supramolecular binding of carbon dioxide in a redox-active porous metal-organic framework

    Science.gov (United States)

    Lu, Zhenzhong; Godfrey, Harry G. W.; da Silva, Ivan; Cheng, Yongqiang; Savage, Mathew; Tuna, Floriana; McInnes, Eric J. L.; Teat, Simon J.; Gagnon, Kevin J.; Frogley, Mark D.; Manuel, Pascal; Rudić, Svemir; Ramirez-Cuesta, Anibal J.; Easun, Timothy L.; Yang, Sihai; Schröder, Martin

    2017-02-01

    Hydrogen bonds dominate many chemical and biological processes, and chemical modification enables control and modulation of host-guest systems. Here we report a targeted modification of hydrogen bonding and its effect on guest binding in redox-active materials. MFM-300(VIII) {[VIII2(OH)2(L)], LH4=biphenyl-3,3',5,5'-tetracarboxylic acid} can be oxidized to isostructural MFM-300(VIV), [VIV2O2(L)], in which deprotonation of the bridging hydroxyl groups occurs. MFM-300(VIII) shows the second highest CO2 uptake capacity in metal-organic framework materials at 298 K and 1 bar (6.0 mmol g-1) and involves hydrogen bonding between the OH group of the host and the O-donor of CO2, which binds in an end-on manner, =1.863(1) Å. In contrast, CO2-loaded MFM-300(VIV) shows CO2 bound side-on to the oxy group and sandwiched between two phenyl groups involving a unique ...c.g.phenyl interaction [3.069(2), 3.146(3) Å]. The macroscopic packing of CO2 in the pores is directly influenced by these primary binding sites.

  8. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Klein-Seetharaman Judith

    2008-02-01

    Full Text Available Abstract Metabotropic glutamate receptors (mGluRs are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%, the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%, the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86% and 8/9 (89% for ArgusLab and 10/14 (71% and 7/9 (78% for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by

  9. Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).

    Science.gov (United States)

    Li, M; Bermak, J C; Wang, Z W; Zhou, Q Y

    2000-03-01

    Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

  10. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Nielsen, Morten M.; Christiansen, Camilla

    2015-01-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch...... to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert...

  11. Thiol-ene immobilisation of carbohydrates onto glass slides as a simple alternative to gold-thiol monolayers, amines or lipid binding.

    Science.gov (United States)

    Biggs, Caroline I; Edmondson, Steve; Gibson, Matthew I

    2015-01-01

    Carbohydrate arrays are a vital tool in studying infection, probing the mechanisms of bacterial, viral and toxin adhesion and the development of new treatments, by mimicking the structure of the glycocalyx. Current methods rely on the formation of monolayers of carbohydrates that have been chemically modified with a linker to enable interaction with a functionalised surface. This includes amines, biotin, lipids or thiols. Thiol-addition to gold to form self-assembled monolayers is perhaps the simplest method for immobilisation as thiolated glycans are readily accessible from reducing carbohydrates in a single step, but are limited to gold surfaces. Here we have developed a quick and versatile methodology which enables the use of thiolated carbohydrates to be immobilised as monolayers directly onto acrylate-functional glass slides via a 'thiol-ene'/Michael-type reaction. By combining the ease of thiol chemistry with glass slides, which are compatible with microarray scanners this offers a cost effective, but also useful method to assemble arrays.

  12. Allosteric enhancers, allosteric agonists and ago-allosteric modulators: where do they bind and how do they act?

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Holst, Birgitte

    2007-01-01

    Many small-molecule agonists also display allosteric properties. Such ago-allosteric modulators act as co-agonists, providing additive efficacy--instead of partial antagonism--and they can affect--and often improve--the potency of the endogenous agonist. Surprisingly, the apparent binding sites...... different binding modes. In another, dimeric, receptor scenario, the endogenous agonist binds to one protomer while the ago-allosteric modulator binds to the other, 'allosteric' protomer. It is suggested that testing for ago-allosteric properties should be an integral part of the agonist drug discovery...... process because a compound that acts with--rather than against--the endogenous agonist could be an optimal agonist drug....

  13. A novel function for the cellulose binding module of cellobiohydrolase I

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A homogeneous cellulose-binding module(CBM)of cellobiohydrolase I(CBHI)from Trichoderma pseudokoningii S-38 was obtained by the limited proteolysis with papain and a series of chromatographs filtration.Analysis of FT-IR spectra demonstrated that the structural changes result from a weakening and splitting of the hydrogen bond network in cellulose by the action of CBMCBHI at 40℃for 24 h.The results of molecular dynamic simulations are consistent with the experimental conclusions, and provide a nanoscopic view of the mechanism that strong and medium H-bonds decreased dramatically when CBM was bound to the cellulose surface.The function of CBMCBHI is not only limited to locating intact CBHI in close proximity with cellulose fibrils,but also is involved in the structural disruption at the fibre surface.The present studies provided considerable evidence for the model of the intramolecular synergy between the catalytic domain and their CBMs.

  14. Carbohydrates Through Animation: Preliminary Step

    Directory of Open Access Journals (Sweden)

    J.K. Sugai

    2004-05-01

    Full Text Available Methods of education are changing, so the educational tools must change too. The developmentof the systems of information and communication gave the opportunity to bring new technology tothe learning process. Modern education needs interactive programs that may be available to theacademic community, in order to ease the learning process and sharing of the knowledge. Then,an educational software on Carbohydrates is being developed using concept maps and FLASH-MXanimations program, and approached through six modules. The introduction of Carbohydrates wasmade by the module Carbohydrates on Nature, which shows the animations gures of a teacher andstudents, visiting a farm, identifying the carbohydrates found in vegetables, animals, and microor-ganisms, integrated by links containing short texts to help understanding the structure and functionof carbohydrates. This module was presented, as pilot experiment, to teachers and students, whichdemonstrated satisfaction, and high receptivity, by using animation and interactivitys program asstrategy to biochemistrys education. The present work is part of the project Biochemistry throughanimation, which is having continuity.

  15. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

    Directory of Open Access Journals (Sweden)

    Riffat I Munir

    Full Text Available Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes, sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199 and carbohydrate binding modules (95 were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  16. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

    Science.gov (United States)

    Munir, Riffat I; Schellenberg, John; Henrissat, Bernard; Verbeke, Tobin J; Sparling, Richard; Levin, David B

    2014-01-01

    Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  17. Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1

    Science.gov (United States)

    Shpigel, Etai; Roiz, Levava; Goren, Raphael; Shoseyov, Oded

    1998-01-01

    Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control. PMID:9701575

  18. Binding of β-VLDL to heparan sulfate proteoglycans requires lipoprotein lipase, whereas apoE only modulates binding affinity

    NARCIS (Netherlands)

    Beer, F. de; Hendriks, W.L.; Vark, L.C. van; Kamerling, S.W.A.; Dijk, K.W. van; Hofker, M.H.; Smelt, A.H.M.; Havekes, L.M.

    1999-01-01

    The binding of β-VLDL to heparan sulfate proteoglycans (HSPG) has been reported to be stimulated by both apoE and lipoprotein lipase (LPL). In the present study we investigated the effect of the isoform and the amount of apoE per particle, as well as the role of LPL on the binding of β-VLDL to HSPG.

  19. γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.

    Science.gov (United States)

    Pozdnyakov, Nikolay; Murrey, Heather E; Crump, Christina J; Pettersson, Martin; Ballard, T Eric; Am Ende, Christopher W; Ahn, Kwangwook; Li, Yue-Ming; Bales, Kelly R; Johnson, Douglas S

    2013-04-05

    γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic β-amyloid peptides (i.e. Aβ42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.

  20. Carbohydrate Binding Module 74 is a novel starch binding domain associated with large and multi-domain α-amylase enzymes

    NARCIS (Netherlands)

    Valk, Vincent; Lammerts van Bueren, Alicia; van der Kaaij, Rachel M; Dijkhuizen, Lubbert

    2016-01-01

    Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C-terminus (Domain 2). Deletion of D

  1. The C-terminal Region of Laminin β Chains Modulates the Integrin Binding Affinities of Laminins*S⃞

    OpenAIRE

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-01-01

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (α, β, and γ), in which three laminin globular modules in the α chain and the Glu residue in the C-terminal tail of the γ chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the β chain is involved in laminin-integrin interactions. We compared the binding affinities of ...

  2. Changes in BQCA Allosteric Modulation of [(3)H]NMS Binding to Human Cortex within Schizophrenia and by Divalent Cations.

    Science.gov (United States)

    Dean, Brian; Hopper, Shaun; Conn, P Jeffrey; Scarr, Elizabeth

    2016-05-01

    Stimulation of the cortical muscarinic M1 receptor (CHRM1) is proposed as a treatment for schizophrenia, a hypothesis testable using CHRM1 allosteric modulators. Allosteric modulators have been shown to change the activity of CHRMs using cloned human CHRMs and CHRM knockout mice but not human CNS, a prerequisite for them working in humans. Here we show in vitro that BQCA, a positive allosteric CHRM1 modulator, brings about the expected change in affinity of the CHRM1 orthosteric site for acetylcholine in human cortex. Moreover, this effect of BQCA is reduced in the cortex of a subset of subjects with schizophrenia, separated into a discrete population because of a profound loss of cortical [(3)H]pirenzepine binding. Surprisingly, there was no change in [(3)H]NMS binding to the cortex from this subset or those with schizophrenia but without a marked loss of cortical CHRM1. Hence, we explored the nature of [(3)H]pirenzepine and [(3)H]NMS binding to human cortex and showed total [(3)H]pirenzepine and [(3)H]NMS binding was reduced by Zn(2+), acetylcholine displacement of [(3)H]NMS binding was enhanced by Mg(2+) and Zn(2+), acetylcholine displacement of [(3)H]pirenzepine was reduced by Mg(2+) and enhanced by Zn(2+), whereas BQCA effects on [(3)H]NMS, but not [(3)H]pirenzepine, binding was enhanced by Mg(2+) and Zn(2+). These data suggest the orthosteric and allosteric sites on CHRMs respond differently to divalent cations and the effects of allosteric modulation of the cortical CHRM1 is reduced in a subset of people with schizophrenia, a finding that may have ramifications for the use of CHRM1 allosteric modulators in the treatment of schizophrenia.

  3. High throughput screening of starch structures using carbohydrate microarrays

    DEFF Research Database (Denmark)

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated...... maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content...... and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers...

  4. An in silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-peptide interactions.

    Directory of Open Access Journals (Sweden)

    Garima Tiwari

    Full Text Available Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K(i or K(d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K(i or K(d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors

  5. Integrative decomposition procedure and Kappa statistics set up ATF2 ion binding module in malignant pleural mesothelioma (MPM)

    Institute of Scientific and Technical Information of China (English)

    Ying SUN; Lin WANG; Lei LIU

    2008-01-01

    Activating transcription factor 2 (ATF2) is a member of the ATF/cyclic AMP-responsive element bind-ing protein family of transcription factors. However, the information concerning ATF2 ion-mediated DNA binding module and function of ATF2 in malignant pleural mesothelioma (MPM) has never been addressed. In this study, by using GRNInfer and GVedit based on linear pro-gramming and a decomposition procedure, with integrated analysis of the function cluster using Kappa statistics and fuzzy heuristic clustering in MPM, we identified one ATF2 ion-mediated DNA binding module involved in invasive function including ATF2 inhibition to target genes FALZ, C20orf31, NME2, PLOD2, RNF10, and RNASEH1, upstream RNF10 and PLOD2 activation to ATF2, upstream RNASEH1 and FALZ inhibition to ATF2 from 40 MPM tumors and 5 normal pleural tissues. Remarkably, our results showed that the predominant effect of ATF2 occupancy is to suppress the activation of target genes on MPM. Importantly, the ATF2 ion-mediated DNA binding module reflects 'mutual' positive and negative feedback regulation mechanism of ATF2 with up-and down-stream genes. It may be useful for developing novel prognostic markers and therapeutic targets in MPM.

  6. Applications of synthetic carbohydrates to chemical biology.

    Science.gov (United States)

    Lepenies, Bernd; Yin, Jian; Seeberger, Peter H

    2010-06-01

    Access to synthetic carbohydrates is an urgent need for the development of carbohydrate-based drugs, vaccines, adjuvants as well as novel drug delivery systems. Besides traditional synthesis in solution, synthetic carbohydrates have been generated by chemoenzymatic methods as well as automated solid-phase synthesis. Synthetic oligosaccharides have proven to be useful for identifying ligands of carbohydrate-binding proteins such as C-type lectins and siglecs using glycan arrays. Furthermore, glyconanoparticles and glycodendrimers have been used for specific targeting of lectins of the immune system such as selectins, DC-SIGN, and CD22. This review focuses on how diverse carbohydrate structures can be synthetically derived and highlights the benefit of synthetic carbohydrates for glycobiology.

  7. New fabrication and applications of carbohydrate arrays.

    Science.gov (United States)

    Huang, Gangliang; Chen, Xin; Xiao, Feng

    2014-01-01

    Carbohydrate arrays are used as high-throughput screening platforms to study the carbohydrate-mediated recognition events for glycobiology. The polysaccharide arrays are easy to fabricate by non-covalently or covalently immobilizing polysaccharides onto array surfaces because polysaccharides have hydrophobic interactions. Oligosaccharides must be derived and covalently or non-covalently immobilized onto array surfaces to fabricate oligosaccharide arrays because they have hydrophilic interactions. At the moment, carbohydrate arrays are mainly used to study the carbohydrate-protein interactions and carbohydrate-binding lectins or antibodies, which are possible to be applied to clinics and diagnoses. This review mainly summed up the new fabrication strategies of carbohydrate arrays and their applications in recent four years.

  8. A role for cysteine 3635 of RYR1 in redox modulation and calmodulin binding

    Science.gov (United States)

    Porter Moore, C.; Zhang, J. Z.; Hamilton, S. L.

    1999-01-01

    Oxidation of the skeletal muscle Ca(2+) release channel (RYR1) increases its activity, produces intersubunit disulfide bonds, and blocks its interaction with calmodulin. Conversely, bound calmodulin protects RYR1 from the effects of oxidants (Zhang, J.-Z., Wu, Y., Williams, B. Y., Rodney, G., Mandel, F., Strasburg, G. M., and Hamilton, S. L. (1999) Am. J. Physiol. 276, Cell Physiol. C46-C53). In addition, calmodulin protects RYR1 from trypsin cleavage at amino acids 3630 and 3637 (Moore, C. P., Rodney, G., Zhang, J.-Z., Santacruz-Toloza, L., Strasburg, G. M., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). The sequence between these two tryptic sites is AVVACFR. Alkylation of RYR1 with N-ethylmaleimide (NEM) blocks both (35)S-apocalmodulin binding and oxidation-induced intersubunit cross-linking. In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit cross-link are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [(3)H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.

  9. A novel fibronectin binding motif in MSCRAMMs targets F3 modules.

    Directory of Open Access Journals (Sweden)

    Sabitha Prabhakaran

    Full Text Available BACKGROUND: BBK32 is a surface expressed lipoprotein and fibronectin (Fn-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21-205 of the lipoprotein. METHODOLOGY/PRINCIPAL FINDINGS: Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence. CONCLUSIONS/SIGNIFICANCE: We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities.

  10. ATP-Binding Cassette Transporters Modulate Both Coelenterazine- and D-Luciferin-Based Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Ruimin Huang

    2011-05-01

    Full Text Available Bioluminescence imaging (BLI of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC transporters on BLI readout, we generated click beetle (cLuc, firefly (fLuc, Renilla (rLuc, and Gaussia (gLuc luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2. In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type–independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.

  11. In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5.

    Science.gov (United States)

    Zysk, John R; Spear, Nathan; Fieles, William; Stein, Mark M; Sygowski, Linda S; King, Megan M; Hoesch, Valerie; Hastings, Richard; Brockel, Becky; Do, Mylinh; Ström, Peter; Gadient, Reto; Chhajlani, Vijay; Elmore, Charles S; Maier, Donna L

    2013-03-01

    The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu(5)) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu(5) compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu(5) and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC(50) of 87 nM with an 89% effect in transfected HEK293 cells and an EC(50) of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu(5) in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)-based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [(3)H]MPEP, the mGlu(5) receptor negative allosteric modulator (NAM), PBPyl exhibited a k(i) value of 34 nM. PBPyl also displaced [(3)H]ABP688, a mGluR(5) receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu(5b) membranes, [(3)H] PBPyl exhibited saturable binding with a K(d) value of 18.6 nM. In competition binding experiments, [(3)H] PBPyl was displaced by high affinity mGlu(5) positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu(5) receptor PAM, this study offers value toward the development of novel PET imaging agents for this important

  12. Macromolecular depletion modulates the binding of red blood cells to activated endothelial cells.

    Science.gov (United States)

    Yang, Yang; Koo, Stephanie; Lin, Cheryl Shuyi; Neu, Björn

    2010-09-01

    Adhesion of red blood cells (RBCs) to endothelial cells (ECs) is usually insignificant but an enhanced adhesion has been observed in various diseases associated with vascular complications. This abnormal adhesion under pathological conditions such as sickle cell disease has been correlated with increased levels of various plasma proteins but the detailed underlying mechanism(s) remains unclear. Usually it is assumed that the proadhesive effects of plasma proteins originate from ligand interactions cross-linking receptors on adjacent cells, but explicit results detailing binding sites or receptors for some proteins (e.g., fibrinogen) on either RBC or EC surfaces that would support this model are missing. In this study, the authors tested whether there is an alternative mechanism. Their results demonstrate that dextran 2 MDa promotes the adhesion of normal RBCs to thrombin-activated ECs and that this effect becomes more pronounced with increasing thrombin concentration or with prolonged thrombin incubation time. It is concluded that depletion interaction originating from nonadsorbing macromolecules (i.e., dextran) can modulate the adhesion of red blood cells to thrombin-activated EC. This study thereby suggests macromolecular depletion as an alternative mechanism for the adhesion-promoting effects of nonadsorbing plasma proteins. These findings should not only aid in getting a better understanding of diseases associated with vascular complications but should also have many potential applications in biomedical or biotechnological areas that require the control of cell-cell or cell surface interactions.

  13. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    Science.gov (United States)

    Kitange, Gaspar J.; Mladek, Ann C.; Schroeder, Mark A.; Pokorny, Jenny C.; Carlson, Brett L.; Zhang, Yuji; Nair, Asha A.; Lee, Jeong-Heon; Yan, Huihuang; Decker, Paul A.; Zhang, Zhiguo; Sarkaria, Jann N.

    2016-01-01

    Summary Here we provide evidence that RBBP4 modulates temozolomide (TMZ) sensitivity through coordinate regulation of 2 key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT) and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51 and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac) and increased H3K9 tri-methylation (H3K9me3). Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin modifying complex that binds within the promoter of MGMT, RAD51 and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. RBBP4/CBP/p300 complex may provide an interesting target for developing therapy sensitizing strategies for GBM and other tumors. PMID:26972001

  14. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    Directory of Open Access Journals (Sweden)

    Gaspar J. Kitange

    2016-03-01

    Full Text Available Here we provide evidence that RBBP4 modulates temozolomide (TMZ sensitivity through coordinate regulation of two key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition, and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51, and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac and increased H3K9 tri-methylation (H3K9me3. Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin-modifying complex that binds within the promoter of MGMT, RAD51, and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. The RBBP4/CBP/p300 complex may provide an interesting target for developing therapy-sensitizing strategies for GBM and other tumors.

  15. The interaction between Histoplasma capsulatum cell wall carbohydrates and host components: relevance in the immunomodulatory role of histoplasmosis

    Directory of Open Access Journals (Sweden)

    Patricia Gorocica

    2009-05-01

    Full Text Available Histoplasma capsulatum is an intracellular fungal pathogen that causes respiratory and systemic disease by proliferating within phagocytic cells. The binding of H. capsulatum to phagocytes may be mediated by the pathogen's cell wall carbohydrates, glucans, which consist of glucose homo and hetero-polymers and whose glycosydic linkage types differ between the yeast and mycelial phases. The ±-1,3-glucan is considered relevant for H. capsulatum virulence, whereas the ²-1,3-glucan is antigenic and participates in the modulation of the host immune response. H. capsulatum cell wall components with lectin-like activity seem to interact with the host cell surface, while host membrane lectin-like receptors can recognize a particular fungal carbohydrate ligand. This review emphasizes the relevance of the main H. capsulatum and host carbohydrate-driven interactions that allow for binding and internalization of the fungal cell into phagocytes and its subsequent avoidance of intracellular elimination.

  16. Studies on the binding of modest modulators of the human enzyme, Sirtuin 6, using STD-NMR.

    Science.gov (United States)

    Bolívar, Beatriz E; Welch, John T

    2017-02-21

    Pyrazinamide (PZA), an essential constituent of short course tuberculosis chemotherapy, binds weakly but selectively to Sirtuin 6 (SIRT6). Despite structural similarities between nicotinamide (NAM), PZA, and pyrazinoic acid (POA), these inhibitors modulate SIRT6 via different mechanisms and binding sites, as suggested by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR). Available experimental evidence, such as that derived from crystal structures and kinetic experiments, is of only limited utility in the elucidation of the mechanistic details of sirtuin inhibition by NAM or other inhibitors. For example, crystallographic structural analysis of sirtuin binding sites does not illuminate differences in binding affinities among sirtuins or capture details of the dynamic process. STD NMR led to findings that not only agreed with the binding kinetics experiments, but also yielded insights into the binding process. The data also illustrate the geometry of the ligand binding epitopes in solution for both the apo- and holoenzyme. Recognition that SIRT6 is affected selectively by PZA, an established clinical agent, suggests that the rational development of more potent and selective NAM surrogates may be possible. These derivatives may be accessible by employing the malleability of this scaffold to assist in the identification by STD-NMR of the motifs that interact with the holo- and apo-enzymes in solution.

  17. Carbohydrates and Diabetes

    Science.gov (United States)

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Carbohydrates and Diabetes KidsHealth > For Teens > Carbohydrates and Diabetes A A A What's in this ... that you should keep track of how many carbohydrates (carbs) you eat. But what exactly are carbohydrates ...

  18. High throughput techniques for discovering new glycine receptor modulators and their binding sites

    Directory of Open Access Journals (Sweden)

    Daniel F Gilbert

    2009-10-01

    Full Text Available The inhibitory glycine receptor (GlyR is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific compounds. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384 well plate using nanogram quantities of cDNA.

  19. Module structure of interphotoreceptor retinoid-binding protein (IRBP may provide bases for its complex role in the visual cycle – structure/function study of Xenopus IRBP

    Directory of Open Access Journals (Sweden)

    Ghosh Debashis

    2007-08-01

    Full Text Available Abstract Background Interphotoreceptor retinoid-binding protein's (IRBP remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling. Results The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each ~300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 μM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites

  20. Fluorescence Resonance Energy Transfer Imaging Reveals that Chemokine-Binding Modulates Heterodimers of CXCR4 and CCR5 Receptors

    OpenAIRE

    2008-01-01

    BACKGROUND: Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation. METHODOLOGY/PRINCIPAL FINDINGS: Using a sensitive Fluorescence Resonance Energy Transfer (FRET) imaging ...

  1. Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B isolectin to alphagalactosyl carbohydrate antigens in the surface phase

    DEFF Research Database (Denmark)

    Kirkeby, Svend; Winter, Harry C; Goldstein, Irwin J

    2004-01-01

    The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4) (GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and lam...

  2. Carbohydrate nanotechnology: hierarchical assembly using nature's other information carrying biopolymers.

    Science.gov (United States)

    Han, Xu; Zheng, Yeting; Munro, Catherine J; Ji, Yiwen; Braunschweig, Adam B

    2015-08-01

    Despite their central role in directing some of the most complex biological processes, carbohydrates--nature's other information carrying biopolymer--have been largely ignored as building blocks for synthetic hierarchical assemblies. The non-stoichiometric binding and astronomical diversity characteristic of carbohydrates could lead to tantalizingly complex assembly algorithms, but these attributes simultaneously increase the difficulty of preparing carbohydrate assemblies and anticipating their behavior. Convergences in biotechnology, nanotechnology, polymer chemistry, surface science, and supramolecular chemistry have led to many recent important breakthroughs in glycan microarrays and synthetic carbohydrate receptors, where the idiosyncrasies of carbohydrate structure and binding are increasingly considered. We hope to inspire more researchers to consider carbohydrate structure, diversity, and binding as attractive tools for constructing synthetic hierarchical assemblies.

  3. Carbohydrate functionalized carbon nanotubes and their applications.

    Science.gov (United States)

    Gorityala, Bala Kishan; Ma, Jimei; Wang, Xin; Chen, Peng; Liu, Xue-Wei

    2010-08-01

    Carbon nanotubes (CNTs) have attracted tremendous attention in biomedical applications due to their molecular size and unique properties. This tutorial review summarizes the strategies to functionalize CNTs with bioactive carbohydrates, which improve their solubility, biocompatibility and biofunctionalities while preserving their desired properties. In addition, studies on the usage of carbohydrate functionalized CNTs to detect bacteria, to bind to specific lectins, to deliver glycomimetic drug molecules into cells and to probe cellular activities as biosensors are reviewed. Improvement in biocompatibility and introduction of bio-functionalities by integration of carbohydrate with CNTs are paving the way to glyconanotechnology and may provide new tools for glycobiological studies.

  4. Sex Steroid Modulation of Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    Science.gov (United States)

    Ockner, Robert K.; Lysenko, Nina; Manning, Joan A.; Monroe, Scott E.; Burnett, David A.

    1980-01-01

    The mechanism by which sex steroids influence very low density hepatic lipoprotein triglyceride production has not been fully elucidated. In previous studies we showed that [14C]oleate utilization and incorporation into triglycerides were greater in hepatocyte suspensions from adult female rats than from males. The sex differences were not related to activities of the enzymes of triglyceride biosynthesis, whereas fatty acid binding protein (FABP) concentration in liver cytosol was greater in females. These findings suggested that sex differences in lipoprotein could reflect a sex steroid influence on the availability of fatty acids for hepatocellular triglyceride biosynthesis. In the present studies, sex steroid effects on hepatocyte [14C]oleate utilization and FABP concentration were investigated directly. Hepatocytes from immature (30-d-old) rats exhibited no sex differences in [14C]oleate utilization. With maturation, total [14C]oleate utilization and triglyceride biosynthesis increased moderately in female cells and decreased markedly in male cells; the profound sex differences in adults were maximal by age 60 d. Fatty acid oxidation was little affected. Rats were castrated at age 30 d, and received estradiol, testosterone, or no hormone until age 60 d, when hepatocyte [14C]oleate utilization was studied. Castration virtually eliminated maturational changes and blunted the sex differences in adults. Estradiol or testosterone largely reproduced the appropriate adult pattern of [14C]oleate utilization regardless of the genotypic sex of the treated animal. In immature females and males, total cytosolic FABP concentrations were similar. In 60-d-old animals, there was a striking correlation among all groups (females, males, castrates, and hormone-treated) between mean cytosolic FABP concentration on the one hand, and mean total [14C]oleate utilization (r = 0.91) and incorporation into triglycerides (r = 0.94) on the other. In 30-d-old animals rates of [14C

  5. Carbohydrate Metabolism Disorders

    Science.gov (United States)

    ... you eat. Food is made up of proteins, carbohydrates, and fats. Chemicals in your digestive system (enzymes) ... metabolic disorder, something goes wrong with this process. Carbohydrate metabolism disorders are a group of metabolic disorders. ...

  6. Carbohydrate restriction and dietary cholesterol modulate the expression of HMG-CoA reductase and the LDL receptor in mononuclear cells from adult men

    Directory of Open Access Journals (Sweden)

    Volek Jeff S

    2007-11-01

    Full Text Available Abstract The liver is responsible for controlling cholesterol homeostasis in the body. HMG-CoA reductase and the LDL receptor (LDL-r are involved in this regulation and are also ubiquitously expressed in all major tissues. We have previously shown in guinea pigs that there is a correlation in gene expression of HMG-CoA reductase and the LDL-r between liver and mononuclear cells. The present study evaluated human mononuclear cells as a surrogate for hepatic expression of these genes. The purpose was to evaluate the effect of dietary carbohydrate restriction with low and high cholesterol content on HMG-CoA reductase and LDL-r mRNA expression in mononuclear cells. All subjects were counseled to consume a carbohydrate restricted diet with 10–15% energy from carbohydrate, 30–35% energy from protein and 55–60% energy from fat. Subjects were randomly assigned to either EGG (640 mg/d additional dietary cholesterol or SUB groups [equivalent amount of egg substitute (0 dietary cholesterol contributions per day] for 12 weeks. At the end of the intervention, there were no changes in plasma total or LDL cholesterol (LDL-C compared to baseline (P > 0.10 or differences in plasma total or LDL-C between groups. The mRNA abundance for HMG-CoA reductase and LDL-r were measured in mononuclear cells using real time PCR. The EGG group showed a significant decrease in HMG-CoA reductase mRNA (1.98 ± 1.26 to 1.32 ± 0.92 arbitrary units P

  7. Modulation of the cellular immune response by a carbohydrate rich fraction from Echinococcus granulosus protoscoleces in infected or immunized Balb/c mice.

    Science.gov (United States)

    Dematteis, S; Pirotto, F; Marqués, J; Nieto, A; Orn, A; Baz, A

    2001-01-01

    Infection of Balb/c mice with Echinococcus granulosus protoscoleces constitutes the model for secondary hydatid infection. The immune response of Balb/c mice infected with E. granulosus is characterized by secretion of antibodies specific for carbohydrate epitopes and production of type-2 cytokines. A role for glycoconjugates in the induction of type-2 responses has been suggested in other host--parasite systems. Although glycoconjugates are immunogenic in E. granulosus infection, the role of these molecules in the establishment of the type-2 response has never been analysed. In this study, a carbohydrate rich fraction (E4+) from E. granulosus protoscoleces was obtained using the monoclonal antibody E492/G1 specific for the moiety Galalpha(1,4)Gal which is widely represented in protoscoleces and other E. granulosus antigenic preparations. The results showed that E4+ was immunogenic in Balb/c mice evoking an antibody response mainly directed against carbohydrate epitopes. In addition, splenocytes from E4+-immunized mice showed suppressed proliferative responses to Con A and E4+ induced IL-10 secretion by E4+-primed and naive splenocytes. The fraction E4+ also was immunogenic in infected mice during early infection. In this case also, splenocytes from infected mice as well as peritoneal cells from infected or naive mice, when stimulated in vitro with E4+, secreted IL-10. Collectively, these results suggest that E4+ may be involved in immunosuppression phenomena and, by stimulating IL-10 secretion, may contribute to the induction and sustaining of the type-2 cytokine response established in early experimental infection.

  8. Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

    Directory of Open Access Journals (Sweden)

    H. S. Euzger

    1999-01-01

    Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

  9. Boronic acids for fluorescence imaging of carbohydrates.

    Science.gov (United States)

    Sun, Xiaolong; Zhai, Wenlei; Fossey, John S; James, Tony D

    2016-02-28

    "Fluorescence imaging" is a particularly exciting and rapidly developing area of research; the annual number of publications in the area has increased ten-fold over the last decade. The rapid increase of interest in fluorescence imaging will necessitate the development of an increasing number of molecular receptors and binding agents in order to meet the demand in this rapidly expanding area. Carbohydrate biomarkers are particularly important targets for fluorescence imaging given their pivotal role in numerous important biological events, including the development and progression of many diseases. Therefore, the development of new fluorescent receptors and binding agents for carbohydrates is and will be increasing in demand. This review highlights the development of fluorescence imaging agents based on boronic acids a particularly promising class of receptors given their strong and selective binding with carbohydrates in aqueous media.

  10. Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module.

    Directory of Open Access Journals (Sweden)

    Chandrika N Deshpande

    Full Text Available The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes.We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators.Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  11. Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

    Energy Technology Data Exchange (ETDEWEB)

    Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan; Hassan, Karl A.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Chang, Changsoo; Labbate, Maurizio; Paulsen, Ian T.; Stokes, H.W.; Curmi, Paul M.G.; Mabbutt, Bridget C. (MIT); (UT-Australia); (Macquarie); (Toronto); (New South)

    2012-02-15

    The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. We report the 1.8 {angstrom} crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  12. Membrane Binding and Modulation of the PDZ Domain of PICK1

    DEFF Research Database (Denmark)

    Erlendsson, Simon; Madsen, Kenneth Lindegaard

    2015-01-01

    Scaffolding proteins serve to assemble protein complexes in dynamic processes by means of specific protein-protein and protein-lipid binding domains. Many of these domains bind either proteins or lipids exclusively; however, it has become increasingly evident that certain domains are capable of b...... lipids. Moreover, we review how these PDZ-membrane interactions are regulated in the case of the synaptic scaffolding protein PICK1 and how this might affect cellular localization and function....

