Galactose

Distribution by Scientific Domains
Distribution within Chemistry

Terms modified by Galactose

  • galactose oxidase
  • galactose residue

  • Selected Abstracts


    Synthesis of Hybrids of D -Glucose and D -Galactose with Pyrrolidine-Based Iminosugars as Glycosidase Inhibitors

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 34 2008
    Venkata Ramana Doddi
    Abstract Sugar,iminosugar hybrid molecules made up of D -glucose and D -galactose with pyrrolidine-based iminosugars, viz. 1,4-dideoxy-1,4-imino- L -xylitol and 1,4-dideoxy-1,4-imino- L -lyxitol, are synthesized from glycal epoxides and found to be moderate glycosidase inhibitors. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source]


    One-Pot Preparation of Polymer,Enzyme,Metallic Nanoparticle Composite Films for High-Performance Biosensing of Glucose and Galactose

    ADVANCED FUNCTIONAL MATERIALS, Issue 11 2009
    Yingchun Fu
    Abstract New polymer,enzyme,metallic nanoparticle composite films with a high-load and a high-activity of immobilized enzymes and obvious electrocatalysis/nano-enhancement effects for biosensing of glucose and galactose are designed and prepared by a one-pot chemical pre-synthesis/electropolymerization (CPSE) protocol. Dopamine (DA) as a reductant and a monomer, glucose oxidase (GOx) or galactose oxidase (GaOx) as the enzyme, and HAuCl4 or H2PtCl6 as an oxidant to trigger DA polymerization and the source of metallic nanoparticles, are mixed to yield polymeric bionanocomposites (PBNCs), which are then anchored on the electrode by electropolymerization of the remaining DA monomer. The prepared PBNC material has good biocompatibility, a highly uniform dispersion of the nanoparticles with a narrow size distribution, and high load/activity of the immobilized enzymes, as verified by transmission/scanning electron microscopy and electrochemical quartz crystal microbalance. The thus-prepared enzyme electrodes show a largely improved amperometric biosensing performance, e.g., a very high detection sensitivity (99 or 129,µA cm,2 mM,1 for glucose for Pt PBNCs on bare or platinized Au), a sub-micromolar limit of detection for glucose, and an excellent durability, in comparison with those based on conventional procedures. Also, the PBNC-based enzyme electrodes work well in the second-generation biosensing mode. The proposed one-pot CPSE protocol may be extended to the preparation of many other functionalized PBNCs for wide applications. [source]


    An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

    BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010
    Niki S.C. Wong
    Abstract Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N -glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-, (IFN-,). Galactose (±uridine), glucosamine (±uridine), and N -acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-, sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP-Hex (,20-fold), UDP-HexNAc (6- to 15-fold) and CMP-sialic acid (30- to 120-fold), respectively. Up-regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine,+,uridine and ManNAc,+,cytidine increased UDP-HexNAc and CMP-sialic acid by another two- to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN-, sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine- and cytidine-activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321,336. © 2010 Wiley Periodicals, Inc. [source]


    Characteristics of Saccharomyces cerevisiae gal1, and gal1,hxk2, mutants expressing recombinant proteins from the GAL promoter

    BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2005
    Hyun Ah Kang
    Abstract Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1, mutant strain was constructed and its induction kinetics investigated. As expected, the gal1, strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05,0.1 g/L). However, the gal1, strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1, mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1, mutant strain, generating gal1,mig1, and gal1,hxk2, double strains. The gal1,hxk2, strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1, strain, whereas the gal1,mig1, strain showed similar patterns to the gal1, strain. Furthermore, the gal1,hxk2, strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1,hxk2, strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae. © 2005 Wiley Periodicals, Inc. [source]


    Oligosaccharide Mimics Containing Galactose and Fucose Specifically Label Tumour Cell Surfaces and Inhibit Cell Adhesion to Fibronectin

