ANALYSIS OF STRUCTURAL ELEMENT OF FAMILY 6 CARBOHYDRATE BINDING MODULE (CTCBM6B OF ALPHA-L-ARABINOFURANOSIDASE FROM CLOSTRIDIUM THERMOCELLUM

Directory of Open Access Journals (Sweden)

Shadab Ahmed

2013-06-01

Full Text Available The amino acid sequence of a family 6 carbohydrate binding module (CtCBM6B from Clostridium thermocellum alpha-L-arabinofuranosidase showed close evolutionary relationship with some other member of family 6 carbohydrate binding modules. The CD spectrum analysis confirmed the secondary structure prediction of CtCBM6B as both showed beta-sheets (44-48% and random coils (52-54% and no alpha-helix. The hydrogen bonding plot of CtCBM6B showed many segments of parallel and anti-parallel beta-strands which was similar to the secondary structure prediction by PSIPRED VIEW. The three dimensional structure of CtCBM6B generated by MODELLER revealed a typical beta-sandwich architecture at its core, characteristic of beta-jelly roll CBM superfamily. The Ramachandran plot analysis by PROCHECK showed that out of 134 residues, 92.9% were in most favoured region, 6.2% in additionally allowed region and only 0.9% in generously allowed region which indicated a stable conformation of 3D model of CtCBM6B. The docking analysis of CtCBM6B for finding putative ligand binding sites showed that it has high binding affinity for arabinobiose, beta-L-arabinofuranose and beta-D-xylopyranose indicated by lower ligand binding energy (-14.28 kcal mol–1, -12.5 kcal mol–1 and -11.3 kcal mol–1, respectively. CtCBM6B also showed appreciable binding affinity with alpha-D-xylopyranose (–10.8 kcal mol–1, beta-L-arabinopyranose (–10.2 kcal mol-1, alpha-L-arabinopyranose (–10.0 kcal mol–1 and alpha-L-arabinofuranose (–8.75 kcal mol–1. The results indicated that CtCBM6B has high potential for binding arabinan, xylans and substituted xylans.

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 ...

1H, 15N and 13C backbone and side-chain resonance assignments of a family 32 carbohydrate-binding module from the Clostridium perfringens NagH.

Science.gov (United States)

Grondin, Julie M; Chitayat, Seth; Ficko-Blean, Elizabeth; Boraston, Alisdair B; Smith, Steven P

2012-10-01

The Gram-positive anaerobe Clostridium perfringens is an opportunistic bacterial pathogen that secretes a battery of enzymes involved in glycan degradation. These glycoside hydrolases are thought to be involved in turnover of mucosal layer glycans, and in the spread of major toxins commonly associated with the development of gastrointestinal diseases and gas gangrene in humans. These enzymes employ multi-modularity and carbohydrate-binding function to degrade extracellular eukaryotic host sugars. Here, we report the full (1)H, (15)N and (13)C chemical shift resonance assignments of the first family 32 carbohydrate-binding module from NagH, a secreted family 84 glycoside hydrolase.

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. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii*

Science.gov (United States)

Das, Subha Narayan; Wagenknecht, Martin; Nareddy, Pavan Kumar; Bhuvanachandra, Bhoopal; Niddana, Ramana; Balamurugan, Rengarajan; Swamy, Musti J.; Moerschbacher, Bruno M.; Podile, Appa Rao

2016-01-01

We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici. Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction. PMID:27405759

Simulation analysis of the cellulase Cel7A carbohydrate binding module on the surface of the cellulose Iβ

Energy Technology Data Exchange (ETDEWEB)

Alekozai, Emal M. [Univ. of Heidelberg (Germany); Univ. of Tennessee, Knoxville, TN (United States); GhattyVenkataKrishna, Pavan K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Uberbacher, Edward C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Crowley, Michael F. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States); Smith, Jeremy C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Cheng, Xiaolin [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)

2013-08-22

The Family 7 cellobiohydrolase (Cel7A) from Trichoderma reesei consists of a carbohydrate-binding module (CBM) joined by a linker to a catalytic domain. Cellulose hydrolysis is limited by the accessibility of Cel7A to crystalline substrates, which is perceived to be primarily mediated by the CBM. The binding of CBM to the cellulose I fiber is characterized by combined Brownian dynamics (BD) and molecular dynamics (MD) simulations. Our results confirm that CBM prefers to dock to the hydrophobic than to the hydrophilic fiber faces. Both electrostatic (ES) and van der Waals (VDW) interactions are required for achieving the observed binding preference. The VDW interactions play a more important role in stabilizing the CBM-fiber binding, whereas the ES interactions contribute through the formation of a number of hydrogen bonds between the CBM and the fiber. At long distances, an ES steering effect is also observed that tends to align the CBM in an antiparallel manner relative to the fiber axis. Moreover, the MD results reveal hindered diffusion of the CBM on all fiber surfaces. The binding of the CBM to the hydrophobic surfaces is found to involve partial dewetting at the CBM-fiber interface coupled with local structural arrangements of the protein. The present simulation results complement and rationalize a large body of previous work and provide detailed insights into the mechanism of the CBM-cellulose fiber interactions.

Cell wall regeneration in Bangia atropurpurea (Rhodophyta) protoplasts observed using a mannan-specific carbohydrate-binding module.

Science.gov (United States)

Umemoto, Yoshiaki; Araki, Toshiyoshi

2010-02-01

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (beta-1,4-mannan, beta-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized beta-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of beta-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP-CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP-CBM27 maintained its binding ability to soluble beta-mannans, while normal GFP could not bind to beta-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP-CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete beta-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, beta-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP-CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP-CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of beta-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.

Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis.

Science.gov (United States)

Liu, Peng; Stajich, Jason E

2015-04-01

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians. Copyright © 2015 Elsevier Inc. All rights reserved.

A role for carbohydrate recognition in mammalian sperm-egg binding

International Nuclear Information System (INIS)

Clark, Gary F.

2014-01-01

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

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.

Structural Basis for Carbohydrate Recognition and Anti-inflammatory Modulation by Gastrointestinal Nematode Parasite Toxascaris leonina Galectin*

Science.gov (United States)

Hwang, Eun Young; Jeong, Mi Suk; Park, Sang Kyun; Ha, Sung Chul; Yu, Hak Sun; Jang, Se Bok

2016-01-01

Toxascaris leonina galectin (Tl-gal) is a galectin-9 homologue protein isolated from an adult worm of the canine gastrointestinal nematode parasite, and Tl-gal-vaccinated challenge can inhibit inflammation in inflammatory bowel disease-induced mice. We determined the first X-ray structures of full-length Tl-gal complexes with carbohydrates (lactose, N-acetyllactosamine, lacto-N-tetraose, sialyllactose, and glucose). Bonds were formed on concave surfaces of both carbohydrate recognition domains (CRDs) in Tl-gal. All binding sites were found in the HXXXR and WGXEER motifs. Charged Arg61/Arg196 and Glu80/Glu215 on the conserved motif of Tl-gal N-terminal CRD and C-terminal CRD are critical amino acids for recognizing carbohydrate binding, and the residues can affect protein folding and structure. The polar amino acids His, Asn, and Trp are also important residues for the interaction with carbohydrates through hydrogen bonding. Hemagglutination activities of Tl-gal were inhibited by interactions with carbohydrates and mutations. We found that the mutation of Tl-gal (E80A/E215A) at the carbohydrate binding region induced protein aggregation and could be caused in many diseases. The short linker region between the N-terminal and C-terminal CRDs of Tl-gal was very stable against proteolysis and maintained its biological activity. This structural information is expected to elucidate the carbohydrate recognition mechanism of Tl-gal and improve our understanding of anti-inflammatory mediators and modulators of immune response. PMID:27742836

Chimeric cellulase matrix for investigating intramolecular synergism between non-hydrolytic disruptive functions of carbohydrate-binding modules and catalytic hydrolysis.

Science.gov (United States)

Wang, Yuguo; Tang, Rentao; Tao, Jin; Wang, Xiaonan; Zheng, Baisong; Feng, Yan

2012-08-24

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis*

Science.gov (United States)

Wang, Yuguo; Tang, Rentao; Tao, Jin; Wang, Xiaonan; Zheng, Baisong; Feng, Yan

2012-01-01

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose. PMID:22778256

The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

Science.gov (United States)

Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

2016-05-09

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity

Defining carbohydrate binding of glucan phosphatases via Affinity gel electrophoresis

DEFF Research Database (Denmark)

Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

2016-01-01

was to determine a technique to measure carbohydrate binding quickly and efficiently. We established a protocol to reproducibly and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE). The results show that the various glucan phosphatases possess differing...

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...

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

Science.gov (United States)

Loris, R; De Greve, H; Dao-Thi, M H; Messens, J; Imberty, A; Wyns, L

2000-08-25

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework. Copyright 2000 Academic Press.

The carbohydrate-binding module (CBM)-like sequence is crucial for rice CWA1/BC1 function in proper assembly of secondary cell wall materials.

Science.gov (United States)

Sato, Kanna; Ito, Sachiko; Fujii, Takeo; Suzuki, Ryu; Takenouchi, Sachi; Nakaba, Satoshi; Funada, Ryo; Sano, Yuzou; Kajita, Shinya; Kitano, Hidemi; Katayama, Yoshihiro

2010-11-01

We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials.

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

Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each ...

Impact of orientation of carbohydrate binding modules family 22 and 6 on the catalytic activity of Thermotoga maritima xylanase XynB.

Science.gov (United States)

Tajwar, Razia; Shahid, Saher; Zafar, Rehan; Akhtar, Muhammad Waheed

2017-11-01

Xylanase XynB of the hyperthermophile Thermotoga maritima, which belongs to glycoside hydrolase family 10 (GH10), does not have an associated carbohydrate binding module (CBM) in the native state. CBM6 and CBM22 from a thermophile Clostridium thermocellum were fused to the catalytic domain of XynB (XynB-C) to determine the effects on activity and other properties. XynB-B22C and XynB-CB22, produced by fusing CBM22 to the N- and C-terminal of XynB-C, showed 1.7- and 3.24-fold increase in activity against the insoluble birchwood xylan, respectively. Similarly, CBM6 when attached to the C-terminal of XynB-C resulted in 2.0-fold increase in activity, whereas its attachment to the N-terminal did not show any increase of activity. XynB-B22C and XynB-CB22 retained all the activity, whereas XynB-B6C and XynB-CB6 lost 17 and 11% of activity, respectively, at 60°C for 4h. Thermostability data and the secondary structure contents obtained by molecular modelling are in agreement with the data from circular dichroism analysis. Molecular modelling analysis showed that the active site residues of the catalytic domain and the binding residues of CBM6 and CBM22 were located on the surface of molecule, except XynB-B6C, where the binding residues were found somewhat buried. In the case of XynB-CB22, the catalytic and the binding residues seem to be located favorably adjacent to each other, thus showing higher increase in activity. This study shows that the active site residues of the catalytic domain and the binding residues of the CBM are arranged in a unique fashion, not reported before. Copyright © 2017 Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

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

1987-01-01

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 μM, comparable to that of GalNAc. The K/sub i,app/ of GalNASA decreased to 40 μm 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 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 125 I-GalNASA exhibited a K/sub d/ of 15-40 μM, 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

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.

Carbohydrate Microarrays in Plant Science

DEFF Research Database (Denmark)

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

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...... 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....

Monovalent cation and amiloride analog modulation of adrenergic ligand binding to the unglycosylated alpha 2B-adrenergic receptor subtype

International Nuclear Information System (INIS)

Wilson, A.L.; Seibert, K.; Brandon, S.; Cragoe, E.J. Jr.; Limbird, L.E.

1991-01-01

The unglycosylated alpha 2B subtype of the alpha 2-adrenergic receptor found in NG-108-15 cells possesses allosteric regulation of adrenergic ligand binding by monovalent cations and 5-amino-substituted amiloride analogs. These findings demonstrate that allosteric modulation of adrenergic ligand binding is not a property unique to the alpha 2A subtype. The observation that amiloride analogs as well as monovalent cations can modulate adrenergic ligand binding to the nonglycosylated alpha 2B subtype indicates that charge shielding due to carbohydrate moieties does not play a role in this allosteric modulation but, rather, these regulatory effects result from interactions of cations and amiloride analogs with the protein moiety of the receptor. Furthermore, the observation that both alpha 2A and alpha 2B receptor subtypes are modulated by amiloride analogs suggests that structural domains that are conserved between the two are likely to be involved in this allosteric modulation

Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering.

Science.gov (United States)

Nakanishi, Akihito; Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

2012-10-23

To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.

Photonic crystal borax competitive binding carbohydrate sensing motif†

Science.gov (United States)

Cui, Qingzhou; Muscatello, Michelle M. Ward; Asher, Sanford A.

2009-01-01

We developed a photonic crystal sensing method for diol containing species such as carbohydrates based on a poly(vinyl alcohol) (PVA) hydrogel containing an embedded crystalline colloidal array (CCA). The polymerized CCA (PCCA) diffracts visible light. We show that in the presence of borax the diffraction wavelength shifts as the concentration of glucose changes. The diffraction shifts result from the competitive binding of glucose to borate, which reduces the concentration of borate bound to the PVA diols. PMID:19381378

Photonic crystal borax competitive binding carbohydrate sensing motif.

Science.gov (United States)

Cui, Qingzhou; Ward Muscatello, Michelle M; Asher, Sanford A

2009-05-01

We developed a photonic crystal sensing method for diol containing species such as carbohydrates based on a poly(vinyl alcohol) (PVA) hydrogel containing an embedded crystalline colloidal array (CCA). The polymerized CCA (PCCA) diffracts visible light. We show that in the presence of borax the diffraction wavelength shifts as the concentration of glucose changes. The diffraction shifts result from the competitive binding of glucose to borate, which reduces the concentration of borate bound to the PVA diols.

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.

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

Science.gov (United States)

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

2000-04-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from Clostridium perfringens.

Directory of Open Access Journals (Sweden)

Julie M Grondin

Full Text Available Clostridium perfringens is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31. This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the CpGH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.

The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module.

Science.gov (United States)

Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H P; Whisstock, James C; Baker, Edward N; Kreikemeyer, Bernd

2012-11-02

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain.

Structure of Dioclea virgata lectin: relations between carbohydrate binding site and nitric oxide production

International Nuclear Information System (INIS)

Delatorre, P.; Gadelha, C.A.A.; Santi-Gadelha, T.; Nobrega, R.B.; Rocha, B.A.M.; Nascimento, K.S.; Naganao, C.S.; Sampaio, A.H.; Cavada, B.S.; Pires, A.F.; Assreuy, A.M.S.

2012-01-01

Full text: Lectins are proteins/glycoproteins with at least one noncatalytic domain binding reversibly to specific monosaccharides or oligosaccharides. By binding to carbohydrate moieties on the cell surface, lectins participate in a range of cellular processes without changing the properties of the carbohydrates involved. The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological proper- ties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. The DvirL diffraction analysis revealed that both the native crystal and the X-Man-complexed form are orthorhombic and belong to space group I222. The cell parameters were: a=65.4 , b=86.6 and c=90.2 (native structure), and a=61.89 , b=87.67 and c=88.78 (X-Man-complexed structure). An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site-ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration. (author)

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...

The Extracellular Protein Factor Epf from Streptococcus pyogenes Is a Cell Surface Adhesin That Binds to Cells through an N-terminal Domain Containing a Carbohydrate-binding Module*

Science.gov (United States)

Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H. P.; Whisstock, James C.; Baker, Edward N.; Kreikemeyer, Bernd

2012-01-01

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain. PMID:22977243

Binding of Human GII.4 Norovirus Virus-Like Particles to Carbohydrates of Romaine Lettuce Leaf Cell Wall Materials

Science.gov (United States)

Esseili, Malak A.

2012-01-01

Norovirus (NoV) genogroup II genotype 4 (GII.4) strains are the dominant cause of the majority of food-borne outbreaks, including those that involve leafy greens, such as lettuce. Since human NoVs use carbohydrates of histo-blood group antigens as receptors/coreceptors, we examined the role of carbohydrates in the attachment of NoV to lettuce leaves by using virus-like particles (VLPs) of a human NoV/GII.4 strain. Immunofluorescence analysis showed that the VLPs attached to the leaf surface, especially to cut edges, stomata, and along minor veins. Binding was quantified using enzyme-linked immunosorbent assay (ELISA) performed on cell wall materials (CWM) from innermost younger leaves and outermost lamina of older leaves. The binding to CWM of older leaves was significantly (P lettuce CWM by utilizing multiple carbohydrate moieties. This binding may enhance virus persistence on the leaf surface and prevent effective decontamination. PMID:22138991

Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

Science.gov (United States)

Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

2004-09-01

We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

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.

Glycobiochemistry of ticks, vectors of infectious diseases: carbohydrate-binding proteins and glycans

Czech Academy of Sciences Publication Activity Database

Grubhoffer, Libor; Hajdušek, Ondřej; Vancová, Marie; Štěrba, Ján; Rudenko, Natalia

2009-01-01

Roč. 276, S1 (2009), s. 141-141 ISSN 1742-464X. [34th FEBS Congress: Life's Molecular Interactions. 04.07.2009-09.07.2009, Prague] R&D Projects: GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518 Keywords : Carbohydrate-binding molecules * Ixodes ricinus * knock-down Subject RIV: EB - Genetics ; Molecular Biology

Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses.

Science.gov (United States)

Chappell, J D; Duong, J L; Wright, B W; Dermody, T S

2000-09-01

The reovirus attachment protein, sigma1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The sigma1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of sigma1 that binds cell surface carbohydrate. Chimeric and truncated sigma1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-sigma1 antibodies, and oligomerization indicates that the chimeric and truncated sigma1 proteins are properly folded. To assess carbohydrate binding, recombinant sigma1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated sigma1 proteins, the sialic acid-binding domain of type 3 sigma1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted beta-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of sigma1 protein purified from virions. In contrast, the homologous region of T1L sigma1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 sigma1 tail. Furthermore, our findings indicate that T1L and T3D sigma1 proteins contain different arrangements of receptor-binding

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.

[Inhibition by cysteine of the carbohydrate-binding activity of lectins from Ricinus communis, Canavalia ensiformis and Euonymus europaeus].

Science.gov (United States)

Dvorkin, V M

1985-10-01

Precipitation induced by different lectins has been studied in the presence of some aminoacids. It was shown that precipitates formed by lectins from Ricinus communis (RCA1), Canavalia ensiformis (Con A), Euonymus europaeus (Eel) in the presence of appropriate carbohydrate-containing molecules disappeared after cysteine addition, like after addition of specific carbohydrate precipitation inhibitors. It is assumed that cysteine residues of RCA1, Con A and Eel lectins are essential for their carbohydrate binding activity.

Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

Science.gov (United States)

Chang, Wei-Chieh; Liu, Kai-Lun; Hsu, Fang-Ciao; Jeng, Shih-Tong; Cheng, Yi-Sheng

2012-01-01

Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (KA) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. PMID:22808208

Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

International Nuclear Information System (INIS)

Lummis, S.C.R.; Johnston, G.A.R.; Nicoletti, G.; Holan, G.

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 [ 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 [ 3 H]diazepam binding are those that are active in displacing [ 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

Modulation of DNA binding by gene-specific transcription factors.

Science.gov (United States)

Schleif, Robert F

2013-10-01

The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

Modulation of the Gastrointestinal Microbiome with Nondigestible Fermentable Carbohydrates To Improve Human Health.

Science.gov (United States)

Deehan, Edward C; Duar, Rebbeca M; Armet, Anissa M; Perez-Muñoz, Maria Elisa; Jin, Mingliang; Walter, Jens

2017-09-01

There is a clear association between the gastrointestinal (GI) microbiome and the development of chronic noncommunicable diseases, providing a rationale for the development of strategies that target the GI microbiota to improve human health. In this article, we discuss the potential of supplementing the human diet with nondigestible fermentable carbohydrates (NDFCs) to modulate the composition, structure, diversity, and metabolic potential of the GI microbiome in an attempt to prevent or treat human disease. The current concepts by which NDFCs can be administered to humans, including prebiotics, fermentable dietary fibers, and microbiota-accessible carbohydrates, as well as the mechanisms by which these carbohydrates exert their health benefits, are discussed. Epidemiological research presents compelling evidence for the health effects of NDFCs, with clinical studies providing further support for some of these benefits. However, rigorously designed human intervention studies with well-established clinical markers and microbial endpoints are still essential to establish (i) the clinical efficiency of specific NDFCs, (ii) the causal role of the GI microbiota in these effects, (iii) the underlying mechanisms involved, and (iv) the degree by which inter-individual differences between GI microbiomes influence these effects. Such studies would provide the mechanistic understanding needed for a systematic application of NDFCs to improve human health via GI microbiota modulation while also allowing the personalization of these dietary strategies.

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.

The starch-binding domain family CBM41 - an in silico analysis of evolutionary relationships

DEFF Research Database (Denmark)

Janeček, Štefan; Majzlová, Katarína; Svensson, Birte

2017-01-01

Within the CAZy database, there are 81 carbohydrate-binding module (CBM) families. A CBM represents a non-catalytic domain in a modular arrangement of glycoside hydrolases (GHs). The present in silico study has been focused on starch-binding domains from the family CBM41 that are usually part...

Expression of the C-terminal family 22 carbohydratebinding module ...

African Journals Online (AJOL)

... not reveal marked cell wall phenotype. In addition, there were no observable changes in the height or the appearance of the transgenic plants expressing the CBM22-2 module. The results indicate that the family 22 carbohydrate binding module is not a potential candidate for use in in planta modification of the cell wall.

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.

Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

Science.gov (United States)

Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

2015-02-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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.

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.

Recognition properties of receptors consisting of imidazole and indole recognition units towards carbohydrates

Directory of Open Access Journals (Sweden)

Monika Mazik

2010-02-01

Full Text Available Compounds 4 and 5, including both 4(5-substituted imidazole or 3-substituted indole units as the entities used in nature, and 2-aminopyridine group as a heterocyclic analogue of the asparagine/glutamine primary amide side chain, were prepared and their binding properties towards carbohydrates were studied. The design of these receptors was inspired by the binding motifs observed in the crystal structures of protein–carbohydrate complexes. 1H NMR spectroscopic titrations in competitive and non-competitive media as well as binding studies in two-phase systems, such as dissolution of solid carbohydrates in apolar media, revealed both highly effective recognition of neutral carbohydrates and interesting binding preferences of these acyclic compounds. Compared to the previously described acyclic receptors, compounds 4 and 5 showed significantly increased binding affinity towards β-galactoside. Both receptors display high β- vs. α-anomer binding preferences in the recognition of glycosides. It has been shown that both hydrogen bonding and interactions of the carbohydrate CH units with the aromatic rings of the receptors contribute to the stabilization of the receptor–carbohydrate complexes. The molecular modeling calculations, synthesis and binding properties of 4 and 5 towards selected carbohydrates are described and compared with those of the previously described receptors.

Carbohydrate epitopes on Haemonchus contortus antigens

NARCIS (Netherlands)

Schallig, H. D.; van Leeuwen, M. A.

1996-01-01

Extracts of infective larvae and adults of the trichostrongylid Haemonchus contortus were studied for the presence of carbohydrate moieties. Several different lectin-binding sites were demonstrated in both stages using a panel of nine lectins. The carbohydrate specificity of the lectins used

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.

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.

Carbohydrate-actuated nanofluidic diode: switchable current rectification in a nanopipette.

Science.gov (United States)

Vilozny, Boaz; Wollenberg, Alexander L; Actis, Paolo; Hwang, Daniel; Singaram, Bakthan; Pourmand, Nader

2013-10-07

Nanofluidic structures share many properties with ligand-gated ion channels. However, actuating ion conductance in artificial systems is a challenge. We have designed a system that uses a carbohydrate-responsive polymer to modulate ion conductance in a quartz nanopipette. The cationic polymer, a poly(vinylpyridine) quaternized with benzylboronic acid groups, undergoes a transition from swollen to collapsed upon binding to monosaccharides. As a result, the current rectification in nanopipettes can be reversibly switched depending on the concentration of monosaccharides. Such molecular actuation of nanofluidic conductance may be used in novel sensors and drug delivery systems.

A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

Science.gov (United States)

Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

1995-04-01

The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

Bivalent Carbohydrate Binding Is Required for Biological Activity of Clitocybe nebularis Lectin (CNL), the N,N′-Diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc)-specific Lectin from Basidiomycete C. nebularis*

Science.gov (United States)

Pohleven, Jure; Renko, Miha; Magister, Špela; Smith, David F.; Künzler, Markus; Štrukelj, Borut; Turk, Dušan; Kos, Janko; Sabotič, Jerica

2012-01-01

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N′-diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency. PMID:22298779

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.

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

Directory of Open Access Journals (Sweden)

Julius W Kim

Full Text Available 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.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.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.

Distribution in rat tissues of modulator-binding protein of particulate nature

International Nuclear Information System (INIS)

Sobue, K.; Muramoto, Y.; Kakiuchi, S.; Yamazaki, R.

1979-01-01

Studies on Ca 2+ -activatable cyclic nucleotide phosphodiesterase led to the discovery of a protein modulator that is required for the activation of this enzyme by Ca 2+ . Later, this protein has been shown to cause the Ca 2+ -dependent activation of several enzymes that include phosphodiesterase, adenylate cyclase, a protein kinase from muscles, phosphorylase b kinase, actomyosin ATPase, membranous ATPase from erythrocytes and nerve synapses. Thus, modulator protein appears to be an intracellular mediator of actions of Ca 2+ . The present work shows the distribution of this particulate modulator-binding component in rat tissues. This paper also describes the labeling of modulator protein with tritium without deteriorating its biological activities and application of this 3 H-modulator protein to the determination of the Ca ++ dependent binding of modulator protein with membranous protein. This technique proves to be useful in studying enzymes or proteins whose functions are regulated by Ca ++ /modulator protein system. (Auth.)

Thermodynamic Characterization of New Positive Allosteric Modulators Binding to the Glutamate Receptor A2 Ligand-Binding Domain

DEFF Research Database (Denmark)

Nørholm, Ann-Beth; Francotte, Pierre; Goffin, Eric

2014-01-01

, and 5a (5-F) and 5b (6-F) are entropy driven. For 5d (8-F), both quantities were equal in size. Thermodynamic integration (TI) and one-step perturbation (OSP) were used to calculate the relative binding affinity of the modulators. The OSP calculations had a higher predictive power than those from TI......,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. Measurements of ligand binding by isothermal titration calorimetry (ITC) showed similar binding affinities for the modulator series at the GluA2 LBD but differences in the thermodynamic driving forces. Binding of 5c (7-F) and 6 (no-F) is enthalpy driven......, and combined with the shorter total simulation time, we found the OSP method to be more effective for this setup. Furthermore, from the molecular dynamics simulations, we extracted the enthalpies and entropies, and along with the ITC data, this suggested that the differences in binding free energies...

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

Science.gov (United States)

Badino, Silke F; Christensen, Stefan J; Kari, Jeppe; Windahl, Michael S; Hvidt, Søren; Borch, Kim; Westh, Peter

2017-08-01

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 10 3 -10 4 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3

International Nuclear Information System (INIS)

Diehl, Carl; Genheden, Samuel; Modig, Kristofer; Ryde, Ulf; Akke, Mikael

2009-01-01

The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used 15 N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the 15 N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes

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

Czech Academy of Sciences Publication Activity Database

Sillam-Dusses, D.; Hanus, Robert; Poulsen, M.; Roy, V.; Favier, M.; Vasseur-Cognet, M.

2016-01-01

Roč. 6, č. 5 (2016), č. článku 160080. ISSN 2046-2441 R&D Projects: GA ČR(CZ) GA14-12774S Institutional support: RVO:61388963 Keywords : reproduction * phenotypic plasticity * carbohydrate-responsive element-binding protein * transcription factor * social insects * lipogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.481, year: 2016 http://rsob.royalsocietypublishing.org/content/6/5/160080

Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

Science.gov (United States)

Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

2013-07-01

Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

A novel carbohydrate-binding surface layer protein from the hyperthermophilic archaeon Pyrococcus horikoshii.

Science.gov (United States)

Goda, Shuichiro; Koga, Tomoyuki; Yamashita, Kenichiro; Kuriura, Ryo; Ueda, Toshifumi

2018-04-08

In Archaea and Bacteria, surface layer (S-layer) proteins form the cell envelope and are involved in cell protection. In the present study, a putative S-layer protein was purified from the crude extract of Pyrococcus horikoshii using affinity chromatography. The S-layer gene was cloned and expressed in Escherichia coli. Isothermal titration calorimetry analyses showed that the S-layer protein bound N-acetylglucosamine and induced agglutination of the gram-positive bacterium Micrococcus lysodeikticus. The protein comprised a 21-mer structure, with a molecular mass of 1,340 kDa, as determined using small-angle X-ray scattering. This protein showed high thermal stability, with a midpoint of thermal denaturation of 79 °C in dynamic light scattering experiments. This is the first description of the carbohydrate-binding archaeal S-layer protein and its characteristics.

Decorin binds myostatin and modulates its activity to muscle cells

International Nuclear Information System (INIS)

Miura, Takayuki; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito; Hennebry, Alex; Berry, Carole J.; Sharma, Mridula; Kambadur, Ravi; Nishimura, Takanori

2006-01-01

Myostatin, a member of TGF-β superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-β and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between myostatin and decorin by surface plasmon assay. Decorin interacted with mature myostatin in the presence of concentrations of Zn 2+ greater than 10 μM, but not in the absence of Zn 2+ . Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K D ) of 2.02 x 10 -8 M and 9.36 x 10 -9 M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap myostatin and modulate its activity to myogenic cells in the ECM

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.

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.

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

Science.gov (United States)

Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

2016-05-13

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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...

Structural and functional insight into the carbohydrate receptor binding of F4 fimbriae-producing enterotoxigenic Escherichia coli.

Science.gov (United States)

Moonens, Kristof; Van den Broeck, Imke; De Kerpel, Maia; Deboeck, Francine; Raymaekers, Hanne; Remaut, Han; De Greve, Henri

2015-03-27

Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4ab, F4ac, and F4ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeGad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D'-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe(150)-Glu(152) and Val(166)-Glu(170) of FaeGad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D'-α1 loop that borders the FaeGad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

Science.gov (United States)

Hatakeyama, Tomomitsu; Ichise, Ayaka; Unno, Hideaki; Goda, Shuichiro; Oda, Tatsuya; Tateno, Hiroaki; Hirabayashi, Jun; Sakai, Hitomi; Nakagawa, Hideyuki

2017-08-01

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure. © 2017 The Protein Society.

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.

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

Science.gov (United States)

Doxey, Andrew C; Cheng, Zhenyu; Moffatt, Barbara A; McConkey, Brendan J

2010-08-03

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. 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. 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.

Species-specific chitin-binding module 18 expansion in the amphibian pathogen Batrachochytrium dendrobatidis.

Science.gov (United States)

Abramyan, John; Stajich, Jason E

2012-01-01

Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity. Amphibian populations are declining worldwide at an unprecedented rate. Although various factors are thought to contribute to this phenomenon, chytridiomycosis has been identified as one of the leading causes. This deadly fungal disease is cause by Batrachochytrium dendrobatidis, a chytrid fungus species unique in its pathogenicity and, furthermore, its specificity to amphibians. Despite more than two decades of research, the biology of this fungus species and its deadly interaction with amphibians had been notoriously difficult to unravel. Due to the alarming rate of worldwide

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus.

Science.gov (United States)

Gao, Xiang; Esseili, Malak A; Lu, Zhongyan; Saif, Linda J; Wang, Qiuhong

2016-05-15

Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed

Membrane proteins bind lipids selectively to modulate their structure and function.

Science.gov (United States)

Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

2014-06-05

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane

New modulated design, docking and synthesis of carbohydrate-conjugate heterobimetallic CuII-SnIV complex as potential topoisomerase II inhibitor: in vitro DNA binding, cleavage and cytotoxicity against human cancer cell lines.