  13. Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

    Science.gov (United States)

    Sharkey, R G; Jover, E; Couraud, F; Baden, D G; Catterall, W A

    1987-03-01

    The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

  14. Guanine Nucleotides Modulate Cell Surface cAMP-Binding Sites in Membranes from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1984-01-01

    D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (Kd = 15 nM, t½ = 15 s). A second class is fast dissociating (t½ about 1 s) and is composed of high affinity binding sites H (Kd ≈ 60 nM), and low a

  15. Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters

    NARCIS (Netherlands)

    Roohparvar, R.; Huser, A.; Zwiers, L.H.; Waard, de M.A.

    2007-01-01

    Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due

  16. The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein-Protein Interaction.

    Science.gov (United States)

    Hung, Yi-Lin; Lee, Hsia-Ju; Jiang, Ingjye; Lin, Shang-Chi; Lo, Wei-Cheng; Lin, Yi-Jan; Sue, Shih-Che

    2015-07-01

    Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the β-barrel and the C-terminal helix bundle. The β1-β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

  17. Oleanolic Acid Diminishes Liquid Fructose-Induced Fatty Liver in Rats: Role of Modulation of Hepatic Sterol Regulatory Element-Binding Protein-1c-Mediated Expression of Genes Responsible for De Novo Fatty Acid Synthesis

    Directory of Open Access Journals (Sweden)

    Changjin Liu

    2013-01-01

    Full Text Available Oleanolic acid (OA, contained in more than 1620 plants and as an aglycone precursor for naturally occurred and synthesized triterpenoid saponins, is used in China for liver disorders in humans. However, the underlying liver-protecting mechanisms remain largely unknown. Here, we found that treatment of rats with OA (25 mg/kg/day, gavage, once daily over 10 weeks diminished liquid fructose-induced excess hepatic triglyceride accumulation without effect on total energy intake. Attenuation of the increased vacuolization and Oil Red O staining area was evident on histological examination of liver in OA-treated rats. Hepatic gene expression profile demonstrated that OA suppressed fructose-stimulated overexpression of sterol regulatory element-binding protein-(SREBP- 1/1c mRNA and nuclear protein. In accord, overexpression of SREBP-1c-responsive genes responsible for fatty acid synthesis was also downregulated. In contrast, overexpressed nuclear protein of carbohydrate response element-binding protein and its target genes liver pyruvate kinase and microsomal triglyceride transfer protein were not altered. Additionally, OA did not affect expression of peroxisome proliferator-activated receptor-gamma- and -alpha and their target genes. It is concluded that modulation of hepatic SREBP-1c-mediated expression of the genes responsible for de novo fatty acid synthesis plays a pivotal role in OA-elicited diminishment of fructose-induced fatty liver in rats.

  18. Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites

    DEFF Research Database (Denmark)

    Kaae, Birgitte Høiriis; Harpsøe, Kasper; Kastrup, Jette Sandholm Jensen;

    2007-01-01

    Dimeric positive allosteric modulators of ionotropic glutamate receptors were designed, synthesized, and characterized pharmacologically in electrophysiological experiments. The designed compounds are dimers of arylpropylsulfonamides and have been constructed without a linker. The monomeric...

  19. Digestion and Interaction of Starches with α-Amylases: I. Mutational analysis of Carbohydrate Binding Sites in barley. II. In Vitro Starch Digestion of Legumes

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch

    2006-01-01

    the hydrolysis of internal 1,4-α-D-glucosidic bonds in starch and related polysaccharides. The present thesis concerns studies of two α-amylases: 1) secondary substrate binding sites in barley α-amylase 1 (AMY1), and 2) the involvement of anti-nutrients in in vitro digestion of starch in legumes by porcine...... of pea starch with different pea protein isolates and fiber has been investigated to learn about the involvement of anti-nutrients for the digestibility of legume starches. All tested samples gave high HI-values, indicating a rapid digestion. In conclusion, the effect of anti-nutrients in legumes...... in morphology between high amylose starch granules and normal starch granules. Legumes (beans, peas, and lentils) are characterised by low blood glucose raising potential, which is proportional to the in vitro starch digestion rates. The high amount of anti-nutritional factors (phytate, proteinaceous inhibitors...

  20. Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Doktor, Thomas K; Borch-Jensen, Jonas;

    2016-01-01

    for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing...... regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP......-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site...

  1. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response.

    Science.gov (United States)

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; Di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D'Andrea, Luca Domenico

    2016-08-08

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.

  2. Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding.

    Science.gov (United States)

    Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu; Fiedler, Jason D; Sligar, Stephen G; Finn, M G; Zwick, Michael B; Dawson, Philip E

    2016-02-18

    The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies.

  3. Ligand binding modulates the structural dynamics and compactness of the major birch pollen allergen.

    Science.gov (United States)

    Grutsch, Sarina; Fuchs, Julian E; Freier, Regina; Kofler, Stefan; Bibi, Marium; Asam, Claudia; Wallner, Michael; Ferreira, Fátima; Brandstetter, Hans; Liedl, Klaus R; Tollinger, Martin

    2014-12-16

    Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.

  4. Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes.

    Science.gov (United States)

    Sinclair, Haydn R; Kemp, Fred; Slegte, Jaap de; Gibson, Glenn R; Rastall, Robert A

    2009-10-12

    We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10mg/mL. The SOS EC(50) value of 5.521mg/mL is high compared with 0.2811microg/mL (182.5rhoM or 1.825x10(-10)M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E((R)) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%, 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease.

  5. Structure-function relations of carbohydrates by neoglycolipid arrays.

    Science.gov (United States)

    Huang, Gang-Liang; Huang, Hua-Liang; Zhang, Hou-Cheng; Wang, Peng-George

    2006-06-01

    The work presented herein is a new noncovalent glycoarray assembly method for microplates created by simply mixing together a carbohydrate and a tetradecylamine. alpha-D-Mannopyranoside, alpha-D-glucopyranoside, and alpha-D-galactopyranoside were utilized in model studies and product formations were detected by lectin binding. The method can be extended to study the steric hindrance effect of carbohydrate-protein interactions, namely the structure-function relations of carbohydrates.

  6. Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

    Science.gov (United States)

    Zhang, Jingjing; Ni, Chen; Yang, Zhenguo; Piontek, Anna; Chen, Huapu; Wang, Sijie; Fan, Yiming; Qin, Zhihai; Piontek, Joerg

    2015-08-01

    Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

  7. The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Nemčovičová, Ivana [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States); Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava (Slovakia); Zajonc, Dirk M., E-mail: dzajonc@liai.org [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States)

    2014-03-01

    The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host

  8. Modulating the function of ATP-binding cassette subfamily G member 2 (ABCG2) with inhibitor cabozantinib.

    Science.gov (United States)

    Zhang, Guan-Nan; Zhang, Yun-Kai; Wang, Yi-Jun; Barbuti, Anna Maria; Zhu, Xi-Jun; Yu, Xin-Yue; Wen, Ai-Wen; Wurpel, John N D; Chen, Zhe-Sheng

    2017-01-25

    Cabozantinib (XL184) is a small molecule tyrosine kinase receptor inhibitor, which targets c-Met and VEGFR2. Cabozantinib has been approved by the Food and Drug Administration to treat advanced medullary thyroid cancer and renal cell carcinoma. In the present study, we evaluated the ability of cabozantinib to modulate the function of the ATP-binding cassette subfamily G member 2 (ABCG2) by sensitizing cells that are resistant to ABCG2 substrate antineoplastic drugs. We used a drug-selected resistant cell line H460/MX20 and three ABCG2 stable transfected cell lines ABCG2-482-R2, ABCG2-482-G2, and ABCG2-482-T7, which overexpress ABCG2. Cabozantinib, at non-toxic concentrations (3 or 5μM), sensitized the ABCG2-overexpressing cells to mitoxantrone, SN-38, and topotecan. Our results indicate that cabozantinib reverses ABCG2-mediated multidrug resistance by antagonizing the drug efflux function of the ABCG2 transporter instead of downregulating its expression. The molecular docking analysis indicates that cabozantinib binds to the drug-binding site of the ABCG2 transporter. Overall, our findings demonstrate that cabozantinib inhibits the ABCG2 transporter function and consequently enhances the effect of the antineoplastic agents that are substrates of ABCG2. Cabozantinib may be a useful agent in anticancer treatment regimens for patients who are resistant to ABCG2 substrate drugs.

  9. Fluorescence resonance energy transfer imaging reveals that chemokine-binding modulates heterodimers of CXCR4 and CCR5 receptors.

    Directory of Open Access Journals (Sweden)

    Nilgun Isik

    Full Text Available BACKGROUND: Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation. METHODOLOGY/PRINCIPAL FINDINGS: Using a sensitive Fluorescence Resonance Energy Transfer (FRET imaging method, we investigated the formation of CCR5 and CXCR4 heterodimers on the plasma membrane of live cells. We found that CCR5 and CXCR4 exist as constitutive heterodimers and ligands of CCR5 and CXCR4 promote different conformational changes within these preexisting heterodimers. Ligands of CCR5, in contrast to a ligand of CXCR4, induced a clear increase in FRET efficiency, indicating that selective ligands promote and stabilize a distinct conformation of the heterodimers. We also found that mutations at C-terminus of CCR5 reduced its ability to form heterodimers with CXCR4. In addition, ligands induce different conformational transitions of heterodimers of CXCR4 and CCR5 or CCR5(STA and CCR5(Delta4. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest a model in which CXCR4 and CCR5 spontaneously form heterodimers and ligand-binding to CXCR4 or CCR5 causes different conformational changes affecting heterodimerization, indicating the complexity of regulation of dimerization/function of these chemokine receptors by ligand binding.

  10. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

    Directory of Open Access Journals (Sweden)

    Romain Pardoux

    Full Text Available To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9TKE(12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d = 25±6 nM to K(d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d = 0.25±0.06 nM. FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as(P-O and ν(s(P-O IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as(UO(2(2+ vibration (from 923 cm(-1 to 908 cm(-1 was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

  11. Carbohydrates in Supramolecular Chemistry.

    Science.gov (United States)

    Delbianco, Martina; Bharate, Priya; Varela-Aramburu, Silvia; Seeberger, Peter H

    2016-02-24

    Carbohydrates are involved in a variety of biological processes. The ability of sugars to form a large number of hydrogen bonds has made them important components for supramolecular chemistry. We discuss recent advances in the use of carbohydrates in supramolecular chemistry and reveal that carbohydrates are useful building blocks for the stabilization of complex architectures. Systems are presented according to the scaffold that supports the glyco-conjugate: organic macrocycles, dendrimers, nanomaterials, and polymers are considered. Glyco-conjugates can form host-guest complexes, and can self-assemble by using carbohydrate-carbohydrate interactions and other weak interactions such as π-π interactions. Finally, complex supramolecular architectures based on carbohydrate-protein interactions are discussed.

  12. Interaction of an S100A9 gene variant with saturated fat and carbohydrates to modulate insulin resistance in 3 populations of different ancestries

    Science.gov (United States)

    BACKGROUND: S100 calcium binding protein A9 (S100A9) has previously been identified as a type 2 diabetes (T2D) gene. However, this finding requires independent validation and more in depth analyses in other populations and ancestries. OBJECTIVES: We aimed to replicate the associations between an S10...

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

    Directory of Open Access Journals (Sweden)

    Christopher J Brown

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

  14. Ligand Binding Modulates the Structural Dynamics and Compactness of the Major Birch Pollen Allergen

    Science.gov (United States)

    Grutsch, Sarina; Fuchs, Julian E.; Freier, Regina; Kofler, Stefan; Bibi, Marium; Asam, Claudia; Wallner, Michael; Ferreira, Fátima; Brandstetter, Hans; Liedl, Klaus R.; Tollinger, Martin

    2014-01-01

    Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens. PMID:25517162

  15. Excess nickel modulates activities of carbohydrate metabolizing enzymes and induces accumulation of sugars by upregulating acid invertase and sucrose synthase in rice seedlings.

    Science.gov (United States)

    Mishra, Pallavi; Dubey, R S

    2013-02-01

    The effects of increasing concentrations of nickel sulfate, NiSO(4) (200 and 400 μM) in the growth medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism were examined in seedlings of the two Indica rice cvs. Malviya-36 and Pant-12. During a 5-20 day growth period of seedlings in sand cultures, with Ni treatment, no definite pattern of alteration in starch level could be observed in the seedlings. In both roots and shoots of the seedlings Ni treatment led to a significant decrease in activities of starch degrading enzymes α-amylase, β-amylase, whereas starch phosphorylase activity increased. The contents of reducing, non-reducing, and total sugars increased in Ni-treated rice seedlings with a concomitant increase in the activities of sucrose degrading enzymes acid invertase and sucrose synthase. However, the activity of sucrose synthesizing enzyme sucrose phosphate synthase declined. These results suggest that Ni toxicity in rice seedlings causes marked perturbation in metabolism of carbohydrates leading to increased accumulation of soluble sugars. Such perturbation could serve as a limiting factor for growth of rice seedlings in Ni polluted environments and accumulating soluble sugars could serve as compatible solutes in the cells under Ni toxicity conditions.

  16. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    Science.gov (United States)

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  17. Identification of Novel Human Breast Carcinoma (MDA-MB-231 Cell Growth Modulators from a Carbohydrate-Based Diversity Oriented Synthesis Library

    Directory of Open Access Journals (Sweden)

    Elena Lenci

    2016-10-01

    Full Text Available The application of a cell-based growth inhibition on a library of skeletally different glycomimetics allowed for the selection of a hexahydro-2H-furo[3,2-b][1,4]oxazine compound as candidate inhibitors of MDA-MB-231 cell growth. Subsequent synthesis of analogue compounds and preliminary biological studies validated the selection of a valuable hit compound with a novel polyhydroxylated structure for the modulation of the breast carcinoma cell cycle mechanism.

  18. Identification of Novel Human Breast Carcinoma (MDA-MB-231) Cell Growth Modulators from a Carbohydrate-Based Diversity Oriented Synthesis Library.

    Science.gov (United States)

    Lenci, Elena; Innocenti, Riccardo; Biagioni, Alessio; Menchi, Gloria; Bianchini, Francesca; Trabocchi, Andrea

    2016-10-20

    The application of a cell-based growth inhibition on a library of skeletally different glycomimetics allowed for the selection of a hexahydro-2H-furo[3,2-b][1,4]oxazine compound as candidate inhibitors of MDA-MB-231 cell growth. Subsequent synthesis of analogue compounds and preliminary biological studies validated the selection of a valuable hit compound with a novel polyhydroxylated structure for the modulation of the breast carcinoma cell cycle mechanism.

  19. Cell Surface Binding and Internalization of Aβ Modulated by Degree of Aggregation

    Directory of Open Access Journals (Sweden)

    David A. Bateman

    2011-01-01

    Full Text Available The amyloid peptides, Aβ40 and Aβ42, are generated through endoproteolytic cleavage of the amyloid precursor protein. Here we have developed a model to investigate the interaction of living cells with various forms of aggregated Aβ40/42. After incubation at endosomal pH 6, we observed a variety of Aβ conformations after 3 (Aβ3, 24 (Aβ24, and 90 hours (Aβ90. Both Aβ4224 and Aβ4024 were observed to rapidly bind and internalize into differentiated PC12 cells, leading to accumulation in the lysosome. In contrast, Aβ40/4290 were both found to only weakly associate with cells, but were observed as the most aggregated using dynamic light scattering and thioflavin-T. Internalization of Aβ40/4224 was inhibited with treatment of monodansylcadaverine, an endocytosis inhibitor. These studies indicate that the ability of Aβ40/42 to bind and internalize into living cells increases with degree of aggregation until it reaches a maximum beyond which its ability to interact with cells diminishes drastically.

  20. Temperature dependence and GABA modulation of (TH)triazolam binding in the rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Earle, M.E.; Concas, A.; Wamsley, J.K.; Yamamura, H.I.

    1987-07-27

    The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. The authors major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of (TH)TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (K/sub d/ = 0.27 +/- 08 nM at 0C; K/sub d/ = 1.96 +/- 0.85 nM at 37C) while the B/sub max/ values remained unchanged (1220 +/- 176 fmoles/mg protein at 0C and 1160 +/- 383 fmoles/mg protein at 37C). Saturation studies of (TH)TZ binding in the presence or absence of GABA (100 M) showed a GABA-shift. At 0C the K/sub d/ values were (K/sub d/ = 0.24 +/- 0.03 nM/-GABA; K/sub d/ = 0.16 +/- 0.04/+GABA) and at 37C the K/sub d/ values were (K/sub d/ = 1.84 +/- 0.44 nM/-GABA; K/sub d/ = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, the authors findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists. 20 references, 3 tables.

  1. Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jason R Gerstner

    Full Text Available Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7 on sleep and long-term memory (LTM formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation "window" that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state.

  2. Cholesterol Oxidase Binds TLR2 and Modulates Functional Responses of Human Macrophages

    Directory of Open Access Journals (Sweden)

    Katarzyna Bednarska

    2014-01-01

    Full Text Available Cholesterol oxidase (ChoD is considered to be an important virulence factor for Mycobacterium tuberculosis (Mtb, but its influence on macrophage activity is unknown. Here we used Nocardia erythropolis ChoD, which is very similar to the Mtb enzyme (70% identity at the amino-acid level, to evaluate the impact of bacterial ChoD on the activity of THP-1-derived macrophages in vitro. We found that ChoD decreased the surface expression of Toll-like receptor type 2 (TLR2 and complement receptor 3 (CR3 on these macrophages. Flow cytometry and confocal microscopy showed that ChoD competed with lipoteichoic acid for ligand binding sites on TLR2 but not on CR3, suggesting that ChoD signaling is mediated via TLR2. Binding of ChoD to the membrane of macrophages had diverse effects on the activity of macrophages, activating p38 mitogen activated kinase and stimulating production of a large amount of interleukin-10. Moreover, ChoD primed macrophages to enhance the production of reactive oxygen species in response to the phorbol myristate acetate, which was reduced by “switching off” TLR-derived signaling through interleukin-1 receptor-associated kinases 1 and 4 inhibition. Our study revealed that ChoD interacts directly with macrophages via TLR2 and influences the biological activity of macrophages during the development of the initial response to infection.

  3. Tight-binding electrons on triangular and kagome lattices under staggered modulated magnetic fields: quantum Hall effects and Hofstadter butterflies

    Energy Technology Data Exchange (ETDEWEB)

    Li Juan; Wang Yifei; Gong Changde, E-mail: yfwang_nju@hotmail.com [Center for Statistical and Theoretical Condensed Matter Physics, and Department of Physics, Zhejiang Normal University, Jinhua 321004 (China)

    2011-04-20

    We consider the tight-binding models of electrons on a two-dimensional triangular lattice and kagome lattice under staggered modulated magnetic fields. Such fields have two components: a uniform-flux part with strength {phi}, and a staggered-flux part with strength {Delta}{phi}. Various properties of the Hall conductances and Hofstadter butterflies are studied. When {phi} is fixed, variation of {Delta}{phi} leads to the quantum Hall transitions and Chern numbers of Landau subbands being redistributed between neighboring pairs. The energy spectra with nonzero {Delta}{phi}s have similar fractal structures but quite different energy gaps compared with the original Hofstadter butterflies of {Delta}{phi} = 0. Moreover, the fan-like structure of Landau levels in the low magnetic field region is also modified appreciably by {Delta}{phi}.

  4. Radioiodinated branched carbohydrates

    Science.gov (United States)

    Goodman, Mark M.; Knapp, Jr., Furn F.

    1989-01-01

    A radioiodinated branched carbohydrate for tissue imaging. Iodine-123 is stabilized in the compound by attaching it to a vinyl functional group that is on the carbohydrate. The compound exhibits good uptake and retention and is promising in the development of radiopharmaceuticals for brain, heart and tumor imaging.

  5. Carbohydrates as allergens.

    Science.gov (United States)

    Commins, Scott P

    2015-01-01

    Complex carbohydrates are effective inducers of Th2 responses, and carbohydrate antigens can stimulate the production of glycan-specific antibodies. In instances where the antigen exposure occurs through the skin, the resulting antibody production can contain IgE class antibody. The glycan-stimulated IgE may be non-specific but may also be antigen specific. This review focuses on the production of cross-reactive carbohydrate determinants, the recently identified IgE antibody response to a mammalian oligosaccharide epitope, galactose-alpha-1,3-galactose (alpha-gal), as well as discusses practical implications of carbohydrates in allergy. In addition, the biological effects of carbohydrate antigens are reviewed in setting of receptors and host recognition.

  6. NITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASE

    KAUST Repository

    Wong, Aloysius Tze

    2014-06-01

    Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

  7. Computerized molecular modeling of carbohydrates

    Science.gov (United States)

    Computerized molecular modleing continues to increase in capability and applicability to carbohydrates. This chapter covers nomenclature and conformational aspects of carbohydrates, perhaps of greater use to carbohydrate-inexperienced computational chemists. Its comments on various methods and studi...

  8. Carbohydrates and Diabetes (For Parents)

    Science.gov (United States)

    ... Old Feeding Your 1- to 2-Year-Old Carbohydrates and Diabetes KidsHealth > For Parents > Carbohydrates and Diabetes ... many kids with diabetes take to stay healthy. Carbohydrates and Blood Sugar The two main forms of ...

  9. Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng; Liu, Xiao-qin; Li, He; Liang, Kai-ni [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China); Miner, Jeffrey N.; Hong, Mei; Kallel, E. Adam; Oeveren, Arjan van; Zhi, Lin [Discovery Research, Ligand Pharmaceuticals Inc., 10275 Science Center Drive, San Diego, California 92121 (United States); Jiang, Tao, E-mail: x-ray@sun5.ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)

    2006-11-01

    Crystal structure of the ligand-binding domain of androgen receptor in complex with LGD2226. The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl) amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 Å was solved and compared with the structure of the AR LBD–R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents.

  10. Does positive selection drive transcription factor binding site turnover? A test with Drosophila cis-regulatory modules.

    Directory of Open Access Journals (Sweden)

    Bin Z He

    2011-04-01

    Full Text Available Transcription factor binding site(s (TFBS gain and loss (i.e., turnover is a well-documented feature of cis-regulatory module (CRM evolution, yet little attention has been paid to the evolutionary force(s driving this turnover process. The predominant view, motivated by its widespread occurrence, emphasizes the importance of compensatory mutation and genetic drift. Positive selection, in contrast, although it has been invoked in specific instances of adaptive gene expression evolution, has not been considered as a general alternative to neutral compensatory evolution. In this study we evaluate the two hypotheses by analyzing patterns of single nucleotide polymorphism in the TFBS of well-characterized CRM in two closely related Drosophila species, Drosophila melanogaster and Drosophila simulans. An important feature of the analysis is classification of TFBS mutations according to the direction of their predicted effect on binding affinity, which allows gains and losses to be evaluated independently along the two phylogenetic lineages. The observed patterns of polymorphism and divergence are not compatible with neutral evolution for either class of mutations. Instead, multiple lines of evidence are consistent with contributions of positive selection to TFBS gain and loss as well as purifying selection in its maintenance. In discussion, we propose a model to reconcile the finding of selection driving TFBS turnover with constrained CRM function over long evolutionary time.

  11. Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity.

    Science.gov (United States)

    Fichorova, Raina N; Yamamoto, Hidemi S; Fashemi, Titilayo; Foley, Evan; Ryan, Stanthia; Beatty, Noah; Dawood, Hassan; Hayes, Gary R; St-Pierre, Guillaume; Sato, Sachiko; Singh, Bibhuti N

    2016-01-08

    Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic, and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phosphoinositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical Trichomonas isolates, and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of Trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES (regulated on activation normal T cell expressed and secreted)). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to Trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live Trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding, Trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galectin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.

  12. Modulation of glutamate transport and receptor binding by glutamate receptor antagonists in EAE rat brain.

    Science.gov (United States)

    Sulkowski, Grzegorz; Dąbrowska-Bouta, Beata; Salińska, Elżbieta; Strużyńska, Lidia

    2014-01-01

    The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.

  13. The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism.

    Science.gov (United States)

    Davis, Warren

    2014-01-01

    The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.

  14. Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan

    Science.gov (United States)

    Kuwabara, Naoyuki; Manya, Hiroshi; Yamada, Takeyuki; Tateno, Hiroaki; Kanagawa, Motoi; Kobayashi, Kazuhiro; Akasaka-Manya, Keiko; Hirose, Yuriko; Mizuno, Mamoru; Ikeguchi, Mitsunori; Toda, Tatsushi; Hirabayashi, Jun; Senda, Toshiya; Endo, Tamao; Kato, Ryuichi

    2016-01-01

    The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose–type GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man structure of α-dystroglycan (α-DG), a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Reduced glycosylation of α-DG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), a glycosyltransferase that participates in the formation of GlcNAc-β1,2-Man glycan, are causally related to muscle-eye-brain disease (MEB), a congenital muscular dystrophy, although the role of POMGnT1 in postphosphoryl modification of GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan remains elusive. Our crystal structures of POMGnT1 agreed with our previous results showing that the catalytic domain recognizes substrate O-mannosylated proteins via hydrophobic interactions with little sequence specificity. Unexpectedly, we found that the stem domain recognizes the β-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-β1,2-Man clustering and also may recruit other enzymes that interact with POMGnT1, e.g., fukutin, which is required for further modification of the GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan. On the basis of our findings, we propose a mechanism for the deficiency in postphosphoryl modification of the glycan observed in POMGnT1-KO mice and MEB patients. PMID:27493216

  15. Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan

    OpenAIRE

    Kuwabara, Naoyuki; Manya, Hiroshi; Yamada, Takeyuki; Tateno, Hiroaki; Kanagawa, Motoi; Kobayashi, Kazuhiro; Akasaka-Manya, Keiko; Hirose, Yuriko; Mizuno, Mamoru; Ikeguchi, Mitsunori; Toda, Tatsushi; Hirabayashi, Jun; Senda, Toshiya; Endo, Tamao; Kato, Ryuichi

    2016-01-01

    Congenital muscular dystrophy (CMD) is caused by hypoglycosylation of α-dystroglycan (α-DG). In some CMD patients, mutations in the gene encoding protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1, POMGnT1, are responsible for such hypoglycosylation. Many CMD-related genes are thought to be involved in the glycosylation of core M3, a specific O-mannose–type structure in α-DG. Although POMGnT1 has long been known to be associated with CMD, its role in the glycosylation of core M3 ...

  16. Adhesive and migratory effects of phosphophoryn are modulated by flanking peptides of the integrin binding motif.

    Directory of Open Access Journals (Sweden)

    Shigeki Suzuki

    Full Text Available Phosphophoryn (PP is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP. Gene duplications in the ancestor dentin matrix protein-1 (DMP-1 genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs. Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH. This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin

  17. Regulatable and Modulable Background Expression Control in Prokaryotic Synthetic Circuits by Auxiliary Repressor Binding Sites.

    Science.gov (United States)

    Merulla, Davide; van der Meer, Jan Roelof

    2016-01-15

    Expression control in synthetic genetic circuitry, for example, for construction of sensitive biosensors, is hampered by the lack of DNA parts that maintain ultralow background yet achieve high output upon signal integration by the cells. Here, we demonstrate how placement of auxiliary transcription factor binding sites within a regulatable promoter context can yield an important gain in signal-to-noise output ratios from prokaryotic biosensor circuits. As a proof of principle, we use the arsenite-responsive ArsR repressor protein from Escherichia coli and its cognate operator. Additional ArsR operators placed downstream of its target promoter can act as a transcription roadblock in a distance-dependent manner and reduce background expression of downstream-placed reporter genes. We show that the transcription roadblock functions both in cognate and heterologous promoter contexts. Secondary ArsR operators placed upstream of their promoter can also improve signal-to-noise output while maintaining effector dependency. Importantly, background control can be released through the addition of micromolar concentrations of arsenite. The ArsR-operator system thus provides a flexible system for additional gene expression control, which, given the extreme sensitivity to micrograms per liter effector concentrations, could be applicable in more general contexts.

  18. The quaternary structure of the recombinant bovine odorant-binding protein is modulated by chemical denaturants.

    Directory of Open Access Journals (Sweden)

    Olga V Stepanenko

    Full Text Available A large group of odorant-binding proteins (OBPs has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer's β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl. Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M. This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

  19. Glutathione selectively modulates the binding of platinum drugs to human copper chaperone Cox17.

    Science.gov (United States)

    Zhao, Linhong; Wang, Zhen; Wu, Han; Xi, Zhaoyong; Liu, Yangzhong

    2015-12-01

    The copper chaperone Cox17 (cytochrome c oxidase copper chaperone) has been shown to facilitate the delivery of cisplatin to mitochondria, which contributes to the overall cytotoxicity of the drug [Zhao et al. (2014) Chem. Commun. 50: , 2667-2669]. Kinetic data indicate that Cox17 has reactivity similar to glutathione (GSH), the most abundant thiol-rich molecule in the cytoplasm. In the present study, we found that GSH significantly modulates the reaction of platinum complexes with Cox17. GSH enhances the reactivity of three anti-cancer drugs (cisplatin, carboplatin and oxaliplatin) to Cox17, but suppresses the reaction of transplatin. Surprisingly, the pre-formed cisplatin-GSH adducts are highly reactive to Cox17; over 90% platinum transfers from GSH to Cox17. On the other hand, transplatin-GSH adducts are inert to Cox17. These different effects are consistent with the drug activity of these platinum complexes. In addition, GSH attenuates the protein aggregation of Cox17 induced by platination. These results indicate that the platinum-protein interactions could be substantially influenced by the cellular environment.

  20. Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence

    Science.gov (United States)

    Mazor, Yariv; Sachsenmeier, Kris F.; Yang, Chunning; Hansen, Anna; Filderman, Jessica; Mulgrew, Kathy; Wu, Herren; Dall’Acqua, William F.

    2017-01-01

    Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected. PMID:28067257

  1. D-Pinitol attenuates 7, 12 dimethylbenz [a] anthracene induced hazards through modulating protein bound carbohydrates, adenosine triphosphatases and lysosomal enzymes during experimental mammary carcinogenesis.

    Science.gov (United States)

    Rengarajan, Thamaraiselvan; Nandakumar, Natarajan; Balasubramanian, Maruthaiveeran Periyasamy

    2012-01-01

    We have reported here that the ameliorative potentials of D-Pinitol during 7, 12-Dimethylbenz [a] anthracene induced experimental breast carcinogenesis. DMBA is a potent organ specific carcinogen which is widely employed to induce mammary carcinoma in rats. D-Pinitol a natural inositol has been reported to found in soybean with many biological functions. The female sprague dawley rats were subjected to carcinogen 7, 12-DMBA and the ameliorative potentials of dietary compound D-Pinitol was investigated with reference to cell surface glycoproteins, lysosomal enzymes and adenosine triphosphatases. Interestingly, administration of D-Pinitol was found to be significantly down regulated the breast tissue glycoproteins and lysosomal enzymes and in contrast the levels of adenosine triphosphatases were remarkably up regulated. Further, the biochemical changes were well reflected and evidenced in the histology of breast and liver tissues. Thus, it can be concluded from the present study that D-Pinitol efficiently attenuates the hazardous consequences of the environmental carcinogen 7,12-DMBA through modulating cell surface glycoproteins, membrane protective role both in lysosomal and ATPase compartment via its antioxidant nature which ultimately results in the findings of future innovative remedies for genotoxin mediated hazards.

  2. Carbohydrates and Depression.

    Science.gov (United States)

    Wurtman, Richard J.; Wurtman, Judith J.