    CHEMBIOCHEM, Issue 2 2005
    Evelyn Y.-L.
    Abstract With the aim of establishing a versatile and easy synthesis of branched saccharides for biological applications, we used molecular-dynamics simulations to model Lewisyto two classes of di- or triantennary saccharide mimetics. One set of mimetics was based on 1,3,5-tris(hydroxymethyl)cyclohexane (TMC) as the core, the other on furan, and both were derivatised with galactose and/or fucose. The TMC-based saccharides were biotinylated, while the furan disaccharides were treated with maleimide-activated biotin in a Diels,Alder fashion to yield oxazatricyclodecanes (OTDs). These were then assayed as cell-surface labels in human colon (SW480 and CaCo-2), liver (PLC), Glia (U333,CG,343) and ovary (SKOV-3) tumour cell lines. Discrete staining patterns were observed in all cells, usually at one or two poles of the cells, particularly with the asymmetric 3-,- L -fucopyranosyloxymethyl-4-,- D -galactopyranosyloxymethyl-OTD. Normal SV40-transformed fibroblasts (SV80) showed no staining. Adhesion of the highly metastatic mouse melanoma line B16,F10 to fibronectin was inhibited by 80,% by the TMC-digalactoside and by 30,% by 3,4-bis-(,- D -galactopyranosyloxymethyl)furan. None of the saccharide mimetics inhibited the adhesion of the less metastatic B16,F1 line. Migration of B16,F10 cells through MatrigelTMwas greatly inhibited by the TMC-digalactoside and weakly inhibited by the TMC-trigalactoside. The saccharide mimetics that had shown the best structural agreements with the terminal saccharides of Lewisyin the molecular dynamics simulation were also the most biologically potent compounds; this underlines the predictive nature of molecular dynamics simulations. The use of the non-saccharide cores enabled us to adapt spacer lengths and terminal saccharides to optimise the structures to bind more avidly to cell-surface lectins. [source]


    FRET-Based Direct and Continuous Monitoring of Human Fucosyltransferases Activity: An Efficient synthesis of Versatile GDP- L -Fucose Derivatives from Abundant d- Galactose,

    CHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2008
    Takahiro Maeda
    Abstract We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6-deoxy-6- N -(2-naphalene-2-yl-acetamide)-,- L -galactopyranos-1-yl-guanosine 5,-diphosphate disodium salt (1), was efficiently synthesized from naturally abundant D -galactopyranose via a key intermediate, 6-azide-1,2,3,4-tetra- O -benzoyl-6-deoxy-,- L -galactopyranose (10). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl-,2,3-LacNAc derivative (2) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis,X (SLex), which is catalyzed by human ,-1,3-fucosyltransferase,VI (FUT-VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate (KM=0.94,,M and Vmax=0.14,,M,min,1) for detecting human FUT-VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT-VI among all of the known GDP-Fuc analogues, including the parent GDP-Fuc. When a dansylated asparagine-linked glycopeptide 20, which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET-based assay with compound 1 could be used to directly monitor the ,1,6-fucosylation at the reducing terminal GlcNAc residue by human FUT-VIII (KM=175,,M and Vmax=0.06,,M/,min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23, which was obtained by a 1,3-dipolar cycloaddition between the disodium salt 16 and 1-ethynyl-naphthalene exhibits highly potent inhibitory effects against the FUT-VI-mediated sialyl Lewis,X synthesis (IC50=5.4,,M). [source]


    A Practical Route to Synthesize 3,4- O -Isopropylidene-2-azidophytosphingosine from D -Galactose

    CHINESE JOURNAL OF CHEMISTRY, Issue 8 2007
    Ning Ding
    Abstract An efficient and practical route to synthesize (2S,3S,4R)-2-azido-3,4- O -isopropyllidene-1,3,4-octadecanetriol from D -galactose in 18% overall yield was described, which required ten steps of reactions and only four times column chromatography purification. [source]


    Tagatose, a new antidiabetic and obesity control drug

    DIABETES OBESITY & METABOLISM, Issue 2 2008
    Y. Lu
    A potentially important new drug for treating type 2 diabetes, tagatose, is now in phase 3 clinical trial. The history, development, additional health benefits, mechanisms of action and the potential for the drug are presented in context with a review of the rapidly growing epidemic of type 2 diabetes and treatments for it. An epimer of fructose, the natural hexose tagatose was originally developed by Spherix Incorporated (formerly Biospherics Inc.) as a low-calorie sugar substitute. Only 20% of orally ingested tagatose is fully metabolized, principally in the liver, following a metabolic pathway identical to that of fructose. Following a decade of studies, tagatose became generally recognized as safe for use in foods and beverages under US FDA regulation. The simple sugar is commercially produced by isomerization of galactose, which is prepared from lactose. Early human studies suggested tagatose as a potential antidiabetic drug through its beneficial effects on postprandial hyperglycaemia and hyperinsulinaemia. A subsequent 14-month trial confirmed its potential for treating type 2 diabetes, and tagatose showed promise for inducing weight loss and raising high-density lipoprotein cholesterol, both important to the control of diabetes and constituting benefits independent of the disease. Furthermore, tagatose was shown to be an antioxidant and a prebiotic, both properties cited in the maintenance and promotion of health. No current therapies for type 2 diabetes provide these multiple health benefits. The predominant side effects of tagatose are gastrointestinal disturbances associated with excessive consumption, generally accommodated within 1- to 2-week period. The health and use potentials for tagatose (branded Naturlose® for this use) are given with respect to current type 2 diabetes drugs and markets. Under an FDA-affirmed protocol, Spherix is currently conducting a phase 3 trial to evaluate a placebo-subtracted treatment effect based on a decrease in HbA1c levels. Side effects, contraindications and possibly beneficial new findings will be carefully monitored. It is hoped that early results of the trial may become available by mid-2008. If a subsequent NDA is successful, tagatose may fill a major health need. [source]