Science.gov (United States)

Tabassum, Sartaj; Afzal, Mohd; Arjmand, Farukh

2014-03-03

New carbohydrate-conjugate heterobimetallic complexes [C₂₂H₅₀N₆O₁₃CuSnCl₂] (3) and [C₂₂H₅₈N₆O₁₇NiSnCl₂] (4) were synthesized from their monometallic analogs [C₂₂H₅₂N₆O₁₃Cu] (1) and [C₂₂H₆₀N₆O₁₇Ni] (2) containing N-glycoside ligand (L). In vitro DNA binding studies of L and complexes (1-4) with CT DNA were carried out by employing various biophysical and molecular docking techniques which revealed that heterobimetallic complex 3 strongly binds to DNA in comparison to 4, monometallic complexes (1 and 2) and the free ligand. Complex 3 cleaves pBR322 DNA via hydrolytic pathway (confirmed by T4 DNA ligase assay) and inhibited Topo-II activity in a dose-dependent manner. Furthermore, complex 3 was docked into the ATPase domain of human-Topo-II in order to probe the possible mechanism of inhibition. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

Science.gov (United States)

Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

2015-01-01

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

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

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.

Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

KAUST Repository

Simone, Giuseppina; Malara, Natalia Maria; Trunzo, Valentina; Perozziello, Gerardo; Neužil, Pavel; Francardi, Marco; Roveda, Laura; Renne, Maria; Prati, Ubaldo; Mollace, Vincenzo; Manz, Andreas; Di Fabrizio, Enzo M.

2013-01-01

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.

Intestinal absorption of copper: influence of carbohydrates.

Science.gov (United States)

Wapnir, R A; Balkman, C

1992-02-01

Macronutrients can modulate the intestinal absorption of trace elements by binding the metal or altering mucosal function. We investigated whether certain simple and complex carbohydrates modify copper (Cu) absorption, using an in vivo perfusion technique in the rat. Corn syrup solids, which contain a mixture of glucose polymers of diverse length, added at either 20 or 50 mosm/kg enhanced Cu absorption from a 31.5 microM (2 mg/liter) Cu solution (128 +/- 11 and 130 +/- 11 pmol/min x cm, respectively, vs 101 +/- 4 pmol/min x cm, P less than 0.05, in the absence of carbohydrate). This was concomitant with a stimulation of net water absorption (1.05 +/- 0.08 and 0.84 +/- 0.08 microliter/min x cm, respectively, vs 0.63 +/- 0.02 microliter/min x cm with no carbohydrate, P less than 0.05). Glucose, fructose, lactose, or sucrose had no influence on Cu absorption, although they altered water exchanges, an effect attributable to a reduction of the outflow component of fluid recirculation. Low concentrations of lactose resulted in a greater accumulation of Cu in the intestinal mucosa (8.75 +/- 0.71 micrograms/g vs 5.77 +/- 0.68 micrograms/g for controls, P less than 0.05). Hence, solutes that moderately stimulate mucosa-to-serosa fluid influx in a progressive manner, such as glucose polymers, may contribute to functionally increase Cu absorption. Conversely, conditions which tend to reduce water inflow or increase water outflow across the small intestinal mucosa, as may occur with high lactose diets or in cases of chronic diarrhea, may have negative effects.

Carbohydrates as T-cell antigens with implications in health and disease.

Science.gov (United States)

Sun, Lina; Middleton, Dustin R; Wantuch, Paeton L; Ozdilek, Ahmet; Avci, Fikri Y

2016-10-01

Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

International Nuclear Information System (INIS)

Li, Ming V.; Chen, Weiqin; Harmancey, Romain N.; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

2010-01-01

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.

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

International Nuclear Information System (INIS)

Kraschnefski, Mark J.; Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von; Blanchard, Helen

2005-01-01

The carbohydrate-binding component (VP8* 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* 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 2 21 and monoclinic P2 1 ) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8* 64–223 structure by molecular replacement

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.

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.

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.

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

Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft with a charact......Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft...... will address surface sites in both barley alpha-amylase 1 and in the related isozyme 2....

Impact of Carbohydrate Restriction on Healthy Adolescent Development.

Science.gov (United States)

Richmond, Hannah M; Duriancik, David M

2017-09-01

Carbohydrate-restricted diets are known for their impact on weight loss; however, research is still required to determine if low-carbohydrate diets are safe for adolescents. Carbohydrates directly stimulate an insulin response, and studies have recently shown that insulin and binding to respective insulin receptors (IRs) are critical in Kisspeptin (Kiss1) neuronal development. These neurons directly stimulate gonadotropin-releasing hormone, which activates the pituitary-gonadal axis during puberty. This information suggests that carbohydrate restriction may delay pubertal development in adolescents due to the impact on insulin and Kiss1 transcription. Studies have observed disturbed insulin metabolism in Type I Diabetics leading to delayed puberty, along with overfeeding stimulating early pubertal onset. Additionally, recent clinical trials bred female mice with IR deletions on Kiss1 neurons and observed delayed vaginal opening and estrus. Current animal research suggests low carbohydrate intake may delay pubertal onset, however additional research is required to determine outcome in human subjects. Copyright© of YS Medical Media ltd.

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

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.

Muscle insulin binding and plasma levels in relation to liver glucokinase activity, glucose metabolism and dietary carbohydrates in rainbow trout.

Science.gov (United States)

Capilla, Encarnación; Médale, Françoise; Navarro, Isabel; Panserat, Stéphane; Vachot, Christiane; Kaushik, Sadasivam; Gutiérrez, Joaquim

2003-01-31

Rainbow trout were fed for 10 weeks with either a carbohydrate-free diet (C-free) or with four experimental diets containing various levels (20 or 40%) and sources of starch (extruded wheat or peas) in order to examine metabolic utilisation of dietary vegetable carbohydrates and its endocrine control. The study was focused on the parameters described as limiting in glucose metabolism in fish. Feeding trials were conducted at 8 and 18 degrees C to establish whether carbohydrate-rich diets can be used in trout farming irrespective of water temperature. At both temperatures, pea diets (especially the highest level) resulted in a feed efficiency as high as the C-free diet. Fish had similar growth rates except when fed the low wheat content diet. Glycaemia values 6 h after feeding were significantly higher in trout fed carbohydrate diets than those given the C-free diet, whereas plasma insulin levels were similar independently of the levels of dietary starch. This study provides the first evidence that glucokinase (GK) activity and mRNA level in trout liver increase in proportion to the content of dietary starch. Nevertheless, these changes were not correlated with plasma insulin levels. Insulin-like growth factor-I (IGF-I) binding and number of receptors in skeletal muscle were consistently higher than those for insulin but no diet-induced differences were found for any of these parameters. Temperature clearly affected the postprandial profile of glucose and insulin, which both showed lower levels 6 h after feeding at 8 degrees C than at 18 degrees C, which was consistent with a lower feed intake. Glucose and insulin levels decreased markedly 24 h after feeding at 18 degrees C, while they were still high at 8 degrees C, an observation concordant with delayed transit rate. These findings indicate satisfactory adaptation of rainbow trout to diets with a relatively high vegetable starch content, especially when provided as extruded peas, and indicate that diets with

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....

A cell wall-degrading esterase of Xanthomonas oryzae requires a unique substrate recognition module for pathogenesis on rice.

Science.gov (United States)

Aparna, Gudlur; Chatterjee, Avradip; Sonti, Ramesh V; Sankaranarayanan, Rajan

2009-06-01

Xanthomonas oryzae pv oryzae (Xoo) causes bacterial blight, a serious disease of rice (Oryza sativa). LipA is a secretory virulence factor of Xoo, implicated in degradation of rice cell walls and the concomitant elicitation of innate immune responses, such as callose deposition and programmed cell death. Here, we present the high-resolution structural characterization of LipA that reveals an all-helical ligand binding module as a distinct functional attachment to the canonical hydrolase catalytic domain. We demonstrate that the enzyme binds to a glycoside ligand through a rigid pocket comprising distinct carbohydrate-specific and acyl chain recognition sites where the catalytic triad is situated 15 A from the anchored carbohydrate. Point mutations disrupting the carbohydrate anchor site or blocking the pocket, even at a considerable distance from the enzyme active site, can abrogate in planta LipA function, exemplified by loss of both virulence and the ability to elicit host defense responses. A high conservation of the module across genus Xanthomonas emphasizes the significance of this unique plant cell wall-degrading function for this important group of plant pathogenic bacteria. A comparison with the related structural families illustrates how a typical lipase is recruited to act on plant cell walls to promote virulence, thus providing a remarkable example of the emergence of novel functions around existing scaffolds for increased proficiency of pathogenesis during pathogen-plant coevolution.

The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View

Directory of Open Access Journals (Sweden)

Umi Baroroh

2017-01-01

Full Text Available Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including α-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, α-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM and the surface-binding site (SBS of α-amylase could facilitate the substrate binding to the enzyme’s active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera α-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various α-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in α-amylase to increase its catalytic activity, especially towards the insoluble starch.

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.

A phylogenetic study of SPBP and RAI1: evolutionary conservation of chromatin binding modules.

Directory of Open Access Journals (Sweden)

Sagar Darvekar

Full Text Available Our genome is assembled into and array of highly dynamic nucleosome structures allowing spatial and temporal access to DNA. The nucleosomes are subject to a wide array of post-translational modifications, altering the DNA-histone interaction and serving as docking sites for proteins exhibiting effector or "reader" modules. The nuclear proteins SPBP and RAI1 are composed of several putative "reader" modules which may have ability to recognise a set of histone modification marks. Here we have performed a phylogenetic study of their putative reader modules, the C-terminal ePHD/ADD like domain, a novel nucleosome binding region and an AT-hook motif. Interactions studies in vitro and in yeast cells suggested that despite the extraordinary long loop region in their ePHD/ADD-like chromatin binding domains, the C-terminal region of both proteins seem to adopt a cross-braced topology of zinc finger interactions similar to other structurally determined ePHD/ADD structures. Both their ePHD/ADD-like domain and their novel nucleosome binding domain are highly conserved in vertebrate evolution, and construction of a phylogenetic tree displayed two well supported clusters representing SPBP and RAI1, respectively. Their genome and domain organisation suggest that SPBP and RAI1 have occurred from a gene duplication event. The phylogenetic tree suggests that this duplication has happened early in vertebrate evolution, since only one gene was identified in insects and lancelet. Finally, experimental data confirm that the conserved novel nucleosome binding region of RAI1 has the ability to bind the nucleosome core and histones. However, an adjacent conserved AT-hook motif as identified in SPBP is not present in RAI1, and deletion of the novel nucleosome binding region of RAI1 did not significantly affect its nuclear localisation.

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.

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

OpenAIRE

Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

2011-01-01

Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

High-glycemic index carbohydrates abrogate the antiobesity effect of fish oil in mice

DEFF Research Database (Denmark)

Hao, Qin; Lillefosse, Haldis Haukås; Fjære, Even

2012-01-01

Fish oil rich in n-3 polyunsaturated fatty acids is known to attenuate diet-induced obesity and adipose tissue inflammation in rodents. Here we aimed to investigate whether different carbohydrate sources modulated the antiobesity effects of fish oil. By feeding C57BL/6J mice isocaloric high-fat d...... metabolic effects of fish oil by demonstrating that high-GI carbohydrates attenuate the antiobesity effects of fish oil.......Fish oil rich in n-3 polyunsaturated fatty acids is known to attenuate diet-induced obesity and adipose tissue inflammation in rodents. Here we aimed to investigate whether different carbohydrate sources modulated the antiobesity effects of fish oil. By feeding C57BL/6J mice isocaloric high...

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

Science.gov (United States)

Allick, Gideon; Bisschop, Peter H; Ackermans, Mariette T; Endert, Erik; Meijer, Alfred J; Kuipers, Folkert; Sauerwein, Hans P; Romijn, Johannes A

2004-12-01

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 with type 2 diabetes using stable isotopes and euglycemic hyperinsulinemic clamps. At basal insulin levels glucose concentrations were 148 +/- 11 and 123 +/- 11 mg/dl (8.2 +/- 0.6 and 6.8 +/- 0.6 mmol/liter) on the high-carbohydrate and high-fat diet, respectively (P carbohydrate diet (1.88 +/- 0.06 vs. 1.55 +/- 0.05 mg/kg.min (10.44 +/- 0.33 vs. 8.61 +/- 0.28 micromol/kg.min) (P carbohydrate and high-fat diet, respectively. We conclude that short-term variations in dietary carbohydrate to fat ratios affect basal glucose metabolism in people with type 2 diabetes merely through modulation of the rate of glycogenolysis, without affecting insulin sensitivity of glucose metabolism.

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

Science.gov (United States)

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

Science.gov (United States)

Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

2016-04-07

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Carbohydrate determinants in ferret conjunctiva are affected by infection with influenza H1N1 virus

DEFF Research Database (Denmark)

Kirkeby, Svend; Martel, Cyril; Aasted, Bent

2013-01-01

Carbohydrates often accomplish as cell-surface receptors for microorganisms and influenza virus preferentially binds to sialic acid through the viral haemagglutinin. The virus may attach not only to the epithelium in the airways, but also to the surface ocular epithelium.......Carbohydrates often accomplish as cell-surface receptors for microorganisms and influenza virus preferentially binds to sialic acid through the viral haemagglutinin. The virus may attach not only to the epithelium in the airways, but also to the surface ocular epithelium....

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.

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.

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.

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

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

Science.gov (United States)

Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

1985-05-01

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

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.

Glycan microarray analysis of the carbohydrate-recognition specificity of native and recombinant forms of the lectin ArtinM

Directory of Open Access Journals (Sweden)

Y. Liu

2015-12-01

Full Text Available 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.

Variation in 12 porcine genes involved in the carbohydrate moiety assembly of glycosphingolipids does not account for differential binding of F4 Escherichia coli and their fimbriae.

Science.gov (United States)

Goetstouwers, Tiphanie; Van Poucke, Mario; Coddens, Annelies; Nguyen, Van Ut; Melkebeek, Vesna; Deforce, Dieter; Cox, Eric; Peelman, Luc J

2014-10-03

Glycosphingolipids (GSLs) are important membrane components composed of a carbohydrate structure attached to a hydrophobic ceramide. They can serve as specific membrane receptors for microbes and microbial products, such as F4 Escherichia coli (F4 ETEC) and isolated F4 fimbriae. The aim of this study was to investigate the hypothesis that variation in genes involved in the assembly of the F4 binding carbohydrate moiety of GSLs (i.e. ARSA, B4GALT6, GAL3ST1, GALC, GBA, GLA, GLB1, GLB1L, NEU1, NEU2, UGCG, UGT8) could account for differential binding of F4 ETEC and their fimbriae. RT-PCR could not reveal any differential expression of the 12 genes in the jejunum of F4 receptor-positive (F4R(+)) and F4 receptor-negative (F4R(-)) pigs. Sequencing the complete open reading frame of the 11 expressed genes (NEU2 was not expressed) identified 72 mutations. Although some of them might have a structural effect, none of them could be associated with a F4R phenotype. We conclude that no regulatory or structural variation in any of the investigated genes is responsible for the genetic susceptibility of pigs towards F4 ETEC.

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.

High-throughput antibody development and retrospective epitope mapping

DEFF Research Database (Denmark)

Rydahl, Maja Gro

the binding profile - in more or less high resolution - of two small molecular probes, 11 carbohydrate binding modules and 24 monoclonal antibodies. This was made possible by combining the HTP multiplexing capacity of carbohydrate microarrays with diverse glycomic tools, to downstream characterize...

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.

Combined enteral infusion of glutamine, carbohydrates, and antioxidants modulates gut protein metabolism in humans.

Science.gov (United States)

Coëffier, Moïse; Claeyssens, Sophie; Lecleire, Stéphane; Leblond, Jonathan; Coquard, Aude; Bôle-Feysot, Christine; Lavoinne, Alain; Ducrotté, Philippe; Déchelotte, Pierre

2008-11-01

Available data suggest that nutrients can affect intestinal protein metabolism, which contributes to the regulation of gut barrier function. We aimed to assess whether an oral nutritional supplement (ONS) containing glutamine (as the dipeptide Ala-Gln), carbohydrates, and antioxidants would modulate duodenal protein metabolism in healthy humans. Thirty healthy control subjects were included and, over a period of 5 h, received by nasogastric tube either saline or ONS providing 11.7 kcal/kg as 0.877 g Ala-Gln/kg, 3.9 g carbohydrates/kg, and antioxidants (29.25 mg vitamin C/kg, 9.75 mg vitamin E/kg, 195 microg beta-carotene/kg, 5.85 mg Se/kg, and 390 microg Zn/kg) or glutamine (0.585 g/kg, 2.34 kcal/kg). Simultaneously, a continuous intravenous infusion of l-[1-(13)C]-leucine was done until endoscopy. Leucine enrichment was assessed by using gas chromatography-mass spectrometric analysis, and mucosal fractional synthesis rate was calculated by using intracellular amino acid enrichment as precursor. Mucosal proteolytic pathways were also evaluated. ONS infusion resulted in a doubling increase (P < 0.01) of duodenal fractional synthesis rate and a significant (P < 0.05) decrease in cathepsin D-mediated proteolysis compared with saline, whereas proteasome and Ca(2+)-dependent activities were unaffected. ONS infusion significantly (P < 0.01) decreased duodenal glutathione but not glutathione disulfide concentrations or the ratio of glutathione to glutathione disulfide. Insulinemia increased after ONS infusion, whereas plasma essential amino acids decreased. Infusion of glutamine alone did not reproduce ONS effects. ONS infusion improves duodenal protein balance in healthy humans. Further investigations are needed to study the origin of these effects and to evaluate ONS supply in stressed persons.

Residues in the 5th module of the low-density lipoprotein receptor that bind apoE and apoB-100

International Nuclear Information System (INIS)

Kroon, P.A.; Zhang, H.-Y.; Smith, R.

2000-01-01

Full text: The low-density lipoprotein receptor (LDLR) binds and removes cholesterol-rich lipoproteins from the circulation. Its ligand-binding (LB) domain consists of seven cysteine-rich LB modules that bind apoB-100 and apoE. These modules fold into well-defined structures with three disulfide bonds, in the presence of Ca 2+ . The 5th module (LB5) is unique in that it is required to bind both apoB-100 and apoE. The aim of the current study was to map residues in human LB5 that are required for ligand binding. This was done by alanine mutagenesis of a series of residues that are conserved in human, mouse, rat and rabbit LB5 (E9, S14, E16, H19, S21, K31, and K33), but not in the other six modules. E37 (R37 in the rabbit) was included, since it has been previously hypothesized to play a role in binding. The variant LB5 modules were first produced as recombinant peptides, and subjected to oxidative folding to determine whether the mutations interfered with Ca 2+ '-dependent folding. Only the S14A and E16A mutations interfered significantly with folding, suggesting that S14 and E16 are required for the structural framework of LB5 and that their substitution in the LDLR may interfere with its folding. The native LDLR and E9A, H19A, S21A, K31A, K33A and E37A LDLRs were expressed in LDLR negative IdlA-7 CHO cells. Labeling with 125 I-lgG-C7 showed that all receptors were expressed on the cell surface. Binding of Dil-labeled LDL (Dil-LDL) and Dil-labeled DMPC, complexed with the N-terminal receptor-binding domain of apoE3 (Dil-E3), at 4 deg C, was used to assess receptor binding. Binding of Dil-E3 (0.1 μ/ml) to the H19A, S21A, K31A, K33A and E37A LDLRs was 65-92% of binding to the native LDLR. In contrast, the E9A LDLR only bound 3% of that of the native LDLR. The binding of Dil-LDL (0.5 Ag/ml) to the E9A LDLR was 23% of that of the native LDLR, while binding to the remaining variant LDLRs ranged from 44-70% of what of the native LDLR. We conclude that (i) E9 of LB5

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.

Pathogenesis and Inhibition of Flaviviruses from a Carbohydrate Perspective

Directory of Open Access Journals (Sweden)

So Young Kim

2017-05-01

Full Text Available Flaviviruses are enveloped, positive single stranded ribonucleic acid (RNA viruses with various routes of transmission. While the type and severity of symptoms caused by pathogenic flaviviruses vary from hemorrhagic fever to fetal abnormalities, their general mechanism of host cell entry is similar. All pathogenic flaviviruses, such as dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Zika virus, bind to glycosaminglycans (GAGs through the putative GAG binding sites within their envelope proteins to gain access to the surface of host cells. GAGs are long, linear, anionic polysaccharides with a repeating disaccharide unit and are involved in many biological processes, such as cellular signaling, cell adhesion, and pathogenesis. Flavivirus envelope proteins are N-glycosylated surface proteins, which interact with C-type lectins, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN through their glycans. In this review, we discuss both host and viral surface receptors that have the carbohydrate components, focusing on the surface interactions in the early stage of flavivirus entry. GAG-flavivirus envelope protein interactions as well as interactions between flavivirus envelope proteins and DC-SIGN are discussed in detail. This review also examines natural and synthetic inhibitors of flaviviruses that are carbohydrate-based or carbohydrate-targeting. Both advantages and drawbacks of these inhibitors are explored, as are potential strategies to improve their efficacy to ultimately help eradicate flavivirus infections.

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.

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

Science.gov (United States)

Srikant, C B; Dahan, A; Craig, C

1990-02-04

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2

The solvation of carbohydrates in dimethylsulfoxide and water

International Nuclear Information System (INIS)

Berger, S.; Diaz, M.D.; Horwat, Ch.

1999-01-01

The solvation of sucrose and other carbohydrates in DMSO and water is probed by intermolecular NOE measurements. The NOE effects are interpreted in terms of specific binding of the solvent to certain sites of the molecules. It is shown that DMSO attaches to specific sites of the sucrose molecule, whereas for water such a clear differentiation cannot be proven. (author)

Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

Science.gov (United States)

Rezabkova, Lenka; Kacirova, Miroslava; Sulc, Miroslav; Herman, Petr; Vecer, Jaroslav; Stepanek, Miroslav; Obsilova, Veronika; Obsil, Tomas

2012-01-01

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the Gtβγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function. PMID:23199924

Multifunctional cellulase and hemicellulase

Energy Technology Data Exchange (ETDEWEB)

Fox, Brian G.; Takasuka, Taichi; Bianchetti, Christopher M.

2015-09-29

A multifunctional polypeptide capable of hydrolyzing cellulosic materials, xylan, and mannan is disclosed. The polypeptide includes the catalytic core (cc) of Clostridium thermocellum Cthe_0797 (CelE), the cellulose-specific carbohydrate-binding module CBM3 of the cellulosome anchoring protein cohesion region (CipA) of Clostridium thermocellum (CBM3a), and a linker region interposed between the catalytic core and the cellulose-specific carbohydrate binding module. Methods of using the multifunctional polypeptide are also disclosed.

A strategy for interaction site prediction between phospho-binding modules and their partners identified from proteomic data.

Science.gov (United States)

Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

2010-12-01

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/.

A Strategy for Interaction Site Prediction between Phospho-binding Modules and their Partners Identified from Proteomic Data*

Science.gov (United States)

Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

2010-01-01

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/. PMID:20733106

Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors§

Science.gov (United States)

Ahmed, Ahmed H.; Oswald, Robert E.

2010-01-01

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 both 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. PMID:20163115

A functional carbohydrate chip platform for analysis of carbohydrate-protein interaction

International Nuclear Information System (INIS)

Seo, Jeong Hyun; Kim, Chang Sup; Hwang, Byeong Hee; Cha, Hyung Joon

2010-01-01

A carbohydrate chip based on glass or other transparent surfaces has been suggested as a potential tool for high-throughput analysis of carbohydrate-protein interactions. Here we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrates by reductive amination, in which reducing sugar groups were coupled with 4-(2-aminoethyl)aniline, which has di-amine groups at both ends. The modified carbohydrates were covalently attached to an amino-reactive NHS-activated glass surface by formation of stable amide bonds. This proposed method was applied for efficient construction of a carbohydrate microarray to analyze carbohydrate-protein interactions. The carbohydrate chip prepared using our method can be successfully used in diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions, and carbohydrate sensor chip or microarray development for diagnosis and screening.

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

Science.gov (United States)

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Computer simulation of protein—carbohydrate complexes: application to arabinose-binding protein and pea lectin

Science.gov (United States)

Rao, V. S. R.; Biswas, Margaret; Mukhopadhyay, Chaitali; Balaji, P. V.

1989-03-01

The CCEM method (Contact Criteria and Energy Minimisation) has been developed and applied to study protein-carbohydrate interactions. The method uses available X-ray data even on the native protein at low resolution (above 2.4 Å) to generate realistic models of a variety of proteins with various ligands. The two examples discussed in this paper are arabinose-binding protein (ABP) and pea lectin. The X-ray crystal structure data reported on ABP-β- L-arabinose complex at 2.8, 2.4 and 1.7 Å resolution differ drastically in predicting the nature of the interactions between the protein and ligand. It is shown that, using the data at 2.4 Å resolution, the CCEM method generates complexes which are as good as the higher (1.7 Å) resolution data. The CCEM method predicts some of the important hydrogen bonds between the ligand and the protein which are missing in the interpretation of the X-ray data at 2.4 Å resolution. The theoretically predicted hydrogen bonds are in good agreement with those reported at 1.7 Å resolution. Pea lectin has been solved only in the native form at 3 Å resolution. Application of the CCEM method also enables us to generate complexes of pea lectin with methyl-α- D-glucopyranoside and methyl-2,3-dimethyl-α- D-glucopyranoside which explain well the available experimental data in solution.

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, ...

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...... of substrate specificities of glycosyltransferases. This review covers the construction of carbohydrate microarrays, detection methods of carbohydrate microarrays and their applications in biological and biomedical research....

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

Science.gov (United States)

Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens.

Science.gov (United States)

Restrepo, B I; Obregón-Henao, A; Mesa, M; Gil, D L; Ortiz, B L; Mejía, J S; Villota, G E; Sanzón, F; Teale, J M

2000-05-01

Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

Carbohydrate Microarray on Glass: a Tool for Carbohydrate-Lectin Interactions

NARCIS (Netherlands)

Tetala, K.K.R.; Giesbers, M.; Visser, G.M.; Sudhölter, E.J.R.; Beek, van T.A.

2007-01-01

A simple method to immobilize carbohydrates on a glass surface to obtain a carbohydrate microarray is described. The array was used to study carbohydrate-lectin interactions. The glass surface was modified with aldehyde terminated linker groups of various chain lengths. Coupling of carbohydrates

Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein

Directory of Open Access Journals (Sweden)

Naofumi Miwa

2015-05-01

Full Text Available Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP, which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

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.

The role of carbohydrates in the radioimmunoassay of human low-molecular-mass kininogen

International Nuclear Information System (INIS)

Turpeinen, U.; Kaerkkaeinen, T.

1985-01-01

The immunoreactivity of human low-molecular-mass kininogen from Cohn plasma fraction IV was investigated after deglycosylations and carbohydrate modifications by radioimmunoassay using the conformation-specific antiserum. Removal of all sialic acids, 44% of amino sugars and 63% of neutral sugars did not alter the immunoreactivity of the protein but the periodate-treated concanavalin A fractions showed strikingly diminished immunoreactivity. A conformational change could account for the observed effect of periodate on the decreased reactivity of the protein in radioimmunoassay. Externally added carbohydrates had no effect on immunoreactivity. The results suggest that the carbohydrate part of kininogen is not involved in the immunoreactivity although it accounts for the observed lectin-binding heterogeneity. (Auth.)

Overlapping binding site for the endogenous agonist, small-molecule agonists, and ago-allosteric modulators on the ghrelin receptor

DEFF Research Database (Denmark)

Holst, Birgitte; Frimurer, Thomas M; Mokrosinski, Jacek

2008-01-01

A library of robust ghrelin receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone......, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing ghrelin's maximal efficacy overlapped with the common....... It is concluded that although each of the ligands in addition exploits other parts of the receptor, a large, common binding site for both small-molecule agonists--including ago-allosteric modulators--and the endogenous agonist is found on the opposing faces of TM-III and -VI of the ghrelin receptor....

Learning about Carbohydrates

Science.gov (United States)

... Videos for Educators Search English Español Learning About Carbohydrates KidsHealth / For Kids / Learning About Carbohydrates Print en ... source of energy for the body. What Are Carbohydrates? There are two major types of carbohydrates (or ...

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 ...

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.

Carbohydrates and the human gut microbiota.

Science.gov (United States)

Chassard, Christophe; Lacroix, Christophe

2013-07-01

Due to its scale and its important role in maintaining health, the gut microbiota can be considered as a 'new organ' inside the human body. Many complex carbohydrates are degraded and fermented by the human gut microbiota in the large intestine to both yield basic energy salvage and impact gut health through produced metabolites. This review will focus on the gut microbes and microbial mechanisms responsible for polysaccharides degradation and fermentation in the large intestine. Gut microbes and bacterial metabolites impact the host at many levels, including modulation of inflammation, and glucose and lipid metabolisms. A complex relationship occurs in the intestine between the human gut microbiota, diet and the host. Research on carbohydrates and gut microbiota composition and functionality is fast developing and will open opportunities for prevention and treatment of obesity, diabetes and other related metabolic disorders through manipulation of the gut ecosystem.

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).

Secbase: database module to retrieve secondary structure elements with ligand binding motifs.

Science.gov (United States)

Koch, Oliver; Cole, Jason; Block, Peter; Klebe, Gerhard

2009-10-01

Secbase is presented as a novel extension module of Relibase. It integrates the information about secondary structure elements into the retrieval facilities of Relibase. The data are accessible via the extended Relibase user interface, and integrated retrieval queries can be addressed using an extended version of Reliscript. The primary information about alpha-helices and beta-sheets is used as provided by the PDB. Furthermore, a uniform classification of all turn families, based on recent clustering methods, and a new helix assignment that is based on this turn classification has been included. Algorithms to analyze the geometric features of helices and beta-strands were also implemented. To demonstrate the performance of the Secbase implementation, some application examples are given. They provide new insights into the involvement of secondary structure elements in ligand binding. A survey of water molecules detected next to the N-terminus of helices is analyzed to show their involvement in ligand binding. Additionally, the parallel oriented NH groups at the alpha-helix N-termini provide special binding motifs to bind particular ligand functional groups with two adjacent oxygen atoms, e.g., as found in negatively charged carboxylate or phosphate groups, respectively. The present study also shows that the specific structure of the first turn of alpha-helices provides a suitable explanation for stabilizing charged structures. The magnitude of the overall helix macrodipole seems to have no or only a minor influence on binding. Furthermore, an overview of the involvement of secondary structure elements with the recognition of some important endogenous ligands such as cofactors shows some distinct preference for particular binding motifs and amino acids.

Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

Science.gov (United States)

Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

2015-03-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry.

Science.gov (United States)

Ferreira Filho, Jaire Alves; Horta, Maria Augusta Crivelente; Beloti, Lilian Luzia; Dos Santos, Clelton Aparecido; de Souza, Anete Pereira

2017-10-12

Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

Diversity of Microbial Carbohydrate-Active enZYmes (CAZYmes) Associated with Freshwater and Soil Samples from Caatinga Biome.

Science.gov (United States)

Andrade, Ana Camila; Fróes, Adriana; Lopes, Fabyano Álvares Cardoso; Thompson, Fabiano L; Krüger, Ricardo Henrique; Dinsdale, Elizabeth; Bruce, Thiago

2017-07-01

Semi-arid and arid areas occupy about 33% of terrestrial ecosystems. However, little information is available about microbial diversity in the semi-arid Caatinga, which represents a unique biome that extends to about 11% of the Brazilian territory and is home to extraordinary diversity and high endemism level of species. In this study, we characterized the diversity of microbial genes associated with biomass conversion (carbohydrate-active enzymes, or so-called CAZYmes) in soil and freshwater of the Caatinga. Our results showed distinct CAZYme profiles in the soil and freshwater samples. Glycoside hydrolases and glycosyltransferases were the most abundant CAZYme families, with glycoside hydrolases more dominant in soil (∼44%) and glycosyltransferases more abundant in freshwater (∼50%). The abundances of individual glycoside hydrolase, glycosyltransferase, and carbohydrate-binding module subfamilies varied widely between soil and water samples. A predominance of glycoside hydrolases was observed in soil, and a higher contribution of enzymes involved in carbohydrate biosynthesis was observed in freshwater. The main taxa associated with the CAZYme sequences were Planctomycetia (relative abundance in soil, 29%) and Alphaproteobacteria (relative abundance in freshwater, 27%). Approximately 5-7% of CAZYme sequences showed low similarity with sequences deposited in non-redundant databases, suggesting putative homologues. Our findings represent a first attempt to describe specific microbial CAZYme profiles for environmental samples. Characterizing these enzyme groups associated with the conversion of carbohydrates in nature will improve our understanding of the significant roles of enzymes in the carbon cycle. We identified a CAZYme signature that can be used to discriminate between soil and freshwater samples, and this signature may be related to the microbial species adapted to the habitat. The data show the potential ecological roles of the CAZYme repertoire and

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.