    1989-01-01

    Describes the symptoms, such as appetite change and mood fluctuation, basic mechanisms, and some treatments of Seasonal Affective Disorder (SAD), Carbohydrate-Craving Obesity (CCO) and Premenstrual Syndrome (PMS). Provides several tables and diagrams, and three reading references. (YP)

  3. Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

    Science.gov (United States)

    Chandrasekaran, E V; Xue, Jun; Xia, Jie; Khaja, Siraj D; Piskorz, Conrad F; Locke, Robert D; Neelamegham, Sriram; Matta, Khushi L

    2016-10-01

    Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucβ1-3 GalNAc and Fucα-1-2 D-Fucβ-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc β-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate

  4. Bioinformatics Identification of Modules of Transcription Factor Binding Sites in Alzheimer's Disease-Related Genes by In Silico Promoter Analysis and Microarrays

    Directory of Open Access Journals (Sweden)

    Regina Augustin

    2011-01-01

    Full Text Available The molecular mechanisms and genetic risk factors underlying Alzheimer's disease (AD pathogenesis are only partly understood. To identify new factors, which may contribute to AD, different approaches are taken including proteomics, genetics, and functional genomics. Here, we used a bioinformatics approach and found that distinct AD-related genes share modules of transcription factor binding sites, suggesting a transcriptional coregulation. To detect additional coregulated genes, which may potentially contribute to AD, we established a new bioinformatics workflow with known multivariate methods like support vector machines, biclustering, and predicted transcription factor binding site modules by using in silico analysis and over 400 expression arrays from human and mouse. Two significant modules are composed of three transcription factor families: CTCF, SP1F, and EGRF/ZBPF, which are conserved between human and mouse APP promoter sequences. The specific combination of in silico promoter and multivariate analysis can identify regulation mechanisms of genes involved in multifactorial diseases.

  5. Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module.

    Science.gov (United States)

    Hebbeln, Peter; Rodionov, Dmitry A; Alfandega, Anja; Eitinger, Thomas

    2007-02-20

    BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only one-third of the widespread bioY genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the alpha-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coli cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue in BioM, demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that BioM, BioN, and BioY proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results substantiate our earlier suggestion of a mechanistically novel group of membrane transporters.

  6. Carbohydrates and T cells: a sweet twosome.

    Science.gov (United States)

    Avci, Fikri Y; Li, Xiangming; Tsuji, Moriya; Kasper, Dennis L

    2013-04-01

    Carbohydrates as T cell-activating antigens have been generating significant interest. For many years, carbohydrates were thought of as T-independent antigens, however, more recent research had demonstrated that mono- or oligosaccharides glycosidically linked to peptides can be recognized by T cells. T cell recognition of these glycopeptides depends on the structure of both peptide and glycan portions of the antigen. Subsequently, it was discovered that natural killer T cells recognized glycolipids when presented by the antigen presenting molecule CD1d. A transformative insight into glycan-recognition by T cells occurred when zwitterionic polysaccharides were discovered to bind to and be presented by MHCII to CD4+ T cells. Based on this latter observation, the role that carbohydrate epitopes generated from glycoconjugate vaccines had in activating helper T cells was explored and it was found that these epitopes are presented to specific carbohydrate recognizing T cells through a unique mechanism. Here we review the key interactions between carbohydrate antigens and the adaptive immune system at the molecular, cellular and systems levels exploring the significant biological implications in health and disease.

  7. Surface characterization of carbohydrate microarrays.

    Science.gov (United States)

    Scurr, David J; Horlacher, Tim; Oberli, Matthias A; Werz, Daniel B; Kroeck, Lenz; Bufali, Simone; Seeberger, Peter H; Shard, Alexander G; Alexander, Morgan R

    2010-11-16

    Carbohydrate microarrays are essential tools to determine the biological function of glycans. Here, we analyze a glycan array by time-of-flight secondary ion mass spectrometry (ToF-SIMS) to gain a better understanding of the physicochemical properties of the individual spots and to improve carbohydrate microarray quality. The carbohydrate microarray is prepared by piezo printing of thiol-terminated sugars onto a maleimide functionalized glass slide. The hyperspectral ToF-SIMS imaging data are analyzed by multivariate curve resolution (MCR) to discern secondary ions from regions of the array containing saccharide, linker, salts from the printing buffer, and the background linker chemistry. Analysis of secondary ions from the linker common to all of the sugar molecules employed reveals a relatively uniform distribution of the sugars within the spots formed from solutions with saccharide concentration of 0.4 mM and less, whereas a doughnut shape is often formed at higher-concentration solutions. A detailed analysis of individual spots reveals that in the larger spots the phosphate buffered saline (PBS) salts are heterogeneously distributed, apparently resulting in saccharide concentrated at the rim of the spots. A model of spot formation from the evaporating sessile drop is proposed to explain these observations. Saccharide spot diameters increase with saccharide concentration due to a reduction in surface tension of the saccharide solution compared to PBS. The multivariate analytical partial least squares (PLS) technique identifies ions from the sugars that in the complex ToF-SIMS spectra correlate with the binding of galectin proteins.

  8. Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

    Directory of Open Access Journals (Sweden)

    Daniel J White

    Full Text Available The EVI1 (ecotropic viral integration site 1 gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196 in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D, which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

  9. ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

    Energy Technology Data Exchange (ETDEWEB)

    Charlton, Hayley K.; Webster, Julie; Kruger, Sarah; Simpson, Fiona; Richards, Ayanthi A. [Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD 4102 (Australia); Whitehead, Jonathan P., E-mail: j.whitehead1@uq.edu.au [Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD 4102 (Australia)

    2010-02-05

    The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2{beta}, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.

  10. Cyclic AMP response element binding protein and brain-derived neurotrophic factor: Molecules that modulate our mood?

    Indian Academy of Sciences (India)

    A Nair; V A Vaidya

    2006-09-01

    Depression is the major psychiatric ailment of our times, afflicting ∼20% of the population. Despite its prevalence, the pathophysiology of this complex disorder is not well understood. In addition, although antidepressants have been in existence for the past several decades, the mechanisms that underlie their therapeutic effects remain elusive. Building evidence implicates a role for the plasticity of specific neuro-circuitry in both the pathophysiology and treatment of depression. Damage to limbic regions is thought to contribute to the etiology of depression and antidepressants have been reported to reverse such damage and promote adaptive plasticity. The molecular pathways that contribute to the damage associated with depression and antidepressant-mediated plasticity are a major focus of scientific enquiry. The transcription factor cyclic AMP response element binding protein (CREB) and the neurotrophin brain-derived neurotrophic factor (BDNF) are targets of diverse classes of antidepressants and are known to be regulated in animal models and in patients suffering from depression. Given their role in neuronal plasticity, CREB and BDNF have emerged as molecules that may play an important role in modulating mood. The purpose of this review is to discuss the role of CREB and BDNF in depression and as targets/mediators of antidepressant action.

  11. Thyroid hormone receptors bind to the promoter of the mouse histone H10 gene and modulate its transcription.

    Science.gov (United States)

    Bauer-Hofmann, R; Alonso, A

    1995-01-01

    It has been shown that the mouse histone H10 promoter contains a DNA element, composed of a direct repeat of the sequence GGTGACC separated by 7 nt, which is able to bind retinoic acid receptors and to modulate transcription of reporter genes following treatment with retinoic acid. We have now investigated whether this DNA motif is also responsive to thyroid hormone. We co-transfected CV-1 monkey kidney cells with chloramphenicol acetyltransferase (CAT) expression plasmids containing either 740 bp of the H10 wild-type promoter or five copies of the repeat element cloned in front of the thymidine kinase promoter and expression vectors for human thyroid hormone receptors (TRs) alpha or beta and retinoid X receptor alpha (RXR alpha). Treatment of transfected cells with triiodothyronine led to a dose-dependent increase in CAT activity. Transfection experiments with increasing amounts of expression vectors for either TR alpha or RXR alpha resulted in up to 6-fold enhancement of CAT transcription. Furthermore, point mutations within the half-sites of the response element of the H10 promoter, as well as deletions within the interspace region, lowered CAT activity to 60-80% of that of the wild-type control. Electrophoretic mobility shift assays showed that the repeat element was able to form retarded complexes with TR alpha homodimers, as well as with TR alpha-RXR alpha heterodimers. Our results suggest that thyroid hormone receptors are involved in the regulation of mouse histone H10 expression. Images PMID:8559662

  12. Identification and Structural Basis of Binding to Host Lung Glycogen by Streptococcal Virulence Factors

    Energy Technology Data Exchange (ETDEWEB)

    Lammerts van Bueren,A.; Higgins, M.; Wang, D.; Burke, R.; Boraston, A.

    2007-01-01

    The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal {alpha}-glucan-metabolizing machinery as virulence factors.

  13. Defined presentation of carbohydrates on a duplex DNA scaffold.

    Science.gov (United States)

    Schlegel, Mark K; Hütter, Julia; Eriksson, Magdalena; Lepenies, Bernd; Seeberger, Peter H

    2011-12-16

    A new method for the spatially defined alignment of carbohydrates on a duplex DNA scaffold is presented. The use of an N-hydroxysuccinimide (NHS)-ester phosphoramidite along with carbohydrates containing an alkylamine linker allows for on-column labeling during solid-phase oligonucleotide synthesis. This modification method during solid-phase synthesis only requires the use of minimal amounts of complex carbohydrates. The covalently attached carbohydrates are presented in the major groove of the B-form duplex DNA as potential substrates for murine type II C-type lectin receptors mMGL1 and mMGL2. CD spectroscopy and thermal melting revealed only minimal disturbance of the overall helical structure. Surface plasmon resonance and cellular uptake studies with bone-marrow-derived dendritic cells were used to assess the capability of these carbohydrate-modified duplexes to bind to mMGL receptors.

  14. PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM.

    Science.gov (United States)

    McCallum, Matthew; Tammam, Stephanie; Little, Dustin J; Robinson, Howard; Koo, Jason; Shah, Megha; Calmettes, Charles; Moraes, Trevor F; Burrows, Lori L; Howell, P Lynne

    2016-05-20

    Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM·PilN(1-12)). Structural analysis, multiangle light scattering coupled with size exclusion chromatography, and bacterial two-hybrid data revealed that PilM forms dimers mediated by the binding of a novel conserved motif in the N terminus of PilM, and binding PilN abrogates this binding interface, resulting in PilM monomerization. Structural comparison of PilM with PilM·PilN(1-12) revealed that upon PilN binding, there is a large domain closure in PilM that alters its ATP binding site. Using biolayer interferometry, we found that the association rate of PilN with PilM is higher in the presence of ATP compared with ADP. Bacterial two-hybrid data suggested the connectivity of the cytoplasmic and inner membrane components of the type IVa pilus machinery in P. aeruginosa, with PilM binding to PilB, PilT, and PilC in addition to PilN. Pull-down experiments demonstrated direct interactions of PilM with PilB and PilT. We propose a working model in which dynamic binding of PilN facilitates functionally relevant structural changes in PilM.

  15. Carbohydrates in therapeutics.

    Science.gov (United States)

    Kilcoyne, Michelle; Joshi, Lokesh

    2007-07-01

    Awareness of the importance of carbohydrates in living systems and medicine is growing due to the increasing understanding of their biological and pharmacological relevance. Carbohydrates are ubiquitous and perform a wide array of biological roles. Carbohydrate-based or -modified therapeutics are used extensively in cardiovascular and hematological treatments ranging from inflammatory diseases and anti-thrombotic treatments to wound healing. Heparin is a well-known and widely used example of a carbohydrate-based drug but will not be discussed as it has been extensively reviewed. We will detail carbohydrate-based and -modified therapeutics, both those that are currently marketed or in various stages of clinical trials and those that are potential therapeutics based on promising preclinical investigations. Carbohydrate-based therapeutics include polysaccharide and oligosaccharide anti-inflammatory, anti-coagulant and anti-thrombotic agents from natural and synthetic sources, some as an alternative to heparin and others which were designed based on known structure-functional relationships. Some of these compounds have multiple biological effects, showing anti-adhesive, anti-HIV and anti-arthrithic activities. Small molecules, derivatives or mimetics of complement inhibitors, are detailed for use in limiting ischemia/ reperfusion injuries. Monosaccharides, both natural and synthetic, have been investigated for their in vivo anti-inflammatory and cardioprotective properties. Modification by glycosylation of natural products, or glycosylation-mimicking modification, has a significant effect on the parent molecule including increased plasma half-life and refining or increasing desired functions. It is hoped that this review will highlight the vast therapeutic potential of these natural bioactive molecules.

  16. Carbohydrates, pollinators, and cycads

    Science.gov (United States)

    Marler, Thomas E; Lindström, Anders J

    2015-01-01

    Cycad biology, ecology, and horticulture decisions are not supported by adequate research, and experiments in cycad physiology in particular have been deficient. Our recent report on free sugar content in a range of cycad taxa and tissues sets the stage for developing continued carbohydrate research. Growth and development of cycad pollen, mediation of the herbivory traits of specialist pollinators, and support of expensive strobilus behavioral traits are areas of cycad pollination biology that would benefit from a greater understanding of the role of carbohydrate relations. PMID:26479502

  17. Carbohydrate metabolism and its regulatory hormones in anorexia nervosa.

    Science.gov (United States)

    Casper, R C

    1996-04-16

    Findings of studies of carbohydrate metabolism in anorexia nervosa are reviewed. Topics covered included fasting blood sugar concentrations; serum insulin concentrations, insulin receptor binding activity, insulin sensitivity, and insulin resistance; plasma ketone bodies and free fatty acids; glucose tolerance tests; growth hormone, cortisol, intestinal hormones, and norepinephrine. Metabolic changes reported in anorexia nervosa are similar to those found in human and animal studies of states of caloric and carbohydrate restriction. Restoration of normal body weight is associated with normalization of virtually all measures. It is concluded that published studies offer no conclusive evidence for a syndrome-specific impairment in carbohydrate metabolism in anorexia nervosa.

  18. Differential modulation in binding of ketoprofen to bovine serum albumin in the presence and absence of surfactants: spectroscopic and calorimetric insights.

    Science.gov (United States)

    Misra, Pinaki P; Kishore, Nand

    2013-07-01

    Surfactants have long been implicated in the unique static and dynamic effect on the structure and function of serum albumins. However, there is very little information on the mode of interactions of drugs to serum albumins in presence of surfactants. The importance of such studies lay in the fact that apart from binding to serum albumins, surfactants are known to radically influence the solvents' micro environment and protein structure. Thus, we have studied the binding of the racemic form of ketoprofen with bovine serum albumin at pH 7.4 in the presence and absence of hexadecyl trimethyl ammonium bromide, sodium dodecyl sulfate, Triton X-100, and NaCl. The structural studies of ketoprofen with bovine serum albumin as investigated by circular dichroism spectroscopy revealed a significant stabilization of bovine serum albumin. However, the combined presence of the surfactants, NaCl and ketoprofen, demonstrated an extremely erratic behavior in terms of stabilization. Further the values of Stern-Volmer and dynamic quenching constant suggested the binding site of ketoprofen to be scattered in the region of domain I B and II A, close to Trp 134. The results of differential scanning calorimetry revealed that the binding of ketoprofen to bovine serum albumin leads to its temperature-dependent separation into two units. The binding parameters of bovine serum albumin obtained from isothermal titration calorimetry in the combined presence of ketoprofen and surfactants/NaCl correlate well with the differential scanning calorimetry studies further confirming the localization of ketoprofen in domain I B and II A. In the combined presence of surfactants, NaCl and ketoprofen, the binding of ketoprofen to bovine serum albumin exhibited altered binding parameters far different from the binding of ketoprofen alone. Overall, the experimental findings strongly indicated positive as well as negative modulation in the binding of ketoprofen to bovine serum albumin in the presence of

  19. Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity.

    Directory of Open Access Journals (Sweden)

    Paulo M F Cunha

    2010-07-01

    Full Text Available Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2 or the zinc-finger transcription factor lame duck (lmd lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.

  20. Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity.

    Science.gov (United States)

    Cunha, Paulo M F; Sandmann, Thomas; Gustafson, E Hilary; Ciglar, Lucia; Eichenlaub, Michael P; Furlong, Eileen E M

    2010-07-01

    Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2) or the zinc-finger transcription factor lame duck (lmd) lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.

  1. KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif.

    Directory of Open Access Journals (Sweden)

    Arnaud D Colantonio

    2011-03-01

    Full Text Available Molecular interactions between killer immunoglobulin-like receptors (KIRs and their MHC class I ligands play a central role in the regulation of natural killer (NK cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02, a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05(+ macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets.

  2. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein.

    Science.gov (United States)

    Davis, Sterenn; Davis, Benjamin M; Richens, Joanna L; Vere, Kelly-Ann; Petrov, Peter G; Winlove, C Peter; O'Shea, Paul

    2015-08-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir's affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers.

  3. IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly.

    Directory of Open Access Journals (Sweden)

    Lisa M Maurer

    Full Text Available Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K of fibronectin (FN stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD motifs in four of the nine FN type 1 (FNI modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3FNI and (5FNI; (7FNI and (9FNI; or (3FNI, (5FNI, (7FNI, and (9FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.

  4. Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation.

    Directory of Open Access Journals (Sweden)

    Vladimir Voynov

    Full Text Available Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation.

  5. Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition

    Energy Technology Data Exchange (ETDEWEB)

    Krejciríková, Veronika; Pachl, Petr; Fábry, Milan; Malý, Petr; Rezácová, Pavlína; Brynda, Jirí (Czech Academy)

    2011-11-18

    Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 {angstrom} resolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K{sub d} value in the micromolar range (K{sub d1} = 600 {+-} 70 {mu}M) and a low-affinity site with K{sub d2} = 28 {+-} 10 mM.

  6. Carbohydrates, Sugar, and Your Child

    Science.gov (United States)

    ... Old Feeding Your 1- to 2-Year-Old Carbohydrates, Sugar, and Your Child KidsHealth > For Parents > Carbohydrates, Sugar, ... a 1-ounce equivalent. previous continue Sizing Up Sugar Foods that are high in added sugar (soda, ...

  7. A low-carbohydrate/high-fat diet improves glucoregulation in type 2 diabetes mellitus by reducing postabsorptive glycogenolysis

    NARCIS (Netherlands)

    Allick, G; Bisschop, PH; Ackermans, MT; Endert, E; Meijer, AJ; Kuipers, F; Sauerwein, HP; Romijn, JA

    2004-01-01

    The aim of this study was to examine the mechanisms by which dietary carbohydrate and fat modulate fasting glycemia. We compared the effects of an eucaloric high-carbohydrate (89% carbohydrate) and high-fat (89% fat) diet on fasting glucose metabolism and insulin sensitivity in seven obese patients

  8. Specific Carbohydrate Diet: Does It Work?

    Science.gov (United States)

    ... Specific Carbohydrate Diet (SCD) Go Back The Specific Carbohydrate Diet (SCD) Email Print + Share There is no ... diet that has received attention is the Specific Carbohydrate Diet. This diet limits poorly digestible carbohydrates to ...

  9. Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

    OpenAIRE

    Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

    2013-01-01

    Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturati...

  10. Prognostic Significance of Carbohydrate Antigen 19-9 in Unresectable Locally Advanced Pancreatic Cancer Treated With Dose-Escalated Intensity Modulated Radiation Therapy and Concurrent Full-Dose Gemcitabine: Analysis of a Prospective Phase 1/2 Dose Escalation Study

    Energy Technology Data Exchange (ETDEWEB)

    Vainshtein, Jeffrey M., E-mail: jvainsh@med.umich.edu [Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan (United States); Schipper, Matthew [Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan (United States); Zalupski, Mark M. [Division of Hematology Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan (United States); Lawrence, Theodore S. [Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan (United States); Abrams, Ross [Department of Radiation Oncology, Rush Medical Center, Chicago, Illinois (United States); Francis, Isaac R. [Department of Radiology, University of Michigan, Ann Arbor, Michigan (United States); Khan, Gazala [Division of Hematology Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan (United States); Leslie, William [Division of Hematology Oncology, Department of Internal Medicine, Rush Medical Center, Chicago, Illinois (United States); Ben-Josef, Edgar [Department of Radiation Oncology, University of Pennsylvania, Philadelphia, Pennsylvania (United States)

    2013-05-01

    Purpose: Although established in the postresection setting, the prognostic value of carbohydrate antigen 19-9 (CA19-9) in unresectable locally advanced pancreatic cancer (LAPC) is less clear. We examined the prognostic utility of CA19-9 in patients with unresectable LAPC treated on a prospective trial of intensity modulated radiation therapy (IMRT) dose escalation with concurrent gemcitabine. Methods and Materials: Forty-six patients with unresectable LAPC were treated at the University of Michigan on a phase 1/2 trial of IMRT dose escalation with concurrent gemcitabine. CA19-9 was obtained at baseline and during routine follow-up. Cox models were used to assess the effect of baseline factors on freedom from local progression (FFLP), distant progression (FFDP), progression-free survival (PFS), and overall survival (OS). Stepwise forward regression was used to build multivariate predictive models for each endpoint. Results: Thirty-eight patients were eligible for the present analysis. On univariate analysis, baseline CA19-9 and age predicted OS, CA19-9 at baseline and 3 months predicted PFS, gross tumor volume (GTV) and black race predicted FFLP, and CA19-9 at 3 months predicted FFDP. On stepwise multivariate regression modeling, baseline CA19-9, age, and female sex predicted OS; baseline CA19-9 and female sex predicted both PFS and FFDP; and GTV predicted FFLP. Patients with baseline CA19-9 ≤90 U/mL had improved OS (median 23.0 vs 11.1 months, HR 2.88, P<.01) and PFS (14.4 vs 7.0 months, HR 3.61, P=.001). CA19-9 progression over 90 U/mL was prognostic for both OS (HR 3.65, P=.001) and PFS (HR 3.04, P=.001), and it was a stronger predictor of death than either local progression (HR 1.46, P=.42) or distant progression (HR 3.31, P=.004). Conclusions: In patients with unresectable LAPC undergoing definitive chemoradiation therapy, baseline CA19-9 was independently prognostic even after established prognostic factors were controlled for, whereas CA19-9 progression

  11. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    Energy Technology Data Exchange (ETDEWEB)

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L. (IIT); (UW-MED)

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  12. Biochemical software: Carbohydrates on Laboratory

    Directory of Open Access Journals (Sweden)

    D.N. Heidrich

    2005-07-01

    Full Text Available Educators around  the  world  are  being  challenged  to  develop  and  design  better and  more  effective strategies for student learning  using a variety  of modern  resources.  In this  present  work, an educa- tional  hypermedia  software  was constructed as a support tool to biochemistry teaching.  Occurrence, structure, main  characteristics and  biological  function  of the  biomolecule  Carbohydrates were pre- sented  through  modules.  The  software was developed  using concept  maps,  ISIS-Draw,  and  FLASH- MX animation program.  The chapter  Carbohydrates on Laboratory illustrates experimental methods of carbohydrates characterization, through  animation of a laboratory scenery.   The  subject was de- veloped showing reactions  as Bial, Benedict, Selliwanoff, Barfoed, Phenol  Sulphuric,  and Iodines, and also enzymatic  reactions  as glucose oxidase and amylase.  There are also links with short texts  in order to help the understanding of the contents  and principles of laboratory practice  as well as background reactions. Application of the software to undergraduate students and high school teachers  showed an excellent  acceptance.   All of them  considered  the  software  a very good learning  tool.  Both  teachers and students welcomed this program  as it is more flexible, and allows the learning in a more individual rhythm. In addition, application of the software would be suitable  to a more effective learning  and it is less expensive than conventional experimental teaching.

  13. Carbohydrates, pollinators, and cycads

    OpenAIRE

    2015-01-01

    Cycad biology, ecology, and horticulture decisions are not supported by adequate research, and experiments in cycad physiology in particular have been deficient. Our recent report on free sugar content in a range of cycad taxa and tissues sets the stage for developing continued carbohydrate research. Growth and development of cycad pollen, mediation of the herbivory traits of specialist pollinators, and support of expensive strobilus behavioral traits are areas of cycad pollination biology th...

  14. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

    Science.gov (United States)

    Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

    2013-12-06

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

  15. Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

    Energy Technology Data Exchange (ETDEWEB)

    Goetz, R.; Dover, K; Laezza, F; Shtraizent, N; Huang, X; Tchetchik, D; Eliseenkova, A; Goldfarb, M; Mohammadi, M; et. al.

    2009-01-01

    Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs.

  16. A synthetic peptide derived from A1 module in CRD4 of human TNF receptor-1 inhibits binding and proinflammatory effect of human TNF-alpha.

    Science.gov (United States)

    Cao, Yingnan; Wang, Zhaohe; Bu, Xianzhang; Tang, Shu; Mei, Zhengrong; Liu, Peiqing

    2009-06-01

    Tumour necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine, which has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Proinflammatory effect of TNF-alpha is activated mainly through human TNF receptor-1 (TNF-R1). However, the role of the fourth cystein-rich domain (CRD4) of TNF-R1 extracellular portion in the interaction of TNF-alpha with TNF-R1 is still unclear. In the present study, binding activity of TNF-alpha to TNF-R1 and protein levels of IkappaB-alpha and nuclear transcription factor kappa B (NF-kappaB) p65 subunit in HeLa cells were measured using enzyme-linked immunosorbent assay (ELISA) and western-blot analysis. Pep 3 (LRENECVS) which was derived from the hydrophilic region of A1 module in CRD4 remarkably inhibited the binding of TNF-alpha to TNF-R1, and also reversed TNF-alpha-induced degradation of IkappaB-alpha and nuclear translocation of NF-kappaB p65 subunit in HeLa cells. Our results confirmed that the hydrophilic region of A1 module in CRD4 participated in the interaction of TNF-alpha with TNF-R1, and demonstrated the potential of small-molecule TNF-alpha extracellular inhibitors targeting at A1 module in CRD4 of TNF-R1 in suppressing proinflammatory effect of TNF-alpha.

  17. Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

    Science.gov (United States)

    Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

    2013-01-01

    Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response. PMID:23746516

  18. Assessing Bacterial Interactions Using Carbohydrate-Based Microarrays

    Directory of Open Access Journals (Sweden)

    Andrea Flannery

    2015-12-01

    Full Text Available Carbohydrates play a crucial role in host-microorganism interactions and many host glycoconjugates are receptors or co-receptors for microbial binding. Host glycosylation varies with species and location in the body, and this contributes to species specificity and tropism of commensal and pathogenic bacteria. Additionally, bacterial glycosylation is often the first bacterial molecular species encountered and responded to by the host system. Accordingly, characterising and identifying the exact structures involved in these critical interactions is an important priority in deciphering microbial pathogenesis. Carbohydrate-based microarray platforms have been an underused tool for screening bacterial interactions with specific carbohydrate structures, but they are growing in popularity in recent years. In this review, we discuss carbohydrate-based microarrays that have been profiled with whole bacteria, recombinantly expressed adhesins or serum antibodies. Three main types of carbohydrate-based microarray platform are considered; (i conventional carbohydrate or glycan microarrays; (ii whole mucin microarrays; and (iii microarrays constructed from bacterial polysaccharides or their components. Determining the nature of the interactions between bacteria and host can help clarify the molecular mechanisms of carbohydrate-mediated interactions in microbial pathogenesis, infectious disease and host immune response and may lead to new strategies to boost therapeutic treatments.

  19. Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

    Science.gov (United States)

    Schroeder, Friedhelm; Petrescu, Anca D; Huang, Huan; Atshaves, Barbara P; McIntosh, Avery L; Martin, Gregory G; Hostetler, Heather A; Vespa, Aude; Landrock, Danilo; Landrock, Kerstin K; Payne, H Ross; Kier, Ann B

    2008-01-01

    Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that

  20. Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

    Science.gov (United States)

    Jadhav, Swati B; Bankar, Sandip B; Granström, Tom; Ojamo, Heikki; Singhal, Rekha S; Survase, Shrikant A

    2015-09-01

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

  1. Probing the electrostatics and pharmacological modulation of sequence-specific binding by the DNA-binding domain of the ETS family transcription factor PU.1: a binding affinity and kinetics investigation.

    Science.gov (United States)

    Munde, Manoj; Poon, Gregory M K; Wilson, W David

    2013-05-27

    Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.

  2. Potential energy surface and binding energy in the presence of an external electric field: modulation of anion-π interactions for graphene-based receptors.

    Science.gov (United States)

    Foroutan-Nejad, Cina; Marek, Radek

    2014-02-14

    Measuring the binding energy or scanning the potential energy surface (PES) of the charged molecular systems in the presence of an external electric field (EEF) requires a careful evaluation of the origin-dependency of the energy of the system and references. Scanning the PES for charged or purely ionic systems for obtaining the intrinsic energy barriers needs careful analysis of the electric work applied on ions by the EEF. The binding energy in the presence of an EEF is different from that in the absence of an electric field as the binding energy is an anisotropic characteristic which depends on the orientation of molecules with respect to the EEF. In this contribution we discuss various aspects of the PES and the concept of binding energy in the presence of an EEF. In addition, we demonstrate that the anion-π bonding properties can be modulated by applying a uniform EEF, which has a more pronounced effect on the larger, more polarizable π-systems. An analogous behavior is presumed for cation-π systems. We predict that understanding the phenomenon introduced in the present account has enormous potential, for example, for separating charged species on the surface of polarizable two-dimensional materials such as graphene or the surface of carbon nanotubes, in desalination of water.

  3. Effects of Carbohydrate Consumption Case Study: carbohydrates in Bread

    Directory of Open Access Journals (Sweden)

    Neacsu N.A.

    2014-12-01

    Full Text Available Carbohydrates perform numerous roles in living organisms; they are an important source of energy. The body uses carbohydrates to make glucose which is the fuel that gives it energy and helps keep everything going. However, excess carbohydrate consumption has negative health effects. Bread is a basic product in our nutrition and it also is a product with a high content of carbohydrates. So, it is important to find out more information on bread and on the recommended bread type best for consumption.

  4. Molecular recognition of surface-immobilized carbohydrates by a synthetic lectin

    Directory of Open Access Journals (Sweden)

    Melanie Rauschenberg

    2014-06-01

    Full Text Available The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of synthetic carbohydrate receptors (“synthetic lectins” constitutes a key advance in biomedical technology. In this article we report a synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontaneously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA, β-D-galactose, β-D-glucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobilized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluorescence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins and that the recognition of synthetic and natural lectins is strictly orthogonal.

  5. Glycidol-carbohydrate hybrids: a new family of DNA alkylating agents.

    Science.gov (United States)

    Toshima, Kazunobu; Okuno, Yukiko; Matsumura, Shuichi

    2003-10-06

    Novel and chiral glycidol-carbohydrate hybrids possessing an epoxy group as a DNA alkylating moiety were designed and synthesized. These artificial hybrids selectively alkylated DNA at the N-7 sites of the guanines and cleaved DNA without any additives. The binding ability of the glycidol was significantly enhanced by the attachment of the carbohydrate.

  6. Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism.

    Science.gov (United States)

    Glaring, Mikkel A; Baumann, Martin J; Abou Hachem, Maher; Nakai, Hiroyuki; Nakai, Natsuko; Santelia, Diana; Sigurskjold, Bent W; Zeeman, Samuel C; Blennow, Andreas; Svensson, Birte

    2011-04-01

    Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.

  7. Low-carbohydrate diets and prostate cancer: how low is "low enough"?

    Science.gov (United States)

    Masko, Elizabeth M; Thomas, Jean A; Antonelli, Jodi A; Lloyd, Jessica C; Phillips, Tameika E; Poulton, Susan H; Dewhirst, Mark W; Pizzo, Salvatore V; Freedland, Stephen J

    2010-09-01

    Previous studies indicate that carbohydrate intake influences prostate cancer biology, as mice fed a no-carbohydrate ketogenic diet (NCKD) had significantly smaller xenograft tumors and longer survival than mice fed a Western diet. As it is nearly impossible for humans to consume and maintain NCKD, we determined whether diets containing 10% or 20% carbohydrate kcal showed similar tumor growth as NCKD. A total of 150 male severe combined immunodeficient mice were fed a Western diet ad libitum, injected with the human prostate cancer cell line LAPC-4, and then randomized 2 weeks later to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate diets. Ten mice not injected were fed an ad libitum low-fat diet (12% fat kcal) serving as the reference in a modified-paired feeding protocol. Mice were sacrificed when tumors reached 1,000 mm(3). Despite consuming extra calories, all mice receiving low-carbohydrate diets were significantly lighter than those receiving a low-fat diet (P Diet did not affect survival (P = 0.34). There were no differences in serum insulin-like growth factor-I or insulin-like growth factor binding protein-3 at sacrifice among the low-carbohydrate arms (P = 0.07 and P = 0.55, respectively). Insulin was significantly lower in the 20% carbohydrate arm (P = 0.03). LAPC-4 xenograft mice fed a low-carbohydrate diet (10-20% carbohydrate kcal) had similar survival as mice consuming NCKD (0% carbohydrate kcal).

