Characterization of melanoidins from a glucose-glycine model system

The properties of melanoidins prepared from glucose and glycine (GG) were investigated by a three step purification protocol consisting of dialysis, gel filtration at high ionic strength and ion metal affinity chromatography. The high molecular weight fraction obtained in the GG system is responsible for 80% of the total brown colour and its antioxidative ability was about 1/4 of that of Trolox measured by the inhibition of linoleic acid oxidation. GG melanoidins have good affinity towards Cu (II) (32% bound to the resin) while it is much lower towards Pb (II) (10%) and Fe (II) (5%). Capillary zone electrophoresis analysis suggests that GG melanoidins are positively charged, although no signal was observed analysing melanoidins by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF/MS).

Effect of pH, temperature, and moisture on the formation of volatile compounds in glycine/glucose model systems

Mixtures of glycine, glucose, and starch were extrusion cooked using sodium hydroxide at 0, 3, and 6 g/L of extruder water feed, 18% moisture, and 120, 150, and 180 degreesC target die temperatures, giving extrudates with pH values of 5.6, 6.8, and 7.4. Freeze-dried equimolar solutions of glucose and glycine were heated either dry or after equilibration to similar to 13% moisture at 180 degreesC in a reaction-tube system designed to mimic the heating profile in an extruder. Volatile compounds were isolated onto Tenax and analyzed by gas chromatography-mass spectrometry. For the extrudates, total yields of volatiles increased with decreasing pH at 180 degreesC, reached a maximum at pH 6.S at 150 degreesC, and increased with increasing pH at 120 degreesC. Amounts increased with temperature at all pH values. Pyrazines were the most abundant class for all sets of conditions (54-79% of total volatiles). Pyrroles, ketones, furans, oxazoles, and pyridines were also identified. Yields of volatiles from the reaction-tube samples increased by > 60% in the moist system. Levels of individual classes also increased in the presence of moisture, except pyrazines, which decreased similar to3.5-fold. Twenty-one of the compounds were common to the reaction-tube samples and the extrudates.

Glucose solvation by the ionic liquid 1,3-dimethylimidazolium chloride: A simulation study

Simulations of beta-glucose in the ionic liquid 1,3-dimethylimidazoliurn chloride have been performed in order to examine the solvation environment of the carbohydrate. Both single molecule and 1:5 glucose:ionic liquid (16.7 wt %) solutions are Studied, and the hydrogen bonding between sugar and solvent is examined. The primary solvation shell around the perimeter of the glucose ring consists predominantly of chloride anions which hydrogen bond to the hydroxyl groups. A small presence of the cation is also found, with the association Occurring through the weakly acidic hydrogen at the 2-position of the imidazolium ring interacting with the oxygen atoms of the sugar secondary hydroxyls. An average chloride coordination number of 4 is found around the glucose for both the single molecule and high concentration Simulations, despite the reduced chloride:glucose ratio in the latter case. In relation to the cation, the glucose molecules occupy positions above and below the plane of the imidazolium ring. Importantly, even at high glucose concentrations, no significant change in the anion-cation interactions and overall liquid structure of the ionic liquid is found, indicating that the glucose is readily accommodated by the solvent at this concentration. Dominant contributions to the sugar-ionic liquid interaction energy come from favorable hydrogen bonding (electrostatic) interactions between hydroxyls and chlorides, although a small favorable van der Waals energy contribution is also seen between the sugar and cations suggesting that the cation could be tailored in order to further improve the dissolution of glucose/cellulose in ionic liquid systems.

Salt-bridge dynamics control substrate-induced conformational change in the membrane transporter GlpT.

Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (Pi) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate:phosphate antiporters.

Acute exercise and impaired glucose tolerance in obese humans

BACKGROUND: Individuals with impaired glucose tolerance (IGT) have a greater risk of developing diabetes and cardiovascular disease compared with those with normal glycemic control. The aim of this study was to examine the effects of acute aerobic exercise on glycemia, regional arterial stiffness, and oxidative stress in obese subjects with IGT.