Hepatocyte heterogeneity in the metabolism of carbohydrates.

Science.gov (United States)

Jungermann, K; Thurman, R G

1992-01-01

the hepatocytes sense the glucagon/insulin gradients via the respective hormone receptors, it is not known how they sense different oxygen tensions; the O2 sensor may be an oxygen-binding heme protein. The zonal separation of glucose release and uptake appears to be important for the liver to operate as a 'glucostat'. Thus, zonation of carbohydrate metabolism develops gradually during the first weeks of life, in part before and in part with weaning, when (in rat and mouse) the fat- and protein-rich but carbohydrate-poor nutrition via milk is replaced by carbohydrate-rich food. Similarly, zonation of carbohydrate metabolism adapts to longer lasting alterations in the need of a 'glucostat', such as starvation, diabetes, portocaval anastomoses or partial hepatectomy.

Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay

Science.gov (United States)

Chiaki Hori; Jill Gaskell; Kiyohiko Igarashi; Masahiro Samejima; David Hibbett; Bernard Henrissat; Dan Cullen

2013-01-01

To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (...

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.

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

Science.gov (United States)

Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

2011-05-01

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

Activation of the carbohydrate response element binding protein (ChREBP) in response to anoxia in the turtle Trachemys scripta elegans.

Science.gov (United States)

Krivoruchko, Anastasia; Storey, Kenneth B

2014-10-01

ChREBP (carbohydrate response element binding protein) is a glucose-responsive transcription factor that is known to be an important regulator of glycolytic and lipogenic genes in response to glucose. We hypothesized that activation of ChREBP could be relevant to anoxia survival by the anoxia-tolerant turtle, Trachemys scripta elegans. Expression of ChREBP in response to 5 and 20h of anoxia was examined using RT-PCR and Western immunoblotting. In addition, subcellular localization and DNA-binding activity of ChREBP protein were assessed and transcript levels of liver pyruvate kinase (LPK), a downstream gene under ChREBP control were quantified using RT-PCR. ChREBP was anoxia-responsive in kidney and liver, with transcript levels increasing by 1.2-1.8 fold in response to anoxia and protein levels increasing by 1.8-1.9 fold. Enhanced nuclear presence under anoxia was also observed in both tissues by 2.2-2.8 fold. A 4.2 fold increase in DNA binding activity of ChREBP was also observed in liver in response to 5h of anoxia. In addition, transcript levels of LPK increased by 2.1 fold in response to 5h of anoxia in the liver. The results suggest that activation of ChREBP in response to anoxia might be a crucial factor for anoxia survival in turtle liver by contributing to elevated glycolytic flux in the initial phases of oxygen limitation. This study provides the first demonstration of activation of ChREBP in response to anoxia in a natural model of anoxia tolerance, further improving our understanding of the molecular nature of anoxia tolerance. Copyright © 2014 Elsevier B.V. All rights reserved.

Glycan Reader: Automated Sugar Identification and Simulation Preparation for Carbohydrates and Glycoproteins

Science.gov (United States)

Jo, Sunhwan; Song, Kevin C.; Desaire, Heather; MacKerell, Alexander D.; Im, Wonpil

2011-01-01

Understanding how glycosylation affects protein structure, dynamics, and function is an emerging and challenging problem in biology. As a first step toward glycan modeling in the context of structural glycobiology, we have developed Glycan Reader and integrated it into the CHARMM-GUI, http://www.charmm-gui.org/input/glycan. Glycan Reader greatly simplifies the reading of PDB structure files containing glycans through (i) detection of carbohydrate molecules, (ii) automatic annotation of carbohydrates based on their three-dimensional structures, (iii) recognition of glycosidic linkages between carbohydrates as well as N-/O-glycosidic linkages to proteins, and (iv) generation of inputs for the biomolecular simulation program CHARMM with the proper glycosidic linkage setup. In addition, Glycan Reader is linked to other functional modules in CHARMM-GUI, allowing users to easily generate carbohydrate or glycoprotein molecular simulation systems in solution or membrane environments and visualize the electrostatic potential on glycoprotein surfaces. These tools are useful for studying the impact of glycosylation on protein structure and dynamics. PMID:21815173

L-selectin-carbohydrate interactions: relevant modifications of the Lewis x trisaccharide.

Science.gov (United States)

Sanders, W J; Katsumoto, T R; Bertozzi, C R; Rosen, S D; Kiessling, L L

1996-11-26

Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. Specifically, three carbohydrate binding proteins termed selectins (E-, P-, and L-selectin) have been shown to be essential for leukocyte rolling along the vascular endothelium, the first step in the recruitment of leukocytes from the blood into inflammatory sites or into secondary lymphoid organs. Although this phenomenon is well-established, little is known about the molecular-level interactions on which it depends. All three selectins recognize sulfated and sialylated derivatives of the Lewis x [Le(x):Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and Lewis a [Le(a): Gal beta 1-->3(Fuc alpha 1-->4)GlcNAc] trisaccharide cores with affinities in the millimolar range, and it is believed that variants of these structures are the carbohydrate determinants of selectin recognition. Recently it was shown that the mucin GlyCAM-1, a secreted physiological ligand for L-selectin, is capped with sulfated derivatives of sialyl Lewis x [sLe(x): Sia alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and that sulfation is required for the high-affinity interaction between GlyCAM-1 and L-selectin. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Our results suggest that 6-sulfo sLe(x) binds to L-selectin with higher affinity than does sLe(x) or 6'-sulfo sLe(x) and that sulfation of sLe(x) capping groups on GlyCAM-1 at the 6-position is important for L-selectin recognition.

Two secondary carbohydrate binding sites on the surface of barley alpha-amylase 1 have distinct functions and display synergy in hydrolysis of starch granules.

Science.gov (United States)

Nielsen, Morten M; Bozonnet, Sophie; Seo, Eun-Seong; Mótyán, János A; Andersen, Joakim M; Dilokpimol, Adiphol; Abou Hachem, Maher; Gyémánt, Gyöngyi; Naested, Henrik; Kandra, Lili; Sigurskjold, Bent W; Svensson, Birte

2009-08-18

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, respectively. Site-directed mutagenesis of Trp(278) and Trp(279), stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr(380) and His(395), making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic beta-cyclodextrin. Compared to that of wild-type AMY1, the K(d) of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15-35 times; furthermore, the k(cat)/K(m) of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2 substitutions eliminated binding to starch granules, and the k(cat)/K(m) of W278A/W279A/Y380A AMY1 was only 0.4% of the wild-type value. Surface plasmon resonance analysis of mutants showed that beta-cyclodextrin binds to SBS2 and SBS1 with K(d,1) and K(d,2) values of 0.07 and 1.40 mM, respectively. A model that accounts for the observed synergy in starch hydrolysis, where SBS1 and SBS2 bind ordered and free alpha-glucan chains, respectively, thus targeting the enzyme 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.

Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

Science.gov (United States)

Venable, Alison; Mitalipova, Maisam; Lyons, Ian; Jones, Karen; Shin, Soojung; Pierce, Michael; Stice, Steven

2005-01-01

Background Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent

Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

Directory of Open Access Journals (Sweden)

Shin Soojung

2005-07-01

Full Text Available Abstract Background Pluripotent human embryonic stem cells (hESCs have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4, to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomatoesculetum lectin (TL, Ricinus communis agglutinin (RCA, and Concanavalin A (Con A bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA and Lotus tetragonolobus lectin (LTL did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L, Vicia villosa agglutinin (VVA, Ulex europaeus agglutinin (UEA, Phaseolus vulgaris erythro-agglutinin (PHA-E, and Maackia amurensis agglutinin (MAA bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the

Gut feedback mechanisms and food intake: a physiological approach to slow carbohydrate bioavailability.

Science.gov (United States)

Zhang, Genyi; Hasek, Like Y; Lee, Byung-Hoo; Hamaker, Bruce R

2015-04-01

Glycemic carbohydrates in foods are an important macronutrient providing the biological fuel of glucose for a variety of physiological processes. A classification of glycemic carbohydrates into rapidly digestible carbohydrate (RDC) and slowly digestible carbohydrate (SDC) has been used to specify their nutritional quality related to glucose homeostasis that is essential to normal functioning of the brain and critical to life. Although there have been many studies and reviews on slowly digestible starch (SDS) and SDC, the mechanisms of their slow digestion and absorption were mostly investigated from the material side without considering the physiological processes of their in vivo digestion, absorption, and most importantly interactions with other food components and the gastrointestinal tract. In this article, the physiological processes modulating the bioavailability of carbohydrates, specifically the rate and extent of their digestion and absorption as well as the related locations, in a whole food context, will be discussed by focusing on the activities of the gastrointestinal tract including glycolytic enzymes and glucose release, sugar sensing, gut hormones, and neurohormonal negative feedback mechanisms. It is hoped that a deep understanding of these physiological processes will facilitate the development of innovative dietary approaches to achieve desired carbohydrate or glucose bioavailability for improved health.

C-terminal low-complexity sequence repeats of Mycobacterium smegmatis Ku modulate DNA binding.

Science.gov (United States)

Kushwaha, Ambuj K; Grove, Anne

2013-01-24

Ku protein is an integral component of the NHEJ (non-homologous end-joining) pathway of DSB (double-strand break) repair. Both eukaryotic and prokaryotic Ku homologues have been characterized and shown to bind DNA ends. A unique feature of Mycobacterium smegmatis Ku is its basic C-terminal tail that contains several lysine-rich low-complexity PAKKA repeats that are absent from homologues encoded by obligate parasitic mycobacteria. Such PAKKA repeats are also characteristic of mycobacterial Hlp (histone-like protein) for which they have been shown to confer the ability to appose DNA ends. Unexpectedly, removal of the lysine-rich extension enhances DNA-binding affinity, but an interaction between DNA and the PAKKA repeats is indicated by the observation that only full-length Ku forms multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer. The C-terminal extension promotes DNA end-joining by T4 DNA ligase, suggesting that the PAKKA repeats also contribute to efficient end-joining. We suggest that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis.

Science.gov (United States)

Lung, Shiu-Cheung; Liao, Pan; Yeung, Edward C; Hsiao, An-Shan; Xue, Yan; Chye, Mee-Len

2017-07-01

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis ( Arabidopsis thaliana ) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1 Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 ( GL2 ), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1 , smo1-1 , and ACBP1 +/- smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1 +/- smo1-1 Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. © 2017 American Society of Plant Biologists. All Rights Reserved.

Inhibition of mannosidase in hybridomas yields monoclonal antibodies with greater capacity for carbohydrate labeling

International Nuclear Information System (INIS)

Simonson, R.B.; Ultee, M.E.; Long, C.G.; Gillette, R.W.; McKearn, T.J.; Rodwell, J.D.

1988-01-01

Labeling an antibody site specifically through its carbohydrate residues preserves more of its antigen-binding activity than does labeling through protein moieties. To boost the amount of immunoglobulin G carbohydrate capable of being labeled, we treated hybridoma cells with a mannosidase inhibitor, deoxymannojirimycin (dMM). Polyacrylamide gel electrophoresis showed formation of a glycoprotein with high mannose content, in that endo-beta-N-acetylglucosaminidase H 3.2.1.96) could digest the antibody from the dMM-treated cells, but not from control cultures. Carbohydrate analysis confirmed this conclusion, indicating that the antibody from the dMM-treated cells had twice as much mannose as did the control antibody. The glucosamine content of the treated-cells' antibodies was half that of the control, and no additional carbohydrate residues were detectable in the antibodies secreted by the dMM-treated cells. We conjugated both the dMM and control antibodies through their carbohydrate to a chelator. In labeling, the dMM antibody conjugate incorporated approximately threefold as much 111 In isotope as the control conjugate. The two labeled antibodies were injected into mice and showed similar organ distributions

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.

Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators.

Science.gov (United States)

Bohl, Casey E; Wu, Zengru; Chen, Jiyun; Mohler, Michael L; Yang, Jun; Hwang, Dong Jin; Mustafa, Suni; Miller, Duane D; Bell, Charles E; Dalton, James T

2008-10-15

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

Science.gov (United States)

Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

2011-11-25

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

Science.gov (United States)

Kang, Hyeran; Bradley, Michael J.; McCullough, Brannon R.; Pierre, Anaëlle; Grintsevich, Elena E.; Reisler, Emil; De La Cruz, Enrique M.

2012-01-01

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments. PMID:23027950

Dietary fiber content influences soluble carbohydrate levels in ruminal fluids.

Science.gov (United States)

Pinder, R S; Patterson, J A; O'Bryan, C A; Crandall, P G; Ricke, S C

2012-01-01

The soluble carbohydrate concentration of ruminal fluid, as affected by dietary forage content (DFC) and/or ruminally undegradable intake protein content (UIPC), was determined. Four ruminally cannulated steers, in a 4 × 4 Latin square design, were offered diets containing high (75 % of DM) or low (25 % of DM) DFC and high (6 % of DM) or low (5 % of DM) UIPC, in a 2 × 2 factorial arrangement. Zinc-treated SBM was the primary UIP source. Soluble hexose concentration (145.1 μM) in ruminal fluid (RF) of steers fed low DFC diets exhibited a higher trend (P = 0.08) than that (124.5 μM) of steers fed high DFC diets. UIPC did not modulate (P = 0.54) ruminal soluble hexose concentrations. Regardless of diet, soluble hexose concentration declined immediately after feeding and did not rise until 3 h after feeding (P ruminal fluid could not be determined. However, unsubstituted xylose and arabinose were excluded. These data indicate that: (i) soluble carbohydrate concentrations remain in ruminal fluid during digestion and fermentation; (ii) slight diurnal changes began after feeding; (iii) DFC influences the soluble carbohydrate concentration in RF; and (iv) UIPC of these diets does not affect the soluble carbohydrate concentration of RF.

Multivalency at Interfaces: Supramolecular Carbohydrate-Functionalized Graphene Derivatives for Bacterial Capture, Release, and Disinfection.

Science.gov (United States)

Qi, Zhenhui; Bharate, Priya; Lai, Chian-Hui; Ziem, Benjamin; Böttcher, Christoph; Schulz, Andrea; Beckert, Fabian; Hatting, Benjamin; Mülhaupt, Rolf; Seeberger, Peter H; Haag, Rainer

2015-09-09

A supramolecular carbohydrate-functionalized two-dimensional (2D) surface was designed and synthesized by decorating thermally reduced graphene sheets with multivalent sugar ligands. The formation of host-guest inclusions on the carbon surface provides a versatile strategy, not only to increase the intrinsic water solubility of graphene-based materials, but more importantly to let the desired biofunctional binding groups bind to the surface. Combining the vital recognition role of carbohydrates and the unique 2D large flexible surface area of the graphene sheets, the addition of multivalent sugar ligands makes the resulting carbon material an excellent platform for selectively wrapping and agglutinating Escherichia coli (E. coli). By taking advantage of the responsive property of supramolecular interactions, the captured bacteria can then be partially released by adding a competitive guest. Compared to previously reported scaffolds, the unique thermal IR-absorption properties of graphene derivatives provide a facile method to kill the captured bacteria by IR-laser irradiation of the captured graphene-sugar-E. coli complex.

The pattern-recognition molecule mannan-binding lectin (MBL) in the pathophysiology of diabetic nephropathy

DEFF Research Database (Denmark)

Axelgaard, Esben; Thiel, Steffen; Hansen, Troels Krarup

to carbohydrates of both specific type and density, which thus provides sufficient binding avidity. The character of MBL binding-sites on host cells remain unknown, but it is speculated that altered protein glycation in diabetes permits MBL binding. Based on new studies using MBL/double knockout C57bl/6j mice, we...

pH Modulates the Binding of EGR1 Transcription Factor to DNA

Science.gov (United States)

Mikles, David C.; Bhat, Vikas; Schuchardt, Brett J.; Deegan, Brian J.; Seldeen, Kenneth L.; McDonald, Caleb B.; Farooq, Amjad

2013-01-01

EGR1 transcription factor orchestrates a plethora of signaling cascades involved in cellular homeostasis and its down-regulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with increasing pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as H382 by virtue of the fact that its substitution to non-ionizable residues abolishes pH-dependence of the binding of EGR1 to DNA. Notably, H382 inserts into the major groove of DNA and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, H382 is predominantly conserved across other members of EGR1 family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating protein-DNA interactions central to this family of transcription factors. Collectively, our findings uncover an unexpected but a key step in the molecular recognition of EGR1 family of transcription factors and suggest that they may act as sensors of pH within the intracellular environment. PMID:23718776

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.

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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Science.gov (United States)

Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

2013-01-01

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity. PMID:23990797

Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

Directory of Open Access Journals (Sweden)

Lifeng Liu

Full Text Available Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1, a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

Science.gov (United States)

Liu, Lifeng; Shang-Guan, Keke; Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

2013-01-01

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

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.

Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans.

Science.gov (United States)

Moye, Zachary D; Son, Minjun; Rosa-Alberty, Ariana E; Zeng, Lin; Ahn, Sang-Joon; Hagen, Stephen J; Burne, Robert A

2016-08-01

) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription of comX and that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increase com gene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans

Science.gov (United States)

Moye, Zachary D.; Son, Minjun; Rosa-Alberty, Ariana E.; Zeng, Lin; Ahn, Sang-Joon

2016-01-01

-stimulating peptide (CSP) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription of comX and that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increase com gene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability. PMID:27260355

Crystallization and preliminary X-ray diffraction analysis of mouse galectin-4 N-terminal carbohydrate recognition domain in complex with lactose

International Nuclear Information System (INIS)

Krejčiříková, Veronika; Fábry, Milan; Marková, Vladimíra; Malý, Petr; Řezáčová, Pavlína; Brynda, Jiří

2008-01-01

Mouse galectin-4 carbohydrate binding domain was overexpressed in E. coli and crystallized in the presence of lactose. The crystals belong to tetragonal space group P42 1 2 and diffraction data were collected to 2.1 Å resolution. Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P42 1 2 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10 Å and preliminary X-ray diffraction data were collected to 3.2 Å resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1 Å. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

Science.gov (United States)

Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

2017-06-05

Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Structural determination of the carbohydrate chains from arthropod and mollusc hemocyanin by means of 500-MHz 1H-NMR spectroscopy

International Nuclear Information System (INIS)

Kuik, J.A. van.

1987-01-01

In this thesis carbohydrate structures of hemocyanins of arthropods and molluscs are studied. Hemocyanins are high-molecular-mass, copper-containing oxygen-transport proteins. The function of these carbohydrate chains are yet still unknown. It is not probable that they play a role in the oxygen-binding processes. They are rather thought to have a function in the build-up of the hemocyanin molecules. 286 refs.; 30 figs.; 25 tabs

The role of carbohydrate in determining the immunochemical properties of the hemagglutinin of influenza A virus

International Nuclear Information System (INIS)

Gitelman, A.K.; Berezin, V.A.; Kharitonenkov, I.G.

1981-01-01

Most of the carbohydrate was removed from influenza with MRC II (H3N2) and its purified hemagglutinin (HA) on treatment with glycosidases, including α-mannosidase, #betta#-N-acetylglucosaminidase, #betta#-galactosidase and α-fucosidase. The release of 50 per cent of the carbohydrate from intact virus particles significantly affected hemagglutinating activity. The ability of untreated and glycosidase-treated virus to inhibit the binding of antibodies directed against the hemagglutinin was almost indistinguishable by competitive radioimmunoassay (RIA). Up to 60 per cent of the carbohydrate from the purified HA of influenza virus could be removed. The antigenicity of glycosidase treated HA molecules decreased 8-fold compared to intact HAs as measured by competitive RIA. In addition, glycosidase digestion of 125 I-labeled HA resulted in a decrease in its reactivity in direct RIA. We conclude that the carbohydrate portion of the HA of influenza virus is not of major importance in defining the antigenicity of HA. (Author)

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.

Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines.

NARCIS (Netherlands)

Dube, N.; Delvin, E.; Yotov, W.; Garofalo, C.; Bendayan, M.; Veerkamp, J.H.; Levy, E.

2001-01-01

Intestinal and liver fatty acid binding proteins (I- and L-FABP) are thought to play a role in enterocyte fatty acid (FA) trafficking. Their modulation by cell differentiation and various potential effectors was investigated in the human Caco-2 cell line. With the acquisition of enterocytic

Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

Science.gov (United States)

Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

2010-01-01

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

Deleted in Malignant Brain Tumors-1 Protein (DMBT1: A Pattern Recognition Receptor with Multiple Binding Sites

Directory of Open Access Journals (Sweden)

Enno C. I. Veerman

2010-12-01

Full Text Available Deleted in Malignant Brain Tumors-1 protein (DMBT1, salivary agglutinin (DMBT1SAG, and lung glycoprotein-340 (DMBT1GP340 are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW. Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

Enhancement of antiviral activity of collectin trimers through cross-linking and mutagenesis of the carbohydrate recognition domain

DEFF Research Database (Denmark)

White, Mitchell R; Boland, Patrick; Tecle, Tesfaldet

2010-01-01

Surfactant protein D (SP-D) plays important roles in innate defense against respiratory viruses [including influenza A viruses (IAVs)]. Truncated trimers composed of its neck and carbohydrate recognition domains (NCRDs) bind various ligands; however, they have minimal inhibitory activity for IAV......., complementary strategies, namely cross-linking of NCRDs through various means and mutagenesis of CRD residues to increase viral binding. These findings may be relevant for antiviral therapy....

Structure of Bacteroides thetaiotaomicron BT2081 at 2.05 Å resolution: the first structural representative of a new protein family that may play a role in carbohydrate metabolism

Energy Technology Data Exchange (ETDEWEB)

Yeh, Andrew P. [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (United States); Abdubek, Polat [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (United States); Astakhova, Tamara [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA (United States); Axelrod, Herbert L. [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (United States); Bakolitsa, Constantina [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA (United States); Cai, Xiaohui [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA (United States); Carlton, Dennis [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (United States); Chen, Connie [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (United States); Chiu, Hsiu-Ju [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (United States); Chiu, Michelle [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (United States); Clayton, Thomas [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (United States); Das, Debanu [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (United States); Deller, Marc C. [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (United States); Duan, Lian; Ellrott, Kyle [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA (United States); Farr, Carol L. [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (United States); Feuerhelm, Julie; Grant, Joanna C. [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (United States); Grzechnik, Anna; Han, Gye Won [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (United States); Jaroszewski, Lukasz [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA (United States); Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA (United States); Jin, Kevin K. [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (United States); Klock, Heath E.; Knuth, Mark W. [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (United States); Kozbial, Piotr [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA (United States); Krishna, S. Sri [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA (United States); Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA (United States); Kumar, Abhinav; Lam, Winnie W. [Joint Center for Structural Genomics, http://www.jcsg.org (United States); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (United States); Marciano, David [Joint Center for Structural Genomics, http://www.jcsg.org (US); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (US); McMullan, Daniel [Joint Center for Structural Genomics, http://www.jcsg.org (US); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (US); Miller, Mitchell D. [Joint Center for Structural Genomics, http://www.jcsg.org (US); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (US); Morse, Andrew T. [Joint Center for Structural Genomics, http://www.jcsg.org (US); Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA (US); Nigoghossian, Edward [Joint Center for Structural Genomics, http://www.jcsg.org (US); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (US); Nopakun, Amanda [Joint Center for Structural Genomics, http://www.jcsg.org (US); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (US); Okach, Linda; Puckett, Christina [Joint Center for Structural Genomics, http://www.jcsg.org (US); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (US); Reyes, Ron [Joint Center for Structural Genomics, http://www.jcsg.org (US); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (US); Tien, Henry J. [Joint Center for Structural Genomics, http://www.jcsg.org (US); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (US); Trame, Christine B.; Bedem, Henry van den [Joint Center for Structural Genomics, http://www.jcsg.org (US); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (US); Weekes, Dana [Joint Center for Structural Genomics, http://www.jcsg.org (US); Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA (US); Wooten, Tiffany [Joint Center for Structural Genomics, http://www.jcsg.org (US); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (US); Xu, Qingping [Joint Center for Structural Genomics, http://www.jcsg.org (US); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (US); Hodgson, Keith O. [Joint Center for Structural Genomics, http://www.jcsg.org (US); Photon Science, SLAC National Accelerator Laboratory, Menlo Park, CA (US); Wooley, John [Joint Center for Structural Genomics, http://www.jcsg.org (US); Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA (US); Elsliger, Marc-André [Joint Center for Structural Genomics, http://www.jcsg.org (US); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (US); Deacon, Ashley M. [Joint Center for Structural Genomics, http://www.jcsg.org (US); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA (US); Godzik, Adam [Joint Center for Structural Genomics, http://www.jcsg.org (US); Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA (US); Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA (US); Lesley, Scott A. [Joint Center for Structural Genomics, http://www.jcsg.org (US); Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA (US); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (US); Wilson, Ian A., E-mail: wilson@scripps.edu [Joint Center for Structural Genomics, http://www.jcsg.org (US); Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA (US)

2010-10-01

The crystal structure of BT2081 from B. thetaiotaomicron reveals a two-domain protein with a putative carbohydrate-binding site in the C-terminal domain. BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP-810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05 Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a β-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a β-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal β-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.

Structure of Bacteroides thetaiotaomicron BT2081 at 2.05 Å resolution: the first structural representative of a new protein family that may play a role in carbohydrate metabolism

International Nuclear Information System (INIS)

Yeh, Andrew P.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Lam, Winnie W.; Marciano, David; McMullan, Daniel; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

2010-01-01

The crystal structure of BT2081 from B. thetaiotaomicron reveals a two-domain protein with a putative carbohydrate-binding site in the C-terminal domain. BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP-810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05 Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a β-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a β-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal β-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft

Molecular Characterization of the RNA-Binding Protein Quaking-a in Megalobrama amblycephala: Response to High-Carbohydrate Feeding and Glucose/Insulin/Glucagon Treatment

Directory of Open Access Journals (Sweden)

Hua-Juan Shi

2018-04-01

Full Text Available The RNA-binding protein quaking-a (Qkia was cloned from the liver of blunt snout bream Megalobrama amblycephala through the rapid amplification of cDNA ends method, with its potential role in glucose metabolism investigated. The full-length cDNA of qkia covered 1,718 bp, with an open reading frame of 1,572 bp, which encodes 383 AA. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (97–99% among most fish and other higher vertebrates. The mRNA of qkia was detected in all examined organs/tissues. Then, the plasma glucose levels and tissue qkia expressions were determined in fish intraperitoneally injected with glucose [1.67 g per kg body weight (BW], insulin (0.052 mg/kg BW, and glucagon (0.075 mg/kg BW respectively, as well as in fish fed two dietary carbohydrate levels (31 and 41% for 12 weeks. Glucose administration induced a remarkable increase of plasma glucose with the highest value being recorded at 1 h. Thereafter, it reduced to the basal value. After glucose administration, qkia expressions significantly decreased with the lowest value being recorded at 1 h in liver and muscle and 8 h in brain, respectively. Then they gradually returned to the basal value. The insulin injection induced a significant decrease of plasma glucose with the lowest value being recorded at 1 h, whereas the opposite was true after glucagon load (the highest value was gained at 4 h. Subsequently, glucose levels gradually returned to the basal value. After insulin administration, the qkia expressions significantly decreased with the lowest value being attained at 2 h in brain and muscle and 1 h in liver, respectively. However, glucagon significantly stimulated the expressions of qkia in tissues with the highest value being gained at 6 h. Moreover, high dietary carbohydrate levels remarkably increased plasma glucose levels, but down-regulated the transcriptions of qkia in tissues. These results indicated that the gene of blunt

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.

Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane

International Nuclear Information System (INIS)

Devirgiliis, Chiara; Gaetani, Sancia; Apreda, Marianna; Bellovino, Diana

2005-01-01

Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH 2 -terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process

Carbohydrate Metabolism Disorders

Science.gov (United States)

... metabolic disorder, something goes wrong with this process. Carbohydrate metabolism disorders are a group of metabolic disorders. Normally your enzymes break carbohydrates down into glucose (a type of sugar). If ...

Modulation of [3H]-glutamate binding by serotonin in the rat hippocampus: An autoradiographic study

International Nuclear Information System (INIS)

Mennini, T.; Miari, A.

1991-01-01

Serotonin (5-HT) added in vitro increased [ 3 H]-glutamate specific binding in the rat hippocampus, reaching statistical significance in layers rich in N-Methyl-D-Aspartate sensitive glutamate receptors. This effect was explained by a significant increase in the apparent affinity of [ 3 H]-glutamate when 5-HT is added in vitro. Two days after lesion of serotonergic afferents to the hippocampus with 5,7- Dihydroxytryptamine [ 3 H]-glutamate binding was significantly decreased in the CA3 region and stratum lacunosum moleculare of the hippocampus, this reduction being reversed by in vitro addition of 10 μM 5-HT. The decrease observed is due to a significant reduction of quisqualate-insensitive (radiatum CA3) and kainate receptors (strata oriens, radiatum, pyramidal of CA3). Five days after lesion [ 3 H]-glutamate binding increased significantly in the CA3 region of the hippocampus but was not different from sham animals in the other hippocampal layers. Two weeks after lesion [ 3 H]-glutamate binding to quisqualate-insensitive receptors was increased in all the hippocampal layers, while kainate and quisqualate-sensitive receptors were not affected. These data are consistent with the possibility that 5-HT is a direct positive modulator of glutamate receptor subtypes

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

DEFF Research Database (Denmark)

Meier, Sebastian; Beeren, Sophie

2014-01-01

We describe a simple method for the simultaneous determination of association constants for a guest binding to seven different hosts in a mixture of more than 20 different oligosaccharides. If the binding parameters are known for one component in the mixture, a single NMR titration suffices...

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...

Antibiotic modulation of the plasminogen binding ability of viridans group streptococci.

Science.gov (United States)

Teles, Cristina; Smith, Andrew; Lang, Sue

2012-01-01

The ability of viridans group streptococci to bind human plasminogen and its subsequent activation into plasmin may contribute to the pathogenesis of infective endocarditis (IE) by leading to a decreased stability of the streptococcal vegetation and facilitating dehiscence of emboli. At levels greater than or equal to their MICs, penicillin, vancomycin, and linezolid are efficacious in the treatment of streptococcal endocarditis. However, at sub-MICs, antibiotics can modulate the expression of bacterial genes, including virulence-associated genes, which can have counterproductive effects on the treatment of endocarditis. The effects of 1/8× and 1/4× MICs of penicillin, vancomycin, and linezolid on the plasminogen binding ability of IE isolates Streptococcus mitis 881/956, Streptococcus oralis 12601, and Streptococcus sanguinis 12403 were assessed phenotypically and the expression of plasminogen receptors α-enolase and glyceraldehyde 3-phosphate dehydrogenase of S. oralis 12601 when exposed to 1/4× MIC of penicillin, was analyzed through quantitative reverse transcription (qRT)-PCR. The plasminogen binding ability of S. mitis 881/956 and S. sanguinis 12403 remained unaffected by exposure to sub-MICs of all of the antibiotics tested, while that of S. oralis 12601 was significantly enhanced by all of the antibiotics tested at sub-MICs. qRT-PCR analysis of S. oralis 12601 demonstrated an upregulation of the eno and gapdh genes, indicating an overexpression of plasminogen receptors. These findings suggest that for some endocarditis isolates, the effect of antibiotic sub-MICs, in addition to a reduced antibacterial effect, may influence the clinical response to nonsurgical therapy. It remains difficult to accurately predict isolate responses to sub-MIC antimicrobials since there appears to be interspecies variation.