  8. Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1alpha mRNA 3'-UTR and modulate HIF-1alpha protein expression.

    Science.gov (United States)

    Hägele, Sonja; Kühn, Uwe; Böning, Melanie; Katschinski, Dörthe M

    2009-01-01

    The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1alpha subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1alpha mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3'-UTR (untranslated region) of HIF-1alpha mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1alpha protein levels mediated by the 3'-UTR of HIF-1alpha mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3'-UTR of HIF-1alpha as well as endogenous HIF-1alpha protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1alpha cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1alpha following insulin stimulation.

  9. The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

    Science.gov (United States)

    Oeste, Clara L; Díez-Dacal, Beatriz; Bray, Francesca; García de Lacoba, Mario; de la Torre, Beatriz G; Andreu, David; Ruiz-Sánchez, Antonio J; Pérez-Inestrosa, Ezequiel; García-Domínguez, Carlota A; Rojas, José M; Pérez-Sala, Dolores

    2011-01-06

    Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2) selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2). Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

  10. Impact of Dietary Carbohydrate and Protein Levels on Carbohydrate Metabolism

    Science.gov (United States)

    Lasker, Denise Ann

    2009-01-01

    The goal of this dissertation was to investigate the impact of changing dietary carbohydrate (CARB) intakes within recommended dietary guidelines on metabolic outcomes specifically associated with glycemic regulations and carbohydrate metabolism. This research utilized both human and animal studies to examine changes in metabolism across a wide…

  11. A Novel Binding Pattern Unique in Two Ligands for One Carbohydrate Recognition Domain in Galectins%半乳糖凝集素糖结合的新模式:一个糖结合域的双重配体结合

    Institute of Scientific and Technical Information of China (English)

    卞乘凤; 张英; 李德峰; 王大成

    2011-01-01

    半乳糖凝集素家族通过糖识别结构域(CRD)可以专一性识别和结合含β-半乳糖的多糖配体来发挥其生物学功能.到目前发现的CRD对β-半乳糖的识别模式是非常保守的,在结构已知的半乳糖凝集素结构中,一个CRD只能结合一个多糖配体分子.最近,通过对人源半乳糖凝聚素-3 CRD与对硝基TF二糖(TFN)复合物的晶体结构解析首次发现,一个CRD可以同时结合2个TFN分子.与这2个TFN分子有双向结合的残基突变体E165A结构分析显示,一个残基的突变引起的结构上的微小变化会使结合位点2丧失结合糖底物的能力,而位点1的配体结合却不受影响.这表明,结合位点1对糖底物保守的识别和结合是基本的、主要的,而结合位点2对于糖有条件的结合,是额外的、次要的.序列比对和立体化学分析显示,参与新位点2结合的关键残基在其他半乳糖凝集素分子中都是保守的,而它们参与糖配体结合并不常见,表明它们作用的发挥是有条件的.可能在复杂寡聚结构的情况下,如有多重分支结构,双重结合位点将有利于对这类配体分子的辨识和结合,已有一系列研究报道,具有分支结构的寡糖与半乳糖分子的亲和势明显高于单价糖配体,与上述分析相一致.对这类双重位点糖结合的可能生物学意义进行了讨论.%Galectins are a protein family with diverse biological functions,which are unique in specifically recognition and binding with β-galactosides as the primary structural basis for its functional performance.So far,all structurally characterized galectins display a conservative binding mode for the β-galactoside-containing carbohydrate ligands,in which one carbohydrate recognition domain (CRD) binds only one ligand.Here a novel binding pattern unique in two carbohydrate ligands for one CRD was reported,which is observed from the structure of Gal-3 CRD complexed with glycan TFN.In this doublet binding

  12. A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.

    Science.gov (United States)

    García-Rodríguez, Luis J; De Piccoli, Giacomo; Marchesi, Vanessa; Jones, Richard C; Edmondson, Ricky D; Labib, Karim

    2015-10-15

    Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.

  13. Acute handling disturbance modulates plasma insulin-like growth factor binding proteins in rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    The effects of acute stressor exposure on proximal (growth hormone; GH) and distal (insulin-like growth factor-I; IGF-I and IGF-binding proteins) components of the somatotropic axis are poorly understood in finfish. We exposed rainbow trout (Oncorhynchus mykiss) to a 5-minute handling disturbance to...

  14. Neuroplastin-65 and a mimetic peptide derived from its homophilic binding site modulate neuritogenesis and neuronal plasticity

    DEFF Research Database (Denmark)

    Owczarek, Sylwia; Soroka, Vladislav; Kiryushko, Darya;

    2011-01-01

    on fibroblast growth factor receptor, p38 mitogen-activated protein kinase, Ca(2+) /calmodulin-dependent protein kinase, and cytoplasmic Ca(2+) concentration. In addition, we show that interference with Np65 homophilic binding by enplastin has an inhibitory effect on Np65-mediated neurite outgrowth in vitro...

  15. Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module

    DEFF Research Database (Denmark)

    Salcini, A E; Confalonieri, S; Doria, M;

    1997-01-01

    EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct...

  16. Binding of a biosynthetic intermediate to AtrA modulates the production of lidamycin by Streptomyces globisporus.

    Science.gov (United States)

    Li, Xingxing; Yu, Tengfei; He, Qing; McDowall, Kenneth J; Jiang, Bingya; Jiang, Zhibo; Wu, Linzhuan; Li, Guangwei; Li, Qinglian; Wang, Songmei; Shi, Yuanyuan; Wang, Lifei; Hong, Bin

    2015-06-01

    The control of secondary production in streptomycetes involves the funneling of environmental and physiological signals to the cluster-situated (transcriptional) regulators (CSRs) of the biosynthetic genes. For some systems, the binding of biosynthetic products to the CSR has been shown to provide negative feedback. Here we show for the production of lidamycin (C-1027), a clinically relevant antitumor agent, by Streptomyces globisporus that negative feedback can extend to a point higher in the regulatory cascade. We show that the DNA-binding activity of the S. globisporus orthologue of AtrA, which was initially described as a transcriptional activator of actinorhodin biosynthesis in S. coelicolor, is inhibited by the binding of heptaene, a biosynthetic intermediate of lidamycin. Additional experiments described here show that S. globisporus AtrA binds in vivo as well as in vitro to the promoter region of the gene encoding SgcR1, one of the CSRs of lidamycin production. The feedback to the pleiotropic regulator AtrA is likely to provide a mechanism for coordinating the production of lidamycin with that of other secondary metabolites. The activity of AtrA is also regulated by actinorhodin. As AtrA is evolutionarily conserved, negative feedback of the type described here may be widespread within the streptomycetes.

  17. PACSINs bind to the TRPV4 cation channel. PACSIN 3 modulates the subcellular localization of TRPV4.

    NARCIS (Netherlands)

    Cuajungco, M.P.; Grimm, C.; Oshima, K.; D'hoedt, D.; Nilius, B.; Mensenkamp, A.R.; Bindels, R.J.M.; Plomann, M.; Heller, S.

    2006-01-01

    TRPV4 is a cation channel that responds to a variety of stimuli including mechanical forces, temperature, and ligand binding. We set out to identify TRPV4-interacting proteins by performing yeast two-hybrid screens, and we isolated with the avian TRPV4 amino terminus the chicken orthologues of mamma

  18. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation.

    Science.gov (United States)

    Cockburn, Darrell; Nielsen, Morten M; Christiansen, Camilla; Andersen, Joakim M; Rannes, Julie B; Blennow, Andreas; Svensson, Birte

    2015-04-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

  19. Humanized mAb H22 binds the human high affinity Fc receptor for IgG (FcgammaRI), blocks phagocytosis, and modulates receptor expression.

    Science.gov (United States)

    Wallace, P K; Keler, T; Coleman, K; Fisher, J; Vitale, L; Graziano, R F; Guyre, P M; Fanger, M W

    1997-10-01

    About 10-15% of patients with immune thrombocytopenic purpura (ITP) cannot be controlled by corticosteroid therapy and splenectomy. For these patients treatment with high-dose IVIgG induces partial or complete responses. The clinical benefits of IVIgG could be due to blockade of Fc receptors for IgG (FcgammaR), because several model systems clearly show that functional FcgammaR are essential for establishment of ITP and related diseases. However, the specific contributions of the three individual classes of FcgammaR remain to be more completely defined. Recently monoclonal antibody (mAb) H22, which recognizes an epitope on FcgammaRI (CD64) outside the ligand binding domain, was humanized by grafting its complementarity determining regions onto human IgG1 constant domains. Because FcgammaRI has a high affinity for human IgG1 antibodies, we predicted mAb H22 would also bind to FcgammaRI through its Fc domain and block FcgammaRI-mediated phagocytosis. These studies demonstrate that mAb H22 blocked phagocytosis of opsonized red blood cells 1000 times more effectively than an irrelevant IgG. Moreover, cross-linking FcgammaRI with mAb H22 rapidly down-modulated FcgammaRI expression on monocytes without affecting other surface antigens. We conclude that because mAb H22 is a humanized mAb that blocks the FcgammaRI ligand binding domain and down-modulates FcgammaRI expression, it is a particularly good candidate for evaluating the role of FcgammaRI in patients with ITP.

  20. Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

    Energy Technology Data Exchange (ETDEWEB)

    Weidmann, Chase A.; Qiu, Chen; Arvola, René M.; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T.; Tanaka Hall, Traci M.; Goldstrohm, Aaron C.

    2016-08-02

    Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation byDrosophilaPumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulatedin vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

  1. Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding.

    Science.gov (United States)

    Hadži-Tašković Šukalović, Vesna; Vuletić, Mirjana; Marković, Ksenija; Cvetić Antić, Tijana; Vučinić, Željko

    2015-01-01

    Comparative biochemical characterization of class III peroxidase activity tightly bound to the cell walls of maize roots was performed. Ionically bound proteins were solubilized from isolated walls by salt washing, and the remaining covalently bound peroxidases were released, either by enzymatic digestion or by a novel alkaline extraction procedure that released covalently bound alkali-resistant peroxidase enzyme. Solubilized fractions, as well as the salt-washed cell wall fragments containing covalently bound proteins, were analyzed for peroxidase activity. Peroxidative and oxidative activities indicated that peroxidase enzymes were predominately associated with walls by ionic interactions, and this fraction differs from the covalently bound one according to molecular weight, isozyme patterns, and biochemical parameters. The effect of covalent binding was evaluated by comparison of the catalytic properties of the enzyme bound to the salt-washed cell wall fragments with the corresponding solubilized and released enzyme. Higher thermal stability, improved resistance to KCN, increased susceptibility to H2O2, stimulated capacity of wall-bound enzyme to oxidize indole-3-acetic acid (IAA) as well as the difference in kinetic parameters between free and bound enzymes point to conformational changes due to covalent binding. Differences in biochemical properties of ionically and covalently bound peroxidases, as well as the modulation of the enzyme properties as a result of covalent binding to the walls, indicate that these two fractions of apoplastic peroxidases play different roles.

  2. Macro Domain from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Is an Efficient ADP-ribose Binding Module: CRYSTAL STRUCTURE AND BIOCHEMICAL STUDIES.

    Science.gov (United States)

    Cho, Chao-Cheng; Lin, Meng-Hsuan; Chuang, Chien-Ying; Hsu, Chun-Hua

    2016-03-01

    The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) encodes the conserved macro domain within non-structural protein 3. However, the precise biochemical function and structure of the macro domain is unclear. Using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the MERS-CoV macro domain as a more efficient adenosine diphosphate (ADP)-ribose binding module than macro domains from other CoVs. Furthermore, the crystal structure of the MERS-CoV macro domain was determined at 1.43-Å resolution in complex with ADP-ribose. Comparison of macro domains from MERS-CoV and other human CoVs revealed structural differences in the α1 helix alters how the conserved Asp-20 interacts with ADP-ribose and may explain the efficient binding of the MERS-CoV macro domain to ADP-ribose. This study provides structural and biophysical bases to further evaluate the role of the MERS-CoV macro domain in the host response via ADP-ribose binding but also as a potential target for drug design.

  3. Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein.

    Science.gov (United States)

    Kuan, Yen-Chou; Hashidume, Tsutomu; Shibata, Takahiro; Uchida, Koji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2017-02-17

    Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.

  4. Synthesis of carbohydrate-based surfactants

    Energy Technology Data Exchange (ETDEWEB)

    Pemberton, Jeanne E.; Polt, Robin L.; Maier, Raina M.

    2016-11-22

    The present invention provides carbohydrate-based surfactants and methods for producing the same. Methods for producing carbohydrate-based surfactants include using a glycosylation promoter to link a carbohydrate or its derivative to a hydrophobic compound.

  5. Controlled Modulation of Serum Protein Binding and Biodistribution of Asymmetric Cyanine Dyes by Variation of the Number of Sulfonate Groups

    Directory of Open Access Journals (Sweden)

    Franziska M. Hamann

    2011-07-01

    Full Text Available To assess the suitability of asymmetric cyanine dyes for in vivo fluoro-optical molecular imaging, a comprehensive study on the influence of the number of negatively charged sulfonate groups governing the hydrophilicity of the DY-67x family of asymmetric cyanines was performed. Special attention was devoted to the plasma protein binding capacity and related pharmacokinetic properties. Four members of the DY-67x cyanine family composed of the same main chromophore, but substituted with a sequentially increasing number of sulfonate groups (n = 1−4; DY-675, DY-676, DY-677, DY-678, respectively, were incubated with plasma proteins dissolved in phosphate-buffered saline. Protein binding was assessed by absorption spectroscopy, gel electrophoresis, ultrafiltration, and dialysis. Distribution of dye in organs was studied by intraveneous injection of 62 nmol dye/kg body weight into mice (n = 12; up to 180 minutes postinjection using whole-body near-infrared fluorescence imaging. Spectroscopic studies, gel electrophoresis, and dialysis demonstrated reduced protein binding with increasing number of sulfonate groups. The bovine serum albumin binding constant of the most hydrophobic dye, DY-675, is 18 times higher than that of the most hydrophilic fluorophore, DY-678. In vivo biodistribution analysis underlined a considerable influence of dye hydrophilicity on biodistribution and excretion pathways, with the more hydrophobic dyes, DY-675 and DY-676, accumulating in the liver, followed by strong fluorescence signals in bile and gut owing to accumulation in feces and comparatively hydrophilic DY-678-COOH accumulating in the bladder. Our results demonstrate the possibility of selectively controlling dye-protein interactions and, thus, biodistribution and excretion pathways via proper choice of the fluorophore's substitution pattern. This underlines the importance of structure-property relationships for fluorescent labels. Moreover, our data could provide the

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

    Science.gov (United States)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin; Korotchenko, Svetlana; Lievens, Patricia Marie-Jeanne; Berezin, Vladimir; Dityatev, Alexander

    2015-12-01

    Our previous studies revealed that L-type voltage-dependent Ca(2+) channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular domains of Cav1.2α1 to identify putative binding sites between the channel and hyaluronic acid or another class of polyanionic glycans, such as heparin/heparan sulfates. None of the tested peptides showed detectable interaction with hyaluronic acid, but two peptides derived from the first pore-forming domain of Cav1.2α1 subunit bound to heparin. At 25 °C the binding of the peptide P7 (MGKMHKTCYN) was at ~50 μM, and that of the peptide P8 (GHGRQCQNGTVCKPGWDGPKHG) was at ~21 μM. The Cav1.2α1 first pore forming segment that contained both peptides maintained a high affinity for heparin (~23 μM), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin-agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity.

  7. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1

    Science.gov (United States)

    El-Diwany, Ramy; Mankowski, Madeleine C.; Wasilewski, Lisa N.; Brady, Jillian K.; Snider, Anna E.; Osburn, William O.; Murrell, Ben; Ray, Stuart C.

    2017-01-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. PMID:28235087

  8. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1.

    Science.gov (United States)

    El-Diwany, Ramy; Cohen, Valerie J; Mankowski, Madeleine C; Wasilewski, Lisa N; Brady, Jillian K; Snider, Anna E; Osburn, William O; Murrell, Ben; Ray, Stuart C; Bailey, Justin R

    2017-02-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.

  9. Impact of dietary polyphenols on carbohydrate metabolism.

    Science.gov (United States)

    Hanhineva, Kati; Törrönen, Riitta; Bondia-Pons, Isabel; Pekkinen, Jenna; Kolehmainen, Marjukka; Mykkänen, Hannu; Poutanen, Kaisa

    2010-03-31

    Polyphenols, including flavonoids, phenolic acids, proanthocyanidins and resveratrol, are a large and heterogeneous group of phytochemicals in plant-based foods, such as tea, coffee, wine, cocoa, cereal grains, soy, fruits and berries. Growing evidence indicates that various dietary polyphenols may influence carbohydrate metabolism at many levels. In animal models and a limited number of human studies carried out so far, polyphenols and foods or beverages rich in polyphenols have attenuated postprandial glycemic responses and fasting hyperglycemia, and improved acute insulin secretion and insulin sensitivity. The possible mechanisms include inhibition of carbohydrate digestion and glucose absorption in the intestine, stimulation of insulin secretion from the pancreatic beta-cells, modulation of glucose release from the liver, activation of insulin receptors and glucose uptake in the insulin-sensitive tissues, and modulation of intracellular signalling pathways and gene expression. The positive effects of polyphenols on glucose homeostasis observed in a large number of in vitro and animal models are supported by epidemiological evidence on polyphenol-rich diets. To confirm the implications of polyphenol consumption for prevention of insulin resistance, metabolic syndrome and eventually type 2 diabetes, human trials with well-defined diets, controlled study designs and clinically relevant end-points together with holistic approaches e.g., systems biology profiling technologies are needed.

  10. Impact of Dietary Polyphenols on Carbohydrate Metabolism

    Directory of Open Access Journals (Sweden)

    Kati Hanhineva

    2010-03-01

    Full Text Available Polyphenols, including flavonoids, phenolic acids, proanthocyanidins and resveratrol, are a large and heterogeneous group of phytochemicals in plant-based foods, such as tea, coffee, wine, cocoa, cereal grains, soy, fruits and berries. Growing evidence indicates that various dietary polyphenols may influence carbohydrate metabolism at many levels. In animal models and a limited number of human studies carried out so far, polyphenols and foods or beverages rich in polyphenols have attenuated postprandial glycemic responses and fasting hyperglycemia, and improved acute insulin secretion and insulin sensitivity. The possible mechanisms include inhibition of carbohydrate digestion and glucose absorption in the intestine, stimulation of insulin secretion from the pancreatic b-cells, modulation of glucose release from the liver, activation of insulin receptors and glucose uptake in the insulin-sensitive tissues, and modulation of intracellular signalling pathways and gene expression. The positive effects of polyphenols on glucose homeostasis observed in a large number of in vitro and animal models are supported by epidemiological evidence on polyphenol-rich diets. To confirm the implications of polyphenol consumption for prevention of insulin resistance, metabolic syndrome and eventually type 2 diabetes, human trials with well-defined diets, controlled study designs and clinically relevant end-points together with holistic approaches e.g., systems biology profiling technologies are needed.

  11. Decarbonylation and dehydrogenation of carbohydrates

    Science.gov (United States)

    Andrews, Mark A.; Klaeren, Stephen A.

    1991-01-01

    Carbohydrates, especially aldose or ketose sugars, including those whose carbonyl group is masked by hemi-acetal or hemi-ketal formation, are decarbonylated by heating the feed carbohydrate together with a transition metal complex in a suitable solvent. Also, primary alcohols, including sugar alditols are simultaneously dehydrogenated and decarbonylated by heating a mixture of rhodium and ruthenium complexes and the alcohol and optionally a hydrogen acceptor in an acceptable solvent. Such defarbonylation and/or dehydrogenation of sugars provides a convenient procedure for the synthesis of certain carbohydrates and may provide a means for the conversion of biomass into useful products.

  12. The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

    DEFF Research Database (Denmark)

    Morrill, Johan; Kulcinskaja, Evelina; Sulewska, Anna Maria;

    2015-01-01

    β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here....... Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme. This is the first example of a low-affinity mannan-specific CBM, which forms a subfamily of CBM10 together with close homologs...... by an exceptionally low Km and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules. These features highlight fine tuning of catalytic and binding...

  13. Chemokine binding protein M3 of murine gammaherpesvirus 68 modulates the host response to infection in a natural host.

    Directory of Open Access Journals (Sweden)

    David J Hughes

    2011-03-01

    Full Text Available Murine γ-herpesvirus 68 (MHV-68 infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus. Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology.

  14. Binding of Candida albicans to Human CEACAM1 and CEACAM6 Modulates the Inflammatory Response of Intestinal Epithelial Cells

    Science.gov (United States)

    Müller, Mario M.; Schäfer, Miriam R.; Clauder, Ann-Katrin; Feer, Sabina; Heyl, Kerstin A.; Stock, Magdalena; Klassert, Tilman E.; Zipfel, Peter F.; Singer, Bernhard B.

    2017-01-01

    ABSTRACT Candida albicans colonizes human mucosa, including the gastrointestinal tract, as a commensal. In immunocompromised patients, C. albicans can breach the intestinal epithelial barrier and cause fatal invasive infections. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM5 (CEA), and CEACAM6 (CD66c) are immunomodulatory receptors expressed on human mucosa and are recruited by bacterial and viral pathogens. Here we show for the first time that a fungal pathogen (i.e., C. albicans) also binds directly to the extracellular domain of human CEACAM1, CEACAM3, CEACAM5, and CEACAM6. Binding was specific for human CEACAMs and mediated by the N-terminal IgV-like domain. In enterocytic C2BBe1 cells, C. albicans caused a transient tyrosine phosphorylation of CEACAM1 and induced higher expression of membrane-bound CEACAM1 and soluble CEACAM6. Lack of the CEACAM1 receptor after short hairpin RNA (shRNA) knockdown abolished CXCL8 (interleukin-8) secretion by C2BBe1 cells in response to C. albicans. In CEACAM1-competent cells, the addition of recombinant soluble CEACAM6 reduced the C. albicans-induced CXCL8 secretion. PMID:28292985

  15. Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

    Science.gov (United States)

    Hughes, David J.; Kipar, Anja; Leeming, Gail H.; Bennett, Elaine; Howarth, Deborah; Cummerson, Joanne A.; Papoula-Pereira, Rita; Flanagan, Brian F.; Sample, Jeffery T.; Stewart, James P.

    2011-01-01

    Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology. PMID:21445235

  16. Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation.

    Science.gov (United States)

    Watanabe, Hitomi; Takeo, Toru; Tojo, Hiromasa; Sakoh, Kazuhito; Berger, Thorsten; Nakagata, Naomi; Mak, Tak W; Kondoh, Gen

    2014-05-01

    Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

  17. Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation.

    Science.gov (United States)

    Feng, Jing; Yang, Weishan; Xie, Zili; Xiang, Fang; Cao, Zhijian; Li, Wenxin; Hu, Hongzhen; Chen, Zongyun; Wu, Yingliang

    2015-06-19

    Among the three extracellular domains of the tetrameric voltage-gated K(+) (Kv) channels consisting of six membrane-spanning helical segments named S1-S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2-4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.

  18. Neoglycoproteins as carbohydrate antigens: synthesis, analysis, and polyclonal antibody response.

    Science.gov (United States)

    Kerékgyártó, Márta; Fekete, Anikó; Szurmai, Zoltán; Kerékgyártó, János; Takács, László; Kurucz, István; Guttman, András

    2013-08-01

    The analysis and polyclonal antibody response for newly synthesized maltose-BSA conjugate neoglycoproteins is described. In this first proof of concept study, a simple carbohydrate antigen, maltose, was linked to BSA by reductive amination. An aglycone spacer was utilized to conserve the intact annular maltose structure and to promote the accessibility of the carbohydrate immunogen hapten during immunization. The neoglycoproteins were investigated by CGE and the number of conjugated maltose residues was determined by MALDI-TOF MS. The neoglycoproteins were then evaluated by immunization of BALB/c mice and the polyclonal antibody response was tested by ELISA as evidence for the presence of sugar-containing epitope-specific antibodies. Selective antibody binding was demonstrated to the synthesized neoglycoproteins with different (low and high) glycosylation degrees suggesting the possible use of this approach to generate antibodies. Moreover, the polyclonal antibody response was not inhibited by maltose or other simple carbohydrates to confirm presence of the neoglycoprotein-specific antibodies.

  19. Kr-pok increases FASN expression by modulating the DNA binding of SREBP-1c and Sp1 at the proximal promoter.

    Science.gov (United States)

    Jeon, Bu-Nam; Kim, Yeon-Sook; Choi, Won-Il; Koh, Dong-In; Kim, Min-Kyeong; Yoon, Jae-Hyeon; Kim, Min-Young; Hur, Benjamin; Paik, Philip Dong-Hyun; Hur, Man-Wook

    2012-04-01

    Kr-pok (kidney cancer-related POZ domain and Krüppel-like protein) is a new proto-oncogenic POZ-domain transcription factor. Fatty acid synthase gene (FASN) encodes one of the key enzymes in fatty acids synthesis and is the only enzyme that synthesizes fatty acids in cancer cells. Sp1 and SREBP-1c are the two major transcription activators of FASN. We investigated whether Kr-pok modulates transcription of the FASN. FASN expression is significantly decreased in Kr-pok knockout murine embryonic fibroblasts. Coimmunoprecipitation, GST fusion protein pull-down, and immunocytochemistry assays show that the zinc-finger domain of Kr-pok interacts directly with the bZIP DNA binding domain of SREBP-1. Electrophoretic mobility shift assay, oligonucleotide pull-down, and chromatin immunoprecipitation assays showed that Kr-pok changes the transcription factor binding dynamics of Sp1 and SREBP-1c to the SRE/E-box elements of the proximal promoter. We found that Kr-pok expression increased during 3T3-L1 preadipocyte differentiation and that FASN expression is decreased by the knockdown of Kr-pok. Kr-pok facilitates the SREBP-1c-mediated preadipocyte differentiation and/or fatty acid synthesis. Kr-pok may act as an important regulator of fatty acid synthesis and may induce rapid cancer cell proliferation by increasing palmitate synthesis.

  20. PARP1 promotes gene expression at the post-transcriptional level by modulating the RNA-binding protein HuR

    Science.gov (United States)

    Ke, Yueshuang; Han, Yanlong; Guo, Xiaolan; Wen, Jitao; Wang, Ke; Jiang, Xue; Tian, Xue; Ba, Xueqing; Boldogh, Istvan; Zeng, Xianlu

    2017-01-01

    Poly(ADP-ribosyl)ation (PARylation) is mainly catalysed by poly-ADP-ribose polymerase 1 (PARP1), whose role in gene transcription modulation has been well established. Here we show that, in response to LPS exposure, PARP1 interacts with the adenylateuridylate-rich element-binding protein embryonic lethal abnormal vision-like 1 (Elavl1)/human antigen R (HuR), resulting in its PARylation, primarily at site D226. PARP inhibition and the D226 mutation impair HuR's PARylation, nucleocytoplasmic shuttling and mRNA binding. Increases in mRNA level or stability of pro-inflammatory cytokines/chemokines are abolished by PARP1 ablation or inhibition, or blocked in D226A HuR-expressing cells. The present study demonstrates a mechanism to regulate gene expression at the post-transcriptional level, and suggests that blocking the interaction of PARP1 with HuR could be a strategy to treat inflammation-related diseases that involve increased mRNA stability. PMID:28272405

  1. White spot syndrome virus IE1 and WSV056 modulate the G1/S transition by binding to the host retinoblastoma protein.

    Science.gov (United States)

    Ran, Xiaozhuo; Bian, Xiaofang; Ji, Yongchang; Yan, Xiumin; Yang, Feng; Li, Fang

    2013-12-01

    DNA viruses often target cellular proteins to modulate host cell cycles and facilitate viral genome replication. However, whether proliferation of white spot syndrome virus (WSSV) requires regulation of the host cell cycle remains unclear. In the present study, we show that two WSSV paralogs, IE1 and WSV056, can interact with Litopenaeus vannamei retinoblastoma (Rb)-like protein (lv-RBL) through the conserved LxCxE motif. Further investigation revealed that IE1 and WSV056 could also bind to Drosophila retinoblastoma family protein 1 (RBF1) in a manner similar to how they bind to lv-RBL. Using the Drosophila RBF-E2F pathway as a model system, we demonstrated that both IE1 and WSV056 could sequester RBF1 from Drosophila E2F transcription factor 1 (E2F1) and subsequently activate E2F1 to stimulate the G1/S transition. Our findings provide the first evidence that WSSV may regulate cell cycle progression by targeting the Rb-E2F pathway.

  2. Transition metals in carbohydrate chemistry

    DEFF Research Database (Denmark)

    Madsen, Robert

    1997-01-01

    This review describes the application of transition metal mediated reactions in carbohydrate synthesis. The different metal mediated transformations are divided into reaction types and illustrated by various examples on monosaccharide derivatives. Carbon-carbon bond forming reactions are further ...

  3. Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat

    Directory of Open Access Journals (Sweden)

    Muller Sylviane

    2008-07-01

    Full Text Available Abstract Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. Conclusion Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

  4. ERα dimerization: a key factor for the weak estrogenic activity of an ERα modulator unable to compete with estradiol in binding assays.

    Science.gov (United States)

    Leclercq, Guy; Laïos, Ioanna; Elie-Caille, Céline; Leiber, Denis; Laurent, Guy; Lesniewska, Eric; Tanfin, Zahra; Jacquot, Yves

    2017-04-01

    Estrothiazine (ESTZ) is a weak estrogen sharing structural similarities with coumestrol. ESTZ failed to compete with [(3)H]17β-estradiol ([(3)H]17β-E2) for binding to the estrogen receptor α (ERα), questioning its ability to interact with the receptor. However, detection by atomic force spectroscopy (AFS) of an ESTZ-induced ERα dimerization has eliminated any remaining doubts. The effect of the compound on the proliferation of ERα-positive and negative breast cancer cells confirmed the requirement of the receptor. The efficiency of ESTZ in MCF-7 cells was weak without any potency to modify the proliferation profile of estradiol and coumestrol. Growth enhancement was associated with a proteasomal degradation of ERα without substantial recruitment of LxxLL coactivators. This may be related to an unusual delay between the acquisition by the receptor of an ERE-binding capacity and the subsequent estrogen-dependent transcription. A complementary ability to enhance TPA-induced AP-1 transcription was observed, even at concentrations insufficient to activate the ERα, suggesting a partly independent mechanism. ESTZ also rapidly and transiently activated ERK1/2 likely through membrane estrogenic pathways provoking a reorganization of the actin network. Finally, the systematic absence of biological responses with an ESTZ derivative unable to induce ERα dimerization stresses the importance of this step in the action of the compound, as reported for conventional estrogens. In view of the existence of many other ERα modulators (endocrine disruptors such as, for example, pesticides, environmental contaminants or phytoestrogens) with extremely weak or similar apparent lack of binding ability, our work may appear as pilot investigation for assessing their mechanism of action.

  5. Estrogen modulates NFκB signaling by enhancing IκBα levels and blocking p65 binding at the promoters of inflammatory genes via estrogen receptor-β.

    Directory of Open Access Journals (Sweden)

    Dongqi Xing

    Full Text Available BACKGROUND: NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17β-estradiol, E2 inhibits expression of these genes in an estrogen receptor (ER-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF-α treated rat aortic smooth muscle cells (RASMCs. This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10-30 min, followed by dramatic increases in IκBα mRNA and protein synthesis (40-60 min. E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP-1 and cytokine-induced neutrophil chemoattractant (CINC-2β genes. ChIP analyses also demonstrated that ERβ can be recruited to the promoters of MCP-1 and CINC-2β during co-treatment with TNF-α and E2. CONCLUSIONS: These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.

  6. Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening.

    Science.gov (United States)

    El-Hawiet, Amr; Shoemaker, Glen K; Daneshfar, Rambod; Kitova, Elena N; Klassen, John S

    2012-01-03

    Applications of a catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10(3) to 10(6) M(-1) range. Ligands with moderate affinity (10(4) to 10(6) M(-1)) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 μM each). Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (~10(3) M(-1)) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of ~10 μM each). However, identification of specific interactions required the use of the reference protein method to correct the mass spectrum for the occurrence of nonspecific carbohydrate-protein binding during the ESI process. The release of the carbohydrate ligands, as ions, was successfully demonstrated using collision-induced dissociation performed on the deprotonated ions of the protein-carbohydrate complexes. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.