    Engineered Pyranose 2-Oxidase: Efficiently Turning Sugars into Electrical Energy

    ELECTROANALYSIS, Issue 7-8 2010
    Oliver Spadiut
    Abstract Due to the recent interest in enzymatic biofuel cells (BFCs), sugar oxidizing enzymes other than the commonly used glucose oxidase are gaining more importance as possible bioelements of implantable microscale-devices, which can, for example, be used in biosensors and pacemakers. In this study we used rational and semi-rational protein design to improve the catalytic activity of the enzyme pyranose 2-oxidase (P2Ox) with its alternative soluble electron acceptors 1,4-benzoquinone and ferricenium ion, which can serve as electron mediators, to possibly boost the power output of enzymatic BFCs. Using a screening assay based on 96-well plates, we identified the variant H450G, which showed lower KM and higher kcat values for both 1,4-benzoquinone and ferricenium ion compared to the wild-type enzyme, when either D -glucose or D -galactose were used as saturating electron donors. Besides this variant, we analyzed the variants V546C and T169G/V546C for their possible application in enzymatic BFCs. The results obtained in homogeneous solution were compared with those obtained when P2Ox was immobilized on the surface of graphite electrodes and either "wired" to an osmium redox polymer or using soluble 1,4-benzoquinone as mediator. According to the spectrophotometrically determined kinetic constants, the possible energy output, measured in flow injection analysis experiments with these variants, increased up to 4-fold compared to systems employing the wild-type enzyme. [source]


    A Lactulose Bienzyme Biosensor Based on Self-Assembled Monolayer Modified Electrodes

    ELECTROANALYSIS, Issue 17 2004
    Susana Campuzano
    Abstract A bienzyme biosensor in which the enzymes ,-galactosidase (,-Gal), fructose dehydrogenase (FDH), and the mediator tetrathiafulvalene (TTF) were coimmobilized by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer on a gold disk electrode, is reported. The working conditions selected were Eapp=+0.10,V and (25±1),°C. The useful lifetime of one single TTF-,-Gal-FDH-MPA-AuE was surprisingly long, 81,days. A linear calibration plot was obtained for lactulose over the 3.0×10,5,1.0×10,3,mol L,1 concentration range, with a limit of detection of 9.6×10,6,mol L,1. The effect of potential interferents (lactose, glucose, galactose, sucrose, and ascorbic acid) on the biosensor response was evaluated. The behavior of the SAM-based biosensor in flow-injection systems in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining lactulose in a pharmaceutical preparation containing a high lactulose concentration, and in different types of milk. Finally, the analytical characteristics of the TTF-,-Gal-FDH-MPA-AuE are critically compared with those reported for other recent enzymatic determinations of lactulose. [source]


    Determination of neutral carbohydrates by CZE with direct UV detection

    ELECTROPHORESIS, Issue 17 2007
    Stella Rovio
    Abstract A new CZE method relying on in-capillary reaction and direct UV detection at the wavelength 270,nm is presented for the simultaneous separation of the neutral carbohydrates xylitol, D -(,)-mannitol, sucrose, D -(+)-fucose, D -(+)-cellobiose, D -(+)-galactose, D -(+)-glucose, L -rhamnose, D -(+)-mannose, D -(,)-arabinose, D -(+)-xylose, and D -(,)-ribose. The alkaline electrolyte solution was prepared of 130,mM sodium hydroxide and 36,mM disodium hydrogen phosphate dihydrate. Separation of the sample mixture was achieved within 35,min. Calibration plots were linear in the range of 0.05,3,mM. Reproducibility of migration times was between 0.3 and 1.1%, and the detection limits for the analytes were 0.02 and 0.05,mM. The optimized method was applied for the determination of neutral monosaccharides in lemon, pineapple, and orange juices and a cognac sample. The methodology is fast since no other sample preparation except dilution is required. [source]