Identification of Determinants of Glucose-Dependent Insulinotropic Polypeptide Receptor That Interact with N-Terminal Biologically Active Region of the Natural Ligand

Glucose-dependent insulinotropic polypeptide receptor (GIPR), a member of family B of the G-protein coupled receptors, is a potential therapeutic target for which discovery of nonpeptide ligands is highly desirable. Structure-activity relationship studies indicated that the N-terminal part of glucose-dependent insulinotropic polypeptide (GIP) is crucial for biological activity. Here, we aimed at identification of residues in the GIPR involved in functional interaction with N-terminal moiety of GIP. A homology model of the transmembrane core of GIPR was constructed, whereas a three-dimensional model of the complex formed between GIP and the N-terminal extracellular domain of GIPR was taken from the crystal structure. The latter complex was docked to the transmembrane domains of GIPR, allowing in silico identification of putative residues of the agonist binding/activation site. All mutants were expressed at the surface of human embryonic kidney 293 cells as indicated by flow cytometry and confocal microscopy analysis of fluorescent GIP binding. Mutation of residues Arg183, Arg190, Arg300, and Phe357 caused shifts of 76-, 71-, 42-, and 16-fold in the potency to induce cAMP formation, respectively. Further characterization of these mutants, including tests with alanine-substituted GIP analogs, were in agreement with interaction of Glu3 in GIP with Arg183 in GIPR. Furthermore, they strongly supported a binding mode of GIP to GIPR in which the N-terminal moiety of GIP was sited within transmembrane helices (TMH) 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5), and Phe357 (TMH6). These data represent an important step toward understanding activation of GIPR by GIP, which should facilitate the rational design of therapeutic agents.

Vitamin C supplementation decreases insulin glycation and improves glucose homeostasis in obese hyperglycemic (ob/ob) mice

The effects of dietary vitamin C supplementation on glucose homeostasis and insulin glycation were examined in adult lean and obese hyperglycemic (ob/ob) mice. In lean mice, supplementation of the drinking water with vitamin C (25 g/L) for 14 days did not affect food intake, fluid intake, glycated hemoglobin, plasma glucose, or plasma insulin concentrations. Total pancreatic insulin content and the percentage of glycated pancreatic insulin were also similar to control lean mice. In ob/ob mice, vitamin C supplementation caused significant reductions by 26% to 48% in food intake and fluid intake, glycated hemoglobin, plasma glucose, and insulin concentrations compared with untreated control ob/ob mice. The total insulin content and the extent of insulin glycation in the pancreas of ob/ob mice were also significantly decreased by 42% to 45% after vitamin C supplementation. This change was accompanied by a significant 80% decrease in the percentage of glycated insulin in the circulation of vitamin C- supplemented ob/ob mice. These data demonstrate that vitamin C supplementation can decrease insulin glycation and ameliorate aspects of the obesity-diabetes syndrome in ob/ob mice. Copyright 2002, Elsevier Science (USA). All rights reserved.

Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia

Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugd(BCAL2946), ugd(BCAM0855) and ugd(BCAM2034), all of which were individually inactivated. Only inactivation of ugd(BCAL2946) resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either ugd(BCAL2946) or ugd(BCAM0855) but not ugd(BCAM2034) was expressed from plasmids. The growth of a conditional ugd(BCAL2946) mutant, created in the Deltaugd(BCAM0855) background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugd(BCAL2946) or ugd(BCAM0855) expressed in trans, but not by ugd(BCAM2034). Biochemical analysis of the purified, recombinant forms of Ugd(BCAL2946) and Ugd(BCAM0855) revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD(+). Purified Ugd(BCAM2034) showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugd(BCAL2946) was 5.4- and 135-fold greater than that of ugd(BCAM0855) and ugd(BCAM2034), respectively. Together, these data indicate that the combined activity of Ugd(BCAL2946) and Ugd(BCAM0855) is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugd(BCAL2946), is required for polymyxin B resistance.

The major facilitator superfamily transporter MdtM contributes to intrinsic resistance of Escherichia coli to quaternary ammonium compounds

OBJECTIVES:
Quaternary ammonium compounds (QACs) are used extensively as biocides and their misuse may be contributing to the development of bacterial resistance. Although the major intrinsic resistance to QACs of Gram-negative bacteria is mediated by the action of tripartite multidrug transporters of the resistance-nodulation-division family, we aimed to test if the promiscuity of the recently characterized major facilitator superfamily multidrug transporter, MdtM, from Escherichia coli enabled it also to function in the efflux of QACs.
METHODS:
The ability of the major facilitator mdtM gene product, when overexpressed from multicopy plasmid, to protect E. coli cells from the toxic effects of a panel of seven QACs was determined using growth inhibition assays in liquid medium. Interaction between QACs and MdtM was studied by a combination of substrate binding assays using purified protein in detergent solution and transport assays using inverted vesicles.
RESULTS:
E. coli cells that overproduced MdtM were less susceptible to the cytotoxic effects of each of the QACs tested compared with cells that did not overproduce the transporter. Purified MdtM bound each QAC with micromolar affinity and the protein utilized the electrochemical proton gradient to transport QACs across the cytoplasmic membrane. Furthermore, the results suggested a functional interaction between MdtM and the tripartite resistance-nodulation-division family AcrAB-TolC efflux system.
CONCLUSIONS:
The results support a hitherto unidentified capacity for a single-component multidrug transporter of the major facilitator superfamily, MdtM, to function in the efflux of a broad range of QACs and thus contribute to the intrinsic resistance of E. coli to these compounds.