  7. Carbohydrate drugs: current status and development prospect.

    Science.gov (United States)

    Zhang, Yan; Wang, Fengshan

    2015-04-01

    In recent years, there has been a great effort devoted to the investigation of the roles of carbohydrates in various essential biological processes and the development of carbohydrates to therapeutic drugs. This review summarizes the carbohydrate drugs which have been recorded in several pharmacopoeias, marketed, and under development. A prospect of the future development of carbohydrate drugs is discussed as well.

  8. Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

    Energy Technology Data Exchange (ETDEWEB)

    2012-06-01

    Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

  9. Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose.

    Directory of Open Access Journals (Sweden)

    Ting-Ying Jiang

    Full Text Available The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21 members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

  10. Entirely Carbohydrate-Based Vaccines: An Emerging Field for Specific and Selective Immune Responses

    Directory of Open Access Journals (Sweden)

    Sharmeen Nishat

    2016-05-01

    Full Text Available Carbohydrates are regarded as promising targets for vaccine development against infectious disease because cell surface glycans on many infectious agents are attributed to playing an important role in pathogenesis. In addition, oncogenic transformation of normal cells, in many cases, is associated with aberrant glycosylation of the cell surface glycan generating tumor associated carbohydrate antigens (TACAs. Technological advances in glycobiology have added a new dimension to immunotherapy when considering carbohydrates as key targets in developing safe and effective vaccines to combat cancer, bacterial infections, viral infections, etc. Many consider effective vaccines induce T-cell dependent immunity with satisfactory levels of immunological memory that preclude recurrence. Unfortunately, carbohydrates alone are poorly immunogenic as they do not bind strongly to the MHCII complex and thus fail to elicit T-cell immunity. To increase immunogenicity, carbohydrates have been conjugated to carrier proteins, which sometimes can impede carbohydrate specific immunity as peptide-based immune responses can negate antibodies directed at the targeted carbohydrate antigens. To overcome many challenges in using carbohydrate-based vaccine design and development approaches targeting cancer and other diseases, zwitterionic polysaccharides (ZPSs, isolated from the capsule of commensal anaerobic bacteria, will be discussed as promising carriers of carbohydrate antigens to achieve desired immunological responses.

  11. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    Science.gov (United States)

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  12. Telomere-binding protein TPP1 modulates telomere homeostasis and confers radioresistance to human colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Lei Yang

    Full Text Available BACKGROUND: Radiotherapy is one of the major therapeutic strategies in cancer treatment. The telomere-binding protein TPP1 is an important component of the shelterin complex at mammalian telomeres. Our previous reports showed that TPP1 expression was elevated in radioresistant cells, but the exact effects and mechanisms of TPP1 on radiosensitivity is unclear. PRINCIPAL FINDINGS: In this study, we found that elevated TPP1 expression significantly correlated with radioresistance and longer telomere length in human colorectal cancer cell lines. Moreover, TPP1 overexpression showed lengthened telomere length and a significant decrease of radiosensitivity to X-rays. TPP1 mediated radioresistance was correlated with a decreased apoptosis rate after IR exposure. Furthermore, TPP1 overexpression showed prolonged G2/M arrest mediated by ATM/ATR-Chk1 signal pathway after IR exposure. Moreover, TPP1 overexpression accelerated the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation. CONCLUSIONS: We demonstrated that elevated expressions of TPP1 in human colorectal cancer cells could protect telomere from DNA damage and confer radioresistance. These results suggested that TPP1 may be a potential target in the radiotherapy of colorectal cancer.

  13. Binding of PAI-1 to endothelial cells stimulated by thymosin beta4 and modulation of their fibrinolytic potential.

    Science.gov (United States)

    Boncela, Joanna; Smolarczyk, Katarzyna; Wyroba, Elzbieta; Cierniewski, Czeslaw S

    2006-01-13

    Our previous studies showed that thymosin beta4 (Tbeta4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with alpha1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tbeta4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-alpha1-acid glycoprotein (AGP) without Tbeta4 treatment. The AGP.PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tbeta4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.

  14. Carbohydrates and endothelial function: is a low-carbohydrate diet or a low-glycemic index diet favourable for vascular health?

    Science.gov (United States)

    Jovanovski, Elena; Zurbau, Andreea; Vuksan, Vladimir

    2015-04-01

    Low-carbohydrate diets have become increasingly popular in both media and clinical research settings. Although they may improve some metabolic markers, their effects on arterial function remain unclear. Endothelial dysfunction is the well-established response to cardiovascular risk factors and a pivotal feature that precedes atherosclerotic diseases. It has been demonstrated that a high carbohydrate-induced hyperglycemia and subsequent oxidative stress acutely worsen the efficacy of the endothelial vasodilatory system. Thus, in theory, a carbohydrate restricted diet may preserve the integrity of the arterial system. This review attempts to provide insight on whether low-carbohydrate diets have a favorable or detrimental impact on vascular function, or it is perhaps the quality of carbohydrate that should direct dietary recommendations. Research to date suggests that diets low in carbohydrate amount may negatively impact vascular endothelial function. Conversely, it appears that maintaining recommended carbohydrate intake with utilization of low glycemic index foods generates a more favorable vascular profile. Understanding these relationships will aid in deciphering the diverging role of modulating quantity and quality of carbohydrates on cardiovascular risk.

  15. Dopaminergic modulation of mitral cell activity in the frog olfactory bulb: a combined radioligand binding-electrophysiological study

    Energy Technology Data Exchange (ETDEWEB)

    Duchamp, A.; Moyse, E.; Delaleu, J.-C.; Coronas, V.; Duchamp-Viret, P. [Laboratoire de Physiologie Neurosensorielle, Universite Claude Bernard and CNRS, F69622 Villeurbanne (France)

    1997-04-28

    Dopamine content in the amphibian olfactory bulb is supplied by interneurons scattered among mitral cells in the external plexiform/mitral cell layer. In mammals, dopamine has been found to be involved in various aspects of bulbar information processing by influencing mitral cell odour responsiveness. Dopamine action in the bulb depends directly on the localization of its receptor targets, found to be mainly of the D{sub 2} type in mammals. The present study assessed, in the frog, both the anatomical localization of D{sub 2}-like, radioligand-labelled receptors of dopamine and the in vivo action of dopamine on unitary mitral cell activity in response to odours delivered over a wide range of concentrations. The [{sup 125}I]iodosulpride-labelled D{sub 2} binding sites were visualized on frozen sagittal sections of frog brains by film radioautography. The sites were found to be restricted to the external plexiform/mitral cell layer; other layers of the olfactory bulb were devoid of specific labelling. Electrophysiological recordings of mitral unit activity revealed that dopamine or its agonist apomorphine induced a drastic reduction of spontaneous firing rate of mitral cells in most cases without altering odour intensity coding properties of these cells. Moreover, pre-treatment with the D{sub 2} antagonist eticlopride blocked the dopamine-induced reduction of mitral cell spontaneous activity.In the frog olfactory bulb, both anatomical localization of D{sub 2}-like receptors and functional data on dopamine involvement in information processing differ from those reported in mammals. This suggests a phylogenetic evolution of dopamine action in the olfactory bulb. In the frog, anatomical data perfectly corroborate electrophysiological results, together strongly suggesting a direct action of dopamine on mitral cells. In a physiologically operating system, such an action would result in a global improvement of signal-to-noise ratio. (Copyright (c) 1997 Elsevier Science B

  16. Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high...

  17. von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII.

    Science.gov (United States)

    Sorvillo, Nicoletta; Hartholt, Robin B; Bloem, Esther; Sedek, Magdalena; ten Brinke, Anja; van der Zwaan, Carmen; van Alphen, Floris P; Meijer, Alexander B; Voorberg, Jan

    2016-03-01

    It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II.

  18. Gender Differences in Carbohydrate Metabolism and Carbohydrate Loading

    Directory of Open Access Journals (Sweden)

    Willoughby Darryn

    2006-06-01

    Full Text Available Abstract Prior to endurance competition, many endurance athletes participate in a carbohydrate loading regimen in order to help delay the onset of fatigue. The "classic" regimen generally includes an intense glycogen depleting training period of approximately two days followed by a glycogen loading period for 3–4 days, ingesting approximately 60–70% of total energy intake as carbohydrates, while the newer method does not consist of an intense glycogen depletion protocol. However, recent evidence has indicated that glycogen loading does not occur in the same manner for males and females, thus affecting performance. The scope of this literature review will include a brief description of the role of estradiol in relation to metabolism and gender differences seen in carbohydrate metabolism and loading.

  19. Maximizing Immune Response to Carbohydrate Antigens on Breast Tumors

    Science.gov (United States)

    2005-08-01

    Emmons, Ph.D. CONTRACTING ORGANIZATION: University of Arkansas for Medical Sciences Little Rock, Arkansas 72205 REPORT DATE: August 2005 TYPE OF REPORT...SUBTITLE 5a. CONTRACT NUMBER Maximizing Immune Response to Carbohydrate Antigens on Breast Tumors 5b. GRANT NUMBER DAMD17-01-1-0366 5c. PROGRAM...binding affinities of peptide and carbohyd- Hollingsworth, M. A. 1997. Oligosaccharides expressed on MUCl rate with I-A’ will be illuminating. However

  20. Glucoamylase starch-binding domain of Aspergillus niger B1: molecular cloning and functional characterization.

    Science.gov (United States)

    Paldi, Tzur; Levy, Ilan; Shoseyov, Oded

    2003-01-01

    Carbohydrate-binding modules (CBMs) are protein domains located within a carbohydrate-active enzyme, with a discrete fold that can be separated from the catalytic domain. Starch-binding domains (SBDs) are CBMs that are usually found at the C-terminus in many amylolytic enzymes. The SBD from Aspergillus niger B1 (CMI CC 324262) was cloned and expressed in Escherichia coli as an independent domain and the recombinant protein was purified on starch. The A. niger B1 SBD was found to be similar to SBD from A. kawachii, A. niger var. awamori and A. shirusami (95-96% identity) and was classified as a member of the CBM family 20. Characterization of SBD binding to starch indicated that it is essentially irreversible and that its affinity to cationic or anionic starch, as well as to potato or corn starch, does not differ significantly. These observations indicate that the fundamental binding area on these starches is essentially the same. Natural and chemically modified starches are among the most useful biopolymers employed in the industry. Our study demonstrates that SBD binds effectively to both anionic and cationic starch. PMID:12646045

  1. Binding and inhibition of drug transport proteins by heparin: a potential drug transporter modulator capable of reducing multidrug resistance in human cancer cells.

    Science.gov (United States)

    Chen, Yunliang; Scully, Michael; Petralia, Gloria; Kakkar, Ajay

    2014-01-01

    A major problem in cancer treatment is the development of resistance to chemotherapeutic agents, multidrug resistance (MDR), associated with increased activity of transmembrane drug transporter proteins which impair cytotoxic treatment by rapidly removing the drugs from the targeted cells. Previously, it has been shown that heparin treatment of cancer patients undergoing chemotherapy increases survival. In order to determine whether heparin is capable reducing MDR and increasing the potency of chemotherapeutic drugs, the cytoxicity of a number of agents toward four cancer cell lines (a human enriched breast cancer stem cell line, two human breast cancer cell lines, MCF-7 and MDA-MB-231, and a human lung cancer cell line A549) was tested in the presence or absence of heparin. Results demonstrated that heparin increased the cytotoxicity of a range of chemotherapeutic agents. This effect was associated with the ability of heparin to bind to several of the drug transport proteins of the ABC and non ABC transporter systems. Among the ABC system, heparin treatment caused significant inhibition of the ATPase activity of ABCG2 and ABCC1, and of the efflux function observed as enhanced intracellular accumulation of specific substrates. Doxorubicin cytoxicity, which was enhanced by heparin treatment of MCF-7 cells, was found to be under the control of one of the major non-ABC transporter proteins, lung resistance protein (LRP). LRP was also shown to be a heparin-binding protein. These findings indicate that heparin has a potential role in the clinic as a drug transporter modulator to reduce multidrug resistance in cancer patients.

  2. A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding.

    Science.gov (United States)

    Gopalakrishnan, Suhasni; Van Emburgh, Beth O; Shan, Jixiu; Su, Zhen; Fields, C Robert; Vieweg, Johannes; Hamazaki, Takashi; Schwartz, Philip H; Terada, Naohiro; Robertson, Keith D

    2009-10-01

    DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the NH(2)-terminal regulatory domain. This variant, which we term DNMT3B3Delta5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Delta5. DNMT3B3Delta5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Delta5 versus DNMT3B3, revealing that DNMT3B3Delta5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Delta5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results show that DNMT3B3Delta5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B.

  3. Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism.

    Science.gov (United States)

    James, P L; Stewart, C E; Rotwein, P

    1996-05-01

    The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo.

  4. The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states

    Directory of Open Access Journals (Sweden)

    Tochiki Keri K

    2012-02-01

    Full Text Available Abstract Background DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2 and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated. Results Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury. Conclusion Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

  5. Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

    Science.gov (United States)

    Wang, Feng; Zha, Wan-sheng; Zhang, Jia-xiang; Li, Shu-long; Wang, Hui; Ye, Liang-ping; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2014-08-17

    Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure.

  6. Variations of Water-Soluble Carbohydrate Contents in Different Age Class Modules of Leymus chinensis Populations in Sandy and Saline-Alkaline Soil on the Songnen Plains of China

    Institute of Scientific and Technical Information of China (English)

    Xue-Mei Ding; Yun-Fei Yang

    2007-01-01

    Leymus chinensis (Trin.) Tzvel. is a rhizomatous pexennial herbage of Gramineae. Reproduction is mainly by vegetative reproduction. Tillering nodes and rhizomes of L. chinensis serve as organs for both vegetative reproduction and nutrient storage. Water-soluble carbohydrate (WSC) contents were measured in tillering nodes, nodes and internodes of rhizomes of different age classes of L. chinensis populations at three development stages, namely the dough ripe stage, the vegetative growth stage after full ripeness, and the withering stage, in two habitats:sandy soil and saline-alkaline soil. The results showed that WSC content in tillering nodes of the three age classes of L. chinensis were all markedly decreased with increasing age in both sandy soil and saline-alkaline soil. A similar trend of changes in WSC contents was observed in the nodes and internodes of rhizomes in different age classes in both habitats. The highest WSC contents were in 2-age-class nodes and internodes of rhizomes, followed by those in the 1 age class, with the lowest WSC contents found in 3-age-class nodes and internodes of rhizomes at the dough ripe and vegetative growth stages after full ripening. In turn, WSC contents decreased with increasing age at the withering stage in both habitats. The WSC content in each age class of internode was higher than that in the node of rhizome at three development stages In both habitats.

  7. Challenges with nonfiber carbohydrate methods.

    Science.gov (United States)

    Hall, M B

    2003-12-01

    Nonfiber carbohydrates (NFC) encompass a compositionally and nutritionally diverse group exclusive of those carbohydrates found in NDF. Their content in feeds has often been described as a single value estimated by difference as 100% of dry matter minus the percentages of CP, NDF (adjusted for CP in NDF), ether extract, and ash. A calculated value was used because of difficulties with assays for individual NFC, but it does not differentiate among nutritionally distinct NFC. Errors in NFC estimation can arise from not accounting for CP in NDF and when multipliers other than 6.25 are appropriate to estimate CP. Analyses that begin to distinguish among NFC are those for starch, soluble fiber (non-NDF, nonstarch polysaccharides), and low molecular weight carbohydrates (mono- and oligosaccharides). Many starch analyses quantify alpha-glucans through specific hydrolysis of alpha-(1 --> 4) and alpha-(1 --> 6) linkages in the glucan, and measurement of released glucose. Incomplete gelatinization and hydrolysis will lead to underestimation of starch content. Starch values are inflated by enzyme preparations that hydrolyze carbohydrates other than alpha-glucan, measurement of all released monosaccharides without specificity for glucose, and failure to exclude free glucose present in the unhydrolyzed sample. Soluble fiber analyses err in a fashion similar to NFC if estimation of CP requires multipliers other than 6.25, or if contaminants such as CP and starch have not been properly accounted. Depolymerization and incomplete precipitation can also decrease soluble fiber estimates. The low molecular weight carbohydrates have been defined as carbohydrates soluble in 78 to 80% ethanol, which separates them from polysaccharides. They can be measured in extracts using broad-spectrum colorimetric assays (phenol-sulfuric acid assay or reducing sugar analysis of acid hydrolyzed samples) or chromatographic methods. Limitations of the colorimetric assays include lack of differentiation

  8. 转录因子碳水化合物反应元件结合蛋白对肝脏糖脂代谢的调节作用%Regulatory role of carbohydrate response element binding protein on hepatic glycolysis and lipogenesis

    Institute of Scientific and Technical Information of China (English)

    周露婷; 章卫平

    2011-01-01

    The liver is the major site of carbohydrate metabolism and lipogenesis. Carbohydrate response element binding protein (ChREBP) is a major transcription factormediating hepatic glycolysis and lipogenesis. The heterodimer formed by ChREBP and Mlx can regulates hepatic expression of glucose-responsive genes required for glucose utilization and de novo lipogenesis. Specific inhibition of liver ChREBP in ob/ob mice can improve hepatic steatosis and insulin resistance. Understanding the roles of ChREBP in hepatic glycolysis and lipogenesis can further explain glucose-induced lipogenesis and may cast new lights on the treatment of metabolic diseases including hepatic steatosis. In this paper we introduce the molecular structure, biological function, and regulatory mechanisms of ChREBP, as well as its relationship with metabolic diseases.%肝脏是体内糖脂代谢的重要器官,转录因子碳水化合物反应元件结合蛋白(ChREBP)是调节肝脏糖酵解及脂肪合成的重要转录因子.ChREBP与Max样蛋白X以异二聚体的形式调控葡萄糖利用及转化为脂肪过程中大量基因的表达.在ob/ob小鼠肝细胞特异性敲除ChREBP基因后能明显改善其脂肪肝及胰岛素抵抗.阐明ChREBP对糖脂代谢的调控机制及其生物学功能,可进一步解释葡萄糖诱导脂肪形成的过程,并有望为脂肪肝等代谢性疾病的干预治疗提供新的思路.本文对ChREBP的结构特征、调控机制、生物学功能及其与疾病的关系等最新进展进行综述.

  9. Transcriptional regulation of the carbohydrate utilization network in Thermotoga maritima

    Directory of Open Access Journals (Sweden)

    Dmitry A Rodionov

    2013-08-01

    Full Text Available Hyperthermophilic bacteria from the Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

  10. Solvent structure improves docking prediction in lectin-carbohydrate complexes.

    Science.gov (United States)

    Gauto, Diego F; Petruk, Ariel A; Modenutti, Carlos P; Blanco, Juan I; Di Lella, Santiago; Martí, Marcelo A

    2013-02-01

    Recognition and complex formation between proteins and carbohydrates is a key issue in many important biological processes. Determination of the three-dimensional structure of such complexes is thus most relevant, but particularly challenging because of their usually low binding affinity. In silico docking methods have a long-standing tradition in predicting protein-ligand complexes, and allow a potentially fast exploration of a number of possible protein-carbohydrate complex structures. However, determining which of these predicted complexes represents the correct structure is not always straightforward. In this work, we present a modification of the scoring function provided by AutoDock4, a widely used docking software, on the basis of analysis of the solvent structure adjacent to the protein surface, as derived from molecular dynamics simulations, that allows the definition and characterization of regions with higher water occupancy than the bulk solvent, called water sites. They mimic the interaction held between the carbohydrate -OH groups and the protein. We used this information for an improved docking method in relation to its capacity to correctly predict the protein-carbohydrate complexes for a number of tested proteins, whose ligands range in size from mono- to tetrasaccharide. Our results show that the presented method significantly improves the docking predictions. The resulting solvent-structure-biased docking protocol, therefore, appears as a powerful tool for the design and optimization of development of glycomimetic drugs, while providing new insights into protein-carbohydrate interactions. Moreover, the achieved improvement also underscores the relevance of the solvent structure to the protein carbohydrate recognition process.

  11. Infiltration anesthetic lidocaine inhibits cancer cell invasion by modulating ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    Science.gov (United States)

    Mammoto, Tadanori; Higashiyama, Shigeki; Mukai, Mutsuko; Mammoto, Akiko; Ayaki, Masako; Mashimo, Takashi; Hayashi, Yukio; Kishi, Yoshihiko; Nakamura, Hiroyuki; Akedo, Hitoshi

    2002-09-01

    Although the mechanism is unknown, infiltration anesthetics are believed to have membrane-stabilizing action. We report here that such a most commonly used anesthetic, lidocaine, effectively inhibited the invasive ability of human cancer (HT1080, HOS, and RPMI-7951) cells at concentrations used in surgical operations (5-20 mM). Ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) from the cell surface plays an important role in invasion by HT1080 cells. Lidocaine reduced the invasion ability of these cells by partly inhibiting the shedding of HB-EGF from the cell surface and modulation of intracellular Ca2+ concentration contributed to this action. The anesthetic action of lidocaine (sodium channel blocking ability) did not contribute to this anti-invasive action. In addition, lidocaine (5-30 mM), infiltrated around the inoculation site, inhibited pulmonary metastases of murine osteosarcoma (LM 8) cells in vivo. These data point to previously unrecognized beneficial actions of lidocaine and suggest that lidocaine might be an ideal infiltration anesthetic for surgical cancer operations.

  12. Transduction of the light signal during complementary chromatic adaptation in the cyanobacterium Calothrix sp. PCC 7601: DNA-binding proteins and modulation by phosphorylation.

    Science.gov (United States)

    Sobczyk, A; Schyns, G; Tandeau de Marsac, N; Houmard, J

    1993-03-01

    The cyanobacterium Calothrix sp. PCC 7601 can adapt its pigment content in response to changes in the incident light wavelength. It synthesizes, as major light-harvesting pigments, either phycocyanin 2 (PC2, encoded by the cpc2 operon) under red light or phycoerythrin (PE, encoded by the cpeBA operon) under green light conditions. The last step of the signal transduction pathway is characterized by a transcriptional control of the expression of these operons. Partially purified protein extracts were used in gel retardation assays and DNase I footprinting experiments to identify the factors that interact with the promoter region of the cpeBA operon. We found that two proteins, RcaA and RcaB, only detected in extracts of cells grown under green light, behave as positive transcriptional factors for the expression of the cpeBA operon. Treatment of the fractions containing RcaA and RcaB with alkaline phosphatase prevents the binding of RcaA but not of RcaB to the cpeBA promoter region. A post-translational modification of RcaA thus modulates its affinity for DNA.

  13. An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation.

    Science.gov (United States)

    Sabbattini, Pierangela; Sjoberg, Marcela; Nikic, Svetlana; Frangini, Alberto; Holmqvist, Per-Henrik; Kunowska, Natalia; Carroll, Tom; Brookes, Emily; Arthur, Simon J; Pombo, Ana; Dillon, Niall

    2014-03-01

    Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states.

  14. Dietary carbohydrate deprivation increases 24-hour nitrogen excretion without affecting postabsorptive hepatic or whole body protein metabolism in healthy men

    NARCIS (Netherlands)

    Bisschop, PH; de Sain-van der Velden, MGM; Stellaard, F; Kuipers, F; Meijer, AJ; Sauerwein, HP; Romijn, JA

    2003-01-01

    Because insulin is an important regulator of protein metabolism, we hypothesized that physiological modulation of insulin secretion, by means of extreme variations in dietary carbohydrate content, affects postabsorptive protein metabolism. Therefore, we studied the effects of three isocaloric diets

  15. Depiction of carbohydrate-active enzyme diversity in Caldicellulosiruptor sp. F32 at the genome level reveals insights into distinct polysaccharide degradation features.

    Science.gov (United States)

    Meng, Dong-Dong; Ying, Yu; Zhang, Kun-Di; Lu, Ming; Li, Fu-Li

    2015-11-01

    Thermophilic bacterium Caldicellulosiruptor sp. F32 can utilize cellulose-, hemicellulose-containing biomass, including unpretreated wheat straw. We have conducted a bioinformatics analysis of the carbohydrate-active enzyme (CAZyme) in the genome of Caldicellulosiruptor sp. F32, which reveals a broad substrate range of the strain. Among 2285 predicted open reading frames (ORFs), 73 (3.2%) CAZyme encoding genes, including 44 glycoside hydrolases (GHs) distributing in 22 GH families, 6 carbohydrate esterases (CEs), 3 polysaccharide lyases (PLs), 21 glycosyl transferases (GTs), and 25 carbohydrate-binding modules (CBMs) were found. An in-depth bioinformatics analysis of CAZyme families that target cellulose, hemicellulose, chitin, pectin, starch, and β-1,3-1,4-glucan degradation were performed to highlight specialized polysaccharide degrading abilities of strain F32. A great number of orthologous multimodular CAZymes of Caldicellulosiruptor sp. F32 were found in other strains of genus Caldicellulosiruptor. While, a portion of the CAZymes of Caldicellulosiruptor sp. F32 showed sequence identity with proteins from strains of genus Clostridium. A thermostable β-glucosidase BlgA synergistically facilitated the enzymatic degradation of Avicel by endo-1,4-β-glucanase CelB, which indicated that the synchronous action of synergism between CAZymes enhanced the lignocellulose degradation by Caldicellulosiruptor sp. F32.

  16. Mechanistic insights into the distribution of carbohydrate clusters on cell membranes revealed by dSTORM imaging

    Science.gov (United States)

    Chen, Junling; Gao, Jing; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

    2016-07-01

    Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the carbohydrate-binding proteins (i.e., galectins) cross-linking their specific carbohydrate ligands. Our results clarify the organizational mechanism of carbohydrates on cell surfaces from their formation, stable existence and size-restriction, which promotes a better understanding of the relationship between the function and distribution of carbohydrates, as well as the structure of cell membranes.Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the

  17. Carbohydrates of human immunodeficiency virus

    DEFF Research Database (Denmark)

    Hansen, J E

    1992-01-01

    Elucidation of the mechanism by which viral infection induces the appearance of carbohydrate neoantigens is highly important. Results from such studies could be expected to be significant for a general understanding of the regulation of glycosylation, and perhaps especially important for the unde...

  18. Racemic carbohydrates - fact or fiction?

    DEFF Research Database (Denmark)

    Senning, Alexander Erich Eugen

    2007-01-01

    Chemical Abstracts Service has developed unsound practices in the naming and handling of simple carbohydrates such as aldopentoses 1, aldohexoses 2, and ketohexoses 3. Typically, the common name glucose is sometimes, inappropriately, interpreted as meaning DL-glucose DL-2d. Thus, a considerable...

  19. Carbohydrate-based immune adjuvants

    Science.gov (United States)

    Petrovsky, Nikolai; Cooper, Peter D

    2011-01-01

    The role for adjuvants in human vaccines has been a matter of vigorous scientific debate, with the field hindered by the fact that for over 80 years, aluminum salts were the only adjuvants approved for human use. To this day, alum-based adjuvants, alone or combined with additional immune activators, remain the only adjuvants approved for use in the USA. This situation has not been helped by the fact that the mechanism of action of most adjuvants has been poorly understood. A relative lack of resources and funding for adjuvant development has only helped to maintain alum’s relative monopoly. To seriously challenge alum’s supremacy a new adjuvant has many major hurdles to overcome, not least being alum’s simplicity, tolerability, safety record and minimal cost. Carbohydrate structures play critical roles in immune system function and carbohydrates also have the virtue of a strong safety and tolerability record. A number of carbohydrate compounds from plant, bacterial, yeast and synthetic sources have emerged as promising vaccine adjuvant candidates. Carbohydrates are readily biodegradable and therefore unlikely to cause problems of long-term tissue deposits seen with alum adjuvants. Above all, the Holy Grail of human adjuvant development is to identify a compound that combines potent vaccine enhancement with maximum tolerability and safety. This has proved to be a tough challenge for many adjuvant contenders. Nevertheless, carbohydrate-based compounds have many favorable properties that could place them in a unique position to challenge alum’s monopoly over human vaccine usage. PMID:21506649

  20. Interactions of carbohydrates and proteins by fluorophore-assisted carbohydrate electrophoresis

    Indian Academy of Sciences (India)

    Gang-Liang Huang; Xin-Ya Mei; Peng-George Wang

    2006-06-01

    A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions is demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE). The procedure is simple and the cost is low. The advantage of this method is that carbohydrate-protein interactions can be easily displayed by FACE, and the carbohydrates do not need to be purified.

  1. Carbohydrate modified diet & insulin sensitizers reduce body weight & modulate metabolic syndrome measures in EMPOWIR (enhance the metabolic profile of women with insulin resistance: a randomized trial of normoglycemic women with midlife weight gain.

    Directory of Open Access Journals (Sweden)

    Harriette R Mogul

    Full Text Available RATIONALE: Progressive midlife weight gain is associated with multiple adverse health outcomes and may represent an early manifestation of insulin resistance in a distinct subset of women. Emerging data implicate hyperinsulinema as a proximate cause of weight gain and support strategies that attenuate insulin secretion. OBJECTIVE: To assess a previously reported novel hypocaloric carbohydrate modified diet alone (D, and in combination with metformin (M and metformin plus low-dose rosiglitazone (MR, in diverse women with midlife weight gain (defined as >20lbs since the twenties, normal glucose tolerance, and hyperinsulinemia. PARTICIPANTS: 46 women, mean age 46.6±1.0, BMI 30.5±0.04 kg/m2, 54.5% white, 22.7% black, 15.9% Hispanic, at 2 university medical centers. METHODS: A dietary intervention designed to reduce insulin excursions was implemented in 4 weekly nutritional group workshops prior to randomization. MAIN OUTCOME MEASURE: Change in 6-month fasting insulin. Pre-specified secondary outcomes were changes in body weight, HOMA-IR, metabolic syndrome (MS measures, leptin, and adiponectin. RESULTS: Six-month fasting insulin declined significantly in the M group: 12.5 to 8.0 µU/ml, p = .026. Mean 6-month weight decreased significantly and comparably in D, M, and MR groups: 4.7, 5.4, and 5.5% (p's.049, .002, and.032. HOMA-IR decreased in M and MR groups (2.5 to 1.6 and 1.9 to 1.3, p's = .054, .013. Additional improvement in MS measures included reduced waist circumference in D and MR groups and increased HDL in the D and M groups. Notably, mean fasting leptin did not decline in a subset of subjects with weight loss (26.15±2.01 ng/ml to 25.99±2.61 ng/ml, p = .907. Adiponectin increased significantly in the MR group (11.1±1.0 to 18.5±7.4, p<.001 Study medications were well tolerated. CONCLUSIONS: These findings suggest that EMPOWIR's easily implemented dietary interventions, alone and in combination with pharmacotherapies that

  2. Analyzing radioligand binding data.

    Science.gov (United States)

    Motulsky, Harvey; Neubig, Richard

    2002-08-01

    Radioligand binding experiments are easy to perform, and provide useful data in many fields. They can be used to study receptor regulation, discover new drugs by screening for compounds that compete with high affinity for radioligand binding to a particular receptor, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling, via measurements of agonist binding and its regulation by ions, nucleotides, and other allosteric modulators. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.

  3. Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

    DEFF Research Database (Denmark)

    Helledie, T; Antonius, M; Sorensen, R V;

    2000-01-01

    Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty...

  4. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Yung-Kuo Lee

    Full Text Available Multidrug resistance (MDR, an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp, MDR-associated protein (MRP 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

  5. Double-stranded RNA-binding protein DRB3 negatively regulates anthocyanin biosynthesis by modulating PAP1 expression in Arabidopsis thaliana.

    Science.gov (United States)

    Sawano, Hikaru; Matsuzaki, Takuma; Usui, Tomoyuki; Tabara, Midori; Fukudome, Akihito; Kanaya, Akihiro; Tanoue, Daichi; Hiraguri, Akihiro; Horiguchi, Gorou; Ohtani, Misato; Demura, Taku; Kozaki, Toshinori; Ishii, Kazuo; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2017-01-01

    The model plant Arabidopsis thaliana has five double-stranded RNA-binding proteins (DRB1-DRB5), two of which, DRB1 and DRB4, are well characterized. In contrast, the functions of DRB2, DRB3 and DRB5 have yet to be elucidated. In this study, we tried to uncover their functions using drb mutants and DRB-over-expressed lines. In over-expressed lines of all five DRB genes, the over-expression of DRB2 or DRB3 (DRB2ox or DRB3ox) conferred a downward-curled leaf phenotype, but the expression profiles of ten small RNAs were similar to that of the wild-type (WT) plant. Phenotypes were examined in response to abiotic stresses. Both DRB2ox and DRB3ox plants exhibited salt-tolerance. When these plants were exposed to cold stress, drb2 and drb3 over-accumulated anthocyanin but DRB2ox and DRB3ox did not. Therefore, the over-expression of DRB2 or DRB3 had pleiotropic effects on host plants. Microarray and deep-sequencing analyses indicated that several genes encoding key enzymes for anthocyanin biosynthesis, including chalcone synthase (CHS), dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS), were down-regulated in DRB3ox plants. When DRB3ox was crossed with the pap1-D line, which is an activation-tagged transgenic line that over-expresses the key transcription factor PAP1 (Production of anthocyanin pigmentation1) for anthocyanin biosynthesis, over-expression of DRB3 suppressed the expression of PAP1, CHS, DFR and ANS genes. DRB3 negatively regulates anthocyanin biosynthesis by modulating the level of PAP1 transcript. Since two different small RNAs regulate PAP1 gene expression, a possible function of DRB3 for small RNA biogenesis is discussed.