    M cells and associated lymphoid tissue of the equine nasopharyngeal tonsil

    EQUINE VETERINARY JOURNAL, Issue 3 2001
    P. KUMAR
    Summary The aim of this study was to characterise the morphological and histochemical features of equine nasopharyngeal tonsillar tissue. Nasal and oropharyngeal tonsillar tissue has been described as the gatekeeper to mucosal immunity because of its strategic location at the entrance to the respiratory and alimentary tracts. A combination of light, scanning and transmission electron microscopy has revealed the presence of follicle-associated epithelium (FAE) overlying lymphoid tissue of the equine nasopharyngeal tonsil caudal to the pharyngeal opening of the guttural pouch. Membranous microvillus (M) cells were identified in the FAE on the basis of short microvilli, an intimate association with lymphocytes, cytoplasmic vimentin filaments and epitopes on the apical surface reactive with lectin GS I-B4 specific for ,-linked galactose. CD4-positive lymphocytes were scattered throughout the lamina propria mucosae as well as forming dense aggregates in the subepithelial part. The central follicular area was heavily populated with B lymphocytes and the dome and parafollicular areas contained both CD4- and CD8-positive lymphocytes. CD8-positive lymphocytes were also present in the epithelium and, together with B lymphocytes, in small numbers in the lamina propria mucosae. These observations indicate that the nasopharyngeal tonsil is potentially an important mucosal immune induction site in the horse and an appropriate target forintranasally administered vaccines. [source]


    Synthesis of Novel gluco - and galacto -Functionalized Platinum Complexes,

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 28 2009
    Janina Möker
    Abstract Cisplatin, carboplatin, oxaliplatin and further derivatives are worldwide established cytostatics for the treatment of a vast range of tumours. These drugs showed extraordinary success; however, side effects and primary or developed secondary resistance of tumour cells represent severe problems, which prompt the development of novel functionalized platinum complexes. Selectively protected monohydroxy derivatives of glucose and galactose could be etherified by ,-halo ethers. Further, Finkelstein reaction and malonate synthesis gave precursor glycoconjugates which were easily transformed into their (diammine)platinum complexes. First tests with different tumour cell lines show biological activity of the gluco -functionalized platinum complex.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source]


    Regioselective C-6 Hydrolysis of Methyl O -Benzoyl-pyranosides Catalysed by Candida Rugosa Lipase

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 10 2009
    Aslan Esmurziev
    Abstract Hydrolysis of six methyl O -benzoyl-pyranosides has been investigated using Candida rugosa lipase in dioxane/buffer mixtures. The lipase catalysed the hydrolysis of all substrates in a regiospecific manner at C-6. The rate of reaction was dependent on pyranoside structure, reaction temperature and scale, dioxane concentration and agitation speed. Starting from their C-6 O -benzoyl precursors, the methyl 2,3,4-tri- O -benzoyl-pyranosides of ,- D -galactose, ,- D -galactose, ,- D -glucose, and methyl 2,3-di- O -benzoyl-,- D -galactopyranoside could be isolated in 85,96,% yield. In hydrolysis of methyl 2,3,4,6-tetra- O -benzoyl-,- D -glucopyranoside and methyl 2,3,4,6-tetra- O -benzoyl-,- D -galactopyranoside substrate inhibition were observed, which in part could be overcome by increasing the reaction volume. Methyl 2,3,4,6-tetra- O -benzoyl-,- D -glucopyranoside and methyl 2,3,4,6-tetra- O -benzoyl-,- D -mannopyranoside were poor substrates for Candida rugosa lipase and low degree of conversion towards products were obtained under all conditions. No acyl migration was detected in any of the products.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source]


    Synthesis of Hybrids of D -Glucose and D -Galactose with Pyrrolidine-Based Iminosugars as Glycosidase Inhibitors

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 34 2008
    Venkata Ramana Doddi
    Abstract Sugar,iminosugar hybrid molecules made up of D -glucose and D -galactose with pyrrolidine-based iminosugars, viz. 1,4-dideoxy-1,4-imino- L -xylitol and 1,4-dideoxy-1,4-imino- L -lyxitol, are synthesized from glycal epoxides and found to be moderate glycosidase inhibitors. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source]


    Glycosylations Directed by the Armed-Disarmed Effect with Acceptors Containing a Single Ester Group

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 23 2007
    Thomas H. Schmidt
    Abstract A selective glycosylation reaction controlled by the armed-disarmed effect is described by the use of phenyl thioglycosides. The donor thioglycoside is fully protected with benzyl ethers while the acceptor thioglycoside contains benzyl ethers at position 2 and 3 and a strongly electron-withdrawing pentafluorobenzoate ester group at position 6. The coupling can be performed with galactose, glucose, mannose, and phthalimide-protected glucosamine to afford the corresponding 1,4-linked disaccharides in good yield. These disaccharides can act as glycosyl donors for an additional coupling reaction in the same pot if another acceptor and more promoter are added. In this way, two consecutiveglycosylations can be achieved to afford trisaccharides in one operation. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source]


    Screening of Garlic Water Extract for Binding Activity with Cholera Toxin B Pentamer by NMR Spectroscopy , An Old Remedy Giving a New Surprise

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 9 2006
    Matteo Politi
    Abstract Binding between a component of the crude hot water extract obtained from Allium sativum crushed bulbs (ASw) and cholera toxin B pentamer (CTB) was detected by STD NMR experiments. Bioassay-oriented fractionation allowed the partial identification of a high molecular weight polysaccharide mainly composed of galactose as the bioactive complex against CTB. This work represents the first example of screening of a medicinal plant by NMR against a specific disease, and corroborates traditional medical uses of the species. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source]


    Synthesis and Comparative Glycosidase Inhibitory Properties of Reducing Castanospermine Analogues

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 14 2005
    Paula Díaz Pérez
    Abstract The feasibility of the intramolecular nucleophilic addition of the nitrogen atom in cyclic (thio)carbamates with a pseudo- C -nucleoside structure to the masked carbonyl group in aldose precursors in the synthesis of reducing (i.e., 5-hydroxy)6-oxaindolizidine frameworks is illustrated by the preparation of the 6- epi, 7- epi, 8- epi and 6,8a-di- epi diastereomers of the potent glycosidase inhibitor (+)-castanospermine. In all cases, the increased anomeric effect caused by the high sp2 character of the pseudoamide-type nitrogen atom resulted in the pseudoanomeric hydroxy group being anchored in an axial orientation in aqueous solution, as in the aglycons in ,-glycosides. These analogs of the natural alkaloid showed a higher selectivity in the inhibition of ,-glucosidases. Structure/glycosidase inhibitory activity studies indicated that inversion of any hydroxy group resulted in a dramatic decrease in the inhibition potency, confirming the critical importance of a correct hydroxylation profile. In the case of (+)-8- epi -6-oxacastanospermine derivatives, with a hydroxylation profile with a structural complementarity to that of D -galactose, a moderate but very selective inhibition of ,-galactosidase was observed, supporting the importance of a defined configuration at pseudoanomeric centres for anomeric specificity. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source]


    The turnover of carbohydrate carbon in a cultivated soil estimated by 13C natural abundances

    EUROPEAN JOURNAL OF SOIL SCIENCE, Issue 4 2006
    D. Derrien
    Summary Understanding the chemical composition of soil organic matter (SOM) requires the determination of the dynamics of each class of compounds. We measured the dynamics of carbon in neutral carbohydrates by use of natural 13C labelling in an experimental wheat and maize sequence extending over 23 years. The isotopic composition of individual neutral monosaccharides was determined in hydrolysed particle-size fractions by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) of trimethylsilyl (TMS) derivatives. The sensitivity in terms of 13C/12C ratios ranged between 1 and 2, depending on the monosaccharide. The age distribution of neutral sugar carbon was very similar to that of total soil carbon. Particulate organic matter (POM) was characterized by the predominance of glucose and xylose of vegetal origin. In POM >,200 µm, the mean age of sugar-C (5 years) was slightly less than that of total carbon (7 years). Xylose was younger than glucose. The fine fraction 0,50 µm contained mainly glucose, arabinose, galactose, xylose, fucose and mannose, which had predominantly microbial origins. The mean age of carbohydrate carbon in the fraction 0,50 µm was between 60 and 100 years and was similar to that of total organic carbon (OC). No difference in the age of carbon between the individual monosaccharides was found. The POM fraction 50,200 µm had an intermediate signature and turnover. Considering the typical lability of carbohydrates, the relatively great age of carbohydrate carbon may be explained by physical or chemical protection from degradation, as well as by recycling of soil organic matter carbon by soil microbes. [source]


    Aspergillus nidulans,-galactosidase of glycoside hydrolase family 36 catalyses the formation of ,-galacto-oligosaccharides by transglycosylation

    FEBS JOURNAL, Issue 17 2010
    Hiroyuki Nakai
    The ,-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic ,-galactosidases and ,-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L,1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with ,-(1,6) regioselectivity from 40 mm 4-nitrophenol ,- d -galactopyranoside, melibiose or raffinose, resulting in a 37,74% yield of 4-nitrophenol ,- d -Galp -(1,6)- d -Galp, ,- d -Galp -(1,6)-,- d -Galp -(1,6)- d -Glcp and ,- d -Galp -(1,6)-,- d -Galp -(1,6)- d -Glcp -(,1,,2)- d -Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol ,- d -galactopyranoside (40 mm), ,-(1,6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39,58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l -arabinose, l -fucose and l -rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l -rhamnose. Structural modelling using Thermotoga maritima GH36 ,-galactosidase as the template and superimposition of melibiose from the complex with human GH27 ,-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred ,-galactosyl to 6-OH of the terminal residue in the ,-linked melibiose, maltose, trehalose, sucrose and turanose in 6,46% yield and the ,-linked lactose, lactulose and cellobiose in 28,38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. ,- d -Galp -(1,6)- d -Manp; ,- d -Galp -(1,6)-,- d -Glcp -(1,4)- d -Glcp; ,- d -Galp -(1,6)-,- d -Galp -(1,4)- d -Fruf; ,- d -Galp -(1,6)- d -Glcp -(,1,,1)- d -Glcp; and ,- d -Galp -(1,6)-,- d -Glcp -(1,3)- d -Fruf. [source]


    Experimental and steady-state analysis of the GAL regulatory system in Kluyveromyces lactis

    FEBS JOURNAL, Issue 14 2010
    Venkat R. Pannala
    The galactose uptake mechanism in yeast is a well-studied regulatory network. The regulatory players in the galactose regulatory mechanism (GAL system) are conserved in Saccharomyces cerevisiae and Kluyveromyces lactis, but the molecular mechanisms that occur as a result of the molecular interactions between them are different. The key differences in the GAL system of K. lactis relative to that of S. cerevisiae are: (a) the autoregulation of KlGAL4; (b) the dual role of KlGal1p as a metabolizing enzyme as well as a galactose-sensing protein; (c) the shuttling of KlGal1p between nucleus and cytoplasm; and (d) the nuclear confinement of KlGal80p. A steady-state model was used to elucidate the roles of these molecular mechanisms in the transcriptional response of the GAL system. The steady-state results were validated experimentally using measurements of ,-galactosidase to represent the expression for genes having two binding sites. The results showed that the autoregulation of the synthesis of activator KlGal4p is responsible for the leaky expression of GAL genes, even at high glucose concentrations. Furthermore, GAL gene expression in K. lactis shows low expression levels because of the limiting function of the bifunctional protein KlGal1p towards the induction process in order to cope with the need for the metabolism of lactose/galactose. The steady-state model of the GAL system of K. lactis provides an opportunity to compare with the design prevailing in S. cerevisiae. The comparison indicates that the existence of a protein, Gal3p, dedicated to the sensing of galactose in S. cerevisiae as a result of genome duplication has resulted in a system which metabolizes galactose efficiently. [source]


    Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma

    FEBS JOURNAL, Issue 3 2007
    Magdalena Kujawa
    We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose,methanol,choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9- S -cysteinyl, 8-,- N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are d -glucose, d -galactose, l -arabinose, and d -xylose. As shown by in situ NMR analysis, d -glucose and d -galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (kcat/Km). The enzyme may play a role in lignocellulose degradation. [source]


    The metabolic role and evolution of l -arabinitol 4-dehydrogenase of Hypocrea jecorina

    FEBS JOURNAL, Issue 10 2004
    Manuela Pail
    l -Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina (anamorph: Trichoderma reesei) has been implicated in the catabolism of l -arabinose, and genetic evidence also shows that it is involved in the catabolism of d -xylose in xylitol dehydrogenase (xdh1) mutants and of d -galactose in galactokinase (gal1) mutants of H. jecorina. In order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity. The resulting enzyme preparation catalyzed the oxidation of pentitols (l -arabinitol) and hexitols (d -allitol, d -sorbitol, l -iditol, l -mannitol) to the same corresponding ketoses as mammalian sorbitol dehydrogenase (SDH), albeit with different catalytic efficacies, showing highest kcat/Km for l -arabinitol. However, it oxidized galactitol and d -talitol at C4 exclusively, yielding l -xylo-3-hexulose and d -arabino-3-hexulose, respectively. Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs. Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for d -sorbitol. A lad1 gene disruptant was almost unable to grow on l -arabinose, grew extremely weakly on l -arabinitol, d -talitol and galactitol, showed reduced growth on d -sorbitol and d -galactose and a slightly reduced growth on d -glucose. The weak growth on l -arabinitol was completely eliminated in a mutant in which the xdh1 gene had also been disrupted. These data show not only that Lad1 is indeed essential for the catabolism of l -arabinose, but also that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolism in fungi. [source]


    Functional analysis of disease-causing mutations in human galactokinase

    FEBS JOURNAL, Issue 8 2003
    David J. Timson
    Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme. One mutant (A198V) had kinetic properties that were essentially wild-type. These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia. [source]


    Transgalactosylation by thermostable ,-glycosidases from Pyrococcus furiosus and Sulfolobus solfataricus

    FEBS JOURNAL, Issue 16 2000
    -glycosides during lactose conversion, Binding interactions of nucleophiles with the galactosylated enzyme intermediate make major contributions to the formation of new
    The hyperthermostable ,-glycosidases from the Archaea Sulfolobus solfataricus (Ss,Gly) and Pyrococcus furiosus (CelB) hydrolyse ,-glycosides of d -glucose or d -galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how specificity is manifested under conditions of kinetically controlled transgalactosylation, the major transfer products formed during the hydrolysis of lactose by these enzymes have been identified, and their appearance and degradation have been determined in dependence of the degree of substrate conversion. CelB and Ss,Gly show a marked preference for making new ,(1,3) and ,(1,6) glycosidic bonds by intermolecular as well as intramolecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic-bond cleavage and release of glucose, is about 2.2 times that of Ss,Gly and yields ,- d -Galp- (1,6)- d -Glc and ,- d -Galp- (1,3)- d -Glc in a molar ratio of ,,1 : 2. The partitioning of galactosylated Ss,Gly between reaction with sugars [kNu (m,1·s,1)] and reaction with water [kwater (s,1)] is about twice that of CelB. It gives a mixture of linear ,- d -glycosides, chiefly trisaccharides at early reaction times, in which the prevailing new glycosidic bonds are ,(1,6) and ,(1,3) for the reactions catalysed by Ss,Gly and CelB, respectively. The accumulation of ,- d -Galp- (1,6)- d -Glc at the end of lactose hydrolysis reflects a 3,10-fold specificity of both enzymes for the hydrolysis of ,(1,3) over ,(1,6) linked glucosides. Galactosyl transfer from Ss,Gly or CelB to d -glucose occurs with partitioning ratios, kNu/kwater, which are seven and >,170 times those for the reactions of the galactosylated enzymes with 1-propanol and 2-propanol, respectively. Therefore, the binding interactions with nucleophiles contribute chiefly to formation of new ,-glycosides during lactose conversion. Likewise, noncovalent interactions with the glucose leaving group govern the catalytic efficiencies for the hydrolysis of lactose by both enzymes. They are almost fully expressed in the rate-limiting first-order rate constant for the galactosyl transfer from the substrate to the enzyme and lead to a positive deviation by ,,2.5 log10 units from structure,reactivity correlations based on the pKa of the leaving group. [source]


    Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase

    FEBS JOURNAL, Issue 24 2000
    Juan C. Díaz Arribas
    Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism-based glycosidase inhibitors. When LPH is treated with 2,,4,-dinitrophenyl 2-deoxy-2-fluoro-,- d -glucopyranoside (1) and 2,,4,-dinitrophenyl-2-deoxy-2-fluoro-,- d -galactopyranoside (2), known mechanism-based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (d -glucal and d -galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett.435, 225,228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem.267, 18744,18752]. [source]


    Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain

    FEBS JOURNAL, Issue 24 2000
    A unique teichoic acid-like polysaccharide, the group O antigen which is a C-polysaccharide in common with pneumococci
    The cell wall of Streptococcus mitis biovar 1 strain SK137 contains the C-polysaccharide known as the common antigen of a closely related species Streptococcus pneumoniae, and a teichoic acid-like polysaccharide with a unique structure. The two polysaccharides are different entities and could be partially separated by gel chromatography. The structures of the two polysaccharides were determined by chemical methods and by NMR spectroscopy. The teichoic acid-like polymer has a heptasaccharide phosphate repeating unit with the following structure: The structure neither contains ribitol nor glycerol phosphate as classical teichoic acids do, thus we have used the expression teichoic acid-like for this polysaccharide. The following structure of the C-polysaccharide repeating unit was established: where AAT is 2-acetamido-4-amino-2,4,6-trideoxy- d -galactose. It has a carbohydrate backbone identical to that of one of the two structures of C-polysaccharide previously identified in S. pneumoniae. C-polysaccharide of S. mitis is characterized by the presence, in each repeating unit, of two residues of phosphocholine and both galactosamine residues in the N-acetylated form. Immunochemical analysis showed that C-polysaccharide constitutes the Lancefield group O antigen. Studies using mAbs directed against the backbone and against the phosphocholine moiety of the C-polysaccharide revealed several different patterns of these epitopes among 95 S. mitis and Streptococcus oralis strains tested and the exclusive presence of the group O antigen in the majority of S. mitis biovar 1 strains. [source]


    Cosmopolitan distribution of phlD -containing dicotyledonous crop-associated biocontrol pseudomonads of worldwide origin

    FEMS MICROBIOLOGY ECOLOGY, Issue 2 2001
    Chunxia Wang
    Abstract In biocontrol fluorescent pseudomonads, phlD encodes a polyketide synthase required for the synthesis of the antifungal compound 2,4-diacetylphloroglucinol (Phl). Here, PCR-restriction fragment length polymorphism analysis was used to compare phlD alleles in 77 dicot-associated pseudomonads originating from various countries worldwide and 10 counterparts from a monocotyledonous host (wheat). The 16 restriction patterns obtained were mostly unrelated to geographic location or dicot host. Cluster analysis distinguished eight phlD clusters at a similarity level of 0.63. One cluster grouped 18 pseudomonads that produced also the antifungal polyketide pyoluteorin but could not assimilate D -galactose, D -galactonate lactone, D -sorbitol, L -arabinose, D -saccharate or D -xylose. These 18 pseudomonads, along with the eight pseudomonads from a second phlD cluster, were the only isolates that failed to deaminase 1-aminocyclopropane-1-carboxylate (ACC), a rare root growth promotion trait. Overall, assessment of phlD polymorphism, ACC deaminase activity and catabolic profiles pointed to a cosmopolitan distribution of Phl-producing biocontrol fluorescent pseudomonads of worldwide origin associated with dicotyledonous crop plants. [source]


    Anticancerogenic effect of a novel chiroinositol-containing polysaccharide from Bifidobacterium bifidum BGN4

    FEMS MICROBIOLOGY LETTERS, Issue 2 2004
    Hyun Ju You
    Abstract Strains of bifidobacteria have many health-promotion effects. Whole cells or cytoplasm extracts of Bifidobacterium bifidum BGN4, isolated from human feces, inhibited the growth of several cancer cell lines. The polysaccharide fraction (BB-pol) extracted from B. bifidum BGN4 had a novel composition, comprising chiroinositol, rhamnose, glucose, galactose, and ribose. Three human colon cancer cell lines were treated with BB-pol: HT-29, HCT-116, and Caco-2. Trypan blue exclusion assay and BrdU incorporation assay showed that BB-pol inhibited the growth of HT-29 and HCT-116 cells but did not inhibit the growth of Caco-2 cells. [source]


    Inhibition of Escherichia coli heat-labile enterotoxin by neoglycoprotein and anti-lectin antibodies which mimic GM1 receptor

    FEMS MICROBIOLOGY LETTERS, Issue 1 2002
    Caroline A Menezes
    Abstract Escherichia coli producing heat-labile enterotoxin is responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality. The B subunits of this toxin are responsible for the binding to the receptor, the complex ganglioside GM1 which has galactose as its terminal sugar. In this study we showed that analogs of galactose (gal) and N -acetylgalactosamine (GalNAc) interfere with the binding of heat-labile toxin to GM1. Antibodies to lectins which mimic sugar structures and neoglycoprotein were employed. These compounds were able to inhibit heat-labile toxin activity efficiently in Vero cells: 37 ,g of IgG-enriched fraction from an antiserum inhibited up to 70% of this activity, and 50% of the binding of heat-labile toxin to GM1. Neoglycoprotein was more efficient than antibodies, since 2.5 ,g of this ligand completely abolished the activity of heat-labile toxin on Vero cells. These data suggest that these molecules could be developed for prophylaxis and diagnosis of diarrhea caused by heat-labile toxin. [source]