Statistical significance of cis-regulatory modules

Directory of Open Access Journals (Sweden)

Smith Andrew D

2007-01-01

Full Text Available Abstract Background It is becoming increasingly important for researchers to be able to scan through large genomic regions for transcription factor binding sites or clusters of binding sites forming cis-regulatory modules. Correspondingly, there has been a push to develop algorithms for the rapid detection and assessment of cis-regulatory modules. While various algorithms for this purpose have been introduced, most are not well suited for rapid, genome scale scanning. Results We introduce methods designed for the detection and statistical evaluation of cis-regulatory modules, modeled as either clusters of individual binding sites or as combinations of sites with constrained organization. In order to determine the statistical significance of module sites, we first need a method to determine the statistical significance of single transcription factor binding site matches. We introduce a straightforward method of estimating the statistical significance of single site matches using a database of known promoters to produce data structures that can be used to estimate p-values for binding site matches. We next introduce a technique to calculate the statistical significance of the arrangement of binding sites within a module using a max-gap model. If the module scanned for has defined organizational parameters, the probability of the module is corrected to account for organizational constraints. The statistical significance of single site matches and the architecture of sites within the module can be combined to provide an overall estimation of statistical significance of cis-regulatory module sites. Conclusion The methods introduced in this paper allow for the detection and statistical evaluation of single transcription factor binding sites and cis-regulatory modules. The features described are implemented in the Search Tool for Occurrences of Regulatory Motifs (STORM and MODSTORM software.

Radiolysis of carbohydrates and of carbohydrate-containing foodstuffs

International Nuclear Information System (INIS)

Diehl, J.F.; Adam, S.; Delincee, H.; Jakubick, V.

1978-01-01

Toxicological evaluation of irradiated foodstuffs requires knowledge of radiation-induced chemical changes. A review of the literature reveals much information on the radiation chemistry of pure substances, e.g., dilute solutions of individual carbohydrates. Much less is known about the interactions of food constituents during irradiation. In an effort to remedy this situation, radiation effects on various compounds have been studied in systems of increasing complexity. In one approach, gas chromatography was used to investigate the radiolysis of tehalose in pure solution and in the presence of amino acids or proteins. In another approach, radiation-induced aggregation of proteins and of [ 14 C]tryptophan with proteins was studied in the absence and presence of carbohydrates (trehalose, starch), emulsified sunfower oil, and a mixture of carbohydrates and emulsified sunflower oil

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…

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.

Histochemistry of lectin-binding sites in Halicryptus spinulosus (Priapulida).

Science.gov (United States)

Busch, A; Schumacher, U; Storch, V

2001-02-01

Priapulida represent one of the phylogenetically oldest multicellular animal groups. In multicellular animals (Metazoa) cell-to-cell and cell-to-matrix interactions are often mediated by carbohydrate residues of glycoconjugates. To analyze the carbohydrate composition of a phylogenetically old species, lectin histochemistry was employed on 5 specimens of the priapulid Halicryptus spinulosus. Many lectins bound to the chitin-containing cuticle, including those specific for carbohydrates other than N-acetylglucosamine, the principle building block of chitin. The connective tissue of the animals contained both N-acetylglucosamine and N-acetylgalactosamine. Mannose residues were widely distributed with the exception of the cuticle, but complex type carbohydrates were not present in the entire animal. Sialic acid residues were only detected in the cuticle and brush border of the intestinal epithelium, while fucose was limited to the cuticle. Thus, the lectin-binding pattern indicated that sugars typical for the linking region of both N- and O-glycoproteins in mammals are also present in H. spinulosus. Carbohydrate residues that are typical for the complex type of N-linked glycans in vertebrates are not present as are carbohydrate residues typical for the termination of O-linked carbohydrate chains. Hence, a truncated form of both N- and O-linked glycosylation is present in H. spinulosus indicating that more complex patterns of glycosylation developed later during evolution.

Carbohydrate-Loading Diet

Science.gov (United States)

... Grape juice (12 ounces) 55 225 Lunch Milk, chocolate, reduced fat (12 ounces) 45 285 4 slices ... usual during carbohydrate loading to get the same benefits as a man does. Despite carbohydrate loading, you ...

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

2016-11-01

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.

Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors

DEFF Research Database (Denmark)

Damgaard, Maria; Al-Khawaja, Anas; Nittegaard-Nielsen, Mia

2017-01-01

-affinity binding and is furthermore reported as an allosteric modulator selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined......-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [(3)H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have...... δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a GHB high...

Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

Science.gov (United States)

Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

2011-09-08

Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids.

Monoclonal antibody-assisted structure-function analysis of the carbohydrate recognition domain of surfactant protein D

DEFF Research Database (Denmark)

Hartshorn, Kevan L; White, Mitchell R; Rynkiewicz, Michael

2010-01-01

Surfactant protein D (SP-D) plays important roles in host defense against a variety of pathogens including influenza A virus (IAV). Ligand binding by SP-D is mediated by the trimeric neck and carbohydrate recognition domain (NCRD). We used monoclonal antibodies (mAbs) against human SP-D and a panel...

The N- and C-terminal carbohydrate recognition domains of Haemonchus contortus galectin bind to distinct receptors of goat PBMC and contribute differently to its immunomodulatory functions in host-parasite interactions.

Science.gov (United States)

Lu, MingMin; Tian, XiaoWei; Yang, XinChao; Yuan, Cheng; Ehsan, Muhammad; Liu, XinChao; Yan, RuoFeng; Xu, LiXin; Song, XiaoKai; Li, XiangRui

2017-09-05

Hco-gal-m is a tandem-repeat galectin isolated from the adult worm of Haemonchus contortus. A growing body of studies have demonstrated that Hco-gal-m could exert its immunomodulatory effects on host peripheral blood mononuclear cells (PBMC) to facilitate the immune evasion. Our previous work revealed that C-terminal and N-terminal carbohydrate recognition domains (CRD) of Hco-gal-m had different sugar binding abilities. However, whether different domains of Hco-gal-m account differently for its multiple immunomodulatory functions in the host-parasite interaction remains to be elucidated. We found that the N-terminal CRD of Hco-gal-m (MNh) and the C-terminal CRD (MCh) could bind to goat peripheral blood mononuclear cells by distinct receptors: transmembrane protein 63A (TMEM63A) was a binding receptor of MNh, while transmembrane protein 147 (TMEM147) was a binding receptor of MCh. In addition, MCh was much more potent than MNh in inhibiting cell proliferation and inducing apoptosis, while MNh was much more effective in inhibiting NO production. Moreover, MNh could suppress the transcription of interferon-γ (IFN-γ), but MCh not. Our data suggested that these two CRDs of Hco-gal-m bind to distinct receptors and contributed differently to its ability to downregulate host immune response. These results will improve our understanding of galectins from parasitic nematodes contributing to the mechanism of parasitic immune evasion and continue to illustrate the diverse range of biological activities attributable to the galectin family.

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.

2013-01-01

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. PMID:24145027

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.

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

Science.gov (United States)

Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

2003-01-01

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

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…

A urokinase receptor-associated protein with specific collagen binding properties

DEFF Research Database (Denmark)

Behrendt, N; Jensen, O N; Engelholm, L H

2000-01-01

membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

Plant α-glucan phosphatases SEX4 and LSF2 display different affinity for amylopectin and amylose

DEFF Research Database (Denmark)

Wilkens, Casper; Auger, Kyle D.; Anderson, Nolan T.

2016-01-01

The plant glucan phosphatases Starch EXcess 4 (SEX4) and Like Sex Four2 (LSF2) apply different starch binding mechanisms. SEX4 contains a carbohydrate binding module, and LSF2 has two surface binding sites (SBSs). We determined KDapp for amylopectin and amylose, and KD for β-cyclodextrin and vali...

Photosynthesis down-regulation precedes carbohydrate accumulation under sink limitation in Citrus.

Science.gov (United States)

Nebauer, Sergio G; Renau-Morata, Begoña; Guardiola, José Luis; Molina, Rosa-Victoria

2011-02-01

Photosynthesis down-regulation due to an imbalance between sources and sinks in Citrus leaves could be mediated by excessive accumulation of carbohydrates. However, there is limited understanding of the physiological role of soluble and insoluble carbohydrates in photosynthesis regulation and the elements triggering the down-regulation process. In this work, the role of non-structural carbohydrates in the regulation of photosynthesis under a broad spectrum of source-sink relationships has been investigated in the Salustiana sweet orange. Soluble sugar and starch accumulation in leaves, induced by girdling experiments, did not induce down-regulation of the photosynthetic rate in the presence of sinks (fruits). The leaf-to-fruit ratio did not modulate photosynthesis but allocation of photoassimilates to the fruits. The lack of strong sink activity led to a decrease in the photosynthetic rate and starch accumulation in leaves. However, photosynthesis down-regulation due to an excess of total soluble sugars or starch was discarded because photosynthesis and stomatal conductance reduction occurred prior to any significant accumulation of these carbohydrates. Gas exchange and fluorescence parameters suggested biochemical limitations to photosynthesis. In addition, the expression of carbon metabolism-related genes was altered within 24 h when strong sinks were removed. Sucrose synthesis and export genes were inhibited, whereas the expression of ADP-glucose pyrophosphorylase was increased to cope with the excess of assimilates. In conclusion, changes in starch and soluble sugar turnover, but not sugar content per se, could provide the signal for photosynthesis regulation. In these conditions, non-stomatal limitations strongly inhibited the photosynthetic rate prior to any significant increase in carbohydrate levels.

Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding*

Science.gov (United States)

Huang, Yiping; Nokhrin, Sergiy; Hassanzadeh-Ghassabeh, Gholamreza; Yu, Corey H.; Yang, Haojun; Barry, Amanda N.; Tonelli, Marco; Markley, John L.; Muyldermans, Serge; Dmitriev, Oleg Y.; Lutsenko, Svetlana

2014-01-01

The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell. PMID:25253690

FHA domains as phospho-threonine binding modules in cell signaling.

Science.gov (United States)

Hammet, Andrew; Pike, Brietta L; McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

2003-01-01

Forkhead-associated (FHA) domains are present in >200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome.

Boronic acid recognition of non-interacting carbohydrates for biomedical applications: increasing fluorescence signals of minimally interacting aldoses and sucralose.

Science.gov (United States)

Resendez, Angel; Halim, Md Abdul; Singh, Jasmeet; Webb, Dominic-Luc; Singaram, Bakthan

2017-11-22

To address carbohydrates that are commonly used in biomedical applications with low binding affinities for boronic acid based detection systems, two chemical modification methods were utilized to increase sensitivity. Modified carbohydrates were analyzed using a two component fluorescent probe based on boronic acid-appended viologen-HPTS (4,4'-o-BBV). Carbohydrates normally giving poor signals (fucose, l-rhamnose, xylose) were subjected to sodium borohydride (NaBH 4 ) reduction in ambient conditions for 1 h yielding the corresponding sugar alcohols from fucose, l-rhamnose and xylose in essentially quantitative yields. Compared to original aldoses, apparent binding affinities were increased 4-25-fold. The chlorinated sweetener and colon permeability marker sucralose (Splenda), otherwise undetectable by boronic acids, was dechlorinated to a detectable derivative by reactive oxygen and hydroxide intermediates by the Fenton reaction or by H 2 O 2 and UV light. This method is specific to sucralose as other common sugars, such as sucrose, do not contain any carbon-chlorine bonds. Significant fluorescence response was obtained for chemically modified sucralose with the 4,4'-o-BBV-HPTS probe system. This proof of principle can be applied to biomedical applications, such as gut permeability, malabsorption, etc.

Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode.

Science.gov (United States)

Nieto, Lidia; Tharun, Inga M; Balk, Mark; Wienk, Hans; Boelens, Rolf; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

2015-11-20

The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the "two-pronged plug two-hole socket" model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain.

Science.gov (United States)

Rust, K; Grosso, L; Zhang, V; Chang, D; Persson, A; Longmore, W; Cai, G Z; Crouch, E

1991-10-01

Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.

Effects of high-carbohydrate and high-fat dietary treatments on measures of heart rate variability and sympathovagal balance.

Science.gov (United States)

Millis, Richard M; Austin, Rachel E; Bond, Vernon; Faruque, Mezbah; Goring, Kim L; Hickey, Brian M; Blakely, Raymond; Demeersman, Ronald E

2009-07-17

We tested the hypothesis that respiratory quotient (RQ) determines sympathovagal balance associated with metabolism of stored and dietary energy substrates. Six 18-20 year-old African-American males were studied after two control pretreatments of fasting and post-treatments of metabolizing high-fat and high-carbohydrate beverages. RQ, heart rate (HR), energy expenditure (EE) and blood pressure (BP) were recorded at rest and repeated 1 h-3 h after ingesting isocaloric high-carbohydrate and high-fat beverages. Sympathovagal modulation of HR was quantified by the low frequency/high frequency (LF/HF) ratio from fast Fourier transform (spectral) analysis of the electrocardiogram RR intervals during paced breathing at 0.2 Hz. Significance of differences of peak post-treatment values from controls was evaluated by analysis of covariance and of correlations by linear regression at Pcarbohydrate and high-fat treatments increased RQ, EE, HR and LF/HF with significant interactions between covariates. LF/HF values were not significant after eliminating covariance of RQ, EE and HR for the control vs. high-fat and for the high-fat vs. high-carbohydrate and after eliminating covariance of EE and HR for the control vs. high-carbohydrate treatments. Across the RQ values, correlations were significant for EE and LF/HF. These findings imply that high RQ and sympathetic modulation produced by metabolizing carbohydrate is associated with high resting energy expenditure. We conclude that respiratory quotient may be an important determinant of the LF/HF ratio in the heart rate variability spectrum, likely, by a respiratory chemosensory mechanism.

The carbohydrate sequence markup language (CabosML): an XML description of carbohydrate structures.

Science.gov (United States)

Kikuchi, Norihiro; Kameyama, Akihiko; Nakaya, Shuuichi; Ito, Hiromi; Sato, Takashi; Shikanai, Toshihide; Takahashi, Yoriko; Narimatsu, Hisashi

2005-04-15

Bioinformatics resources for glycomics are very poor as compared with those for genomics and proteomics. The complexity of carbohydrate sequences makes it difficult to define a common language to represent them, and the development of bioinformatics tools for glycomics has not progressed. In this study, we developed a carbohydrate sequence markup language (CabosML), an XML description of carbohydrate structures. The language definition (XML Schema) and an experimental database of carbohydrate structures using an XML database management system are available at http://www.phoenix.hydra.mki.co.jp/CabosDemo.html kikuchi@hydra.mki.co.jp.

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.

Carbohydrates and Diabetes (For Parents)

Science.gov (United States)

... Staying Safe Videos for Educators Search English Español 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 ...

Combined albumin and bicarbonate adversely affects equine sperm-oviduct binding

NARCIS (Netherlands)

Leemans, Bart; Gadella, Bart M; Stout, Tom Arjun Edgar; Sostaric, Edita; De Schauwer, Catharina; Nelis, Hilde Maria; Hoogewijs, Maarten; Van Soom, Ann

2016-01-01

In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. D-galactose has previously been

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

Science.gov (United States)

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 by Drosophila Pumilio (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 regulated in 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.

Shielding voices: The modulation of binding processes between voice features and response features by task representations.

Science.gov (United States)

Bogon, Johanna; Eisenbarth, Hedwig; Landgraf, Steffen; Dreisbach, Gesine

2017-09-01

Vocal events offer not only semantic-linguistic content but also information about the identity and the emotional-motivational state of the speaker. Furthermore, most vocal events have implications for our actions and therefore include action-related features. But the relevance and irrelevance of vocal features varies from task to task. The present study investigates binding processes for perceptual and action-related features of spoken words and their modulation by the task representation of the listener. Participants reacted with two response keys to eight different words spoken by a male or a female voice (Experiment 1) or spoken by an angry or neutral male voice (Experiment 2). There were two instruction conditions: half of participants learned eight stimulus-response mappings by rote (SR), and half of participants applied a binary task rule (TR). In both experiments, SR instructed participants showed clear evidence for binding processes between voice and response features indicated by an interaction between the irrelevant voice feature and the response. By contrast, as indicated by a three-way interaction with instruction, no such binding was found in the TR instructed group. These results are suggestive of binding and shielding as two adaptive mechanisms that ensure successful communication and action in a dynamic social environment.

Development of gastrointestinal surface. VIII. Lectin identification of carbohydrate differences

International Nuclear Information System (INIS)

Pang, K.Y.; Bresson, J.L.; Walker, W.A.

1987-01-01

Binding of microvillus membranes (MVM) from newborn and adult rats by concanavalin A (Con A), Ulex europaeus (UEA I), Dolichos bifluorus (DBA), and Triticum vulgaris (WGA) was examined to determine the availability of carbohydrate-containing sites for these lectins on the intestinal surface during development. Consistent patterns of differences in the reaction of MVM with these lectins were found. Con A and UEA had much higher reactivities to MVM of adult than newborn rats. 125 I-labeled-UEA gel overlay experiments revealed the abundance of UEA-binding sites in MVM of adult rat in contrast to the two binding sites in MVM of a newborn rat. DBA bound only to MVM of the adults, and very few binding sites were found in immature MVM. In contrast to these lectins, WGA binding was much higher in MVM of the newborns and decreased with maturation. Additional experiments on the age dependence of UEA and DBA reactivities revealed that the most striking changes occur in animals from 2 to 2 wk of age. In MVM from 2-wk-old rats, there were only 13.9% and < 0.2% of the adult binding capacities for UEA and DBA, respectively. By the time the animals were 4 wk old, the binding capacity for UEA had attained close to the level of the adults, whereas for DBA it reached 71.3% of the adult value. These results provide definite evidence of changes in the intestinal surface during perinatal development

Development of gastrointestinal surface. VIII. Lectin identification of carbohydrate differences

Energy Technology Data Exchange (ETDEWEB)

Pang, K.Y.; Bresson, J.L.; Walker, W.A.

1987-05-01

Binding of microvillus membranes (MVM) from newborn and adult rats by concanavalin A (Con A), Ulex europaeus (UEA I), Dolichos bifluorus (DBA), and Triticum vulgaris (WGA) was examined to determine the availability of carbohydrate-containing sites for these lectins on the intestinal surface during development. Consistent patterns of differences in the reaction of MVM with these lectins were found. Con A and UEA had much higher reactivities to MVM of adult than newborn rats. /sup 125/I-labeled-UEA gel overlay experiments revealed the abundance of UEA-binding sites in MVM of adult rat in contrast to the two binding sites in MVM of a newborn rat. DBA bound only to MVM of the adults, and very few binding sites were found in immature MVM. In contrast to these lectins, WGA binding was much higher in MVM of the newborns and decreased with maturation. Additional experiments on the age dependence of UEA and DBA reactivities revealed that the most striking changes occur in animals from 2 to 2 wk of age. In MVM from 2-wk-old rats, there were only 13.9% and < 0.2% of the adult binding capacities for UEA and DBA, respectively. By the time the animals were 4 wk old, the binding capacity for UEA had attained close to the level of the adults, whereas for DBA it reached 71.3% of the adult value. These results provide definite evidence of changes in the intestinal surface during perinatal development.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis1[OPEN

Science.gov (United States)

Hsiao, An-Shan; Xue, Yan

2017-01-01

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1. Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 (GL2), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1, smo1-1, and ACBP1+/−smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1+/−smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. PMID:28500265

Exponential increase in postprandial blood-glucose exposure with increasing carbohydrate loads using a linear carbohydrate-to-insulin ratio.

Science.gov (United States)

Marran, K J; Davey, B; Lang, A; Segal, D G

2013-04-10

Postprandial glucose excursions contribute significantly to average blood glucose, glycaemic variability and cardiovascular risk. Carbohydrate counting is a method of insulin dosing that balances carbohydrate load to insulin dose using a fixed ratio. Many patients and current insulin pumps calculate insulin delivery for meals based on a linear carbohydrate-to-insulin relationship. It is our hypothesis that a non-linear relationship exists between the amounts of carbohydrate consumed and the insulin required to cover it. To document blood glucose exposure in response to increasing carbohydrate loads on fixed carbohydrate-to-insulin ratios. Five type 1 diabetic subjects receiving insulin pump therapy with good control were recruited. Morning basal rates and carbohydrate- to-insulin ratios were optimised. A Medtronic glucose sensor was used for 5 days to collect data for area-under-the-curve (AUC) analysis, during which standardised meals of increasing carbohydrate loads were consumed. Increasing carbohydrate loads using a fixed carbohydrate-to-insulin ratio resulted in increasing glucose AUC. The relationship was found to be exponential rather than linear. Late postprandial hypoglycaemia followed carbohydrate loads of >60 g and this was often followed by rebound hyperglycaemia that lasted >6 hours. A non-linear relationship exists between carbohydrates consumed and the insulin required to cover them. This has implications for control of postprandial blood sugars, especially when consuming large carbohydrate loads. Further studies are required to look at the optimal ratios, duration and type of insulin boluses required to cover increasing carbohydrate loads.

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

Directory of Open Access Journals (Sweden)

Christophe Moreau

2016-11-01

Full Text Available 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.

Mannose-binding Lectin and the Risk of HIV Transmission and Disease Progression in Children A Systematic Review

NARCIS (Netherlands)

Israëls, Joël; Scherpbier, Henriette J.; Frakking, Florine N. J.; van de Wetering, Marianne D.; Kremer, Leontien C. M.; Kuijpers, Taco W.

2012-01-01

Background: Mannose-binding lectin (MBL) can activate the complement system by binding to carbohydrates, such as those presented on the HIV virion surface. It is unclear whether genetically determined MBL deficiency is related to vertical HIV transmission and disease progression in HIV-infected

gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]-t-butylbicyclophosphorothionate binding to cerebellar granule cells

International Nuclear Information System (INIS)

Gallo, V.; Wise, B.C.; Vaccarino, F.; Guidotti, A.

1985-01-01

t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, the authors have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [ 35 S]TBPS. At 25 degrees C, and in a modified Locke solution, the [ 35 S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [ 35 S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [ 35 S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [ 35 S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [ 35 S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [ 35 S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

Science.gov (United States)

Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

2017-07-18

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

Modulation of telomere binding proteins: a future area of research for skin protection and anti-aging target.

Science.gov (United States)

Imbert, Isabelle; Botto, Jean-Marie; Farra, Claude D; Domloge, Nouha

2012-06-01

Telomere shortening is considered as one of the main characteristics of cellular aging by limiting cellular division. Besides the fundamental advances through the discoveries of telomere and telomerase, which were recognized by a Nobel Prize, telomere protection remains an essential area of research. Recently, it was evidenced that studying the cross-talks between the proteins associated with telomere should provide a better understanding of the mechanistic basis for telomere-associated aging phenotypes. In this review, we discuss the current knowledge on telomere shortening, telomerase activity, and the essential role of telomere binding proteins in telomere stabilization and telomere-end protection. This review highlights the capacity of telomere binding proteins to limit cellular senescence and to maintain skin tissue homeostasis, which is of key importance to reduce accelerated tissue aging. Future studies addressing telomere protection and limitation of DNA damage response in human skin should include investigations on telomere binding proteins. As little is known about the expression of telomere binding proteins in human skin and modulation of their expression with aging, it remains an interesting field of skin research and a key area for future skin protection and anti-aging developments. © 2012 Wiley Periodicals, Inc.

[Performance enhancement by carbohydrate intake during sport: effects of carbohydrates during and after high-intensity exercise].

Science.gov (United States)

Beelen, Milou; Cermak, Naomi M; van Loon, Luc J C

2015-01-01

Endogenous carbohydrate availability does not provide sufficient energy for prolonged moderate to high-intensity exercise. Carbohydrate ingestion during high-intensity exercise can therefore enhance performance.- For exercise lasting 1 to 2.5 hours, athletes are advised to ingest 30-60 g of carbohydrates per hour.- Well-trained endurance athletes competing for longer than 2.5 hours at high intensity can metabolise up to 90 g of carbohydrates per hour, provided that a mixture of glucose and fructose is ingested.- Athletes participating in intermittent or team sports are advised to follow the same strategies but the timing of carbohydrate intake depends on the type of sport.- If top performance is required again within 24 hours after strenuous exercise, the advice is to supplement endogenous carbohydrate supplies quickly within the first few hours post-exercise by ingesting large amounts of carbohydrate (1.2 g/kg/h) or a lower amount of carbohydrate (0.8 g/kg/h) with a small amount of protein (0.2-0.4 g/kg/h).

Fat and carbohydrate metabolism during submaximal exercise in children.

Science.gov (United States)

Aucouturier, Julien; Baker, Julien S; Duché, Pascale

2008-01-01

During exercise, the contribution of fat and carbohydrate to energy expenditure is largely modulated by the intensity of exercise. Age, a short- or long-term diet enriched in carbohydrate or fat substrate stores, training and gender are other factors that have also been found to affect this balance. These factors have been extensively studied in adults from the perspective of improving performance in athletes, or from a health perspective in people with diseases. During the last decade, lifestyle changes associated with high-energy diets rich in lipid and reduced physical activity have contributed to the increase in childhood obesity. This lifestyle change has emerged as a serious health problem favouring the early development of cardiovascular diseases, insulin resistance or type 2 diabetes mellitus. Increasing physical activity levels in young people is important to increase energy expenditure and promote muscle oxidative capacity. Therefore, it is surprising that the regulation of balance between carbohydrate and lipid use during exercise has received much less attention in children than in adults. In this review, we have focused on the factors that affect carbohydrate and lipid metabolism during exercise and have identified areas that may be relevant in explaining the higher contribution of lipid to energy expenditure in children when compared with adults. Low muscle glycogen content is possibly associated with a low activity of glycolytic enzymes and high oxidative capacity, while lower levels of sympathoadrenal hormones are likely to favour lipid metabolism in children. Changes in energetic metabolism occurring during adolescence are also dependent on pubertal events with an increase in testosterone in boys and estrogen and progesterone in girls. The profound effects of ovarian hormones on carbohydrate and fat metabolism along with their effects on oxidative enzymes could explain that differences in substrate metabolism have not always been observed between

Methodological challenges in carbohydrate analyses

Directory of Open Access Journals (Sweden)

Mary Beth Hall

2007-07-01

Full Text Available Carbohydrates can provide up to 80% of the dry matter in animal diets, yet their specific evaluation for research and diet formulation is only now becoming a focus in the animal sciences. Partitioning of dietary carbohydrates for nutritional purposes should reflect differences in digestion and fermentation characteristics and effects on animal performance. Key challenges to designating nutritionally important carbohydrate fractions include classifying the carbohydrates in terms of nutritional characteristics, and selecting analytical methods that describe the desired fraction. The relative lack of information on digestion characteristics of various carbohydrates and their interactions with other fractions in diets means that fractions will not soon be perfectly established. Developing a system of carbohydrate analysis that could be used across animal species could enhance the utility of analyses and amount of data we can obtain on dietary effects of carbohydrates. Based on quantities present in diets and apparent effects on animal performance, some nutritionally important classes of carbohydrates that may be valuable to measure include sugars, starch, fructans, insoluble fiber, and soluble fiber. Essential to selection of methods for these fractions is agreement on precisely what carbohydrates should be included in each. Each of these fractions has analyses that could potentially be used to measure them, but most of the available methods have weaknesses that must be evaluated to see if they are fatal and the assay is unusable, or if the assay still may be made workable. Factors we must consider as we seek to analyze carbohydrates to describe diets: Does the assay accurately measure the desired fraction? Is the assay for research, regulatory, or field use (affects considerations of acceptable costs and throughput? What are acceptable accuracy and variability of measures? Is the assay robust (enhances accuracy of values? For some carbohydrates, we

Cats and Carbohydrates: The Carnivore Fantasy?

Directory of Open Access Journals (Sweden)

Adronie Verbrugghe

2017-11-01

Full Text Available The domestic cat’s wild ancestors are obligate carnivores that consume prey containing only minimal amounts of carbohydrates. Evolutionary events adapted the cat’s metabolism and physiology to this diet strictly composed of animal tissues and led to unique digestive and metabolic peculiarities of carbohydrate metabolism. The domestic cat still closely resembles its wild ancestor. Although the carnivore connection of domestic cats is well recognised, little is known about the precise nutrient profile to which the digestive physiology and metabolism of the cat have adapted throughout evolution. Moreover, studies show that domestic cats balance macronutrient intake by selecting low-carbohydrate foods. The fact that cats evolved consuming low-carbohydrate prey has led to speculations that high-carbohydrate diets could be detrimental for a cat’s health. More specifically, it has been suggested that excess carbohydrates could lead to feline obesity and diabetes mellitus. Additionally, the chances for remission of diabetes mellitus are higher in cats that consume a low-carbohydrate diet. This literature review will summarise current carbohydrate knowledge pertaining to digestion, absorption and metabolism of carbohydrates, food selection and macronutrient balancing in healthy, obese and diabetic cats, as well as the role of carbohydrates in prevention and treatment of obesity and diabetes mellitus.

Major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the determination of Schistosoma mansoni schistosomulum surface antigenicity

International Nuclear Information System (INIS)

Omer-ali, P.; Magee, A.I.; Kelly, C.; Simpson, A.J.G.

1986-01-01

A radioimmunoassay that makes use of whole Schistosomula and 125 I-labeled protein A has been used to characterize and to quantify the binding of antisera to the surface of 3 hr mechanically transformed schistosomula of Schistosoma mansoni. This technique facilitates the determination of epitopes on the schistosomula in addition to those detected by surface labeling and immunoprecipitation. By using this technique, it has been demonstrated that there is a much greater binding to the parasite surface of antibodies from chronically infected mice (CMS) than of antibodies from mice infected with highly irradiated cercariae (VMS), and CMS recognizes epitopes that VMS does not. Treatment of the surface of the schistosomula with trifluoromethanesulphonic acid and sodium metaperiodate has suggested that the discrepancy of the binding between the two sera is due to the recognition of a large number of additional epitopes by CMS, which are carbohydrate in nature. Some of the carbohydrate epitopes are expressed on the previously described surface glycoprotein antigens of M/sub r/ 200,000, 38,000, and 17,000

Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose

DEFF Research Database (Denmark)

Kirkeby, S

2016-01-01

FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini...... to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human...

Modulation of ocular surface glycocalyx barrier function by a galectin-3 N-terminal deletion mutant and membrane-anchored synthetic glycopolymers.

Directory of Open Access Journals (Sweden)

Jerome Mauris

Full Text Available BACKGROUND: Interaction of transmembrane mucins with the multivalent carbohydrate-binding protein galectin-3 is critical to maintaining the integrity of the ocular surface epithelial glycocalyx. This study aimed to determine whether disruption of galectin-3 multimerization and insertion of synthetic glycopolymers in the plasma membrane could be used to modulate glycocalyx barrier function in corneal epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Abrogation of galectin-3 biosynthesis in multilayered cultures of human corneal epithelial cells using siRNA, and in galectin-3 null mice, resulted in significant loss of corneal barrier function, as indicated by increased permeability to the rose bengal diagnostic dye. Addition of β-lactose, a competitive carbohydrate inhibitor of galectin-3 binding activity, to the cell culture system, transiently disrupted barrier function. In these experiments, treatment with a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, but not full-length galectin-3, prevented the recovery of barrier function to basal levels. As determined by fluorescence microscopy, both cellobiose- and lactose-containing glycopolymers incorporated into apical membranes of corneal epithelial cells, independently of the chain length distribution of the densely glycosylated, polymeric backbones. Membrane incorporation of cellobiose glycopolymers impaired barrier function in corneal epithelial cells, contrary to their lactose-containing counterparts, which bound to galectin-3 in pull-down assays. CONCLUSIONS/SIGNIFICANCE: These results indicate that galectin-3 multimerization and surface recognition of lactosyl residues is required to maintain glycocalyx barrier function at the ocular surface. Transient modification of galectin-3 binding could be therapeutically used to enhance the efficiency of topical drug delivery.

Red pitaya juice supplementation ameliorates energy balance homeostasis by modulating obesity-related genes in high-carbohydrate, high-fat diet-induced metabolic syndrome rats.

Science.gov (United States)

Ramli, Nurul Shazini; Ismail, Patimah; Rahmat, Asmah

2016-07-26

Red pitaya (Hylocereus polyrhizus) or known as buah naga merah in Malay belongs to the cactus family, Cactaceae. Red pitaya has been shown to give protection against liver damage and may reduce the stiffness of the heart. Besides, the beneficial effects of red pitaya against obesity have been reported; however, the mechanism of this protection is not clear. Therefore, in the present study, we have investigated the red pitaya-targeted genes in obesity using high-carbohydrate, high-fat diet-induced metabolic syndrome rat model. A total of four groups were tested: corn-starch (CS), corn-starch + red pitaya juice (CRP), high-carbohydrate, high-fat (HCHF) and high-carbohydrate, high-fat + red pitaya juice (HRP). The intervention with 5 % red pitaya juice was continued for 8 weeks after 8 weeks initiation of the diet. Retroperitoneal, epididymal and omental fat pads were collected and weighed. Plasma concentration of IL-6 and TNF-α were measured using commercial kits. Gene expression analysis was conducted using RNA extracted from liver samples. A total of eighty-four genes related to obesity were analyzed using PCR array. The rats fed HCHF-diet for 16 weeks increased body weight, developed excess abdominal fat deposition and down-regulated the expression level of IL-1α, IL-1r1, and Cntfr as compared to the control group. Supplementation of red pitaya juice for 8 weeks increased omental and epididymal fat but no change in retroperitoneal fat was observed. Red pitaya juice reversed the changes in energy balance homeostasis in liver tissues by regulation of the expression levels of Pomc and Insr. The increased protein expression levels of IL-6 and TNF-α in HCHF group and red pitaya treated rats confirmed the results of gene expression. Collectively, this study revealed the usefulness of this diet-induced rat model and the beneficial effects of red pitaya on energy balance homeostasis by modulating the anorectic, orexigenic and energy expenditure related

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.

Phenol-Sulfuric Acid Method for Total Carbohydrates

Science.gov (United States)

Nielsen, S. Suzanne

The phenol-sulfuric acid method is a simple and rapid colorimetric method to determine total carbohydrates in a sample. The method detects virtually all classes of carbohydrates, including mono-, di-, oligo-, and polysaccharides. Although the method detects almost all carbohydrates, the absorptivity of the different carbohydrates varies. Thus, unless a sample is known to contain only one carbohydrate, the results must be expressed arbitrarily in terms of one carbohydrate.

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

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

Complexes of natural carbohydrates with metal cations

International Nuclear Information System (INIS)

Alekseev, Yurii E; Garnovskii, Alexander D; Zhdanov, Yu A

1998-01-01

Data on the interaction of natural carbohydrates (mono-, oligo-, and poly-saccharides, amino sugars, and natural organic acids of carbohydrate origin) with metal cations are surveyed and described systematically. The structural diversity of carbohydrate metal complexes, caused by some specific features of carbohydrates as ligands, is demonstrated. The influence of complex formation on the chemical properties of carbohydrates is discussed. It is shown that the formation of metal complexes plays an important role in the configurational and conformational analysis of carbohydrates. The practical significance of the coordination interaction in the series of carbohydrate ligands is demonstrated. The bibliography includes 571 references.

Binding activity of patterned concanavalin A studied by atomic force microscopy

International Nuclear Information System (INIS)

Lebed, Kateryna; Pyka-Fosciak, Grazyna; Raczkowska, Joanna; Lekka, Malgorzata; Styczen, Jan

2005-01-01

The mode of protein immobilization plays a crucial role in the preparation of protein microarrays used for a wide spectrum of applications in analytical biochemistry. The microcontact printing technique was used to form a protein pattern using concanavalin A (Con A) since Con A belongs to a group of proteins widely used in analytical assays due to their selectivity as regards different kinds of carbohydrates. Atomic force microscopy was used to image surface topography, delivering information about the quality of the protein pattern. The force spectroscopy mode was used to verify the functional activity of deposited proteins via determination of the forces of interaction between Con A and carboxypeptidase Y bearing carbohydrate structure recognized by Con A. The calculated binding force between Con A and CaY was 105 ± 2 pN and it was compared with that measured for Con A deposited directly from the protein solution. The similarity of the value obtained for the interaction force was independent of the mode of protein deposition, thereby verifying that the microcontact printing technique did not influence the carbohydrate binding activity of Con A. The correlation between the surface topography of patterned samples and adhesion maps obtained showed the possible use of AFM for studying the chemical properties of different regions of the micropatterns produced

Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

Directory of Open Access Journals (Sweden)

Y.-H. Percival Zhang

2011-01-01

Full Text Available The hydrogen economy presents an appealing energy future but its implementation must solve numerous problems ranging from low-cost sustainable production, high-density storage, costly infrastructure, to eliminating safety concern. The use of renewable carbohydrate as a high-density hydrogen carrier and energy source for hydrogen production is possible due to emerging cell-free synthetic biology technology—cell-free synthetic pathway biotransformation (SyPaB. Assembly of numerous enzymes and co-enzymes in vitro can create complicated set of biological reactions or pathways that microorganisms or catalysts cannot complete, for example, C6H10O5 (aq + 7 H2O (l à 12 H2 (g + 6 CO2 (g (PLoS One 2007, 2:e456. Thanks to 100% selectivity of enzymes, modest reaction conditions, and high-purity of generated hydrogen, carbohydrate is a promising hydrogen carrier for end users. Gravimetric density of carbohydrate is 14.8 H2 mass% if water can be recycled from proton exchange membrane fuel cells or 8.33% H2 mass% without water recycling. Renewable carbohydrate can be isolated from plant biomass or would be produced from a combination of solar electricity/hydrogen and carbon dioxide fixation mediated by high-efficiency artificial photosynthesis mediated by SyPaB. The construction of this carbon-neutral carbohydrate economy would address numerous sustainability challenges, such as electricity and hydrogen storage, CO2 fixation and long-term storage, water conservation, transportation fuel production, plus feed and food production.

Renewable Hydrogen Carrier - Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

Science.gov (United States)

2011-01-31

combinations have been investigated for the production of hydrogen from biomass carbohydrate. Chemical catalysis approaches include pyrolysis [19...temperature. High fructose corn syrup, low-cost sucrose replacement, is made by stabilized glucose isomerase, which can work at ~60 °C for even about two...gasoline, vegetable oil vs. biodiesel, corn kernels vs. ethanol [31,109]. Given a price of $0.18/kg carbohydrate (i.e., $10.6/GJ) [2,44], the hydrogen

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.

Protein metabolism in obese patients during very low-calorie mixed diets containing different amounts of proteins and carbohydrates.

Science.gov (United States)

Pasquali, R; Casimirri, F; Melchionda, N

1987-12-01

To assess long-term nitrogen sparing capacity of very low-calorie mixed diets, we administered two isoenergetic (2092KJ) liquid formula regimens of different composition for 8 weeks to two matched groups of massively obese patients (group 1: proteins 60 g, carbohydrate 54 g; group 2: proteins 41 g, carbohydrates 81 g). Weight loss was similar in both groups. Daily nitrogen balance (g) during the second month resulted more a negative in group 2 with respect to group 1. However, within the groups individual nitrogen sparing capacity varied markedly; only a few in group 1 and one in group 2 were able to attain nitrogen equilibrium throughout the study. Daily urine excretion of 3-methylhistidine fell significantly in group 1 but did not change in group 2. Unlike total proteins, albumins, and transferrin, serum levels of retinol-binding protein, thyroxin-binding globulin, and complement-C3 fell significantly in both groups but per cent variations of complement-C3 were more pronounced in the first group. Prealbumin levels fell persistently in group 1 and transiently in group 2. The results indicate that even with this type of diet an adequate amount of dietary protein represents the most important factor in minimizing whole body protein catabolism during long-term semistarvation in massively obese patients. Moreover, they confirm the possible role of dietary carbohydrates in the regulation of some visceral protein metabolism.

A novel fibronectin binding motif in MSCRAMMs targets F3 modules.

Directory of Open Access Journals (Sweden)

Sabitha Prabhakaran

Full Text Available 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.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.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.

Osmotic stress regulates the strength and kinetics of sugar binding to the maltoporin channel

International Nuclear Information System (INIS)

Gurnev, Philip A; Bezrukov, Sergey M; Harries, Daniel; Adrian Parsegian, V

2010-01-01

We study the effect of osmotic stress, exerted by salts, on carbohydrate binding to the sugar-specific bacterial channel maltoporin. When the channel is reconstituted into planar lipid bilayers, single events of its occlusion by sugar are seen as transient interruptions in the flow of small ions. We find that, for most salts, changes in the free energy of maltoporin-sugar binding vary linearly with solution osmotic pressure. Such a change in binding with solution osmolarity indicates that for each salt a constant number of salt-excluding water molecules is released upon sugar-maltoporin association at all salt concentrations. We find that larger numbers of water molecules are released upon binding of the cyclic carbohydrate β-cyclodextrin (CD) than upon binding of the corresponding linear homologue maltoheptaose (m7). Remarkably, the extent to which salts affect the binding constants and rates depends sensitively on the type of salt; dehydration in solutions of different anions corresponds to the Hofmeister series. In sodium sulfate solutions, CD and m7 respectively release about 120 and 35 salt-excluding water molecules; in sodium chloride solutions, 35 and 15 waters. No water release is observed with sodium bromide. Finally, by adding adamantane, known to form an inclusion complex with CD, we can infer that CD not only dehydrates but also undergoes a conformational change upon binding to the channel. As a practical outcome, our results also demonstrate how osmotic stress can improve single-molecule detection of different solutes using protein-based nanopores.

Melatonin redirects carbohydrates metabolism during sugar starvation in plant cells.

Science.gov (United States)

Kobylińska, Agnieszka; Borek, Sławomir; Posmyk, Małgorzata M

2018-05-01

Recent studies have shown that melatonin is an important molecule in plant physiology. It seems that the most important is that melatonin efficacy eliminates oxidative stress (direct and indirect antioxidant) and moreover induce plant stress reaction and switch on different defence strategies (preventively and interventively actions). In this report, the impact of exogenous melatonin on carbohydrate metabolism in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells during sugar starvation was examined. We analysed starch concentration, α-amylase and PEPCK activity as well as proteolytic activity in culture media. It has been shown that BY-2 cell treatment with 200 nM of melatonin improved viability of sugar-starved cells. It was correlated with higher starch content and phosphoenolpyruvate carboxykinase (PEPCK) activity. The obtained results revealed that exogenous melatonin under specific conditions (stress) can play regulatory role in sugar metabolism, and it may modulate carbohydrate concentration in etiolated BY-2 cells. Moreover, our results confirmed the hypothesis that if the starch is synthesised even in sugar-starved cells, it is highly probable that melatonin shifts the BY-2 cell metabolism on gluconeogenesis pathway and allows for synthesis of carbohydrates from nonsugar precursors, that is amino acids. These points to another defence strategy that was induced by exogenous melatonin applied in plants to overcome adverse environmental conditions. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

pH modulates the binding of early growth response protein 1 transcription factor to DNA.

Science.gov (United States)

Mikles, David C; Bhat, Vikas; Schuchardt, Brett J; Deegan, Brian J; Seldeen, Kenneth L; McDonald, Caleb B; Farooq, Amjad

2013-08-01

The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating the protein-DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment. © 2013 FEBS.

Serum levels of mannan-binding lectin in chickens prior to and during experimental infection with avian infectious bronchitis virus

DEFF Research Database (Denmark)

Juul-Madsen, H.R.; Munch, M.; Handberg, Kurt

2003-01-01

Mannan-binding lectin (MBL) is a glycoprotein and a member of the C-type lectin super family, the collectin family, and the acute phase protein family. The MBL exerts its function by directly binding to microbial surfaces through its carbohydrate recognition domains, followed by direct opsonizati...

Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens

Directory of Open Access Journals (Sweden)

Yew Margaret

2007-03-01

Full Text Available Abstract Background Natural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling. Results The VH4-59 gene encodes antibodies in the VH4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding. Conclusion Xenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy

Predicting water-soluble carbohydrates and ethanol-soluble carbohydrates in cool-season grasses with near-infrared reflectance spectroscopy

Science.gov (United States)

Grazing animals may require a high or low total nonstructural carbohydrate diet for optimal health and production. Understanding how nonstructural carbohydrates fluctuate in Kentucky pastures and being able to quantify and monitor nonstructural carbohydrates in a timely manner will greatly aid in m...

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

of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5. The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser...... of modulators BPAM97 (2) and BPAM344 (3) into a hydroxyl group (BPAM557 (4) and BPAM521 (5), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from −4.9 (2) and −7.5 (3) to −6.2 (4) and −14.5 (5), but also a less favorable binding entropy (−TΔS, kcal/mol) from −2.3 (2) and −1.3 (3) to −0......-497 to the hydroxyl group of 5, whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5. In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation...

Synthesis of Sulochrin-125I and Its Binding Affinity as α-Glucosidase Inhibitor using Radioligand Binding Assay (RBA Method

Directory of Open Access Journals (Sweden)

W. Lestari

2014-04-01

Full Text Available Most of diabetics patients have type 2 diabetes mellitus or non insulin dependent diabetes mellitus. Treatment type 2 diabetes mellitus can be done by inhibiting α-glucosidase enzyme which converts carbohydrates into glucose. Sulochrin is one of the potential compounds which can inhibit the function of α-glucosidase enzyme. This study was carried out to obtain data of sulochrin binding with α-glucosidase enzyme as α-glucosidase inhibitor using Radioligand Binding Assay (RBA method. Primary reagent required in RBA method is labeled radioactive ligand (radioligand. In this study, the radioligand was sulochrin-125I and prior to sulochrin-125I synthesis, the sulochrin-I was synthesized. Sulochrin-I and sulochrin-125I were synthesized and their bindings were studied using Radioligand Binding Assay method. Sulochrin-I was synthesized with molecular formula C17H15O7I and molecular weight 457.9940. Sulochrin-125I was synthesized from sulochrin-I by isotope exchange method. From the RBA method, dissociation constant (Kd and maximum binding (Bmax were obtained 26.316 nM and Bmax 9.302 nM respectively. This low Kd indicated that sulochrin was can bind to α-glucosidase

Discovery and design of carbohydrate-based therapeutics.

Science.gov (United States)

Cipolla, Laura; Araújo, Ana C; Bini, Davide; Gabrielli, Luca; Russo, Laura; Shaikh, Nasrin

2010-08-01

Till now, the importance of carbohydrates has been underscored, if compared with the two other major classes of biopolymers such as oligonucleotides and proteins. Recent advances in glycobiology and glycochemistry have imparted a strong interest in the study of this enormous family of biomolecules. Carbohydrates have been shown to be implicated in recognition processes, such as cell-cell adhesion, cell-extracellular matrix adhesion and cell-intruder recognition phenomena. In addition, carbohydrates are recognized as differentiation markers and as antigenic determinants. Due to their relevant biological role, carbohydrates are promising candidates for drug design and disease treatment. However, the growing number of human disorders known as congenital disorders of glycosylation that are being identified as resulting from abnormalities in glycan structures and protein glycosylation strongly indicates that a fast development of glycobiology, glycochemistry and glycomedicine is highly desirable. The topics give an overview of different approaches that have been used to date for the design of carbohydrate-based therapeutics; this includes the use of native synthetic carbohydrates, the use of carbohydrate mimics designed on the basis of their native counterpart, the use of carbohydrates as scaffolds and finally the design of glyco-fused therapeutics, one of the most recent approaches. The review covers mainly literature that has appeared since 2000, except for a few papers cited for historical reasons. The reader will gain an overview of the current strategies applied to the design of carbohydrate-based therapeutics; in particular, the advantages/disadvantages of different approaches are highlighted. The topic is presented in a general, basic manner and will hopefully be a useful resource for all readers who are not familiar with it. In addition, in order to stress the potentialities of carbohydrates, several examples of carbohydrate-based marketed therapeutics are given

Global microarray analysis of carbohydrate use in alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5.

Directory of Open Access Journals (Sweden)

Yajian Song

Full Text Available The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources.

Global Microarray Analysis of Carbohydrate Use in Alkaliphilic Hemicellulolytic Bacterium Bacillus sp. N16-5

Science.gov (United States)

Song, Yajian; Xue, Yanfen; Ma, Yanhe

2013-01-01

The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources. PMID:23326578

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission.

Science.gov (United States)

Falk, Magnus; Alcalde, Miguel; Bartlett, Philip N; De Lacey, Antonio L; Gorton, Lo; Gutierrez-Sanchez, Cristina; Haddad, Raoudha; Kilburn, Jeremy; Leech, Dónal; Ludwig, Roland; Magner, Edmond; Mate, Diana M; Conghaile, Peter Ó; Ortiz, Roberto; Pita, Marcos; Pöller, Sascha; Ruzgas, Tautgirdas; Salaj-Kosla, Urszula; Schuhmann, Wolfgang; Sebelius, Fredrik; Shao, Minling; Stoica, Leonard; Sygmund, Cristoph; Tilly, Jonas; Toscano, Miguel D; Vivekananthan, Jeevanthi; Wright, Emma; Shleev, Sergey

2014-01-01

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission.

Directory of Open Access Journals (Sweden)

Magnus Falk

Full Text Available Here for the first time, we detail self-contained (wireless and self-powered biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor, and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

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......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...... therapy with glycosylation enzyme inhibitors will, however, require the development of more specific and less toxic compounds. If carbohydrate antigens can elicit a neutralizing immune response in vivo, the possibility exists that carbohydrate neoantigens can be utilized in the construction of a vaccine...

Carbohydrates digestion and metabolism in the spiny lobster (Panulirus argus): biochemical indication for limited carbohydrate utilization.

Science.gov (United States)

Rodríguez-Viera, Leandro; Perera, Erick; Montero-Alejo, Vivian; Perdomo-Morales, Rolando; García-Galano, Tsai; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

2017-01-01

As other spiny lobsters, Panulirus argus is supposed to use preferentially proteins and lipids in energy metabolism, while carbohydrates are well digested but poorly utilized. The aim of this study was to evaluate the effect of dietary carbohydrate level on digestion and metabolism in the spiny lobster P. argus . We used complementary methodologies such as post-feeding flux of nutrients and metabolites, as well as measurements of α-amylase expression and activity in the digestive tract. Lobsters readily digested and absorbed carbohydrates with a time-course that is dependent on their content in diet. Lobster showed higher levels of free glucose and stored glycogen in different tissues as the inclusion of wheat flour increased. Modifications in intermediary metabolism revealed a decrease in amino acids catabolism coupled with a higher use of free glucose as carbohydrates rise up to 20%. However, this effect seems to be limited by the metabolic capacity of lobsters to use more than 20% of carbohydrates in diets. Lobsters were not able to tightly regulate α-amylase expression according to dietary carbohydrate level but exhibited a marked difference in secretion of this enzyme into the gut. Results are discussed to highlight the limitations to increasing carbohydrate utilization by lobsters. Further growout trials are needed to link the presented metabolic profiles with phenotypic outcomes.

Carbohydrates digestion and metabolism in the spiny lobster (Panulirus argus: biochemical indication for limited carbohydrate utilization

Directory of Open Access Journals (Sweden)

Leandro Rodríguez-Viera

2017-11-01

Full Text Available As other spiny lobsters, Panulirus argus is supposed to use preferentially proteins and lipids in energy metabolism, while carbohydrates are well digested but poorly utilized. The aim of this study was to evaluate the effect of dietary carbohydrate level on digestion and metabolism in the spiny lobster P. argus. We used complementary methodologies such as post-feeding flux of nutrients and metabolites, as well as measurements of α-amylase expression and activity in the digestive tract. Lobsters readily digested and absorbed carbohydrates with a time-course that is dependent on their content in diet. Lobster showed higher levels of free glucose and stored glycogen in different tissues as the inclusion of wheat flour increased. Modifications in intermediary metabolism revealed a decrease in amino acids catabolism coupled with a higher use of free glucose as carbohydrates rise up to 20%. However, this effect seems to be limited by the metabolic capacity of lobsters to use more than 20% of carbohydrates in diets. Lobsters were not able to tightly regulate α-amylase expression according to dietary carbohydrate level but exhibited a marked difference in secretion of this enzyme into the gut. Results are discussed to highlight the limitations to increasing carbohydrate utilization by lobsters. Further growout trials are needed to link the presented metabolic profiles with phenotypic outcomes.

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

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

Dietary carbohydrate deprivation increases 24-hour nitrogen excretion without affecting postabsorptive hepatic or whole body protein metabolism in healthy men

NARCIS (Netherlands)

Bisschop, P. H.; de Sain-van der Velden, M. G. M.; Stellaard, F.; Kuipers, F.; Meijer, A. J.; Sauerwein, H. P.; Romijn, J. A.

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

Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

DEFF Research Database (Denmark)

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

2017-01-01

is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super......-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate...

Characterisation of the effect of ion channel modulators on I1-imidazoline binding sites in bovine adrenal medulla

International Nuclear Information System (INIS)

Musgrave, I.F.; Kotsopoulos, D.; Hughes, R.A.

1998-01-01

Full text: The structure of I 1 -imidazoline binding sites is still unknown and we have proposed that they represent ion channels (i). In these experiments we characterised the effects of the known ion channel modulators methyltriphenylphosphonium (MTPP), 4-aminopyridine (4-AP) and tetraethyl ammonium (TEA) on [ 3 H] clonidine binding in bovine adrenal medullary membranes as these membranes have a relatively well defined I 1 -imidazoline binding site (Molderings et al, 1993). Membranes from bovine adrenal medulla's were prepared by a minor modification of the method of Rapier et al. [ 3 H] Clonidine binding was performed by the method of Ernsberger et al (3), with [ 3 H] clonidine (62 Ci/mmol) used at a final concentration of 5 nM. [ 3 H] Clonidine binding was displaced from bovine adrenal medullary membranes by adrenergic drugs with the order of potency being oxymetazoline > clonidine > moxonidine = idazoxan >> yonimbine. This order of potency is consistent with previous studies of I 1 -imidazoline binding sites (4). Non-linear curve fitting to this data was consistent with a single site model. Both TEA and 4-AP displaced [ H] clonidine with similar potency to its effect on ion channels, TEA having a EC>> of 54 ± 0.3 μM (n=3). The displacement of [ 3 H] clonidine produced by both TEA and 4-AP also fitted to a single site model. Displacement of [ 3 H] clonidine by MTPP fitted a two site model (p 1 -imidazoline binding sites defined with [ 3 H] clonidine may represent ion channels. We have used this data to perform molecular modelling and have determined a common conformation of I 1 -prefering ligands which will aid in the development of I 1 -selective ligands in the future. Copyright (1998) Australian Neuroscience Society

Who is the carbohydrate?

Directory of Open Access Journals (Sweden)

Paulo Enrique Cuevas Mestanza

2017-10-01

Full Text Available Biochemistry is a complex science that studies biomolecules and their interactions in metabolic pathways in living beings. Due to the large amount of contents against the short period to apply them, only expositive classes are not enough to arouse the interest of students and solve questions. In this perspective, is very important to develop new educational tools to improve the understanding of these contents. “Who is the carbohydrate?” It is a didactic game created to review the structural and functional relationship of carbohydrates. Based on the classic “Guess who?” The objective of the player or group is to first find out the opponent's carbohydrate name.

New insight into structure/function relationships in plant alpha-amylase family GH13 members

DEFF Research Database (Denmark)

Seo, Eun-Seong; Andersen, Joakim Mark; Nielsen, Morten Munch

2010-01-01

Two carbohydrate binding surface sites (SBSs) on barley α-amylase 1 (AMY1) of glycoside hydrolase family 13 (GH13) displayed synergy in interactions with starch granules, thus being pivotal for hydrolysis of supramolecular substrates. Mutational analysis showed that SBS1 is more critical for the ......Two carbohydrate binding surface sites (SBSs) on barley α-amylase 1 (AMY1) of glycoside hydrolase family 13 (GH13) displayed synergy in interactions with starch granules, thus being pivotal for hydrolysis of supramolecular substrates. Mutational analysis showed that SBS1 is more critical...... binding domains (SBDs) mediate binding to starch granules. SBDs are currently categorised into 9 carbohydrate binding module (CBM) families. A novel CBM20 subfamily encountered in regulatory enzymes possesses characteristically low affinity for β-CD. Although α-amylase is essential for starch mobilisation...... in germinating barley seeds, efficient degradation requires the concerted action of α-amylase, β-amylase, limit dextrinase (LD) and possibly α-glucosidase. Limit dextrinase (LD) is encoded by a single gene and represents the sole debranching activity during germination. Recent expression of functional LD...

Modifying the Dietary Carbohydrate-to-Protein Ratio Alters the Postprandial Macronutrient Oxidation Pattern in Liver of AMPK-Deficient Mice.

Science.gov (United States)

Chalvon-Demersay, Tristan; Even, Patrick C; Chaumontet, Catherine; Piedcoq, Julien; Viollet, Benoit; Gaudichon, Claire; Tomé, Daniel; Foretz, Marc; Azzout-Marniche, Dalila

2017-09-01

Background: Hepatic AMP-activated kinase (AMPK) activity is sensitive to the dietary carbohydrate-to-protein ratio. However, the role of AMPK in metabolic adaptations to variations in dietary macronutrients remains poorly understood. Objective: The objective of this study was to determine the role of hepatic AMPK in the adaptation of energy metabolism in response to modulation of the dietary carbohydrate-to-protein ratio. Methods: Male 7-wk-old wild-type (WT) and liver AMPK-deficient (knockout) mice were fed either a normal-protein and normal-carbohydrate diet (NP-NC; 14% protein, 76% carbohydrate on an energy basis), a low-protein and high-carbohydrate diet (LP-HC; 5% protein, 85% carbohydrate), or a high-protein and low-carbohydrate diet (HP-LC; 55% protein, 35% carbohydrate) for 3 wk. During this period, after an overnight fast, metabolic parameters were measured and indirect calorimetry was performed in mice during the first hours after refeeding a 1-g calibrated meal of their own diet in order to investigate lipid and carbohydrate metabolism. Results: Knockout mice fed an LP-HC or HP-LC meal exhibited 24% and 8% lower amplitudes in meal-induced carbohydrate and lipid oxidation changes. By contrast, knockout mice fed an NP-NC meal displayed normal carbohydrate and lipid oxidation profiles. These mice exhibited a transient increase in hepatic triglycerides and a decrease in hepatic glycogen. These changes were associated with a 650% higher secretion of fibroblast growth factor 21 (FGF21) 2 h after refeeding. Conclusions: The consequences of hepatic AMPK deletion depend on the dietary carbohydrate-to-protein ratio. In mice fed the NP-NC diet, deletion of AMPK in the liver led to an adaptation of liver metabolism resulting in increased secretion of FGF21. These changes possibly compensated for the absence of hepatic AMPK, as these mice exhibited normal postprandial changes in carbohydrate and lipid oxidation. By contrast, in mice fed the LP-HC and HP-LC diets, the

Mannobiose Binding Induces Changes in Hydrogen Bonding and Protonation States of Acidic Residues in Concanavalin A As Revealed by Neutron Crystallography

Energy Technology Data Exchange (ETDEWEB)

Gerlits, Oksana O. [UT/ORNL; Coates, Leighton [Biology; Woods, Robert J. [Complex; Kovalevsky, Andrey [Biology

2017-08-30

Plant lectins are carbohydrate-binding proteins with various biomedical applications. Concanavalin A (Con A) holds promise in treating cancerous tumors. To better understand the Con A carbohydrate binding specificity, we obtained a room-temperature neutron structure of this legume lectin in complex with a disaccharide Manα1–2Man, mannobiose. The neutron structure afforded direct visualization of the hydrogen bonding between the protein and ligand, showing that the ligand is able to alter both protonation states and interactions for residues located close to and distant from the binding site. An unprecedented low-barrier hydrogen bond was observed forming between the carboxylic side chains of Asp28 and Glu8, with the D atom positioned equidistant from the oxygen atoms having an O···D···O angle of 101.5°.

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

Science.gov (United States)

Polireddy, Kishore; Khan, Mohiuddin Md Taimur; Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J; Haynes, Mark K; Bologa, Cristian G; Oprea, Tudor I; Tegos, George P; Sklar, Larry A; Krishnamurthy, Partha

2012-01-01

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

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�

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

International Nuclear Information System (INIS)

Nemčovičová, Ivana; Zajonc, Dirk M.

2014-01-01

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 6 G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor interactions

Gastrointestinal transit of extruded or pelletized diets in pacu fed distinct inclusion levels of lipid and carbohydrate

Directory of Open Access Journals (Sweden)

Claucia Aparecida Honorato

2014-11-01

Full Text Available The objective of this work was to evaluate the effect of pelletized or extruded diets, with different levels of carbohydrate and lipid, on the gastrointestinal transit time (GITT and its modulation in pacu (Piaractus mesopotamicus. One hundred and eighty pacu juveniles were fed with eight isonitrogenous diets containing two carbohydrate levels (40 and 50% and two lipid levels (4 and 8%. Four diets were pelletized and four were extruded. Carbohydrate and lipid experimental levels caused no changes to the bolus transit time. However, the bolus permanence time was related to diet processing. Fish fed pelletized diets exhibited the highest gastrointestinal transit time. Regression analysis of bolus behavior for pelletized and extruded diets with 4% lipid depicted different fits. GITT regression analysis of fish fed 8% lipid was fitted to a cubic equation and displayed adjustments of food permanence, with enhanced utilization of the diets, either with extruded or pelletized diets. GITT of fish fed extruded diets with 4% lipid was adjusted to a linear equation. The GITT of pacu depends on the diet processing and is affected by dietary levels of lipid and carbohydrate.

Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT genomic binding patterns discerns cell-specific cis-regulatory modules

Directory of Open Access Journals (Sweden)

Kang Keunsoo

2013-01-01

Full Text Available Abstract Background Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types. Results We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM, which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. Conclusions Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors.

Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy.

Science.gov (United States)

Peláez, Jesús; Long, Julie A

2007-01-01

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.

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

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...

Minimally refined biomass fuel. [carbohydrate-water-alcohol mixture

Energy Technology Data Exchange (ETDEWEB)

Pearson, R.K.; Hirschfeld, T.B.

1981-03-26

A minimally refined fluid composition, suitable as a fuel mixture and derived from biomass material, is comprised of one or more water-soluble carbohydrates such as sucrose, one or more alcohols having less than four carbons, and water. The carbohydrate provides the fuel source; water-solubilizes the carbohydrate; and the alcohol aids in the combustion of the carbohydrate and reduces the viscosity of the carbohydrate/water solution. Because less energy is required to obtain the carbohydrate from the raw biomass than alcohol, an overall energy savings is realized compared to fuels employing alcohol as the primary fuel.

Technological aspects of functional food-related carbohydrates.

NARCIS (Netherlands)

Voragen, A.G.J.

1998-01-01

Carbohydrates in food occur as natural constituents or are added as ingredients or additives. The most important endogenous carbohydrates in food are starch, depolymerized starch, sucrose, lactose, glucose, fructose and sorbitol (digestible) and carbohydrates such as raffinose, stachyose, resistant

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.

Expression of binding properties of Gal/GalNAc reactive lectins by mammalian glycotopes (an updated report).

Science.gov (United States)

Wu, A M

2001-01-01

Expression of the binding properties of Gal/GalNAc specific lectins, based on the affinity of decreasing order of mammalian glycotopes (determinants) rather than monosaccharide inhibition pattern, is probably one of the best ways to express carbohydrate specifity and should facilitate the selection of lectins as structural probes for studying mammalian glycobiology. Eleven mammalian structural units have been selected to express the binding domain of applied lectins. They are: 1. F, GalNAcalpha1 --> 3GalNAc; 2. A, GalNAcalpha1 --> 3Gal; 3. T, Galbeta1 --> 3GalNAc; 4. I, Galbeta 1 --> 3GlcNAc; 5. II, Galbeta1 --> 4GlcNAc; 6. B, Galalpha1 --> 3Gal; 7. E, Galalpha1--> 4Gal; 8. L, Galbeta1 --> 4Glc; 9. P, GalNAcbeta1 --> 3Gal; 10. S, GalNAcbeta1 --> 4Gal and 11. Tn, GalNAcalpha1 --> 4Ser (Thr) of the peptide chain. Thus, the carbohydrate specificity of Gal/GalNAc reactive lectins can be divided into classes according to their highest affinity for the above disaccharides and/or Tn residue. Examples of the binding properties of these lectins can be demonstrated by Ricimus communis agglutinin (RCA1), grouped as II specific lectin and its binding property is II > I > B > T; Ahrus precatorius agglutinin (APA), classified as T and its carbohydrate specificity is T > I/II > E > B > Tn; Artocarpus integrifolia (jacalin, AIL), as T/Tn specific and its binding reactivity is T > Tn > I (II) and Geodia cydonium (GCL), as F/A specific, and with affinity for F > Ah [GalNAcalpha1-->43(L(Fuc)alpha1-->2)Gal] > I > L. Due to the multiple reactivity of lectins toward mammalian glycotopes, the possible existence of different combining sites or subsites in the same molecule has to be examined, and the differential binding properties of these combining sites (if any) have to be characterized. To establish the relationship among the amino acid sequences of the combining sites of plant lectins and mammalian glycotopes should be an important direction to be addressed in lectinology.

Detection of secondary binding sites in proteins using fragment screening.

Science.gov (United States)

Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

2015-12-29

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

Thyroxine binding to serum thyronine-binding globulin in thyroidectomized adult and normal neonatal rats

International Nuclear Information System (INIS)

Young, R.A.; Meyers, B.; Alex, S.; Fang, S.L.; Braverman, L.E.

1988-01-01

The amount of tracer [125I]T4 bound to serum thyronine-binding globulin (TBG) was measured by polyacrylamide gel electrophoresis in adult thyroidectomized (TX) rats and normal 1-day to 4-week-old rat puts. Thyroidectomy was associated with the appearance of significant amounts of [125I]T4 binding to serum TBG in lean rats, but not in obese Zucker rats. Treatment of the TX rats in vivo with replacement doses of T4 prevented this increase in TBG binding, but enrichment of serum from TX rats with T4 did not. Significant amounts of tracer [125I]T4 binding to TBG was present in serum from 1- to 3-week-old normal rat pups, but not in 1-day- or 4-week-old pups. There were significantly higher levels of TBG binding of [125I]T4 in serum from 2-week-old rat pups raised in litters of 16 pups compared to those raised in litters of 4 pups. All manipulations that result in the appearance of TBG in rat serum also result in either weight loss or a slowing in the rate of growth, suggesting that the appearance of TBG in rat serum has a nutritional component. This possibility is further supported by the observations that increases in TBG binding of [125I]T4 are not found in obese Zucker rats fed a low protein-high carbohydrate diet for 14 days or fasted for 7 days, or after thyroidectomy, perhaps owing to the large stores of fuel in the obese rat

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.

Randomized controlled trial of changes in dietary carbohydrate/fat ratio and simple vs complex carbohydrates on body weight and blood lipids: the CARMEN study. The Carbohydrate Ratio Management in European National diets.

Science.gov (United States)

Saris, W H; Astrup, A; Prentice, A M; Zunft, H J; Formiguera, X; Verboeket-van de Venne, W P; Raben, A; Poppitt, S D; Seppelt, B; Johnston, S; Vasilaras, T H; Keogh, G F

2000-10-01

To investigate the long-term effects of changes in dietary carbohydrate/fat ratio and simple vs complex carbohydrates. Randomized controlled multicentre trial (CARMEN), in which subjects were allocated for 6 months either to a seasonal control group (no intervention) or to one of three experimental groups: a control diet group (dietary intervention typical of the average national intake); a low-fat high simple carbohydrate group; or a low-fat high complex carbohydrate group. Three hundred and ninety eight moderately obese adults. The change in body weight was the primary outcome; changes in body composition and blood lipids were secondary outcomes. Body weight loss in the low-fat high simple carbohydrate and low-fat high complex carbohydrate groups was 0.9 kg (P Fat mass changed by -1.3kg (Plow-fat high simple carbohydrate, low-fat high complex carbohydrate and control diet groups, respectively. Changes in blood lipids did not differ significantly between the dietary treatment groups. Our findings suggest that reduction of fat intake results in a modest but significant reduction in body weight and body fatness. The concomitant increase in either simple or complex carbohydrates did not indicate significant differences in weight change. No adverse effects on blood lipids were observed. These findings underline the importance of this dietary change and its potential impact on the public health implications of obesity.

Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

DEFF Research Database (Denmark)

Kirkeby, Svend; André, Sabine; Gabius, Hans-Joachim

2004-01-01

OBJECTIVES: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic alphagalactose (Galalpha) antigens in the doner tissue. DESIGN AND METHODS: We have measured the binding potentials of Galalpha detecting lectins and antibodies, including...

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.

Vacuum ultraviolet photoionization of carbohydrates and nucleotides

International Nuclear Information System (INIS)

Shin, Joong-Won; Bernstein, Elliot R.

2014-01-01

Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5 ′ -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

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

Directory of Open Access Journals (Sweden)

Kishore Polireddy

Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Bitopic Ligands and Metastable Binding Sites

DEFF Research Database (Denmark)

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

2017-01-01

of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

Cell surface carbohydrates as prognostic markers in human carcinomas

DEFF Research Database (Denmark)

Dabelsteen, Erik

1996-01-01

Tumour development is usually associated with changes in cell surface carbohydrates. These are often divided into changes related to terminal carbohydrate structures, which include incomplete synthesis and modification of normally existing carbohydrates, and changes in the carbohydrate core...... structure. The latter includes chain elongation of both glycolipids and proteins, increased branching of carbohydrates in N-linked glycoproteins, and blocked synthesis of carbohydrates in O-linked mucin-like glycoproteins. In mature organisms, expression of distinct carbohydrates is restricted to specific...... cell types; within a given tissue, variation in expression may be related to cell maturation. Tumour-associated carbohydrate structures often reflect a certain stage of cellular development; most of these moieties are structures normally found in other adult or embryonic tissues. There is no unique...

Dietary carbohydrates and glucose metabolism in diabetic patients.

Science.gov (United States)

Parillo, M; Riccardi, G

1995-12-01

Dietary carbohydrates represent one of the major sources of energy for the human body. However, the main (if not the only) therapy for diabetes since ancient times has been based on reducing dietary carbohydrates drastically because of their effects on blood glucose levels. The introduction of insulin in the 1920s and then of oral hypoglycaemic drugs led to various studies evaluating the biochemical characteristics of carbohydrates and their effects on glucose metabolism in diabetic patients. This review considers the role of dietary carbohydrates in the diet of diabetic patients in the light of the most recent studies and provides a short summary of the biochemistry of carbohydrates and the physiology of carbohydrate digestion.

Generic tools to assess genuine carbohydrate specific effects on in vitro immune modulation exemplified by β-glucans

DEFF Research Database (Denmark)

Rieder, Anne; Grimmer, Stine; Aachmann, Finn L.

2013-01-01

Even if carbohydrate preparations from plant/fungal sources have a high degree of purity, observed immune-stimulation may be caused by minute sample contaminations. Using the example of different β-glucans we present a range of analytical tools crucial for validation of possible immune-stimulator...

Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

Science.gov (United States)

Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

2016-07-01

Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection.

Mice lacking pituitary tumor transforming gene show elevated exposure of DGalNAc carbohydrate determinants

Directory of Open Access Journals (Sweden)

Lutsyk A. D.

2012-04-01

Full Text Available Aim. To investigate the influence of pituitary tumor transforming gene (pttg-1 knockout on glycome of parenchimal organs by means of lectin histochemistry. Methods. DGalNAc, DGlcNAc, NeuNAc carbohydrate determinants were labelled with soybean agglutinin (SBA and wheat germ agglutinin (WGA, conjugated to peroxidase, with subsequent visualization of the lectin-binding sites with diaminobenzidine. The testes and kidneys of murine strain BL6/C57 with the pttg-1 gene knockout (PTTG-KO were compared to the wild type (PTTG-WT animals, both groups 1 month of age. Results. Knockout of the pttg-1 gene was accompanied by enhanced exposure of the DGalNAc sugar residues within the Golgi complex of secondary spermatocytes, in a brush border of renal tubules and on the lumenal surface of collecting ducts. Conclusions. This study suggests that knockout of the pttg-1 gene may lead to the changes in carbohydrate processing in mammalian organism.

Characterization of carbohydrate structures of bovine MUC15 and distribution of the mucin in bovine milk

DEFF Research Database (Denmark)

Pallesen, Lone Tjener; Pedersen, Lise Refstrup Linnebjerg; Petersen, Torben Ellebæk

2007-01-01

-containing fractions as well, such as skim milk and whey. Compositional and structural studies of the carbohydrates of bovine milk MUC15 showed that the glycans are composed of fucose, galactose, mannose, N-acetylgalactosamine, N-acetylglycosamine, and sialic acid. The carbohydrate was shown to constitute 65......% of the total molecular weight, and the molar ratios of the individual sugars to protein of the O-linked glycans were determined. The glycan structures of MUC15 were further studied by enzymatic deglycosylation experiments using different endo- and exoglycosidases as well as a panel of lectins. The N......-linked glycans. By comparing the results of peanut agglutinin lectin binding, enzymatic deglycosylation, and monosaccharide composition analysis, we concluded that bovine MUC15 also contains more complex O-glycans containing high amounts N-acetylglucosamine residues. Furthermore, a small subset of the O...

A metagenome-derived thermostable β-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

Science.gov (United States)

Angelov, Angel; Pham, Vu Thuy Trang; Übelacker, Maria; Brady, Silja; Leis, Benedikt; Pill, Nicole; Brolle, Judith; Mechelke, Matthias; Moerch, Matthias; Henrissat, Bernard; Liebl, Wolfgang

2017-12-11

The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

Analysis and validation of carbohydrate three-dimensional structures

International Nuclear Information System (INIS)

Lütteke, Thomas

2009-01-01

The article summarizes the information that is gained from and the errors that are found in carbohydrate structures in the Protein Data Bank. Validation tools that can locate these errors are described. Knowledge of the three-dimensional structures of the carbohydrate molecules is indispensable for a full understanding of the molecular processes in which carbohydrates are involved, such as protein glycosylation or protein–carbohydrate interactions. The Protein Data Bank (PDB) is a valuable resource for three-dimensional structural information on glycoproteins and protein–carbohydrate complexes. Unfortunately, many carbohydrate moieties in the PDB contain inconsistencies or errors. This article gives an overview of the information that can be obtained from individual PDB entries and from statistical analyses of sets of three-dimensional structures, of typical problems that arise during the analysis of carbohydrate three-dimensional structures and of the validation tools that are currently available to scientists to evaluate the quality of these structures

Crosstalk between innate and adaptive immune responses to infectious bronchitis virus after vaccination and challenge of chickens varying in serum mannose-binding lectin concentrations

DEFF Research Database (Denmark)

Juul-Madsen, Helle R.; Norup, Liselotte R.; Jørgensen, Poul Henrik

2011-01-01

Mannose-binding lectin (MBL), a C-type collectin with structural similarities to C1q, is an innate pattern-recognition molecule that is sequestered to sites of inflammation and infections. MBL selectively binds distinct chemical patterns, including carbohydrates expressed on all kinds of pathogen...

Aminooxylated Carbohydrates: Synthesis and Applications.

Science.gov (United States)

Pifferi, Carlo; Daskhan, Gour Chand; Fiore, Michele; Shiao, Tze Chieh; Roy, René; Renaudet, Olivier

2017-08-09

Among other classes of biomolecules, carbohydrates and glycoconjugates are widely involved in numerous biological functions. In addition to addressing the related synthetic challenges, glycochemists have invested intense efforts in providing access to structures that can be used to study, activate, or inhibit these biological processes. Over the past few decades, aminooxylated carbohydrates have been found to be key building blocks for achieving these goals. This review provides the first in-depth overview covering several aspects related to the syntheses and applications of aminooxylated carbohydrates. After a brief introduction to oxime bonds and their relative stabilities compared to related C═N functions, synthetic aspects of oxime ligation and methodologies for introducing the aminooxy functionality onto both glycofuranosyls and glycopyranosyls are described. The subsequent section focuses on biological applications involving aminooxylated carbohydrates as components for the construcion of diverse architectures. Mimetics of natural structures represent useful tools for better understanding the features that drive carbohydrate-receptor interaction, their biological output and they also represent interesting structures with improved stability and tunable properties. In the next section, multivalent structures such as glycoclusters and glycodendrimers obtained through oxime ligation are described in terms of synthetic design and their biological applications such as immunomodulators. The second-to-last section discusses miscellaneous applications of oxime-based glycoconjugates, such as enantioselective catalysis and glycosylated oligonucleotides, and conclusions and perspectives are provided in the last section.

A Universal Protocol for Photochemical Covalent Immobilization of Intact Carbohydrates for the Preparation of Carbohydrate Microarrays

Science.gov (United States)

Wang, Huibin; Zhang, Yiming; Yuan, Xun; Chen, Yi; Yan, Mingdi

2010-01-01

A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, non-reducing sugars such as alditols and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose and their oligo- and polysaccharides were confirmed using surface plasmon resonance imaging and laser-induced fluorescence imaging. PMID:21138274

Role of Fatty Acid-Binding Protein 2 Ala54Thr Genotype on Weight Loss and Cardiovascular Risk Factors after a High-Protein/Low-Carbohydrate versus a Standard Hypocaloric Diet during 9 Months.

Science.gov (United States)

de Luis, Daniel Antonio; Izaola, Olatz; de la Fuente, Beatriz; Primo, David; Romero, Enrique

2015-01-01

It has been found that the expression of fatty acid-binding protein 2 gene mRNA is under dietary control. The polymorphism Ala54Thr of this protein was associated with high insulin resistance. The aim of our study was to investigate the influence of Thr54 polymorphism on metabolic response, weight loss and serum adipokine levels secondary to high-protein/low-carbohydrate vs. standard hypocaloric diets during 9 months. A population of 193 obese subjects was analyzed in a randomized trial. A nutritional evaluation was performed at the beginning and at the end of a 9-month period in which subjects received 1 of 2 diets (diet HP: high-protein/low-carbohydrate vs. diet S: standard diet). With both diets and in both genotype groups, body mass index, weight, fat mass, waist circumference, systolic blood pressure and leptin levels decreased. With both diets and only in wild genotype (diet HP vs. diet S), glucose (-6.2 ± 2.1 vs. -4.9 ± 2.0 mg/dl; p diet HP than HS. With both diets and only in the wild genotype, total cholesterol and LDL-total cholesterol levels decreased. Carriers of Thr54 allele have a different metabolic response after weight loss than wild type non-A carriers obese, with a lack of decrease of LDL-cholesterol, glucose, insulin levels and HOMA-R. © 2015 S. Karger AG, Basel.

Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody.

Science.gov (United States)

Bruehl, R E; Bertozzi, C R; Rosen, S D

2000-10-20

Sulfated forms of sialyl-Le(X) containing Gal-6-SO(4) or GlcNAc-6-SO(4) have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta1-->4GlcNAc) core of sulfosialyl-Le(X), however, remains unsettled. Using a panel of sulfated lactose (Galbeta1-->4Glc) neoglycolipids as substrates in direct binding assays, we found that 6',6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6'- and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6', 6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO(4) and GlcNAc-6-SO(4) may contribute to L-selectin recognition, either as components of sulfosialyl-Le(X) capping groups or in internal structures. By contrast, only GlcNAc-6-SO(4) appears to contribute to MECA-79 binding.

Qualitative Carbohydrate Analysis using Alkaline Potassium ...

Indian Academy of Sciences (India)

IAS Admin

CLASSROOM. 285. RESONANCE | March 2016. Qualitative Carbohydrate Analysis using Alkaline. Potassium Ferricyanide. Keywords. Alkaline potassium ferricyanide, qualitative ... Carbohydrates form a distinct class of organic compounds often .... Laboratory Techniques: A contemporary Approach, W B Saunders Com-.

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

is not universal and is in fact rare among some families of enzymes. In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. Such a site is termed a surface (or secondary) binding site (SBS). SBSs have been...

Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

International Nuclear Information System (INIS)

Rodriguez-Munoz, Rafael; Villarreal-Silva, Marcela; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Mondragon, Monica; Mondragon, Ricardo; Cerna, Joel; Cisneros, Bulmaro

2008-01-01

The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in Center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation

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.

What Happened to the IGF Binding Proteins?

Science.gov (United States)

Bach, Leon A

2018-02-01

Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential. Copyright © 2018 Endocrine Society.

Utilization of carbohydrates by radiation processing

International Nuclear Information System (INIS)

Kume, T.; Nagasawa, N.; Yoshii, F.

2002-01-01

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

Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor

International Nuclear Information System (INIS)

Imai, Y.; Singer, M.S.; Fennie, C.; Lasky, L.A.; Rosen, S.D.

1991-01-01

Lymphocyte attachment to high endothelial venules within lymph nodes is mediated by the peripheral lymph node homing receptor (pnHR), originally defined on mouse lymphocytes by the MEL-14 mAb. The pnHR is a calcium-dependent lectin-like receptor, a member of the LEC-CAM family of adhesion proteins. Here, using a soluble recombinant form of the homing receptor, we have identified an endothelial ligand for the pnHR as an ∼ 50-kD sulfated, fucosylated, and sialylated glycoprotein, which we designate Sgp50 (sulfated glycoprotein of 50 kD). Recombinant receptor binding to this lymph node-specific glycoprotein requires calcium and is inhibitable by specific carbohydrates and by MEL-14 mAb. Sialylation of the component is required for binding. Additionally, the glycoprotein is precipitated by MECA-79, an adhesion-blocking mAb reactive with lymph node HEV. A related glycoprotein of ∼ 90 kD (designated as Sgp90) is also identified

Renal function following long-term weight loss in individuals with abdominal obesity on a very-low-carbohydrate diet vs high-carbohydrate diet.

Science.gov (United States)

Brinkworth, Grant D; Buckley, Jonathan D; Noakes, Manny; Clifton, Peter M

2010-04-01

A frequently cited concern of very-low-carbohydrate diets is the potential for increased risk of renal disease associated with a higher protein intake. However, to date, no well-controlled randomized studies have evaluated the long-term effects of very-low-carbohydrate diets on renal function. To study this issue, renal function was assessed in 68 men and women with abdominal obesity (age 51.5+/-7.7 years, body mass index [calculated as kg/m(2)] 33.6+/-4.0) without preexisting renal dysfunction who were randomized to consume either an energy-restricted ( approximately 1,433 to 1,672 kcal/day), planned isocaloric very-low-carbohydrate (4% total energy as carbohydrate [14 g], 35% protein [124 g], 61% fat [99 g]), or high-carbohydrate diet (46% total energy as carbohydrate [162 g], 24% protein [85 g], 30% fat [49 g]) for 1 year. Body weight, serum creatinine, estimated glomerular filtration rate and urinary albumin excretion were assessed before and after 1 year (April 2006-July 2007). Repeated measures analysis of variance was conducted. Weight loss was similar in both groups (very-low-carbohydrate: -14.5+/-9.7 kg, high-carbohydrate: -11.6+/-7.3 kg; P=0.16). By 1 year, there were no changes in either group in serum creatinine levels (very-low-carbohydrate: 72.4+/-15.1 to 71.3+/-13.8 mumol/L, high-carbohydrate: 78.0+/-16.0 to 77.2+/-13.2 mumol/L; P=0.93 time x diet effect) or estimated glomerular filtration rate (very-low-carbohydrate: 90.0+/-17.0 to 91.2+/-17.8 mL/min/1.73 m(2), high-carbohydrate: 83.8+/-13.8 to 83.6+/-11.8 mL/min/1.73 m(2); P=0.53 time x diet effect). All but one participant was classified as having normoalbuminuria at baseline, and for these participants, urinary albumin excretion values remained in the normoalbuminuria range at 1 year. One participant in high-carbohydrate had microalbuminuria (41.8 microg/min) at baseline, which decreased to a value of 3.1 microg/min (classified as normoalbuminuria) at 1 year. This study provides preliminary

Carbohydrate Nutrition and Team Sport Performance.

Science.gov (United States)

Williams, Clyde; Rollo, Ian

2015-11-01

The common pattern of play in 'team sports' is 'stop and go', i.e. where players perform repeated bouts of brief high-intensity exercise punctuated by lower intensity activity. Sprints are generally 2-4 s long and recovery between sprints is of variable length. Energy production during brief sprints is derived from the degradation of intra-muscular phosphocreatine and glycogen (anaerobic metabolism). Prolonged periods of multiple sprints drain muscle glycogen stores, leading to a decrease in power output and a reduction in general work rate during training and competition. The impact of dietary carbohydrate interventions on team sport performance have been typically assessed using intermittent variable-speed shuttle running over a distance of 20 m. This method has evolved to include specific work to rest ratios and skills specific to team sports such as soccer, rugby and basketball. Increasing liver and muscle carbohydrate stores before sports helps delay the onset of fatigue during prolonged intermittent variable-speed running. Carbohydrate intake during exercise, typically ingested as carbohydrate-electrolyte solutions, is also associated with improved performance. The mechanisms responsible are likely to be the availability of carbohydrate as a substrate for central and peripheral functions. Variable-speed running in hot environments is limited by the degree of hyperthermia before muscle glycogen availability becomes a significant contributor to the onset of fatigue. Finally, ingesting carbohydrate immediately after training and competition will rapidly recover liver and muscle glycogen stores.

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...

Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations.

OpenAIRE

Leedle, J A; Hespell, R B

1980-01-01

A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates were developed for enumerating rumen bacteria. The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium. These colonies were pinpoint size (less than or equal to mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added. With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than...

Perceived hunger is lower and weight loss is greater in overweight premenopausal women consuming a low-carbohydrate/high-protein vs high-carbohydrate/low-fat diet.

Science.gov (United States)

Nickols-Richardson, Sharon M; Coleman, Mary Dean; Volpe, Joanne J; Hosig, Kathy W

2005-09-01

The impact of a low-carbohydrate/high-protein diet compared with a high-carbohydrate/low-fat diet on ratings of hunger and cognitive eating restraint were examined. Overweight premenopausal women consumed a low-carbohydrate/high-protein (n=13) or high-carbohydrate/low-fat diet (n=15) for 6 weeks. Fasting body weight (BW) was measured and the Eating Inventory was completed at baseline, weeks 1 to 4, and week 6. All women experienced a reduction in BW (Plow-carbohydrate/high-protein vs high-carbohydrate/low-fat group at week 6 (Plow-carbohydrate/high-protein but not in the high-carbohydrate/low-fat group from baseline to week 6. In both groups, self-rated cognitive eating restraint increased (Plow-carbohydrate/high-protein group may have contributed to a greater percentage of BW loss.

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

Czech Academy of Sciences Publication Activity Database

Krejčiříková, Veronika; Pachl, Petr; Fábry, Milan; Malý, Petr; Řezáčová, Pavlína; Brynda, Jiří

2011-01-01

Roč. 67, Pt3 (2011), 204-211 ISSN 0907-4449 R&D Projects: GA ČR GA203/09/0820; GA ČR GA304/03/0090; GA ČR GA301/07/0600 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50520701; CEZ:AV0Z40550506 Keywords : S-type lectins * carbohydrate binding * molecular recognition Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 12.619, year: 2011

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...

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

DEFF Research Database (Denmark)

Teillet, F; Lacroix, M; Thiel, Steffen

2007-01-01

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To ident......Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2....... To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19...... and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3...

Effect of Carbohydrate, Caffeine, and Carbohydrate + Caffeine Mouth Rinsing on Intermittent Running Performance in Collegiate Male Lacrosse Athletes.

Science.gov (United States)

Dolan, Patrick; Witherbee, Kyle E; Peterson, Kimi M; Kerksick, Chad M

2017-09-01

Dolan, P, Witherbee, KE, Peterson, KM, and Kerksick, CM. Effect of carbohydrate, caffeine, and carbohydrate + caffeine mouth rinsing on intermittent running performance in collegiate male lacrosse athletes. J Strength Cond Res 31(9): 2473-2479, 2017-Recently, an interest has developed in the potential to rinse the oral cavity with key nutrients to impact various types of exercise and presumably sporting performance. Although multiple studies examining carbohydrate mouth rinsing have been completed, conflicting evidence surrounding caffeine mouth rinsing persists, and no research has explored its ability to impact high-intensity, intermittent running performance. This study investigated the independent and synergistic ability of carbohydrate and caffeine mouth rinsing to improve intermittent running performance. The Yo-Yo Intermittent Recovery Test-Level 1 (Yo-Yo Level 1) was completed in 10 collegiate (National Collegiate Athletic Association [NCAA] Division II) male lacrosse players after a 10-second mouth rinse with a solution of either carbohydrate (CHO), caffeine (CAF), carbohydrate + caffeine (CHO + CAF), placebo (H2O), or a no rinse control (CON). No significant improvements in Yo-Yo IRT-1 performance were found (p > 0.05). Perceptual indications of effort (i.e., rating of their perceived exertion [RPE]) were significantly lower (p ≤ 0.05) in CHO and CHO + CAF when compared with CON after speed level 11. Interestingly, RPE levels were nonsignificantly lower in all but one level of the Yo-Yo Level 1 for CHO in comparison with other groups. Carbohydrate and caffeine mouth rinsing seems to exert no impact on running performance before maximal intermittent running in a group of male collegiate lacrosse players.

Requirement for the Phospho-H2AX Binding Module of Crb2 in Double-Strand Break Targeting and Checkpoint Activation▿

Science.gov (United States)

Sanders, Steven L.; Arida, Ahmad R.; Phan, Funita P.

2010-01-01

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability. PMID:20679488

Requirement for the phospho-H2AX binding module of Crb2 in double-strand break targeting and checkpoint activation.

Science.gov (United States)

Sanders, Steven L; Arida, Ahmad R; Phan, Funita P

2010-10-01

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.

Optical absorption of irradiated carbohydrates

International Nuclear Information System (INIS)

Supe, A.A.; Tiliks, Yu.E.

1994-01-01

The optical absorption spectra of γ-irradiated carbohydrates (glucose, lactose, sucrose, maltose, and starch) and their aqueous solutions were studied. The comparison of the data obtained with the determination of the concentrations of molecular and radical products of radiolysis allows the absorption bands with maxima at 250 and 310 nm to be assigned to the radicals trapped in the irradiated carbohydrates

Linking Bacillus cereus Genotypes and Carbohydrate Utilization Capacity.

Directory of Open Access Journals (Sweden)

Alicja K Warda

Full Text Available We characterised carbohydrate utilisation of 20 newly sequenced Bacillus cereus strains isolated from food products and food processing environments and two laboratory strains, B. cereus ATCC 10987 and B. cereus ATCC 14579. Subsequently, genome sequences of these strains were analysed together with 11 additional B. cereus reference genomes to provide an overview of the different types of carbohydrate transporters and utilization systems found in B. cereus strains. The combined application of API tests, defined growth media experiments and comparative genomics enabled us to link the carbohydrate utilisation capacity of 22 B. cereus strains with their genome content and in some cases to the panC phylogenetic grouping. A core set of carbohydrates including glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose could be used by all strains, whereas utilisation of other carbohydrates like xylose, galactose, and lactose, and typical host-derived carbohydrates such as fucose, mannose, N-acetyl-galactosamine and inositol is limited to a subset of strains. Finally, the roles of selected carbohydrate transporters and utilisation systems in specific niches such as soil, foods and the human host are discussed.

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-...

Host-adaptation of Francisella tularensis alters the bacterium's surface-carbohydrates to hinder effectors of innate and adaptive immunity.

Directory of Open Access Journals (Sweden)

Tiffany M Zarrella

Full Text Available The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.F. tularensis undergoes host-adaptation which

A web server for analysis, comparison and prediction of protein ligand binding sites.

Science.gov (United States)

Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

2016-03-25

One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

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

Association of carbohydrate and fat intake with metabolic syndrome.

Science.gov (United States)

Kwon, Yu-Jin; Lee, Hye-Sun; Lee, Ji-Won

2018-04-01

In Asia, dietary pattern has been changed with increased intake of refined carbohydrates, sugar, and saturated fat, while the prevalence of metabolic syndrome (MetS) is on the rise. However, it remains unclear whether a high-carbohydrate or a high-fat diet is more metabolically harmful, and the optimal amount of carbohydrates and fat has not been determined. The aim of our study was to examine the role of carbohydrate and fat intake in MetS in a Korean population. Data were obtained from a large, population-based, cross-sectional study (6737 males and 8845 females). The subjects were divided into nine groups based on carbohydrate and fat proportion, and multiple logistic regression analysis was performed after adjusting for confounding variables. Regardless of fat intake, the risk of MetS significantly increased in males with higher carbohydrate proportions (of total energy intake). In females, the risk of MetS was significantly elevated only in those with both the highest carbohydrate proportion and lowest fat proportion. A high carbohydrate proportion was associated with a higher prevalence of MetS in males, and a high carbohydrate proportion combined with a low fat proportion was associated with MetS in females. Our results indicate that reduction of excessive carbohydrate intake paired with an adequate fat intake, taking into consideration optimal types of fat, is useful for MetS prevention. Longitudinal studies are needed to clarify the optimal types and amounts of carbohydrate and fat proportions as well as the mechanism underlying these relationships. Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

The mannan-binding lectin pathway of complement activation: biology and disease association

DEFF Research Database (Denmark)

Petersen, Steen Vang; Thiel, S; Jensenius, J C

2001-01-01

Mannan-binding lectin (MBL) is a plasma protein found in association with several serine proteases (MASPs) forming the MBL complex. MBL recognises carbohydrate structures arranged in a particular geometry, such as those found on the surface of micro-organisms. When bound to e.g. bacteria the MBL...... as an initiator of the host response against potential pathogenic micro-organisms. Udgivelsesdato: 2001-Aug...

Quantifying Protein-Carbohydrate Interactions Using Liquid Sample Desorption Electrospray Ionization Mass Spectrometry

Science.gov (United States)

Yao, Yuyu; Shams-Ud-Doha, Km; Daneshfar, Rambod; Kitova, Elena N.; Klassen, John S.

2015-01-01

The application of liquid sample desorption electrospray ionization mass spectrometry (liquid sample DESI-MS) for quantifying protein-carbohydrate interactions in vitro is described. Association constants for the interactions between lysozyme and β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc and β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc, and between a single chain antibody and α-D-Galp-(1 → 2)-[α-D-Abep-(1 → 3)]-α-D-Manp-OCH3 and β-D-Glcp-(1 → 2)-[α-D-Abep-(1 → 3)]-α-D-Manp-OCH3 measured using liquid sample DESI-MS were found to be in good agreement with values measured by isothermal titration calorimetry and the direct ESI-MS assay. The reference protein method, which was originally developed to correct ESI mass spectra for the occurrence of nonspecific ligand-protein binding, was shown to reliably correct liquid sample DESI mass spectra for nonspecific binding. The suitability of liquid sample DESI-MS for quantitative binding measurements carried out using solutions containing high concentrations of the nonvolatile biological buffer phosphate buffered saline (PBS) was also explored. Binding of lysozyme to β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-D-GlcNAc in aqueous solutions containing up to 1× PBS was successfully monitored using liquid sample DESI-MS; with ESI-MS the binding measurements were limited to concentrations less than 0.02 X PBS.

Crystal structure of reovirus attachment protein σ1 in complex with sialylated oligosaccharides.

Science.gov (United States)

Reiter, Dirk M; Frierson, Johnna M; Halvorson, Elizabeth E; Kobayashi, Takeshi; Dermody, Terence S; Stehle, Thilo

2011-08-01

Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α-helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties.

Crystal structure of reovirus attachment protein σ1 in complex with sialylated oligosaccharides.

Directory of Open Access Journals (Sweden)

Dirk M Reiter

2011-08-01

Full Text Available Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α-helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties.

A lectin histochemical study on carbohydrate moieties of the gonadotropin-like substance in the epithelial cells of Hatschek's pit of Branchiostoma belcheri

Science.gov (United States)

Fang, Y. Q.; Welsch, U.

1997-03-01

The present light microscopic lectin, histochemical study suggests for the first time that the vertebrate gonadotropin-like substance in the basal part of the epithelial cells of Hatschek's pit is a sialic acid-containing glycoprotein. The binding intensity of the epithelial cells in Hatschek's pit to 6 lectins ( Limulus polyphemus agglutinin (LPA), Wheat germ agglutinin (WGA), Helix pomatia agglutinin (HPA), Concanavalin A (Con A), Ulex europaeus agglutinin I (UEA I) and Ricinus communis agglutinin I (RCA I)) indicate that the carbohydrate composition of the gonadotrophic glycoprotein is similar to that of mammals and fish, and that N-acetyl-D-galactosamine, sialic acid, glucosamine, D-mannose and L-fucose are components of the carbohydrate portion.

Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-β-d-xylanase 10B in complex with xylohexaose

Energy Technology Data Exchange (ETDEWEB)

Najmudin, Shabir, E-mail: shabir@dq.fct.unl.pt [REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal); Pinheiro, Benedita A. [CIISA - Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa (Portugal); Romão, Maria J. [REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal); Prates, José A. M.; Fontes, Carlos M. G. A., E-mail: shabir@dq.fct.unl.pt [CIISA - Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa (Portugal); REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal)

2008-08-01

The N-terminal moiety of C. thermocellum endo-1,4-β-d-xylanase 10B, comprising a carbohydrate-binding module (CBM22-1) and a GH10 E337A mutant domain, has been crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3{sub 2}21, contain a dimer in the asymmetric unit and diffract to beyond 2.0 Å resolution. The cellulosome of Clostridium thermocellum is a highly organized multi-enzyme complex of cellulases and hemicellulases involved in the hydrolysis of plant cell-wall polysaccharides. The bifunctional multi-modular xylanase Xyn10B is one of the hemicellulase components of the C. thermocellum cellulosome. The enzyme contains an internal glycoside hydrolase family 10 catalytic domain (GH10) and a C-terminal family 1 carbohydrate esterase domain (CE1). The N-terminal moiety of Xyn10B (residues 32–551), comprising a carbohydrate-binding module (CBM22-1) and the GH10 E337A mutant, was crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3{sub 2}21 and contain a dimer in the asymmetric unit. The crystals diffracted to beyond 2.0 Å resolution.

The effect of dietary carbohydrate on gastroesophageal reflux disease.

Science.gov (United States)

Wu, Keng-Liang; Kuo, Chung-Mou; Yao, Chih-Chien; Tai, Wei-Chen; Chuah, Seng-Kee; Lim, Chee-Sang; Chiu, Yi-Chun

2018-01-12

Acid changes in gastroesophageal reflux with vary component in the food have less been studied, especially carbohydrate. We plan to clarify the effect of different carbohydrate density on low esophageal acid and reflux symptoms of patients with gastroesophgeal reflux disease. Twelve patients (52 ± 12 years old; five female) with gastroesophageal reflux disease were recruited for the prospective crossover study. Each patient was invited for panendoscope, manometry and 24 h pH monitor. The two formulated liquid meal, test meal A: 500 ml liquid meal (containing 84.8 g carbohydrate) and B: same volume liquid meal (but 178.8 g carbohydrate) were randomized supplied as lunch or dinner. Reflux symptoms were recorded. There are significant statistic differences in more Johnson-DeMeester score (p = 0.019), total reflux time (%) (p = 0.028), number of reflux periods (p = 0.026) and longest reflux (p = 0.015) after high carbohydrate diet than low carbohydrate. Total reflux time and number of long reflux periods more than 5 min are significant more after high carbohydrate diet. More acid reflux symptoms are found after high carbohydrate diet. High carbohydrate diet could induce more acid reflux in low esophagus and more reflux symptoms in patients with gastroesophageal reflux disease. Copyright © 2018. Published by Elsevier B.V.

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein.

Science.gov (United States)

Haarmeyer, Carolyn N; Smith, Matthew D; Chundawat, Shishir P S; Sammond, Deanne; Whitehead, Timothy A

2017-04-01

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28-0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low

Thermoactivation of a cellobiohydrolase

DEFF Research Database (Denmark)

Westh, Peter; Borch, Kim; Sørensen, Trine Holst

2018-01-01

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activ...

Acute Effects of Carbohydrate Supplementation on Intermittent Sports Performance.

Science.gov (United States)

Baker, Lindsay B; Rollo, Ian; Stein, Kimberly W; Jeukendrup, Asker E

2015-07-14

Intermittent sports (e.g., team sports) are diverse in their rules and regulations but similar in the pattern of play; that is, intermittent high-intensity movements and the execution of sport-specific skills over a prolonged period of time (~1-2 h). Performance during intermittent sports is dependent upon a combination of anaerobic and aerobic energy systems, both of which rely on muscle glycogen and/or blood glucose as an important substrate for energy production. The aims of this paper are to review: (1) potential biological mechanisms by which carbohydrate may impact intermittent sport performance; (2) the acute effects of carbohydrate ingestion on intermittent sport performance, including intermittent high-intensity exercise capacity, sprinting, jumping, skill, change of direction speed, and cognition; and (3) what recommendations can be derived for carbohydrate intake before/during exercise in intermittent sports based on the available evidence. The most researched intermittent sport is soccer but some sport-specific studies have also been conducted in other sports (e.g., rugby, field hockey, basketball, American football, and racquet sports). Carbohydrate ingestion before/during exercise has been shown in most studies to enhance intermittent high-intensity exercise capacity. However, studies have shown mixed results with regards to the acute effects of carbohydrate intake on sprinting, jumping, skill, change of direction speed, and cognition. In most of these studies the amount of carbohydrate consumed was ~30-60 g/h in the form of a 6%-7% carbohydrate solution comprised of sucrose, glucose, and/or maltodextrin. The magnitude of the impact that carbohydrate ingestion has on intermittent sport performance is likely dependent on the carbohydrate status of the individual; that is, carbohydrate ingestion has the greatest impact on performance under circumstances eliciting fatigue and/or hypoglycemia. Accordingly, carbohydrate ingestion before and during a game

Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).

Science.gov (United States)

Zhou, Li; Yeo, Alan T; Ballarano, Carmine; Weber, Urs; Allen, Karen N; Gilmore, Thomas D; Whitty, Adrian

2014-12-23

Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated homodimer comprising mostly α-helix. It has been shown that a NEMO fragment spanning residues 44-111, which contains the IKKα/β binding site, is structurally disordered in the absence of bound IKKβ. Herein we show that enforcing dimerization of NEMO1-120 or NEMO44-111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized to bind IKKβ with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to the denatured state by up to 3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO-L107C bound endogenous IKKβ in A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKβ binding domain of NEMO possesses an ordered structure in the unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full-length NEMO protein. The stability of the NEMO coiled coil is maintained by strong interhelix interactions in the region centered on residue 54. The disulfide-linked constructs we describe herein may be useful for crystallization of NEMO's IKKβ binding domain in the absence of bound IKKβ, thereby facilitating the structural characterization of small-molecule inhibitors.

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

Energy Technology Data Exchange (ETDEWEB)

Makyio, Hisayoshi [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan); Shimabukuro, Junpei; Suzuki, Tatsuya [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Imamura, Akihiro; Ishida, Hideharu [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Kiso, Makoto [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Ando, Hiromune, E-mail: hando@gifu-u.ac.jp [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Kato, Ryuichi, E-mail: ryuichi.kato@kek.jp [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan)

2016-08-26

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

International Nuclear Information System (INIS)

Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Ando, Hiromune; Kato, Ryuichi

2016-01-01

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

Science.gov (United States)

Dilworth, David; Bonnafous, Pierre; Edoo, Amiirah Bibi; Bourbigot, Sarah; Pesek-Jardim, Francy; Gudavicius, Geoff; Serpa, Jason J.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.

2017-01-01

Abstract Prolyl isomerases are defined by a catalytic domain that facilitates the cis–trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets. PMID:29036638

Very low-carbohydrate versus isocaloric high-carbohydrate diet in dietary obese rats.

Science.gov (United States)

Axen, Kathleen V; Axen, Kenneth

2006-08-01

The effects of a very low-carbohydrate (VLC), high-fat (HF) dietary regimen on metabolic syndrome were compared with those of an isocaloric high-carbohydrate (HC), low-fat (LF) regimen in dietary obese rats. Male Sprague-Dawley rats, made obese by 8 weeks ad libitum consumption of an HF diet, developed features of the metabolic syndrome vs. lean control (C) rats, including greater visceral, subcutaneous, and hepatic fat masses, elevated plasma cholesterol levels, impaired glucose tolerance, and fasting and post-load insulin resistance. Half of the obese rats (VLC) were then fed a popular VLC-HF diet (Weeks 9 and 10 at 5% and Weeks 11 to 14 at 15% carbohydrate), and one-half (HC) were pair-fed an HC-LF diet (Weeks 9 to 14 at 60% carbohydrate). Energy intakes of pair-fed VLC and HC rats were less than C rats throughout Weeks 9 to 14. Compared with HC rats, VLC rats exhibited impaired insulin and glycemic responses to an intraperitoneal glucose load at Week 10 and lower plasma triacylglycerol levels but retarded loss of hepatic, retroperitoneal, and total body fat at Week 14. VLC, HC, and C rats no longer differed in body weight, plasma cholesterol, glucose tolerance, or fasting insulin resistance at Week 14. Progressive decreases in fasting insulin resistance in obese groups paralleled concomitant reductions in hepatic, retroperitoneal, and total body fat. When energy intake was matched, the VLC-HF diet provided no advantage in weight loss or in improving those components of the metabolic syndrome induced by dietary obesity and may delay loss of hepatic and visceral fat as compared with an HC-LF diet.

Liver Fatty Acid Composition and Inflammation in Mice Fed with High-Carbohydrate Diet or High-Fat Diet.

Science.gov (United States)

da Silva-Santi, Lorena Gimenez; Antunes, Marina Masetto; Caparroz-Assef, Silvana Martins; Carbonera, Fabiana; Masi, Laureane Nunes; Curi, Rui; Visentainer, Jesuí Vergílio; Bazotte, Roberto Barbosa

2016-10-29

Both high-carbohydrate diet (HCD) and high-fat diet (HFD) modulate liver fat accumulation and inflammation, however, there is a lack of data on the potential contribution of carbohydrates and lipids separately. For this reason, the changes in liver fatty acid (FA) composition in male Swiss mice fed with HCD or HFD were compared, at the time points 0 (before starting the diets), and after 7, 14, 28 or 56 days. Activities of stearoyl-CoA desaturase-1 (SCD-1), ∆-6 desaturase (D6D), elongases and de novo lipogenesis (DNL) were estimated. Liver mRNA expression of acetyl-CoA carboxylase 1 (ACC1) was evaluated as an additional indicator of the de novo lipogenesis. Myeloperoxidase activity, nitric oxide (NO) production, and mRNA expressions of F4/80, type I collagen, interleukin (IL)-6, IL-1β, IL-10, and tumor necrosis factor-α (TNF-α) were measured as indication of the liver inflammatory state. The HCD group had more intense lipid deposition, particularly of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs). This group also showed higher DNL, SCD-1, and D6D activities associated with increased NO concentration, as well as myeloperoxidase activity. Livers from the HFD group showed higher elongase activity, stored more polyunsaturated fatty acids (PUFAs) and had a lower omega-6/omega-3 fatty acid ( n -6/ n -3) ratio. In conclusion, liver lipid accumulation, fatty acids (FA) composition and inflammation were modulated by the dietary composition of lipids and carbohydrates. The HCD group had more potent lipogenic and inflammatory effects in comparison with HFD.

Liver Fatty Acid Composition and Inflammation in Mice Fed with High-Carbohydrate Diet or High-Fat Diet

Directory of Open Access Journals (Sweden)

Lorena Gimenez da Silva-Santi

2016-10-01

Full Text Available Both high-carbohydrate diet (HCD and high-fat diet (HFD modulate liver fat accumulation and inflammation, however, there is a lack of data on the potential contribution of carbohydrates and lipids separately. For this reason, the changes in liver fatty acid (FA composition in male Swiss mice fed with HCD or HFD were compared, at the time points 0 (before starting the diets, and after 7, 14, 28 or 56 days. Activities of stearoyl-CoA desaturase-1 (SCD-1, ∆-6 desaturase (D6D, elongases and de novo lipogenesis (DNL were estimated. Liver mRNA expression of acetyl-CoA carboxylase 1 (ACC1 was evaluated as an additional indicator of the de novo lipogenesis. Myeloperoxidase activity, nitric oxide (NO production, and mRNA expressions of F4/80, type I collagen, interleukin (IL-6, IL-1β, IL-10, and tumor necrosis factor-α (TNF-α were measured as indication of the liver inflammatory state. The HCD group had more intense lipid deposition, particularly of saturated fatty acids (SFAs and monounsaturated fatty acids (MUFAs. This group also showed higher DNL, SCD-1, and D6D activities associated with increased NO concentration, as well as myeloperoxidase activity. Livers from the HFD group showed higher elongase activity, stored more polyunsaturated fatty acids (PUFAs and had a lower omega-6/omega-3 fatty acid (n-6/n-3 ratio. In conclusion, liver lipid accumulation, fatty acids (FA composition and inflammation were modulated by the dietary composition of lipids and carbohydrates. The HCD group had more potent lipogenic and inflammatory effects in comparison with HFD.

Allosteric modulation of G-protein coupled receptors

DEFF Research Database (Denmark)

Jensen, Anders A.; Spalding, Tracy A

2004-01-01

are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites....... In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines...... the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands....

DoGlycans-Tools for Preparing Carbohydrate Structures for Atomistic Simulations of Glycoproteins, Glycolipids, and Carbohydrate Polymers for GROMACS

DEFF Research Database (Denmark)

Danne, Reinis; Poojari, Chetan; Martinez-Seara, Hector

2017-01-01

Carbohydrates constitute a structurally and functionally diverse group of biological molecules and macromolecules. In cells they are involved in, e.g., energy storage, signaling, and cell-cell recognition. All of these phenomena take place in atomistic scales, thus atomistic simulation would...... be the method of choice to explore how carbohydrates function. However, the progress in the field is limited by the lack of appropriate tools for preparing carbohydrate structures and related topology files for the simulation models. Here we present tools that fill this gap. Applications where the tools...

Differential effects of simple vs. complex carbohydrates on VLDL secretion rates and HDL metabolism in the guinea pig.

Science.gov (United States)

Fernandez, M L; Abdel-Fattah, G; McNamara, D J

1995-04-28

Guinea pigs were fed isocaloric diets containing 52% (w/w) carbohydrate, either sucrose or starch, to investigate effects of simple vs. complex carbohydrates on plasma VLDL and HDL metabolism. Plasma cholesterol concentrations were not different between dietary groups while plasma triacylglycerol (TAG) and VLDL cholesterol levels were significantly increased in animals fed the sucrose diet (P < 0.05). Hepatic VLDL TAG secretion rates measured following intravenous injection of Triton WR-1339 were not affected by carbohydrate type whereas the rate of apo B secretion was 1.9-fold higher in sucrose fed animals (P < 0.02). Nascent VLDL from the sucrose group contained less TAG per apo B suggesting that the higher plasma TAG in animals fed simple carbohydrates results from increased secretion of VLDL particles with lower TAG content. Sucrose fed animals exhibited higher concentrations of hepatic free cholesterol (P < 0.01) while hepatic TAG levels and acyl CoA:cholesterol acyltransferase (ACAT) activity were not different between groups. Plasma HDL cholesterol concentrations and composition, and plasma lecithin cholesterol acyltransferase (LCAT) activity were not affected by diet yet there was a positive correlation between HDL cholesteryl ester content and LCAT activities (r = 0.70, P < 0.05). Hepatic membranes from the sucrose group had a higher hepatic HDL binding protein number (Bmax) with no changes in the dissociation constant (Kd). These results suggest that at the same carbohydrate energy intake, simple sugars induce modest changes in HDL metabolism while VLDL metabolism is affected at multiple sites, as indicated by the higher concentrations of hepatic cholesterol, dissociation in the synthesis rates of VLDL components, and compositional changes in nascent and mature VLDL.

Carbohydrate absorption from one serving of fruit juice in young children: age and carbohydrate composition effects.

Science.gov (United States)

Nobigrot, T; Chasalow, F I; Lifshitz, F

1997-04-01

To test the hypotheses that: the efficiency of carbohydrate absorption in childhood increases with age, and decreased carbohydrate absorption occurs more frequently with juices containing more fructose than glucose and/or sorbitol than with juices which contain equal amounts of fructose and glucose and are sorbitol-free. One hundred and four healthy children were recruited from the Ambulatory Center at Maimonides Children's Center. They were assigned to one of three age groups: approximately 1, 3 and 5 years of age. Each child received one age-specific dose (by randomization) of one of four juices: a) pear juice which contains fructose in excess to glucose and a large amount of sorbitol; b) apple juice which is similar to pear juice in its fructose to glucose ratio but contains four times less sorbitol than pear juice; c) white grape juice or d) purple grape juice both of which contain equal amounts of fructose and glucose and are sorbitol-free. Breath hydrogen excretion (BH2) was utilized as the index of carbohydrate absorption. It was measured in fasting children and at 30-minute intervals for 3 hours after drinking the single serving of juice. Multiple breath hydrogen related parameters were quantified and results were expressed as: BH2 peak, area under the curve, and degree of carbohydrate malabsorption. After the test, parents completed a questionnaire and recorded signs and symptoms of intestinal malabsorption for 24 hours. Pear juice related BH2 levels were significantly higher among children 1 and 3 years of age as compared to the levels achieved after the other juices. Apple juice related BH2 levels were significantly higher only among the youngest age group of children. There was no significant difference in carbohydrate absorption among the 5 year old children regardless of the juice consumed. Incomplete carbohydrate absorption (BH2 peak above 20 ppm) occurred more frequently after pear juice consumption (84%) than after apple juice (41%) or grape juice

The TROVE module: a common element in Telomerase, Ro and Vault ribonucleoproteins.

Science.gov (United States)

Bateman, Alex; Kickhoefer, Valerie

2003-10-16

Ribonucleoproteins carry out a variety of important tasks in the cell. In this study we show that a number of these contain a novel module, that we speculate mediates RNA-binding. The TROVE module--Telomerase, Ro and Vault module--is found in TEP1 and Ro60 the protein components of three ribonucleoprotein particles. This novel module, consisting of one or more domains, may be involved in binding the RNA components of the three RNPs, which are telomerase RNA, Y RNA and vault RNA. A second conserved region in these proteins is shown to be a member of the vWA domain family. The vWA domain in TEP1 is closely related to the previously recognised vWA domain in VPARP a second component of the vault particle. This vWA domain may mediate interactions between these vault components or bind as yet unidentified components of the RNPs. This work suggests that a number of ribonucleoprotein components use a common RNA-binding module. The TROVE module is also found in bacterial ribonucleoproteins suggesting an ancient origin for these ribonucleoproteins.

Single tag for total carbohydrate analysis.

Science.gov (United States)

Anumula, Kalyan Rao

2014-07-15

Anthranilic acid (2-aminobenzoic acid, 2-AA) has the remarkable property of reacting rapidly with every type of reducing carbohydrate. Reactivity of 2-AA with carbohydrates in aqueous solutions surpasses all other tags reported to date. This unique capability is attributed to the strategically located -COOH which accelerates Schiff base formation. Monosaccharides, oligosaccharides (N-, O-, and lipid linked and glycans in secretory fluids), glycosaminoglycans, and polysaccharides can be easily labeled with 2-AA. With 2-AA, labeling is simple in aqueous solutions containing proteins, peptides, buffer salts, and other ingredients (e.g., PNGase F, glycosidase, and transferase reaction mixtures). In contrast, other tags require relatively pure glycans for labeling in anhydrous dimethyl sulfoxide-acetic acid medium. Acidic conditions are known to cause desialylation, thus requiring a great deal of attention to sample preparation. Simpler labeling is achieved with 2-AA within 30-60 min in mild acetate-borate buffered solution. 2-AA provides the highest sensitivity and resolution in chromatographic methods for carbohydrate analysis in a simple manner. Additionally, 2-AA is uniquely qualified for quantitative analysis by mass spectrometry in the negative mode. Analyses of 2-AA-labeled carbohydrates by electrophoresis and other techniques have been reported. Examples cited here demonstrate that 2-AA is the universal tag for total carbohydrate analysis. Copyright © 2014 Elsevier Inc. All rights reserved.

A comparison of low-carbohydrate vs. high-carbohydrate diets: energy restriction, nutrient quality and correlation to body mass index.

Science.gov (United States)

Bowman, Shanthy A; Spence, Joseph T

2002-06-01

To evaluate free-living adults' diets that ranged from very low to high amounts of carbohydrate for their energy content, nutritional quality and correlation to Body Mass Index. Adults ages 19 years and older, who had complete dietary intake data on day-1 of the USDA's 1994 to 1996 Continuing Survey of Food Intakes by Individuals (CSFII 1994-1996), were divided into four groups--very low, low, moderate and high carbohydrate--based on the percent total energy from carbohydrate. Mean energy, nutrient, food intakes and Body Mass Index values were compared among the groups. SUDAAN software package was used for the data analysis and pair-wise mean comparisons (p low-fat foods, grain products and fruits. This group also had the lowest sodium intake. Adults eating a high-carbohydrate diet are more likely to have Body Mass Index values below 25. A study of diets of free-living adults in the U.S. showed that diets high in carbohydrate were both energy restrictive and nutritious and may be adopted for successful weight management.

The case for low carbohydrate diets in diabetes management

Directory of Open Access Journals (Sweden)

McFarlane Samy I

2005-07-01

Full Text Available Abstract A low fat, high carbohydrate diet in combination with regular exercise is the traditional recommendation for treating diabetes. Compliance with these lifestyle modifications is less than satisfactory, however, and a high carbohydrate diet raises postprandial plasma glucose and insulin secretion, thereby increasing risk of CVD, hypertension, dyslipidemia, obesity and diabetes. Moreover, the current epidemic of diabetes and obesity has been, over the past three decades, accompanied by a significant decrease in fat consumption and an increase in carbohydrate consumption. This apparent failure of the traditional diet, from a public health point of view, indicates that alternative dietary approaches are needed. Because carbohydrate is the major secretagogue of insulin, some form of carbohydrate restriction is a prima facie candidate for dietary control of diabetes. Evidence from various randomized controlled trials in recent years has convinced us that such diets are safe and effective, at least in short-term. These data show low carbohydrate diets to be comparable or better than traditional low fat high carbohydrate diets for weight reduction, improvement in the dyslipidemia of diabetes and metabolic syndrome as well as control of blood pressure, postprandial glycemia and insulin secretion. Furthermore, the ability of low carbohydrate diets to reduce triglycerides and to increase HDL is of particular importance. Resistance to such strategies has been due, in part, to equating it with the popular Atkins diet. However, there are many variations and room for individual physician planning. Some form of low carbohydrate diet, in combination with exercise, is a viable option for patients with diabetes. However, the extreme reduction of carbohydrate of popular diets (

Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

Science.gov (United States)

Brauser, Annemarie; Schroeder, Indra; Gutsmann, Thomas; Cosentino, Cristian; Moroni, Anna; Winterhalter, Mathias

2012-01-01

One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg2+ reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg2+-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg2+. Analysis of the resulting amplitude histograms with β distributions revealed the rate constants of blocking (kOB) and unblocking (kBO) in the range of 1,000 to 120,000 s−1. As expected for a bimolecular reaction, kOB was proportional to blocker concentration and kBO independent of it. kOB was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being ∼25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg2+ and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg2+. The difference in the accessibility of the binding sites also explains the dependency of kOB on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding. PMID:22689827

Effect of phytooestrogen - coumestrol and estrone on some aspects of carbohydrate metabolism in ovariectomized female rats

International Nuclear Information System (INIS)

Nogowski, L.; Nowak, K.W.; Mackowiak, P.

1992-01-01

The purpose of the presented study is a comparison of phytooestrogen -coumestrol and estrone effects on carbohydrate metabolism in ovariectomized female rats and to examine the partition of pancreatic hormones in changes of this metabolism. Administration of coumestrol diminished muscle glycogen in investigated animals. There were no significant changes in insulin and glucagon blood level but decrease in the specific insulin binding in the insulin receptor activity in this tissue and it could be a cause of glycogen deficiency. (author). 28 refs, 4 tabs

Activation of the transcription factor carbohydrate-responsive element-binding protein by glucose leads to increased pancreatic beta cell differentiation in rats.

Science.gov (United States)

Soggia, A; Flosseau, K; Ravassard, P; Szinnai, G; Scharfmann, R; Guillemain, G

2012-10-01

Pancreatic cell development is a tightly controlled process. Although information is available regarding the mesodermal signals that control pancreatic development, little is known about the role of environmental factors such as nutrients, including glucose, on pancreatic development. We previously showed that glucose and its metabolism through the hexosamine biosynthesis pathway (HBP) promote pancreatic endocrine cell differentiation. Here, we analysed the role of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) in this process. This transcription factor is activated by glucose, and has been recently described as a target of the HBP. We used an in vitro bioassay in which pancreatic endocrine and exocrine cells develop from rat embryonic pancreas in a way that mimics in vivo pancreatic development. Using this model, gain-of-function and loss-of-function experiments were undertaken. ChREBP was produced in the endocrine lineage during pancreatic development, its abundance increasing with differentiation. When rat embryonic pancreases were cultured in the presence of glucose or xylitol, the production of ChREBP targets was induced. Concomitantly, beta cell differentiation was enhanced. On the other hand, when embryonic pancreases were cultured with inhibitors decreasing ChREBP activity or an adenovirus producing a dominant-negative ChREBP, beta cell differentiation was reduced, indicating that ChREBP activity was necessary for proper beta cell differentiation. Interestingly, adenovirus producing a dominant-negative ChREBP also reduced the positive effect of N-acetylglucosamine, a substrate of the HBP acting on beta cell differentiation. Our work supports the idea that glucose, through the transcription factor ChREBP, controls beta cell differentiation from pancreatic progenitors.

"Click" saccharide/beta-lactam hybrids for lectin inhibition.

Science.gov (United States)

Palomo, Claudio; Aizpurua, Jesus M; Balentová, Eva; Azcune, Itxaso; Santos, J Ignacio; Jiménez-Barbero, Jesús; Cañada, Javier; Miranda, José Ignacio

2008-06-05

Hybrid glycopeptide beta-lactam mimetics designed to bind lectins or carbohydrate recognition domains in selectins have been prepared according to a "shape-modulating linker" design. This approach was implemented using the azide-alkyne "click" cycloaddition reaction, and as shown by NMR/MD experiments, binding of the resulting mimetics to Ulex Europaeus Lectin-1 (UEL-1) occurred after a "bent-to-extended" conformational change around a partially rotatable triazolylmethylene moiety.

Crystal structure of CbpF, a bifunctional choline-binding protein and autolysis regulator from Streptococcus pneumoniae.

Science.gov (United States)

Molina, Rafael; González, Ana; Stelter, Meike; Pérez-Dorado, Inmaculada; Kahn, Richard; Morales, María; Moscoso, Miriam; Campuzano, Susana; Campillo, Nuria E; Mobashery, Shahriar; García, José L; García, Pedro; Hermoso, Juan A

2009-03-01

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline-binding proteins. The three-dimensional structure of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non-consensus choline-binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well-defined modules. The carboxy-terminal module is involved in cell wall binding and the amino-terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.

Incident microalbuminuria and complement factor mannan-binding lectin-associated protein 19 in people with newly diagnosed type 1 diabetes

DEFF Research Database (Denmark)

Ostergaard, J A; Thiel, S; Hoffmann-Petersen, I T

2017-01-01

BACKGROUND: Evidence links the lectin pathway of complement activation to diabetic kidney disease. Upon carbohydrate-recognition by pattern-recognition molecules, e.g., mannan-binding lectin (MBL), the MBL-associated serine protease (MASP-2) is activated and initiates the complement cascade. The ...

Effects of flow changes on radiotracer binding: Simultaneous measurement of neuroreceptor binding and cerebral blood flow modulation.

Science.gov (United States)

Sander, Christin Y; Mandeville, Joseph B; Wey, Hsiao-Ying; Catana, Ciprian; Hooker, Jacob M; Rosen, Bruce R

2017-01-01

The potential effects of changes in blood flow on the delivery and washout of radiotracers has been an ongoing question in PET bolus injection studies. This study provides practical insight into this topic by experimentally measuring cerebral blood flow (CBF) and neuroreceptor binding using simultaneous PET/MRI. Hypercapnic challenges (7% CO 2 ) were administered to non-human primates in order to induce controlled increases in CBF, measured with pseudo-continuous arterial spin labeling. Simultaneously, dopamine D 2 /D 3 receptor binding of [ 11 C]raclopride or [ 18 F]fallypride was monitored with dynamic PET. Experiments showed that neither time activity curves nor quantification of binding through binding potentials ( BP ND ) were measurably affected by CBF increases, which were larger than two-fold. Simulations of experimental procedures showed that even large changes in CBF should have little effect on the time activity curves of radiotracers, given a set of realistic assumptions. The proposed method can be applied to experimentally assess the flow sensitivity of other radiotracers. Results demonstrate that CBF changes, which often occur due to behavioral tasks or pharmacological challenges, do not affect PET [ 11 C]raclopride or [ 18 F]fallypride binding studies and their quantification. The results from this study suggest flow effects may have limited impact on many PET neuroreceptor tracers with similar properties.

Acute Effects of Carbohydrate Supplementation on Intermittent Sports Performance

Directory of Open Access Journals (Sweden)

Lindsay B. Baker

2015-07-01

Full Text Available Intermittent sports (e.g., team sports are diverse in their rules and regulations but similar in the pattern of play; that is, intermittent high-intensity movements and the execution of sport-specific skills over a prolonged period of time (~1–2 h. Performance during intermittent sports is dependent upon a combination of anaerobic and aerobic energy systems, both of which rely on muscle glycogen and/or blood glucose as an important substrate for energy production. The aims of this paper are to review: (1 potential biological mechanisms by which carbohydrate may impact intermittent sport performance; (2 the acute effects of carbohydrate ingestion on intermittent sport performance, including intermittent high-intensity exercise capacity, sprinting, jumping, skill, change of direction speed, and cognition; and (3 what recommendations can be derived for carbohydrate intake before/during exercise in intermittent sports based on the available evidence. The most researched intermittent sport is soccer but some sport-specific studies have also been conducted in other sports (e.g., rugby, field hockey, basketball, American football, and racquet sports. Carbohydrate ingestion before/during exercise has been shown in most studies to enhance intermittent high-intensity exercise capacity. However, studies have shown mixed results with regards to the acute effects of carbohydrate intake on sprinting, jumping, skill, change of direction speed, and cognition. In most of these studies the amount of carbohydrate consumed was ~30–60 g/h in the form of a 6%–7% carbohydrate solution comprised of sucrose, glucose, and/or maltodextrin. The magnitude of the impact that carbohydrate ingestion has on intermittent sport performance is likely dependent on the carbohydrate status of the individual; that is, carbohydrate ingestion has the greatest impact on performance under circumstances eliciting fatigue and/or hypoglycemia. Accordingly, carbohydrate ingestion before

Expedient synthesis of C-aryl carbohydrates by consecutive biocatalytic benzoin and aldol reactions.

Science.gov (United States)

Hernández, Karel; Parella, Teodor; Joglar, Jesús; Bujons, Jordi; Pohl, Martina; Clapés, Pere

2015-02-16

The introduction of aromatic residues connected by a C-C bond into the non-reducing end of carbohydrates is highly significant for the development of innovative structures with improved binding affinity and selectivity (e.g., C-aril-sLex). In this work, an expedient asymmetric "de novo" synthetic route to new aryl carbohydrate derivatives based on two sequential stereoselectively biocatalytic carboligation reactions is presented. First, the benzoin reaction of aromatic aldehydes to dimethoxyacetaldehyde is conducted, catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I. Then, the α-hydroxyketones formed are reduced by using NaBH4 yielding the anti diol. After acetal hydrolysis, the aldol addition of dihydroxyacetone, hydroxyacetone, or glycolaldehyde catalyzed by the stereocomplementary D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase is performed. Both aldolases accept unphosphorylated donor substrates, avoiding the need of handling the phosphate group that the dihydroxyacetone phosphate-dependent aldolases require. In this way, 6-C-aryl-L-sorbose, 6-C-aryl-L-fructose, 6-C-aryl-L-tagatose, and 5-C-aryl-L-xylose derivatives are prepared by using this methodology. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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...... number of CA names and registry numbers have been created for non-existing racemic carbohydrates and linked to irrelevant references which, moreover, in many cases cannot be retrieved by the SciFinder Scholar program....

Discrete and structurally unique proteins (tāpirins) mediate attachment of extremely thermophilic Caldicellulosiruptor species to cellulose.

Science.gov (United States)

Blumer-Schuette, Sara E; Alahuhta, Markus; Conway, Jonathan M; Lee, Laura L; Zurawski, Jeffrey V; Giannone, Richard J; Hettich, Robert L; Lunin, Vladimir V; Himmel, Michael E; Kelly, Robert M

2015-04-24

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins ("tāpirins," origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose. �

Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

DEFF Research Database (Denmark)

Meschi, Joseph; Crouch, Erika C; Skolnik, Paul

2005-01-01

The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate......-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate...

Modulation of [3H]-dopamine binding by cholecystokinin octapeptide (CCK-8)

International Nuclear Information System (INIS)

Murphy, R.B.; Schuster, D.I.

1982-01-01

Cholecystokinin-octapeptide (CCK-8) is a putative neurotransmitter which has been demonstrated previously to occur in midbrain dopamine neurones. We observe that CCK-8 causes changes in both the affinity and density of binding sites for [ 3 H]-dopamine in rat striatal homogenates, in vitro, upon incubation with the peptide at a concentration of 1 micromolar. A dose-response study of the competetion of CCK-8 with [ 3 H]-dopamine binding indicates an IC50 for the peptide of 450 nM; desulfated CCK-8 and the related peptide caerulin are at least 4-fold less active than CCK-8. CCK-8 was also administered to rats in a separate study; the binding of [ 3 H]-dopamine was evaluated to homogenates of striata and olfactory tubercles obtained from these animals, which had been treated with systemic injection at a dose of 20 micrograms/kg, daily, for four days. A decrease in the number of striatal binding sites for the radioligand was observed, with a concomitant increase in the number of binding sites in the olfactory tubercle. These data collectively suggest a possible regulatory role for CCK-8 in the ascending dopamine systems

Microalgal carbohydrates. An overview of the factors influencing carbohydrates production, and of main bioconversion technologies for production of biofuels

Energy Technology Data Exchange (ETDEWEB)

Markou, Giorgos; Georgakakis, Dimitris [Agricultural Univ. of Athens (Greece). Dept. of Natural Resources Management and Agricultural Engineering; Angelidaki, Irini [Technical Univ. of Denmark, Lyngby (Denmark). Dept. of Environmental Engineering

2012-11-15

Microalgal biomass seems to be a promising feedstock for biofuel generation. Microalgae have relative high photosynthetic efficiencies, high growth rates, and some species can thrive in brackish water or seawater and wastewater from the food- and agro-industrial sector. Today, the main interest in research is the cultivation of microalgae for lipids production to generate biodiesel. However, there are several other biological or thermochemical conversion technologies, in which microalgal biomass could be used as substrate. However, the high protein content or the low carbohydrate content of the majority of the microalgal species might be a constraint for their possible use in these technologies. Moreover, in the majority of biomass conversion technologies, carbohydrates are the main substrate for production of biofuels. Nevertheless, microalgae biomass composition could be manipulated by several cultivation techniques, such as nutrient starvation or other stressed environmental conditions, which cause the microalgae to accumulate carbohydrates. This paper attempts to give a general overview of techniques that can be used for increasing the microalgal biomass carbohydrate content. In addition, biomass conversion technologies, related to the conversion of carbohydrates into biofuels are discussed. (orig.)

Validation of lignocellulosic biomass carbohydrates determination via acid hydrolysis.

Science.gov (United States)

Zhou, Shengfei; Runge, Troy M

2014-11-04

This work studied the two-step acid hydrolysis for determining carbohydrates in lignocellulosic biomass. Estimation of sugar loss based on acid hydrolyzed sugar standards or analysis of sugar derivatives was investigated. Four model substrates (starch, holocellulose, filter paper and cotton) and three levels of acid/material ratios (7.8, 10.3 and 15.4, v/w) were studied to demonstrate the range of test artifacts. The method for carbohydrates estimation based on acid hydrolyzed sugar standards having the most satisfactory carbohydrate recovery and relative standard deviation. Raw material and the acid/material ratio both had significant effect on carbohydrate hydrolysis, suggesting the acid to have impacts beyond a catalyst in the hydrolysis. Following optimal procedures, we were able to reach a carbohydrate recovery of 96% with a relative standard deviation less than 3%. The carbohydrates recovery lower than 100% was likely due to the incomplete hydrolysis of substrates, which was supported by scanning electron microscope (SEM) images. Copyright © 2014 Elsevier Ltd. All rights reserved.

Whey or Casein Hydrolysate with Carbohydrate for Metabolism and Performance in Cycling.

Science.gov (United States)

Oosthuyse, T; Carstens, M; Millen, A M E

2015-07-01

The protein type most suitable for ingestion during endurance exercise is undefined. This study compared co-ingestion of either 15 g/h whey or casein hydrolysate with 63 g/h fructose: maltodextrin (0.8:1) on exogenous carbohydrate oxidation, exercise metabolism and performance. 2 h postprandial, 8 male cyclists ingested either: carbohydrate-only, carbohydrate-whey hydrolysate, carbohydrate-casein hydrolysate or placebo-water in a crossover, double-blind design during 2 h of exercise at 60%W max followed by a 16-km time trial. Data were evaluated by magnitude-based inferential statistics. Exogenous carbohydrate oxidation, measured from (13)CO2 breath enrichment, was not substantially influenced by co-ingestion of either protein hydrolysate. However, only co-ingestion of carbohydrate-casein hydrolysate substantially decreased (98% very likely decrease) total carbohydrate oxidation (mean±SD, 242±44; 258±47; 277±33 g for carbohydrate-casein, carbohydrate-whey and carbohydrate-only, respectively) and substantially increased (93% likely increase) total fat oxidation (92±14; 83±27; 73±19 g) compared with carbohydrate-only. Furthermore, only carbohydrate-casein hydrolysate ingestion resulted in a faster time trial (-3.6%; 90% CI: ±3.2%) compared with placebo-water (95% likely benefit). However, neither protein hydrolysate enhanced time trial performance when compared with carbohydrate-only. Under the conditions of this study, ingesting carbohydrate-casein, but not carbohydrate-whey hydrolysate, favourably alters metabolism during prolonged moderate-strenuous cycling without substantially altering cycling performance compared with carbohydrate-only. © Georg Thieme Verlag KG Stuttgart · New York.

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...

Total dissolved carbohydrate in Mahi river estuary

Digital Repository Service at National Institute of Oceanography (India)

Bhosle, N.B.; Rokade, M.A.; Zingde, M.D.

Total dissolved carbohydrate varied from 4.37-15 mg l-1 and 3.71-15.95 mg l-1 in the surface and bottom samples respectively. Highest concentration of carbohydrate was observed at station 1 which decreased downward upto Station 6 which showed...

Chiral reagents in glycosylation and modification of carbohydrates.

Science.gov (United States)

Wang, Hao-Yuan; Blaszczyk, Stephanie A; Xiao, Guozhi; Tang, Weiping

2018-02-05

Carbohydrates play a significant role in numerous biological events, and the chemical synthesis of carbohydrates is vital for further studies to understand their various biological functions. Due to the structural complexity of carbohydrates, the stereoselective formation of glycosidic linkages and the site-selective modification of hydroxyl groups are very challenging and at the same time extremely important. In recent years, the rapid development of chiral reagents including both chiral auxiliaries and chiral catalysts has significantly improved the stereoselectivity for glycosylation reactions and the site-selectivity for the modification of carbohydrates. These new tools will greatly facilitate the efficient synthesis of oligosaccharides, polysaccharides, and glycoconjugates. In this tutorial review, we will summarize these advances and highlight the most recent examples.

Bindings in working memory: The role of object-based attention.

Science.gov (United States)

Gao, Zaifeng; Wu, Fan; Qiu, Fangfang; He, Kaifeng; Yang, Yue; Shen, Mowei

2017-02-01

Over the past decade, it has been debated whether retaining bindings in working memory (WM) requires more attention than retaining constituent features, focusing on domain-general attention and space-based attention. Recently, we proposed that retaining bindings in WM needs more object-based attention than retaining constituent features (Shen, Huang, & Gao, 2015, Journal of Experimental Psychology: Human Perception and Performance, doi: 10.1037/xhp0000018 ). However, only unitized visual bindings were examined; to establish the role of object-based attention in retaining bindings in WM, more emperical evidence is required. We tested 4 new bindings that had been suggested requiring no more attention than the constituent features in the WM maintenance phase: The two constituent features of binding were stored in different WM modules (cross-module binding, Experiment 1), from auditory and visual modalities (cross-modal binding, Experiment 2), or temporally (cross-time binding, Experiments 3) or spatially (cross-space binding, Experiments 4-6) separated. In the critical condition, we added a secondary object feature-report task during the delay interval of the change-detection task, such that the secondary task competed for object-based attention with the to-be-memorized stimuli. If more object-based attention is required for retaining bindings than for retaining constituent features, the secondary task should impair the binding performance to a larger degree relative to the performance of constituent features. Indeed, Experiments 1-6 consistently revealed a significantly larger impairment for bindings than for the constituent features, suggesting that object-based attention plays a pivotal role in retaining bindings in WM.

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)

The TROVE module: A common element in Telomerase, Ro and Vault ribonucleoproteins

Directory of Open Access Journals (Sweden)

Bateman Alex

2003-10-01

Full Text Available Abstract Background Ribonucleoproteins carry out a variety of important tasks in the cell. In this study we show that a number of these contain a novel module, that we speculate mediates RNA-binding. Results The TROVE module – Telomerase, Ro and Vault module – is found in TEP1 and Ro60 the protein components of three ribonucleoprotein particles. This novel module, consisting of one or more domains, may be involved in binding the RNA components of the three RNPs, which are telomerase RNA, Y RNA and vault RNA. A second conserved region in these proteins is shown to be a member of the vWA domain family. The vWA domain in TEP1 is closely related to the previously recognised vWA domain in VPARP a second component of the vault particle. This vWA domain may mediate interactions between these vault components or bind as yet unidentified components of the RNPs. Conclusions This work suggests that a number of ribonucleoprotein components use a common RNA-binding module. The TROVE module is also found in bacterial ribonucleoproteins suggesting an ancient origin for these ribonucleoproteins.

Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

KAUST Repository

Li, Lixin

2013-07-01

Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp. © 2013 The Author.

Dietary composition modulates brain mass and solubilizable Aβ levels in a mouse model of aggressive Alzheimer's amyloid pathology

Directory of Open Access Journals (Sweden)

Buxbaum Joseph D

2009-10-01

Full Text Available Abstract Objective Alzheimer's disease (AD is a progressive neurodegenerative disease of the central nervous system (CNS. Recently, an increased interest in the role diet plays in the pathology of AD has resulted in a focus on the detrimental effects of diets high in cholesterol and fat and the beneficial effects of caloric restriction. The current study examines how dietary composition modulates cerebral amyloidosis and neuronal integrity in the TgCRND8 mouse model of AD. Methods From 4 wks until 18 wks of age, male and female TgCRND8 mice were maintained on one of four diets: (1 reference (regular commercial chow; (2 high fat/low carbohydrate custom chow (60 kcal% fat/30 kcal% protein/10 kcal% carbohydrate; (3 high protein/low carbohydrate custom chow (60 kcal% protein/30 kcal% fat/10 kcal% carbohydrate; or (4 high carbohydrate/low fat custom chow (60 kcal% carbohydrate/30 kcal% protein/10 kcal% fat. At age 18 wks, mice were sacrificed, and brains studied for (a wet weight; (b solubilizable Aβ content by ELISA; (c amyloid plaque burden; (d stereologic analysis of selected hippocampal subregions. Results Animals receiving a high fat diet showed increased brain levels of solubilizable Aβ, although we detected no effect on plaque burden. Unexpectedly, brains of mice fed a high protein/low carbohydrate diet were 5% lower in weight than brains from all other mice. In an effort to identify regions that might link loss of brain mass to cognitive function, we studied neuronal density and volume in hippocampal subregions. Neuronal density and volume in the hippocampal CA3 region of TgCRND8 mice tended to be lower in TgCRND8 mice receiving the high protein/low carbohydrate diet than in those receiving the regular chow. Neuronal density and volume were preserved in CA1 and in the dentate gyrus. Interpretation Dissociation of Aβ changes from brain mass changes raises the possibility that diet plays a role not only in modulating amyloidosis but also in

Radiolabeled Peptide Scaffolds for PET/SPECT - Optical in Vivo Imaging of Carbohydrate-Lectin Interactions

Energy Technology Data Exchange (ETDEWEB)

Deutscher, Susan

2014-09-30

The objective of this research is to develop phage display-selected peptides into radio- and fluoresecently- labeled scaffolds for the multimodal imaging of carbohydrate-lectin interactions. While numerous protein and receptor systems are being explored for the development of targeted imaging agents, the targeting and analysis of carbohydrate-lectin complexes in vivo remains relatively unexplored. Antibodies, nanoparticles, and peptides are being developed that target carbohydrate-lectin complexes in living systems. However, antibodies and nanoparticles often suffer from slow clearance and toxicity problems. Peptides are attractive alternative vehicles for the specific delivery of radionuclides or fluorophores to sites of interest in vivo, although, because of their size, uptake and retention may be less than antibodies. We have selected high affinity peptides that bind a specific carbohydrate-lectin complex involved in cell-cell adhesion and cross-linking using bacteriophage (phage) display technologies (1,2). These peptides have allowed us to probe the role of these antigens in cell adhesion. Fluorescent versions of the peptides have been developed for optical imaging and radiolabeled versions have been used in single photon emission computed tomography (SPECT) and positron emission tomography (PET) in vivo imaging (3-6). A benefit in employing the radiolabeled peptides in SPECT and PET is that these imaging modalities are widely used in living systems and offer deep tissue sensitivity. Radiolabeled peptides, however, often exhibit poor stability and high kidney uptake in vivo. Conversely, optical imaging is sensitive and offers good spatial resolution, but is not useful for deep tissue penetration and is semi-quantitative. Thus, multimodality imaging that relies on the strengths of both radio- and optical- imaging is a current focus for development of new in vivo imaging agents. We propose a novel means to improve the efficacy of radiolabeled and fluorescently

Differential usage of storage carbohydrates in the CAM bromeliad Aechmea 'Maya' during acclimation to drought and recovery from dehydration.

Science.gov (United States)

Ceusters, Johan; Borland, Anne M; Londers, Elsje; Verdoodt, Veerle; Godts, Christof; De Proft, Maurice P

2009-02-01

CAM requires a substantial investment of resources into storage carbohydrates to account for nocturnal CO(2) uptake, thereby restricting carbohydrate partitioning to other metabolic activities, including dark respiration, growth and acclimation to abiotic stress. Flexible modulation of carbon flow to the different competing sinks under changing environmental conditions is considered a key determinant for the growth, productivity and ecological success of the CAM pathway. The aim of the present study was to examine how shifts in carbohydrate partitioning could assure maintenance of photosynthetic integrity and a positive carbon balance under conditions of increasing water deprivation in CAM species. Measurements of gas exchange, leaf water relations, malate, starch and soluble sugar (glucose, fructose and sucrose) contents were made in leaves of the CAM bromeliad Aechmea 'Maya' over a 6-month period of drought and subsequently over a 2-month period of recovery from drought. Results indicated that short-term influences of water stress were minimized by elevating the level of respiratory recycling, and carbohydrate pools were maintained at the expense of export for growth while providing a comparable nocturnal carbon gain to that in well-watered control plants. Longer term drought resulted in a disproportionate depletion of key carbohydrate reserves. Sucrose, which was of minor importance for providing substrate for the dark reactions under well-watered conditions, became the major source of carbohydrate for nocturnal carboxylation as drought progressed. Flexibility in terms of the major carbohydrate source used to sustain dark CO(2) uptake is therefore considered a crucial factor in meeting the carbon and energy demands under limiting environmental conditions. Recovery from CAM-idling was found to be dependent on the restoration of the starch pool, which was used predominantly for provision of substrate for nocturnal carboxylation, while net carbon export was limited

Blood Triglycerides Levels and Dietary Carbohydrate Indices in Healthy Koreans

Directory of Open Access Journals (Sweden)

Hye Sook Min

2016-05-01

Full Text Available Objectives: Previous studies have obtained conflicting findings regarding possible associations between indices measuring carbohydrate intake and dyslipidemia, which is an established risk factor of coronary heart disease. In the present study, we examined cross-sectional associations between carbohydrate indices, including the dietary glycemic index (GI, glycemic load (GL, total amount of carbohydrates, and the percentage of energy from carbohydrates, and a range of blood lipid parameters. Methods: This study included 1530 participants (554 men and 976 women from 246 families within the Healthy Twin Study. We analyzed the associations using a generalized linear mixed model to control for familial relationships. Results: Levels of the Apo B were inversely associated with dietary GI, GL, and the amount of carbohydrate intake for men, but these relationships were not significant when fat-adjusted values of the carbohydrate indices were used. Triglyceride levels were positively associated with dietary GI and GL in women, and this pattern was more notable in overweight participants (body mass index [BMI] ≥25 kg/m2. However, total, low-density lipoprotein and high-density lipoprotein cholesterol levels were not significantly related with carbohydrate intake overall. Conclusions: Of the blood lipid parameters we investigated, only triglyceride levels were positively related with dietary carbohydrate indices among women participants in the Healthy Twin Study, with an interactive role observed for BMI. However, these associations were not observed in men, suggesting that the association between blood lipid levels and carbohydrate intake depends on the type of lipid, specific carbohydrate indices, gender, and BMI.

Effect of dietary carbohydrate source on the development of obesity in agouti transgenic mice.

Science.gov (United States)

Morris, Kristin L; Zemel, Michael B

2005-01-01

Our objective was to evaluate the effects of a qualitative change in dietary carbohydrate source on body weight and adiposity in a rodent model of diet-induced obesity. We evaluated the effects of high-fat diets (basal) varying in carbohydrate source in aP2-agouti transgenic mice. In the ad libitum study, animals were given free access to the basal diet or one of four test diets for 6 weeks. In two of the diets, dietary carbohydrate was derived from a single source: mung bean noodles (MUNG) or rolled oats (ROLL). The remaining diets were designed to mimic commercially available instant oatmeal with added sugar (IO-S) or flavored instant oatmeal (IO-F). In the energy-restricted study, animals were given ad libitum access to the basal diet for 6 weeks. Subsequently, animals were assigned to one of six treatment groups for 6 weeks. One group was continued on the basal diet ad libitum. The remaining groups were maintained with energy restriction (70% ad libitum) on either the basal, MUNG, ROLL, IO-S, or IO-F diet. Subcutaneous fat pad mass was significantly higher (p<0.05) in the energy-restricted basal and IO-S groups compared with the energy-restricted ROLL diet. Similarly, visceral fat pad mass was significantly lower with ROLL and MUNG diets (p<0.05 for both) compared with basal and IO-S diets, and the insulin:glucose ratio was reduced (by 23% to 34%, p<0.05) in these two diets compared with all others. In ad libitum-fed animals, liver fatty acid synthase expression was 43% to 62% lower (p<0.05) with ROLL and MUNG diets compared with all others. These data suggest that a qualitative change in dietary carbohydrate source modulates body weight and adiposity.

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

Directory of Open Access Journals (Sweden)

Irina V. Gorudko

2013-07-01

Myeloperoxidase (MPO is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist. In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE. Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

HuR/ELAVL1 RNA binding protein modulates interleukin-8 induction by muco-active ribotoxin deoxynivalenol

International Nuclear Information System (INIS)

Choi, Hye Jin; Yang, Hyun; Park, Seong Hwan; Moon, Yuseok

2009-01-01

HuR/Elav-like RNA binding protein 1 (ELAVL1) positively regulates mRNA stability of AU-rich elements (ARE)-containing transcript such as pro-inflammatory cytokines. Ribotoxic stresses can trigger the production of pro-inflammatory mediators by enhancing mRNA stability and the transcriptional activity. We investigated the effects of ribotoxin deoxynivalenol (DON) on HuR translocation and its involvement in the regulation of the pro-inflammatory interleukin-8 (IL-8) mRNA stability. Exposure to the muco-active DON induced nuclear export of both endogenous and exogenous HuR RNA binding protein in human intestinal epithelial cells. Moreover, the interference with HuR protein production suppressed ribotoxic DON-induced IL-8 secretion and its mRNA stability. Cytoplasmic HuR protein interacted with IL-8 mRNA and the complex stabilization was due to the presence of 3'-untranslated region of the transcript. Partly in terms of IL-8-modulating transcription factors, HuR protein was demonstrated to be positively and negatively associated with DON-induced early growth response gene 1 (EGR-1) and activating transcription factor 3 (ATF3), respectively. HuR was a critical mechanistic link between ribotoxic stress and the pro-inflammatory cytokine production, and may have a broader functional significance with regard to mucosal insults since ribotoxic stress responses are also produced upon interactions with the diverse environment of gut.

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.

Carbohydrate deficient transferrin (CDT) in alcoholic cirrhosis: a kinetic study

DEFF Research Database (Denmark)

Henriksen, Jens Henrik; Grønbaek, M; Møller, Søren

1997-01-01

BACKGROUND/AIMS: Carbohydrate deficient transferrin has been introduced as a marker of excessive alcohol intake. The present study was undertaken in order to measure the circulating level of carbohydrate deficient transferrin in patients with alcoholic cirrhosis and to assess arteriovenous kinetics...... of carbohydrate deficient transferrin in liver and kidney. METHODS/RESULTS: The median value of serum carbohydrate deficient transferrin was 16.0 U/l in patients with alcoholic cirrhosis (n = 41), and this value was not significantly different from that of a normal control group (median 17.4 U/l, n = 55, ns......). Carbohydrate deficient transferrin was significantly higher in patients with cirrhosis and high current alcohol intake than in abstaining patients (20 vs. 14 U/l, p 50 g/day) had a significantly higher carbohydrate deficient transferrin...

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

International Nuclear Information System (INIS)

Wang, Feng; Liu, Xiao-qin; Li, He; Liang, Kai-ni; Miner, Jeffrey N.; Hong, Mei; Kallel, E. Adam; Oeveren, Arjan van; Zhi, Lin; Jiang, Tao

2006-01-01

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

IGF binding proteins.

Science.gov (United States)

Bach, Leon A

2017-12-18

Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

Dietary non-digestible carbohydrates and the resistance to intestinal infections

NARCIS (Netherlands)

Bruggencate, ten S.J.M.

2004-01-01

Keywords: Non-digestible carbohydrates, prebiotics, inulin, FOS, calcium, microflora, short-chain fatty acids, mucin, intestinal permeability, salmonella, infection, rat, humanDietary non-digestible carbohydrates and the resistance to intestinal infectionsNon-digestible carbohydrates (NDC) stimulate

Evaluation of certain crop residues for carbohydrate and protein fractions by cornell net carbohydrate and protein system

Directory of Open Access Journals (Sweden)

Venkateswarulu Swarna

2015-06-01

Full Text Available Four locally available crop residues viz., jowar stover (JS, maize stover (MS, red gram straw (RGS and black gram straw (BGS were evaluated for carbohydrate and protein fractions using Cornell Net Carbohydrate and Protein (CNCP system. Lignin (% NDF was higher in legume straws as compared to cereal stovers while Non-structural carbohydrates (NSC (% DM followed the reverse trend. The carbohydrate fractions A and B1 were higher in BGS while B2 was higher in MS as compared to other crop residues. The unavailable cell wall fraction (C was higher in legume straws when compared to cereal stovers. Among protein fractions, B1 was higher in legume straws when compared to cereal stovers while B2 was higher in cereal stovers as compared to legume straws. Fraction B3 largely, bypass protein was highest in MS as compared to other crop residues. Acid detergent insoluble crude protein (ADICP (% CP or unavailable protein fraction C was lowest in MS and highest in BGS. It is concluded that MS is superior in nutritional value for feeding ruminants as compared to other crop residues.

The use of fast atom bombardment and laser desorption mass spectrometry in the analysis of complex carbohydrates

International Nuclear Information System (INIS)

Egge, H.; Peter-Katalinic, J.; Karas, M.; Stahl, B.

1991-01-01

Oligosaccharides occurring free in secretions or bound to lipid or protein, are known to modulate the biological response in many living systems. The structural characterization of these highly diverse oligosaccharides, that may be further complicated by the occurrence of non-carbohydrate substituents such as alkyl, acyl, sulfate, or phosphate groups, for example, represents the first step towards a rational approach that is able to relate structure to function. The structural delineation of carbohydrate residues at defined sites of attachment is especially important in recombinant glycoproteins because the type and extent of glycosylation affect their biological properties. In recent years the development of soft ionization procedures and the increase in mass range above 10,000 mass units at full acceleration, together with the development of highly sensitive detectors, has allowed the analysis of glycans containing more than 30 sugar units in the nano-and subnanomolar range. (author)

Method for the direct determination of available carbohydrates in low-carbohydrate products using high-performance anion exchange chromatography.

Science.gov (United States)

Ellingson, David; Potts, Brian; Anderson, Phillip; Burkhardt, Greg; Ellefson, Wayne; Sullivan, Darryl; Jacobs, Wesley; Ragan, Robert

2010-01-01

An improved method for direct determination of available carbohydrates in low-level products has been developed and validated for a low-carbohydrate soy infant formula. The method involves modification of an existing direct determination method to improve specificity, accuracy, detection levels, and run times through a more extensive enzymatic digestion to capture all available (or potentially available) carbohydrates. The digestion hydrolyzes all common sugars, starch, and starch derivatives down to their monosaccharide components, glucose, fructose, and galactose, which are then quantitated by high-performance anion-exchange chromatography with photodiode array detection. Method validation consisted of specificity testing and 10 days of analyzing various spike levels of mixed sugars, maltodextrin, and corn starch. The overall RSD was 4.0% across all sample types, which contained within-day and day-to-day components of 3.6 and 3.4%, respectively. Overall average recovery was 99.4% (n = 10). Average recovery for individual spiked samples ranged from 94.1 to 106% (n = 10). It is expected that the method could be applied to a variety of low-carbohydrate foods and beverages.

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

Directory of Open Access Journals (Sweden)

Clara L Oeste

2011-01-01

Full Text Available 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.

Detecting Elusive Intermediates in Carbohydrate Conversion: A Dynamic Ensemble of Acyclic Glucose-Catalyst Complexes

DEFF Research Database (Denmark)

Meier, Sebastian; Karlsson, Magnus; Jensen, Pernille Rose

2017-01-01

within few seconds prior to reaching a steady state. Exchange between the acyclic intermediates increases at conditions that favor epimerization. Species accounting for less than 0.05% of total glucose can be monitored with sub-second time resolution to allow kinetic analysis of intermediate formation...... and catalytic conversion. Epimerization occurs 2-3 orders of magnitude-fold faster than the binding of acyclic glucose to the catalyst at near-optimum reaction conditions. The current study brings insight in to the nature of acyclic intermediate-catalyst complexes of very low population and into experimental...... strategies for characterizing very minor intermediates in carbohydrate conversion to value-added compounds....

Characterization of the Organic Component of Low-Molecular-Weight Chromium-Binding Substance and Its Binding of Chromium123

Science.gov (United States)

Chen, Yuan; Watson, Heather M.; Gao, Junjie; Sinha, Sarmistha Halder; Cassady, Carolyn J.; Vincent, John B.

2011-01-01

Chromium was proposed to be an essential element over 50 y ago and was shown to have therapeutic potential in treating the symptoms of type 2 diabetes; however, its mechanism of action at a molecular level is unknown. One chromium-binding biomolecule, low-molecular weight chromium-binding substance (LMWCr or chromodulin), has been found to be biologically active in in vitro assays and proposed as a potential candidate for the in vivo biologically active form of chromium. Characterization of the organic component of LMWCr has proven difficult. Treating bovine LMWCr with trifluoroacetic acid followed by purification on a graphite powder micro-column generates a heptapeptide fragment of LMWCr. The peptide sequence of the fragment was analyzed by MS and tandem MS (MS/MS and MS/MS/MS) using collision-induced dissociation and post-source decay. Two candidate sequences, pEEEEGDD and pEEEGEDD (where pE is pyroglutamate), were identified from the MS/MS experiments; additional tandem MS suggests the sequence is pEEEEGDD. The N-terminal glutamate residues explain the inability to sequence LMWCr by the Edman method. Langmuir isotherms and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to apoLMWCr. The sequence pEEEEGDD was found to bind 4 chromic ions per peptide with nearly identical cooperativity and binding constants to those of apoLMWCr. This work should lead to further studies elucidating or eliminating a potential role for LMWCr in treating the symptoms of type 2 diabetes and other conditions resulting from improper carbohydrate and lipid metabolism. PMID:21593351

Therapeutic potential of carbohydrates as regulators of macrophage activation.

Science.gov (United States)

Lundahl, Mimmi L E; Scanlan, Eoin M; Lavelle, Ed C

2017-12-15

It is well established for a broad range of disease states, including cancer and Mycobacterium tuberculosis infection, that pathogenesis is bolstered by polarisation of macrophages towards an anti-inflammatory phenotype, known as M2. As these innate immune cells are relatively long-lived, their re-polarisation to pro-inflammatory, phagocytic and bactericidal "classically activated" M1 macrophages is an attractive therapeutic approach. On the other hand, there are scenarios where the resolving inflammation, wound healing and tissue remodelling properties of M2 macrophages are beneficial - for example the successful introduction of biomedical implants. Although there are numerous endogenous and exogenous factors that have an impact on the macrophage polarisation spectrum, this review will focus specifically on prominent macrophage-modulating carbohydrate motifs with a view towards highlighting structure-function relationships and therapeutic potential. Copyright © 2017 Elsevier Inc. All rights reserved.

Cerebral carbohydrate cost of physical exertion in humans

DEFF Research Database (Denmark)

Dalsgaard, Mads K; Ogoh, Shigehiko; Dawson, Ellen A

2004-01-01

Above a certain level of cerebral activation the brain increases its uptake of glucose more than that of O(2), i.e., the cerebral metabolic ratio of O(2)/(glucose + 12 lactate) decreases. This study quantified such surplus brain uptake of carbohydrate relative to O(2) in eight healthy males who...... to exhaustion (15.8 +/- 1.7 min; P carbohydrate was not substantiated...... and, consequently, exhaustive exercise involves a brain surplus carbohydrate uptake of a magnitude comparable with its glycogen content....

Characterization of carbohydrate fractions and fermentation quality ...

African Journals Online (AJOL)

This experiment was carried out to evaluate the effects of adding fast-sile (FS), previous fermented juice (PFJ), sucrose (S) or fast-sile + sucrose (FS + S) on the fermentation characteristics and carbohydrates fractions of alfalfa silages by the Cornell net carbohydrates and proteins systems (CNCPS). Silages quality were well ...