  6. Disorders of carbohydrate digestion and absorption.

    Science.gov (United States)

    Heitlinger, L A; Lebenthal, E

    1988-04-01

    The carbohydrate malabsorptive syndromes are frequently seen by pediatricians. The congenital deficiency states are quite rare, but adult type hypolactasia and lactose intolerance following rotavirus infection are recognized with increasing frequency by primary care physicians. Therapy for these disorders involves identification of the offending carbohydrate, removal of the carbohydrate from the diet, and exclusion of other entities that may result in carbohydrate malabsorption but not respond to its removal from the diet. Prognosis for both the primary and secondary carbohydrate malabsorption syndromes is excellent. Compliance with diets for those pediatric patients who will require lifelong therapy remains problematic.

  7. Starch-binding domains in the post-genome era.

    Science.gov (United States)

    Machovic, M; Janecek, S

    2006-12-01

    Starch belongs to the most abundant biopolymers on Earth. As a source of energy, starch is degraded by a large number of various amylolytic enzymes. However, only about 10% of them are capable of binding and degrading raw starch. These enzymes usually possess a distinct sequence-structural module, the so-called starchbinding domain (SBD). In general, all carbohydrate-binding modules (CBMs) have been classified into the CBM families. In this sequence-based classification the individual types of SBDs have been placed into seven CBM families: CBM20, CBM21, CBM25, CBM26, CBM34, CBM41 and CBM45. The family CBM20, known also as a classical C-terminal SBD of microbial amylases, is the most thoroughly studied. The three-dimensional structures have already been determined by X-ray crystallography or nuclear magnetic resonance for SBDs from five CBM families (20, 25, 26, 34 and 41), and the structure of the CBM21 has been modelled. Despite differences among the amino acid sequences, the fold of a distorted beta-barrel seems to be conserved together with a similar way of substrate binding (mainly stacking interactions between aromatic residues and glucose rings). SBDs have recently been discovered in many non-amylolytic proteins. These may, for example, have regulatory functions in starch metabolism in plants or glycogen metabolism in mammals. SBDs have also found practical uses.

  8. Carbohydrate Metabolism in Submariner Personnel

    Science.gov (United States)

    1983-06-01

    metabolism the Wilkerson Point System, for glucose values, used in conjunction with patterns of insulin response described by Kraft(4) serves as the means...amount of exercise and carbohydrate metabolism characteristics occurred in both submariners and non-submariners. An inverse relationship also seems to...individuals(7). In the present study a significant negative correlation was also found between exercise vs one and two hour postprandial glucose and two hour

  9. Carbohydrates Through Animation: Preliminary Step

    OpenAIRE

    J.K. Sugai; M.S.R. Figueiredo; ANTÔNIO, R. V.; P. M. Oliveira; V.A Cardoso; Ricardo, J.; Merino, E.; L.F Figueiredo; D.N. Heidrich

    2004-01-01

    Methods of education are changing, so the educational tools must change too. The developmentof the systems of information and communication gave the opportunity to bring new technology tothe learning process. Modern education needs interactive programs that may be available to theacademic community, in order to ease the learning process and sharing of the knowledge. Then,an educational software on Carbohydrates is being developed using concept maps and FLASH-MXanimations program, and approach...

  10. Levorotatory carbohydrates and xylitol subdue Streptococcus mutans and Candida albicans adhesion and biofilm formation.

    Science.gov (United States)

    Brambilla, Eugenio; Ionescu, Andrei C; Cazzaniga, Gloria; Ottobelli, Marco; Samaranayake, Lakshman P

    2016-05-01

    Dietary carbohydrates and polyols affect the microbial colonization of oral surfaces by modulating adhesion and biofilm formation. The aim of this study was to evaluate the influence of a select group of l-carbohydrates and polyols on either Streptococcus mutans or Candida albicans adhesion and biofilm formation in vitro. S. mutans or C. albicans suspensions were inoculated on polystyrene substrata in the presence of Tryptic soy broth containing 5% of the following compounds: d-glucose, d-mannose, l-glucose, l-mannose, d- and l-glucose (raceme), d- and l-mannose (raceme), l-glucose and l-mannose, sorbitol, mannitol, and xylitol. Microbial adhesion (2 h) and biofilm formation (24 h) were evaluated using MTT-test and Scanning Electron Microscopy (SEM). Xylitol and l-carbohydrates induced the lowest adhesion and biofilm formation in both the tested species, while sorbitol and mannitol did not promote C. albicans biofilm formation. Higher adhesion and biofilm formation was noted in both organisms in the presence of d-carbohydrates relative to their l-carbohydrate counterparts. These results elucidate, hitherto undescribed, interactions of the individually tested strains with l- and d-carbohydrates, and how they impact fungal and bacterial colonization. In translational terms, our data raise the possibility of using l-form of carbohydrates and xylitol for dietary control of oral plaque biofilms.

  11. A systematic study of chemogenomics of carbohydrates.

    Science.gov (United States)

    Gu, Jiangyong; Luo, Fang; Chen, Lirong; Yuan, Gu; Xu, Xiaojie

    2014-03-04

    Chemogenomics focuses on the interactions between biologically active molecules and protein targets for drug discovery. Carbohydrates are the most abundant compounds in natural products. Compared with other drugs, the carbohydrate drugs show weaker side effects. Searching for multi-target carbohydrate drugs can be regarded as a solution to improve therapeutic efficacy and safety. In this work, we collected 60 344 carbohydrates from the Universal Natural Products Database (UNPD) and explored the chemical space of carbohydrates by principal component analysis. We found that there is a large quantity of potential lead compounds among carbohydrates. Then we explored the potential of carbohydrates in drug discovery by using a network-based multi-target computational approach. All carbohydrates were docked to 2389 target proteins. The most potential carbohydrates for drug discovery and their indications were predicted based on a docking score-weighted prediction model. We also explored the interactions between carbohydrates and target proteins to find the pathological networks, potential drug candidates and new indications.

  12. Carbohydrates

    Science.gov (United States)

    ... products, and do not help you feel as satisfied. FIBER High-fiber foods include: Whole grains, such ... ADAM Health Solutions. About MedlinePlus Site Map FAQs Customer Support Get email updates Subscribe to RSS Follow ...

  13. MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells

    Directory of Open Access Journals (Sweden)

    Yamashita Shunichi

    2011-03-01

    Full Text Available Abstract Background Esophageal squamous cell carcinoma (ESCC is often diagnosed at later stages until they are incurable. MicroRNA (miR is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC. Methods Total RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR. Results Based on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2

  14. Microfibril-associated Protein 4 Binds to Surfactant Protein A (SP-A) and Colocalizes with SP-A in the Extracellular Matrix of the Lung

    DEFF Research Database (Denmark)

    Schlosser, Anders; Thomsen, Theresa H.; Shipley, J. Michael;

    2006-01-01

    Pulmonary surfactant protein A (SP-A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro-organisms, and mediates microbial lysis and clearance. SP-A also modulates multiple immune-related functions including cytokine production and chemotaxis...... for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril-associated protein 4 (MFAP4) and SP-A. MFAP4 is a collagen-binding molecule containing a C-terminal fibrinogen-like domain and a N-terminal located integrin-binding motif. We produced recombinant MFAP4...... with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium-dependent binding between MFAP4 and human SP-A1 and SP-A2. No binding was seen to recombinant SP...

  15. Low-carbohydrate nutrition and metabolism.

    Science.gov (United States)

    Westman, Eric C; Feinman, Richard D; Mavropoulos, John C; Vernon, Mary C; Volek, Jeff S; Wortman, James A; Yancy, William S; Phinney, Stephen D

    2007-08-01

    The persistence of an epidemic of obesity and type 2 diabetes suggests that new nutritional strategies are needed if the epidemic is to be overcome. A promising nutritional approach suggested by this thematic review is carbohydrate restriction. Recent studies show that, under conditions of carbohydrate restriction, fuel sources shift from glucose and fatty acids to fatty acids and ketones, and that ad libitum-fed carbohydrate-restricted diets lead to appetite reduction, weight loss, and improvement in surrogate markers of cardiovascular disease.

  16. Digestion of carbohydrates in the pig.

    Science.gov (United States)

    Drochner, W

    1993-01-01

    A review of carbohydrate digestion in the pig is given. The cascade of digestion in the mouth, stomach, small and large intestine is described. Principles of enzymatic and fermentative digestion according to new results with fistulated animals are discussed. The efficacy and quality of fermentation in the large intestine depending on level and quality of carbohydrates in the diet are demonstrated. Some aspects of energetical efficacy of hindgut digestion are discussed. Dietetic effects of carbohydrates are described.

  17. Regioselective azidotrimethylsilylation of carbohydrates and applications thereof.

    Science.gov (United States)

    L, Mallikharjuna Rao; Yousuf, Syed Khalid; Mukherjee, Debaraj; Taneja, Subhash Chandra

    2012-12-07

    Azidotrimethylsilylation of carbohydrates (monosaccharides and disaccharides) has been achieved in high yields under Mitsunobu conditions. The azidation of carbohydrates is effected at 0 °C essentially only at the primary alcoholic position in mono, di- and triols in protected/unprotected glycosides, whereas the remaining secondary hydroxyl groups got silylated. Surprisingly, no azidation of the secondary hydroxyls was observed in all the carbohydrate substrates. Applications of the methodology for the synthesis of amino sugars, triazoles and azasugars are reported.

  18. Genome-Wide DNA Binding of GBF1 IsModulated by Its Heterodimerizing ProteinPartners, HY5 and HYH

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Dear Editor, In today's post-genomic era where direct targets of manytranscription factors have been identified, it is becomingincreasingly evident that transcriptional networks are verycomplex. Heterodimerization of transcription factors is oneof the several methods by which these complex transcrip-tional networks are formed. By heterodimerization, DNA-binding specificity and affinity, transactivation properties,and ultimately cell physiology might be altered (Naar et al.,2001). The formation of heterodimers has the potential torecognize additional binding sites and increase the rangeof DNA-binding specificity (Foster et al., 1994). Further, het-erodimerization also allows the production of new proteinconfigurations. For example, the protein STF1 from soybeancan dimerize with GBF proteins and this dimerization bringstogether the acidic region from STF1 and the proline-richregion of the GBF proteins into one binding element (Cheonget al., 1998). These results highlight the importance and/orconsequences of heterodimerization of transcription factorsat particular locus. However, to understand the complex tran-scriptional networks, it is important to investigate that howheterodimerization affects the whole-genome-wide bind-ing and transcriptional properties of a transcription factor.Here in this study, we have investigated genome-wide DNAbinding of bZIP transcription factor GBF1, and analyzed theimportance of its heterodimerization with HY5 and HYH forits genome-wide binding. We have found that GBF1 bindingsites are enriched within the 1-kb regions upstream to thetranscription start sites of target genes. Moreover, the bind-ings of GBF1 to most of its targets are largely dependent onHY5, while HYH only affects the binding of GBF1 to somespecific sites.

  19. The effects of carbohydrate variation in isocaloric diets on glycogenolysis and gluconeogenesis in healthy men

    NARCIS (Netherlands)

    Bisschop, PH; Arias, AMP; Ackermans, MT; Endert, E; Pijl, H; Kuipers, F; Meijer, AJ; Sauerwein, HP; Romijn, JA

    2000-01-01

    To evaluate the effect of dietary carbohydrate content on postabsorptive glucose metabolism, we quantified gluconeogenesis and glycogenolysis after 11 days of high carbohydrate (85% carbohydrate), control (44% carbohydrate), and very low carbohydrate (2% carbohydrate) diets in six healthy men. Diets

  20. Synthesis of Bioinspired Carbohydrate Amphiphiles that Promote and Inhibit Biofilms.

    Science.gov (United States)

    Dane, Eric L; Ballok, Alicia E; O'Toole, George A; Grinstaff, Mark W

    2014-02-01

    The synthesis and characterization of a new class of bioinspired carbohydrate amphiphiles that modulate Pseudomonas aeruginosa biofilm formation are reported. The carbohydrate head is an enantiopure poly-amido-saccharide (PAS) prepared by a controlled anionic polymerization of β-lactam monomers derived from either glucose or galactose. The supramolecular assemblies formed by PAS amphiphiles are investigated in solution using fluorescence assays and dynamic light scattering. Dried samples are investigated using X-ray, infrared spectroscopy, and transmission electron microscopy. Additionally, the amphiphiles are evaluated for their ability to modulate biofilm formation by the Gram-negative bacterium Pseudomonas aeruginosa. Remarkably, from a library of eight amphiphiles, we identify a structure that promotes biofilm formation and two structures that inhibit biofilm formation. Using biological assays and electron microscopy, we relate the chemical structure of the amphiphiles to the observed activity. Materials that modulate the formation of biofilms by bacteria are important both as research tools for microbiologists to study the process of biofilm formation and for their potential to provide new drug candidates for treating biofilm-associated infections.

  1. Regioselective monodeprotection of peracetylated carbohydrates.

    Science.gov (United States)

    Filice, Marco; Guisan, Jose M; Terreni, Marco; Palomo, Jose M

    2012-10-01

    This protocol describes the regioselective deprotection of single hydroxyls in peracetylated monosaccharides and disaccharides by enzymatic or chemoenzymatic strategies. The introduction of a one-pot enzymatic step by using immobilized biocatalysts obviates the requirement to carry out tedious workups and time-consuming purifications. By using this straightforward protocol, different per-O-acetylated glycopyranosides (mono- or disaccharides, 1-substituted or glycals) can be transformed into a whole set of differentially monodeprotected 1-alcohols, 3-alcohols, 4-alcohols and 6-alcohols in high yields. These tailor-made glycosyl acceptors can then be used for stereoselective glycosylation for oligosaccharide and glycoderivative synthesis. They have been successfully used as building blocks to synthesize tailor-made di- and trisaccharides involved in the structure of lacto-N-neo-tetraose and precursors of the tumor-associated carbohydrate antigen T and the antitumoral drug peracetylated β-naphtyl-lactosamine. We are able to prepare a purified monoprotected carbohydrate in between 1 and 4 d. With this protocol, the small library of monodeprotected products can be synthesized in 1-2 weeks.

  2. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    Science.gov (United States)

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-02-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

  3. Structure-activity relationships in carbohydrates revealed by their hydration.

    Science.gov (United States)

    Maugeri, Laura; Busch, Sebastian; McLain, Sylvia E; Pardo, Luis Carlos; Bruni, Fabio; Ricci, Maria Antonietta

    2016-12-21

    One of the more intriguing aspects of carbohydrate chemistry is that despite having very similar molecular structures, sugars have very different properties. For instance, there is a sensible difference in sweet taste between glucose and trehalose, even though trehalose is a disaccharide that comprised two glucose units, suggesting a different ability of these two carbohydrates to bind to sweet receptors. Here we have looked at the hydration of specific sites and at the three-dimensional configuration of water molecules around three carbohydrates (glucose, cellobiose, and trehalose), combining neutron diffraction data with computer modelling. Results indicate that identical chemical groups can have radically different hydration patterns depending on their location on a given molecule. These differences can be linked with the specific activity of glucose, cellobiose, and trehalose as a sweet substance, as building block of cellulose fiber, and as a bioprotective agent, respectively. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

  4. Fat/carbohydrate ratio but not energy density determines snack food intake and activates brain reward areas.

    Science.gov (United States)

    Hoch, Tobias; Kreitz, Silke; Gaffling, Simone; Pischetsrieder, Monika; Hess, Andreas

    2015-05-14

    The snack food potato chips induces food intake in ad libitum fed rats, which is associated with modulation of the brain reward system and other circuits. Here, we show that food intake in satiated rats is triggered by an optimal fat/carbohydrate ratio. Like potato chips, an isocaloric fat/carbohydrate mixture influenced whole brain activity pattern of rats, affecting circuits related e.g. to reward/addiction, but the number of modulated areas and the extent of modulation was lower compared to the snack food itself.

  5. Fat/carbohydrate ratio but not energy density determines snack food intake and activates brain reward areas

    OpenAIRE

    Hoch, Tobias; Kreitz, Silke; Gaffling, Simone; Pischetsrieder, Monika; Hess, Andreas

    2015-01-01

    The snack food potato chips induces food intake in ad libitum fed rats, which is associated with modulation of the brain reward system and other circuits. Here, we show that food intake in satiated rats is triggered by an optimal fat/carbohydrate ratio. Like potato chips, an isocaloric fat/carbohydrate mixture influenced whole brain activity pattern of rats, affecting circuits related e.g. to reward/addiction, but the number of modulated areas and the extent of modulation was lower compared t...

  6. Binding of copper(II) ions to the polyproline II helices of PEVK modules of the giant elastic protein titin as revealed by ESI-MS, CD, and NMR.

    Science.gov (United States)

    Ma, Kan; Wang, Kuan

    2003-10-01

    Titin, a family of giant elastic proteins, constitutes an elastic sarcomere matrix in striated muscle. In the I-band region of the sarcomere, the titin PEVK segment acts as a molecular spring to generate elasticity as well as sites of adhesion with parallel thin filaments. Previously, we reported that PEVK consists of tandem repeats of 28 residue modules and that the "polyproline II-coil" motif is the fundamental conformational motif of the PEVK module. In order to characterize the factors that may affect and alter the PPII-coil conformational motifs, we have initiated a systematic study of the interaction with divalent cations (Cu2+, Ca2+, Zn2+, and Ni2+) and a conformational profile of PEVK peptides (a representative 28-mer peptide PR: PEPPKEVVPEKKAPVAPPKKPEVPPVKV and its subfragments PR1: kvPEPPKEVVPE, PR2: VPEKKAPVAPPK, PR3: KPEVPPVKV). UV-Vis absorption difference spectra and CD spectra showed that Cu2+ bound to PR1 with high affinity (20 microM), while its binding to PR2 and PR3 as well as the binding of other cations to all four peptides were of lower affinity (>100 microM). Conformational studies by CD revealed that Cu2+ binding to PR1 resulted in a polyproline II to turn transition up to a 1:2 PR1/Cu2+ ratio and a coil to turn transition at higher Cu2+ concentration. ESI-MS provided the stoichiometry of PEVK peptide-Cu2+ complexes at both low and high ion strength, confirming the specific high affinity binding of Cu2+ to PR1 and PR. Furthermore, NMR and ESI-MS/MS fragmentation analysis elucidated the binding sites of the PEVK peptide-Cu2+ complexes at (-2)KVPE2, 8VPE10, 13APV15, and 22EVP24. A potential application of Cu2+ binding in peptide sequencing by mass spectrometry was also revealed. We conclude that Cu2+ binds and bends PEVK peptides to a beta-turn-like structure at specific sites. The specific targeting of Cu2+ towards PPII is likely to be of significant value in elucidating the roles of PPII in titin elasticity as well as in interactions of

  7. Copper Binding and Subsequent Aggregation of α-Synuclein Are Modulated by N-Terminal Acetylation and Ablated by the H50Q Missense Mutation.

    Science.gov (United States)

    Mason, Rebecca J; Paskins, Aimee R; Dalton, Caroline F; Smith, David P

    2016-08-30

    The Parkinson's disease-associated protein α-synuclein exhibits significant conformational heterogeneity. Bacterially expressed α-synuclein is known to bind to copper, resulting in the formation of aggregation-prone compact conformations. However, in vivo, α-synuclein undergoes acetylation at its N-terminus. Here the effect of this modification and the pathological H50Q mutation on copper binding and subsequent conformational transitions were investigated by electrospray ionization-ion mobility spectrometry-mass spectrometry. We demonstrate that acetylation perturbs the ability of α-synuclein to bind copper and that the H50Q missense mutation in the presence of N-terminal acetylation prevents copper binding. These modifications and mutations prevent the formation of the most compact conformations and inhibit copper-induced aggregation.

  8. The effect of carbohydrate and fat variation in euenergetic diets on postabsorptive free fatty acid release

    NARCIS (Netherlands)

    Bisschop, PH; Ackermans, MT; Endert, E; Ruiter, AFC; Meijer, AJ; Kuipers, F; Sauerwein, HP; Romijn, JA

    2002-01-01

    Diet composition and energy content modulate free fatty acid (FFA) release. The aim of this study was to evaluate the dose-response effects of euenergetic variations in dietary carbohydrate and fat content on postabsorptive FFA release. The rate of appearance (R-a) of palmitate was measured by infus

  9. Schizophrenia, gluten, and low-carbohydrate, ketogenic diets: a case report and review of the literature

    OpenAIRE

    Westman Eric C; Kraft Bryan D

    2009-01-01

    Abstract We report the unexpected resolution of longstanding schizophrenic symptoms after starting a low-carbohydrate, ketogenic diet. After a review of the literature, possible reasons for this include the metabolic consequences from the elimination of gluten from the diet, and the modulation of the disease of schizophrenia at the cellular level.

  10. Potential of Peptides as Inhibitors and Mimotopes: Selection of Carbohydrate-Mimetic Peptides from Phage Display Libraries

    Directory of Open Access Journals (Sweden)

    Teruhiko Matsubara

    2012-01-01

    Full Text Available Glycoconjugates play various roles in biological processes. In particular, oligosaccharides on the surface of animal cells are involved in virus infection and cell-cell communication. Inhibitors of carbohydrate-protein interactions are potential antiviral drugs. Several anti-influenza drugs such as oseltamivir and zanamivir are derivatives of sialic acid, which inhibits neuraminidase. However, it is very difficult to prepare a diverse range of sugar derivatives by chemical synthesis or by the isolation of natural products. In addition, the pathogenic capsular polysaccharides of bacteria are carbohydrate antigens, for which a safe and efficacious method of vaccination is required. Phage-display technology has been improved to enable the identification of peptides that bind to carbohydrate-binding proteins, such as lectins and antibodies, from a large repertoire of peptide sequences. These peptides are known as “carbohydrate-mimetic peptides (CMPs” because they mimic carbohydrate structures. Compared to carbohydrate derivatives, it is easy to prepare mono- and multivalent peptides and then to modify them to create various derivatives. Such mimetic peptides are available as peptide inhibitors of carbohydrate-protein interactions and peptide mimotopes that are conjugated with adjuvant for vaccination.

  11. Wood adhesives containing proteins and carbohydrates

    Science.gov (United States)

    In recent years there has been resurgent interest in using biopolymers as sustainable and environmentally friendly ingredients in wood adhesive formulations. Among them, proteins and carbohydrates are the most commonly used. In this chapter, an overview is given of protein-based and carbohydrate-...

  12. Mastering ectomycorrhizal symbiosis: the impact of carbohydrates.

    Science.gov (United States)

    Nehls, Uwe

    2008-01-01

    Mycorrhiza formation is the consequence of a mutualistic interaction between certain soil fungi and plant roots that helps to overcome nutritional limitations faced by the respective partners. In symbiosis, fungi contribute to tree nutrition by means of mineral weathering and mobilization of nutrients from organic matter, and obtain plant-derived carbohydrates as a response. Support with easily degradable carbohydrates seems to be the driving force for fungi to undergo this type of interaction. As a consequence, the fungal hexose uptake capacity is strongly increased in Hartig net hyphae of the model fungi Amanita muscaria and Laccaria bicolor. Next to fast carbohydrate uptake and metabolism, storage carbohydrates are of special interest. In functional A. muscaria ectomycorrhizas, expression and activity of proteins involved in trehalose biosynthesis is mainly localized in hyphae of the Hartig net, indicating an important function of trehalose in generation of a strong carbon sink by fungal hyphae. In symbiosis, fungal partners receive up to approximately 19 times more carbohydrates from their hosts than normal leakage of the root system would cause, resulting in a strong carbohydrate demand of infected roots and, as a consequence, a more efficient plant photosynthesis. To avoid fungal parasitism, the plant seems to have developed mechanisms to control carbohydrate drain towards the fungal partner and link it to the fungus-derived mineral nutrition. In this contribution, current knowledge on fungal strategies to obtain carbohydrates from its host and plant strategies to enable, but also to control and restrict (under certain conditions), carbon transfer are summarized.

  13. Carbohydrate clearance receptors in transfusion medicine

    DEFF Research Database (Denmark)

    Sørensen, Anne Louise Tølbøll; Clausen, Henrik; Wandall, Hans H

    2012-01-01

    Complex carbohydrates play important functions for circulation of proteins and cells. They provide protective shields and refraction from non-specific interactions with negative charges from sialic acids to enhance circulatory half-life. For recombinant protein therapeutics carbohydrates are espe...

  14. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Joong-Won, E-mail: jshin@govst.edu [Division of Science, Governors State University, University Park, Illinois 60484-0975 (United States); Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States); Bernstein, Elliot R., E-mail: erb@lamar.colostate.edu [Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States)

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  15. Influence of carbohydrates on the interaction of procyanidin B3 with trypsin.

    Science.gov (United States)

    Gonçalves, Rui; Mateus, Nuno; De Freitas, Victor

    2011-11-09

    The biological properties of procyanidins, in particular their inhibition of digestive enzymes, have received much attention in the past few years. Dietary carbohydrates are an environmental factor that is known to affect the interaction of procyanidins with proteins. This work aimed at understanding the effect of ionic food carbohydrates (polygalacturonic acid, arabic gum, pectin, and xanthan gum) on the interaction between procyanidins and trypsin. Physical-chemical techniques such as saturation transfer difference-NMR (STD-NMR) spectroscopy, fluorescence quenching, and nephelometry were used to evaluate the interaction process. Using STD-NMR, it was possible to identify the binding of procyanidin B3 to trypsin. The tested carbohydrates prevented the association of procyanidin B3 and trypsin by a competition mechanism in which the ionic character of carbohydrates and their ability to encapsulate procyanidins seem crucial leading to a reduction in STD signal and light scattering and to a recovery of the proteins intrinsic fluorescence. On the basis of these results, it was possible to grade the carbohydrates in their aggregation inhibition ability: XG > PA > AG ≫ PC. These effects may be relevant since the coingestion of procyanidins and ionic carbohydrates are frequent and furthermore since these might negatively affect the antinutritional properties ascribed to procyanidins in the past.

  16. Derivatization Reaction of Carbohydrates with Urea as the Reagent and Fluorimetric Determination of Carbohydrates

    Institute of Scientific and Technical Information of China (English)

    YANG,Jing-He(杨景和); CAO,Xi-Hui(曹西慧); WANG,Min(王敏); WU,Xia(吴霞); SUN,Chang-Xia(孙长侠)

    2002-01-01

    It is found that in the presence of sulfuric acid carbohydrates condense with urea to afford the condensation products, which emit fluorescence. Under optimum conditions, the fluorescence intensities of system are proportional to the concentrations of carbohydrates. Based on this linear relationship,quantitative determination of kinds of carbohydrates has been made. Among an the carbohydrates tested, the sensitivity of α-rhamnose is the highest and its limits of detection reaches 3.5 × 10-8 mol/L. So α-rhamnose can be selectively determed in the presence of other carbohydrates. A interaction mechanism is also discussed.

  17. Industrial applications of marine carbohydrates.

    Science.gov (United States)

    Sudha, Prasad N; Aisverya, S; Nithya, R; Vijayalakshmi, K

    2014-01-01

    Biomaterials have been used increasingly in various fields, such as drug delivery, imaging, and tissue engineering. The main reason justifying the widespread use of biomaterials relies on its valuable and low-cost source of new drugs. Current research goals are focused on identifying more potent and specific compounds with antitumor, immunomodulatory, antihyperlipidemic, anticoagulant, and antiviral activities. The increasing knowledge of structural analysis and chemical modifications enables the use of these marine carbohydrates in a newer way for the human welfare. This chapter focuses on the recent developments related to industrial and biomedical applications using chitin, chitosan, alginate, agar, and carrageenan derivatives and reports the main advances published over the last 10-15 years.

  18. Nondigestible carbohydrates and mineral bioavailability.

    Science.gov (United States)

    Greger, J L

    1999-07-01

    Generally, fiber and compounds associated with fiber in cereal products (e.g., phytates) have been found to reduce the apparent absorption of minerals (such as calcium, magnesium, zinc and manganese) in humans, livestock and animal models. The effects of "soluble" forms of fiber (specifically pectins, gums, resistant starches, lactulose, oligofructose and inulin) on mineral absorption are more difficult to characterize. The addition of these soluble forms of fiber has been found in various studies to add viscosity to the gut contents, promote fermentation and the production of volatile fatty acids in the cecum, have a trophic effect on the ceca of animals and increase serum enteroglucagon concentrations. Thus it is not surprising that the addition of soluble forms of fiber to diets often has been found to improve absorption of minerals. This may reflect absorption of electrolytes from the large intestine. Future work should address the mechanisms by which ingestion of nondigestible carbohydrates improves mineral absorption in humans.

  19. Stacking interactions between carbohydrate and protein quantified by combination of theoretical and experimental methods.

    Directory of Open Access Journals (Sweden)

    Michaela Wimmerová

    Full Text Available Carbohydrate-receptor interactions are an integral part of biological events. They play an important role in many cellular processes, such as cell-cell adhesion, cell differentiation and in-cell signaling. Carbohydrates can interact with a receptor by using several types of intermolecular interactions. One of the most important is the interaction of a carbohydrate's apolar part with aromatic amino acid residues, known as dispersion interaction or CH/π interaction. In the study presented here, we attempted for the first time to quantify how the CH/π interaction contributes to a more general carbohydrate-protein interaction. We used a combined experimental approach, creating single and double point mutants with high level computational methods, and applied both to Ralstonia solanacearum (RSL lectin complexes with α-L-Me-fucoside. Experimentally measured binding affinities were compared with computed carbohydrate-aromatic amino acid residue interaction energies. Experimental binding affinities for the RSL wild type, phenylalanine and alanine mutants were -8.5, -7.1 and -4.1 kcal x mol(-1, respectively. These affinities agree with the computed dispersion interaction energy between carbohydrate and aromatic amino acid residues for RSL wild type and phenylalanine, with values -8.8, -7.9 kcal x mol(-1, excluding the alanine mutant where the interaction energy was -0.9 kcal x mol(-1. Molecular dynamics simulations show that discrepancy can be caused by creation of a new hydrogen bond between the α-L-Me-fucoside and RSL. Observed results suggest that in this and similar cases the carbohydrate-receptor interaction can be driven mainly by a dispersion interaction.

  20. Ion mobility studies of carbohydrates as group I adducts: isomer specific collisional cross section dependence on metal ion radius.

    Science.gov (United States)

    Huang, Yuting; Dodds, Eric D

    2013-10-15

    Carbohydrates play numerous critical roles in biological systems. Characterization of oligosaccharide structures is essential to a complete understanding of their functions in biological processes; nevertheless, their structural determination remains challenging in part due to isomerism. Ion mobility spectrometry provides the means to resolve gas phase ions on the basis of their shape-to-charge ratios, thus providing significant potential for separation and differentiation of carbohydrate isomers. Here, we report on the determination of collisional cross sections for four groups of isomeric carbohydrates (including five isomeric disaccharides, four isomeric trisaccharides, two isomeric pentasaccharides, and two isomeric hexasaccharides) as their group I metal ion adducts (i.e., [M + Li](+), [M + Na](+), [M + K](+), [M + Rb](+), and [M + Cs](+)). In all, 65 collisional cross sections were measured, the great majority of which have not been previously reported. As anticipated, the collisional cross sections of the carbohydrate metal ion adducts generally increase with increasing metal ion radius; however, the collisional cross sections were found to scale with the group I cation size in isomer specific manners. Such measurements are of substantial analytical value, as they illustrate how the selection of charge carrier influences carbohydrate ion mobility determinations. For example, certain pairs of isomeric carbohydrates assume unique collisional cross sections upon binding one metal ion, but not another. On the whole, these data suggest a role for the charge carrier as a probe of carbohydrate structure and thus have significant implications for the continued development and application of ion mobility spectrometry for the distinction and resolution of isomeric carbohydrates.