The molecular dynamics of Trypanosoma brucei UDP-galactose 4'-epimerase:a drug target for African sleeping sickness

During the past century, several epidemics of human African trypanosomiasis, a deadly disease caused by the protist Trypanosoma brucei, have afflicted sub-Saharan Africa. Over 10 000 new victims are reported each year, with hundreds of thousands more at risk. As current drug treatments are either highly toxic or ineffective, novel trypanocides are urgently needed. The T. brucei galactose synthesis pathway is one potential therapeutic target. Although galactose is essential for T. brucei survival, the parasite lacks the transporters required to intake galactose from the environment. UDP-galactose 4'-epimerase (TbGalE) is responsible for the epimerization of UDP-glucose to UDP-galactose and is therefore of great interest to medicinal chemists. Using molecular dynamics simulations, we investigate the atomistic motions of TbGalE in both the apo and holo states. The sampled conformations and protein dynamics depend not only on the presence of a UDP-sugar ligand, but also on the chirality of the UDP-sugar C4 atom. This dependence provides important insights into TbGalE function and may help guide future computer-aided drug discovery efforts targeting this protein.

On the Mechanism of Lewis Acid Catalyzed Glucose Transformations in Ionic Liquids

A complementary computational and experimental study of the reactivity of Lewis acidic CrCl2, CuCl2 and FeCl2 catalysts towards glucose activation in dialkylimidazolium chloride ionic liquids is performed. The selective dehydration of glucose to 5-hydroxymethylfurfural (HMF) proceeds through the intermediate formation of fructose. Although chromium(II) and copper(II) chlorides are able to dehydrate fructose with high HMF selectivity, reasonable HMF yields from glucose are only obtained with CrCl2 as the catalyst. Glucose conversion by CuCl2 is not selective, while FeCl2 catalyst does not activate sugar molecules. These differences in reactivity are rationalized on the basis of in situ X-ray absorption spectroscopy measurements and the results of density functional theory calculations. The reactivity in glucose dehydration and HMF selectivity are determined by the behavior of the ionic liquid-mediated Lewis acid catalysts towards the initial activation of the sugar molecules. The formation of a coordination complex between the Lewis acidic Cr2+ center and glucose directs glucose transformation into fructose. For Cu2+ the direct coordination of sugar to the copper(II) chloride complex is unfavorable. Glucose deprotonation by a mobile Cl- ligand in the CuCl42- complex initiates the nonselective conversion. In the course of the reaction the Cu2+ ions are reduced to Cu+. Both paths are prohibited for the FeCl2 catalyst.

A New Stereocontrolled Synthetic Route to (-)-Echinosporin from D-Glucose via Padwa Allenylsulfone [3+2]-Anionic Cycloadditive Elimination

A new formal total synthesis of (-)-echinosporin has been developed based upon the Padwa [3 + 2]-cycloadditive elimination reaction of allenylsulfone 4 with the D-glucose-derived enone 14 which provides cycloadduct 12.

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.

In silico prediction of the effects of mutations in the human UDP-galactose 4'-epimerase gene:Towards a predictive framework for type III galactosemia

The enzyme UDP-galactose 4'-epimerase (GALE) catalyses the reversible epimerisation of both UDP-galactose and UDP-N-acetyl-galactosamine. Deficiency of the human enzyme (hGALE) is associated with type III galactosemia. The majority of known mutations in hGALE are missense and private thus making clinical guidance difficult. In this study a bioinformatics approach was employed to analyse the structural effects due to each mutation using both the UDP-glucose and UDP-N-acetylglucosamine bound structures of the wild-type protein. Changes to the enzyme's overall stability, substrate/cofactor binding and propensity to aggregate were also predicted. These predictions were found to be in good agreement with previous in vitro and in vivo studies when data was available and allowed for the differentiation of those mutants that severely impair the enzyme's activity against UDP-galactose. Next this combination of techniques were applied to another twenty-six reported variants from the NCBI dbSNP database that have yet to be studied to predict their effects. This identified p.I14T, p.R184H and p.G302R as likely severely impairing mutations. Although severely impaired mutants were predicted to decrease the protein's stability, overall predicted stability changes only weakly correlated with residual activity against UDP-galactose. This suggests other protein functions such as changes in cofactor and substrate binding may also contribute to the mechanism of impairment. Finally this investigation shows that this combination of different in silico approaches is useful in predicting the effects of mutations and that it could be the basis of an initial prediction of likely clinical severity when new hGALE mutants are discovered.