  1. Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3'UTR in a manner that requires transcription.

    Science.gov (United States)

    Roe, David F; Craviso, Gale L; Waymire, Jack C

    2004-01-05

    Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 microM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated cells chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3'UTR responsible for stabilization using labeled TH 3'UTR probes and electro-mobility shift assays shows the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Both changes are prevented by actinomycin D. Characterization of the protein binding to the TH UTR probes indicates SMC is disrupted by polyribonucleotides, poly (A) and poly (U), while binding to FMC is reduced by poly (CU). Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains three proteins. Northwesterns yielded similar results. Comparison of DMPP-induced protein binding with the poly C binding protein (PCBP) involved in hypoxia induced rat PC12 TH mRNA stability indicates none of the bovine UTR binding proteins are the PCBP. Thus, nicotinic stimulation produces a transcription-dependent increase in TH mRNA half-life that is mediated by previously unrecognized TH mRNA binding proteins.

  2. Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense.

    Directory of Open Access Journals (Sweden)

    Mario Waespy

    Full Text Available Fourteen different active Trypanosoma congolense trans-sialidases (TconTS, 11 variants of TconTS1 besides TconTS2, TconTS3 and TconTS4, have been described. Notably, the specific transfer and sialidase activities of these TconTS differ by orders of magnitude. Surprisingly, phylogenetic analysis of the catalytic domains (CD grouped each of the highly active TconTS together with the less active enzymes. In contrast, when aligning lectin-like domains (LD, the highly active TconTS grouped together, leading to the hypothesis that the LD of TconTS modulates its enzymatic activity. So far, little is known about the function and ligand specificity of these LDs. To explore their carbohydrate-binding potential, glycan array analysis was performed on the LD of TconTS1, TconTS2, TconTS3 and TconTS4. In addition, Saturation Transfer Difference (STD NMR experiments were done on TconTS2-LD for a more detailed analysis of its lectin activity. Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD. Interestingly, terminal mannose residues are not acceptor substrates for TconTS activity. This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself. Experimental evidence for such a scenario is presented.

  3. NMR investigations of protein-carbohydrate interactions : Studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N ',N ''-triacetylchitotriose

    NARCIS (Netherlands)

    Asensio, JL; Siebert, HC; von der Lieth, CW; Laynez, J; Bruix, M; Soedjanaamadja, UM; Beintema, JJ; Canada, FJ; Gabius, HJ; Jimenez-Barbero, J

    2000-01-01

    Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N',N"-triacetylchitotriose has been an

  4. Application of surface plasmon resonance for the detection of carbohydrates, glycoconjugates, and measurement of the carbohydrate-specific interactions: a comparison with conventional analytical techniques. A critical review.

    Science.gov (United States)

    Safina, Gulnara

    2012-01-27

    Carbohydrates (glycans) and their conjugates with proteins and lipids contribute significantly to many biological processes. That makes these compounds important targets to be detected, monitored and identified. The identification of the carbohydrate content in their conjugates with proteins and lipids (glycoforms) is often a challenging task. Most of the conventional instrumental analytical techniques are time-consuming and require tedious sample pretreatment and utilising various labeling agents. Surface plasmon resonance (SPR) has been intensively developed during last two decades and has received the increasing attention for different applications, from the real-time monitoring of affinity bindings to biosensors. SPR does not require any labels and is capable of direct measurement of biospecific interaction occurring on the sensing surface. This review provides a critical comparison of modern analytical instrumental techniques with SPR in terms of their analytical capabilities to detect carbohydrates, their conjugates with proteins and lipids and to study the carbohydrate-specific bindings. A few selected examples of the SPR approaches developed during 2004-2011 for the biosensing of glycoforms and for glycan-protein affinity studies are comprehensively discussed.

  5. The cancer glycome: carbohydrates as mediators of metastasis.

    Science.gov (United States)

    Glavey, Siobhan V; Huynh, Daisy; Reagan, Michaela R; Manier, Salomon; Moschetta, Michele; Kawano, Yawara; Roccaro, Aldo M; Ghobrial, Irene M; Joshi, Lokesh; O'Dwyer, Michael E

    2015-07-01

    Glycosylation is a frequent post-translational modification which results in the addition of carbohydrate determinants, "glycans", to cell surface proteins and lipids. These glycan structures form the "glycome" and play an integral role in cell-cell and cell-matrix interactions through modulation of adhesion and cell trafficking. Glycosylation is increasingly recognized as a modulator of the malignant phenotype of cancer cells, where the interaction between cells and the tumor micro-environment is altered to facilitate processes such as drug resistance and metastasis. Changes in glycosylation of cell surface adhesion molecules such as selectin ligands, integrins and mucins have been implicated in the pathogenesis of several solid and hematological malignancies, often with prognostic implications. In this review we focus on the functional significance of alterations in cancer cell glycosylation, in terms of cell adhesion, trafficking and the metastatic cascade and provide insights into the prognostic and therapeutic implications of recent findings in this fast-evolving niche.

  6. Distribution of a 69-kD laminin-binding protein in aortic and microvascular endothelial cells: modulation during cell attachment, spreading, and migration

    DEFF Research Database (Denmark)

    Yannariello-Brown, J; Wewer, U; Liotta, L;

    1988-01-01

    Affinity chromatography and immunolocalization techniques were used to investigate the mechanism(s) by which endothelial cells interact with the basement membrane component laminin. Bovine aortic endothelial cells (BAEC) membranes were solubilized and incubated with a laminin-Sepharose affinity...... column. SDS-PAGE analysis of the eluted proteins identified a 69-kD band as the major binding protein, along with minor components migrating at 125, 110, 92, 85, 75, 55, and 30 kD. Polyclonal antibodies directed against a peptide sequence of the 69-kD laminin-binding protein isolated from human tumor......, with a granular perinuclear distribution and in linear arrays throughout the cell. During migration a redistribution from diffuse to predominanately linear arrays that co-distributed with actin microfilaments was noted in double-label experiments. The 69-kD laminin-binding protein colocalized with actin filaments...

  7. ICAT Inhibits beta-Catenin Binding to Tcf/Lef-Family Transcription Factors and in the General Coactivator p300 Using Independent Structural Modules

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, D. L.

    2002-01-01

    In the canonical Wnt signaling pathway, {beta}-catenin activates target genes through its interactions with Tcf/Lef-family transcription factors and additional transcriptional coactivators. The crystal structure of ICAT, an inhibitor of {beta}-catenin-mediated transcription, bound to the armadillo repeat domain of {beta}-catenin, has been determined. ICAT contains an N-terminal helilical domain that binds to repeats 11 and 12 of {beta}-catenin, and an extended C-terminal region that binds to repeats 5-10 in a manner similar that of Tcfs and other {beta}-catenin ligands. Full-length ICAT dissociates complexes of {beta}-catenin, Lef-1, and the transcriptional coactivator p300, whereas the helical domain alone selectively blocks binding to p300. The C-terminal armadillo repeats of {beta}-catenin may be an attractive target for compounds designed to disrupt aberrant {beta}-catenin-mediated transcription associated with various cancers.

  8. Carbohydrate-mediated inhibition of ice recrystallization in cryopreserved human umbilical cord blood.

    Science.gov (United States)

    Wu, Luke K; Tokarew, Jacqueline M; Chaytor, Jennifer L; von Moos, Elizabeth; Li, Yuhua; Palii, Carmen; Ben, Robert N; Allan, David S

    2011-01-01

    Cryopreservation of human umbilical cord blood (UCB) typically involves the cryoprotectant dimethylsulfoxide (DMSO), however, infusional toxicity and reductions in cell viability remain a concern. Ice recrystallization (IR) is an important source of cryopreservation-induced cellular injury and limits the stem cell dose in UCB units. Carbohydrates have wide-ranging intrinsic IR inhibition (IRI) activity related to structural properties. We investigated the impact of carbohydrate IRI on cell viability, induction of apoptosis and hematopoietic progenitor function in cryopreserved UCB. Mononuclear cells (MNCs) from UCB were cryopreserved in storage media containing specific carbohydrates (200mM) and compared to 5% DMSO. Samples were analyzed under conditions of high IR ('slow' thaw) and low IR ('fast' thaw). Thawed samples were analyzed for viability and apoptosis by flow cytometry and hematopoietic function using colony-forming unit (CFU) assays. IRI of carbohydrate solutions was determined using the 'splat cooling' assay. Greater IRI capacity of carbohydrates correlated with increased yield of viable MNCs (r(2)=0.92, p=0.004) and CD34(+) cells (r(2)=0.96, p=0.019) after thawing under conditions of high IR. The correlations were less apparent under conditions of low IR. Carbohydrates with greater IRI modulate the induction of early apoptosis during thawing, especially in CD34+ cells (r(2)=0.96, p=0.0001) as compared to total mononuclear cells (p=0.006), and preserve CFU capacity in vitro (r(2)=0.92, p=<0.0001). Our results suggest that carbohydrates with potent IRI increase the yield of non-apoptotic and functional hematopoietic progenitors and provide a foundation for the development of novel synthetic carbohydrates with enhanced IRI properties to improve cryopreservation of UCB.

  9. D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons.

    Directory of Open Access Journals (Sweden)

    Khursheed A Wani

    Full Text Available Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1 required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.

  10. D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons.

    Science.gov (United States)

    Wani, Khursheed A; Catanese, Mary; Normantowicz, Robyn; Herd, Muriel; Maher, Kathryn N; Chase, Daniel L

    2012-01-01

    Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1) required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.

  11. Glycan microarray analysis of the carbohydrate-recognition specificity of native and recombinant forms of the lectin ArtinM.

    Science.gov (United States)

    Liu, Y; Cecílio, N T; Carvalho, F C; Roque-Barreira, M C; Feizi, T

    2015-12-01

    This article contains data related to the researc.h article entitled "Yeast-derived ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM" by Cecílio et al. (2015) [1]. ArtinM, a D-mannose-binding lectin isolated from the seeds of Artocarpus heterophyllus, exerts immunomodulatory and regenerative activities through its Carbohydrate Recognition Domain (CRD) (Souza et al., 2013; Mariano et al., 2014 [2], [3]). The limited availability of the native lectin (n-ArtinM) led us to characterize a recombinant form of the protein, obtained by expression in Saccharomyces cerevisiae (y-ArtinM). We compared the carbohydrate-binding specificities of y-ArtinM and n-ArtinM by analyzing the binding of biotinylated preparations of the two lectin forms using a neoglycolipid (NGL)-based glycan microarray. Data showed that y-ArtinM mirrored the specificity exhibited by n-ArtinM.

  12. Effect of anti-carbohydrate antibodies on HIV infection in a monocytic cell line (U937)

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Clausen, H;

    1991-01-01

    . This study therefore investigated the neutralization of HIV in a monocytic cell line (U937) using mAbs against these carbohydrate gp120-epitopes. While antibodies against one of the epitopes (AI) neutralized infection of U937 cells despite binding to the Fc-receptor, one mAb against the sialosyl-Tn epitope...... enhanced infection. This enhancement was independent of complement and could be blocked by mAb Leu3a against the CD4-receptor. The study indicated that enhancement of infection in monocytic cells can occur by the same anti-carbohydrate antibodies that neutralize infection in lymphocytes, and that antibody...

  13. Carbohydrate Detection and Lectin Isolation from Tegumental Tissue of Fasciola hepatica

    Directory of Open Access Journals (Sweden)

    MB Molaei Rad

    2010-02-01

    Full Text Available "nBackground: Fascioliasis is a chronic hepatic disease and may be resulted from mechani­cal/molecular parasite adhesion to host liver tissue. The aim of this study was to detect surface car­bohydrate and lectin, carbohydrate-binding protein isolation that might be responsible of this molecular binding."nMethods: The present experimental work was conducted in the Department of Medical Parasitol­ogy and Mycology, School of Public Health, Tehran University of Medical Sciences, Te­hran, Iran.  Fasciola hepatica parasites were collected from abattoir (Saman, Tehran, Iran and surface mannose-carbohydrate was detected by fluorescein isothiocyanate (FITC conju­gated lectin (Lentil. Lectin of tegumental tissue from F. hepatica was isolated by affinity chroma­tography and detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE."nResults: Mannose carbohydrate was observed on the surface of tegumental tissue from para­site under fluorescence microscope. Carbohydrate-binding protein or lectin with MW of 50 kDa also was isolated from homogenized tegument of helminth."nConclusion: These results are important for understanding of molecular pathogenesis of F. hepat­ica at the chronic phase of fascioliasis

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

    DEFF Research Database (Denmark)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin

    2015-01-01

    M), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin–agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1...... rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity....

  15. GTP is an allosteric modulator of the interaction between the guanylate-binding protein 1 and the prosurvival kinase PIM1.

    Science.gov (United States)

    Persico, Marco; Petrella, Lella; Orteca, Nausicaa; Di Dato, Antonio; Mariani, Marisa; Andreoli, Mirko; De Donato, Marta; Scambia, Giovanni; Novellino, Ettore; Ferlini, Cristiano; Fattorusso, Caterina

    2015-02-16

    GBP1 and PIM1 are known to interact with a molar ratio 1:1. GBP1:PIM1 binding initiates a signaling pathway that induces resistance to common chemotherapeutics such as paclitaxel. Since GBP1 is a large GTPase which undergoes conformational changes in a nucleotide-dependent manner, we investigated the effect of GTP/GDP binding on GBP1:PIM1 interaction by using computational and biological studies. It resulted that only GTP decreases the formation of the GBP1:PIM1 complex through an allosteric mechanism, putting the bases for the identification of new compounds potentially able to revert resistance to paclitaxel.

  16. Multivalent ligand presentation as a central concept to study intricate carbohydrate-protein interactions.

    Science.gov (United States)

    Jayaraman, Narayanaswamy

    2009-12-01

    Studying the weak binding affinities between carbohydrates and proteins has been a central theme in sustained efforts to uncover intricate details of this class of biomolecular interaction. The amphiphilic nature of most carbohydrates, the competing nature of the surrounding water molecules to a given protein receptor site and the receptor binding site characteristics led to the realization that carbohydrates are required to exert favorable interactions, primarily through clustering of the ligands. The clustering of sugar ligands has been augmented using many different innovative molecular scaffolds. The synthesis of clustered ligands also facilitates fine-tuning of the spatial and topological proximities between the ligands, so as to allow the identification of optimal molecular features for significant binding affinity enhancements. The kinetic and thermodynamic parameters have been delineated in many instances, thereby allowing an ability to correlate the multivalent presentation and the observed ligand-receptor interaction profiles. This critical review presents various multivalent ligands, synthetic and semisynthetic, and mechanisms by which the weak binding affinities are overcome, and the ligand-receptor complexation leads to significantly enhanced binding affinities (157 references).

  17. Carbohydrates mediate sperm-ovum adhesion and triggering of the acrosome reaction

    Institute of Scientific and Technical Information of China (English)

    DaulatR.P.Tulsiani

    2000-01-01

    The fertilization process is the net result of a complex sequence of events that collectively result in the fusion of the opposite gametes. The male gamete undergoes continuous morphological and biochemical modifications during sperm development in the testis (spermatogenesis), maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are able to recognize and bind to the bioactive glycan residue(s) on the ovum's extracellular coat, the zona pellucida (ZP). Sperm-zona binding in the mouse and several other species is believed to take place in two stages. First, capacitated (acrosome-intact) spermatozoa loosely and reversibly adhere to the zona-intact ovum. In the second stage tight irreversible binding occurs. Both types of bindings are attributed to the presence of glycan- binding proteins (receptors) on the sperm plasma membrane and their complementary bioactive glycan units (ligands) on the surface of the ZP. The carbohydrate-mediated adhesion event initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be prerequisite which allows the hypemctivated acrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum. This review focuses on the role of carbohydrate residues in sperm-ovum interaction, and triggering of the acrosome reaction. I have attempted to discuss extensive progress that has been made to enhance our understanding of the well programmed multiple molecular events necessary for successful fertilization. This review will identify these events, and discuss the functional significance of carbohydrates in these events.

  18. Specific binding of photoaffinity-labeling peptidomimetics of Pro-Leu-Gly-NH2 to the dopamine D2L receptor: evidence for the allosteric modulation of the dopamine receptor.

    Science.gov (United States)

    Mann, Amandeep; Verma, Vaneeta; Basu, Dipannita; Skoblenick, Kevin J; Beyaert, Michael G R; Fisher, Abigail; Thomas, Nancy; Johnson, Rodney L; Mishra, Ram K

    2010-09-01

    The present study was undertaken to investigate the mechanistic role of l-prolyl-l-leucyl-glycinamide (PLG) in modulating agonist binding to the dopamine D(2L) receptor. Competition and displacement assays indicate that the photoaffinity-labeling peptidomimetics of PLG, 3(R)-[(4(S)-(4-azido-2-hydroxy-benzoyl) amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1a) and 3(R)-[(4(S)-(4-azido-2-hydroxy-5-iodo-benzoyl)amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1b) bind at the same site as PLG. Autoradiography was used to establish the covalent binding of [(125)I]-1b to an approximately 51kDa protein in bovine striatal membranes. Western blot analysis with a dopamine D(2L)-specific antibody, in combination with autoradiography, following a two-dimensional gel separation, suggested this approximately 51kDa protein to be the dopamine D(2L) receptor. Further evidence for binding of 1b to dopamine D(2L) was provided by samples immunoprecipitated with the D(2L) antibody. These samples were analyzed by western blotting in parallel with autoradiography of [(125)I]-1b labeled protein. Both methods revealed bands at approximately 51kDa. Furthermore, PLG is shown to compete with 1b for binding to the dopamine D(2L) receptor as determined by autoradiography, as well as competition experiments with PLG and 1a. Collectively, these findings suggest the successful development of a photoaffinity-labeling agent, compound 1b, that has been used to elucidate the interaction of PLG specifically with the dopamine D(2L) receptor.

  19. 抗菌肽人β防御素3融合糖类结合域对葡萄球菌的抑制作用%Inhibition role of fusing antimicrobial peptides humanβ-defensin 3 and carbohydrate binding domain on staphylococcus

    Institute of Scientific and Technical Information of China (English)

    黄洁雯; 郭晓奎; 李擎天

    2014-01-01

    目的:探讨抗菌肽人β防御素3融合糖类结合域(hBD3-CBD)对金黄色葡萄球菌 N315、表皮葡萄球菌35984的抑制作用。方法以直接杀伤和分子生物学方法检测 hBD3-CBD 对金黄色葡萄球菌 N315和表皮葡萄球菌35984菌株的抑制作用以及对细菌关键基因表达的影响。结果直接杀菌作用显示,抗菌肽 hBD3以及 hBD3-CBD 对金黄色葡萄球菌 N315、表皮葡萄球菌35984均有显著的抑制作用;hBD3-CBD 的抑制作用强于 hBD3;hBD3-CBD 抑制作用的稳定性亦强于 hBD3。在金黄色葡萄球菌 N315、表皮葡萄球菌35984的关键基因表达检测中,hBD3-CBD 对葡萄球菌的 agr 和 mecA 基因表达有显著抑制作用,还抑制表皮葡萄球菌 icaA 基因的表达和促进 icaR 基因表达,这说明 hBD3-CBD 能够抑制表皮葡萄球菌的生物膜形成。结论抗菌肽的融合策略对于改善抗菌肽的抑菌效能意义重大,并为其未来的应用带来更多希望。%Objective To explore the inhibition of fusing antimicrobial peptides humanβ-defensin 3 and carbohydrate binding do-main on Staphylococcus aureus N315 and Staphylococcus epidermidis 35984.Methods The direct bactericidal test and other molec-ular biology methods were adopted to detect the inhibition role on Staphylococcus aureus strain N315 and Staphylococcus epidermi-dis strain 35984 and the influence on the key genes expression.Results The direct bactericidal test demonstrated that antimicrobial peptides hBD3 and hBD3-CBD had significantly inhibitory effects on staphylococcus aureus N315 and staphylococcus epidermidis 35984;the inhibitory effects of hBD3-CBD was stronger than that of hBD3;the stability of the inhibitory effects of hBD3-CBD also stronger than that of hBD3.In the key gene expression test,there were significant inhibitions on the agr and mecA gene expressions in Staphylococcus aureus N315 and Staphylococcus epidermidis 35984 by hBD3-CBD.At the same time,hBD3-CBD

  20. Lectin-carbohydrate interactions on nanoporous gold monoliths.

    Science.gov (United States)

    Tan, Yih Horng; Fujikawa, Kohki; Pornsuriyasak, Papapida; Alla, Allan J; Ganesh, N Vijaya; Demchenko, Alexei V; Stine, Keith J

    2013-07-01

    also confirmed using atomic force microscopy (AFM). The results highlight the potential for application of carbohydrate modified np-Au monoliths to glycoscience and glycotechnology and demonstrate that they can be used for capture and release of carbohydrate binding proteins in significant quantities.

  1. Binding of Streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence.

    Science.gov (United States)

    Agarwal, Vaibhav; Sroka, Magdalena; Fulde, Marcus; Bergmann, Simone; Riesbeck, Kristian; Blom, Anna M

    2014-05-30

    The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.

  2. Positive versus negative modulation of different endogenous chemokines for CC-chemokine receptor 1 by small molecule agonists through allosteric versus orthosteric binding

    DEFF Research Database (Denmark)

    Jensen, Pia C; Thiele, Stefanie; Ulven, Trond

    2008-01-01

    5 and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand...

  3. Modulated Binding of SATB1, a Matrix Attachment Region Protein, to the AT-Rich Sequence Flanking the Major Breakpoint Region of BCL2

    Science.gov (United States)

    Ramakrishnan, Meera; Liu, Wen-Man; DiCroce, Patricia A.; Posner, Aleza; Zheng, Jian; Kohwi-Shigematsu, Terumi; Krontiris, Theodore G.

    2000-01-01

    The t(14,18) chromosomal translocation that occurs in human follicular lymphoma constitutively activates the BCL2 gene and disrupts control of apoptosis. Interestingly, 70% of the t(14,18) translocations are confined to three 15-bp clusters positioned within a 150-bp region (major breakpoint region or [MBR]) in the untranslated portion of terminal exon 3. We analyzed DNA-protein interactions in the MBR, as these may play some role in targeting the translocation to this region. An 87-bp segment (87MBR) immediately 3′ to breakpoint cluster 3 was essential for DNA-protein interaction monitored with mobility shift assays. We further delineated a core binding region within 87MBR: a 33-bp, very AT-rich sequence highly conserved between the human and mouse BCL2 gene (37MBR). We have purified and identified one of the core factors as the matrix attachment region (MAR) binding protein, SATB1, which is known to bind to AT-rich sequences with a high propensity to unwind. Additional factors in nuclear extracts, which we have not yet characterized further, increased SATB1 affinity for the 37MBR target four- to fivefold. Specific binding activity within 37MBR displayed cell cycle regulation in Jurkat T cells, while levels of SATB1 remained constant throughout the cell cycle. Finally, we demonstrated in vivo binding of SATB1 to the MBR, strongly suggesting the BCL2 major breakpoint region is a MAR. We discuss the potential consequences of our observations for both MBR fragility and regulatory function. PMID:10629043

  4. Modulation of ligand-heme reactivity by binding pocket residues demonstrated in cytochrome c' over the femtosecond-second temporal range.

    Science.gov (United States)

    Russell, Henry J; Hardman, Samantha J O; Heyes, Derren J; Hough, Michael A; Greetham, Gregory M; Towrie, Michael; Hay, Sam; Scrutton, Nigel S

    2013-12-01

    The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide (NO) and carbon monoxide (CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue (R124) and a distal leucine residue (L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124A variant. In the R124A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range.

  5. Utilization of carbohydrates by radiation processing

    Energy Technology Data Exchange (ETDEWEB)

    Kume, T. E-mail: kume@taka.jaeri.go.jp; Nagasawa, N.; Yoshii, F

    2002-03-01

    Upgrading and utilization of carbohydrates such as chitosan, sodium alginate, carrageenan, cellulose, pectin have been investigated for recycling these bio-resources and reducing the environmental pollution. These carbohydrates were easily degraded by irradiation and various kinds of biological activities such as anti-microbial activity, promotion of plant growth, suppression of heavy metal stress, phytoalexins induction, etc. were induced. On the other hand, some carbohydrate derivatives, carboxymethylcellulose and carboxymethylstarch, could be crosslinked under certain radiation condition and produce the biodegradable hydrogel for medical and agricultural use.

  6. pRB binds to and modulates the transrepressing activity of the E1A-regulated transcription factor p120E4F

    NARCIS (Netherlands)

    Fajas, L.; Paul, C.; Zugasti, O.; Cam, L. Le; Polanowska, J.; Fabbrizio, E.; Medema, R.H.; Vignais, M.-L.; Sardet, C.

    2000-01-01

    The retinoblastoma protein pRB is involved in the transcriptional control of genes essential for cell cycle progression and differentiation. pRB interacts with different transcription factors and thereby modulates their activity by sequestration, corepression, or activation. We report that pRB, but

  7. A peptide motif from the second fibronectin module of the neural cell adhesion molecule, NCAM, NLIKQDDGGSPIRHY, is a binding site for the FGF receptor

    DEFF Research Database (Denmark)

    Jacobsen, Jacob Hedemand; Kiselyov, Vladislav; Bock, Elisabeth

    2008-01-01

    The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) a...

  8. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function.

    Science.gov (United States)

    Chen, D; Zhang, Z; Li, M; Wang, W; Li, Y; Rayburn, E R; Hill, D L; Wang, H; Zhang, R

    2007-08-01

    As a major negative regulator of p53, the MDM2 oncogene plays an important role in carcinogenesis and tumor progression. MDM2 promotes p53 proteasomal degradation and negatively regulates p53 function. The mechanisms by which the MDM2-p53 interaction is regulated are not fully understood, although several MDM2-interacting molecules have recently been identified. To search for novel MDM2-binding partners, we screened a human prostate cDNA library by the yeast two-hybrid assay using full-length MDM2 protein as the bait. Among the candidate proteins, ribosomal protein S7 was identified and confirmed as a novel MDM2-interacting protein. Herein, we demonstrate that S7 binds to MDM2, in vitro and in vivo, and that the interaction between MDM2 and S7 leads to modulation of MDM2-p53 binding by forming a ternary complex among MDM2, p53 and S7. This results in the stabilization of p53 protein through abrogation of MDM2-mediated p53 ubiquitination. Consequently, S7 overexpression increases p53 transactivational activities, induces apoptosis, and inhibits cell proliferation. The identification of S7 as a novel MDM2-interacting partner contributes to elucidation of the complex regulation of the MDM2-p53 interaction and has implications in cancer prevention and therapy.

  9. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

    Directory of Open Access Journals (Sweden)

    John M Morrison

    2012-03-01

    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  10. Carbohydrate feeding and exercise: effect of beverage carbohydrate content.

    Science.gov (United States)

    Murray, R; Seifert, J G; Eddy, D E; Paul, G L; Halaby, G A

    1989-01-01

    The purpose of this study was to determine the effect of ingesting fluids of varying carbohydrate content upon sensory response, physiologic function, and exercise performance during 1.25 h of intermittent cycling in a warm environment (Tdb = 33.4 degrees C). Twelve subjects (7 male, 5 female) completed four separate exercise sessions; each session consisted of three 20 min bouts of cycling at 65% VO2max, with each bout followed by 5 min rest. A timed cycling task (1200 pedal revolutions) completed each exercise session. Immediately prior to the first 20 min cycling bout and during each rest period, subjects consumed 2.5 ml.kg BW-1 of water placebo (WP), or solutions of 6%, 8%, or 10% sucrose with electrolytes (20 mmol.l-1 Na+, 3.2 mmol.l-1 K+). Beverages were administered in double blind, counterbalanced order. Mean (+/- SE) times for the 1200 cycling task differed significantly: WP = 13.62 +/- 0.33 min, *6% = 13.03 +/- 0.24 min, 8% = 13.30 +/- 0.25 min, 10% = 13.57 +/- 0.22 min (* = different from WP and 10%, P less than 0.05). Compared to WP, ingestion of the CHO beverages resulted in higher plasma glucose and insulin concentrations, and higher RER values during the final 20 min of exercise (P less than 0.05). Markers of physiologic function and sensory perception changed similarly throughout exercise; no differences were observed among subjects in response to beverage treatments for changes in plasma concentrations of lactate, sodium, potassium, for changes in plasma volume, plasma osmolality, rectal temperature, heart rate, oxygen uptake, rating of perceived exertion, or for indices of gastrointestinal distress, perceived thirst, and overall beverage acceptance.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. DcR3 binds to ovarian cancer via heparan sulfate proteoglycans and modulates tumor cells response to platinum with corresponding alteration in the expression of BRCA1

    Directory of Open Access Journals (Sweden)

    Connor Joseph P

    2012-05-01

    Full Text Available Abstract Background Overcoming platinum resistance is a major obstacle in the treatment of Epithelial Ovarian Cancer (EOC. In our previous work Decoy Receptor 3 (DcR3 was found to be related to platinum resistance. The major objective of this work was to define the cellular interaction of DcR3 with EOC and to explore its effects on platinum responsiveness. Methods We studied cell lines and primary cultures for the expression of and the cells ability to bind DcR3. Cells were cultured with DcR3 and then exposed to platinum. Cell viability was determined by MTT assay. Finally, the cells molecular response to DcR3 was studied using real time RT-PCR based differential expression arrays, standard RT-PCR, and Western blot. Results High DcR3 in the peritoneal cavity of women with EOC is associated with significantly shorter time to first recurrence after platinum based therapy (p = 0.02. None-malignant cells contribute DcR3 in the peritoneal cavity. The cell lines studied do not secrete DcR3; however they all bind exogenous DcR3 to their surface implying that they can be effected by DcR3 from other sources. DcR3s protein binding partners are minimally expressed or negative, however, all cells expressed the DcR3 binding Heparan Sulfate Proteoglycans (HSPGs Syndecans-2, and CD44v3. DcR3 binding was inhibited by heparin and heparinase. After DcR3 exposure both SKOV-3 and OVCAR-3 became more resistant to platinum with 15% more cells surviving at high doses. On the contrary CaOV3 became more sensitive to platinum with 20–25% more cell death. PCR array analysis showed increase expression of BRCA1 mRNA in SKOV-3 and OVCAR-3 and decreased BRCA1 expression in CaOV-3 after exposure to DcR3. This was confirmed by gene specific real time PCR and Western blot analysis. Conclusions Non-malignant cells contribute to the high levels of DcR3 in ovarian cancer. DcR3 binds readily to EOC cells via HSPGs and alter their responsiveness to platinum chemotherapy. The

  12. Carbohydrate Moieties and Cytoenzymatic Characterization of Hemocytes in Whiteleg Shrimp Litopenaeus vannamei

    Science.gov (United States)

    Velázquez, Edwin; Rodríguez-Jaramillo, Carmen; Ascencio, Felipe

    2016-01-01

    Hemocytes represent one of the most important defense mechanisms against foreign material in Crustacea and are also involved in a variety of other physiological responses. Fluorescent lectin-binding assays and cytochemical reactions were used to identify specificity and distribution of carbohydrate moieties and presence of several hydrolytic enzymes, in hemocytes of whiteleg shrimp Litopenaeus vannamei. Two general classes of circulating hemocytes (granular and agranular) exist in L. vannamei, which express carbohydrates residues for FITC-conjugated lectins WGA, LEA, and PNA; UEA and Con-A were not observed. Enzymatic studies indicated that acid phosphatase, nonspecific esterase, and specific esterases were present; alkaline phosphatase was not observed. The enzymes and carbohydrates are useful tools in hemocyte classification and cellular defense mechanism studies. PMID:27833641

  13. Carbohydrate Moieties and Cytoenzymatic Characterization of Hemocytes in Whiteleg Shrimp Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    Norma Estrada

    2016-01-01

    Full Text Available Hemocytes represent one of the most important defense mechanisms against foreign material in Crustacea and are also involved in a variety of other physiological responses. Fluorescent lectin-binding assays and cytochemical reactions were used to identify specificity and distribution of carbohydrate moieties and presence of several hydrolytic enzymes, in hemocytes of whiteleg shrimp Litopenaeus vannamei. Two general classes of circulating hemocytes (granular and agranular exist in L. vannamei, which express carbohydrates residues for FITC-conjugated lectins WGA, LEA, and PNA; UEA and Con-A were not observed. Enzymatic studies indicated that acid phosphatase, nonspecific esterase, and specific esterases were present; alkaline phosphatase was not observed. The enzymes and carbohydrates are useful tools in hemocyte classification and cellular defense mechanism studies.

  14. Workshop to establish databases of carbohydrate spectra

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The workshop was organized to formulate guidelines for establishing spectral databases of complex carbohydrates. The databases will enable the scientific community to avoid the great waste of research effort and funds that frequently occurs when carbohydrate chemists are forced to duplicate the structural characterization of previously characterized complex carbohydrates. Chemists waste their effort on repetitive characterizations because in the absence of spectral databases they are unaware they are analyzing a known molecule until they have completely determined its structure. Chemists will be able to avoid much of this wasted effort when the collections of mass and of nuclear magnetic resonance (NMR) spectra initiated at the workshop are subsequently developed into searchable databases. Then scientists only need query the databases with the spectrum or with information defining the spectrum of an unidentified carbohydrate to find out if it has been previously characterized.

  15. Carbohydrates in pig nutrition - Recent advances

    DEFF Research Database (Denmark)

    Knudsen, Knud Erik Bach; Lærke, Helle Nygaard; Ingerslev, Anne Krog

    2016-01-01

    The dietary carbohydrates are a diverse group of substances with a range of chemical, physical, and physiological properties. The primary chemical classification of carbohydrates is by molecular size (degree of polymerization [DP]), the type of linkage (α or β), and composition of individual...... in the small intestine. Some types of soluble NSP are found to interact with intestinal mucus and produce a layer that significantly delays the transport of lipid digestion products. Potentially, the same may be the case for proteinous compounds. The delay in the transport of the nutrients to the gut...... monomers. This approach divides carbohydrates into 3 main groups, sugars (DP1–2), oligosaccharides (DP3–9), and polysaccharides (DP ≥ 10), the latter being further divided into starch (α-1:4,1,6-D-glucans) and nonstarch polysaccharides (NSP). Dietary fiber (DF) recently has been defined as carbohydrate...

  16. The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis

    DEFF Research Database (Denmark)

    Danen, Erik H J; Sonneveld, Petra; Brakebusch, Cord

    2002-01-01

    We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels...... receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions....

  17. sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

    DEFF Research Database (Denmark)

    Christner, Martin; Heinze, Constanze; Busch, Michael;

    2012-01-01

    Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant‐associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related...... virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed...

  18. Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic.

    Science.gov (United States)

    Karnik, Rucha; Grefen, Christopher; Bayne, Robert; Honsbein, Annegret; Köhler, Tim; Kioumourtzoglou, Dimitrios; Williams, Mary; Bryant, Nia J; Blatt, Michael R

    2013-04-01

    The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual "handshaking" mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.

  19. The Origin of the Constant Carbohydrate Diet

    Directory of Open Access Journals (Sweden)

    Charles Herbert Read

    2009-01-01

    Full Text Available The Constant Carbohydrate diet, based entirely on carbohydrate exchanges, is now widely used in the dietary treatment of diabetes mellitus. Being based on sound scientific principles and simple in design, the Constant Carabohydrate diet is appropriate for all those having diabetes mellitus, young or old, no matter their ethncity. This report describes why and how it was developed in 1951. Its simplicity makes it adaptable to all ethnic diets.

  20. The Origin of the Constant Carbohydrate Diet

    Directory of Open Access Journals (Sweden)

    Read CharlesHerbert

    2008-01-01

    Full Text Available The Constant Carbohydrate diet, based entirely on carbohydrate exchanges, is now widely used in the dietary treatment of diabetes mellitus. Being based on sound scientific principles and simple in design, the Constant Carabohydrate diet is appropriate for all those having diabetes mellitus, young or old, no matter their ethncity. This report describes why and how it was developed in 1951. Its simplicity makes it adaptable to all ethnic diets.

  1. Conversion of carbohydrates to levulinic acid esters

    DEFF Research Database (Denmark)

    2014-01-01

    The present invention relates to the field of converting carbohydrates into levulinic acid, a platform chemical for many chemical end products. More specifically the invention relates to a method for converting carbohydrates such as mono-, di- or polysaccharides, obtained from for example biomass...... production into a suitable levulinic acid ester in the presence of a zeolite or zeotype catalyst and a suitable alcohol, and the ester may be further converted into levulinic acid if desired....

  2. Molecular simulations of carbohydrates and protein-carbohydrate interactions: motivation, issues and prospects.

    Science.gov (United States)

    Fadda, Elisa; Woods, Robert J

    2010-08-01

    The characterization of the 3D structure of oligosaccharides, their conjugates and analogs is particularly challenging for traditional experimental methods. Molecular simulation methods provide a basis for interpreting sparse experimental data and for independently predicting conformational and dynamic properties of glycans. Here, we summarize and analyze the issues associated with modeling carbohydrates, with a detailed discussion of four of the most recently developed carbohydrate force fields, reviewed in terms of applicability to natural glycans, carbohydrate-protein complexes and the emerging area of glycomimetic drugs. In addition, we discuss prospectives and new applications of carbohydrate modeling in drug discovery.

  3. Metabolic engineering of monoclonal antibody carbohydrates for antibody-drug conjugation.

    Science.gov (United States)

    Okeley, Nicole M; Toki, Brian E; Zhang, Xinqun; Jeffrey, Scott C; Burke, Patrick J; Alley, Stephen C; Senter, Peter D

    2013-10-16

    The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

  4. A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding

    OpenAIRE

    Gopalakrishnan, Suhasni; Van Emburgh, Beth O.; Shan, Jixiu; Su, Zhen; Fields, C. Robert; Vieweg, Johannes; Hamazaki, Takashi; Schwartz, Philip H; Terada, Naohiro; Robertson, Keith D.

    2009-01-01

    DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe her...

  5. Bacterial Adhesion of Streptococcus suis to Host Cells and Its Inhibition by Carbohydrate Ligands

    Directory of Open Access Journals (Sweden)

    Sauli Haataja

    2013-07-01

    Full Text Available Streptococcus suis is a Gram-positive bacterium, which causes sepsis and meningitis in pigs and humans. This review examines the role of known S. suis virulence factors in adhesion and S. suis carbohydrate-based adhesion mechanisms, as well as the inhibition of S. suis adhesion by anti-adhesion compounds in in vitro assays. Carbohydrate-binding specificities of S. suis have been identified, and these studies have shown that many strains recognize Galα1-4Gal-containing oligosaccharides present in host glycolipids. In the era of increasing antibiotic resistance, new means to treat infections are needed. Since microbial adhesion to carbohydrates is important to establish disease, compounds blocking adhesion could be an alternative to antibiotics. The use of oligosaccharides as drugs is generally hampered by their relatively low affinity (micromolar to compete with multivalent binding to host receptors. However, screening of a library of chemically modified Galα1-4Gal derivatives has identified compounds that inhibit S. suis adhesion in nanomolar range. Also, design of multivalent Galα1-4Gal-containing dendrimers has resulted in a significant increase of the inhibitory potency of the disaccharide. The S. suis adhesin binding to Galα1-4Gal-oligosaccharides, Streptococcal adhesin P (SadP, was recently identified. It has a Galα1-4Gal-binding N-terminal domain and a C-terminal LPNTG-motif for cell wall anchoring. The carbohydrate-binding domain has no homology to E. coli P fimbrial adhesin, which suggests that these Gram-positive and Gram-negative bacterial adhesins recognizing the same receptor have evolved by convergent evolution. SadP adhesin may represent a promising target for the design of anti-adhesion ligands for the prevention and treatment of S. suis infections.

  6. Collagen and Stretch Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells

    Science.gov (United States)

    Perrone, Carmen E.; Fenwick-Smith, Daniela; Vandenburgh, Herman H.

    1995-01-01

    Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

  7. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Julien

    Full Text Available New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664 reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.

  8. Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung

    OpenAIRE

    Chuang, Pao-Tien; Kawcak, T'Nay; McMahon, Andrew P.

    2003-01-01

    Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue is how negative regulation of Hh signaling might contribute to generating differential responses over tens of cell diameters. In cells that respond to Hh, two proteins that are up-regulated are Patched1 (Ptch1), the Hh receptor, a general target in both invertebrate and vertebrate organisms, and Hip1, a Hh-binding protein that is vertebrate specific. To address the developmental role of Hip1 i...

  9. Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene, GmDREB3, in soybean (Glycine max L.)

    OpenAIRE

    Chen, Ming; Xu, Zhaoshi; Xia, Lanqin; Li, Liancheng; Cheng, Xianguo; Dong, Jianhui; Wang, Qiaoyan; Ma, Youzhi

    2008-01-01

    DREB (dehydration-responsive element-binding protein) transcription factors have important roles in the stress-related regulation network in plants. A DREB orthologue, GmDREB3, belonging to the A-5 subgroup of the DREB subfamily, was isolated from soybean using the RACE (rapid amplification of cDNA ends) method. Northern blot analysis showed that expression of GmDREB3 in soybean seedlings was induced following cold stress treatment for 0.5 h and was not detected after 3 h. However, it was not...

  10. Metabolic aspects of low carbohydrate diets and exercise

    OpenAIRE

    Peters Sandra; LeBlanc Paul

    2004-01-01

    Abstract Following a low carbohydrate diet, there is a shift towards more fat and less carbohydrate oxidation to provide energy to skeletal muscle, both at rest and during exercise. This review summarizes recent work on human skeletal muscle carbohydrate and fat metabolic adaptations to a low carbohydrate diet, focusing mainly on pyruvate dehydrogenase and pyruvate dehydrogenase kinase, and how these changes relate to the capacity for carbohydrate oxidation during exercise.

  11. Metabolic aspects of low carbohydrate diets and exercise

    Directory of Open Access Journals (Sweden)

    Peters Sandra

    2004-01-01

    Full Text Available Abstract Following a low carbohydrate diet, there is a shift towards more fat and less carbohydrate oxidation to provide energy to skeletal muscle, both at rest and during exercise. This review summarizes recent work on human skeletal muscle carbohydrate and fat metabolic adaptations to a low carbohydrate diet, focusing mainly on pyruvate dehydrogenase and pyruvate dehydrogenase kinase, and how these changes relate to the capacity for carbohydrate oxidation during exercise.

  12. Glycosylated Conductive Polymer: A Multimodal Biointerface for Studying Carbohydrate-Protein Interactions.

    Science.gov (United States)

    Zeng, Xiangqun; Qu, Ke; Rehman, Abdul

    2016-09-20

    Carbohydrate-protein interactions occur through glycoproteins, glycolipids, or polysaccharides displayed on the cell surface with lectins. However, studying these interactions is challenging because of the complexity and heterogeneity of the cell surface, the inherent structural complexity of carbohydrates, and the typically weak affinities of the binding reactions between the lectins and monovalent carbohydrates. The lack of chromophores and fluorophores in carbohydrate structures often drives such investigations toward fluorescence labeling techniques, which usually require tedious and complex synthetic work to conjugate fluorescent tags with additional risk of altering the reaction dynamics. Probing these interactions directly on the cell surface is even more difficult since cells could be too fragile for labeling or labile dynamics could be affected by the labeled molecules that may interfere with the cellular activities, resulting in unwanted cell responses. In contrast, label-free biosensors allow real-time monitoring of carbohydrate-protein interactions in their natural states. A prerequisite, though, for this strategy to work is to mimic the coding information on potential interactions of cell surfaces onto different biosensing platforms, while the complementary binding process can be transduced into a useful signal noninvasively. Through carbohydrate self-assembled monolayers and glycopolymer scaffolds, the multivalency of the naturally existing simple and complex carbohydrates can be mimicked and exploited with label-free readouts (e.g., optical, acoustic, mechanical, electrochemical, and electrical sensors), yet such inquiries reflect only limited aspects of complicated biointeraction processes due to the unimodal transduction. In this Account, we illustrate that functionalized glycosylated conductive polymer scaffolds are the ideal multimodal biointerfaces that not only simplify the immobilization process for surface fabrication via electrochemical

  13. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    Science.gov (United States)

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  14. TBL2 is a novel PERK-binding protein that modulates stress-signaling and cell survival during endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Yoshinori Tsukumo

    Full Text Available Under ER stress, PKR-like ER-resident kinase (PERK phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4. This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta-like 2 (TBL2 as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway.

  15. New modulated design and synthesis of chiral CuII/SnIV bimetallic potential anticancer drug entity: In vitro DNA binding and pBR322 DNA cleavage activity

    Science.gov (United States)

    Tabassum, Sartaj; Sharma, Girish Chandra; Arjmand, Farukh

    2012-05-01

    A new chiral ligand scaffold L derived from (R)-2-amino-2-phenyl ethanol and diethyl oxalate was isolated and thoroughly characterized by various spectroscopic methods. The ligand L was allowed to react with CuCl2·2H2O and NiCl2·6H2O to achieve monometallic complexes 1 and 2, respectively. Subsequently modulation of 1 and 2 was carried out in the presence of SnCl4·5H2O to obtain heterobimetallic potential drug candidates 3 and 4 possessing (CuII/SnIV and NiII/SnIV) metallic cores, respectively and characterized by elemental analysis and spectroscopic data including 1H, 13C and 119Sn NMR in case of 3 and 4. In vitro DNA binding studies revealed that complex 3 avidly binds to DNA as quantified by Kb and Ksv values. Complex 3 exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322 DNA and the cleavage activity of 3 was significantly enhanced in the presence of activators and follows the order H2O2 > Asc > MPA > GSH. Complex 3 cleave pBR322 DNA via hydrolytic pathway and accessible to major groove of DNA.

  16. Potential effect of ultrasound on carbohydrates.

    Science.gov (United States)

    Bera, Smritilekha; Mondal, Dhananjoy; Martin, Jacob T; Singh, Man

    2015-06-17

    The use of ultrasound has emerged as one of the most useful alternative energy sources for the synthesis of carbohydrate-derived biologically and pharmaceutically potential compounds. Spectacular advances have been made in the field of sonication-assisted organic reactions, which are known for producing superior yields, enhanced reactivity of the reactant, improved stereoselectivity, and shortened reaction times. Orthogonal protection-deprotection reactions and/or modification and manipulation of functional groups in carbohydrates are common synthetic steps in carbohydrate chemistry. These reaction steps can be driven by the ultrasonic energy generated by acoustic cavitation via the formation and subsequent collapse of ultrasound-induced bubbles. The ultrasound-assisted synthesis of differently functionalised monosaccharides is useful in a wide variety of applications of carbohydrate chemistry such as the glycosylation of oligosaccharides, one pot domino reactions, thioglycoside syntheses, azidoglycoside syntheses, 1,3-dipolar cycloaddition reactions, and syntheses of natural products. This review article covers ultrasound-mediated reactions on carbohydrates that have been described in the literature since 2000.

  17. Saturated fat, carbohydrate, and cardiovascular disease.

    Science.gov (United States)

    Siri-Tarino, Patty W; Sun, Qi; Hu, Frank B; Krauss, Ronald M

    2010-03-01

    A focus of dietary recommendations for cardiovascular disease (CVD) prevention and treatment has been a reduction in saturated fat intake, primarily as a means of lowering LDL-cholesterol concentrations. However, the evidence that supports a reduction in saturated fat intake must be evaluated in the context of replacement by other macronutrients. Clinical trials that replaced saturated fat with polyunsaturated fat have generally shown a reduction in CVD events, although several studies showed no effects. An independent association of saturated fat intake with CVD risk has not been consistently shown in prospective epidemiologic studies, although some have provided evidence of an increased risk in young individuals and in women. Replacement of saturated fat by polyunsaturated or monounsaturated fat lowers both LDL and HDL cholesterol. However, replacement with a higher carbohydrate intake, particularly refined carbohydrate, can exacerbate the atherogenic dyslipidemia associated with insulin resistance and obesity that includes increased triglycerides, small LDL particles, and reduced HDL cholesterol. In summary, although substitution of dietary polyunsaturated fat for saturated fat has been shown to lower CVD risk, there are few epidemiologic or clinical trial data to support a benefit of replacing saturated fat with carbohydrate. Furthermore, particularly given the differential effects of dietary saturated fats and carbohydrates on concentrations of larger and smaller LDL particles, respectively, dietary efforts to improve the increasing burden of CVD risk associated with atherogenic dyslipidemia should primarily emphasize the limitation of refined carbohydrate intakes and a reduction in excess adiposity.

  18. Curdlan sulphate modulates protein synthesis and enhances NF-κB and C/EBP binding activity in HepG2 cells

    Directory of Open Access Journals (Sweden)

    A. Guzdek

    1997-01-01

    Full Text Available In human hepatoma HepG2 cell line curdlan sulphate enhances basal and interleukin-6-stimulated fibrinogen and antichymotrypsin (ACT synthesis, slightly increases basal ceruloplasmin production and exerts only minor effects on alpha-1-proteinase inhibitor and transferrin. Curdlan sulphate may, at least in part, affect protein synthesis at a pretranslational level, as the expression of ACT mRNA was found to be increased, whereas intracellular enzyme, manganese superoxide dismutase mRNA level was decreased in the cell culture treated with curdlan sulphate. Gel mobility shift analysis revealed that curdlan sulphate increases the DNA binding activity of NF-κB and C/EBP, suggesting that these transcription factors may participate in the regulatory effects of curdlan sulphate in HepG2 cells.

  19. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen.

    Science.gov (United States)

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka

    2015-03-24

    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  20. Modulation of calcium oxalate dihydrate growth by selective crystal-face binding of phosphorylated osteopontin and polyaspartate peptide showing occlusion by sectoral (compositional) zoning.

    Science.gov (United States)

    Chien, Yung-Ching; Masica, David L; Gray, Jeffrey J; Nguyen, Sarah; Vali, Hojatollah; McKee, Marc D

    2009-08-28

    Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin/uropontin (OPN) are commonly found in kidney stones. To investigate the effects of OPN on COD growth, COD crystals were grown with phosphorylated OPN or a polyaspartic acid-rich peptide of OPN (DDLDDDDD, poly-Asp(86-93)). Crystals grown with OPN showed increased dimensions of the {110} prismatic faces attributable to selective inhibition at this crystallographic face. At high concentrations of OPN, elongated crystals with dominant {110} faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp(86-93) dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp(86-93) followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into {110} crystal sectors. Computational modeling of poly-Asp(86-93) adsorption to COD {110} and {101} surfaces also suggests increased stabilization of the COD {110} surface and negligible change to the natively stable {101} surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN. In summary, OPN and its poly-Asp(86-93) sequence similarly affect COD mineral growth; the {110} crystallographic faces become enhanced and dominant attributable to {110} face inhibition by the protein/peptide, and peptides can incorporate into the mineral phase. We, thus, conclude that the poly-Asp(86-93) domain is central to the OPN ability to interact with the {110} faces of COD, where it binds to inhibit crystal growth with subsequent intracrystalline incorporation (occlusion).

  1. Multi-organ expression profiling uncovers a gene module in coronary artery disease involving transendothelial migration of leukocytes and LIM domain binding 2: the Stockholm Atherosclerosis Gene Expression (STAGE study.

    Directory of Open Access Journals (Sweden)

    Sara Hägg

    2009-12-01

    Full Text Available Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD. The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE study was to determine whether there are functionally associated genes (rather than individual genes important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue and atherosclerotic and unaffected arterial wall (n = 40/tissue isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes. In the second step (performed within tissue clusters, one atherosclerotic lesion (n = 49/48 and one visceral fat (n = 59 cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015. The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54 relating to carotid stenosis (P = 0.04, 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10(-27 and-30. Genes in the transendothelial migration of leukocytes (TEML pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module. In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004. The transcription co-factor LIM domain binding 2 (LDB2 was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2

  2. Carbon monoxide binding to the heme group at the dimeric interface modulates structure and copper accessibility in the Cu,Zn superoxide dismutase from Haemophilus ducreyi: in silico and in vitro evidences.

    Science.gov (United States)

    Chillemi, Giovanni; De Santis, Serena; Falconi, Mattia; Mancini, Giordano; Migliorati, Valentina; Battistoni, Andrea; Pacello, Francesca; Desideri, Alessandro; D'Angelo, Paola

    2012-01-01

    X-ray absorption near-edge structure (XANES) spectroscopy and molecular dynamics (MD) simulations have been jointly applied to the study of the Cu,Zn superoxide dismutase from Haemophilus ducreyi (HdSOD) in interaction with the carbon monoxide molecule. The configurational flexibility of the Fe(II)-heme group, intercalated between the two subunits, has been sampled by MD simulations and included in the XANES data analysis without optimization in the structural parameter space. Our results provide an interpretation of the observed discrepancy in the Fe-heme distances as detected by extended X-ray absorption fine structure (EXAFS) spectroscopy and the classical XANES analysis, in which the structural parameters are optimized in a unique structure. Moreover, binding of the CO molecule to the heme induces a long range effect on the Cu,Zn active site, as evidenced by both MD simulations and in vitro experiments. MD simulation of the CO bound system, in fact, highlighted a structural rearrangement of the protein-protein hydrogen bond network in the region of the Cu,Zn active site, correlated with an increase in water accessibility at short distance from the copper atom. In line, in vitro experiments evidenced an increase of copper accessibility to a chelating agent when the CO molecule binds to the heme group, as compared to a heme deprived HdSOD. Altogether, our results support the hypothesis that the HdSOD is a heme-sensor protein, in which binding to small gaseous molecules modulates the enzyme superoxide activity as an adaptive response to the bacterial environment.

  3. Galectin-3-Binding and Metastasis

    Science.gov (United States)

    Nangia-Makker, Pratima; Balan, Vitaly; Raz, Avraham

    2013-01-01

    i. Summary Galectin-3 is a member of a family of carbohydrate-binding proteins. It is present in the nucleus, the cytoplasm and also extracellular matrix of many normal and neoplastic cell types. Arrays of reports show an upregulation of this protein in transformed and metastatic cell lines (1, 2). Moreover, in many human carcinomas, an increased expression of galectin-3 correlates with progressive tumor stages (3–6). Several lines of analysis have demonstrated that the galectins participate in cell-cell and cell-matrix interactions by recognizing and binding complimentary glycoconjugates and thereby play a crucial role in normal and pathological processes. Elevated expression of the protein is associated with an increased capacity for anchorage-independent growth, homotypic aggregation, and tumor cell lung colonization (7–9). In this chapter we describe the methods of purification of galectin-3 from transformed E. coli and some of the commonly used functional assays for analyzing galectin-3 binding. PMID:22674139

  4. Nutraceutical and pharmacological implications of marine carbohydrates.

    Science.gov (United States)

    Pallela, Ramjee

    2014-01-01

    Current day's research has been focusing much on the potential pharmacological or nutraceutical agents of selective health benefits with less toxicity. As a consequence of increased demand of nutritional supplements of great medicinal values, development of therapeutic agents from natural sources, in particular, marine environment are being considered much important. A diverse array of marine natural products containing medicinally useful nutritional substances, i.e., marine nutraceuticals have been focused to the benefit of mankind. Carbohydrates, by being constituted in considerable amount of many marine organisms display several nutraceutical and pharmaceutical behavior to defend from various diseases. Moreover, the carbohydrates from algae as well as from shellfish wastes, like chitosan and its derivatives, showed tremendous applications in biology and biomedicine. In the current chapter, several of marine carbohydrates from various marine flora and fauna have been covered with their applications and prospects in the development of nutraceuticals and pharmaceuticals.

  5. Investigation of Carbohydrate Recognition via Computer Simulation

    Directory of Open Access Journals (Sweden)

    Quentin R. Johnson

    2015-04-01

    Full Text Available Carbohydrate recognition by proteins, such as lectins and other (biomolecules, can be essential for many biological functions. Recently, interest has arisen due to potential protein and drug design and future bioengineering applications. A quantitative measurement of carbohydrate-protein interaction is thus important for the full characterization of sugar recognition. We focus on the aspect of utilizing computer simulations and biophysical models to evaluate the strength and specificity of carbohydrate recognition in this review. With increasing computational resources, better algorithms and refined modeling parameters, using state-of-the-art supercomputers to calculate the strength of the interaction between molecules has become increasingly mainstream. We review the current state of this technique and its successful applications for studying protein-sugar interactions in recent years.

  6. Inhibiting oral intoxication of botulinum neurotoxin A complex by carbohydrate receptor mimics.

    Science.gov (United States)

    Lee, Kwangkook; Lam, Kwok-Ho; Kruel, Anna-Magdalena; Mahrhold, Stefan; Perry, Kay; Cheng, Luisa W; Rummel, Andreas; Jin, Rongsheng

    2015-12-01

    Botulinum neurotoxins (BoNTs) cause the disease botulism manifested by flaccid paralysis that could be fatal to humans and animals. Oral ingestion of the toxin with contaminated food is one of the most common routes for botulism. BoNT assembles with several auxiliary proteins to survive in the gastrointestinal tract and is subsequently transported through the intestinal epithelium into the general circulation. Several hemagglutinin proteins form a multi-protein complex (HA complex) that recognizes host glycans on the intestinal epithelial cell surface to facilitate BoNT absorption. Blocking carbohydrate binding to the HA complex could significantly inhibit the oral toxicity of BoNT. Here, we identify lactulose, a galactose-containing non-digestible sugar commonly used to treat constipation, as a prototype inhibitor against oral BoNT/A intoxication. As revealed by a crystal structure, lactulose binds to the HA complex at the same site where the host galactose-containing carbohydrate receptors bind. In vitro assays using intestinal Caco-2 cells demonstrated that lactulose inhibits HA from compromising the integrity of the epithelial cell monolayers and blocks the internalization of HA. Furthermore, co-administration of lactulose significantly protected mice against BoNT/A oral intoxication in vivo. Taken together, these data encourage the development of carbohydrate receptor mimics as a therapeutic intervention to prevent BoNT oral intoxication.

  7. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. PPIP5K1 modulates ligand competition between diphosphoinositol polyphosphates and PtdIns(3,4,5)P3 for polyphosphoinositide-binding domains.

    Science.gov (United States)

    Gokhale, Nikhil A; Zaremba, Angelika; Janoshazi, Agnes K; Weaver, Jeremy D; Shears, Stephen B

    2013-08-01

    We describe new signalling consequences for PPIP5K1 (diphosphoinositol pentakisphosphate kinase type 1)-mediated phosphorylation of InsP6 and 5-InsP7 to 1-InsP7 and InsP8. In NIH 3T3 cells, either hyperosmotic stress or receptor activation by PDGF (platelet-derived growth factor) promoted translocation of PPIP5K1 from the cytoplasm to the plasma membrane. The PBD1 (polyphosphoinositide-binding domain) in PPIP5K1 recapitulated that translocation. Mutagenesis of PBD1 to reduce affinity for PtdIns(3,4,5)P3 prevented translocation. Using surface plasmon resonance, we found that PBD1 association with vesicular PtdIns(3,4,5)P3 was inhibited by InsP6 and diphosphoinositol polyphosphates. However, the inhibition by PPIP5K1 substrates (IC50: 5-InsP7=5 μM and InsP6=7 μM) was substantially more potent than that of the PPIP5K1 products (IC50: InsP8=32 μM and 1-InsP7=43 μM). This rank order of ligand competition with PtdIns(3,4,5)P3 was also exhibited by the PH (pleckstrin homology) domains of Akt (also known as protein kinase B), GRP1 (general receptor for phosphoinositides 1) and SIN1 (stress-activated protein kinase-interaction protein 1). We propose that, in vivo, PH domain binding of InsP6 and 5-InsP7 suppresses inappropriate signalling ('noise') from stochastic increases in PtdIns(3,4,5)P3. That restraint may be relieved by localized depletion of InsP6 and 5-InsP7 at the plasma membrane following PPIP5K1 recruitment. We tested this hypothesis in insulin-stimulated L6 myoblasts, using mTOR (mechanistic/mammalian target of rapamycin)-mediated phosphorylation of Akt on Ser473 as a readout for SIN1-mediated translocation of mTORC (mTOR complex) 2 to the plasma membrane [Zoncu, Efeyan and Sabatini (2011) Nat. Rev. Mol. Cell Biol. 12, 21-35]. Knockdown of PPIP5K1 expression was associated with a 40% reduction in Ser473 phosphorylation. A common feature of PtdIns(3,4,5)P3-based signalling cascades may be their regulation by PPIP5K1.

  9. Capillary electrophoresis-mass spectrometry of carbohydrates.

    Science.gov (United States)

    Zaia, Joseph

    2013-01-01

    The development of methods for capillary electrophoresis (CE) with on-line mass spectrometric detection (CE/MS) is driven by the need for accurate, robust, and sensitive glycomics analysis for basic biomedicine, biomarker discovery, and analysis of recombinant protein therapeutics. One important capability is to profile glycan mixtures with respect to the patterns of substituents including sialic acids, acetate, sulfate, phosphate, and other groups. There is additional need for an MS-compatible separation system capable of resolving carbohydrate isomers. This chapter summarizes applications of CS/MS to analysis of carbohydrates, glycoproteins, and glycopeptides that have appeared since 2008. Readers are referred to recent comprehensive reviews covering earlier publications.

  10. Synthesis of chiral dopants based on carbohydrates.

    Science.gov (United States)

    Tsuruta, Toru; Koyama, Tetsuo; Yasutake, Mikio; Hatano, Ken; Matsuoka, Koji

    2014-07-01

    Chiral dopants based on carbohydrates for nematic liquid crystals were synthesized from D-glucose, and their helical twisting power (HTP) values were evaluated. The chiral dopants induced helices in the host nematic liquid crystals. An acetyl derivative having an ether-type glycosidic linkage between carbohydrate and a mesogenic moiety showed the highest HTP value of 10.4 μm(-1), while an acetyl derivative having an anomeric ester-type linkage did not show any HTP. It was surprising that this molecule had no HTP despite the presence of chirality in the molecule. A relationship between HTP and specific rotation was not observed in this study.

  11. Retinoids modulate expression of the endocytic partners megalin, cubilin, and disabled-2 and uptake of vitamin D-binding protein in human mammary cells.

    Science.gov (United States)

    Chlon, Timothy M; Taffany, David A; Welsh, Joellen; Rowling, Matthew J

    2008-07-01

    The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells.

  12. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination.

    Science.gov (United States)

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R; Guerrero, Justin A; Shen, Tian; Casali, Paolo

    2017-02-08

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S-S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52(+/+) counterparts, which display normal CSR, Rad52(-/-) B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S-S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S-S recombination, as emphasized by the significantly greater CSR reduction in Rad52(-/-) versus Rad52(+/+) B cells on Ku86 knockdown.

  13. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

    Science.gov (United States)

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R.; Guerrero, Justin A.; Shen, Tian; Casali, Paolo

    2017-01-01

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52−/− B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S–S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S–S recombination, as emphasized by the significantly greater CSR reduction in Rad52−/− versus Rad52+/+ B cells on Ku86 knockdown. PMID:28176781

  14. Pathways for Modulating Exosome Lipids Identified By High-Density Lipoprotein-Like Nanoparticle Binding to Scavenger Receptor Type B-1.

    Science.gov (United States)

    Angeloni, Nicholas L; McMahon, Kaylin M; Swaminathan, Suchitra; Plebanek, Michael P; Osman, Iman; Volpert, Olga V; Thaxton, C Shad

    2016-03-11

    Exosomes are produced by cells to mediate intercellular communication, and have been shown to perpetuate diseases, including cancer. New tools are needed to understand exosome biology, detect exosomes from specific cell types in complex biological media, and to modify exosomes. Our data demonstrate a cellular pathway whereby membrane-bound scavenger receptor type B-1 (SR-B1) in parent cells becomes incorporated into exosomes. We tailored synthetic HDL-like nanoparticles (HDL NP), high-affinity ligands for SR-B1, to carry a fluorescently labeled phospholipid. Data show SR-B1-dependent transfer of the fluorescent phospholipid from HDL NPs to exosomes. Modified exosomes are stable in serum and can be directly detected using flow cytometry. As proof-of-concept, human serum exosomes were found to express SR-B1, and HDL NPs can be used to label and isolate them. Ultimately, we discovered a natural cellular pathway and nanoparticle-receptor pair that enables exosome modulation, detection, and isolation.

  15. Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Science.gov (United States)

    Kazi, Julhash U; Rönnstrand, Lars

    2012-01-01

    Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient s