Structure and Mechanism of ArnA: Conformational Change Implies Ordered Dehydrogenase Mechanism in Key Enzyme for Polymyxin Resistance

PubMed Central

Gatzeva-Topalova, Petia Z.; May, Andrew P.; Sousa, Marcelo C.

2010-01-01

Summary The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors. PMID:15939024

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis.

PubMed

Kärkönen, Anna; Fry, Stephen C

2006-03-01

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K (m) (for UDP-glucose) 0.5-1.0 mM; there was also a minor activity with an unusually high K (m) of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K (m) values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E(L), E(M) and E(H) respectively). E(M) was the single major contributor to extractable UDPGDH activity when assayed at 0.6-9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E(L)-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E(H) activity is not due to ADH. At 30 microM UDP-glucose, 20-150 microM UDP-xylose inhibited UDPGDH activity, whereas 5-15 microM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.

Discovery and Biochemical Characterization of the UDP-Xylose Biosynthesis Pathway in Sphaerobacter thermophilus.

PubMed

Gu, Bin; Laborda, Pedro; Wei, Shuang; Duan, Xu-Chu; Song, Hui-Bo; Liu, Li; Voglmeir, Josef

2016-01-01

The biosynthesis of UDP-xylose requires the stepwise oxidation/ decarboxylation of UDP-glucose, which is catalyzed by the enzymes UDPglucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS). UDPxylose biosynthesis is ubiquitous in animals and plants. However, only a few UGD and UXS isoforms of bacterial origin have thus far been biochemically characterized. Sphaerobacter thermophilus DSM 20745 is a bacterium isolated from heated sewage sludge, and therefore can be a valuable source of thermostable enzymes of biotechnological interest. However, no biochemical characterizations of any S. thermophilus enzymes have yet been reported. Herein, we describe the cloning and characterization of putative UGD (StUGD) and UXS (StUXS) isoforms from this organism. HPLC- and plate reader-based activity tests of the recombinantly expressed StUGD and StUXS showed that they are indeed active enzymes. Both StUGD and StUXS showed a temperature optimum of 70°C, and a reasonable thermal stability up to 60°C. No metal ions were required for enzymatic activities. StUGD had a higher pH optimum than StUXS. The simple purification procedures and the thermotolerance of StUGD and StUXS make them valuable biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose at elevated temperatures. The biosynthetic potential of StUGD was further exemplified in a coupled enzymatic reaction with an UDP-glucuronosyltransferase, allowing the glucuronylation of the natural model substrate bilirubin.

Advanced Processing for Biomedical Informatics (APBI)

DTIC Science & Technology

2009-10-01

phosphatidic acid phosphatase type 2 domain containing 1A PPAPDC1A 1 96051 2.71E-06 4.39E-05 4.55 8.14 12.1 205030_at fatty acid binding protein 7...W81XWH‐06‐2‐0072    Principal Investigator: Craig D. Shriver, COL MC    54    209355_s_at phosphatidic acid phosphatase type 2B PPAP2B 8613 1.62E-06...Investigator: Craig D. Shriver, COL MC    24    209711_at solute carrier family 35 (UDP- glucuronic acid /UDP-N- acetylgalactosamine dual transporter), member

Synthesis of UDP-apiose in Bacteria: The marine phototroph Geminicoccus roseus and the plant pathogen Xanthomonas pisi.

PubMed

Smith, James Amor; Bar-Peled, Maor

2017-01-01

The branched-chain sugar apiose was widely assumed to be synthesized only by plant species. In plants, apiose-containing polysaccharides are found in vascularized plant cell walls as the pectic polymers rhamnogalacturonan II and apiogalacturonan. Apiosylated secondary metabolites are also common in many plant species including ancestral avascular bryophytes and green algae. Apiosyl-residues have not been documented in bacteria. In a screen for new bacterial glycan structures, we detected small amounts of apiose in methanolic extracts of the aerobic phototroph Geminicoccus roseus and the pathogenic soil-dwelling bacteria Xanthomonas pisi. Apiose was also present in the cell pellet of X. pisi. Examination of these bacterial genomes uncovered genes with relatively low protein homology to plant UDP-apiose/UDP-xylose synthase (UAS). Phylogenetic analysis revealed that these bacterial UAS-like homologs belong in a clade distinct to UAS and separated from other nucleotide sugar biosynthetic enzymes. Recombinant expression of three bacterial UAS-like proteins demonstrates that they actively convert UDP-glucuronic acid to UDP-apiose and UDP-xylose. Both UDP-apiose and UDP-xylose were detectable in cell cultures of G. roseus and X. pisi. We could not, however, definitively identify the apiosides made by these bacteria, but the detection of apiosides coupled with the in vivo transcription of bUAS and production of UDP-apiose clearly demonstrate that these microbes have evolved the ability to incorporate apiose into glycans during their lifecycles. While this is the first report to describe enzymes for the formation of activated apiose in bacteria, the advantage of synthesizing apiose-containing glycans in bacteria remains unknown. The characteristics of bUAS and its products are discussed.

The UDP-glucuronosyltransferases of the blood-brain barrier: their role in drug metabolism and detoxication

PubMed Central

Ouzzine, Mohamed; Gulberti, Sandrine; Ramalanjaona, Nick; Magdalou, Jacques; Fournel-Gigleux, Sylvie

2014-01-01

UDP-glucuronosyltransferases (UGTs) form a multigenic family of membrane-bound enzymes expressed in various tissues, including brain. They catalyze the formation of β-D-glucuronides from structurally unrelated substances (drugs, other xenobiotics, as well as endogenous compounds) by the linkage of glucuronic acid from the high energy donor, UDP-α-D-glucuronic acid. In brain, UGTs actively participate to the overall protection of the tissue against the intrusion of potentially harmful lipophilic substances that are metabolized as hydrophilic glucuronides. These metabolites are generally inactive, except for important pharmacologically glucuronides such as morphine-6-glucuronide. UGTs are mainly expressed in endothelial cells and astrocytes of the blood brain barrier (BBB). They are also associated to brain interfaces devoid of BBB, such as circumventricular organ, pineal gland, pituitary gland and neuro-olfactory tissues. Beside their key-role as a detoxication barrier, UGTs play a role in the steady-state of endogenous compounds, like steroids or dopamine (DA) that participate to the function of the brain. UGT isoforms of family 1A, 2A, 2B and 3A are expressed in brain tissues to various levels and are known to present distinct but overlapping substrate specificity. The importance of these enzyme species with regard to the formation of toxic, pharmacologically or physiologically relevant glucuronides in the brain will be discussed. PMID:25389387

Role of UDP-N-Acetylglucosamine (GlcNAc) and O-GlcNAcylation of Hyaluronan Synthase 2 in the Control of Chondroitin Sulfate and Hyaluronan Synthesis*

PubMed Central

Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Karousou, Eugenia; Viola, Manuela; Bartolini, Barbara; Hascall, Vincent C.; Tammi, Markku; De Luca, Giancarlo; Passi, Alberto

2012-01-01

Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1–3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t½ >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies. PMID:22887999

Vitamin C. Biosynthesis, recycling and degradation in mammals.

PubMed

Linster, Carole L; Van Schaftingen, Emile

2007-01-01

Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate.

The UDP-glycosyltransferase (UGT) superfamily expressed in humans, insects and plants: Animal-plant arms-race and co-evolution.

PubMed

Bock, Karl Walter

2016-01-01

UDP-glycosyltransferases (UGTs) are major phase II enzymes of a detoxification system evolved in all kingdoms of life. Lipophilic endobiotics such as hormones and xenobiotics including phytoalexins and drugs are conjugated by vertebrates mainly with glucuronic acid, by invertebrates and plants mainly with glucose. Plant-herbivore arms-race has been the major driving force for evolution of large UGT and other enzyme superfamilies. The UGT superfamily is defined by a common protein structure and signature sequence of 44 amino acids responsible for binding the UDP moiety of the sugar donor. Plants developed toxic phytoalexins stored as glucosides. Upon herbivore attack these conjugates are converted to highly reactive compounds. In turn, animals developed large families of UGTs in their intestine and liver to detoxify these phytoalexins. Interestingly, phytoalexins, exemplified by quercetin glucuronides and glucosinolate-derived isocyanates, are known insect attractant pigments in plants, and antioxidants, anti-inflammatory and chemopreventive compounds of humans. It is to be anticipated that phytochemicals may provide a rich source in beneficial drugs. Copyright © 2015. Published by Elsevier Inc.

Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae.

PubMed

Ikushiro, Shinichi; Nishikawa, Miyu; Masuyama, Yuuka; Shouji, Tadashi; Fujii, Miharu; Hamada, Masahiro; Nakajima, Noriyuki; Finel, Moshe; Yasuda, Kaori; Kamakura, Masaki; Sakaki, Toshiyuki

2016-07-05

Glucuronidation is one of the most common pathways in mammals for detoxification and elimination of hydrophobic xenobiotic compounds, including many drugs. Metabolites, however, can form active or toxic compounds, such as acyl glucuronides, and their safety assessment is often needed. The absence of efficient means for in vitro synthesis of correct glucuronide metabolites frequently limits such toxicological analyses. To overcome this hurdle we have developed a new approach, the essence of which is a coexpression system containing a human, or another mammalian UDP-glucuronosyltransferases (UGTs), as well as UDP-glucose-6-dehydrogenase (UGDH), within the budding yeast, Saccharomyces cerevisiae. The system was first tested using resting yeast cells coexpressing UGDH and human UGT1A6, 7-hydroxycoumarin as the substrate, in a reaction medium containing 8% glucose, serving as a source of UDP-glucuronic acid. Glucuronides were readily formed and recovered from the medium. Subsequently, by selecting suitable mammalian UGT enzyme for the coexpression system we could obtain the desired glucuronides of various compounds, including molecules with multiple conjugation sites and acyl glucuronides of several carboxylic acid containing drugs, namely, mefenamic acid, flufenamic acid, and zomepirac. In conclusion, a new and flexible yeast system with mammalian UGTs has been developed that exhibits a capacity for efficient production of various glucuronides, including acyl glucuronides.

Genome-Wide Identification and Expression Analysis of the UGlcAE Gene Family in Tomato.

PubMed

Ding, Xing; Li, Jinhua; Pan, Yu; Zhang, Yue; Ni, Lei; Wang, Yaling; Zhang, Xingguo

2018-05-27

The UGlcAE has the capability of interconverting UDP-d-galacturonic acid and UDP-d-glucuronic acid, and UDP-d-galacturonic acid is an activated precursor for the synthesis of pectins in plants. In this study, we identified nine UGlcAE protein-encoding genes in tomato. The nine UGlcAE genes that were distributed on eight chromosomes in tomato, and the corresponding proteins contained one or two trans-membrane domains. The phylogenetic analysis showed that SlUGlcAE genes could be divided into seven groups, designated UGlcAE1 to UGlcAE6 , of which the UGlcAE2 were classified into two groups. Expression profile analysis revealed that the SlUGlcAE genes display diverse expression patterns in various tomato tissues. Selective pressure analysis indicated that all of the amino acid sites of SlUGlcAE proteins are undergoing purifying selection. Fifteen stress-, hormone-, and development-related elements were identified in the upstream regions (0.5 kb) of these SlUGlcAE genes. Furthermore, we investigated the expression patterns of SlUGlcAE genes in response to three hormones (indole-3-acetic acid (IAA), gibberellin (GA), and salicylic acid (SA)). We detected firmness, pectin contents, and expression levels of UGlcAE family genes during the development of tomato fruit. Here, we systematically summarize the general characteristics of the SlUGlcAE genes in tomato, which could provide a basis for further function studies of tomato UGlcAE genes.

Trichothecenes Mycotoxin Studies

DTIC Science & Technology

1986-02-01

15-monoacetoxyscirpenol with C-UDP-glucuronic acid (data not shown). We were unable to hydrolyze this metabolite by incu- bation with either bovine ...then, this metabolite appears to be non- toxic. This coupled with our findings that this glucuronide is not hydrolyzed by E. coli or bovine liver a...leakage or violation of the skin patches by two of the animals toward the end of these early experiments. Because of their small size and higher level of

Carbohydrate metabolism changes in Prunus persica gummosis infected with Lasiodiplodia theobromae.

PubMed

Li, Z; Gao, L; Wang, Y T; Zhu, W; Ye, J L; Li, G H

2014-05-01

Peach gummosis represents a significant global disease of stone fruit trees and a major disease in the south peach production area of the Yangtze River of China. In this study, the carbohydrate composition of peach shoots during infection by Lasiodiplodia theobromae was examined. The expression of genes related to metabolic enzymes was also investigated. Control wounded and noninoculated tissue, lesion tissue, and wounded and inoculated surrounding lesion tissue of peach shoots were analyzed. Soluble sugars, glucose, mannose, arabinose, and xylose significantly increased in inoculated tissues of peach shoots compared with control tissues at different times after inoculation. Accumulation of polysaccharides was also observed by section observation and periodic acid Schiff's reagent staining during infection. Analysis using quantitative reverse-transcription polymerase chain reaction revealed that the abundance of key transcripts on the synthesis pathway of uridine diphosphate (UDP)-D-glucuronate, UDP-D-galactose, and UDP-D-arabinose increased but the synthesis of L-galactose and guanosine diphosphate-L-galactose were inhibited. After inoculation, the transcript levels of sugar transport-related genes (namely, SUT, SOT, GMT, and UGT) was induced. These changes in sugar content and gene expression were directly associated with peach gum polysaccharide formation and may be responsible for the symptoms of peach gummosis.

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

PubMed Central

Bethke, Gerit; Thao, Amanda; Xiong, Guangyan; Hatsugai, Noriyuki; Katagiri, Fumiaki; Pauly, Markus

2016-01-01

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-d-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-d-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions. PMID:26813622

Biosynthetic elongation of isolated teichuronic acid polymers via glucosyl- and N-acetylmannosaminuronosyltransferases from solubilized cytoplasmic membrane fragments of Micrococcus luteus.

PubMed Central

Hildebrandt, K M; Anderson, J S

1990-01-01

Cytoplasmic membrane fragments of Micrococcus luteus catalyze in vitro biosynthesis of teichuronic acid from uridine diphosphate D-glucose (UDP-glucose), uridine diphosphate N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), and uridine diphosphate N-acetyl-D-glucosamine. Membrane fragments solubilized with Thesit (dodecyl alcohol polyoxyethylene ether) can utilize UDP-glucose and UDP-ManNAcA to effect elongation of teichuronic acid isolated from native cell walls. When UDP-glucose is the only substrate supplied, the detergent-solubilized glucosyltransferase incorporates a single glucosyl residue onto each teichuronic acid acceptor. When both UDP-glucose and UDP-ManNAcA are supplied, the glucosyltransferase and the N-acetylmannosaminuronosyltransferase act cooperatively to elongate the teichuronic acid acceptor by multiple additions of the disaccharide repeat unit. As shown by polyacrylamide gel electrophoresis, low-molecular-weight fractions of teichuronic acid are converted to higher-molecular-weight polymers by the addition of as many as 17 disaccharide repeat units. Images PMID:2118507

Synthesis of aryl azide derivatives of UDP-GlcNAc and UDP-GalNAc and their use for the affinity labeling of glycosyltransferases and the UDP-HexNAc pyrophosphorylase.

PubMed

Zeng, Y; Shabalin, Y; Szumilo, T; Pastuszak, I; Drake, R R; Elbein, A D

1996-07-15

The chemical synthesis and utilization of two photoaffinity analogs, 125I-labeled 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc and -UDP-GalNAc, is described. Starting with either UDP-GlcNAc or UDP-GalNAc, the synthesis involved the preparation of the 5-mercuri-UDP-HexNAc and then attachment of an allylamine to the 5 position to give 5-(3-amino)allyl-UDP-HexNAc. This was followed by acylation with N-hydroxysuccinimide p-aminosalicylic acid to form the final product, i.e., 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc or UDP-GalNAc. These products could then be iodinated with chloramine T to give the 125I-derivatives. Both the UDP-GlcNAc and the UDP-GalNAc derivatives reacted in a concentration-dependent manner with a highly purified UDP-HexNAc pyrophosphorylase, and both specifically labeled the subunit(s) of this protein. The labeling of the protein by the UDP-GlcNAc derivative was inhibited in dose-dependent fashion by either unlabeled UDP-GlcNAc or unlabeled UDP-GalNAc. Likewise, labeling with the UDP-GalNAc probe was blocked by either UDP-GlcNAc or UDP-GalNAc. The UDP-GlcNAc probe also specifically labeled a partially purified preparation of GlcNAc transferase I.

Measurement of glucuronidation by isolated rat liver cells using [14C]fructose.

PubMed

Dawson, J; Knowles, R G; Pogson, C I

1992-03-03

We have developed a simple and sensitive method for the study of the relative rates of glucuronidation of compounds, in isolated liver cells, based on the incorporation of 14C from fructose into glucuronide conjugates. Liver cells from fasted rats are used to minimize any reduction of the specific activity by glycogenolysis. Although rates of glucuronidation are lower in isolated liver cells from fasted rats than in those from fed rats, because of a reduction in the concentration of UDP-glucuronic acid, it is possible to compare the rates of glucuronidation of different compounds. Radiolabelled glucuronides are separated from [14C]fructose and [14C]glucose, produced by the liver cells, by normal-phase HPLC on a polar amino-cyano column. The specific activity of the glucuronide was found to be approximately 50% of that of the [14C]fructose. Absolute amounts of glucuronide can be determined by measuring the specific activity of the [14C]glucose, also produced by liver cells from fructose, which reflects that of the glucose-6-phosphate and hence the UDP-glucuronic acid used for glucuronidation, although for the measurement of relative rates this would not be necessary. We have used this method to examine the kinetics of the glucuronidation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The method should be applicable to the study of the rates of glucuronidation of a range of aglycones and, unlike other methods, does not require glucuronide standards or radiolabelled aglycone.

Inhibition of UDP-glucose dehydrogenase by 6-thiopurine and its oxidative metabolites: Possible mechanism for its interaction within the bilirubin excretion pathway and 6TP associated liver toxicity.

PubMed

Weeramange, Chamitha J; Binns, Cassie M; Chen, Chixiang; Rafferty, Ryan J

2018-03-20

6-Thiopurine (6TP) is an actively prescribed drug in the treatment of various diseases ranging from Crohn's disease and other inflammatory diseases to acute lymphocytic leukemia and non-Hodgkin's leukemia. While 6TP has beneficial therapeutic uses, severe toxicities are also reported with its use, such as jaundice and liver toxicity. While numerous investigations into the mode in which toxicity originates has been undertaken. None have investigated the effects of inhibition towards UDP-Glucose Dehydrogenase (UDPGDH), an oxidative enzyme responsible for UDP-glucuronic acid (UDPGA) formation or UDP-Glucuronosyl transferase (UGT1A1), which is responsible for the conjugation of bilirubin with UDPGA for excretion. Failure to excrete bilirubin leads to jaundice and liver toxicity. We proposed that either 6TP or its primary oxidative excretion metabolites inhibit one or both of these enzymes, resulting in the observed toxicity from 6TP administration. Inhibition analysis of these purines revealed that 6-thiopurine has weak to no inhibition towards UDPGDH with a K i of 288 μM with regard to varying UDP-glucose, but 6-thiouric (primary end metabolite, fully oxidized at carbon 2 and 8, and highly retained by the body) has a near six-fold increased inhibition towards UDPGDH with a K i of 7 μM. Inhibition was also observed by 6-thioxanthine (oxidized at carbon 2) and 8-OH-6TP with K i values of 54 and 14 μM, respectively. Neither 6-thiopurine or its excretion metabolites were shown to inhibit UGT1A1. Our results show that the C2 and C8 positions of 6TP are pivotal in said inhibition towards UDPGDH and have no effect upon UGT1A1, and that blocking C8 could lead to new analogs with reduced, if not eliminated jaundice and liver toxicities. Copyright © 2017 Elsevier B.V. All rights reserved.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

PubMed

Benini, Stefano; Toccafondi, Mirco; Rejzek, Martin; Musiani, Francesco; Wagstaff, Ben A; Wuerges, Jochen; Cianci, Michele; Field, Robert A

2017-11-01

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystal structure of product-bound complex of UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pampa, K.J., E-mail: sagarikakj@gmail.com; Lokanath, N.K.; Girish, T.U.

Highlights: • Determined the structure of UDP-D-ManNAcADH to a resolution of 1.55 Å. • First complex structure of PhUDP-D-ManNAcADH with UDP-D-ManMAcA. • The monomeric structure consists of three distinct domains. • Cys258 acting as catalytic nucleophilic and Lys204 acts as acid/base catalyst. • Oligomeric state plays an important role for the catalytic function. - Abstract: UDP-N-acetyl-D-mannosamine dehydrogenase (UDP-D-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-D-mannosamine (UDP-D-ManNAc) to Uridine-diphospho-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA) through twofold oxidation of NAD{sup +}. In order to reveal the structural features of the Pyrococcus horikoshii UDP-D-ManNAcADH, we have determined the crystal structure of the product-bound enzyme bymore » X-ray diffraction to resolution of 1.55 Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-D-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.« less

N-glycosylation in Archaea: on the coordinated actions of Haloferax volcanii AglF and AglM.

PubMed

Yurist-Doutsch, Sophie; Magidovich, Hilla; Ventura, Valeria V; Hitchen, Paul G; Dell, Anne; Eichler, Jerry

2010-02-01

Like Eukarya and Bacteria, Archaea are also capable of performing N-glycosylation. In the halophilic archaeon Haloferax volcanii, N-glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S-layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N-glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N-glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP-glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose-1-phosphate uridyltransferase, UDP-glucuronic acid was generated from glucose-1-phosphate and UTP in a NAD(+)-dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N-glycosylation process.

Biosynthesis of UDP-GlcNAc, UndPP-GlcNAc and UDP-GlcNAcA Involves Three Easily Distinguished 4-Epimerase Enzymes, Gne, Gnu and GnaB

PubMed Central

Cunneen, Monica M.; Liu, Bin; Wang, Lei; Reeves, Peter R.

2013-01-01

We have undertaken an extensive survey of a group of epimerases originally named Gne, that were thought to be responsible for inter-conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc). The analysis builds on recent work clarifying the specificity of some of these epimerases. We find three well defined clades responsible for inter-conversion of the gluco- and galacto-configuration at C4 of different N-acetylhexosamines. Their major biological roles are the formation of UDP-GalNAc, UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) and undecaprenyl pyrophosphate-N-acetylgalactosamine (UndPP-GalNAc) from the corresponding glucose forms. We propose that the clade of UDP-GlcNAcA epimerase genes be named gnaB and the clade of UndPP-GlcNAc epimerase genes be named gnu, while the UDP-GlcNAc epimerase genes retain the name gne. The Gne epimerases, as now defined after exclusion of those to be named GnaB or Gnu, are in the same clade as the GalE 4-epimerases for inter-conversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). This work brings clarity to an area that had become quite confusing. The identification of distinct enzymes for epimerisation of UDP-GlcNAc, UDP-GlcNAcA and UndPP-GlcNAc will greatly facilitate allocation of gene function in polysaccharide gene clusters, including those found in bacterial genome sequences. A table of the accession numbers for the 295 proteins used in the analysis is provided to enable the major tree to be regenerated with the inclusion of additional proteins of interest. This and other suggestions for annotation of 4-epimerase genes will facilitate annotation. PMID:23799153

Biotransformation of Flavokawains A, B, and C, Chalcones from Kava (Piper methysticum), by Human Liver Microsomes.

PubMed

Zenger, Katharina; Agnolet, Sara; Schneider, Bernd; Kraus, Birgit

2015-07-22

The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4' and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2'-O-glucuronide, FKB-2'-O-glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo.

Modulation of hyaluronan synthase activity in cellular membrane fractions.

PubMed

Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

2009-10-30

Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, F.C.; Brown, R.M. Jr.; Drake, R.R. Jr.

1990-03-25

Photoaffinity labeling of purified cellulose synthase with (beta-32P)5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of (beta-32P)5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.

Molecular cloning and tissue-specific transcriptional regulation of the first peroxidase family member, Udp1, in stinging nettle (Urtica dioica).

PubMed

Douroupi, Triantafyllia G; Papassideri, Issidora S; Stravopodis, Dimitrios J; Margaritis, Lukas H

2005-12-05

A full-length cDNA clone, designated Udp1, was isolated from Urtica dioica (stinging nettle), using a polymerase chain reaction based strategy. The putative Udp1 protein is characterized by a cleavable N-terminal signal sequence, likely responsible for the rough endoplasmic reticulum entry and a 310 amino acids mature protein, containing all the important residues, which are evolutionary conserved among different members of the plant peroxidase family. A unique structural feature of the Udp1 peroxidase is defined into the short carboxyl-terminal extension, which could be associated with the vacuolar targeting process. Udp1 peroxidase is differentially regulated at the transcriptional level and is specifically expressed in the roots. Interestingly, wounding and ultraviolet radiation stress cause an ectopic induction of the Udp1 gene expression in the aerial parts of the plant. A genomic DNA fragment encoding the Udp1 peroxidase was also cloned and fully sequenced, revealing a structural organization of three exons and two introns. The phylogenetic relationships of the Udp1 protein to the Arabidopsis thaliana peroxidase family members were also examined and, in combination with the homology modelling approach, dictated the presence of distinct structural elements, which could be specifically involved in the determination of substrate recognition and subcellular localization of the Udp1 peroxidase.

In Vitro Biosynthesis and Chemical Identification of UDP-N-acetyl-d-quinovosamine (UDP-d-QuiNAc)*

PubMed Central

Li, Tiezheng; Simonds, Laurie; Kovrigin, Evgenii L.; Noel, K. Dale

2014-01-01

N-acetyl-d-quinovosamine (2-acetamido-2,6-dideoxy-d-glucose, QuiNAc) occurs in the polysaccharide structures of many Gram-negative bacteria. In the biosynthesis of QuiNAc-containing polysaccharides, UDP-QuiNAc is the hypothetical donor of the QuiNAc residue. Biosynthesis of UDP-QuiNAc has been proposed to occur by 4,6-dehydration of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) to UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose followed by reduction of this 4-keto intermediate to UDP-QuiNAc. Several specific dehydratases are known to catalyze the first proposed step. A specific reductase for the last step has not been demonstrated in vitro, but previous mutant analysis suggested that Rhizobium etli gene wreQ might encode this reductase. Therefore, this gene was cloned and expressed in Escherichia coli, and the resulting His6-tagged WreQ protein was purified. It was tested for 4-reductase activity by adding it and NAD(P)H to reaction mixtures in which 4,6-dehydratase WbpM had acted on the precursor substrate UDP-GlcNAc. Thin layer chromatography of the nucleotide sugars in the mixture at various stages of the reaction showed that WbpM converted UDP-GlcNAc completely to what was shown to be its 4-keto-6-deoxy derivative by NMR and that addition of WreQ and NADH led to formation of a third compound. Combined gas chromatography-mass spectrometry analysis of acid hydrolysates of the final reaction mixture showed that a quinovosamine moiety had been synthesized after WreQ addition. The two-step reaction progress also was monitored in real time by NMR. The final UDP-sugar product after WreQ addition was purified and determined to be UDP-d-QuiNAc by one-dimensional and two-dimensional NMR experiments. These results confirmed that WreQ has UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose 4-reductase activity, completing a pathway for UDP-d-QuiNAc synthesis in vitro. PMID:24817117

Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines

PubMed Central

Smith, Claire M.; Fry, Stephen C.; Gough, Kevin C.; Patel, Alexandra J. F.; Glenn, Sarah; Goldrick, Marie; Roberts, Ian S.; Andrew, Peter W.

2014-01-01

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections. PMID:24498433

Recent advances in the in silico modelling of UDP glucuronosyltransferase substrates.

PubMed

Sorich, Michael J; Smith, Paul A; Miners, John O; Mackenzie, Peter I; McKinnon, Ross A

2008-01-01

UDP glucurononosyltransferases (UGT) are a superfamily of enzymes that catalyse the conjugation of a range of structurally diverse drugs, environmental and endogenous chemicals with glucuronic acid. This process plays a significant role in the clearance and detoxification of many chemicals. Over the last decade the regulation and substrate profiles of UGT isoforms have been increasingly characterised. The resulting data has facilitated the prototyping of ligand based in silico models capable of predicting, and gaining insights into, binding affinity and the substrate- and regio- selectivity of glucuronidation by UGT isoforms. Pharmacophore modelling has produced particularly insightful models and quantitative structure-activity relationships based on machine learning algorithms result in accurate predictions. Simple structural chemical descriptors were found to capture much of the chemical information relevant to UGT metabolism. However, quantum chemical properties of molecules and the nucleophilic atoms in the molecule can enhance both the predictivity and chemical intuitiveness of structure-activity models. Chemical diversity analysis of known substrates has shown some bias towards chemicals with aromatic and aliphatic hydroxyl groups. Future progress in in silico development will depend on larger and more diverse high quality metabolic datasets. Furthermore, improved protein structure data on UGTs will enable the application of structural modelling techniques likely leading to greater insight into the binding and reactive processes of UGT catalysed glucuronidation.

Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.

PubMed

Liger, D; Masson, A; Blanot, D; van Heijenoort, J; Parquet, C

1996-01-01

The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan. It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-MurNAc) and L-alanine. The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc. The murC gene product appears as a 50-kDa protein accounting for ca. 50% of total cell proteins. A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene. The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol. The K(m) values for substrates UDP-N-acetylmuramic acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively. The specificity of the enzyme for its substrates was investigated with various analogues. Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) from Haemophilus influenzae

PubMed Central

Mol, Clifford D.; Brooun, Alexei; Dougan, Douglas R.; Hilgers, Mark T.; Tari, Leslie W.; Wijnands, Robert A.; Knuth, Mark W.; McRee, Duncan E.; Swanson, Ronald V.

2003-01-01

UDP-N-acetylmuramic acid:l-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg2+ and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-l-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn2+ have been determined to 1.85- and 1.7-Å resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the γ-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates. PMID:12837790

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae.

PubMed

Mol, Clifford D; Brooun, Alexei; Dougan, Douglas R; Hilgers, Mark T; Tari, Leslie W; Wijnands, Robert A; Knuth, Mark W; McRee, Duncan E; Swanson, Ronald V

2003-07-01

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.

UDP-4-Keto-6-Deoxyglucose, a Transient Antifungal Metabolite, Weakens the Fungal Cell Wall Partly by Inhibition of UDP-Galactopyranose Mutase

PubMed Central

Ma, Liang; Salas, Omar; Bowler, Kyle

2017-01-01

ABSTRACT Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a Ki of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs. PMID:29162710

Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity

PubMed Central

Hesse, Lars; Bostock, Julieanne; Dementin, Sebastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Chopra, Ian

2003-01-01

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):l-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:l-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (l-alanine, l-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide. PMID:14594822

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity.

PubMed

Hesse, Lars; Bostock, Julieanne; Dementin, Sebastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Chopra, Ian

2003-11-01

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thoden, James B.; Holden, Hazel M.

2010-09-08

2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3{prime} hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD{sup +} to NADH. This enzyme has been shown to usemore » {alpha}-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and {alpha}-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both {alpha}-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3{prime}, respectively) abutting the re face of the cofactor. They are positioned {approx}3 {angstrom} from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.« less

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis.

PubMed

Hirotani, M; Kuroda, R; Suzuki, H; Yoshikawa, T

2000-05-01

A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53,094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4'-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments.

The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

PubMed Central

Shulami, Smadar; Gat, Orit; Sonenshein, Abraham L.; Shoham, Yuval

1999-01-01

A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer. PMID:10368143

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms*

PubMed Central

Nakajima, Kazuki; Ito, Emi; Ohtsubo, Kazuaki; Shirato, Ken; Takamiya, Rina; Kitazume, Shinobu; Angata, Takashi; Taniguchi, Naoyuki

2013-01-01

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using 13C6-glucose and 13C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with 13C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a 13C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using 13C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism. PMID:23720760

Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

PubMed Central

Ramos, Ana; Boels, Ingeborg C.; de Vos, Willem M.; Santos, Helena

2001-01-01

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation. PMID:11133425

Introducing the "TCDD-inducible AhR-Nrf2 gene battery".

PubMed

Yeager, Ronnie L; Reisman, Scott A; Aleksunes, Lauren M; Klaassen, Curtis D

2009-10-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the "AhR gene battery." However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug-processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 mug/kg, ip) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug-processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid-synthesizing gene UDP-glucose dehydrogenase, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 in WT mice but not in Nrf2-null mice. Therefore, the present study demonstrates the novel finding that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice.

Structure and function of nucleotide sugar transporters: Current progress.

PubMed

Hadley, Barbara; Maggioni, Andrea; Ashikov, Angel; Day, Christopher J; Haselhorst, Thomas; Tiralongo, Joe

2014-06-01

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure-function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

The elaborate route for UDP-arabinose delivery into the Golgi of plants

DOE PAGES

Rautengarten, Carsten; Birdseye, Devon; Pattathil, Sivakumar; ...

2017-04-03

In plants, L-Arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDPAraf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transportedmore » back into the lumen. During this step it is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Therefore, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgilocalized UDP-Araf transporters in Arabidopsis. The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo.« less

The elaborate route for UDP-arabinose delivery into the Golgi of plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rautengarten, Carsten; Birdseye, Devon; Pattathil, Sivakumar

In plants, L-Arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDPAraf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transportedmore » back into the lumen. During this step it is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Therefore, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgilocalized UDP-Araf transporters in Arabidopsis. The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo.« less

Biochemistry of metalocenes. The organ distribution of hydroxyacetyl (/sup 103/Ru)ruthenocene and its glucuronide in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, A.J.; Macha, J.; Wenzel, M.

1980-01-01

Hydroxyacetyl(/sup 103/Ru)ruthenocene and its o-glucuronide were prepared in vitro by incubation of acetyl(/sup 103/Ru)ruthenocene with rat-liver homogenate, NADPH, and UDP-glucuronate. The factors affecting hydroxylation and glucuronidation in vitro were optimized for acetylruthenocene. Hydroxyacetyl(/sup 103/Ru)ruthenocene glucuronide showed no affinity for the adrenal glands, but after iv administration of hydroxyacetyl(/sup 103/Ru)ruthenocene there was a distinct accumulation of Ru-103 in adrenals, similar to that found after administration of acetyl(/sup 103/Ru)ruthenocene.

Double layer zinc-UDP coordination polymers: structure and properties.

PubMed

Qiu, Qi-Ming; Gu, Leilei; Ma, Hongwei; Yan, Li; Liu, Minghua; Li, Hui

2018-05-17

A homochiral Zn-UDP coordination polymer with an alternating parallel ABAB sequence was constructed and studied by X-ray single crystal diffraction analysis. Its crystal structure shows that there are potentially open sites in the 2D layers. The activation of the sites makes the coordination polymer a fluorescent sensor for novel heterogeneous detection of amino acids.

Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate.

PubMed

Moss, Angela K; Hamarneh, Sulaiman R; Mohamed, Mussa M Rafat; Ramasamy, Sundaram; Yammine, Halim; Patel, Palak; Kaliannan, Kanakaraju; Alam, Sayeda N; Muhammad, Nur; Moaven, Omeed; Teshager, Abeba; Malo, Nondita S; Narisawa, Sonoko; Millán, José Luis; Warren, H Shaw; Hohmann, Elizabeth; Malo, Madhu S; Hodin, Richard A

2013-03-15

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.

In vitro synthesis of intermediates involved in the assembly of enterobacterial common antigen (ECA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barr, K.; Wolski, S.; Kroto, J.

1986-05-01

ECA is a cell surface antigen found in all bacteria belonging to the family Enterobacteriaceae. The serological specificity of ECA is determined by a linear heteropolysaccharide comprised of trisaccharide repeat units; the component sugars are N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcUA), and 4-acetamido-D-fucose (Fuc4NAc). In vivo studies have suggested that GlcNAc-pyrophosphorylundecaprenol (GlcNAc-PP-lipid) is an intermediate in ECA synthesis. More recently, they have demonstrated UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase activity in cell envelope preparations of E. coli. Radioactivity from UDP-(/sup 3/H)Glc-NAc was incorporated into endogenous lipid acceptor, and the labeled product was characterized as GlcNAc-PP-lipid (lipid I). Transferase activity was inhibited by tunicamycin andmore » UMP, but it was unaffected by UDP. The reaction was reversible, and the synthesis of UDP-(/sup 3/H)GlcNAc from UMP and (/sup 3/H)GlcNAc-PP-lipid was also sensitive to tunicamycin. The simultaneous addition of UDP-(/sup 14/C)ManNAcUA and UDP-(/sup 3/H)GlcNAc to cell envelope preparations resulted in the synthesis of a more polar lipid (lipid II) that contained both labeled sugars in equimolar amounts. Synthesis of lipid II was dependent on prior synthesis of lipid I. Accordingly, (/sup 3/H)GlcNAc-PP-lipid that had been synthesized in vivo served as an acceptor in vitro of ManNAcUA residues from UDP-ManNAcUA. Lipid II has been tentatively identified as ManNAcUA-GlcNAc-pyrophosphorylundecaprenol.« less

Effect of the Ratio of Non-fibrous Carbohydrates to Neutral Detergent Fiber and Protein Structure on Intake, Digestibility, Rumen Fermentation, and Nitrogen Metabolism in Lambs

PubMed Central

Ma, T.; Tu, Y.; Zhang, N. F.; Deng, K. D.; Diao, Q. Y.

2015-01-01

This study aimed to investigate the effect of the ratio of non-fibrous carbohydrates to neutral detergent fibre (NFC/NDF) and undegraded dietary protein (UDP) on rumen fermentation and nitrogen metabolism in lambs. Four Dorper×thin-tailed Han crossbred lambs, averaging 62.3±1.9 kg of body weight and 10 mo of age, were randomly assigned to four dietary treatments of combinations of two levels of NFC/NDF (1.0 and 1.7) and two levels of UDP (35% and 50% of crude protein [CP]). Duodenal nutrient flows were measured with dual markers of Yb and Co, and microbial N (MN) synthesis was estimated using 15N. High UDP decreased organic matter (OM) intake (p = 0.002) and CP intake (p = 0.005). Ruminal pH (p<0.001), ammonia nitrogen (NH3-N; p = 0.008), and total volatile fatty acids (p<0.001) were affected by dietary NFC/NDF. The ruminal concentration of NH3-N was also affected by UDP (p<0.001). The duodenal flow of total MN (p = 0.007) was greater for lambs fed the high NFC/NDF diet. The amount of metabolisable N increased with increasing dietary NFC:NDF (p = 0.02) or UDP (p = 0.04). In conclusion, the diets with high NFC/NDF (1.7) and UDP (50% of CP) improved metabolisable N supply to lambs. PMID:26323398

Effect of the Ratio of Non-fibrous Carbohydrates to Neutral Detergent Fiber and Protein Structure on Intake, Digestibility, Rumen Fermentation, and Nitrogen Metabolism in Lambs.

PubMed

Ma, T; Tu, Y; Zhang, N F; Deng, K D; Diao, Q Y

2015-10-01

This study aimed to investigate the effect of the ratio of non-fibrous carbohydrates to neutral detergent fibre (NFC/NDF) and undegraded dietary protein (UDP) on rumen fermentation and nitrogen metabolism in lambs. Four Dorper×thin-tailed Han crossbred lambs, averaging 62.3±1.9 kg of body weight and 10 mo of age, were randomly assigned to four dietary treatments of combinations of two levels of NFC/NDF (1.0 and 1.7) and two levels of UDP (35% and 50% of crude protein [CP]). Duodenal nutrient flows were measured with dual markers of Yb and Co, and microbial N (MN) synthesis was estimated using (15)N. High UDP decreased organic matter (OM) intake (p = 0.002) and CP intake (p = 0.005). Ruminal pH (p<0.001), ammonia nitrogen (NH3-N; p = 0.008), and total volatile fatty acids (p<0.001) were affected by dietary NFC/NDF. The ruminal concentration of NH3-N was also affected by UDP (p<0.001). The duodenal flow of total MN (p = 0.007) was greater for lambs fed the high NFC/NDF diet. The amount of metabolisable N increased with increasing dietary NFC:NDF (p = 0.02) or UDP (p = 0.04). In conclusion, the diets with high NFC/NDF (1.7) and UDP (50% of CP) improved metabolisable N supply to lambs.

Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate

PubMed Central

Hamarneh, Sulaiman R.; Mohamed, Mussa M. Rafat; Ramasamy, Sundaram; Yammine, Halim; Patel, Palak; Kaliannan, Kanakaraju; Alam, Sayeda N.; Muhammad, Nur; Moaven, Omeed; Teshager, Abeba; Malo, Nondita S.; Narisawa, Sonoko; Millán, José Luis; Warren, H. Shaw; Hohmann, Elizabeth; Malo, Madhu S.; Hodin, Richard A.

2013-01-01

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP. PMID:23306083

Coding and transmission of subband coded images on the Internet

NASA Astrophysics Data System (ADS)

Wah, Benjamin W.; Su, Xiao

2001-09-01

Subband-coded images can be transmitted in the Internet using either the TCP or the UDP protocol. Delivery by TCP gives superior decoding quality but with very long delays when the network is unreliable, whereas delivery by UDP has negligible delays but with degraded quality when packets are lost. Although images are delivered currently over the Internet by TCP, we study in this paper the use of UDP to deliver multi-description reconstruction-based subband-coded images. First, in order to facilitate recovery from UDP packet losses, we propose a joint sender-receiver approach for designing optimized reconstruction-based subband transform (ORB-ST) in multi-description coding (MDC). Second, we carefully evaluate the delay-quality trade-offs between the TCP delivery of SDC images and the UDP and combined TCP/UDP delivery of MDC images. Experimental results show that our proposed ORB-ST performs well in real Internet tests, and UDP and combined TCP/UDP delivery of MDC images provide a range of attractive alternatives to TCP delivery.

Leishmania UDP-sugar pyrophosphorylase: the missing link in galactose salvage?

PubMed

Damerow, Sebastian; Lamerz, Anne-Christin; Haselhorst, Thomas; Führing, Jana; Zarnovican, Patricia; von Itzstein, Mark; Routier, Françoise H

2010-01-08

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

Identification of a Direct Biosynthetic Pathway for UDP-N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

PubMed

Dadashipour, Mohammad; Iwamoto, Mariko; Hossain, Mohammad Murad; Akutsu, Jun-Ichi; Zhang, Zilian; Kawarabayasi, Yutaka

2018-05-15

Most organisms, from Bacteria to Eukarya , synthesize UDP- N -acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP- N -acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea , the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii , predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii , and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways. IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially. Copyright © 2018 American Society for Microbiology.

Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC.

PubMed

Nakajima, Kazuki; Kitazume, Shinobu; Angata, Takashi; Fujinawa, Reiko; Ohtsubo, Kazuaki; Miyoshi, Eiji; Taniguchi, Naoyuki

2010-07-01

Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases.

PubMed

Decker, Daniel; Kleczkowski, Leszek A

2017-01-01

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP- N -acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P ( K m values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P ( K m of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P ( K m of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P ( K m of 1 mM) and, to some extent, D-Glc-1-P ( K m of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

PubMed Central

Decker, Daniel; Kleczkowski, Leszek A.

2017-01-01

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products. PMID:28970843

Genetic alteration of UDP-rhamnose metabolism in Botrytis cinerea leads to the accumulation of UDP-KDG that adversely affects development and pathogenicity.

PubMed

Ma, Liang; Salas, Omar; Bowler, Kyle; Oren-Young, Liat; Bar-Peled, Maor; Sharon, Amir

2017-02-01

Botrytis cinerea is a model plant-pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)-glucose, the major fungal wall nucleotide sugar precursor, to UDP-rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose-containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP-rhamnose pathway intermediate UDP-4-keto-6-deoxy-glucose (UDP-KDG) in hyphae of the Δbcer strain. UDP-KDG could not be detected in hyphae of the wild-type strain, indicating fast conversion to UDP-rhamnose by the BcEr enzyme. The correlation between high UDP-KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP-KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP-KDG may lead to the development of new antifungal drugs. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Attachment of UDP-hexosamines to the ribosomes isolated from rat liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kopacz-Jodczyk, T.; Paszkiewicz-Gadek, A.; Galasinski, W.

1988-06-01

The binding of UDP-N-acetylhexosamines with purified ribosomes was studied and it was found that the radioactive nucleotides can be attached to these particles. The radioactivity of the purified ribosomal pellet depends on the amounts of ribosomes and UDP-N-acetylhexosamines. Some characteristics of the binding system indicate that the attachment of UDP-sugar to ribosome does not require the participation of glycosyltransferases. The results of the competition experiment would suggest that there are specific sites on ribosomes for the binding of UDP-N-acetylglucosamine.

The attachment of UDP-hexosamines to the ribosomes isolated from rat liver.

PubMed

Kopacz-Jodczyk, T; Paszkiewicz-Gadek, A; Gałasiński, W

1988-06-01

The binding of UDP-N-acetylhexosamines with purified ribosomes was studied and it was found that the radioactive nucleotides can be attached to these particles. The radioactivity of the purified ribosomal pellet depends on the amounts of ribosomes and UDP-N-acetylhexosamines. Some characteristics of the binding system indicate that the attachment of UDP-sugar to ribosome does not require the participation of glycosyltransferases. The results of the competition experiment would suggest that there are specific sites on ribosomes for the binding of UDP-N-acetylglucosamine.

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis, glycogen storage, and detoxification

PubMed Central

Iverson, Sonya V.; Eriksson, Sofi; Xu, Jianqiang; Prigge, Justin R.; Talago, Emily A.; Meade, Tesia A.; Meade, Erin S.; Capecchi, Mario R.; Arnér, Elias S.J.; Schmidt, Edward E.

2013-01-01

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug-metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however their constitutive metabolic state supported more robust GSH biosynthesis-, glutathionylation-, and glucuronidation-systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage. PMID:23743293

Some pharmacological properties of uridine nucleotides

PubMed Central

Smith, M. W.

1964-01-01

Uridine di-, tri- and monophosphates (UDP, UTP and UMP) contracted the goldfish intestine preparation in that order of decreasing potency. Adenosine triphosphate (ATP) sensitized the gut to UTP and UDP but not to UMP. The fluoro-derivatives of UMP and UTP behaved like the unsubstituted nucleotides on the goldfish intestine but the main effect of 6-azaUDP and large amounts of uracil and uridine was to cause a relaxation. Structure-action relationships are discussed on the basis of these findings. UDPglucose and UDPacetylglucosamine each contracted the goldfish intestine but they were 500-times less active than UDP. Other smooth muscle preparations (tortoise jejunum, rat uterus, guinea-pig ileum and the fowl rectal caecum) contracted to UTP and UDP, but large amounts were needed. The cardiovascular effects in rats of UMP, UDP and UTP were complex and mediated mainly through an action on the peripheral blood vessels. In rats treated with phenoxybenzamine, UMP raised the blood pressure while UDP and UTP first lowered then raised the blood pressure. The fall in blood pressure was not abolished by pronethalol or atropine. The uridine phosphates affected the rat isolated heart only under hypoxic conditions. UTP and UDP dilated the blood vessels of the rabbit ear and UTP was six-times more effective than ATP. UTP and UDP were equiactive in increasing the force of beat of the frog isolated heart. UMP also had an effect if large amounts were given. PMID:14190461

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases.

PubMed

Soya, Naoto; Shoemaker, Glen K; Palcic, Monica M; Klassen, John S

2009-11-01

The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

Gene expression patterns and catalytic properties of UDP-D-glucose 4-epimerases from barley (Hordeum vulgare L.).

PubMed

Zhang, Qisen; Hrmova, Maria; Shirley, Neil J; Lahnstein, Jelle; Fincher, Geoffrey B

2006-02-15

UGE (UDP-Glc 4-epimerase or UDP-Gal 4-epimerase; EC 5.1.3.2) catalyses the interconversion of UDP-Gal and UDP-Glc. Both nucleotide sugars act as activated sugar donors for the biosynthesis of cell wall polysaccharides such as cellulose, xyloglucans, (1,3;1,4)-beta-D-glucan and pectins, together with other biologically significant compounds including glycoproteins and glycolipids. Three members of the HvUGE (barley UGE) gene family, designated HvUGE1, HvUGE2 and HvUGE3, have been characterized. Q-PCR (quantitative real-time PCR) showed that HvUGE1 mRNA was most abundant in leaf tips and mature roots, but its expression levels were relatively low in basal leaves and root tips. The HvUGE2 gene was transcribed at significant levels in all organs examined, while HvUGE3 mRNA levels were very low in all the organs. Heterologous expression of a near full-length cDNA confirmed that HvUGE1 encodes a functional UGE. A non-covalently bound NAD+ was released from the enzyme after denaturing with aqueous ethanol and was identified by its spectrophotometric properties and by electrospray ionization MS. The K(m) values were 40 microM for UDP-Gal and 55 muM for UDP-Glc. HvUGE also catalyses the interconversion of UDP-GalNAc and UDP-GlcNAc, although it is not known if this has any biological significance. A three-dimensional model of the HvUGE revealed that its overall structural fold is highly conserved compared with the human UGE and provides a structural rationale for its ability to bind UDP-GlcNAc.

Nucleotide and Nucleotide Sugar Analysis by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry on Surface-Conditioned Porous Graphitic Carbon

PubMed Central

2010-01-01

We examined the analysis of nucleotides and nucleotide sugars by chromatography on porous graphitic carbon with mass spectrometric detection, a method that evades contamination of the MS instrument with ion pairing reagent. At first, adenosine triphosphate (ATP) and other triphosphate nucleotides exhibited very poor chromatographic behavior on new columns and could hardly be eluted from columns previously cleaned with trifluoroacetic acid. Satisfactory performance of both new and older columns could, however, be achieved by treatment with reducing agent and, unexpectedly, hydrochloric acid. Over 40 nucleotides could be detected in cell extracts including many isobaric compounds such as ATP, deoxyguanosine diphosphate (dGTP), and phospho-adenosine-5′-phosphosulfate or 3′,5′-cyclic adenosine 5'-monophosphate (AMP) and its much more abundant isomer 2′,3′-cylic AMP. A fast sample preparation procedure based on solid-phase extraction on carbon allowed detection of very short-lived analytes such as cytidine 5'-monophosphate (CMP)-2-keto-deoxy-octulosonic acid. In animal cells and plant tissues, about 35 nucleotide sugars were detected, among them rarely considered metabolites such as uridine 5'-diphosphate (UDP)-l-arabinopyranose, UDP-l-arabinofuranose, guanosine 5'-diphosphate (GDP)-l-galactofuranose, UDP-l-rhamnose, and adenosine diphosphate (ADP)-sugars. Surprisingly, UDP-arabinopyranose was also found in Chinese hamster ovary (CHO) cells. Due to the unique structural selectivity of graphitic carbon, the method described herein distinguishes more nucleotides and nucleotide sugars than previously reported approaches. PMID:21043458

Effects of inter-packet spacing on the delivery of multimedia content

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kapadia, A. C.; Feng, A. C.; Feng, W. C.

2001-01-01

Streaming multimedia content with UDP has become increasingly popular over distributed systems such as the Internet. However, because UDP does not possess any congestion-control mechanism and most best-effort trafic is served by the congestion-controlled TCP, UDP flows steal bandwidth from TCP to the point that TCP flows can starve for network resources. Furthermore, such applications may cause the Internet infrastructure to eventually suffer from congestion collapse because UDP trafic does not self-regulate itself. To address this problem, next-generation Internet routers will implement active queue-management schemes to punish malicious traffic, e.g., non-adaptive UDP flows, and to the improve the performance ofmore » congestion-controlled traffic, e.g., TCP flows. The arrival of such routers will cripple the performance of today's UDP-based multimedia applications. So, in this paper, we introduce the notion of inter-packet spacing with control feedback to enable these UDP-based applications to perform well in the next-generation Internet while being adaptive and self-regulating. When compared with traditional UDP-based multimedia streaming, we illustrate that our counterintuitive, interpacket-spacing scheme with control feedback can reduce packet loss by 90% without adversely affecting delivered throughput. Keywords: network protocol, multimedia, packet spacing, rate-adjusting congestion control.« less

Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple.

PubMed

Jugdé, Hélène; Nguy, Danny; Moller, Isabel; Cooney, Janine M; Atkinson, Ross G

2008-08-01

The dihydrochalcone phlorizin (phloretin 2'-glucoside) contributes to the flavor, color and health benefits of apple fruit and processed products. A genomics approach was used to identify the gene MdPGT1 in apple (Malus x domestica) with homology to the UDP-glycosyltransferase 88 family of uridine diphosphate glycosyltransferases that show specificity towards flavonoid substrates. Expressed sequence tags for MdPGT1 were found in all tissues known to produce phlorizin including leaf, flower and fruit. However, the highest expression was measured by quantitative PCR in apple root tissue. The recombinant MdPGT1 enzyme expressed in Escherichia coli glycosylated phloretin in the presence of [(3)H]-UDP-glucose, but not other apple antioxidants, including quercetin, naringenin and cyanidin. The product of phloretin and UDP-glucose co-migrated with an authentic phlorizin standard. LC/MS indicated that MdPGT1 could glycosylate phloretin in the presence of three sugar donors: UDP-glucose, UDP-xylose and UDP-galactose. This is the first report of functional characterization of a UDP-glycosyltransferase that utilizes a dihydrochalcone as its primary substrate.

Motor Learning Enhances Use-Dependent Plasticity

PubMed Central

2017-01-01

Motor behaviors are shaped not only by current sensory signals but also by the history of recent experiences. For instance, repeated movements toward a particular target bias the subsequent movements toward that target direction. This process, called use-dependent plasticity (UDP), is considered a basic and goal-independent way of forming motor memories. Most studies consider movement history as the critical component that leads to UDP (Classen et al., 1998; Verstynen and Sabes, 2011). However, the effects of learning (i.e., improved performance) on UDP during movement repetition have not been investigated. Here, we used transcranial magnetic stimulation in two experiments to assess plasticity changes occurring in the primary motor cortex after individuals repeated reinforced and nonreinforced actions. The first experiment assessed whether learning a skill task modulates UDP. We found that a group that successfully learned the skill task showed greater UDP than a group that did not accumulate learning, but made comparable repeated actions. The second experiment aimed to understand the role of reinforcement learning in UDP while controlling for reward magnitude and action kinematics. We found that providing subjects with a binary reward without visual feedback of the cursor led to increased UDP effects. Subjects in the group that received comparable reward not associated with their actions maintained the previously induced UDP. Our findings illustrate how reinforcing consistent actions strengthens use-dependent memories and provide insight into operant mechanisms that modulate plastic changes in the motor cortex. SIGNIFICANCE STATEMENT Performing consistent motor actions induces use-dependent plastic changes in the motor cortex. This plasticity reflects one of the basic forms of human motor learning. Past studies assumed that this form of learning is exclusively affected by repetition of actions. However, here we showed that success-based reinforcement signals could affect the human use-dependent plasticity (UDP) process. Our results indicate that learning augments and interacts with UDP. This effect is important to the understanding of the interplay between the different forms of motor learning and suggests that reinforcement is not only important to learning new behaviors, but can shape our subsequent behavior via its interaction with UDP. PMID:28143961

Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC.

PubMed

Kochanowski, N; Blanchard, F; Cacan, R; Chirat, F; Guedon, E; Marc, A; Goergen, J-L

2006-01-15

Analysis of intracellular nucleotide and nucleotide sugar contents is essential in studying protein glycosylation of mammalian cells. Nucleotides and nucleotide sugars are the donor substrates of glycosyltransferases, and nucleotides are involved in cellular energy metabolism and its regulation. A sensitive and reproducible ion-pair reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed, allowing the direct and simultaneous detection and quantification of some essential nucleotides and nucleotide sugars. After a perchloric acid extraction, 13 molecules (8 nucleotides and 5 nucleotide sugars) were separated, including activated sugars such as UDP-glucose, UDP-galactose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine. To validate the analytical parameters, the reproducibility, linearity of calibration curves, detection limits, and recovery were evaluated for standard mixtures and cell extracts. The developed method is capable of resolving picomolar quantities of nucleotides and nucleotide sugars in a single chromatographic run. The HPLC method was then applied to quantify intracellular levels of nucleotides and nucleotide sugars of Chinese hamster ovary (CHO) cells cultivated in a bioreactor batch process. Evolutions of the titers of nucleotides and nucleotide sugars during the batch process are discussed.

Cytosolic invertase contributes to the supply of substrate for cellulose biosynthesis in developing wood.

PubMed

Rende, Umut; Wang, Wei; Gandla, Madhavi Latha; Jönsson, Leif J; Niittylä, Totte

2017-04-01

Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5'-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined. We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula × tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis. Suppression of CIN activity by 38-55% led to a 9-13% reduction in crystalline cellulose. The changes in cellulose were reflected in reduced diameter of acid-insoluble cellulose microfibrils and increased glucose release from wood upon enzymatic digestion of cellulose. Reduced CIN activity decreased the amount of the cellulose biosynthesis precursor UDP-glucose in developing wood, pointing to the likely cause of the cellulose phenotype. The findings suggest that CIN activity has an important role in the cellulose biosynthesis of trees, and indicate that cellulose biosynthesis in wood relies on a quantifiable UDP-glucose pool. The results also introduce a concept of altering cellulose microfibril properties by modifying substrate supply to cellulose biosynthesis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Design and synthesis of novel N-benzylidenesulfonohydrazide inhibitors of MurC and MurD as potential antibacterial agents.

PubMed

Frlan, Rok; Kovac, Andreja; Blanot, Didier; Gobec, Stanislav; Pecar, Slavko; Obreza, Ales

2008-01-11

A series of novel N-benzylidenesulfonohydrazide compounds were designed and synthesized as inhibitors of UDP-N-acetylmuramic acid: L-alanine ligase (MurC) and UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (MurD) from E. coli, involved in the biosynthesis of bacterial cell-walls. Some compounds possessed inhibitory activity against both enzymes with IC(50) values as low as 30 microM. In addition, a new, one-pot synthesis of amidobenzaldehydes is reported.

Identification of UDP glucosyltransferases from the aluminum-resistant tree Eucalyptus camaldulensis forming β-glucogallin, the precursor of hydrolyzable tannins.

PubMed

Tahara, Ko; Nishiguchi, Mitsuru; Frolov, Andrej; Mittasch, Juliane; Milkowski, Carsten

2018-08-01

In the highly aluminum-resistant tree Eucalyptus camaldulensis, hydrolyzable tannins are proposed to play a role in internal detoxification of aluminum, which is a major factor inhibiting plant growth on acid soils. To understand and modulate the molecular mechanisms of aluminum detoxification by hydrolyzable tannins, the biosynthetic genes need to be identified. In this study, we identified and characterized genes encoding UDP-glucose:gallate glucosyltransferase, which catalyzes the formation of 1-O-galloyl-β-d-glucose (β-glucogallin), the precursor of hydrolyzable tannins. By homology-based cloning, seven full-length candidate cDNAs were isolated from E. camaldulensis and expressed in Escherichia coli as recombinant N-terminal His-tagged proteins. Phylogenetic analysis classified four of these as UDP glycosyltransferase (UGT) 84A subfamily proteins (UGT84A25a, -b, UGT84A26a, -b) and the other three as UGT84J subfamily proteins (UGT84J3, -4, -5). In vitro enzyme assays showed that the UGT84A proteins catalyzed esterification of UDP-glucose and gallic acid to form 1-O-galloyl-β-d-glucose, whereas the UGT84J proteins were inactive. Further analyses with UGT84A25a and -26a indicated that they also formed 1-O-glucose esters of other structurally related hydroxybenzoic and hydroxycinnamic acids with a preference for hydroxybenzoic acids. The UGT84A genes were expressed in leaves, stems, and roots of E. camaldulensis, regardless of aluminum stress. Taken together, our results suggest that the UGT84A subfamily enzymes of E. camaldulensis are responsible for constitutive production of 1-O-galloyl-β-d-glucose, which is the first step of hydrolyzable tannin biosynthesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

THE URBAN DISPERSION PROGRAM ( UDP ) NYC MSG05 EXPERIMENT

EPA Science Inventory

The multi-organizational Urban Dispersion Program (UDP) has been conducting tracer release experiments at various locations within the United States. In March 2005 the UDP conducted the first NYC based experiment called Madison Square Garden -05 (MSG05). The field study involved ...

Joint Mobile Network Operations: Routing Design and Quality of Service Configuration

DTIC Science & Technology

2007-09-01

EF service for the desktop VTC application, CU- SeeMe , which uses UDP packets on ports 7648 and 7649. We also might want to provide AF service to...between commanders. In this case, the example application used is CU- SeeMe , which operates through UDP on ports 7648, 7649, or 24032. The required...range 7648 7649 access-list 101 permit udp any any eq 24032 Matches all CU- SeeMe traffic from any host access-list 102 permit udp 192.168.32.0

Cytoplasmic peptidoglycan intermediate levels in Staphylococcus aureus.

PubMed

Vemula, Harika; Ayon, Navid J; Gutheil, William G

2016-02-01

Intracellular cytoplasmic peptidoglycan (PG) intermediate levels were determined in Staphylococcus aureus during log-phase growth in enriched media. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode, and amine intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode. Levels of UDP-linked intermediates in S. aureus varied from 1.4 μM for UDP-GlcNAc-Enolpyruvyate to 1200 μM for UDP-MurNAc. Levels of amine intermediates (L-Ala, D-Ala, D-Ala-D-Ala, L-Glu, D-Glu, and L-Lys) varied over a range of from 860 μM for D-Ala-D-Ala to 30-260 mM for the others. Total PG was determined from the D-Glu content of isolated PG, and used to estimate the rate of PG synthesis (in terms of cytoplasmic metabolite flux) as 690 μM/min. The total UDP-linked intermediates pool (2490 μM) is therefore sufficient to sustain growth for 3.6 min. Comparison of UDP-linked metabolite levels with published pathway enzyme characteristics demonstrates that enzymes on the UDP-branch range from >80% saturation for MurA, Z, and C, to <5% saturation for MurB. Metabolite levels were compared with literature values for Escherichia coli, with the major difference in UDP-intermediates being the level of UDP-MurNAc, which was high in S. aureus (1200 μM) and low in E. coli (45 μM). Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Lightweight UDP Pervasive Protocol in Smart Home Environment Based on Labview

NASA Astrophysics Data System (ADS)

Kurniawan, Wijaya; Hannats Hanafi Ichsan, Mochammad; Rizqika Akbar, Sabriansyah; Arwani, Issa

2017-04-01

TCP (Transmission Control Protocol) technology in a reliable environment was not a problem, but not in an environment where the entire Smart Home network connected locally. Currently employing pervasive protocols using TCP technology, when data transmission is sent, it would be slower because they have to perform handshaking process in advance and could not broadcast the data. On smart home environment, it does not need large size and complex data transmission between monitoring site and monitoring center required in Smart home strain monitoring system. UDP (User Datagram Protocol) technology is quick and simple on data transmission process. UDP can broadcast messages because the UDP did not require handshaking and with more efficient memory usage. LabVIEW is a programming language software for processing and visualization of data in the field of data acquisition. This paper proposes to examine Pervasive UDP protocol implementations in smart home environment based on LabVIEW. UDP coded in LabVIEW and experiments were performed on a PC and can work properly.

Globally maximizing, locally minimizing: unsupervised discriminant projection with applications to face and palm biometrics.

PubMed

Yang, Jian; Zhang, David; Yang, Jing-Yu; Niu, Ben

2007-04-01

This paper develops an unsupervised discriminant projection (UDP) technique for dimensionality reduction of high-dimensional data in small sample size cases. UDP can be seen as a linear approximation of a multimanifolds-based learning framework which takes into account both the local and nonlocal quantities. UDP characterizes the local scatter as well as the nonlocal scatter, seeking to find a projection that simultaneously maximizes the nonlocal scatter and minimizes the local scatter. This characteristic makes UDP more intuitive and more powerful than the most up-to-date method, Locality Preserving Projection (LPP), which considers only the local scatter for clustering or classification tasks. The proposed method is applied to face and palm biometrics and is examined using the Yale, FERET, and AR face image databases and the PolyU palmprint database. The experimental results show that UDP consistently outperforms LPP and PCA and outperforms LDA when the training sample size per class is small. This demonstrates that UDP is a good choice for real-world biometrics applications.

Identification of eukaryotic UDP-galactopyranose mutase inhibitors using the ThermoFAD assay.

PubMed

Martín Del Campo, Julia S; Eckshtain-Levi, Meital; Sobrado, Pablo

2017-11-04

Aspergillus fumigatus is a human pathogen responsible for deadly infections in immune-compromised patients. A potential strategy for treating A. fumigatus infections is by targeting the biosynthesis of cell wall components, such as galactofuranase, which is absent in humans. Galactofuranose biosynthesis is initiated by the flavoenzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM requires the reduced form of the flavin for activity, which is obtained by reacting with NADPH. We aimed to identify inhibitors of UGM by screening a kinase inhibitor library using ThermoFAD, a flavin fluorescence thermal shift assay. The screening assay identified flavopiridol as a compound that increased the melting temperature of A. fumigatus UGM. Further characterization showed that flavopiridol is a non-competitive inhibitor of UGM and docking studies suggest that it binds in the active site. This compound does not inhibit the prokaryotic UGM from Mycobacteria tuberculosis. Copyright © 2017 Elsevier Inc. All rights reserved.

UDP-Glucuronosyltransferase Expression in Mouse Liver Is Increased in Obesity- and Fasting-Induced Steatosis

PubMed Central

Xu, Jialin; Kulkarni, Supriya R.; Li, Liya

2012-01-01

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lepob/ob (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance. PMID:22031624

UDP-glucuronosyltransferase expression in mouse liver is increased in obesity- and fasting-induced steatosis.

PubMed

Xu, Jialin; Kulkarni, Supriya R; Li, Liya; Slitt, Angela L

2012-02-01

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lep(ob/ob) (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance.

Both UDP N-acetylglucosamine pyrophosphorylases of Tribolium castaneum are critical for molting, survival, and fecundity

USDA-ARS?s Scientific Manuscript database

A bioinformatics search of the genome of the red flour beetle, Tribolium castaneum, resulted in the identification of two genes encoding proteins closely related to UDP-N-acetylglucosamine pyrophosphorylases (UAP), which provide the activated precursor, UDP-N-acetylglucosamine, for the synthesis of ...

Cell wall composition and digestibility alterations in Brachypodium distachyon acheived through reduced expression of the UDP-arabinopyranose mutase

USDA-ARS?s Scientific Manuscript database

Plant cell-wall polysaccharide biosynthesis requires nucleotide-activated sugars. The prominent grass cell wall sugars, glucose (Glc), xylose (Xyl), and arabinose (Ara), are biosynthetically related via the UDP-sugar interconversion pathway. RNA-seq analysis of Brachypodium distachyon UDP-sugar inte...

Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

PubMed

Park, Joohae; Tefsen, Boris; Heemskerk, Marc J; Lagendijk, Ellen L; van den Hondel, Cees A M J J; van Die, Irma; Ram, Arthur F J

2015-11-02

Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter. Based on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively. The two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the Golgi. A. niger contains two genes encoding UDP-Galf-transporters. Deletion and localization studies indicate that UgtA and UgtB have redundant functions in the biosynthesis of Galf-containing glycoconjugates.

Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

PubMed Central

Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L.; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W.; Klock, Heath E.; Miller, Mitchell D.; Godzik, Adam; Lesley, Scott A.; Deacon, Ashley M.; Mengin-Lecreulx, Dominique; Wilson, Ian A.

2011-01-01

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships. PMID:21445265

Structure and function of the first full-length murein peptide ligase (Mpl) cell wall recycling protein.

PubMed

Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Mengin-Lecreulx, Dominique; Wilson, Ian A

2011-03-18

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In gram-negative bacteria, ∼30-60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.

Long range molecular dynamics study of interactions of the eukaryotic glucosamine-6-phosphate synthase with fructose-6-phosphate and UDP-GlcNAc.

PubMed

Miszkiel, Aleksandra; Wojciechowski, Marek

2017-11-01

Glucosamine-6-phosphate synthase (EC 2.6.1.16) is responsible for catalysis of the first and practically irreversible step in hexosamine metabolism. The final product of this pathway, uridine 5' diphospho N-acetyl-d-glucosamine (UDP-GlcNAc), is an essential substrate for assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin resistance in type II diabetes, which makes of it a potential target for anti-fungal, anti-bacterial and anti-diabetic therapy. The crystal structure of isomerase domain from human pathogenic fungus Candida albicans has been solved recently but it doesn't reveal the molecular mechanism details of inhibition taking place under UDP-GlcNAc influence, the unique feature of eukaryotic enzyme. The following study is a continuation of the previous research based on comparative molecular dynamics simulations of the structures with and without the enzyme's physiological inhibitor (UDP-GlcNAc) bound. The models used for this study included fructose-6-phosphate, one of the enzyme's substrates in its binding pocket. The simulation results studies demonstrated differences in mobility of the compared structures. Some amino acid residues were determined, for which flexibility is evidently different between the models. Importantly, it has been confirmed that the most fixed residues are related to the inhibitor binding process and to the catalysis reaction. The obtained results constitute an important step towards understanding of the inhibition that GlcN-6-P synthase is subjected by UDP-GlcNAc molecule. Copyright © 2017 Elsevier Inc. All rights reserved.

Performance of a veterinary urine dipstick paddle system for diagnosis and identification of urinary tract infections in dogs and cats.

PubMed

Ybarra, Winnie L; Sykes, Jane E; Wang, Yenlie; Byrne, Barbara A; Westropp, Jodi L

2014-04-01

To evaluate the performance of a veterinary urine dipstick paddle (UDP) for diagnosis and identification of urinary tract infection (UTI) in dogs and cats. Prospective, randomized, blinded study. 207 urine specimens. UDPs were inoculated by 2 investigators and incubated according to manufacturer's instructions. Results, including presence or absence of bacterial growth, organism counts, and identification of uropathogens, were compared between investigators and with microbiology laboratory results. A subset of UDPs with bacterial growth was submitted to the laboratory for confirmation. The laboratory reported 64 (30.9%) specimens had growth of bacteria. Bacterial growth was reported for 63 (30.4%) and 58 (28.0%) of the UDPs by investigators 1 and 2, respectively. Sensitivity and specificity of the UDP for detection of bacterial growth were 97.3% and 98.6%, respectively, for investigator 1 and 89.1% and 99.3%, respectively, for investigator 2. For UPDs with ≥ 10(5) colony-forming units/mL, organism counts correlated well between the laboratory and investigators 1 (r = 0.95) and 2 (r = 0.89). Pathogen identification was not always accurate. Only 25 of 33 (75.8%) UDPs submitted for confirmation yielded bacteria consistent with those isolated from the original bacterial culture of urine. The veterinary UDP system was a sensitive test for screening patients for bacterial UTI, but uropathogen identification was not always accurate. When UDPs have bacterial growth, a fresh urine specimen should be submitted to the laboratory to confirm the identity of the organisms and to permit antimicrobial susceptibility testing.

Packet spacing : an enabling mechanism for delivering multimedia content in computational grids /

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, A. C.; Feng, W. C.; Belford, Geneva G.

2001-01-01

Streaming multimedia with UDP has become increasingly popular over distributed systems like the Internet. Scientific applications that stream multimedia include remote computational steering of visualization data and video-on-demand teleconferencing over the Access Grid. However, UDP does not possess a self-regulating, congestion-control mechanism; and most best-efort traflc is served by congestion-controlled TCF! Consequently, UDP steals bandwidth from TCP such that TCP$ows starve for network resources. With the volume of Internet traffic continuing to increase, the perpetuation of UDP-based streaming will cause the Internet to collapse as it did in the mid-1980's due to the use of non-congestion-controlled TCP. To address thismore » problem, we introduce the counterintuitive notion of inter-packet spacing with control feedback to enable UDP-based applications to perform well in the next-generation Internet and computational grids. When compared with traditional UDP-based streaming, we illustrate that our approach can reduce packet loss over SO% without adversely afecting delivered throughput. Keywords: network protocol, multimedia, packet spacing, streaming, TCI: UDlq rate-adjusting congestion control, computational grid, Access Grid.« less

Phrenic nerve decompression for the management of unilateral diaphragmatic paralysis – preoperative evaluation and operative technique

PubMed Central

Hoshide, Reid; Brown, Justin

2017-01-01

Background: Unilateral diaphragmatic paralysis (UDP) can be a very disabling, typically causing shortness of breath and reduced exercise tolerance. We present a case of a surgical decompression of the phrenic nerve of a patient who presented with UDP, which occurred following cervical spine surgery. Methods: The workup for the etiology of UDP demonstrated paradoxical movement on “sniff test” and notably impaired pulmonary function tests. Seven months following the onset of the UDP, he underwent a surgical decompression of the phrenic nerve at the level of the anterior scalene. Results: He noted rapid symptomatic improvement following surgery and reversal of the above noted objective findings was documented. At his 4-year follow-up, he had complete resolution of his clinical symptoms. Repeated physiologic testing of his respiratory function had shown a complete reversal of his UDP. Conclusions: Anatomical compression of the phrenic nerve by redundant neck vasculature should be considered in the differential diagnosis of UDP. Here we demonstrated the techniques in workup and surgical management, with both subjective and objective evidence of success. PMID:29184705

Phrenic nerve decompression for the management of unilateral diaphragmatic paralysis - preoperative evaluation and operative technique.

PubMed

Hoshide, Reid; Brown, Justin

2017-01-01

Unilateral diaphragmatic paralysis (UDP) can be a very disabling, typically causing shortness of breath and reduced exercise tolerance. We present a case of a surgical decompression of the phrenic nerve of a patient who presented with UDP, which occurred following cervical spine surgery. The workup for the etiology of UDP demonstrated paradoxical movement on "sniff test" and notably impaired pulmonary function tests. Seven months following the onset of the UDP, he underwent a surgical decompression of the phrenic nerve at the level of the anterior scalene. He noted rapid symptomatic improvement following surgery and reversal of the above noted objective findings was documented. At his 4-year follow-up, he had complete resolution of his clinical symptoms. Repeated physiologic testing of his respiratory function had shown a complete reversal of his UDP. Anatomical compression of the phrenic nerve by redundant neck vasculature should be considered in the differential diagnosis of UDP. Here we demonstrated the techniques in workup and surgical management, with both subjective and objective evidence of success.

Regulatory insights into the production of UDP-N-acetylglucosamine by Lactobacillus casei

PubMed Central

Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio; Yebra, María J.

2012-01-01

UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU. PMID:22825354

Biochemical and Structural Characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: Enzymes Required for the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thoden, James B.; Holden, Hazel M.

2011-12-22

The unusual sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, or ManNAc3NAcA, has been observed in the lipopolysaccharides of both pathogenic and nonpathogenic Gram-negative bacteria. It is added to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates UDP-ManNAc3NAcA. Five enzymes are ultimately required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetylglucosamine. The second enzyme in the pathway, encoded by the wlba gene and referred to as WlbA, catalyzes the NAD-dependent oxidation of the C-3' hydroxyl group of the UDP-linked sugar. Here we describe a combined structural and functional investigation of the WlbA enzymes from Bordetella pertussis and Chromobacterium violaceum. For this investigation,more » ternary structures were determined in the presence of NAD(H) and substrate to 2.13 and 1.5 {angstrom} resolution, respectively. Both of the enzymes display octameric quaternary structures with their active sites positioned far apart. The octamers can be envisioned as tetramers of dimers. Kinetic studies demonstrate that the reaction mechanisms for these enzymes are sequential and that they do not require {alpha}-ketoglutarate for activity. These results are in sharp contrast to those recently reported for the WlbA enzymes from Pseudomonas aeruginosa and Thermus thermophilus, which function via ping-pong mechanisms that involve {alpha}-ketoglutarate. Taken together, the results reported here demonstrate that there are two distinct families of WlbA enzymes, which differ with respect to amino acid sequences, quaternary structures, active site architectures, and kinetic mechanisms.« less

Biochemical and Structural Characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: Enzymes Required for the Biosynthesis of 2,3-Diacetamido-2,3-Dideoxy-d-Mannuronic Acid¶

PubMed Central

Thoden, James B.; Holden, Hazel M.

2011-01-01

The unusual sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, or ManNAc3NAcA1, has been observed in the lipopolysaccharides of both pathogenic and nonpathogenic Gram-negative bacteria. It is added to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates, UDP-ManNAc3NAcA. Five enzymes are ultimately required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetylglucosamine. The second enzyme in the pathway, encoded by the wlba gene and referred to as WlbA, catalyzes the NAD-dependent oxidation of the C-3' hydroxyl group of the UDP-linked sugar. Here we describe a combined structural and functional investigation of the WlbA enzymes from Bordetella pertussis and Chromobacterium violaceum. For this investigation, ternary structures were determined in the presence of NAD(H) and substrate to 2.13 Å and 1.5 Å resolution, respectively. Both of the enzymes display octameric quaternary structures with their active sites positioned far apart. The octamers can be envisioned as tetramers of dimers. Kinetic studies demonstrate that the reaction mechanisms for these enzymes are sequential and that they do not require α-ketoglutarate for activity. These results are in sharp contrast to those recently reported for the WlbA enzymes from Pseudomonas aeruginosa and Thermus thermophilus, which function via ping-pong mechanisms that involve α-ketoglutarate. Taken together the results reported here demonstrate that there are two distinct families of WlbA enzymes, which differ with respect to amino acid sequences, quaternary structures, active site architectures, and kinetic mechanisms. PMID:21241053

Highly sulfated hexasaccharide sequences isolated from chondroitin sulfate of shark fin cartilage: insights into the sugar sequences with bioactivities.

PubMed

Mizumoto, Shuji; Murakoshi, Saori; Kalayanamitra, Kittiwan; Deepa, Sarama Sathyaseelan; Fukui, Shigeyuki; Kongtawelert, Prachya; Yamada, Shuhei; Sugahara, Kazuyuki

2013-02-01

Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates and are linear polysaccharides consisting of variously sulfated repeating disaccharides, [-4GlcUAβ1-3GalNAcβ1-](n), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. CS chains containing D-disaccharide units [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)] are involved in the development of cerebellar Purkinje cells and neurite outgrowth-promoting activity through interaction with a neurotrophic factor, pleiotrophin, resulting in the regulation of signaling. In this study, to obtain further structural information on the CS chains containing d-disaccharide units involved in brain development, oligosaccharides containing D-units were isolated from a shark fin cartilage. Seven novel hexasaccharide sequences, ΔO-D-D, ΔA-D-D, ΔC-D-D, ΔE-A-D, ΔD-D-C, ΔE-D-D and ΔA-B-D, in addition to three previously reported sequences, ΔC-A-D, ΔC-D-C and ΔA-D-A, were isolated from a CS preparation of shark fin cartilage after exhaustive digestion with chondroitinase AC-I, which cannot act on the galactosaminidic linkages bound to D-units. The symbol Δ stands for a 4,5-unsaturated bond of uronic acids, whereas A, B, C, D, E and O represent [GlcUA-GalNAc(4-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(4-O-sulfate)], [GlcUA-GalNAc(6-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)], [GlcUA-GalNAc(4-O-, 6-O-sulfate)] and [GlcUA-GalNAc], respectively. In binding studies using an anti-CS monoclonal antibody, MO-225, the epitopes of which are involved in cerebellar development in mammals, novel epitope structures, ΔA-D-A, ΔA-D-D and ΔA-B-D, were revealed. Hexasaccharides containing two consecutive D-units or a B-unit will be useful for the structural and functional analyses of CS chains particularly in the neuroglycobiological fields.

Antibiotic Effects on Methicillin-Resistant Staphylococcus aureus Cytoplasmic Peptidoglycan Intermediate Levels and Evidence for Potential Metabolite Level Regulatory Loops.

PubMed

Vemula, Harika; Ayon, Navid J; Burton, Alloch; Gutheil, William G

2017-06-01

Cytoplasmic peptidoglycan (PG) precursor levels were determined in methicillin-resistant Staphylococcus aureus (MRSA) after exposure to several cell wall-targeting antibiotics. Three experiments were performed: (i) exposure to 4× MIC levels (acute); (ii) exposure to sub-MIC levels (subacute); (iii) a time course experiment of the effect of vancomycin. In acute exposure experiments, fosfomycin increased UDP-GlcNAc, as expected, and resulted in substantially lower levels of total UDP-linked metabolite accumulation relative to other pathway inhibitors, indicating reduced entry into this pathway. Upstream inhibitors (fosfomycin, d-cycloserine, or d-boroalanine) reduced UDP-MurNAc-pentapeptide levels by more than fourfold. Alanine branch inhibitors (d-cycloserine and d-boroalanine) reduced d-Ala-d-Ala levels only modestly (up to 4-fold) but increased UDP-MurNAc-tripeptide levels up to 3,000-fold. Downstream pathway inhibitors (vancomycin, bacitracin, moenomycin, and oxacillin) increased UDP-MurNAc-pentapeptide levels up to 350-fold and UDP-MurNAc-l-Ala levels up to 80-fold, suggesting reduced MurD activity by downstream inhibitor action. Sub-MIC exposures demonstrated effects even at 1/8× MIC which strongly paralleled acute exposure changes. Time course data demonstrated that UDP-linked intermediate levels respond rapidly to vancomycin exposure, with several intermediates increasing three- to sixfold within minutes. UDP-linked intermediate level changes were also multiphasic, with some increasing, some decreasing, and some increasing and then decreasing. The total (summed) UDP-linked intermediate pool increased by 1,475 μM/min during the first 10 min after vancomycin exposure, providing a revised estimate of flux in this pathway during logarithmic growth. These observations outline the complexity of PG precursor response to antibiotic exposure in MRSA and indicate likely sites of regulation (entry and MurD). Copyright © 2017 American Society for Microbiology.

A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

PubMed

Shapiro, Adam B; Livchak, Stephania; Gao, Ning; Whiteaker, James; Thresher, Jason; Jahić, Haris; Huang, Jian; Gu, Rong-Fang

2012-03-01

A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

Evaluation of UDP-GlcN derivatives for selective labeling of 5-(hydroxymethyl)cytosine.

PubMed

Dai, Nan; Bitinaite, Jurate; Chin, Hang-Gyeong; Pradhan, Sriharsa; Corrêa, Ivan R

2013-11-04

5-(hydroxymethyl)cytosine (5-hmC) is a newly identified oxidative product of 5-methylcytosine (5-mC) in the mammalian genome, and is believed to be an important epigenetic marker influencing a variety of biological processes. In addition to its relatively low abundance, the fluctuation of 5-hmC levels over time during cell development poses a formidable challenge for its accurate mapping and quantification. Here we describe a specific chemoenzymatic approach to 5-hmC detection in DNA samples by using new uridine 5'-diphosphoglucosamine (UDP-GlcN) probes. Our approach requires modification of the glucose moiety of UDP-Glc with small amino groups and transfer of these glucose derivatives to the hydroxy moiety of 5-hmC by using T4 phage glucosyltransferases. We evaluated the transfer efficiencies of three glucosyltransferases (wild-type α- and β-GTs and a Y261L mutant β-GT) with five different UDP-Glc derivatives containing functionalized groups for subsequent bioconjugation and detection. Our results indicate that UDP-6-N3 -Glc, UDP-6-GlcN, and UDP-2-GlcN can be transferred by β-GT with efficiencies similar to that seen with the native UDP-Glc cofactor. 6-N3 -Glc- and 6-GlcN-containing oligonucleotides were selectively labeled with reactive fluorescent probes. In addition, a 2 kb DNA fragment modified with 2-GlcN groups was specifically detected by use of a commercially available antiglucosamine antibody. Alternative substrates for β-GT and correlated glycosyltransferases might prove useful for the study of the function and dynamics of 5-hmC and other modified nucleotides, as well as for multiplex analysis. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cloning and Expression Analysis of a UDP-Galactose/Glucose Pyrophosphorylase from Melon Fruit Provides Evidence for the Major Metabolic Pathway of Galactose Metabolism in Raffinose Oligosaccharide Metabolizing Plants1

PubMed Central

Dai, Nir; Petreikov, Marina; Portnoy, Vitaly; Katzir, Nurit; Pharr, David M.; Schaffer, Arthur A.

2006-01-01

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by α-galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism. PMID:16829585

Binding of uridine 5'-diphosphate in the "basic patch" of the zinc deacetylase LpxC and implications for substrate binding.

PubMed

Gennadios, Heather A; Christianson, David W

2006-12-26

LpxC is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of lipid A, a vital component of the outer membrane of Gram-negative bacteria. Accordingly, the inhibition of LpxC is an attractive strategy for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC from Aquifex aeolicus complexed with uridine 5'-diphosphate (UDP), and the 3.1 A resolution structure of LpxC complexed with pyrophosphate. The X-ray crystal structure of the LpxC-UDP complex provides the first view of interactions likely to be exploited by the substrate UDP group in the "basic patch" of the active site. The diphosphate group of UDP makes hydrogen bond interactions with strictly conserved residue K239 as well as solvent molecules. The ribose moiety of UDP interacts with partially conserved residue E197. The UDP uracil group hydrogen bonds with both the backbone NH group and the backbone carbonyl group of E160, and with the backbone NH group of K162 through an intervening water molecule. Finally, the alpha-phosphate and uracil groups of UDP interact with R143 and R262 through intervening water molecules. The structure of LpxC complexed with pyrophosphate reveals generally similar intermolecular interactions in the basic patch. Unexpectedly, diphosphate binding in both complexes is accompanied by coordination to an additional zinc ion, resulting in the identification of a new metal-binding site termed the E-site. The structures of the LpxC-UDP and LpxC-pyrophosphate complexes provide new insights with regard to substrate recognition in the basic patch and metal ion coordination in the active site of LpxC.

The binding of decomposition products of UDP-galactose to the microsomes and polyribosomes isolated from rat liver.

PubMed

Kopacz-Jodczyk, T; Gałasiński, W

1987-10-01

UDP-D-[U-14C]galactose is decomposed to [U-14C]galactose-1-phosphate and [U-14C]galactose by rat liver microsomal and crude polyribosomal fractions, under conditions commonly used to assay of glycosyltransferase activities. UDP-D-[U-14C]galactose, at neutral pH, is also chemically degraded to the [U-14C]galactose-1,2-cyclic phosphate. The 1,2-cyclic phosphate derivative of galactose also exists in the commercial UDP-D-[U-14C]galactose. It is a very important finding that products of the UDP-D-[U-14C]galactose decomposition are tightly, although nonenzymatically, bound to tested subcellular fractions and may create a false impression of protein glycosylation. The application of controls containing all radioactive substances present in suitable samples is recommended in order to avoid incorrect interpretations of the results.

Binding of decomposition products of UDP-galactose to the microsomes and polyribosomes isolated from rat liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kopacz-Jodczyk, T.; Galasinski, W.

1987-10-01

UDP-D-(U-/sup 14/C)galactose is decomposed to (U-/sup 14/C)galactose-1-phosphate and (U-/sup 14/C)galactose by rat liver microsomal and crude polyribosomal fractions, under conditions commonly used to assay of glycosyltransferase activities. UDP-D-(U-/sup 14/C)galactose, at neutral pH, is also chemically degraded to the (U-/sup 14/C)galactose-1,2-cyclic phosphate. The 1,2-cyclic phosphate derivative of galactose also exists in the commercial UDP-D-(U-/sup 14/C)galactose. It is a very important finding that products of the UDP-D-(U-/sup 14/C)galactose decomposition are tightly, although nonenzymatically, bound to tested subcellular fractions and may create a false impression of protein glycosylation. The application of controls containing all radioactive substances present in suitable samples is recommended inmore » order to avoid incorrect interpretations of the results.« less

A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

PubMed Central

Führing, Jana Indra; Cramer, Johannes Thomas; Schneider, Julia; Baruch, Petra; Gerardy-Schahn, Rita; Fedorov, Roman

2015-01-01

In mammals, UDP-glucose pyrophosphorylase (UGP) is the only enzyme capable of activating glucose-1-phosphate (Glc-1-P) to UDP-glucose (UDP-Glc), a metabolite located at the intersection of virtually all metabolic pathways in the mammalian cell. Despite the essential role of its product, the molecular basis of UGP function is poorly understood. Here we report the crystal structure of human UGP in complex with its product UDP-Glc. Beyond providing first insight into the active site architecture, we describe the substrate binding mode and intermolecular interactions in the octameric enzyme that are crucial to its activity. Importantly, the quaternary mechanism identified for human UGP in this study may be common for oligomeric sugar-activating nucleotidyltransferases. Elucidating such mechanisms is essential for understanding nucleotide sugar metabolism and opens the perspective for the development of drugs that specifically inhibit simpler organized nucleotidyltransferases in pathogens. PMID:25860585

Biosynthesis of a (1. -->. 4)-. beta. -D-glucan. [Lupinus albus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brummond, D.O.

1983-01-01

An enzymatic activity isolated from Lupinus albus that produced an insoluble (1..-->..4)-..beta..-D-glucan from UDP-D-glucose has been solubilized and partially purified. Some of the properties of the enzyme system have been characterized. A proposed sequence of reactions between UDP-D-glucose and the final dextran may involve a (1..-->..4)-..beta..-linked polysaccharide bonded to UDP.

The participation of ribosomes in protein glycosylation. Interaction of the ribosome-UDP-N-acetyl-glucosamine complex with dolichol phosphate.

PubMed

Paszkiewicz-Gadek, A; Porowska, H; Gałasiński, W

1992-01-01

UDP-N-acetylglucosamine can be bound by pure ribosomes. The part of N-acetylglucosamine-1-P can be transferred from the complex ribosome-UDP-N-acetylglucosamine onto dolichol phosphate. Evidence is presented that N-acetylglucosamine bound to dolichol phosphate can be transferred to the nascent peptide synthesized on the ribosome.

Mechanism of ribonucleotide reductase from Herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ator, M.A.; Stubbe, J.; Spector, T.

1986-03-15

Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of (3'-3H)ADP, (3'-H)UDP, and (5-3H) UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of (3'-3H)ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of (beta-32P)ClUDP with the reductase resulted in themore » production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase.« less

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

PubMed

Friedman, Aaron J; Durrant, Jacob D; Pierce, Levi C T; McCorvie, Thomas J; Timson, David J; McCammon, J Andrew

2012-08-01

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. © 2012 John Wiley & Sons A/S.

Identification of novel inhibitors against UDP-galactopyranose mutase to combat leishmaniasis.

PubMed

Kashif, Mohammad; Tabrez, Shams; Husein, Atahar; Arish, Mohd; Kalaiarasan, Ponnusamy; Manna, Partha P; Subbarao, Naidu; Akhter, Yusuf; Rub, Abdur

2018-03-01

Leishmania, a protozoan parasite that causes leishmaniasis, affects 1-2 million people every year worldwide. Leishmaniasis is a vector born disease and characterized by a diverse group of clinical syndromes. Current treatment is limited because of drug resistance, high cost, poor safety, and low efficacy. The urgent need for potent agents against Leishmania has led to significant advances in the development of novel antileishmanial drugs. β-galactofuranose (β-Galf) is an important component of Leishmanial cell surface matrix and plays a critical role in the pathogenesis of parasite. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) which acts as the precursor for β-Galf synthesis. Due to its absence in human, this enzyme is selected as the potential target in search of new antileishmanial drugs. Three dimensional protein structure model of Leishmania major UGM (LmUGM) has been homology modeled using Trypanosoma cruzi UGM (TcUGM) as a template. The stereochemistry was validated further. We selected already reported active compounds from PubChem database to target the LmUGM. Three compounds (6064500, 44570814, and 6158954) among the top hit occupied the UDP binding site of UGM suggested to work as a possible inhibitor for it. In vitro antileishmanial activity assay was performed with the top ranked inhibitor, 6064500. The 6064500 molecule has inhibited the growth of Leishmania donovani promastigotes significantly. Further, at similar concentrations it has exhibited significantly lesser toxicity than standard drug miltefosine hydrate in mammalian cells. © 2017 Wiley Periodicals, Inc.

The Nutrient-Sensing Hexosamine Biosynthetic Pathway as the Hub of Cancer Metabolic Rewiring.

PubMed

Chiaradonna, Ferdinando; Ricciardiello, Francesca; Palorini, Roberta

2018-06-02

Alterations in glucose and glutamine utilizing pathways and in fatty acid metabolism are currently considered the most significant and prevalent metabolic changes observed in almost all types of tumors. Glucose, glutamine and fatty acids are the substrates for the hexosamine biosynthetic pathway (HBP). This metabolic pathway generates the "sensing molecule" UDP- N -Acetylglucosamine (UDP-Glc N Ac). UDP-Glc N Ac is the substrate for the enzymes involved in protein N - and O -glycosylation, two important post-translational modifications (PTMs) identified in several proteins localized in the extracellular space, on the cell membrane and in the cytoplasm, nucleus and mitochondria. Since protein glycosylation controls several key aspects of cell physiology, aberrant protein glycosylation has been associated with different human diseases, including cancer. Here we review recent evidence indicating the tight association between the HBP flux and cell metabolism, with particular emphasis on the post-transcriptional and transcriptional mechanisms regulated by the HBP that may cause the metabolic rewiring observed in cancer. We describe the implications of both protein O - and N -glycosylation in cancer cell metabolism and bioenergetics; focusing our attention on the effect of these PTMs on nutrient transport and on the transcriptional regulation and function of cancer-specific metabolic pathways.

Inhibitory effects of Aphanizomenon flos-aquae constituents on human UDP-glucose dehydrogenase activity.

PubMed

Scoglio, Stefano; Lo Curcio, Valeria; Catalani, Simona; Palma, Francesco; Battistelli, Serafina; Benedetti, Serena

2016-12-01

The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability. Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested. Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD(+). In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells. Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.

Identification of the UDP-glucose-4-epimerase required for galactofuranose biosynthesis and galactose metabolism in A. niger.

PubMed

Park, Joohae; Tefsen, Boris; Arentshorst, Mark; Lagendijk, Ellen; van den Hondel, Cees Amjj; van Die, Irma; Ram, Arthur Fj

2014-01-01

Galactofuranose (Gal f )-containing glycoconjugates are important to secure the integrity of the cell wall of filamentous fungi. Mutations that prevent the biosynthesis of Gal f -containing molecules compromise cell wall integrity. In response to cell wall weakening, the cell wall integrity (CWI)-pathway is activated to reinforce the strength of the cell wall. Activation of CWI-pathway in Aspergillus niger is characterized by the specific induction of the agsA gene, which encodes a cell wall α-glucan synthase. In this study, we screened a collection of cell wall mutants with an induced expression of agsA for defects in Gal f biosynthesis using a with anti-Gal f antibody (L10). From this collection of mutants, we previously identified mutants in the UDP-galactopyranose mutase encoding gene ( ugmA ). Here, we have identified six additional UDP-galactopyranose mutase ( ugmA ) mutants and one mutant (named mutant #41) in an additional complementation group that displayed strongly reduced Gal f -levels in the cell wall. By using a whole genome sequencing approach, 21 SNPs in coding regions were identified between mutant #41 and its parental strain which changed the amino acid sequence of the encoded proteins. One of these mutations was in gene An14g03820, which codes for a putative UDP-glucose-4-epimerase (UgeA). The A to G mutation in this gene causes an amino acid change of Asn to Asp at position 191 in the UgeA protein. Targeted deletion of ugeA resulted in an even more severe reduction of Gal f in N-linked glucans, indicating that the UgeA protein in mutant #41 is partially active. The ugeA gene is also required for growth on galactose despite the presence of two UgeA homologs in the A. niger genome. By using a classical mutant screen and whole genome sequencing of a new Gal f -deficient mutant, the UDP-glucose-4-epimerase gene ( ugeA ) has been identified. UgeA is required for the biosynthesis of Gal f as well as for galactose metabolism in Aspergillus niger .

UDP-arabinopyranose mutase 3 is required for pollen wall morphogenesis in rice (Oryza sativa).

PubMed

Sumiyoshi, Minako; Inamura, Takuya; Nakamura, Atsuko; Aohara, Tsutomu; Ishii, Tadashi; Satoh, Shinobu; Iwai, Hiroaki

2015-02-01

l-Arabinose is one of the main constituents of cell wall polysaccharides such as pectic rhamnogalacturonan I (RG-I), glucuronoarabinoxylans and other glycoproteins. It is found predominantly in the furanose form rather than in the thermodynamically more stable pyranose form. UDP-L-arabinofuranose (UDP-Araf), rather than UDP-L-arabinopyranose (UDP-Arap), is a sugar donor for the biosynthesis of arabinofuranosyl (Araf) residues. UDP-arabinopyranose mutases (UAMs) have been shown to interconvert UDP-Araf and UDP-Arap and are involved in the biosynthesis of polysaccharides including Araf. The UAM gene family has three members in Oryza sativa. Co-expression network in silico analysis showed that OsUAM3 expression was independent from OsUAM1 and OsUAM2 co-expression networks. OsUAM1 and OsUAM2 were expressed ubiquitously throughout plant development, but OsUAM3 was expressed primarily in reproductive tissue, particularly at the pollen cell wall formation developmental stage. OsUAM3 co-expression networks include pectin catabolic enzymes. To determine the function of OsUAMs in reproductive tissues, we analyzed RNA interference (RNAi)-knockdown transformants (OsUAM3-KD) specific for OsUAM3. OsUAM3-KD plants grew normally and showed abnormal phenotypes in reproductive tissues, especially in terms of the pollen cell wall and exine. In addition, we examined modifications of cell wall polysaccharides at the cellular level using antibodies against polysaccharides including Araf. Immunolocalization of arabinan using the LM6 antibody showed low levels of arabinan in OsUAM3-KD pollen grains. Our results suggest that the function of OsUAM3 is important for synthesis of arabinan side chains of RG-I and is required for reproductive developmental processes, especially the formation of the cell wall in pollen. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A Conserved UDP-Glucose Dehydrogenase Encoded outside the hasABC Operon Contributes to Capsule Biogenesis in Group A Streptococcus

PubMed Central

Cole, Jason N.; Aziz, Ramy K.; Kuipers, Kirsten; Timmer, Anjuli M.; Nizet, Victor

2012-01-01

Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host. PMID:22961854

Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans

DOE PAGES

Willis, Jonathan D.; Smith, James A.; Mazarei, Mitra; ...

2016-10-26

Switchgrass (Panicum virgatum L.) is a C 4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. The expression ofmore » a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.« less

Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willis, Jonathan D.; Smith, James A.; Mazarei, Mitra

Switchgrass (Panicum virgatum L.) is a C 4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. The expression ofmore » a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.« less

Identification and Partial Characterization of a Novel UDP-N-Acetylenolpyruvoylglucosamine Reductase/UDP-N-Acetylmuramate:l-Alanine Ligase Fusion Enzyme from Verrucomicrobium spinosum DSM 4136(T).

PubMed

Naqvi, Kubra F; Patin, Delphine; Wheatley, Matthew S; Savka, Michael A; Dobson, Renwick C J; Gan, Han Ming; Barreteau, Hélène; Blanot, Didier; Mengin-Lecreulx, Dominique; Hudson, André O

2016-01-01

The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/C Vs ) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44-46°C. Its apparent K m values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum.

Identification and Partial Characterization of a Novel UDP-N-Acetylenolpyruvoylglucosamine Reductase/UDP-N-Acetylmuramate:l-Alanine Ligase Fusion Enzyme from Verrucomicrobium spinosum DSM 4136T

PubMed Central

Naqvi, Kubra F.; Patin, Delphine; Wheatley, Matthew S.; Savka, Michael A.; Dobson, Renwick C. J.; Gan, Han Ming; Barreteau, Hélène; Blanot, Didier; Mengin-Lecreulx, Dominique; Hudson, André O.

2016-01-01

The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/CVs) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44–46°C. Its apparent Km values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum. PMID:27047475

Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

PubMed Central

Shi, Xueyan; Dick, Ryan A.; Ford, Kevin A.; Casida, John E.

2009-01-01

Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19 and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19 and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than that of TMX. Imidacloprid (IMI), CLO and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI and 4-hydroxy-thiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid and CLO metabolism in vivo in mice, elevating the brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites. PMID:19391582

Corrective action investigation plan for Corrective Action Unit Number 423: Building 03-60 Underground Discharge Point, Tonopah Test Range, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1997-10-27

This Corrective Action Investigation Plan (CAIP) contains the environmental sample collection objectives and the criteria for conducting site investigation activities at Corrective Action Unit (CAU) Number 423, the Building 03-60 Underground Discharge Point (UDP), which is located in Area 3 at the Tonopah Test Range (TTR). The TTR, part of the Nellis Air Force Range, is approximately 225 kilometers (140 miles) northwest of Las Vegas, Nevada. CAU Number 423 is comprised of only one Corrective Action Site (CAS) which includes the Building 03-60 UDP and an associated discharge line extending from Building 03-60 to a point approximately 73 meters (240more » feet) northwest. The UDP was used between approximately 1965 and 1990 to dispose of waste fluids from the Building 03-60 automotive maintenance shop. It is likely that soils surrounding the UDP have been impacted by oil, grease, cleaning supplies and solvents as well as waste motor oil and other automotive fluids released from the UDP.« less

An Impact Assessment Model for Distributed Adaptive Security Situation Assessment

DTIC Science & Technology

2005-01-01

the cargo manifest can be either a 56K modem-based TCP/IP connection (the oval labeled internet) or a 40K wireless modem connection ( cell phone ) that...via a UDP connection on the 40K wireless modem ( cell phone ). For each resource, either alternative may be used to achieve the same goal, but some...Manifests Comm-in Comp- power Comm- out JTF Internet (TCP-IP) Cell phone (TCP-IP) Internet (UDP) Cell phone (UDP) Manual Computer 4

[The effect of uridine and uridine nucleotides on isolated rat heart performance in regional myocardial ischemia].

PubMed

Eliseev, V V; Rodionova, O M; Sapronov, N S; Selizarova, N O

2002-01-01

We studied the effects of uridine, uridine-5'-monophosphate (UMP), uridine-5'-diphosphate (UDP) and uridine-5'-triphosphate on contractility, coronary flow and heart rate in isolated perfused rat hearts under 60-minute regional ischemia of the left ventricle. All the compounds (50 mumol/l) induced a positive inotropic effect but had no effect on the heart rate. Uridine and UMP prevented the development of the contracture. UDP and especially UTP increased coronary flow. Probably, a protective effect of uridine and UMP is due to activation of myocardial glycogen synthesis while favourable effects of UDP and UTP on contractility and coronary flow are explained by their influence on P2U-receptors of cardiomyocytes. In addition, coronary dilatation induced by UDP and UTP promoted the reduction of the damaged zone.

Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-β-d-glucoside

PubMed Central

Michlmayr, Herbert; Malachová, Alexandra; Varga, Elisabeth; Kleinová, Jana; Lemmens, Marc; Newmister, Sean; Rayment, Ivan; Berthiller, Franz; Adam, Gerhard

2015-01-01

Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-β-d-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON), and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins. PMID:26197338

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase.

PubMed

Schmölzer, Katharina; Lemmerer, Martin; Gutmann, Alexander; Nidetzky, Bernd

2017-04-01

Nucleotide sugar-dependent ("Leloir") glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5'-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/g cell dry weight ) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 g product /L, 86% yield (based on UDP), and a total turnover number of 103 g UDP-glc /g cell dry weight at a space-time yield of 10 g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924-928. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

PubMed

Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian; Wagtberg Sen, Jette; Rasmussen, Søren Kofoed; Kontoravdi, Cleo; Weilguny, Dietmar; Andersen, Mikael Rørdam

2015-03-01

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP-Gal biosynthesis, which was observed to be both cell line and cultivation condition-dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality. © 2014 Wiley Periodicals, Inc.

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vetting,M.; Frantom, P.; Blanchard, J.

2008-01-01

The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-l-myo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-l-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97 rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-l-myo-inositol-1-phosphate. The structuremore » highlights the residues important in binding of UDP-N-acetylglucosamine and 1-l-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the {beta}-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-l-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond.« less

Examining the Effect of Organizational Roles in Shaping Network Traffic Activity

DTIC Science & Technology

2012-08-01

absolute value, and are presented in Table 3. Role Correlation Feature Admin 0.3004 bpp 0.2845 portsPerFlow 0.2063 addrDist -0.1869...OS Correlation Feature XP 0.4783 notTcpUdp 0.2867 addrDist -0.2389 bpp 0.1933 protocol -0.1852 flowInt Windows 7 0.3884 portDist 0.2367...addrDist 0.2001 direction 0.1751 bpp 0.1653 portsPerFlow Mac -0.2376 notTcpUdp 0.1978 UDP 0.1885 duration -0.1783 addrDist -0.1736 countEmpties

Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maruyama, Daisuke; Nishitani, Yuichi; Nonaka, Tsuyoshi

2006-12-01

UDP-N-acetylglucosamine pyrophosphorylase was purified and crystallized and X-ray diffraction data were collected to 2.3 Å resolution. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the productmore » complex belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å.« less

[beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose.

PubMed Central

Li, L.; Drake, R. R.; Clement, S.; Brown, R. M.

1993-01-01

Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed. PMID:12231766

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

PubMed Central

Kapuria, Vaibhav; Röhrig, Ute F.; Bhuiyan, Tanja; Borodkin, Vladimir S.; van Aalten, Daan M.F.; Zoete, Vincent; Herr, Winship

2016-01-01

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc), O-linked-GlcNAc transferase (OGT) catalyzes Ser/Thr O-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase–protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase–protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT. PMID:27056667

Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase.

PubMed

Jackson, R G; Lim, E K; Li, Y; Kowalczyk, M; Sandberg, G; Hoggett, J; Ashford, D A; Bowles, D J

2001-02-09

Biochemical characterization of recombinant gene products following a phylogenetic analysis of the UDP-glucosyltransferase (UGT) multigene family of Arabidopsis has identified one enzyme (UGT84B1) with high activity toward the plant hormone indole-3-acetic acid (IAA) and three related enzymes (UGT84B2, UGT75B1, and UGT75B2) with trace activities. The identity of the IAA conjugate has been confirmed to be 1-O-indole acetyl glucose ester. A sequence annotated as a UDP-glucose:IAA glucosyltransferase (IAA-UGT) in the Arabidopsis genome and expressed sequence tag data bases given its similarity to the maize iaglu gene sequence showed no activity toward IAA. This study describes the first biochemical analysis of a recombinant IAA-UGT and provides the foundation for future genetic approaches to understand the role of 1-O-indole acetyl glucose ester in Arabidopsis.

Effects of Curcuma xanthorrhiza Extracts and Their Constituents on Phase II Drug-metabolizing Enzymes Activity.

PubMed

Salleh, Nurul Afifah Mohd; Ismail, Sabariah; Ab Halim, Mohd Rohaimi

2016-01-01

Curcuma xanthorrhiza is a native Indonesian plant and traditionally utilized for a range of illness including liver damage, hypertension, diabetes, and cancer. The study determined the effects of C. xanthorrhiza extracts (ethanol and aqueous) and their constituents (curcumene and xanthorrhizol) on UDP-glucuronosyltransferase (UGT) and glutathione transferase (GST) activities. The inhibition studies were evaluated both in rat liver microsomes and in human recombinant UGT1A1 and UGT2B7 enzymes. p-nitrophenol and beetle luciferin were used as the probe substrates for UGT assay while 1-chloro-2,4-dinitrobenzene as the probe for GST assay. The concentrations of extracts studied ranged from 0.1 to 1000 μg/mL while for constituents ranged from 0.01 to 500 μM. In rat liver microsomes, UGT activity was inhibited by the ethanol extract (IC 50 =279.74 ± 16.33 μg/mL). Both UGT1A1 and UGT2B7 were inhibited by the ethanol and aqueous extracts with IC 50 values ranging between 9.59-22.76 μg/mL and 110.71-526.65 μg/Ml, respectively. Rat liver GST and human GST Pi-1 were inhibited by ethanol and aqueous extracts, respectively (IC 50 =255.00 ± 13.06 μg/mL and 580.80 ± 18.56 μg/mL). Xanthorrhizol was the better inhibitor of UGT1A1 (IC 50 11.30 ± 0.27 μM) as compared to UGT2B7 while curcumene did not show any inhibition. For GST, both constituents did not show any inhibition. These findings suggest that C. xanthorrhiza have the potential to cause herb-drug interaction with drugs that are primarily metabolized by UGT and GST enzymes. Findings from this study would suggest which of Curcuma xanthorrhiza extracts and constituents that would have potential interactions with drugs which are highly metabolized by UGT and GST enzymes. Further clinical studies can then be designed if needed to evaluate the in vivo pharmacokinetic relevance of these interactions Abbreviations Used : BSA: Bovine serum albumin, CAM: Complementary and alternative medicine, cDNA: Complementary deoxyribonucleic acid, CDNB: 1-Chloro-2,4-dinitrobenzene, CuSO4.5H2O: Copper(II) sulfate pentahydrate, CXEE: Curcuma xanthorrhiza ethanol extract, CXAE: Curcuma xanthorrhiza aqueous extract, GC-MS: Gas chromatography-mass spectroscopy, GSH: Glutathione, GST: Glutathione S-transferase, KCl: Potassium chloride, min: Minutes, MgCl 2 : Magnesium chloride, mg/mL: Concentration (weight of test substance in milligrams per volume of test concentration), mM: Milimolar, Na 2 CO 3 : Sodium carbonate, NaOH: Sodium hydroxide, nmol: nanomol, NSAIDs: Non-steroidal antiinflammatory drug, p-NP: para-nitrophenol, RLU: Relative light unit, SEM: Standard error of mean, UDPGA: UDP-glucuronic acid, UGT: UDP-glucuronosyltransferase.

The influence of an intramolecular hydrogen bond in differential recognition of inhibitory acceptor analogs by human ABO(H) blood group A and B glycosyltransferases.

PubMed

Nguyen, Hoa P; Seto, Nina O L; Cai, Ye; Leinala, Eeva K; Borisova, Svetlana N; Palcic, Monica M; Evans, Stephen V

2003-12-05

Human ABO(H) blood group glycosyltransferases GTA and GTB catalyze the final monosaccharide addition in the biosynthesis of the human A and B blood group antigens. GTA and GTB utilize a common acceptor, the H antigen disaccharide alpha-l-Fucp-(1-->2)-beta-d-Galp-OR, but different donors, where GTA transfers GalNAc from UDP-GalNAc and GTB transfers Gal from UDP-Gal. GTA and GTB are two of the most homologous enzymes known to transfer different donors and differ in only 4 amino acid residues, but one in particular (Leu/Met-266) has been shown to dominate the selection between donor sugars. The structures of the A and B glycosyltransferases have been determined to high resolution in complex with two inhibitory acceptor analogs alpha-l-Fucp(1-->2)-beta-d-(3-deoxy)-Galp-OR and alpha-l-Fucp-(1-->2)-beta-d-(3-amino)-Galp-OR, in which the 3-hydroxyl moiety of the Gal ring has been replaced by hydrogen or an amino group, respectively. Remarkably, although the 3-deoxy inhibitor occupies the same conformation and position observed for the native H antigen in GTA and GTB, the 3-amino analog is recognized differently by the two enzymes. The 3-amino substitution introduces a novel intramolecular hydrogen bond between O2' on Fuc and N3' on Gal, which alters the minimum-energy conformation of the inhibitor. In the absence of UDP, the 3-amino analog can be accommodated by either GTA or GTB with the l-Fuc residue partially occupying the vacant UDP binding site. However, in the presence of UDP, the analog is forced to abandon the intramolecular hydrogen bond, and the l-Fuc residue is shifted to a less ordered conformation. Further, the residue Leu/Met-266 that was thought important only in distinguishing between donor substrates is observed to interact differently with the 3-amino acceptor analog in GTA and GTB. These observations explain why the 3-deoxy analog acts as a competitive inhibitor of the glycosyltransferase reaction, whereas the 3-amino analog displays complex modes of inhibition.

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase-A computational perspective on severe epimerase-deficiency galactosemia.

PubMed

Timson, David J; Lindert, Steffen

2013-09-10

UDP-galactose 4'-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD+ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia. Copyright © 2013 Elsevier B.V. All rights reserved.

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium.

PubMed

Carneiro, Inês; Timóteo, M Alexandrina; Silva, Isabel; Vieira, Cátia; Baldaia, Catarina; Ferreirinha, Fátima; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

2014-07-01

Despite the abundant expression of the UDP-sensitive P2Y6 receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood. We compared the effects of UDP and of the selective P2Y6 receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [(3) H]-ACh overflow experiments. Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y6 receptor antagonist, MRS2578. Effects mediated by urothelial P2Y6 receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y1 receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [(3) H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y1 receptor antagonist, MRS2179. The P2Y6 receptor agonist increased threefold ATP levels in the voided fluid. Activation of P2Y6 receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y1 receptors. © 2014 The British Pharmacological Society.

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner*

PubMed Central

Gagnon, Susannah M. L.; Meloncelli, Peter J.; Zheng, Ruixiang B.; Haji-Ghassemi, Omid; Johal, Asha R.; Borisova, Svetlana N.; Lowary, Todd L.; Evans, Stephen V.

2015-01-01

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the “tucked under” conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be “isosteric” with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB. PMID:26374898

Identification of endoplasmic reticulum proteins involved in glycan assembly: synthesis and characterization of P3-(4-azidoanilido)uridine 5'-triphosphate, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins.

PubMed Central

Rancour, D M; Menon, A K

1998-01-01

Much of the enzymic machinery required for the assembly of cell surface carbohydrates is located in the endoplasmic reticulum (ER) of eukaryotic cells. Structural information on these proteins is limited and the identity of the active polypeptide(s) is generally unknown. This paper describes the synthesis and characteristics of a photoaffinity reagent that can be used to identify and analyse members of the ER glycan assembly apparatus, specifically those glycosyltransferases, nucleotide phosphatases and nucleotide-sugar transporters that recognize uridine nucleotides or UDP-sugars. The photoaffinity reagent, P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP), was synthesized easily from commercially available precursors. AAUTP inhibited the activity of ER glycosyltransferases that utilize UDP-GlcNAc and UDP-Glc, indicating that it is recognized by UDP-sugar-binding proteins. In preliminary tests AAUTP[alpha-32P] labelled bovine milk galactosyltransferase, a model UDP-sugar-utilizing enzyme, in a UV-light-dependent, competitive and saturable manner. When incubated with rat liver ER vesicles, AAUTP[alpha-32P] labelled a discrete subset of ER proteins; labelling was light-dependent and metal ion-specific. Photolabelling of intact ER vesicles with AAUTP[alpha-32P] caused selective incorporation of radioactivity into proteins with cytoplasmically disposed binding sites; UDP-Glc:glycoprotein glucosyltransferase, a lumenal protein, was labelled only when the vesicle membrane was disrupted. These data indicate that AAUTP is a membrane topological probe of catalytic sites in target proteins. Strategies for using AAUTP to identify and study novel ER proteins involved in glycan assembly are discussed. PMID:9677326

M-type K+ currents in rat cultured thoracolumbar sympathetic neurones and their role in uracil nucleotide-evoked noradrenaline release

PubMed Central

Nörenberg, W; von Kügelgen, I; Meyer, A; Illes, P; Starke, K

2000-01-01

Cultured sympathetic neurones are depolarized and release noradrenaline in response to extracellular ATP, UDP and UTP. We examined the possibility that, in neurones cultured from rat thoracolumbar sympathetic ganglia, inhibition of the M-type potassium current might underlie the effects of UDP and UTP. Reverse transcriptase-polymerase chain reaction indicated that the cultured cells contained mRNA for P2Y2-, P2Y4- and P2Y6-receptors as well as for the KCNQ2- and KCNQ3-subunits which have been suggested to assemble into M-channels. In cultures of neurones taken from newborn as well as from 10 day-old rats, oxotremorine, the M-channel blocker Ba2+ and UDP all released previously stored [3H]-noradrenaline. The neurones possessed M-currents, the kinetic properties of which were similar in neurones from newborn and 9–12 day-old rats. UDP, UTP and ATP had no effect on M-currents in neurones prepared from newborn rats. Oxotremorine and Ba2+ substantially inhibited the current. ATP also had no effect on the M-current in neurones prepared from 9–12 day-old rats. Oxotremorine and Ba2+ again caused marked inhibition. In contrast to cultures from newborn animals, UDP and UTP attenuated the M-current in neurones from 9–12 day-old rats; however, the maximal inhibition was less than 30%. The results indicate that inhibition of the M-current is not involved in uracil nucleotide-induced transmitter release from rat cultured sympathetic neurones during early development. M-current inhibition may contribute to release at later stages, but only to a minor extent. The mechanism leading to noradrenaline release by UDP and UTP remains unknown. PMID:10683196

Genetic basis of coaggregation receptor polysaccharide biosynthesis in Streptococcus sanguinis and related species.

PubMed

Yang, J; Yoshida, Y; Cisar, J O

2014-02-01

Interbacterial adhesion between streptococci and actinomyces promotes early dental plaque biofilm development. Recognition of coaggregation receptor polysaccharides (RPS) on strains of Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis by Actinomyces spp. type 2 fimbriae is the principal mechanism of these interactions. Previous studies of genetic loci for synthesis of RPS (rps) and RPS precursors (rml, galE1 and galE2) in S. gordonii 38 and S. oralis 34 revealed differences between these strains. To determine whether these differences are strain-specific or species-specific, we identified and compared loci for polysaccharide biosynthesis in additional strains of these species and in several strains of the previously unstudied species, S. sanguinis. Genes for synthesis of RPS precursors distinguished the rps loci of different streptococci. Hence, rml genes for synthesis of TDP-L-Rha were in rps loci of S. oralis strains but at other loci in S. gordonii and S. sanguinis. Genes for two distinct galactose epimerases were also distributed differently. Hence, galE1 for epimerization of UDP-Glc and UDP-Gal was in galactose operons of S. gordonii and S. sanguinis strains but surprisingly, this gene was not present in S. oralis. Moreover, galE2 for epimerization of both UDP-Glc and UDP-Gal and UDP-GlcNAc and UDP-GalNAc was at a different locus in each species, including rps operons of S. sanguinis. The findings provide insight into cell surface properties that distinguish different RPS-producing streptococci and open an approach for identifying these bacteria based on the arrangement of genes for synthesis of polysaccharide precursors. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Man o' War Mutation in UDP-α-D-Xylose Synthase Favors the Abortive Catalytic Cycle and Uncovers a Latent Potential for Hexamer Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walsh, Jr., Richard M.; Polizzi, Samuel J.; Kadirvelraj, Renuka

The man o’ war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-D-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ~16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies showmore » that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-D-4-keto-xylose, is not reduced to the final product, UDP-α-D-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.« less

Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations▿ †

PubMed Central

Mitsuya, Yumi; Varghese, Vici; Wang, Chunlin; Liu, Tommy F.; Holmes, Susan P.; Jayakumar, Prerana; Gharizadeh, Baback; Ronaghi, Mostafa; Klein, Daniel; Fessel, W. Jeffrey; Shafer, Robert W.

2008-01-01

T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method—ultradeep pyrosequencing (UDPS; 454 Life Sciences)—to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants (“revertant” samples) compared with samples from untreated persons that lack such revertants (“control” samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants. PMID:18715933

The Undiagnosed Diseases Program Integrated Collaboration System (UDPICS): One Program’s Experience Developing Custom Software to Support Research for Complex-Disease Families

PubMed Central

Guzman, Jessica; Lee, Elizabeth; Draper, David; Valivullah, Zaheer; Yu, Guoyun; Sincan, Murat; Gahl, William A.; Adams, David R.

2015-01-01

The Undiagnosed Diseases Program (UDP) was started in 2008 with the goals of making diagnoses and facilitating related translational research. The individuals and families seen by the UDP are often unique and medically complex. Approximately 40% of UDP cases are pediatric. The Undiagnosed Diseases Program Integrated Collaboration System (UDPICS) was designed to create a collaborative workspace for researchers, clinicians and families. We describe our progress in developing the system to date, focusing on design rationale, challenges and issues that are likely to be common in the development of similar systems in the future. PMID:27417368

Xylosylation of Phenolic Hydroxyl Groups of the Monomeric Lignin Model Compounds 4-Methylguaiacol and Vanillyl Alcohol by Coriolus versicolor

PubMed Central

Kondo, Ryuichiro; Yamagami, Hikari; Sakai, Kokki

1993-01-01

When 4-methylguaiacol (MeG), a phenolic lignin model compound, was added to a culture that was inoculated with Coriolus versicolor, it was bioconverted into 2-methoxy-4-methylphenyl β-d-xyloside (MeG-Xyl). The phenolic hydroxyl group of vanillyl alcohol was much more extensively xylosylated than the alcoholic hydroxyl group. When a mixture of MeG and commercial UDP-xylose was incubated with cell extracts of mycelia, transformation of UDP-xylose into MeG-Xyl was observed. This result suggested that UDP-xylosyltransferase was involved in the xylosylation of phenolic hydroxyl groups of lignin model compounds. PMID:16348869

Cloning and expression of a novel UDP-GlcNAc:alpha-D-mannoside beta1,2-N-acetylglucosaminyltransferase homologous to UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.

PubMed Central

Zhang, Wenli; Betel, Doron; Schachter, Harry

2002-01-01

A TBLASTN search with human UDP-GlcNAc:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) as a probe identified human and mouse Unigenes encoding a protein similar to human GnT I (34% identity over 340 amino acids). The recombinant protein converted Man(alpha1-6)[Man(alpha1-3)]Man(beta1-)O-octyl to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-)O-octyl, the reaction catalysed by GnT I. The enzyme also added GlcNAc to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-)O-octyl (the substrate for beta-1,2-N-acetylglucosaminyltransferase II), Man(alpha1-)O-benzyl [with K(m) values of approximately 0.3 and >30 mM for UDP-GlcNAc and Man(alpha1-)O-benzyl respectively] and the glycopeptide CYA[Man(alpha1-)O-T]AV (K(m) approximately 12 mM). The product formed with Man(alpha1-)O-benzyl was identified as GlcNAc(beta1-2)Man(alpha1-)O-benzyl by proton NMR spectroscopy. The enzyme was named UDP-GlcNAc:alpha-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). The human gene mapped to chromosome 1. Northern-blot analysis showed a 3.3 kb message with a wide tissue distribution. The cDNA has a 1980 bp open reading frame encoding a 660 amino acid protein with a type-2 domain structure typical of glycosyltransferases. Man(beta1-)O-octyl, Man(beta1-)O-p-nitrophenyl and GlcNAc(beta1-2)Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-4)GlcNAc(beta1-4)GlcNAc(beta1-)O-Asn were not acceptors, indicating that GnT I.2 is specific for alpha-linked terminal Man and does not have N-acetylglucosaminyltransferase III, IV, V, VII or VIII activities. CYA[Man(alpha1-)O-T]AV was between three and seven times more effective as an acceptor than the other substrates, suggesting that GnT I.2 may be responsible for the synthesis of the GlcNAc(beta1-2)Man(alpha1-)O-Ser/Thr moiety on alpha-dystroglycan and other O-mannosylated proteins. PMID:11742540

Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis

USDA-ARS?s Scientific Manuscript database

UDP-glucosyltransferase (UGT) is a key enzyme during anthocyanin biosynthesis by catalyzing glucosylation of anthocyanins so as to increase their solubility and accumulation. Previously it has been shown that preharvest spray of calcium chloride enhances anthocyanin accumulation in strawberry fruit ...

Preparation and evaluation of unitary doses of propafenone used in children with supraventricular tachycardia: a pilot study.

PubMed

Flores Pérez, J; Ramírez Mendiola, B; Flores Pérez, C; García Álvarez, R; Juárez Olguín, H

2013-01-01

The aim was to prepare and evaluate unitary doses of propafenone (UDP) used in children with supraventricular tachycardia. UDP were prepared from four brands of tablets at doses of propafenone, 11, 25 and 90 mg, used in the Cardiology Service of this Institute. The stability of doses was determined at 20±5°C and 40°C for up to day 30. Besides, a weight variation test was performed. Plasma levels of propafenone were determined at steady state in 3 children diagnosed with supraventricular tachycardia under treatment with UDP. Concentrations of drug in blood were measured using a high pressure liquid chromatography method, previously validated. The stability of UDP, showed no significant statistical differences (p > 0.05) between doses or brands up to day 30, at both temperatures. The coefficient of variation from the weight variation was less than 6%. The plasma levels of propafenone at steady state were: patient 1, 31.57 ng/ml; patient 2, 226.46 ng/ml; and patient 3, 221.29 ng/ml. The actual administered dose for the patients could vary up to 6%, and doses prepared from different brands of tablets remain stables for up to day 30 at both temperatures. UDP is a temporal, safe and alternative option when pediatrics formulation of this drug is lacking.

The NIH Undiagnosed Diseases Program and Network: Applications to modern medicine

PubMed Central

Gahl, William A.; Mulvihill, John J.; Toro, Camilo; Markello, Thomas C.; Wise, Anastasia L.; Ramoni, Rachel B.; Adams, David R.; Tifft, Cynthia J.

2017-01-01

Introduction The inability of some seriously and chronically ill individuals to receive a definitive diagnosis represents an unmet medical need. In 2008, the NIH Undiagnosed Diseases Program (UDP) was established to provide answers to patients with mysterious conditions that long eluded diagnosis and to advance medical knowledge. Patients admitted to the NIH UDP undergo a five-day hospitalization, facilitating highly collaborative clinical evaluations and a detailed, standardized documentation of the individual’s phenotype. Bedside and bench investigations are tightly coupled. Genetic studies include commercially available testing, single nucleotide polymorphism microarray analysis, and family exomic sequencing studies. Selected gene variants are evaluated by collaborators using informatics, in vitro cell studies, and functional assays in model systems (fly, zebrafish, worm, or mouse). Insights from the UDP In seven years, the UDP received 2954 complete applications and evaluated 863 individuals. Nine vignettes (two unpublished) illustrate the relevance of an undiagnosed diseases program to complex and common disorders, the coincidence of multiple rare single gene disorders in individual patients, newly recognized mechanisms of disease, and the application of precision medicine to patient care. Conclusions The UDP provides examples of the benefits expected to accrue with the recent launch of a national Undiagnosed Diseases Network (UDN). The UDN should accelerate rare disease diagnosis and new disease discovery, enhance the likelihood of diagnosing known diseases in patients with uncommon phenotypes, improve management strategies, and advance medical research. PMID:26846157

Overlapping and distinct roles of Aspergillus fumigatus UDP-glucose 4-epimerases in galactose metabolism and the synthesis of galactose-containing cell wall polysaccharides.

PubMed

Lee, Mark J; Gravelat, Fabrice N; Cerone, Robert P; Baptista, Stefanie D; Campoli, Paolo V; Choe, Se-In; Kravtsov, Ilia; Vinogradov, Evgeny; Creuzenet, Carole; Liu, Hong; Berghuis, Albert M; Latgé, Jean-Paul; Filler, Scott G; Fontaine, Thierry; Sheppard, Donald C

2014-01-17

The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.

Determinants and expansion of specificity in a trichothecene UDP-glucosyltransferase from Oryza sativa

USDA-ARS?s Scientific Manuscript database

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease...

Peptidoglycan precursor from Fusobacterium nucleatum contains lanthionine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fredriksen, A.; Vasstrand, E.N.; Jensen, H.B.

1991-01-01

Fusobacterium nucleatum was grown in the presence of ({sup 14}C)UDP. By means of sequential precipitation and chromatographic separation of the cytoplasmic content, a peptidoglycan ({sup 14}C)UDP pentapeptide containing lanthionine was isolated. This finding indicates that lanthionine is synthesized and incorporated as such during the assembly of the peptidoglycan.

Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni

PubMed Central

Shibata, Hitoshi; Sonoke, Satoru; Ochiai, Hideo; Nishihashi, Hideji; Yamada, Masaharu

1991-01-01

To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-O-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction I, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-O-methylsteviol, but not to iso-steviol and 13-O-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-O-glucopyranoside. Enzyme fraction II, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP-glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13, 19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-O-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-O-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A. PMID:16667943

A bacterial-type ABC transporter is involved in aluminum tolerance in rice.

PubMed

Huang, Chao Feng; Yamaji, Naoki; Mitani, Namiki; Yano, Masahiro; Nagamura, Yoshiaki; Ma, Jian Feng

2009-02-01

Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose. Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall.

Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC.

PubMed

Marmor, S; Petersen, C P; Reck, F; Yang, W; Gao, N; Fisher, S L

2001-10-09

The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala). The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue. The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states. Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI). Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site. The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.

Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-l-alanine ligase.

PubMed

Ehmann, David E; Demeritt, Julie E; Hull, Kenneth G; Fisher, Stewart L

2004-05-06

UDP-N-acetylmuramyl-l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC(50) of 2.3 microM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP-N-acetyl-muramic acid (UNAM) with a K(i) of 4.5 microM against ATP and 6.3 microM against UNAM. Fluorescence binding experiments yielded a K(d) of 3.1 microM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding.

New Method To Generate Enzymatically Deficient Clostridium difficile Toxin B as an Antigen for Immunization

PubMed Central

Genth, Harald; Selzer, Jörg; Busch, Christian; Dumbach, Jürgen; Hofmann, Fred; Aktories, Klaus; Just, Ingo

2000-01-01

The family of the large clostridial cytotoxins, encompassing Clostridium difficile toxins A and B as well as the lethal and hemorrhagic toxins from Clostridium sordellii, monoglucosylate the Rho GTPases by transferring a glucose moiety from the cosubstrate UDP-glucose. Here we present a new detoxification procedure to block the enzyme activity by treatment with the reactive UDP-2′,3′-dialdehyde to result in alkylation of toxin A and B. Alkylation is likely to occur in the catalytic domain, because the native cosubstrate UDP-glucose completely protected the toxins from inactivation and the alkylated toxin competes with the native toxin at the cell receptor. Alkylated toxins are good antigens resulting in antibodies recognizing only the C-terminally located receptor binding domain, whereas formaldehyde treatment resulted in antibodies recognizing both the receptor binding domain and the catalytic domain, indicating that the catalytic domain is concealed under native conditions. Antibodies against the native catalytic domain (amino acids 1 through 546) and those holotoxin antibodies recognizing the catalytic domain inhibited enzyme activity. However, only antibodies against the receptor binding domain protected intact cells from the cytotoxic activity of toxin B, whereas antibodies against the catalytic domain were protective only when inside the cell. PMID:10678912

Flexibility and mutagenic resiliency of glycosyltransferases.

PubMed

Bay, Marie Lund; Cuesta-Seijo, Jose A; Weadge, Joel T; Persson, Mattias; Palcic, Monica M

2014-10-01

The human blood group A and B antigens are synthesized by two highly homologous enzymes, glycosyltransferase A (GTA) and glycosyltransferase B (GTB), respectively. These enzymes catalyze the transfer of either GalNAc or Gal from their corresponding UDP-donors to αFuc1-2βGal-R terminating acceptors. GTA and GTB differ at only four of 354 amino acids (R176G, G235S, L266M, G268A), which alter the donor specificity from UDP-GalNAc to UDP-Gal. Blood type O individuals synthesize truncated or non-functional enzymes. The cloning, crystallization and X-ray structure elucidations for GTA and GTB have revealed key residues responsible for donor discrimination and acceptor binding. Structural studies suggest that numerous conformational changes occur during the catalytic cycle. Over 300 ABO alleles are tabulated in the blood group antigen mutation database (BGMUT) that provides a framework for structure-function studies. Natural mutations are found in all regions of GTA and GTB from the active site, flexible loops, stem region and surfaces remote from the active site. Our characterizations of natural mutants near a flexible loop (V175M), on a remote surface site (P156L), in the metal binding motif (M212V) and near the acceptor binding site (L232P) demonstrate the resiliency of GTA and GTB to mutagenesis.

vanC Cluster of Vancomycin-Resistant Enterococcus gallinarum BM4174

PubMed Central

Arias, Cesar A.; Courvalin, Patrice; Reynolds, Peter E.

2000-01-01

Glycopeptide-resistant enterococci of the VanC type synthesize UDP-muramyl-pentapeptide[d-Ser] for cell wall assembly and prevent synthesis of peptidoglycan precursors ending in d-Ala. The vanC cluster of Enterococcus gallinarum BM4174 consists of five genes: vanC-1, vanXYC, vanT, vanRC, and vanSC. Three genes are sufficient for resistance: vanC-1 encodes a ligase that synthesizes the dipeptide d-Ala-d-Ser for addition to UDP-MurNAc-tripeptide, vanXYC encodes a d,d-dipeptidase–carboxypeptidase that hydrolyzes d-Ala-d-Ala and removes d-Ala from UDP-MurNAc-pentapeptide[d-Ala], and vanT encodes a membrane-bound serine racemase that provides d-Ser for the synthetic pathway. The three genes are clustered: the start codons of vanXYC and vanT overlap the termination codons of vanC-1 and vanXYC, respectively. Two genes which encode proteins with homology to the VanS-VanR two-component regulatory system were present downstream from the resistance genes. The predicted amino acid sequence of VanRC exhibited 50% identity to VanR and 33% identity to VanRB. VanSC had 40% identity to VanS over a region of 308 amino acids and 24% identity to VanSB over a region of 285 amino acids. All residues with important functions in response regulators and histidine kinases were conserved in VanRC and VanSC, respectively. Induction experiments based on the determination of d,d-carboxypeptidase activity in cytoplasmic extracts confirmed that the genes were expressed constitutively. Using a promoter-probing vector, regions upstream from the resistance and regulatory genes were identified that have promoter activity. PMID:10817725

The Universal Design for Play Tool: Establishing Validity and Reliability

ERIC Educational Resources Information Center

Ruffino, Amy Goetz; Mistrett, Susan G.; Tomita, Machiko; Hajare, Poonam

2006-01-01

The Universal Design for Play (UDP) Tool is an instrument designed to evaluate the presence of universal design (UD) features in toys. This study evaluated its psychometric properties, including content validity, construct validity, and test-retest reliability. The UDP tool was designed to assist in selecting toys most appropriate for children…

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, Eric E.; Roessler, Paul G.

1999-01-01

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

Improving UDP/IP Transmission Without Increasing Congestion

NASA Technical Reports Server (NTRS)

Burleigh, Scott

2006-01-01

Datagram Retransmission (DGR) is a computer program that, within certain limits, ensures the reception of each datagram transmitted under the User Datagram Protocol/Internet Protocol. [User Datagram Protocol (UDP) is considered unreliable because it does not involve a reliability-ensuring connection-initiation dialogue between sender and receiver. UDP is well suited to issuing of many small messages to many different receivers.] Unlike prior software for ensuring reception of UDP datagrams, DGR does not contribute to network congestion by retransmitting data more frequently as an ever-increasing number of messages and acknowledgements is lost. Instead, DGR does just the opposite: DGR includes an adaptive timeout-interval- computing component that provides maximum opportunity for reception of acknowledgements, minimizing retransmission. By monitoring changes in the rate at which message-transmission transactions are completed, DGR detects changes in the level of congestion and responds by imposing varying degrees of delay on the transmission of new messages. In addition, DGR maximizes throughput by not waiting for acknowledgement of a message before sending the next message. All DGR communication is asynchronous, to maximize efficient utilization of network connections. DGR manages multiple concurrent datagram transmission and acknowledgement conversations.

Purification, crystallization and preliminary X-ray analysis of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC).

PubMed

Deva, Taru; Pryor, KellyAnn D; Leiting, Barbara; Baker, Edward N; Smith, Clyde A

2003-08-01

UDP-N-acetylmuramoyl:L-alanine ligase (MurC) is involved in the pathway leading from UDP-N-glucosamine to the UDP-N-acetylmuramoyl:pentapeptide unit, which is the building block for the peptidoglycan layer found in all bacterial cell walls. The pathways leading to the biosynthesis of the peptidoglycan layer are important targets for the development of novel antibiotics, since animal cells do not contain these pathways. MurC is the first of four similar ATP-dependent amide-bond ligases which share primary and tertiary structural similarities. The crystal structures of three of these have been determined by X-ray crystallography, giving insights into the binding of the carbohydrate substrate and the ATP. Diffraction-quality crystals of the enzyme MurC have been obtained in both native and selenomethionine forms and X-ray diffraction data have been collected at the Se edge at a synchrotron source. The crystals are orthorhombic, with unit-cell parameters a = 73.9, b = 93.6, c = 176.8 A, and diffraction has been observed to 2.6 A resolution.

Alteration of cell wall polysaccharides through transgenic expression of UDP-Glc 4-epimerase-encoding genes in potato tubers.

PubMed

Huang, Jie-Hong; Kortstee, Anne; Dees, Dianka C T; Trindade, Luisa M; Schols, Henk A; Gruppen, Harry

2016-08-01

Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyzes the conversion of UDP-glucose to UDP-galactose. Cell wall materials from the cv. Kardal (wild-type, background) and two UGE transgenic lines (UGE 45-1 and UGE 51-16) were isolated and fractionated. The galactose (Gal) content (mg/100g tuber) from UGE 45-1 transgenic line was 38% higher than that of wild-type, and resulted in longer pectin side chains. The Gal content present in UGE 51-16 was 17% lower than that of wild-type, although most pectin populations maintained the same level of Gal. Both UGE transgenic lines showed unexpectedly a decrease in acetylation and an increase in methyl-esterification of pectin. Both UGE transgenic lines showed similar proportions of homogalacturonan and rhamnogalacturonan I within pectin backbone as the wild-type, except for the calcium-bound pectin fraction exhibiting relatively less rhamnogalacturonan I. Next to pectin modification, xyloglucan populations from both transgenic lines were altered resulting in different XSGG and XXGG proportion in comparison to wild-type. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.

PubMed

Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica M; Peters, Thomas

2017-07-04

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H, 15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H, 15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[ 13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

PubMed Central

Carneiro, Inês; Timóteo, M Alexandrina; Silva, Isabel; Vieira, Cátia; Baldaia, Catarina; Ferreirinha, Fátima; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

2014-01-01

BACKGROUND AND PURPOSE Despite the abundant expression of the UDP-sensitive P2Y6 receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood. EXPERIMENTAL APPROACH We compared the effects of UDP and of the selective P2Y6 receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [3H]-ACh overflow experiments. KEY RESULTS Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y6 receptor antagonist, MRS2578. Effects mediated by urothelial P2Y6 receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y1 receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [3H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y1 receptor antagonist, MRS2179. The P2Y6 receptor agonist increased threefold ATP levels in the voided fluid. CONCLUSIONS AND IMPLICATIONS Activation of P2Y6 receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y1 receptors. PMID:24697602

Microglia P2Y₆ receptors mediate nitric oxide release and astrocyte apoptosis.

PubMed

Quintas, Clara; Pinho, Diana; Pereira, Clara; Saraiva, Lucília; Gonçalves, Jorge; Queiroz, Glória

2014-09-03

During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis. Co-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining. UTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia. In LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(β1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end

PubMed Central

Baggenstoss, Bruce A; Washburn, Jennifer L

2017-01-01

Abstract Class I hyaluronan synthases (HAS) assemble [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-β1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcβ1,4)2–5[GlcUA(β1,3)GlcNAc]2–6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2–3 series members. Since lyase releases dehydro-oligos (dHn; M−18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2–6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2–5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(β1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-β1,4)3–4]. PMID:28138013

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(β1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end.

PubMed

Weigel, Paul H; Baggenstoss, Bruce A; Washburn, Jennifer L

2017-06-01

Class I hyaluronan synthases (HAS) assemble [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-β1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcβ1,4)2-5[GlcUA(β1,3)GlcNAc]2-6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2-3 series members. Since lyase releases dehydro-oligos (dHn; M-18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2-6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2-5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(β1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-β1,4)3-4]. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Phylogenetic analysis of pectin-related gene families in Physcomitrella patens and nine other plant species yields evolutionary insights into cell walls

PubMed Central

2014-01-01

Background Pectins are acidic sugar-containing polysaccharides that are universally conserved components of the primary cell walls of plants and modulate both tip and diffuse cell growth. However, many of their specific functions and the evolution of the genes responsible for producing and modifying them are incompletely understood. The moss Physcomitrella patens is emerging as a powerful model system for the study of plant cell walls. To identify deeply conserved pectin-related genes in Physcomitrella, we generated phylogenetic trees for 16 pectin-related gene families using sequences from ten plant genomes and analyzed the evolutionary relationships within these families. Results Contrary to our initial hypothesis that a single ancestral gene was present for each pectin-related gene family in the common ancestor of land plants, five of the 16 gene families, including homogalacturonan galacturonosyltransferases, polygalacturonases, pectin methylesterases, homogalacturonan methyltransferases, and pectate lyase-like proteins, show evidence of multiple members in the early land plant that gave rise to the mosses and vascular plants. Seven of the gene families, the UDP-rhamnose synthases, UDP-glucuronic acid epimerases, homogalacturonan galacturonosyltransferase-like proteins, β-1,4-galactan β-1,4-galactosyltransferases, rhamnogalacturonan II xylosyltransferases, and pectin acetylesterases appear to have had a single member in the common ancestor of land plants. We detected no Physcomitrella members in the xylogalacturonan xylosyltransferase, rhamnogalacturonan I arabinosyltransferase, pectin methylesterase inhibitor, or polygalacturonase inhibitor protein families. Conclusions Several gene families related to the production and modification of pectins in plants appear to have multiple members that are conserved as far back as the common ancestor of mosses and vascular plants. The presence of multiple members of these families even before the divergence of other important cell wall-related genes, such as cellulose synthases, suggests a more complex role than previously suspected for pectins in the evolution of land plants. The presence of relatively small pectin-related gene families in Physcomitrella as compared to Arabidopsis makes it an attractive target for analysis of the functions of pectins in cell walls. In contrast, the absence of genes in Physcomitrella for some families suggests that certain pectin modifications, such as homogalacturonan xylosylation, arose later during land plant evolution. PMID:24666997

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, E.E.; Roessler, P.G.

1999-07-27

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

Simulating Real-World Exposures during Emergency Events: Studying Effects of Indoor and Outdoor Releases in the Urban Dispersion Project in Upper Manhattan, NY

EPA Science Inventory

A prospective personal exposure study, involving indoor and outdoor releases, was conducted in upper Midtown Manhattan in New York City as part of the Urban Dispersion Program (UDP) focusing on atmospheric dispersion of chemicals in complex urban settings. The UDP experiments inv...

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: Activity enhancement by alamethicin, a pore-forming peptide

EPA Science Inventory

An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosph...

{open_quotes}The effects of diabetes on the activity of the enzyme glutamine: fructose-6-phosphate amindotransferase{close_quotes}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, S.P.

1994-12-31

Hexsoamine synthetic pathway (HexNSP) controls the supply of essential substrates for glycoprotein synthesis. In vitro studies suggest that increased flux of glucose via the hexsoamine synthetic pathway may play a role in glucose induced insulin resistance of glucose transport. Glutamine: fructose-6-phosphate amindotransferase (GFAT) controls flux into the hexsoamine synthetic pathway; the major products are UDPN-acetylhexosamines (UDP.HexNac=UDP.GlcNAc= UDP.GalNac). I examined whether diabetes ({approximately} 7 days post intravenous streptozotocin, and genetically linked) affects the activity of glutamine: fructose-6-phosphate in rat and mouse skeletal muscle in vivo. Nucleotide linked HexNAc were analyzed by high pressure liquid chromatography(HPLC) in deproteinized hind limb muscle extracts.

Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells

PubMed Central

McAlroy, H L; Ahmed, S; Day, S M; Baines, D L; Wong, H Y; Yip, C Y; Ko, W H; Wilson, S M; Collett, A

2000-01-01

Apical ATP, ATP, UTP and UDP evoked transient increases in short circuit current (ISC, a direct measure of transepithelial ion transport) in confluent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors. Experiments using cells grown on glass coverslips showed that ATP and UTP consistently increased intracellular free calcium ([Ca2+]i) whilst sensitivity to UDP was variable. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. Messenger RNA transcripts corresponding to the P2Y2, P2Y4 and P2Y6 receptors genes were detected in cells grown on Transwell membranes by the reverse transcriptase–polymerase chain reaction. Identical results were obtained for cells grown on glass. Experiments in which ISC and [Ca2+]i were monitored simultaneously in cells on Transwell membranes, confirmed that apical ATP and UTP increased both parameters and showed that the UDP-evoked increase in ISC was accompanied by a [Ca2+]i-signal. Ionomycin consistently increased [Ca2+]i in such polarized cells but caused no discernible change in ISC. However, subsequent application of apical ATP or UTP evoked a small rise in ISC but no rise in [Ca2+]i. UDP evoked no such response. As well as evoking increases in [Ca2+]i, the ATP/UTP-sensitive receptors present in Caco-2 cells thus allow direct control over ion channels in the apical membrane. The UDP-sensitive receptors, however, appear to simply evoke a rise in [Ca2+]i. PMID:11139443

Identification and characterization of glycosyltransferases involved in the synthesis of the side chains of the cell wall pectic polysaccharide rhamnogalacturonan II

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Neill, Malcolm

Our goal was to gain insight into the genes and proteins involved in the biosynthesis of rhamnogalacturonan II (RG-II), a borate cross-linked and structurally conserved pectic polysaccharide present in the primary cell walls of all vascular plants. The research conducted during the funding period established that (i) Avascular plants have the ability to synthesize UDP-apiose but lack the glycosyltransferase machinery required to synthesize RG-II or other apiose-containing cell wall glycans. (ii) RG-II structure is highly conserved in the Lemnaceae (duckweeds and relatives). However, the structures of other wall pectins and hemicellulose have changed substantial during the diversification of the Lemnaceae.more » This supports the notion that a precise structure of RG-II must be maintained to allow borate cross-linking to occur in a controlled manner. (iii) Enzymes involved in the conversion of UDP-GlcA to UDP-Api, UDP-Xyl, and UDP-Ara may have an important role in controlling the composition of duckweed cell walls. (iv) RG-II exists as the borate ester cross-linked dimer in the cell walls of soybean root hairs and roots. Thus, RG-II is present in the walls of plants cells that grow by tip or by expansive growth. (v) A reduction in RG-II cross-linking in the maize tls1 mutant, which lacks a borate channel protein, suggests that the growth defects observed in the mutant are, at least in part, due to defects in the cell wall.« less

High-resolution crystal structures and STD NMR mapping of human ABO(H) blood group glycosyltransferases in complex with trisaccharide reaction products suggest a molecular basis for product release.

PubMed

Gagnon, Susannah M L; Legg, Max S G; Sindhuwinata, Nora; Letts, James A; Johal, Asha R; Schuman, Brock; Borisova, Svetlana N; Palcic, Monica M; Peters, Thomas; Evans, Stephen V

2017-10-01

The human ABO(H) blood group A- and B-synthesizing glycosyltransferases GTA and GTB have been structurally characterized to high resolution in complex with their respective trisaccharide antigen products. These findings are particularly timely and relevant given the dearth of glycosyltransferase structures collected in complex with their saccharide reaction products. GTA and GTB utilize the same acceptor substrates, oligosaccharides terminating with α-l-Fucp-(1→2)-β-d-Galp-OR (where R is a glycolipid or glycoprotein), but use distinct UDP donor sugars, UDP-N-acetylgalactosamine and UDP-galactose, to generate the blood group A (α-l-Fucp-(1→2)[α-d-GalNAcp-(1→3)]-β-d-Galp-OR) and blood group B (α-l-Fucp-(1→2)[α-d-Galp-(1→3)]-β-d-Galp-OR) determinant structures, respectively. Structures of GTA and GTB in complex with their respective trisaccharide products reveal a conflict between the transferred sugar monosaccharide and the β-phosphate of the UDP donor. Mapping of the binding epitopes by saturation transfer difference NMR measurements yielded data consistent with the X-ray structural results. Taken together these data suggest a mechanism of product release where monosaccharide transfer to the H-antigen acceptor induces active site disorder and ejection of the UDP leaving group prior to trisaccharide egress. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effects of plasma glycosyltransferase on the ABO(H) blood group antigens of human von Willebrand factor.

PubMed

Kano, Taiki; Kondo, Kazunao; Hamako, Jiharu; Matsushita, Fumio; Sakai, Kazuya; Matsui, Taei

2018-04-04

Von Willebrand factor (VWF) is one of the plasma protein carrying ABO(H) blood group antigens, but the combining process of these antigens is not clear. In the present study, we examined whether plasma glycosyltransferase affects the blood group antigens on VWF. VWF expressing H-antigen (H-VWF) from blood group O and bovine serum albumin conjugated with H-antigen (H-BSA) were incubated with recombinant α1-3-N-acetylgalactosaminyltransferase (rA-transferase) and A-plasma with or without an additional UDP-GalNAc. Transformed antigens were detected by western blotting and ELISA, using an anti-A antibody. Both H-VWF and H-BSA acquired the A-antigen after incubation with rA-transferase and UDP-GalNAc. Incubation with A-plasma very weakly converted the H-antigen on BSA and VWF to A-antigen only in the presence of supplemented UDP-GalNAc. This conversion was enhanced on desialylation of H-VWF. These results indicate that sugar chains of plasma VWF can be modified by the external glycosyltransferase, but that plasma glycosyltransferase has no effect on the blood group antigens of VWF due to its low activity and the lack of donor sugars. Further, sialic acid residues of VWF may exert a protective effect against post-translational glycosylation. Our results clearly exclude the possibility that blood group antigens of VWF are constructed extracellularly in plasma.

Role of the Heat Sink Layer Ta for Ultrafast Spin Dynamic Process in Amorphous TbFeCo Thin Films

NASA Astrophysics Data System (ADS)

Ren, Y.; Zhang, Z. Z.; Min, T.; Jin, Q. Y.

The ultrafast demagnetization processes (UDP) in Ta (t nm)/TbFeCo (20 nm) films have been studied using the time-resolved magneto-optical Kerr effect (TRMOKE). With a fixed pump fluence of 2 mJ/cm2, for the sample without a Ta underlayer (t=0nm), we observed the UDP showing a two-step decay behavior, with a relatively longer decay time (τ2) around 3.0 ps in the second step due to the equilibrium of spin-lattice relaxation following the 4f occupation. As a 10nm Ta layer is deposited, the two-step demagnetization still exists while τ2 decreases to ˜1.9ps. Nevertheless, the second-step decay (τ2=0ps) disappears as the Ta layer thickness is increased up to 20 nm, only the first-step UDP occurs within 500 fs, followed by a fast recovery process. The rapid magnetization recovery rate strongly depends on the pump fluence. We infer that the Ta layer provides conduction electrons involving the thermal equilibrium of spin-lattice interaction and serves as heat bath taking away energy from spins of TbFeCo alloy film in UDP.

Constraints of nonresponding flows based on cross layers in the networks

NASA Astrophysics Data System (ADS)

Zhou, Zhi-Chao; Xiao, Yang; Wang, Dong

2016-02-01

In the active queue management (AQM) scheme, core routers cannot manage and constrain user datagram protocol (UDP) data flows by the sliding window control mechanism in the transport layer due to the nonresponsive nature of such traffic flows. However, the UDP traffics occupy a large part of the network service nowadays which brings a great challenge to the stability of the more and more complex networks. To solve the uncontrollable problem, this paper proposes a cross layers random early detection (CLRED) scheme, which can control the nonresponding UDP-like flows rate effectively when congestion occurs in the access point (AP). The CLRED makes use of the MAC frame acknowledgement (ACK) transmitting congestion information to the sources nodes and utilizes the back-off windows of the MAC layer throttling data rate. Consequently, the UDP-like flows data rate can be restrained timely by the sources nodes in order to alleviate congestion in the complex networks. The proposed CLRED can constrain the nonresponsive flows availably and make the communication expedite, so that the network can sustain stable. The simulation results of network simulator-2 (NS2) verify the proposed CLRED scheme.

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP).

PubMed

Liu, Jun; Zou, Yang; Guan, Wanyi; Zhai, Yafei; Xue, Mengyang; Jin, Lan; Zhao, Xueer; Dong, Junkai; Wang, Wenjun; Shen, Jie; Wang, Peng George; Chen, Min

2013-07-01

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparative Analysis of Particle Swarm and Differential Evolution via Tuning on Ultrasmall Titanium Oxide Nanoclusters

NASA Astrophysics Data System (ADS)

Inclan, Eric; Lassester, Jack; Geohegan, David; Yoon, Mina

Optimization algorithms (OA) coupled with numerical methods enable researchers to identify and study (meta) stable nanoclusters without the control restrictions of empirical methods. An algorithm's performance is governed by two factors: (1) its compatibility with an objective function, (2) the dimension of a design space, which increases with cluster size. Although researchers often tune an algorithm's user-defined parameters (UDP), tuning is not guaranteed to improve performance. In this research, Particle Swarm (PSO) and Differential Evolution (DE), are compared by tuning their UDP in a multi-objective optimization environment (MOE). Combined with a Kolmogorov Smirnov test for statistical significance, the MOE enables the study of the Pareto Front (PF), made of the UDP settings that trade-off between best performance in energy minimization (``effectiveness'') based on force-field potential energy, and best convergence rate (``efficiency''). By studying the PF, this research finds that UDP values frequently suggested in the literature do not provide best effectiveness for these methods. Additionally, monotonic convergence is found to significantly improve efficiency without sacrificing effectiveness for very small systems, suggesting better compatibility. Work is supported by the U.S. Department of Energy, Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division.

Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461.

PubMed Central

Videira, P; Fialho, A; Geremia, R A; Breton, C; Sá-Correia, I

2001-01-01

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929-939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH(2) and -CO(2)H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH(2) (GelK(NH2)) and -CO(2)H (GelK(COOH)) halves were overexpressed in Escherichia coli. GelK and GelK(NH2) were present in both the soluble and membrane fractions of E. coli, whereas GelK(COOH) was only present in the soluble fraction. GelK catalysed the transfer of [(14)C]glucuronic acid from UDP-[(14)C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by beta-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PP(i)-lipid moiety is needed for enzymic activity. Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily. PMID:11513745

Peptide Epimerization Machineries Found in Microorganisms.

PubMed

Ogasawara, Yasushi; Dairi, Tohru

2018-01-01

D-Amino acid residues have been identified in peptides from a variety of eukaryotes and prokaryotes. In microorganisms, UDP- N -acetylmuramic acid pentapeptide (UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelate-D-Ala-D-Ala), a unit of peptidoglycan, is a representative. During its biosynthesis, D-Ala and D-Glu are generally supplied by racemases from the corresponding isomers. However, we recently identified a unique unidirectional L-Glu epimerase catalyzing the epimerization of the terminal L-Glu of UDP-MurNAc-L-Ala-L-Glu. Several such enzymes, introducing D-amino acid resides into peptides via epimerization, have been reported to date. This includes a L-Ala-D/L-Glu epimerase, which is possibly used during peptidoglycan degradation. In bacterial primary metabolisms, to the best of our knowledge, these two machineries are the only examples of peptide epimerization. However, a variety of peptides containing D-amino acid residues have been isolated from microorganisms as secondary metabolites. Their biosynthetic mechanisms have been studied and three different peptide epimerization machineries have been reported. The first is non-ribosomal peptide synthetase (NRPS). Excellent studies with dissected modules of gramicidin synthetase and tyrocidine synthetase revealed the reactions of the epimerization domains embedded in the enzymes. The obtained information is still utilized to predict epimerization domains in uncharacterized NRPSs. The second includes the biosynthetic enzymes of lantibiotics, which are ribosome-dependently supplied peptide antibiotics containing polycyclic thioether amino acids (lanthionines). A mechanism for the formation of the D-Ala moiety in lanthionine by two enzymes, dehydratases catalyzing the conversion of L-Ser into dehydroalanine and enzymes catalyzing nucleophilic attack of the thiol of cysteine into dehydroalanine, was clarified. Similarly, the formation of a D-Ala residue by reduction of the dehydroalanine residue was also reported. The last type of machinery includes radical- S -adenosylmethionine (rSAM)-dependent enzymes, which catalyze a variety of radical-mediated chemical transformations. In the biosynthesis of polytheonamide, a marine sponge-derived and ribosome-dependently supplied peptide composed of 48 amino acids, a rSAM enzyme (PoyD) is responsible for unidirectional epimerizations of multiple different amino acids in the precursor peptide. In this review, we briefly summarize the discovery and current mechanistic understanding of these peptide epimerization enzymes.

Identification of the mpl gene encoding UDP-N-acetylmuramate: L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase in Escherichia coli and its role in recycling of cell wall peptidoglycan.

PubMed Central

Mengin-Lecreulx, D; van Heijenoort, J; Park, J T

1996-01-01

A gene, mpl, encoding UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelat e ligase was recognized by its amino acid sequence homology with murC as the open reading frame yjfG present at 96 min on the Escherichia coli map. The existence of such an enzymatic activity was predicted from studies indicating that reutilization of the intact tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate occurred and accounted for well over 30% of new cell wall synthesis. Murein tripeptide ligase activity could be demonstrated in crude extracts, and greatly increased activity was produced when the gene was cloned and expressed under control of the trc promoter. A null mutant totally lacked activity but was viable, showing that the enzyme is not essential for growth. PMID:8808921

Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF.

PubMed

Sova, Matej; Kovac, Andreja; Turk, Samo; Hrast, Martina; Blanot, Didier; Gobec, Stanislav

2009-12-01

Enzymes involved in the biosynthesis of bacterial peptidoglycan represent important targets for development of new antibacterial drugs. Among them, Mur ligases (MurC to MurF) catalyze the formation of the final cytoplasmic precursor UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid. We present the design, synthesis and biological evaluation of a series of phosphorylated hydroxyethylamines as new type of small-molecule inhibitors of Mur ligases. We show that the phosphate group attached to the hydroxyl moiety of the hydroxyethylamine core is essential for good inhibitory activity. The IC(50) values of these inhibitors were in the micromolar range, which makes them a promising starting point for the development of multiple inhibitors of Mur ligases as potential antibacterial agents. In addition, 1-(4-methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate 7a was discovered as one of the best inhibitors of MurE described so far.

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins--towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation.

PubMed

Daskalova, Sasha M; Radder, Josiah E; Cichacz, Zbigniew A; Olsen, Sam H; Tsaprailis, George; Mason, Hugh; Lopez, Linda C

2010-08-24

Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins. The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform. Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae*

PubMed Central

Asención Diez, Matías D.; Miah, Farzana; Stevenson, Clare E. M.; Lawson, David M.; Iglesias, Alberto A.; Bornemann, Stephen

2017-01-01

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli. However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae. The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate. PMID:27903647

Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an α-Gal epitope

PubMed Central

2010-01-01

Background Oligosaccharides containing a terminal Gal-α1,3-Gal moiety are collectively known as α-Gal epitopes. α-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the α-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the α-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the α-Gal epitope, Gal-α1,3-Lac. Results Agrobacterium sp. ATCC 31749 was engineered to produce Gal-α1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:α1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the α-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting α-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-α1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-α1,3-Lac. Conclusions The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of α-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the α-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides. PMID:20067629

Characterization and mutational analysis of the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis.

PubMed

Beerens, Koen; Soetaert, Wim; Desmet, Tom

2013-09-01

UDP-hexose 4-epimerases are important enzymes that play key roles in various biological pathways, including lipopolysaccharide biosynthesis, galactose metabolism through the Leloir pathway, and biofilm formation. Unfortunately, the determinants of their substrate specificity are not yet fully understood. They can be classified into three groups, with groups 1 and 3 preferring non-acetylated and acetylated UDP-hexoses, respectively, whereas members of group 2 are equally active on both types of substrates. In this study, the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis (mGalE) was functionally expressed in Escherichia coli and thoroughly characterized. The enzyme was found to be thermostable, displaying its highest activity at 70 °C and having a half-life of 23 min at 60 °C. Activity could be detected on both acetylated and non-acetylated UDP-hexoses, meaning that this epimerase belongs to group 2. This observation correlates well with the identity of the so-called "gatekeeper" residue (Ser279), which has previously been suggested to influence substrate specificity (Schulz et al., J Biol Chem 279:32796-32803, 2004). Furthermore, substituting this serine to a tyrosine brings about a significant preference for non-acetylated sugars, thereby demonstrating that a single residue can determine substrate specificity among type 1 and type 2 epimerases. In addition, two consecutive glycine residues (Gly118 and Gly119) were identified as a unique feature of GalE enzymes from Thermus species, and their importance for activity as well as affinity was confirmed by mutagenesis. Finally, homology modeling and mutational analysis has revealed that the enzyme's catalytic triad contains a threonine residue (Thr117) instead of the usual serine.

Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle

2015-10-15

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM hasmore » several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 {angstrom} to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.« less

Biophysical Characterization and Activity of Lymphostatin, a Multifunctional Virulence Factor of Attaching and Effacing Escherichia coli *

PubMed Central

Cassady-Cain, Robin L.; Blackburn, Elizabeth A.; Alsarraf, Husam; Dedic, Emil; Bease, Andrew G.; Böttcher, Bettina; Jørgensen, René; Wear, Martin; Stevens, Mark P.

2016-01-01

Attaching and effacing Escherichia coli cause diarrhea and typically produce lymphostatin (LifA), an inhibitor of mitogen-activated proliferation of lymphocytes and pro-inflammatory cytokine synthesis. A near-identical factor (Efa1) has been reported to mediate adherence of E. coli to epithelial cells. An amino-terminal region of LifA shares homology with the catalytic domain of the large clostridial toxins, which are retaining glycosyltransferases with a DXD motif involved in binding of a metal ion. Understanding the mode(s) of action of lymphostatin has been constrained by difficulties obtaining a stably transformed plasmid expression clone. We constructed a tightly inducible clone of enteropathogenic E. coli O127:H6 lifA for affinity purification of lymphostatin. The purified protein inhibited mitogen-activated proliferation of bovine T lymphocytes in the femtomolar range. It is a monomer in solution and the molecular envelope was determined using both transmission electron microscopy and small-angle x-ray scattering. Domain architecture was further studied by limited proteolysis. The largest proteolytic fragment containing the putative glycosyltransferase domain was tested in isolation for activity against T cells, and was not sufficient for activity. Tryptophan fluorescence studies indicated thatlymphostatin binds uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) but not UDP-glucose (UDP-Glc). Substitution of the predicted DXD glycosyltransferase motif with alanine residues abolished UDP-GlcNAc binding and lymphostatin activity, although other biophysical properties were unchanged. The data indicate that lymphostatin has UDP-sugar binding potential that is critical for activity, and is a major leap toward identifying the nature and consequences of modifications of host cell factors. PMID:26786100

A multi-port 10GbE PCIe NIC featuring UDP offload and GPUDirect capabilities.

NASA Astrophysics Data System (ADS)

Ammendola, Roberto; Biagioni, Andrea; Frezza, Ottorino; Lamanna, Gianluca; Lo Cicero, Francesca; Lonardo, Alessandro; Martinelli, Michele; Stanislao Paolucci, Pier; Pastorelli, Elena; Pontisso, Luca; Rossetti, Davide; Simula, Francesco; Sozzi, Marco; Tosoratto, Laura; Vicini, Piero

2015-12-01

NaNet-10 is a four-ports 10GbE PCIe Network Interface Card designed for low-latency real-time operations with GPU systems. To this purpose the design includes an UDP offload module, for fast and clock-cycle deterministic handling of the transport layer protocol, plus a GPUDirect P2P/RDMA engine for low-latency communication with NVIDIA Tesla GPU devices. A dedicated module (Multi-Stream) can optionally process input UDP streams before data is delivered through PCIe DMA to their destination devices, re-organizing data from different streams guaranteeing computational optimization. NaNet-10 is going to be integrated in the NA62 CERN experiment in order to assess the suitability of GPGPU systems as real-time triggers; results and lessons learned while performing this activity will be reported herein.

Deletion of the Lymantria dispar multicapsid nucleopolyhedrovirus ecdysteroid UDP-glucosyl transferase gene enhances viral killing speed in the last instar of the gypsy moth

Treesearch

James M. Slavicek; Holly J.R. Popham; C.I. Riegel

1999-01-01

The Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) is used on a limited basis as a gypsy moth (L. dispar) control agent. In an effort to improve the efficacy (i.e., killing speed) of the LdMNPV, we generated a recombinant viral strain (vEGT-) that does not produce the enzyme ecdysteroid UDP-glucosyltransferase (EGT). We...

Molecular dynamics simulations of glycosyltransferase LgtC.

PubMed

Snajdrová, Lenka; Kulhánek, Petr; Imberty, Anne; Koca, Jaroslav

2004-04-02

Molecular dynamics simulations have been performed on fully solvated alpha-(1-->4)-galactosyltransferase LgtC from Neisseria meningitidis with and without the donor substrate UDP-Gal and in the presence of the manganese ion. The analysis of the trajectories revealed a limited movement in the loop X (residues 75-80) and a larger conformational change in the loop Y (residues 246-251) in the simulation, when UDP-Gal was not present. In this case, the loops X and Y open by almost 10A, exposing the active site to the solvent. The 'hinge region' responsible for the opening is composed of residues 246-247. We have also analyzed the behavior of the manganese ion in the simulations. The coordination number is 6 when UDP-Gal is present and it increases to 7 when it is absent. In the latter case, three water molecules become coordinated to the ion. In both cases, the coordination is very stable implying that the manganese ion is tightly bound in the active site of the enzyme even if UDP-Gal is not present. Further analysis of the structural water molecules location confirmed that the mobility of water molecules in the active site and the accessibility of this site for solvent are higher in the absence of the substrate.

Alkaloids and leishmania donovani UDP-galactopyarnose mutase: Anovel approach in drug designing against Visceral leishmaniasis.

PubMed

Srivastava, Ankita; Chandra, Deepak

2017-06-05

The unsatisfactory treatment options for Visceral Leishmaniasis (VL), needs identification of new drug targets. Among natural products, Alkaloids have been proved to be highly effective against number of diseases. In Leishmania UDP-galactopyranose mutase (UGM) is a critical enzyme required for cell wall synthesis and thus a drug target for structure based drug designing against L. donovani. To build the homology model of UDP galactopyranse mutase and investigate the interaction of selected alkaloids with this modeled UDP galactopyranose mutase by molecular docking. Since there is no crystal structure record has been found with this protein, a homology modeling was performed and a three dimensional structure of L. donovani UGM was created using MODELLER v9.9, structure quality was validated using PROCHECK and QMEAN programs which confirms that the structure is reliable. Further Molecular docking was performed with previously reported 15 alkaloids. It was found that Protopine shows a binding energy of -12.39Kcal/mole, binds at Flavin adenine dinucleotide (FAD) biding site. Concluding that Protopine, an alkaloid could interrupt the functional aspect of L. donovani UGM and thus may be useful for drug designing studies. These finding would contribute to the understanding of effect of drug on the parasite. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Development of an in vivo glucosylation platform by coupling production to growth: Production of phenolic glucosides by a glycosyltransferase of Vitis vinifera.

PubMed

De Bruyn, Frederik; De Paepe, Brecht; Maertens, Jo; Beauprez, Joeri; De Cocker, Pieter; Mincke, Stein; Stevens, Christian; De Mey, Marjan

2015-08-01

Glycosylation of small molecules can significantly alter their properties such as solubility, stability, and/or bioactivity, making glycosides attractive and highly demanded compounds. Consequently, many biotechnological glycosylation approaches have been developed, with enzymatic synthesis and whole-cell biocatalysis as the most prominent techniques. However, most processes still suffer from low yields, production rates and inefficient UDP-sugar formation. To this end, a novel metabolic engineering strategy is presented for the in vivo glucosylation of small molecules in Escherichia coli W. This strategy focuses on the introduction of an alternative sucrose metabolism using sucrose phosphorylase for the direct and efficient generation of glucose 1-phosphate as precursor for UDP-glucose formation and fructose, which serves as a carbon source for growth. By targeted gene deletions, a split metabolism is created whereby glucose 1-phosphate is rerouted from the glycolysis to product formation (i.e., glucosylation). Further, the production pathway was enhanced by increasing and preserving the intracellular UDP-glucose pool. Expression of a versatile glucosyltransferase from Vitis vinifera (VvGT2) enabled the strain to efficiently produce 14 glucose esters of various hydroxycinnamates and hydroxybenzoates with conversion yields up to 100%. To our knowledge, this fast growing (and simultaneously producing) E. coli mutant is the first versatile host described for the glucosylation of phenolic acids in a fermentative way using only sucrose as a cheap and sustainable carbon source. © 2015 Wiley Periodicals, Inc.

Comparison of the UDP-N-Acetylmuramate:l-Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

PubMed Central

Mahapatra, Sebabrata; Crick, Dean C.; Brennan, Patrick J.

2000-01-01

In the peptidoglycan of Mycobacterium leprae, l-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:l-alanine ligase) of M. leprae showed Km and Vmax for l-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine. PMID:11073931

Multicluster

DTIC Science & Technology

1993-03-01

CLUSTER A CLUSTER B .UDP D "Orequeqes ProxyDistribute 0 Figure 4-4: HOSTALL Implementation HOST_ALL is implemented as follows. The kernel looks up the...it includes the HOSTALL request as an argument. The generic CronusHost object is managed by the Cronus Kernel. A kernel that receives a ProxyDistnbute...request uses its cached service information to send the HOSTALL request to each host in its cluster via UDP. If the kernel has no cached information

Predictive Displays for High Latency Teleoperation

DTIC Science & Technology

2016-08-04

PREDICTIVE DISPLAYS FOR HIGH LATENCY TELEOPERATION” Analysis of existing approach 3 C om m s. C hannel Vehicle OCU D Throttle, Steer, Brake D Video ...presents opportunity mitigate outgoing latency. • Video is not governed by physics, however, video is dependent on the state of the vehicle, which...Commands, estimates UDP: H.264 Video UDP: Vehicle state • C++ implementation • 2 threads • OpenCV for image manipulation • FFMPEG for video decoding

Distributed Cognition and Process Management Enabling Individualized Translational Research: The NIH Undiagnosed Diseases Program Experience

PubMed Central

Links, Amanda E.; Draper, David; Lee, Elizabeth; Guzman, Jessica; Valivullah, Zaheer; Maduro, Valerie; Lebedev, Vlad; Didenko, Maxim; Tomlin, Garrick; Brudno, Michael; Girdea, Marta; Dumitriu, Sergiu; Haendel, Melissa A.; Mungall, Christopher J.; Smedley, Damian; Hochheiser, Harry; Arnold, Andrew M.; Coessens, Bert; Verhoeven, Steven; Bone, William; Adams, David; Boerkoel, Cornelius F.; Gahl, William A.; Sincan, Murat

2016-01-01

The National Institutes of Health Undiagnosed Diseases Program (NIH UDP) applies translational research systematically to diagnose patients with undiagnosed diseases. The challenge is to implement an information system enabling scalable translational research. The authors hypothesized that similar complex problems are resolvable through process management and the distributed cognition of communities. The team, therefore, built the NIH UDP integrated collaboration system (UDPICS) to form virtual collaborative multidisciplinary research networks or communities. UDPICS supports these communities through integrated process management, ontology-based phenotyping, biospecimen management, cloud-based genomic analysis, and an electronic laboratory notebook. UDPICS provided a mechanism for efficient, transparent, and scalable translational research and thereby addressed many of the complex and diverse research and logistical problems of the NIH UDP. Full definition of the strengths and deficiencies of UDPICS will require formal qualitative and quantitative usability and process improvement measurement. PMID:27785453

X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa

DOE PAGES

Welner, Ditte Hededam; Tsai, Alex Yi-Lin; DeGiovanni, Andy M.; ...

2017-03-29

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstratemore » DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.« less

Study of atmospheric scattering and absorbing aerosols at 550 nm over nearby western Indian tropical sites of Thar Desert effected region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vyas, B. M., E-mail: bmvyas@yahoo.com; Saxenna, Abhishek; Panwar, Chhagan

The first time experimental results based on spaced satellite observations of different kinds of aerosols properties have been described over two different contrast environmental conditions locations in western tropical Indian region specifically first at Jaisalmer (26.90°N, 69.90°E, 220 m above mean sea level (amsl)) located in central Thar dessert vicinity of western Indian site over Indian Thar Desert region and another at Udaipur (24.6° N, 73.7° E, 560 m amsl) site concerning to semi-urban and semi arid place of hilly areas. The daily values of aerosols optical depth absorption at 500nm (AOD abs 500nm), aerosols optical depth extinction at 500nmmore » (AOD ext 500nm) along with aerosols optical depth at 500nmon (AOD 500nm) of eleven year period from Jan., 2004 to Dec., 2014 are basis of primary database of the present investigation. From the synthesis if the above database and the basis of rigorous statistical approach, following some of interesting facts are noted (i) larger annual monthly AOD variation of 0.93 is noted over JSM when compared to observed annual monthly change in AOD cycle, over UDP, of only 0.50 clearly indicating the more impact of desert influence activities about more than double times over JSM than UDP (ii) The higher abundance of absorbing aerosols occurrences about two time higher are seen in JSM in comparison to UDP. It indicates the clear evidence of strong optical absorption properties of useful solar mid visible wavelength at 550nm as the results of presence of more availability of dust aerosols as mineral natural type in pre-monsoon to post-monsoon over JSM which is also more predominant over JSM than the UDP region located far away from desert activity regime (iii) The greater sharing of extinction solar radiation effect on aerosols are more effective in pre-monsoon in UDP in reference to over JSM, where as in case of UDP, the aerosols effect through the scattering mechanism gradually reduce from monsoon to winter months as compared to observed over JSM. The more detailed analysis other important results are also discussed thoroughly in this paper.« less

Optimized UDP-glucuronosyltransferase (UGT) activity assay for trout liver S9 fractions

EPA Pesticide Factsheets

This publication provides an optimized UGT assay for trout liver S9 fractions which can be used to perform in vitro-in vivo extrapolations of measured UGT activityThis dataset is associated with the following publication:Ladd, M., P. Fitzsimmons , and J. Nichols. Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: Activity enhancement by alamethicin, a pore-forming peptide. XENOBIOTICA. Taylor & Francis, Inc., Philadelphia, PA, USA, 46(12): 1066-1075, (2016).

Strategies for Transporting Data Between Classified and Unclassified Networks

DTIC Science & Technology

2016-03-01

datagram protocol (UDP) must be used. The UDP is typically used when speed is a higher priority than data integrity, such as in music or video streaming ...and the exit point of data are separate and can be tightly controlled. This does effectively prevent the comingling of data and is used in industry to...perform functions such as streaming video and audio from secure to insecure networks (ref. 1). A second disadvantage lies in the fact that the

Parallel increase of dolichol pathway and nucleotide pyrophosphatases in rat hepatocytes by dexamethasone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sarkar, M.; Mookerjea, S.

1986-05-01

Incorporation of (/sup 14/C)-mannose to dolichol phosphate mannose, dolichol pyrophosphate oligosaccharide and N-linked glycoproteins in cultured hepatocytes was increased by dexamethasone. Nucleotide pyrophosphatases are now measured to investigate possible control of glycosylation by the nucleotide sugar pools. Dexamethasone caused about 2 fold increase of UDP-GlcNAc and GDP-Man pyrophosphatase activity which is evident as early as 4 hr and increased up to 12 hr of incubation. The K/sub m/ for UDP-GlcNAc and GDP-Man were respectively 0.43 mM and 0.47 mM in homogenate membrane and the values remained unchanged by dexamethasone treatment. However the V/sub max/ of the enzymes were increased withmore » both UDP-GlcNAc and GDP-Man. The broad pH optima of the enzymes (pH 8 to 10) indicated their alkaline nature. Mixing experiments of the cell homogenates from control and dexamethasone treated cells showed that UDP-GlcNAc and GDP-Man pyrophosphatase activities were additive which ruled out the possibility of presence of any activator or removal of any inhibitor due to dexamethasone. The parallel increase of nucleotide pyrophosphatase and dolichol linked pathway by dexamethasone does not support the possibility that stimulation of glycoprotein synthesis by dexamethasone is mediated by transfer of nucleotide sugars towards dolichol saccharides.« less

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta

PubMed Central

Guns, Pieter-Jan D F; Korda, András; Crauwels, Herta M; Van Assche, Tim; Robaye, Bernard; Boeynaems, Jean-Marie; Bult, Hidde

2005-01-01

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5′ triphosphate (ATP), uridine 5′ triphosphate (UTP), uridine 5′ diphosphate (UDP); >90%) or partial (adenosine 5′ diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP∼UTP∼ADP>adenosine 5′-[γ-thio] triphosphate (ATPγS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53±0.07) was compatible with literature, but the pKb for UTP (5.19±0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2′-deoxy-N6-methyladenosine3′,5′-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation. PMID:15997227

Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

PubMed Central

Asención Diez, Matías D.; Peirú, Salvador; Demonte, Ana M.; Gramajo, Hugo

2012-01-01

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high Vmax in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (Vmax of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium. PMID:22210767

Biochemical and Molecular Characterization of a Flavonoid 3-O-glycosyltransferase Responsible for Anthocyanins and Flavonols Biosynthesis in Freesia hybrida

PubMed Central

Sun, Wei; Liang, Lingjie; Meng, Xiangyu; Li, Yueqing; Gao, Fengzhan; Liu, Xingxue; Wang, Shucai; Gao, Xiang; Wang, Li

2016-01-01

The glycosylation of flavonoids increases their solubility and stability in plants. Flowers accumulate anthocyanidin and flavonol glycosides which are synthesized by UDP-sugar flavonoid glycosyltransferases (UFGTs). In our previous study, a cDNA clone (Fh3GT1) encoding UFGT was isolated from Freesia hybrida, which was preliminarily proved to be invovled in cyanidin 3-O-glucoside biosynthesis. Here, a variety of anthocyanin and flavonol glycosides were detected in flowers and other tissues of F. hybrida, implying the versatile roles of Fh3GT1 in flavonoids biosynthesis. To further unravel its multi-functional roles, integrative analysis between gene expression and metabolites was investigated. The results showed expression of Fh3GT1 was positively related to the accumulation of anthocyanins and flavonol glycosides, suggesting its potential roles in the biosynthesis of both flavonoid glycosides. Subsequently, biochemical analysis results revealed that a broad range of flavonoid substrates including flavonoid not naturally occurred in F. hybrida could be recognized by the recombinant Fh3GT1. Both UDP-glucose and UDP-galactose could be used as sugar donors by recombinant Fh3GT1, although UDP-galactose was transferred with relatively low activity. Furthermore, regiospecificity analysis demonstrated that Fh3GT1 was able to glycosylate delphinidin at the 3-, 4-′, and 7- positions in a sugar-dependent manner. And the introduction of Fh3GT1 into Arabidopsis UGT78D2 mutant successfully restored the anthocyanins and flavonols phenotypes caused by lost-of-function of the 3GT, indicating that Fh3GT1 functions as a flavonoid 3-O-glucosyltransferase in vivo. In summary, these results demonstrate that Fh3GT1 is a flavonoid 3-O-glycosyltransferase using UDP-glucose as the preferred sugar donor and may involve in flavonoid glycosylation in F. hybrida. PMID:27064818

Biochemical and Molecular Characterization of a Flavonoid 3-O-glycosyltransferase Responsible for Anthocyanins and Flavonols Biosynthesis in Freesia hybrida.

PubMed

Sun, Wei; Liang, Lingjie; Meng, Xiangyu; Li, Yueqing; Gao, Fengzhan; Liu, Xingxue; Wang, Shucai; Gao, Xiang; Wang, Li

2016-01-01

The glycosylation of flavonoids increases their solubility and stability in plants. Flowers accumulate anthocyanidin and flavonol glycosides which are synthesized by UDP-sugar flavonoid glycosyltransferases (UFGTs). In our previous study, a cDNA clone (Fh3GT1) encoding UFGT was isolated from Freesia hybrida, which was preliminarily proved to be invovled in cyanidin 3-O-glucoside biosynthesis. Here, a variety of anthocyanin and flavonol glycosides were detected in flowers and other tissues of F. hybrida, implying the versatile roles of Fh3GT1 in flavonoids biosynthesis. To further unravel its multi-functional roles, integrative analysis between gene expression and metabolites was investigated. The results showed expression of Fh3GT1 was positively related to the accumulation of anthocyanins and flavonol glycosides, suggesting its potential roles in the biosynthesis of both flavonoid glycosides. Subsequently, biochemical analysis results revealed that a broad range of flavonoid substrates including flavonoid not naturally occurred in F. hybrida could be recognized by the recombinant Fh3GT1. Both UDP-glucose and UDP-galactose could be used as sugar donors by recombinant Fh3GT1, although UDP-galactose was transferred with relatively low activity. Furthermore, regiospecificity analysis demonstrated that Fh3GT1 was able to glycosylate delphinidin at the 3-, 4-', and 7- positions in a sugar-dependent manner. And the introduction of Fh3GT1 into Arabidopsis UGT78D2 mutant successfully restored the anthocyanins and flavonols phenotypes caused by lost-of-function of the 3GT, indicating that Fh3GT1 functions as a flavonoid 3-O-glucosyltransferase in vivo. In summary, these results demonstrate that Fh3GT1 is a flavonoid 3-O-glycosyltransferase using UDP-glucose as the preferred sugar donor and may involve in flavonoid glycosylation in F. hybrida.

Elucidation of the biosynthesis of the di-C-glycosylflavone isoschaftoside, an allelopathic component from Desmodium spp. that inhibits Striga spp. development.

PubMed

Hamilton, Mary L; Kuate, Serge P; Brazier-Hicks, Melissa; Caulfield, John C; Rose, Ruth; Edwards, Robert; Torto, Baldwyn; Pickett, John A; Hooper, Antony M

2012-12-01

Isoschaftoside, an allelopathic di-C-glycosylflavone from Desmodium spp. root exudates, is biosynthesised through sequential glucosylation and arabinosylation of 2-hydroxynaringenin with UDP-glucose and UDP-arabinose. Complete conversion to the flavone requires chemical dehydration implying a dehydratase enzyme has a role in vivo to complete the biosynthesis. The C-glucosyltransferase has been partially characterised and its activity demonstrated in highly purified fractions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Riegert, Alexander S.; Thoden, James B.; Schoenhofen, Ian C.

Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc. The pathway for its biosynthesis, initiating with UDP-GlcNAc, requires three enzymes referred to as PglF, PglE, and PlgD. The focus of this investigation is on PglF, an NAD+-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PglF catalyzes the first step in the pathway, namely, themore » dehydration of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose. Most members of the SDR superfamily contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in PglF, this residue is a methionine. Here we describe a detailed structural and functional investigation of PglF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 Å or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that does not require the typically conserved tyrosine residue.« less

MurD ligase from E. coli: Tetrahedral intermediate formation study by hybrid quantum mechanical/molecular mechanical replica path method.

PubMed

Perdih, Andrej; Hodoscek, Milan; Solmajer, Tom

2009-02-15

MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase), a three-domain bacterial protein, catalyses a highly specific incorporation of D-glutamate to the cytoplasmic intermediate UDP-N-acetyl-muramoyl-L-alanine (UMA) utilizing ATP hydrolysis to ADP and P(i). This reaction is part of a biosynthetic path yielding bacterial peptidoglycan. On the basis of structural studies of MurD complexes, a stepwise catalytic mechanism was proposed that commences with a formation of the acyl-phosphate intermediate, followed by a nucleophilic attack of D-glutamate that, through the formation of a tetrahedral reaction intermediate and subsequent phosphate dissociation, affords the final product, UDP-N-acetyl-muramoyl-L-alanine-D-glutamate (UMAG). A hybrid quantum mechanical/molecular mechanical (QM/MM) molecular modeling approach was utilized, combining the B3LYP QM level of theory with empirical force field simulations to evaluate three possible reaction pathways leading to tetrahedral intermediate formation. Geometries of the starting structures based on crystallographic experimental data and tetrahedral intermediates were carefully examined together with a role of crucial amino acids and water molecules. The replica path method was used to generate the reaction pathways between the starting structures and the corresponding tetrahedral reaction intermediates, offering direct comparisons with a sequential kinetic mechanism and the available structural data for this enzyme. The acquired knowledge represents new and valuable information to assist in the ongoing efforts leading toward novel inhibitors of MurD as potential antibacterial drugs. (c) 2008 Wiley-Liss, Inc.

Glycyrrhetinic acid exhibits strong inhibitory effects towards UDP-glucuronosyltransferase (UGT) 1A3 and 2B7.

PubMed

Huang, Yin-Peng; Cao, Yun-Feng; Fang, Zhong-Ze; Zhang, Yan-Yan; Hu, Cui-Min; Sun, Xiao-Yu; Yu, Zhen-Wen; Zhu, Xu; Hong, Mo; Yang, Lu; Sun, Hong-Zhi

2013-09-01

The aim of the present study is to evaluate the inhibitory effects of liver UDP-glucuronosyltransferases (UGTs) by glycyrrhizic acid and glycyrrhetinic acid, which are the bioactive ingredients isolated from licorice. The results showed that glycyrrhetinic acid exhibited stronger inhibition towards all the tested UGT isoforms, indicating that the deglycosylation process played an important role in the inhibitory potential towards UGT isoforms. Furthermore, the inhibition kinetic type and parameters were determined for the inhibition of glycyrrhetinic acid towards UGT1A3 and UGT2B7. Data fitting using Dixon and Lineweaver-Burk plots demonstrated that the inhibition of UGT1A3 and UGT2B7 by glycyrrhetinic acid was best fit to competitive and noncompetitive type, respectively. The second plot using the slopes from Lineweaver-Burk plots versus glycyrrhetinic acid concentrations was employed to calculate the inhibition kinetic parameters (K(i)), and the values were calculated to be 0.2 and 1.7 μM for UGT1A3 and UGT2B7, respectively. All these results remind us the possibility of UGT inhibition-based herb-drug interaction. However, the explanation of these in vitro parameters should be paid more caution due to complicated factors, including the probe substrate-dependent UGT inhibition behaviour, environmental factors affecting the abundance of herbs' ingredients, and individual difference of pharmacokinetic factors. Copyright © 2012 John Wiley & Sons, Ltd.

A Novel Eliminase from a Marine Bacterium That Degrades Hyaluronan and Chondroitin Sulfate*

PubMed Central

Han, Wenjun; Wang, Wenshuang; Zhao, Mei; Sugahara, Kazuyuki; Li, Fuchuan

2014-01-01

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ4,5HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications. PMID:25122756

A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

PubMed

Han, Wenjun; Wang, Wenshuang; Zhao, Mei; Sugahara, Kazuyuki; Li, Fuchuan

2014-10-03

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic polymorphism of UDP-glucuronosyltransferase (UGT2B15) and glucuronidation of paracetamol in healthy population.

PubMed

Mehboob, Huma; Iqbal, Tahira; Jamil, Amer; Khaliq, Tanweer

2016-05-01

Inter individual variability in polymorphic UDP-glucuronosyltransferase (UGT2B15) has been associated with varied glucuronidation level. The present project was designed to determine the genetic polymorphism of UDP-glucuronosyltransferase (UGT2B15) and glucuronidation of paracetamol in healthy (male=59 and female=50) population. The association between genotype (UGT2B15) and phenotype (paracetamol glucuronidation) has been evaluated. According to trimodal model, genotypes and phenotypes were categorized as fast, intermediate and slow glucuronidators. Presence of wild type allele illustrated a UGT2B15 genotype as fast glucuronidator. The glucuronidation status was investigated by HPLC analysis of paracetamol. Ratio of paracetamol glucuronide to paracetamol was determined with two antimodes at glucuronidation ratio of 0.3 and 1.8. In our study, 7% and 12% of population was distributed as slow glucuronidators by phenotype and genotype, respectively and association between phenotype and genotype was good for analysis of glucuronidation status as displayed by kappa value (0.792).

A review on transport layer protocol performance for delivering video on an adhoc network

NASA Astrophysics Data System (ADS)

Suherman; Suwendri; Al-Akaidi, Marwan

2017-09-01

The transport layer protocol is responsible for the end to end data transmission. Transmission control protocol (TCP) provides a reliable connection and user datagram protocol (UDP) offers fast but unguaranteed data transfer. Meanwhile, the 802.11 (wireless fidelity/WiFi) networks have been widely used as internet hotspots. This paper evaluates TCP, TCP variants and UDP performances for video transmission on an adhoc network. The transport protocol - medium access cross-layer is proposed by prioritizing TCP acknowledgement to reduce delay. The NS-2 evaluations show that the average delays increase linearly for all the evaluated protocols and the average packet losses grow logarithmically. UDP produces the lowest transmission delay; 5.4% and 5.8% lower than TCP and TCP variant, but experiences the highest packet loss. Both TCP and TCP Vegas maintain packet loss as low as possible. The proposed cross-layer successfully decreases TCP and TCP Vegas delay about 0.12 % and 0.15%, although losses remain similar.

Bioactivation of carboxylic acid compounds by UDP-Glucuronosyltransferases to DNA-damaging intermediates: role of glycoxidation and oxidative stress in genotoxicity.

PubMed

Sallustio, Benedetta C; Degraaf, Yvette C; Weekley, Josephine S; Burcham, Philip C

2006-05-01

Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin > gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P < 0.05 Kruskal-Wallis). Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51 x and 2.57 x control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10 x more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and clofibric acid, induced DNA damage in isolated hepatocytes via glucuronidation- dependent pathways. These findings suggest acyl glucuronides are able to access and damage nuclear DNA via iron-catalyzed glycation/glycoxidative processes.

Distribution of NTPDase5 and NTPDase6 and the regulation of P2Y receptor signalling in the rat cochlea

PubMed Central

O’Keeffe, Mary G.; Thorne, Peter R.; Housley, Gary D.; Robson, Simon C.

2010-01-01

Membrane-bound ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) in the inner ear regulate complex extracellular purinergic type-2 (P2) receptor signalling pathways through hydrolysis of extracellular nucleoside 5′-triphosphates and diphosphates. This study investigated the distribution of NTPDase5 and NTPDase6, two intracellular members of the E-NTPDase family, and linked this to regulation of P2 receptor signalling in the adult rat cochlea. These extracellular ectonucleotidases preferentially hydrolyse nucleoside 5′-diphosphates such as UDP and GDP. Expression of both enzymes at mRNA and protein level was detected in cochlear tissues and there was in vivo release of soluble NTPDase5 and 6 into cochlear fluids. Strong NTPDase5 immunostaining was found in the spiral ganglion neurones and supporting Deiters’ cells of the organ of Corti, while NTPDase6 was confined to the inner hair cells. Upregulation of NTPDase5 after exposure to loud sound indicates a dynamic role for NTPDase5 in cochlear response to stress, whereas NTPDase6 may have more limited extracellular roles. Noise-induced upregulation of co-localised UDP-preferring P2Y6 receptors in the spiral ganglion neurons further supports the involvement of NTPDase5 in regulation of P2Y receptor signalling. Noise stress also induced P2Y14 (UDP- and UDP-glucose preferring) receptor expression in the root processes of the outer sulcus cells, but this was not associated with localization of the E-NTPDases. PMID:20806016

Activation of pollen tube callose synthase by detergents. Evidence for different mechanisms of action.

PubMed Central

Li, H; Bacic, A; Read, S M

1997-01-01

In pollen tubes of Nicotiana alata, a membrane-bound, Ca(2+)-independent callose synthase (CalS) is responsible for the biosynthesis of the (1,3)-beta-glucan backbone of callose, the main cell wall component. Digitonin increases CalS activity 3- to 4-fold over a wide range of concentrations, increasing the maximum initial velocity without altering the Michaelis constant for UDP-glucose. The CalS activity that requires digitonin for assay (the latent CalS activity) is not inhibited by the membrane-impermeant, active site-directed reagent UDP-pyridoxal when the reaction is conducted in the absence of digitonin. This is consistent with digitonin increasing CalS activity by the permeabilization of membrane vesicles. A second group of detergents, including 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Zwittergent 3-16, and 1-alpha-lysolecithin, activate pollen tube CalS 10- to 15-fold, but only over a narrow range of concentrations just below their respective critical micellar concentrations. This activation could not be attributed to any particular chemical feature of these detergents. CHAPS increases maximum initial velocity and decreases the Michaelis constant for UDP-glucose and activates CalS even in the presence of permeabilizing concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate that activation by CHAPS occurs by recruitment of previously inactive CalS molecules to the pool of active enzyme. The activation of pollen tube CalS by these detergents therefore resembles activation of the enzyme by trypsin. PMID:9276948

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae.

PubMed

Asención Diez, Matías D; Miah, Farzana; Stevenson, Clare E M; Lawson, David M; Iglesias, Alberto A; Bornemann, Stephen

2017-01-20

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Liver glucose metabolism in humans

PubMed Central

Adeva-Andany, María M.; Pérez-Felpete, Noemi; Fernández-Fernández, Carlos; Donapetry-García, Cristóbal; Pazos-García, Cristina

2016-01-01

Information about normal hepatic glucose metabolism may help to understand pathogenic mechanisms underlying obesity and diabetes mellitus. In addition, liver glucose metabolism is involved in glycosylation reactions and connected with fatty acid metabolism. The liver receives dietary carbohydrates directly from the intestine via the portal vein. Glucokinase phosphorylates glucose to glucose 6-phosphate inside the hepatocyte, ensuring that an adequate flow of glucose enters the cell to be metabolized. Glucose 6-phosphate may proceed to several metabolic pathways. During the post-prandial period, most glucose 6-phosphate is used to synthesize glycogen via the formation of glucose 1-phosphate and UDP–glucose. Minor amounts of UDP–glucose are used to form UDP–glucuronate and UDP–galactose, which are donors of monosaccharide units used in glycosylation. A second pathway of glucose 6-phosphate metabolism is the formation of fructose 6-phosphate, which may either start the hexosamine pathway to produce UDP-N-acetylglucosamine or follow the glycolytic pathway to generate pyruvate and then acetyl-CoA. Acetyl-CoA may enter the tricarboxylic acid (TCA) cycle to be oxidized or may be exported to the cytosol to synthesize fatty acids, when excess glucose is present within the hepatocyte. Finally, glucose 6-phosphate may produce NADPH and ribose 5-phosphate through the pentose phosphate pathway. Glucose metabolism supplies intermediates for glycosylation, a post-translational modification of proteins and lipids that modulates their activity. Congenital deficiency of phosphoglucomutase (PGM)-1 and PGM-3 is associated with impaired glycosylation. In addition to metabolize carbohydrates, the liver produces glucose to be used by other tissues, from glycogen breakdown or from de novo synthesis using primarily lactate and alanine (gluconeogenesis). PMID:27707936

Lightweight active router-queue management for multimedia networking

NASA Astrophysics Data System (ADS)

Parris, Mark; Jeffay, Kevin; Smith, F. D.

1998-12-01

The Internet research community is promoting active queue management in routers as a proactive means of addressing congestion in the Internet. Active queue management mechanisms such as Random Early Detection (RED) work well for TCP flows but can fail in the presence of unresponsive UDP flows. Recent proposals extend RED to strongly favor TCP and TCP-like flows and to actively penalize `misbehaving' flows. This is problematic for multimedia flows that, although potentially well-behaved, do not, or can not, satisfy the definition of a TCP-like flow. In this paper we investigate an extension to RED active queue management called Class-Based Thresholds (CBT). The goal of CBT is to reduce congestion in routers and to protect TCP from all UDP flows while also ensuring acceptable throughput and latency for well-behaved UDP flows. CBT attempts to realize a `better than best effort' service for well-behaved multimedia flows that is comparable to that achieved by a packet or link scheduling discipline, however, CBT does this by queue management rather than by scheduling. We present results of experiments comparing our mechanisms to plain RED and to FRED, a variant of RED designed to ensure fair allocation of bandwidth amongst flows. We also compare CBT to a packet scheduling scheme. The experiments show that CBT (1) realizes protection for TCP, and (2) provides throughput and end-to-end latency for tagged UDP flows, that is better than that under FRED and RED and comparable to that achieved by packet scheduling. Moreover CBT is a lighter-weight mechanism than FRED in terms of its state requirements and implementation complexity.

Leloir Glycosyltransferases and Natural Product Glycosylation: Biocatalytic Synthesis of the C-Glucoside Nothofagin, a Major Antioxidant of Redbush Herbal Tea

PubMed Central

Bungaruang, Linda; Gutmann, Alexander; Nidetzky, Bernd

2013-01-01

Nothofagin is a major antioxidant of redbush herbal tea and represents a class of bioactive flavonoid-like C-glycosidic natural products. We developed an efficient enzymatic synthesis of nothofagin based on a one-pot coupled glycosyltransferase-catalyzed transformation that involves perfectly selective 3′-C-β-d-glucosylation of naturally abundant phloretin and applies sucrose as expedient glucosyl donor. C-Glucosyltransferase from Oryza sativa (rice) was used for phloretin C-glucosylation from uridine 5′-diphosphate (UDP)-glucose, which was supplied continuously in situ through conversion of sucrose and UDP catalyzed by sucrose synthase from Glycine max (soybean). In an evaluation of thermodynamic, kinetic, and stability parameters of the coupled enzymatic reactions, poor water solubility of the phloretin acceptor substrate was revealed as a major bottleneck of conversion efficiency. Using periodic feed of phloretin controlled by reaction progress, nothofagin concentrations (45 mM; 20 g l−1) were obtained that vastly exceed the phloretin solubility limit (5–10 mM). The intermediate UDP-glucose was produced from catalytic amounts of UDP (1.0 mM) and was thus recycled 45 times in the process. Benchmarked against comparable glycosyltransferase-catalyzed transformations (e.g., on quercetin), the synthesis of nothofagin has achieved intensification in glycosidic product formation by up to three orders of magnitude (μM→mM range). It thus makes a strong case for the application of Leloir glycosyltransferases in biocatalytic syntheses of glycosylated natural products as fine chemicals. PMID:24415961

Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation.

PubMed

Robles-Martinez, Leobarda; Mendez, Tavis L; Apodaca, Jennifer; Das, Siddhartha

2017-01-01

The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2-3 fold higher than of GlcCer. Although both activities follow Michaelis-Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production. Copyright © 2016 Elsevier B.V. All rights reserved.

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

PubMed

Meyer, Benjamin H; Shams-Eldin, Hosam; Albers, Sonja-Verena

2017-01-01

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D 100 ), IV (F 220 ) and V (F 264 ) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Hsp70-GlcNAc-binding activity is released by stress, proteasome inhibition, and protein misfolding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guinez, Celine; Mir, Anne-Marie; Leroy, Yves

2007-09-21

Numerous recent works strengthen the idea that the nuclear and cytosolic-specific O-GlcNAc glycosylation protects cells against injuries. We have first investigated O-GlcNAc level and Hsp70-GlcNAc-binding activity (HGBA) behaviour after exposure of HeLa and HepG{sub 2} cells to a wide variety of stresses. O-GlcNAc and HGBA responses were different according to the stress and according to the cell. HGBA was released for almost all stresses, while O-GlcNAc level was modified either upwards or downwards, depending to the stress. Against all expectations, we demonstrated that energy charge did not significantly vary with stress whereas UDP-GlcNAc pools were more dramatically affected even ifmore » differences in UDP-GlcNAc contents were not correlated with O-GlcNAc variations suggesting that O-GlcNAc transferase is itself finely regulated during cell injury. Finally, HGBA could be triggered by proteasome inhibition and by L-azetidine-2-carboxylic acid (a proline analogue) incorporation demonstrating that protein misfolding is one of the key-activator of this Hsp70 property.« less

Genetic dissection of floridean starch synthesis in the cytosol of the model dinoflagellate Crypthecodinium cohnii

PubMed Central

Dauvillée, David; Deschamps, Philippe; Ral, Jean-Philippe; Plancke, Charlotte; Putaux, Jean-Luc; Devassine, Jimi; Durand-Terrasson, Amandine; Devin, Aline; Ball, Steven G.

2009-01-01

Starch defines an insoluble semicrystalline form of storage polysaccharides restricted to Archaeplastida (red and green algae, land plants, and glaucophytes) and some secondary endosymbiosis derivatives of the latter. While green algae and land-plants store starch in plastids by using an ADP-glucose-based pathway related to that of cyanobacteria, red algae, glaucophytes, cryptophytes, dinoflagellates, and apicomplexa parasites store a similar type of polysaccharide named floridean starch in their cytosol or periplast. These organisms are suspected to store their floridean starch from UDP-glucose in a fashion similar to heterotrophic eukaryotes. However, experimental proof of this suspicion has never been produced. Dinoflagellates define an important group of both photoautotrophic and heterotrophic protists. We now report the selection and characterization of a low starch mutant of the heterotrophic dinoflagellate Crypthecodinium cohnii. We show that the sta1-1 mutation of C. cohnii leads to a modification of the UDP-glucose-specific soluble starch synthase activity that correlates with a decrease in starch content and an alteration of amylopectin structure. These experimental results validate the UDP-glucose-based pathway proposed for floridean starch synthesis. PMID:19940244

Survey of O-GlcNAc level variations in Xenopus laevis from oogenesis to early development.

PubMed

Dehennaut, Vanessa; Lefebvre, Tony; Leroy, Yves; Vilain, Jean-Pierre; Michalski, Jean-Claude; Bodart, Jean-François

2009-04-01

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I-V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.

Alternative methods for the median lethal dose (LD(50)) test: the up-and-down procedure for acute oral toxicity.

PubMed

Rispin, Amy; Farrar, David; Margosches, Elizabeth; Gupta, Kailash; Stitzel, Katherine; Carr, Gregory; Greene, Michael; Meyer, William; McCall, Deborah

2002-01-01

The authors have developed an improved version of the up-and-down procedure (UDP) as one of the replacements for the traditional acute oral toxicity test formerly used by the Organisation for Economic Co-operation and Development member nations to characterize industrial chemicals, pesticides, and their mixtures. This method improves the performance of acute testing for applications that use the median lethal dose (classic LD50) test while achieving significant reductions in animal use. It uses sequential dosing, together with sophisticated computer-assisted computational methods during the execution and calculation phases of the test. Staircase design, a form of sequential test design, can be applied to acute toxicity testing with its binary experimental endpoints (yes/no outcomes). The improved UDP provides a point estimate of the LD50 and approximate confidence intervals in addition to observed toxic signs for the substance tested. It does not provide information about the dose-response curve. Computer simulation was used to test performance of the UDP without the need for additional laboratory validation.

The metabolism of galactose in the human gastric mucous membrane.

PubMed

Kopacz-Jodczyk, T; Zwierz, K; Gałasiński, W

1984-12-01

After incubating pieces of human gastric mucous membrane with radioactive galactose, labeled metabolites of glycolysis (FDP,PEP,pyruvate):hexose and hexosamine intermediates in glycoconjugate biosynthesis (gal-1P, UDP-gal,acetylated hexosamines, and their phosphate esters), amino acids (glycine, alanine, and serine), and oxoglutarate as a metabolite of the citric acid cycle were isolated from the acid-soluble fraction. These results suggest that galactose in the human gastric mucous membrane is epimerized to glucose and metabolized in the glycolytic pathway together with oxidation in the citric acid cycle and in the direction of glycoconjugate biosynthesis.

A UDP-Glucose:Monoterpenol Glucosyltransferase Adds to the Chemical Diversity of the Grapevine Metabolome1[W

PubMed Central

Bönisch, Friedericke; Frotscher, Johanna; Stanitzek, Sarah; Rühl, Ernst; Wüst, Matthias; Bitz, Oliver; Schwab, Wilfried

2014-01-01

Terpenoids represent one of the major classes of natural products and serve different biological functions. In grape (Vitis vinifera), a large fraction of these compounds is present as nonvolatile terpene glycosides. We have extracted putative glycosyltransferase (GT) sequences from the grape genome database that show similarity to Arabidopsis (Arabidopsis thaliana) GTs whose encoded proteins glucosylate a diversity of terpenes. Spatial and temporal expression levels of the potential VvGT genes were determined in five different grapevine varieties. Heterologous expression and biochemical assays of candidate genes led to the identification of a UDP-glucose:monoterpenol β-d-glucosyltransferase (VvGT7). The VvGT7 gene was expressed in various tissues in accordance with monoterpenyl glucoside accumulation in grape cultivars. Twelve allelic VvGT7 genes were isolated from five cultivars, and their encoded proteins were biochemically analyzed. They varied in substrate preference and catalytic activity. Three amino acids, which corresponded to none of the determinants previously identified for other plant GTs, were found to be important for enzymatic catalysis. Site-specific mutagenesis along with the analysis of allelic proteins also revealed amino acids that impact catalytic activity and substrate tolerance. These results demonstrate that VvGT7 may contribute to the production of geranyl and neryl glucoside during grape ripening. PMID:24784757

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus.

PubMed

Patin, Delphine; Boniface, Audrey; Kovač, Andreja; Hervé, Mireille; Dementin, Sébastien; Barreteau, Hélène; Mengin-Lecreulx, Dominique; Blanot, Didier

2010-12-01

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His(6)-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted L-Ala, L-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for L-Ala. S. aureus MurE was very specific for L-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and L-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (L-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and L-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Indole-3-acetic acid UDP-glucosyltransferase from immature seeds of pea is involved in modification of glycoproteins.

PubMed

Ostrowski, Maciej; Hetmann, Anna; Jakubowska, Anna

2015-09-01

The glycosylation of auxin is one of mechanisms contributing to hormonal homeostasis. The enzyme UDPG: indole-3-ylacetyl-β-D-glucosyltransferase (IAA glucosyltransferase, IAGlc synthase) catalyzes the reversible reaction: IAA+UDPG↔1-O-IA-glucose+UDP, which is the first step in the biosynthesis of IAA-ester conjugates in monocotyledonous plants. In this study, we report IAA-glucosyltransferase isolated using a biochemical approach from immature seed of pea (Pisum sativum). The enzyme was purified by PEG fractionation, DEAE-Sephacel anion-exchange chromatography and preparative PAGE. LC-MS/MS analysis of tryptic peptides of the enzyme revealed the high identity with maize IAGlc synthase, but lack of homology with other IAA-glucosyltransferases from dicots. Biochemical characterization showed that of several acyl acceptors tested, the enzyme had the highest activity on IAA as the glucosyl acceptor (Km=0.52 mM, Vmax=161 nmol min(-1), kcat/Km=4.36 mM s(-1)) and lower activity on indole-3-propionic acid and 1-naphthalene acetic acid. Whereas indole-3-butyric acid and indole-3-propionic acid were competitive inhibitors of IAGlc synthase, D-gluconic acid lactone, an inhibitor of β-glucosidase activity, potentiated the enzyme activity at the optimal concentration of 0.3mM. Moreover, we demonstrated that the 1-O-IA-glucose synthesized by IAGlc synthase is the substrate for IAA labeling of glycoproteins from pea seeds indicating a possible role of this enzyme in the covalent modification of a class of proteins by a plant hormone. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of the urease-derived peptide Jaburetox on the central nervous system of Triatoma infestans (Insecta: Heteroptera).

PubMed

Galvani, Gerónimo L; Fruttero, Leonardo L; Coronel, María F; Nowicki, Susana; Demartini, Diogo R; Defferrari, Marina S; Postal, Melissa; Canavoso, Lilián E; Carlini, Célia R; Settembrini, Beatriz P

2015-02-01

Triatoma infestans is the main vector of Chagas'disease in Southern Cone countries. In triatomines, symptoms suggesting neurotoxicity were observed after treatment with Jaburetox (Jbtx), the entomotoxic peptide obtained from jackbean urease. Here, we study its effect in the central nervous system (CNS) of this species. Immunohistochemistry, Western blots, immunoprecipitation, two-dimensional electrophoresis, tandem mass spectrometry and enzymatic assays were performed. Anti-Jbtx antibody labeled somata of the antennal lobe only in Jbtx-treated insects. Western blot assays of nervous tissue using the same antibody reacted with a 61kDa protein band only in peptide-injected insects. Combination of immunoprecipitation, two-dimensional electrophoresis and tandem mass spectrometry identified UDP-N-acetylglucosamine pyrophosphorylase (UDP-GlcNAcP) as a molecular target for Jbtx. The activity of UDP-GlcNAcP increased significantly in the CNS of Jbtx-treated insects. The effect of Jbtx on the activity of nitric oxide synthase (NOS) and NO production was investigated as NO is a recognized messenger molecule in the CNS of T. infestans. NOS activity and NO levels decreased significantly in CNS homogenates of Jbtx-treated insects. UDP-GlcNAcP is a molecular target of Jbtx. Jbtx impaired the activity of T. infestans nitrergic system, which may be related with early behavioral effects. We report that the CNS of Triatoma infestans is a target for the entomotoxic peptide and propose that a specific area of the brain is involved. Besides potentially providing tools for control strategies of Chagas' disease vectors our data may be relevant in various fields of research as insect physiology, neurobiology and protein function. Copyright © 2014 Elsevier B.V. All rights reserved.

Extracellular nucleotides potentiate the cytosolic Ca2+, but not cyclic adenosine 3', 5'-monophosphate response to parathyroid hormone in rat osteoblastic cells.

PubMed

Kaplan, A D; Reimer, W J; Feldman, R D; Dixon, S J

1995-04-01

Binding to PTH to its cell surface receptor activates both adenylyl cyclase and phospholipase-C, leading to elevation of cytosolic cAMP and free Ca2+. We have shown previously that extracellular nucleotides interact with P2U and P2Y subtypes of purinoceptor on osteoblastic cells, both linked to Ca2+ mobilization. In the present study, we investigated possible interactions between nucleotide and PTH signaling pathways in osteoblastic cells. The cytosolic free Ca2+ concentration ([Ca2+]i) of UMR-106 osteoblastic cells was monitored by fluorescence spectrophotometry. PTH (0.01-1 microM; bovine 1-84 or human 1-34) induced a small transient elevation of [Ca2+]i, lasting less than 1 min. A number of nucleotides, including ATP, UTP, and UDP, induced transient elevation of [Ca2+]i and potentiated the subsequent Ca2+ response to PTH. Of the nucleotides tested, UDP was the most effective at potentiating the PTH-induced Ca2+ transient. Treatment of cells with UDP (100 microM for 2.5 min), but not inorganic phosphate or uridine, reversibly potentiated the Ca2+ response to PTH (0.1 microM) by 11 +/- 2-fold (mean +/- SEM; n = 39). In contrast, UDP did not affect the cAMP response to PTH, indicating a selective action on Ca2+ signaling. Potentiation of the Ca2+ signal was still observed in the absence of extracellular Ca2+, establishing that nucleotides enhance PTH-induced release of Ca2+ from intracellular stores. Studies using selective purinoceptor agonists suggest that potentiation of PTH signaling is mediated by the P2U receptor subtype. In vivo, nucleotides released during trauma or inflammation may modulate PTH-induced Ca2+ signaling in osteoblasts.

Functional Expression of Enterobacterial O-Polysaccharide Biosynthesis Enzymes in Bacillus subtilis

PubMed Central

Schäffer, Christina; Wugeditsch, Thomas; Messner, Paul; Whitfield, Chris

2002-01-01

The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-α-d-14C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli. PMID:12324313

INCORPORATION OF PHOSPHATE INTO GLYCOGEN BY GLYCOGEN SYNTHASE

PubMed Central

Contreras, Christopher J.; Segvich, Dyann M.; Mahalingan, Krishna; Chikwana, Vimbai M.; Kirley, Terence L.; Hurley, Thomas D.; DePaoli-Roach, Anna A.; Roach, Peter J.

2016-01-01

The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab 13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab 17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of 32P from [β-32P]UDP-glucose to glycogen by glycogen synthase. The 32P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The 32P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-32P]UDP generated in the reaction. Furthermore, [32P]UDP did not bind non-covalently to glycogen. The 32P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, perhaps C3, phosphorylation. PMID:27036853

Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y6 activation of the phospholipase C/inositol trisphosphate pathway

PubMed Central

Yu, Weiqun; Sun, Xiaofeng; Robson, Simon C.; Hill, Warren G.

2013-01-01

Bladder dysfunction characterized by abnormal bladder smooth muscle (BSM) contractions is pivotal to the disease process in overactive bladder, urge incontinence, and spinal cord injury. Purinergic signaling comprises one key pathway in modulating BSM contractility, but molecular mechanisms remain unclear. Here we demonstrate, using myography, that activation of P2Y6 by either UDP or a specific agonist (MRS 2693) induced a sustained increase in BSM tone (up to 2 mN) in a concentration-dependent manner. Notably, activation of P2Y6 enhanced ATP-mediated BSM contractile force by up to 45%, indicating synergistic interactions between P2X and P2Y signaling. P2Y6-activated responses were abolished by phospholipase C (PLC) and inositol trisphosphate (IP3) receptor antagonists U73122 and xestospongin C, demonstrating involvement of the PLC/IP3 signal pathway. Mice null for Entpd1, an ectonucleotidase on BSM, demonstrated increased force generation on P2Y6 activation (150%). Thus, in vivo perturbations to purinergic signaling resulted in altered P2Y6 activity and bladder contractility. We conclude that UDP, acting on P2Y6, regulates BSM tone and in doing so selectively maximizes P2X1-mediated contraction forces. This novel neurotransmitter pathway may play an important role in urinary voiding disorders characterized by abnormal bladder motility.—Yu, W., Sun, X., Robson, S. C., Hill, W. G. Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y6 activation of the phospholipase C/inositol trisphosphate pathway. PMID:23362118

Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy

PubMed Central

Kottom, Theodore J.; Hebrink, Deanne M.; Jenson, Paige E.; Ramirez-Prado, Jorge H.

2017-01-01

N-acetylglucosamine (GlcNAc) serves as an essential structural sugar on the cell surface of organisms. For example, GlcNAc is a major component of bacterial peptidoglycan, it is an important building block of fungal cell walls, including a major constituent of chitin and mannoproteins, and it is also required for extracellular matrix generation by animal cells. Herein, we provide evidence for a uridine diphospho (UDP)–GlcNAc pathway in Pneumocystis species. Using an in silico search of the Pneumocystis jirovecii and P. murina (Pm) genomic databases, we determined the presence of at least four proteins implicated in the Saccharomyces cerevisiae UDP-GlcNAc biosynthetic pathway. These genes, termed GFA1, GNA1, AGM1, and UDP-GlcNAc pyrophosphorylase (UAP1), were either confirmed to be present in the Pneumocystis genomes by PCR, or, in the case of Pm uap1 (Pmuap1), functionally confirmed by direct enzymatic activity assay. Expression analysis using quantitative PCR of Pneumocystis pneumonia in mice demonstrated abundant expression of the Pm uap1 transcript. A GlcNAc-binding recombinant protein and a novel GlcNAc-binding immune detection method both verified the presence of GlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using high-performance gas chromatography/mass spectrometry documented the presence of GlcNAc glycosyl residues. Pc was shown to synthesize GlcNAc in vitro. The competitive UDP-GlcNAc substrate synthetic inhibitor, nikkomycin Z, suppressed incorporation of GlcNAc by Pc preparations. Finally, treatment of rats with Pneumocystis pneumonia using nikkomycin Z significantly reduced organism burdens. Taken together, these data support an important role for GlcNAc generation in the cell surface of Pneumocystis organisms. PMID:27632412

Genome-wide analysis of family-1 UDP glycosyltransferases (UGT) and identification of UGT genes for FHB resistance in wheat (Triticum aestivum L.).

PubMed

He, Yi; Ahmad, Dawood; Zhang, Xu; Zhang, Yu; Wu, Lei; Jiang, Peng; Ma, Hongxiang

2018-04-19

Fusarium head blight (FHB), a devastating disease in wheat worldwide, results in yield loses and mycotoxin, such as deoxynivalenol (DON), accumulation in infected grains. DON also facilitates the pathogen colonization and spread of FHB symptoms during disease development. UDP-glycosyltransferase enzymes (UGTs) are known to contribute to detoxification and enhance FHB resistance by glycosylating DON into DON-3-glucoside (D3G) in wheat. However, a comprehensive investigation of wheat (Triticum aestivum) UGT genes is still lacking. In this study, we carried out a genome-wide analysis of family-1 UDP glycosyltransferases in wheat based on the PSPG conserved box that resulted in the identification of 179 putative UGT genes. The identified genes were clustered into 16 major phylogenetic groups with a lack of phylogenetic group K. The UGT genes were invariably distributed among all the chromosomes of the 3 genomes. At least 10 intron insertion events were found in the UGT sequences, where intron 4 was observed as the most conserved intron. The expression analysis of the wheat UGT genes using both online microarray data and quantitative real-time PCR verification suggested the distinct role of UGT genes in different tissues and developmental stages. The expression of many UGT genes was up-regulated after Fusarium graminearum inoculation, and six of the genes were further verified by RT-qPCR. We identified 179 UGT genes from wheat using the available sequenced wheat genome. This study provides useful insight into the phylogenetic structure, distribution, and expression patterns of family-1 UDP glycosyltransferases in wheat. The results also offer a foundation for future work aimed at elucidating the molecular mechanisms underlying the resistance to FHB and DON accumulation.

Engineering Sialic Acid Synthesis Ability in Insect Cells.

PubMed

Viswanathan, Karthik; Narang, Someet; Betenbaugh, Michael J

2015-01-01

Insect cells lack the ability to synthesize the sialic acid donor molecule CMP-sialic acid or its precursor, sialic acid. In this chapter, we describe a method to engineer CMP-sialic acid synthesis capability into Spodoptera frugiperda (Sf9) cells, a prototypical insect cell line, by recombinant expression of sialic acid synthesis pathway genes using baculovirus technology. Co-expression of a sialuria mutant UDP-GlcNAc-2-epimerase/ManNAc kinase (EKR263L), wild-type sialic acid 9-phosphate synthase (SAS), and wild-type CMP-sialic acid synthetase (CSAS) in the presence of GlcNAc leads to synthesis of CMP-sialic acids synthesis to support sialylation of N-glycans on glycoproteins.

Use Of Transgenic Mice In UDP-Glucuronosyltransferase (UGT) Studies

PubMed Central

Ou, Zhimin; Huang, Min; Zhao, Lizi; Xie, Wen

2009-01-01

Transgenic mouse models are useful to understand the function and regulation of drug metabolizing enzymes in vivo. This article is intended to describe the general strategies and to discuss specific examples on how to use transgenic, gene knockout, and humanized mice to study the function as well as genetic and pharmacological regulation of UDP-glucuronosyltransferases (UGTs). The physiological and pharmacological implications of transcription factor-mediated UGT regulation will also be discussed. The UGT-regulating transcription factors to be discussed in this article include nuclear hormone receptors (NRs), aryl hydrocarbon receptor (AhR), and nuclear factor erythroid 2-related factor 2 (Nrf2). PMID:20070245

Regioselectivity of Human UDP-Glucuronosyltransferase Isozymes in Flavonoid Biotransformation by Metal Complexation and Tandem Mass Spectrometry

PubMed Central

Robotham, Scott A.; Brodbelt, Jennifer S.

2011-01-01

Based on reactions with five flavonoids, the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC-MS/MS fragmentation patterns of [Co(II)(flavonoid – H)(4,7-diphenyl-1,10-phenanthroline)2]+ complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme. The various UGT enzymes, including eight UGT1A and four UGT2B, displayed a remarkable range of selectivities, both in terms of the positions of glucuronidation and relative reactivity with flavanones versus flavonols. PMID:21889496

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem.

PubMed

Patin, Delphine; Bostock, Julieanne; Chopra, Ian; Mengin-Lecreulx, Dominique; Blanot, Didier

2012-06-01

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses.

PubMed

Geisler, Matt; Wilczynska, Malgorzata; Karpinski, Stanislaw; Kleczkowski, Leszek A

2004-11-01

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.

Genes for seed longevity in barley identified by genomic analysis on Near Isogenic Lines.

PubMed

Wozny, Dorothee; Kramer, Katharina; Finkemeier, Iris; Acosta, Ivan F; Koornneef, Maarten

2018-05-09

Genes controlling differences in seed longevity between two barley (Hordeum vulgare) accessions were identified by combining quantitative genetics 'omics' technologies in Near Isogenic Lines (NILs). The NILs were derived from crosses between the spring barley landraces L94 from Ethiopia and Cebada Capa from Argentina. A combined transcriptome and proteome analysis on mature, non-aged seeds of the two parental lines and the L94 NILs by RNA-sequencing and total seed proteomic profiling identified the UDP-glycosyltransferase MLOC_11661.1 as candidate gene for the QTL on 2H, and the NADP-dependent malic enzyme (NADP-ME) MLOC_35785.1 as possible downstream target gene. To validate these candidates, they were expressed in Arabidopsis under the control of constitutive promoters to attempt complementing the T-DNA knock-out line nadp-me1. Both the NADP-ME MLOC_35785.1 and the UDP-glycosyltransferase MLOC_11661.1 were able to rescue the nadp-me1 seed longevity phenotype. In the case of the UDP-glycosyltransferase, with high accumulation in NILs, only the coding sequence of Cebada Capa had a rescue effect. This article is protected by copyright. All rights reserved.

Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Payyavula, Raja S.; Tschaplinski, Timothy J.; Jawdy, Sara

Background: UDP-glucose pyrophopharylase (UGPase) is a sugar metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and uridine triphosphate glucose. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in woody plants such as Populus is poorly understood. Results: We characterized the functional role of UGPase in Populus deltoides by overexpressing a native gene. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of UGPase results in perturbations inmore » primary as well as secondary metabolism resulting in reduced sugar and starch levels and increased phenolics such as caffeoyl- and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced. Conclusions: These results demonstrate that UGPase plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism outside of cell wall biosynthesis of Populus.« less

Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus

DOE PAGES

Payyavula, Raja S.; Tschaplinski, Timothy J.; Jawdy, Sara; ...

2014-10-07

Background: UDP-glucose pyrophopharylase (UGPase) is a sugar metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and uridine triphosphate glucose. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in woody plants such as Populus is poorly understood. Results: We characterized the functional role of UGPase in Populus deltoides by overexpressing a native gene. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of UGPase results in perturbations inmore » primary as well as secondary metabolism resulting in reduced sugar and starch levels and increased phenolics such as caffeoyl- and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced. Conclusions: These results demonstrate that UGPase plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism outside of cell wall biosynthesis of Populus.« less

Inhibition of UDP-glucuronosyltransferase (UGT)-mediated glycyrrhetinic acid 3-O-glucuronidation by polyphenols and triterpenoids.

PubMed

Koyama, Mayuko; Shirahata, Tatsuya; Hirashima, Rika; Kobayashi, Yoshinori; Itoh, Tomoo; Fujiwara, Ryoichi

2017-08-01

Glycyrrhetinic acid (GA) is an active metabolite of glycyrrhizin, which is a main constituent in licorice (Glycyrrhiza glabra). While GA exhibits a wide variety of pharmacological activities in the body, it is converted to a toxic metabolite GA 3-O-glucuronide by hepatic UDP-glucuronosyltransferases (UGTs). To avoid the development of the toxic metabolite-induced pseudohyperaldosteronism (pseudoaldosteronism), there is a limitation in maximum daily dosage of licorice and in combined usage of other glycyrrhizin-containing natural medicine. In this study, we investigated the inhibitory effects of various polyphenols and triterpenoids on the UGT-mediated GA 3-O-glucuronidation. In human liver microsomes, UGT-mediated GA glucuronidation was significantly inhibited by protopanaxadiol with an IC 50 value of 59.2 μM. Isoliquiritigenin, rosmarinic acid, alisol B, alisol acetate, and catechin moderately inhibited the GA glucuronidation with IC 50 values of 96.4 μM, 125 μM, 160 μM, 163 μM, and 164 μM. Other tested 19 polyphenols and triterpenoids, including liquiritigenin, did not inhibit UGT-mediated GA glucuronidation in human liver microsomes. Our data indicate that relatively higher dosage of licorice can be used without a risk of developing pseudohyperaldosteronism in combination of natural medicine containing protopanaxadiol such as Panax ginseng. Furthermore, supplemental protopanaxadiol and isoliquiritigenin might be useful in preventing licorice-inducing pseudoaldosteronism. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Identification of an unintended consequence of Nrf2-directed cytoprotection against a key tobacco carcinogen plus a counteracting chemopreventive intervention

PubMed Central

Paonessa, Joseph D.; Ding, Yi; Randall, Kristen L.; Munday, Rex; Argoti, Dayana; Vouros, Paul; Zhang, Yuesheng

2011-01-01

Nrf2 is a major cytoprotective gene and is a key chemopreventive target against cancer and other diseases. Here we show that Nrf2 faces a dilemma in defense against 4-aminobiphenyl (ABP), a major human bladder carcinogen from tobacco smoke and other environmental sources. While Nrf2 protected mouse liver against ABP (which is metabolically activated in liver), the bladder level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the predominant ABP-DNA adduct formed in bladder cells and tissues, was markedly higher in Nrf2+/+ mice than in Nrf2−/− mice after ABP exposure. Notably, Nrf2 protected bladder cells against ABP in vitro. Mechanistic investigations showed that the dichotomous effects of Nrf2 could be explained at least partly by upregulation of UDP-glucuronosyltransferase (UGT). Nrf2 promoted conjugation of ABP with glucuronic acid in the liver, increasing urinary excretion of the conjugate. While glucuronidation of ABP and its metabolites is a detoxification process, these conjugates, which are excreted in urine, are known to be unstable in acidic urine, leading to delivery of the parent compounds to bladder. Hence, while higher liver UGT activity may protect the liver against ABP it increases bladder exposure to ABP. These findings raise concerns of potential bladder toxicity when Nrf2-activating chemopreventive agents are used in humans exposed to ABP, especially in smokers. We further demonstrate that 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) significantly inhibits dG-C8-ABP formation in bladder cells and tissues, but does not appear to significantly modulate ABP-catalyzing UGT in liver. Thus, CPDT exemplifies a counteracting solution to the dilemma posed by Nrf2. PMID:21487034

N-glycolyl groups of nonhuman chondroitin sulfates survive in ancient fossils.

PubMed

Bergfeld, Anne K; Lawrence, Roger; Diaz, Sandra L; Pearce, Oliver M T; Ghaderi, Darius; Gagneux, Pascal; Leakey, Meave G; Varki, Ajit

2017-09-26

Biosynthesis of the common mammalian sialic acid N -glycolylneuraminic acid (Neu5Gc) was lost during human evolution due to inactivation of the CMAH gene, possibly expediting divergence of the Homo lineage, due to a partial fertility barrier. Neu5Gc catabolism generates N -glycolylhexosamines, which are potential precursors for glycoconjugate biosynthesis. We carried out metabolic labeling experiments and studies of mice with human-like Neu5Gc deficiency to show that Neu5Gc degradation is the metabolic source of UDP-GlcNGc and UDP-GalNGc and the latter allows an unexpectedly selective incorporation of N -glycolyl groups into chondroitin sulfate (CS) over other potential glycoconjugate products. Partially N -glycolylated-CS was chemically synthesized as a standard for mass spectrometry to confirm its natural occurrence. Much lower amounts of GalNGc in human CS can apparently be derived from Neu5Gc-containing foods, a finding confirmed by feeding Neu5Gc-rich chow to human-like Neu5Gc-deficient mice. Unlike the case with Neu5Gc, N -glycolyl-CS was also stable enough to be detectable in animal fossils as old as 4 My. This work opens the door for investigating the biological and immunological significance of this glycosaminoglycan modification and for an "ancient glycans" approach to dating of Neu5Gc loss during the evolution of Homo .

Structure-based design of diverse inhibitors of Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase: combined molecular docking, dynamic simulation, and biological activity.

PubMed

Soni, Vijay; Suryadevara, Priyanka; Sriram, Dharmarajan; Kumar, Santhosh; Nandicoori, Vinay Kumar; Yogeeswari, Perumal

2015-07-01

Persistent nature of Mycobacterium tuberculosis is one of the major factors which make the drug development process monotonous against this organism. The highly lipophilic cell wall, which constituting outer mycolic acid and inner peptidoglycan layers, acts as a barrier for the drugs to enter the bacteria. The rigidity of the cell wall is imparted by the peptidoglycan layer, which is covalently linked to mycolic acid by arabinogalactan. Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) serves as the starting material in the biosynthesis of this peptidoglycan layers. This UDP-GlcNAc is synthesized by N-acetylglucosamine-1-phosphate uridyltransferase (GlmU(Mtb)), a bi-functional enzyme with two functional sites, acetyltransferase site and uridyltransferase site. Here, we report design and screening of nine inhibitors against UTP and NAcGlc-1-P of uridyltransferase active site of glmU(Mtb). Compound 4 was showing good inhibition and was selected for further analysis. The isothermal titration calorimetry (ITC) experiments showed the binding energy pattern of compound 4 to the uridyltransferase active site is similar to that of substrate UTP. In silico molecular dynamics (MD) simulation studies, for compound 4, carried out for 10 ns showed the protein-compound complex to be stable throughout the simulation with relative rmsd in acceptable range. Hence, these compounds can serve as a starting point in the drug discovery processes against Mycobacterium tuberculosis.

Screening of recombinant glycosyltransferases reveals the broad acceptor specificity of stevia UGT-76G1.

PubMed

Dewitte, Griet; Walmagh, Maarten; Diricks, Margo; Lepak, Alexander; Gutmann, Alexander; Nidetzky, Bernd; Desmet, Tom

2016-09-10

UDP-glycosyltransferases (UGTs) are a promising class of biocatalysts that offer a sustainable alternative for chemical glycosylation of natural products. In this study, we aimed to characterize plant-derived UGTs from the GT-1 family with an emphasis on their acceptor promiscuity and their potential application in glycosylation processes. Recombinant expression in E. coli provided sufficient amounts of enzyme for the in-depth characterization of the salicylic acid UGT from Capsella rubella (UGT-SACr) and the stevia UGT from Stevia rebaudiana (UGT-76G1Sr). The latter was found to have a remarkably broad specificity with activities on a wide diversity of structures, from aliphatic and branched alcohols, over small phenolics to larger flavonoids, terpenoids and even higher glycoside compounds. As an example for its industrial potential, the glycosylation of curcumin was thoroughly evaluated. Under optimized conditions, 96% of curcumin was converted within 24h into the corresponding curcumin β-glycosides. In addition, the reaction was performed in a coupled system with sucrose synthase from Glycine max, to enable the cost-efficient (re)generation of UDP-Glc from sucrose as abundant and renewable resource. Copyright © 2016 Elsevier B.V. All rights reserved.

Spontaneous mutations of the UDP-glucose:flavonoid 3-O-glucosyltransferase gene confers pale- and dull-colored flowers in the Japanese and common morning glories.

PubMed

Morita, Yasumasa; Ishiguro, Kanako; Tanaka, Yoshikazu; Iida, Shigeru; Hoshino, Atsushi

2015-09-01

UDP-glucose:flavonoid 3- O -glucosyltransferase is essential for maintaining proper production quantity, acylation, and glucosylation of anthocyanin, and defects cause pale and dull flower pigmentation in morning glories. The Japanese (Ipomoea nil) and the common (I. purpurea) morning glory display bright blue and dark purple flowers, respectively. These flowers contain acylated and glucosylated anthocyanin pigments, and a number of flower color mutants have been isolated in I. nil. Of these, the duskish mutants of I. nil produce pale- and dull-colored flowers. We found that the Duskish gene encodes UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT). The duskish-1 mutation is a frameshift mutation caused by a 4-bp insertion, and duskish-2 is an insertion of a DNA transposon, Tpn10, at 1.3 kb upstream of the 3GT start codon. In the duskish-2 mutant, excision of Tpn10 is responsible for restoration of the expression of the 3GT gene. The recombinant 3GT protein displays expected 3GT enzymatic activities to catalyze 3-O-glucosylation of anthocyanidins in vitro. Anthocyanin analysis of a duskish-2 mutant and its germinal revertant showing pale and normal pigmented flowers, respectively, revealed that the mutation caused around 80 % reduction of anthocyanin accumulation. We further characterized two I. purpurea mutants showing pale brownish-red flowers, and found that they carry the same frameshift mutation in the 3GT gene. Most of the flower anthocyanins in the mutants were previously found to be anthocyanidin 3-O-glucosides lacking several caffeic acid and glucose moieties that are attached to the anthocyanins in the wild-type plants. These results indicated that 3GT is essential not only for production, but also for proper acylation and glucosylation, of anthocyanin in the morning glories.

Cloning, Expression and Characterization of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Wolbachia Endosymbiont of Human Lymphatic Filarial Parasite Brugia malayi

PubMed Central

Shahab, Mohd; Verma, Meenakshi; Pathak, Manisha; Mitra, Kalyan; Misra-Bhattacharya, Shailja

2014-01-01

Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (K m) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds. PMID:24941309

Purification, crystallization and preliminary X-ray diffraction studies of N-acetylglucosamine-phosphate mutase from Candida albicans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishitani, Yuichi; Maruyama, Daisuke; Nonaka, Tsuyoshi

2006-04-01

Preliminary X-ray diffraction studies on N-acetylglucosamine-phosphate mutase from C. albicans are reported. N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the α-d-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 60.2, b = 130.2, c = 78.0 Å, β = 106.7°. The crystals diffract X-rays to beyond 1.8 Å resolution using synchrotron radiation.

Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE enzyme ternary complex.

PubMed

Ruane, Karen M; Lloyd, Adrian J; Fülöp, Vilmos; Dowson, Christopher G; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I

2013-11-15

Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

Effect of various alanine analogues on the L-alanine-adding enzyme from Escherichia coli.

PubMed

Liger, D; Blanot, D; van Heijenoort, J

1991-05-01

An extract from Escherichia coli containing the L-alanine-adding enzyme with a high specific activity was prepared. Several compounds structurally related to L-alanine were tested as inhibitors of this activity. Intact amino and carboxyl groups were necessary for an interaction with the enzyme. Certain halogenated (haloalanines) or unsaturated (L-vinylglycine, L-propargylglycine, 3-cyano-L-alanine) amino acids were good inhibitors. Radioactive glycine, serine and 1-aminoethylphosphonic acid were tested as substrates. Whereas glycine or L-serine gave rise to the formation of the corresponding nucleotide product, no synthesis of UDP-N-acetylmuramyl-L-1-aminoethylphosphonic acid could be detected.

Role of extrahepatic UDP-glucuronosyltransferase 1A1: Advances in understanding breast milk-induced neonatal hyperbilirubinemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Ryoichi, E-mail: fujiwarar@pharm.kitasato-u.ac.jp; Maruo, Yoshihiro; Chen, Shujuan

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepaticmore » tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression. - Highlights: • Breast-feeding can be a factor for the development of neonatal hyperbilirubinemia. • UDP-glucuronosyltransferase (UGT) 1A1 is the sole bilirubin-metabolizing enzyme. • Extrahepatic UGT1A1 plays an important role in bilirubin metabolism. • We discuss the potential mechanism of breast milk-induced neonatal jaundice.« less

Comparison of the mechanism of cellulose biosynthesis in plants and bacteria. [Acetobacter xylinum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delmer, D.P.; Benziman, M.; Klein, A.S.

1983-01-01

Results of recent studies on the mechanism of cellulose biosynthesis in higher plants and in the bacterium Acetobacter xylinum are compared and contrasted. In higher plants, the synthesizing complex is thought to be mobile in the fluid-mosaic plasma membrane, whereas it is stationary in cells of A. xylinum. Similar patterns of sensitivity to inhibitors of cellulose synthesis as well as to changes in transmembrane electrical potential are shared by both plants and A. xylinum. In vivo tracer studies with both types of organisms support the concept the UDP-glucose is a precursor of cellulose. A characterization of additional compounds which maymore » serve as precursors in A. xylinum beyond the level of UDP-glucose is described. UDP-glucose:..beta..-glucan synthetases have been solubilized from plants and A. xylinum. Attempts at purification of solubilized soybean glucan synthetases indicate that a factor(s) is lost during purification which is necessary for activity; studies described elsewhere indicate that the A. xylinum ..beta..-1,4-glucan synthetase requires a protein factor for GTP activation. The stimulatory effect of polyethylene glycol (PEG) on glucan synthetases from both plants and A. xylinum may relate to stabilization by PEG of enzyme-factor associations. 34 references, 3 figures, 3 tables.« less

Structure of human O-GlcNAc transferase and its complex with a peptide substrate

PubMed Central

Lazarus, Michael B.; Nam, Yunsun; Jiang, Jiaoyang; Sliz, Piotr; Walker, Suzanne

2010-01-01

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that couples metabolic status to the regulation of a wide variety of cellular signaling pathways by acting as a nutrient sensor1. OGT catalyzes the transfer of N-acetyl-glucosamine from UDP-GlcNAc to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins2,3, including numerous transcription factors4, tumor suppressors, kinases5, phosphatases1, and histone-modifying proteins6. Aberrant O-GlcNAcylation by OGT has been linked to insulin resistance7, diabetic complications8, cancer9 and neurodegenerative diseases including Alzheimer’s10. Despite the importance of OGT, the details of how it recognizes and glycosylates its protein substrates are largely unknown. We report here two crystal structures of human OGT, as a binary complex with UDP (2.8 A) and a ternary complex with UDP and a peptide substrate (1.95 A). The structures provide clues to the enzyme mechanism, show how OGT recognizes target peptide sequences, and reveal the fold of the unique domain between the two halves of the catalytic region. This information will accelerate the rational design of biological experiments to investigate OGT’s functions and the design of inhibitors for use as cellular probes and to assess its potential as a therapeutic target. PMID:21240259

Identification and characterization of UDP-glucose:Phloretin 4'-O-glycosyltransferase from Malus x domestica Borkh.

PubMed

Yahyaa, Mosaab; Davidovich-Rikanati, Rachel; Eyal, Yoram; Sheachter, Alona; Marzouk, Sally; Lewinsohn, Efraim; Ibdah, Mwafaq

2016-10-01

Apples (Malus x domestica Brokh.) are among the world's most important food crops with nutritive and medicinal importance. Many of the health beneficial properties of apple fruit are suggested to be due to (poly)phenolic metabolites, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the sweet tasting dihydrochalcones, such as trilobatin, are unknown. To identify candidate genes for involvement in the glycosylation of dihydrochalcones, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Bacillus subtilis phloretin glycosyltransferase. Herein reported is the identification and functional characterization of a Malus x domestica gene encoding phloretin-4'-O-glycosyltransferase designated MdPh-4'-OGT. Recombinant MdPh-4'-OGT protein glycosylates phloretin in the presence of UDP-glucose into trilobatin in vitro. Its apparent Km values for phloretin and UDP-glucose were 26.1 μM and 1.2 mM, respectively. Expression analysis of the MdPh-4'-OGT gene indicated that its transcript levels showed significant variation in apple tissues of different developmental stages. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

Bengoechea, José Antonio; Pinta, Elise; Salminen, Tiina; Oertelt, Clemens; Holst, Otto; Radziejewska-Lebrecht, Joanna; Piotrowska-Seget, Zofia; Venho, Reija; Skurnik, Mikael

2002-08-01

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.

Initiating an undiagnosed diseases program in the Western Australian public health system.

PubMed

Baynam, Gareth; Broley, Stephanie; Bauskis, Alicia; Pachter, Nicholas; McKenzie, Fiona; Townshend, Sharron; Slee, Jennie; Kiraly-Borri, Cathy; Vasudevan, Anand; Hawkins, Anne; Schofield, Lyn; Helmholz, Petra; Palmer, Richard; Kung, Stefanie; Walker, Caroline E; Molster, Caron; Lewis, Barry; Mina, Kym; Beilby, John; Pathak, Gargi; Poulton, Cathryn; Groza, Tudor; Zankl, Andreas; Roscioli, Tony; Dinger, Marcel E; Mattick, John S; Gahl, William; Groft, Stephen; Tifft, Cynthia; Taruscio, Domenica; Lasko, Paul; Kosaki, Kenjiro; Wilhelm, Helene; Melegh, Bela; Carapetis, Jonathan; Jana, Sayanta; Chaney, Gervase; Johns, Allison; Owen, Peter Wynn; Daly, Frank; Weeramanthri, Tarun; Dawkins, Hugh; Goldblatt, Jack

2017-05-03

New approaches are required to address the needs of complex undiagnosed diseases patients. These approaches include clinical genomic diagnostic pipelines, utilizing intra- and multi-disciplinary platforms, as well as specialty-specific genomic clinics. Both are advancing diagnostic rates. However, complementary cross-disciplinary approaches are also critical to address those patients with multisystem disorders who traverse the bounds of multiple specialties and remain undiagnosed despite existing intra-specialty and genomic-focused approaches. The diagnostic possibilities of undiagnosed diseases include genetic and non-genetic conditions. The focus on genetic diseases addresses some of these disorders, however a cross-disciplinary approach is needed that also simultaneously addresses other disorder types. Herein, we describe the initiation and summary outcomes of a public health system approach for complex undiagnosed patients - the Undiagnosed Diseases Program-Western Australia (UDP-WA). Briefly the UDP-WA is: i) one of a complementary suite of approaches that is being delivered within health service, and with community engagement, to address the needs of those with severe undiagnosed diseases; ii) delivered within a public health system to support equitable access to health care, including for those from remote and regional areas; iii) providing diagnoses and improved patient care; iv) delivering a platform for in-service and real time genomic and phenomic education for clinicians that traverses a diverse range of specialties; v) retaining and recapturing clinical expertise; vi) supporting the education of junior and more senior medical staff; vii) designed to integrate with clinical translational research; and viii) is supporting greater connectedness for patients, families and medical staff. The UDP-WA has been initiated in the public health system to complement existing clinical genomic approaches; it has been targeted to those with a specific diagnostic need, and initiated by redirecting existing clinical and financial resources. The UDP-WA supports the provision of equitable and sustainable diagnostics and simultaneously supports capacity building in clinical care and translational research, for those with undiagnosed, typically rare, conditions.

Incorporation of phosphate into glycogen by glycogen synthase.

PubMed

Contreras, Christopher J; Segvich, Dyann M; Mahalingan, Krishna; Chikwana, Vimbai M; Kirley, Terence L; Hurley, Thomas D; DePaoli-Roach, Anna A; Roach, Peter J

2016-05-01

The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [β-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation. Copyright © 2016 Elsevier Inc. All rights reserved.

Metabolism of the Folate Precursor p-Aminobenzoate in Plants

PubMed Central

Eudes, Aymerick; Bozzo, Gale G.; Waller, Jeffrey C.; Naponelli, Valeria; Lim, Eng-Kiat; Bowles, Dianna J.; Gregory, Jesse F.; Hanson, Andrew D.

2008-01-01

Plants produce p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. In plant tissues, however, pABA is known to occur predominantly as its glucose ester (pABA-Glc), and the role of this metabolite in folate synthesis has not been defined. In this study, the UDP-glucose:pABA acyl-glucosyltransferase (pAGT) activity in Arabidopsis extracts was found to reside principally (95%) in one isoform with an apparent Km for pABA of 0.12 mm. Screening of recombinant Arabidopsis UDP-glycosyltransferases identified only three that recognized pABA. One of these (UGT75B1) exhibited a far higher kcat/Km value than the others and a far lower apparent Km for pABA (0.12 mm), suggesting its identity with the principal enzyme in vivo. Supporting this possibility, ablation of UGT75B1 reduced extractable pAGT activity by 95%, in vivo [14C]pABA glucosylation by 77%, and the endogenous pABA-Glc/pABA ratio by 9-fold. The Keq for the pABA esterification reaction was found to be 3 × 10-3. Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDP-glucose/UDP ratios, this Keq value allowed estimation that only 4% of cytosolic pABA is esterified. That pABA-Glc predominates in planta therefore implies that it is sequestered away from the cytosol and, consistent with this possibility, vacuoles isolated from [14C]pABA-fed pea leaves were estimated to contain≥88% of the [14C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [3H]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis. PMID:18385129

Empowering human cardiac progenitor cells by P2Y14 nucleotide receptor overexpression.

PubMed

Khalafalla, Farid G; Kayani, Waqas; Kassab, Arwa; Ilves, Kelli; Monsanto, Megan M; Alvarez, Roberto; Chavarria, Monica; Norman, Benjamin; Dembitsky, Walter P; Sussman, Mark A

2017-12-01

Autologous cardiac progenitor cell (CPC) therapy is a promising approach for treatment of heart failure (HF). There is an unmet need to identify inherent deficits in aged/diseased human CPCs (hCPCs) derived from HF patients in the attempts to augment their regenerative capacity prior to use in the clinical setting. Here we report significant functional correlations between phenotypic properties of hCPCs isolated from cardiac biopsies of HF patients, clinical parameters of patients and expression of the P2Y 14 purinergic receptor (P2Y 14 R), a crucial detector for extracellular UDP-sugars released during injury/stress. P2Y 14 R is downregulated in hCPCs derived from HF patients with lower ejection fraction or diagnosed with diabetes. Augmenting P2Y 14 R expression levels in aged/diseased hCPCs antagonizes senescence and improves functional responses. This study introduces purinergic signalling modulation as a potential strategy to rejuvenate and improve phenotypic characteristics of aged/functionally compromised hCPCs prior to transplantation in HF patients. Autologous cardiac progenitor cell therapy is a promising alternative approach to current inefficient therapies for heart failure (HF). However, ex vivo expansion and pharmacological/genetic modification of human cardiac progenitor cells (hCPCs) are necessary interventions to rejuvenate aged/diseased cells and improve their regenerative capacities. This study was designed to assess the potential of improving hCPC functional capacity by targeting the P2Y 14 purinergic receptor (P2Y 14 R), which has been previously reported to induce regenerative and anti-senescence responses in a variety of experimental models. c-Kit + hCPCs were isolated from cardiac biopsies of multiple HF patients undergoing left ventricular assist device implantation surgery. Significant correlations existed between the expression of P2Y 14 R in hCPCs and clinical parameters of HF patients. P2Y 14 R was downregulated in hCPCs derived from patients with a relatively lower ejection fraction and patients diagnosed with diabetes. hCPC lines with lower P2Y 14 R expression did not respond to P2Y 14 R agonist UDP-glucose (UDP-Glu) while hCPCs with higher P2Y 14 R expression showed enhanced proliferation in response to UDP-Glu stimulation. Mechanistically, UDP-Glu stimulation enhanced the activation of canonical growth signalling pathways ERK1/2 and AKT. Restoring P2Y 14 R expression levels in functionally compromised hCPCs via lentiviral-mediated overexpression improved proliferation, migration and survival under stress stimuli. Additionally, P2Y 14 R overexpression reversed senescence-associated morphology and reduced levels of molecular markers of senescence p16 INK4a , p53, p21 and mitochondrial reactive oxygen species. Findings from this study unveil novel biological roles of the UDP-sugar receptor P2Y 14 in hCPCs and suggest purinergic signalling modulation as a promising strategy to improve phenotypic properties of functionally impaired hCPCs. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Novel Proinflammatory Function of Renal Intercalated Cells.

PubMed

Breton, Sylvie; Brown, Dennis

2018-01-01

Serious and often fatal acute kidney injury (AKI) is frequently seen after major surgery, local and remote organ damage, and sepsis. It is associated with uncontrolled inflammation, and is usually diagnosed only after the kidneys have gone through significant and often irreversible damage. During our work involving another type of kidney disease that leads to acid-base disorders of the blood, we unexpectedly found high levels of a protein called the P2Y14 "purinergic" receptor, in specialized kidney epithelial cells called intercalated cells (ICs). These cells are responsible for maintaining whole body acid-base balance by regulating the secretion of excess protons into the urine, which normalizes blood pH. However, it turns out that the P2Y14 receptor in these cells responds to a molecule called uridine diphosphate (UDP)-glucose, which is a danger signal released by damaged cells anywhere in the body. When UDP-glucose reaches the kidney, it stimulates ICs to produce chemoattractant cytokines; this results in renal inflammation and contributes to the onset of AKI. Key Message: Thus, our work now points to ICs as key mediators of renal inflammation and AKI, following surgery and/or damage to remote organs, sepsis, and also local insults to the kidney itself. The link between the proton secreting ICs of the kidney and AKI is an example of how a fundamental research project with a defined aim, in this case understanding acid-base homeostasis, can lead to a novel observation that has unexpected but major implications in another area of human health. © 2018 The Author(s) Published by S. Karger AG, Basel.

In vitro and in vivo genotoxicity assessment of the dopamine receptor antagonist molindone hydrochloride.

PubMed

Krishna, Gopala; Gopalakrishnan, Gopa; Goel, Saryu

2016-05-01

Molindone hydrochloride is a dihydroindolone neuroleptic with dopamine D2 and D5 receptor antagonist activity. As an integral component of its preclinical safety evaluation, molindone hydrochloride was evaluated in a series of in vitro and in vivo genetic toxicology assays. In the bacterial reverse gene mutation assays employing four Salmonella tester strains (TA98, TA100, TA1535, and TA1537) and the E. coli tester strain WP2uvrA, molindone hydrochloride was negative in all strains, except TA100, in which it induced a positive response (up to 3-fold) in the presence of rat liver S9. With human S9, a small (2-fold), but nonreproducible, increase in revertants was observed in TA100 at the highest concentration of molindone tested (5,000 µg/plate). The mutagenicity was completely abrogated by the addition of glutathione and UDP-glucuronic acid to rat liver S9, suggesting detoxification of the mutagenic metabolite(s) by Phase II conjugation reactions, pathways commonly operational in humans. Molindone hydrochloride did not induce chromosomal aberrations in human lymphocyte cultures, did not elicit a positive response in a rat bone marrow micronucleus test for clastogencity/aneugenicity, and did not give a positive response in the rat liver comet assay for DNA damage. Collectively, the weight of evidence from these studies, combined with a large margin of safety and efficient detoxification through Phase II conjugation supports the interpretation that molindone hydrochloride does not pose a genotoxic risk to humans at the anticipated clinical dose levels. © 2016 Wiley Periodicals, Inc.

Glycosyltransferases in the Golgi membranes of onion stem

PubMed Central

Powell, Janet T.; Brew, Keith

1974-01-01

Cell fractions consisting largely of Golgi membranes were prepared from the meristematic region of the onion. Several enzyme activities were found to be localized in these fractions: inosine diphosphatase, galactosyltransferases and glucosyltransferases. The fractions catalysed the transfer of [14C]galactose from UDP-galactose to endogenous and cell-sap acceptors, to N-acetylglucosamine and to ovalbumin. In the presence of bovine α-lactalbumin, transfer to glucose (lactose synthesis) was catalysed. [14C]Glucose was transferred from UDP-glucose to endogenous and cell-sap acceptors, to cellobiose and to fructose (sucrose synthesis). All these activities were latent, being potentiated by detergents (Triton X-100 or sodium deoxycholate). The characteristics of some of these enzyme activities are described and their biological significance is discussed. ImagesPLATE 1 PMID:4374190

First results of GEN-AU: Cloning of Deoxynivalenol- and Zearalenone-inactivating UDP-glucosyltransferase genes fromArabidopsis thaliana and expression in yeast for production of mycotoxin-glucosides.

PubMed

Poppenberger, B; Berthiller, F; Lucyshyn, D; Schuhmacher, R; Krska, R; Adam, G

2005-06-01

First results of the GEN-AU pilot project "Fusarium virulence and plant resistance mechanisms" are reported. Employing genetically engineered yeast strains we have been able to clone genes from the model plantArabidopsis thaliana encoding UDP-glucosyltransferases which can inactivate deoxynivalenol (DON) and zearalenone (ZON). The structure of the metabolites produced by the transformed yeast strains were determined by LC-MS/MS as DON-3O-glucoside and ZON-4O-glucoside, respectively. ZON and derivatives added to glucosyltransferase expressing yeast cultures are converted into the corresponding glucosides in very high yield, opening an efficient way to produce reference materials for these masked mycotoxins.

Staphylococcus aureus MurC participates in L-alanine recognition via histidine 343, a conserved motif in the shallow hydrophobic pocket.

PubMed

Kurokawa, Kenji; Nishida, Satoshi; Ishibashi, Mihoko; Mizumura, Hikaru; Ueno, Kohji; Yutsudo, Takashi; Maki, Hideki; Murakami, Kazuhisa; Sekimizu, Kazuhisa

2008-03-01

UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had K(m) values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K(m) values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.

Cross regulation between mTOR signaling and O-GlcNAcylation.

PubMed

Very, Ninon; Steenackers, Agata; Dubuquoy, Caroline; Vermuse, Jeanne; Dubuquoy, Laurent; Lefebvre, Tony; El Yazidi-Belkoura, Ikram

2018-06-01

The hexosamine biosynthetic pathway (HBP) integrates glucose, amino acids, fatty acids and nucleotides metabolisms for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is the nucleotide sugar donor for O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) processes. O-GlcNAc transferase (OGT) is the enzyme which transfers the N-acetylglucosamine (O-GlcNAc) residue onto target proteins. Several studies previously showed that glucose metabolism dysregulations associated with obesity, diabetes or cancer correlated with an increase of OGT expression and global O-GlcNAcylation levels. Moreover, these diseases present an increased activation of the nutrient sensing mammalian target of rapamycin (mTOR) pathway. Other works demonstrate that mTOR regulates protein O-GlcNAcylation in cancer cells through stabilization of OGT. In this context, we studied the cross-talk between these two metabolic sensors in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells. We report that levels of OGT and O-GlcNAcylation are increased in obese mice colon tissues and colon cancer cells and are associated with a higher activation of mTOR signaling. In parallel, treatments with mTOR regulators modulate OGT and O-GlcNAcylation levels in both normal and colon cancer cells. However, deregulation of O-GlcNAcylation affects mTOR signaling activation only in cancer cells. Thus, a crosstalk exists between O-GlcNAcylation and mTOR signaling in contexts of metabolism dysregulation associated to obesity or cancer.

Influence of casein hydrolysates on exopolysaccharide synthesis by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.

PubMed

Zhang, Qingli; Yang, Bao; Brashears, Mindy M; Yu, Zhimin; Zhao, Mouming; Liu, Ning; Li, Yinjuan

2014-05-01

A lot of interesting research has been undertaken to enhance the yield of exopolysaccharides (EPS) produced by lactic acid bacteria (LAB). The objective of this study was to determine the influence of casein hydrolysates (CH) with molecular weight less than 3 kDa on cell viability, EPS synthesis and the enzyme activity involved in EPS synthesis during the co-culturing of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus in MRS broth for 72 h at 37 ± 0.1 °C. The highest EPS yield (150.1 mg L⁻¹) was obtained on CH prepared with papain (CHP) at 48 h. At 24 h, EPS were composed of galactose, glucose and rhamnose in a molar ratio of 1.0:2.4:1.5. The monosaccharide composition changed with extension of the fermentation time. The activities of α-phosphoglucomutase, uridine 5'-diphosphate (UDP)-glucose pyrophosphorylase and UDP-galactose 4-epimerase were associated with EPS synthesis. Moreover, the activities of β-phosphoglucomutase and deoxythymadine 5'-diphosphate (dTDP)-glucose pyrophosphorylase involved in rhamnose synthesis were very low at the exponential growth phase and could not be detected during other given periods. The influence of different CH (<3 kDa) on LAB viability, EPS production, EPS monomeric composition and activity levels of key metabolic enzymes was distinct. Besides, their influence was related to the distribution of amino acids. © 2013 Society of Chemical Industry.

Glutathione S-transferases and UDP-glycosyltransferases Are Involved in Response to Aluminum Stress in Flax

PubMed Central

Dmitriev, Alexey A.; Krasnov, George S.; Rozhmina, Tatiana A.; Kishlyan, Natalya V.; Zyablitsin, Alexander V.; Sadritdinova, Asiya F.; Snezhkina, Anastasiya V.; Fedorova, Maria S.; Yurkevich, Olga Y.; Muravenko, Olga V.; Bolsheva, Nadezhda L.; Kudryavtseva, Anna V.; Melnikova, Nataliya V.

2016-01-01

About 30% of the world's ice-free land area is occupied by acid soils. In soils with pH below 5, aluminum (Al) releases to the soil solution, and becomes highly toxic for plants. Therefore, breeding of varieties that are resistant to Al is needed. Flax (Linum usitatissimum L.) is grown worldwide for fiber and seed production. Al toxicity in acid soils is a serious problem for flax cultivation. However, very little is known about mechanisms of flax resistance to Al and the genetics of this resistance. In the present work, we sequenced 16 transcriptomes of flax cultivars resistant (Hermes and TMP1919) and sensitive (Lira and Orshanskiy) to Al, which were exposed to control conditions and aluminum treatment for 4, 12, and 24 h. In total, 44.9–63.3 million paired-end 100-nucleotide reads were generated for each sequencing library. Based on the obtained high-throughput sequencing data, genes with differential expression under aluminum exposure were revealed in flax. The majority of the top 50 up-regulated genes were involved in transmembrane transport and transporter activity in both the Al-resistant and Al-sensitive cultivars. However, genes encoding proteins with glutathione transferase and UDP-glycosyltransferase activity were in the top 50 up-regulated genes only in the flax cultivars resistant to aluminum. For qPCR analysis in extended sampling, two UDP-glycosyltransferases (UGTs), and three glutathione S-transferases (GSTs) were selected. The general trend of alterations in the expression of the examined genes was the up-regulation under Al stress, especially after 4 h of Al exposure. Moreover, in the flax cultivars resistant to aluminum, the increase in expression was more pronounced than that in the sensitive cultivars. We speculate that the defense against the Al toxicity via GST antioxidant activity is the probable mechanism of the response of flax plants to aluminum stress. We also suggest that UGTs could be involved in cell wall modification and protection from reactive oxygen species (ROS) in response to Al stress in L. usitatissimum. Thus, GSTs and UGTs, probably, play an important role in the response of flax to Al via detoxification of ROS and cell wall modification. PMID:28066475

Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

PubMed

Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Yu, Xiao-Bin; Jiang, Yun; Lin, Chi-Chung

2011-07-01

Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target.

PubMed

Cova, Marta; López-Gutiérrez, Borja; Artigas-Jerónimo, Sara; González-Díaz, Aida; Bandini, Giulia; Maere, Steven; Carretero-Paulet, Lorenzo; Izquierdo, Luis

2018-03-05

Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.

The cardioprotective effect of uridine and uridine-5'-monophosphate: the role of the mitochondrial ATP-dependent potassium channel.

PubMed

Krylova, Irina B; Kachaeva, Evgeniya V; Rodionova, Olga M; Negoda, Alexander E; Evdokimova, Nataliya R; Balina, Maria I; Sapronov, Nikolay S; Mironova, Galina D

2006-07-01

The activity of mitochondrial ATP-dependent potassium channel (mitoKATP) of rat heart and liver mitochondria was shown to decrease during aging. This partially explains the increase of risk of ischemia at a mature age since mitoKATP activation provides cardioprotection. We demonstrated that uridine-5'-diphosphate (UDP) possesses the property to activate mitoKATP. At a concentration of 30 microM, it reactivated mitoKATP in mitochondria, and 5-hydroxydecanoate (5-HD) eliminated this effect. In experimental animals, UDP precursors uridine and uridine-5'-monophosphate (UMP) (both 30 mg/kg, administered intravenously 5 min before coronary occlusion) decreased the myocardium ischemic alteration index (1.9 and 3.5 times, respectively) and the T-wave amplitude within 60 min after occlusion. Both effects were inhibited by Glibenclamide (Glib) and 5-HD. UMP and uridine decreased the number of premature ventricular beats 5.6 and 1.9 times and the duration of ventricular tachycardia 9.4 and 4.1 times, respectively. Glib and 5-HD inhibited the anti-arrhythmic parameters, 5-HD being less effective. Uridine and UMP decreased the duration of fibrillation 10.8 and 3.6 times, respectively, and this effect was not abolished by Glib and 5-HD. Thus, uridine and UMP, which are the precursors of UDP in the cell, possess cardioprotective properties. MitoKATP prevents mainly ischemic injuries and partially rhythm disorders.

A UDP-glucosyltransferase functions in both acylphloroglucinol glucoside and anthocyanin biosynthesis in strawberry (Fragaria × ananassa).

PubMed

Song, Chuankui; Zhao, Shuai; Hong, Xiaotong; Liu, Jingyi; Schulenburg, Katja; Schwab, Wilfried

2016-03-01

Physiologically active acylphloroglucinol (APG) glucosides were recently found in strawberry (Fragaria sp.) fruit. Although the formation of the APG aglycones has been clarified, little is known about APG glycosylation in plants. In this study we functionally characterized ripening-related glucosyltransferase genes in Fragaria by comprehensive biochemical analyses of the encoded proteins and by a RNA interference (RNAi) approach in vivo. The allelic proteins UGT71K3a/b catalyzed the glucosylation of diverse hydroxycoumarins, naphthols and flavonoids as well as phloroglucinols, enzymatically synthesized APG aglycones and pelargonidin. Total enzymatic synthesis of APG glucosides was achieved by co-incubation of recombinant dual functional chalcone/valerophenone synthase and UGT71K3 proteins with essential coenzyme A esters and UDP-glucose. An APG glucoside was identified in strawberry fruit which has not yet been reported in other plants. Suppression of UGT71K3 activity in transient RNAi-silenced fruits led to a loss of pigmentation and a substantial decrease of the levels of various APG glucosides and an anthocyanin. Metabolite analyses of transgenic fruits confirmed UGT71K3 as a UDP-glucose:APG glucosyltransferase in planta. These results provide the foundation for the breeding of fruits with improved health benefits and for the biotechnological production of bioactive natural products. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro.

PubMed

Qi, Jieqiong; Wang, Ruihong; Zeng, Yazhen; Yu, Wengong; Gu, Yuchao

2017-08-09

O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3',5,5'-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.

Novel mechanism of network protection against the new generation of cyber attacks

NASA Astrophysics Data System (ADS)

Milovanov, Alexander; Bukshpun, Leonid; Pradhan, Ranjit

2012-06-01

A new intelligent mechanism is presented to protect networks against the new generation of cyber attacks. This mechanism integrates TCP/UDP/IP protocol stack protection and attacker/intruder deception to eliminate existing TCP/UDP/IP protocol stack vulnerabilities. It allows to detect currently undetectable, highly distributed, low-frequency attacks such as distributed denial-of-service (DDoS) attacks, coordinated attacks, botnet, and stealth network reconnaissance. The mechanism also allows insulating attacker/intruder from the network and redirecting the attack to a simulated network acting as a decoy. As a result, network security personnel gain sufficient time to defend the network and collect the attack information. The presented approach can be incorporated into wireless or wired networks that require protection against known and the new generation of cyber attacks.

Role of UDP-glucuronosyltransferase isoforms in 13-cis retinoic acid metabolism in humans.

PubMed

Rowbotham, Sophie E; Illingworth, Nicola A; Daly, Ann K; Veal, Gareth J; Boddy, Alan V

2010-07-01

13-cis Retinoic acid (13cisRA, isotretinoin) is an important drug in both dermatology, and the treatment of high-risk neuroblastoma. 13cisRA is known to undergo cytochrome P450-mediated oxidation, mainly by CYP2C8, but phase II metabolic pathways have not been characterized. In the present study, the glucuronidation activities of human liver (HLM) and intestinal microsomes (HIM), as well as a panel of human UDP-glucuronosyltransferases (UGTs) toward both 13cisRA and the 4-oxo metabolite, 4-oxo 13cisRA, were compared using high-performance liquid chromatography. Both HLM and, to a greater extent, HIM catalyzed the glucuronidation of 13cisRA and 4-oxo 13cisRA. Based on the structures of 13cisRA and 4-oxo 13cisRA, the glucuronides formed are conjugated at the terminal carboxylic acid. Further analysis revealed that UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A9 were the major isoforms responsible for the glucuronidation of both substrates. For 13cisRA, a pronounced substrate inhibition was observed with individual UGTs and with HIM. UGT1A3 exhibited the highest rate of activity toward both substrates, and a high rate of activity toward 13cisRA glucuronidation was also observed with UGT1A7. However, for both substrates, K(m) values were above concentrations reported in clinical studies. Therefore, UGT1A9 is likely to be the most important enzyme in the glucuronidation of both substrates as this enzyme had the lowest K(m) and is expressed in both the intestine and at high levels in the liver.

SU-F-T-335: Piecewise Uniform Dose Prescription and Optimization Based On PET/CT Images

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, G; Liu, J

Purpose: In intensity modulated radiation therapy (IMRT), the tumor target volume is given a uniform dose prescription, which does not consider the heterogeneous characteristics of tumor such as hypoxia, clonogen density, radiosensitivity, tumor proliferation rate and so on. Our goal is to develop a nonuniform target dose prescription method which can spare organs at risk (OARs) better and does not decrease the tumor control probability (TCP). Methods: We propose a piecewise uniform dose prescription (PUDP) based on PET/CT images of tumor. First, we propose to delineate biological target volumes (BTV) and sub-biological target volumes (sub-BTVs) by our Hierarchical Mumford-Shah Vectormore » Model based on PET/CT images of tumor. Then, in order to spare OARs better, we make the BTV mean dose minimized while restrict the TCP to a constant. So, we can get a general formula for determining an optimal dose prescription based on a linearquadratic model (LQ). However, this dose prescription is high heterogeneous, it is very difficult to deliver by IMRT. Therefore we propose to use the equivalent uniform dose (EUD) in each sub-BTV as its final dose prescription, which makes a PUDP for the BTV. Results: We have evaluated the IMRT planning of a patient with nasopharyngeal carcinoma respectively using PUDP and UDP. The results show that the highest and mean doses inside brain stem are 48.425Gy and 19.151Gy respectively when the PUDP is used for IMRT planning, while they are 52.975Gy and 20.0776Gy respectively when the UDP is used. Both of the resulting TCPs(0.9245, 0.9674) are higher than the theoretical TCP(0.8739), when 70Gy is delivered to the BTV. Conclusion: Comparing with the UDP, the PUDP can spare the OARs better while the resulting TCP by PUDP is not significantly lower than by UDP. This work was supported in part by National Natural Science Foundation of China undergrant no.61271382 and by the foundation for construction of scientific project platform forthe cancer hospital of Hunan province.« less

Phylogenomic analysis of UDP glycosyltransferase 1 multigene family in Linum usitatissimum identified genes with varied expression patterns.

PubMed

Barvkar, Vitthal T; Pardeshi, Varsha C; Kale, Sandip M; Kadoo, Narendra Y; Gupta, Vidya S

2012-05-08

The glycosylation process, catalyzed by ubiquitous glycosyltransferase (GT) family enzymes, is a prevalent modification of plant secondary metabolites that regulates various functions such as hormone homeostasis, detoxification of xenobiotics and biosynthesis and storage of secondary metabolites. Flax (Linum usitatissimum L.) is a commercially grown oilseed crop, important because of its essential fatty acids and health promoting lignans. Identification and characterization of UDP glycosyltransferase (UGT) genes from flax could provide valuable basic information about this important gene family and help to explain the seed specific glycosylated metabolite accumulation and other processes in plants. Plant genome sequencing projects are useful to discover complexity within this gene family and also pave way for the development of functional genomics approaches. Taking advantage of the newly assembled draft genome sequence of flax, we identified 137 UDP glycosyltransferase (UGT) genes from flax using a conserved signature motif. Phylogenetic analysis of these protein sequences clustered them into 14 major groups (A-N). Expression patterns of these genes were investigated using publicly available expressed sequence tag (EST), microarray data and reverse transcription quantitative real time PCR (RT-qPCR). Seventy-three per cent of these genes (100 out of 137) showed expression evidence in 15 tissues examined and indicated varied expression profiles. The RT-qPCR results of 10 selected genes were also coherent with the digital expression analysis. Interestingly, five duplicated UGT genes were identified, which showed differential expression in various tissues. Of the seven intron loss/gain positions detected, two intron positions were conserved among most of the UGTs, although a clear relationship about the evolution of these genes could not be established. Comparison of the flax UGTs with orthologs from four other sequenced dicot genomes indicated that seven UGTs were flax diverged. Flax has a large number of UGT genes including few flax diverged ones. Phylogenetic analysis and expression profiles of these genes identified tissue and condition specific repertoire of UGT genes from this crop. This study would facilitate precise selection of candidate genes and their further characterization of substrate specificities and in planta functions.

Phylogenomic analysis of UDP glycosyltransferase 1 multigene family in Linum usitatissimum identified genes with varied expression patterns

PubMed Central

2012-01-01

Background The glycosylation process, catalyzed by ubiquitous glycosyltransferase (GT) family enzymes, is a prevalent modification of plant secondary metabolites that regulates various functions such as hormone homeostasis, detoxification of xenobiotics and biosynthesis and storage of secondary metabolites. Flax (Linum usitatissimum L.) is a commercially grown oilseed crop, important because of its essential fatty acids and health promoting lignans. Identification and characterization of UDP glycosyltransferase (UGT) genes from flax could provide valuable basic information about this important gene family and help to explain the seed specific glycosylated metabolite accumulation and other processes in plants. Plant genome sequencing projects are useful to discover complexity within this gene family and also pave way for the development of functional genomics approaches. Results Taking advantage of the newly assembled draft genome sequence of flax, we identified 137 UDP glycosyltransferase (UGT) genes from flax using a conserved signature motif. Phylogenetic analysis of these protein sequences clustered them into 14 major groups (A-N). Expression patterns of these genes were investigated using publicly available expressed sequence tag (EST), microarray data and reverse transcription quantitative real time PCR (RT-qPCR). Seventy-three per cent of these genes (100 out of 137) showed expression evidence in 15 tissues examined and indicated varied expression profiles. The RT-qPCR results of 10 selected genes were also coherent with the digital expression analysis. Interestingly, five duplicated UGT genes were identified, which showed differential expression in various tissues. Of the seven intron loss/gain positions detected, two intron positions were conserved among most of the UGTs, although a clear relationship about the evolution of these genes could not be established. Comparison of the flax UGTs with orthologs from four other sequenced dicot genomes indicated that seven UGTs were flax diverged. Conclusions Flax has a large number of UGT genes including few flax diverged ones. Phylogenetic analysis and expression profiles of these genes identified tissue and condition specific repertoire of UGT genes from this crop. This study would facilitate precise selection of candidate genes and their further characterization of substrate specificities and in planta functions. PMID:22568875

Comparative Effects of Fructose and Glucose on Lipogenic Gene Expression and Intermediary Metabolism in HepG2 Liver Cells

PubMed Central

Fiehn, Oliver; Adams, Sean H.

2011-01-01

Consumption of large amounts of fructose or sucrose increases lipogenesis and circulating triglycerides in humans. Although the underlying molecular mechanisms responsible for this effect are not completely understood, it is possible that as reported for rodents, high fructose exposure increases expression of the lipogenic enzymes fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC-1) in human liver. Since activation of the hexosamine biosynthesis pathway (HBP) is associated with increases in the expression of FAS and ACC-1, it raises the possibility that HBP-related metabolites would contribute to any increase in hepatic expression of these enzymes following fructose exposure. Thus, we compared lipogenic gene expression in human-derived HepG2 cells after incubation in culture medium containing glucose alone or glucose plus 5 mM fructose, using the HBP precursor 10 mM glucosamine (GlcN) as a positive control. Cellular metabolite profiling was conducted to analyze differences between glucose and fructose metabolism. Despite evidence for the active uptake and metabolism of fructose by HepG2 cells, expression of FAS or ACC-1 did not increase in these cells compared with those incubated with glucose alone. Levels of UDP-N-acetylglucosamine (UDP-GlcNAc), the end-product of the HBP, did not differ significantly between the glucose and fructose conditions. Exposure to 10 mM GlcN for 10 minutes to 24 hours resulted in 8-fold elevated levels of intracellular UDP-GlcNAc (P<0.001), as well as a 74–126% increase in FAS (P<0.05) and 49–95% increase in ACC-1 (P<0.01) expression above controls. It is concluded that in HepG2 liver cells cultured under standard conditions, sustained exposure to fructose does not result in an activation of the HBP or increased lipogenic gene expression. Should this scenario manifest in human liver in vivo, it would suggest that high fructose consumption promotes triglyceride synthesis primarily through its action to provide lipid precursor carbon and not by activating lipogenic gene expression. PMID:22096489

Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.

PubMed

Zawadzke, Laura E; Norcia, Michael; Desbonnet, Charlene R; Wang, Hong; Freeman-Cook, Kevin; Dougherty, Thomas J

2008-02-01

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex*

PubMed Central

Ruane, Karen M.; Lloyd, Adrian J.; Fülöp, Vilmos; Dowson, Christopher G.; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I.

2013-01-01

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly. PMID:24064214

Unique regulatory properties of the UDP-glucose:. beta. -1,4-glucan synthetase of Acetobacter xylinum. [Acetobacter xylinum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benziman, M.; Aloni, Y.; Delmer, D.P.

1983-01-01

Conditions have been found for an extremely efficient transfer of glucose from UDP-glucose to a cellulosic ..beta..-1,4-glucan product, using enzyme preparations derived from cells of Acetobacter xylinum. Membrane fractions obtained by rupturing cells in the presence of 20% (w/v) polyethylene glycol-4000 (PEG-4000) exhibited UDP-glucose:..beta..-1,4-glucan synthetase activity 3- to 10-fold higher than those previously reported. Enzyme prepared in this fashion also shows a further marked activation by GTP. The activation (apparent K/sub alpha/ = 35 ..mu..M) is quite specific for GTP. A variety of other nucleotides and nucleotide derivatives had no effect on activity. Guanosine-5'-(lambda-thio)triphosphate, an analog of GTP, is evenmore » more efficient than GTP (K/sub alpha/ = 17 ..mu..M). Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor essential for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself is devoid of synthetase activity and does not stimulate activity of the enzyme in the absence of GTP. Under optimal conditions, in the presence of GTP, factor, and PEG-4000, initial rates of enzyme activity that are 200 times higher than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose. 26 references, 3 figures, 3 tables.« less

The mechanism of synthesis of a mixed-linkage (1-->3), (1-->4)beta-D-glucan in maize. Evidence for multiple sites of glucosyl transfer in the synthase complex

PubMed

Buckeridge; Vergara; Carpita

1999-08-01

We examined the mechanism of synthesis in vitro of (1-->3), (1-->4)beta-D-glucan (beta-glucan), a growth-specific cell wall polysaccharide found in grasses and cereals. beta-Glucan is composed primarily of cellotriosyl and cellotetraosyl units linked by single (1-->3)beta-linkages. The ratio of cellotriosyl and cellotetraosyl units in the native polymer is strictly controlled at between 2 and 3 in all grasses, whereas the ratios of these units in beta-glucan formed in vitro vary from 1.5 with 5 &mgr;M UDP-glucose (Glc) to over 11 with 30 mM substrate. These results support a model in which three sites of glycosyl transfer occur within the synthase complex to produce the cellobiosyl-(1-->3)-D-glucosyl units. We propose that failure to fill one of the sites results in the iterative addition of one or more cellobiosyl units to produce the longer cellodextrin units in the polymer. Variations in the UDP-Glc concentration in excised maize (Zea mays) coleoptiles did not result in wide variations in the ratios of cellotriosyl and cellotetraosyl units in beta-glucan synthesized in vivo, indicating that other factors control delivery of UDP-Glc to the synthase. In maize sucrose synthase is enriched in Golgi membranes and plasma membranes and may be involved in the control of substrate delivery to beta-glucan synthase and cellulose synthase.

4-Methylumbelliferone Diminishes Catabolically Activated Articular Chondrocytes and Cartilage Explants via a Mechanism Independent of Hyaluronan Inhibition*

PubMed Central

Ishizuka, Shinya; Askew, Emily B.; Ishizuka, Naoko; Knudson, Cheryl B.; Knudson, Warren

2016-01-01

Depletion of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. This loss is often accompanied by a coordinate loss in another glycosaminoglycan, hyaluronan. Chondrocytes experimentally depleted of cell-associated hyaluronan respond by switching to a pro-catabolic metabolism that includes enhanced production of endogenous inflammatory mediators and increased synthesis of matrix metalloproteinases. Hyaluronan turnover is also increased. Together, such a response provides for possible establishment of a self-perpetuating spiral of events that maintains or prolongs the pro-catabolic state. Chondrocytes or cartilage can also be activated by treatment with pro-inflammatory cytokines and mediators such as IL-1β, TNFα, LPS, fibronectin fragments, and hyaluronan oligosaccharides. To determine the mechanism of chondrocyte activation due to hyaluronan loss, a depletion method was required that did not include degrading the hyaluronan. In recent years, several laboratories have used the coumarin derivative, 4-methylumbelliferone, as a potent inhibitor of hyaluronan biosynthesis, due in part to its ability to sequester intracellular UDP-glucuronic acid and inhibition of hyaluronan synthase transcription. However, contrary to our expectation, although 4-methylumbelliferone was indeed an inhibitor of hyaluronan biosynthesis, this depletion did not give rise to an activation of chondrocytes or cartilage. Rather, 4-methylumbelliferone directly and selectively blocked gene products associated with the pro-catabolic metabolic state of chondrocytes and did so through a mechanism preceding and independent of hyaluronan inhibition. These data suggest that 4-methylumbelliferone has additional useful applications to block pro-inflammatory cell activation events but complicates how it is used for defining functions related to hyaluronan. PMID:27129266

Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides.

PubMed

Gufford, Brandon T; Graf, Tyler N; Paguigan, Noemi D; Oberlies, Nicholas H; Paine, Mary F

2015-11-01

Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-β-D-glucuronide (15.7 mg), silybin A-5-O-β-D-glucuronide (1.6 mg), and silybin A-4´´-O-β-D-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides

PubMed Central

Gufford, Brandon T.; Graf, Tyler N.; Paguigan, Noemi D.; Oberlies, Nicholas H.

2015-01-01

Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-β-d-glucuronide (15.7 mg), silybin A-5-O-β-d-glucuronide (1.6 mg), and silybin A-4´´-O-β-d-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action. PMID:26316643

Development of a one-pot assay for screening and identification of Mur pathway inhibitors in Mycobacterium tuberculosis

PubMed Central

Eniyan, Kandasamy; Kumar, Anuradha; Rayasam, Geetha Vani; Perdih, Andrej; Bajpai, Urmi

2016-01-01

The cell wall of Mycobacterium tuberculosis (Mtb) consists of peptidoglycan, arabinogalactan and mycolic acids. The cytoplasmic steps in the peptidoglycan biosynthetic pathway, catalyzed by the Mur (A-F) enzymes, involve the synthesis of UDP-n-acetylmuramyl pentapeptide, a key precursor molecule required for the formation of the peptidoglycan monomeric building blocks. Mur enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be promising Mtb drug targets. However, the caveat is that most of the current assays utilize a single Mur enzyme, thereby identifying inhibitors against only one of the enzymes. Here, we report development of a one-pot assay that reconstructs the entire Mtb Mur pathway in vitro and has the advantage of eliminating the requirement for nucleotide intermediates in the pathway as substrates. The MurA-MurF enzymes were purified and a one-pot assay was developed through optimization of successive coupled enzyme assays using UDP-n-acetylglucosamine as the initial sugar substrate. The assay is biochemically characterized and optimized for high-throughput screening of molecules that could disrupt multiple targets within the pathway. Furthermore, we have validated the assay by performing it to identify D-Cycloserine and furan-based benzene-derived compounds with known Mur ligase inhibition as inhibitors of Mtb MurE and MurF. PMID:27734910

Species differences in drug glucuronidation: Humanized UDP-glucuronosyltransferase 1 mice and their application for predicting drug glucuronidation and drug-induced toxicity in humans

PubMed Central

Fujiwara, Ryoichi; Yoda, Emiko; Tukey, Robert H.

2018-01-01

More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans. PMID:29079228

Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.

PubMed

Liger, D; Masson, A; Blanot, D; van Heijenoort, J; Parquet, C

1995-05-15

The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli was over-produced in strains harbouring recombinant plasmids bearing the murC gene under the control of the lac or trc promoter. Plasmid pAM1005, in which the promoter and ribosome-binding site region of murC were removed and in which the gene was directly under the control of promoter trc, led to a 2000-fold amplification of the L-alanine-adding activity after induction by isopropyl-thio-beta-D-galactopyranoside. The murC gene product was visualized as a 50-kDa protein accounting for approximately 50% of the cell protein. A two-step purification led to 1 g of a homogeneous protein from an 18-1 culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the murC gene. The presence of 2-mercaptoethanol and glycerol was essential for the stability of the enzyme. The Km values for UDP-N-acetylmuramic acid, L-alanine and ATP/Mg2+ were estimated at 100, 20 and 450 microM, respectively. Under the optimal in vitro conditions a turnover number of 928 min-1 was calculated and a copy number/cell of 600 could be roughly estimated. The specificity of the enzyme for its substrates was investigated with various analogues. The enzyme also catalysed the reverse reaction.

A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides

PubMed Central

Ciocchini, Andrés E.; Guidolin, L. Soledad; Casabuono, Adriana C.; Couto, Alicia S.; Iñón de Iannino, Nora; Ugalde, Rodolfo A.

2007-01-01

Cyclic β-1,2-glucans (CβG) are osmolyte homopolysaccharides with a cyclic β-1,2-backbone of 17–25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the CβG synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked β-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a β-1,2-glucan phosphorylase present at the CβG synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the β-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this “length-controlling” phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides. PMID:17921247

Optimization of a UDP-glucuronosyltransferase assay for trout ...

EPA Pesticide Factsheets

An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosphoglucuronic acid (UDPGA; a necessary cofactor), alamethicin (a pore-forming agent added to eliminate latency), and substrate (p-nitrophenol). Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. All studies were performed under physiological conditions (pH 7.8, 11 °C) to support ongoing development of methods for extrapolating in vitro rates of biotransformation to the intact animal. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. The purpose of the present study was to optimize an existing in vitro assay for hepatic UGT activity in rainbow trout. The original assay, adapted here for use with trout S9 fractions, was updated by incorporating a membrane disrupting agent (alamethicin) to reduce latency. Additional experiments were conducted to evaluate

UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells

PubMed Central

Liñán-Rico, Andromeda; Ochoa-Cortes, Fernando; Zuleta-Alarcon, Alix; Alhaj, Mazin; Tili, Esmerina; Enneking, Josh; Harzman, Alan; Grants, Iveta; Bergese, Sergio; Christofi, Fievos L.

2017-01-01

Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca2+-dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca2+imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca2+oscillations in BON. UTP evoked a biphasic concentration-dependent Ca2+response. Cells responded in the order of UTP, ATP > UTPγS > UDP >> MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y4, 50% cells), UDP (P2Y6, 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca2+responses were blocked with inhibitors of PLC, IP3R, SERCA Ca2+pump, La3+sensitive Ca2+channels or chelation of intracellular free Ca2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca2+pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca2+currents (ICa), Vm-depolarization and inhibited IK (not IA) currents. An IKv7.2/7.3 K+ channel blocker XE-991 mimicked UTP-induced Vm-depolarization and blocked UTP-responses. XE-991 blocked IK and UTP caused further reduction. La3+ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca2+buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y4/P2Y6R. Zero-Ca2+buffer augmented Ca2+responses and 5-HT release. Conclusion: UTP activates a predominant P2Y4R pathway to trigger Ca2+oscillations via internal Ca2+mobilization through a PLC/IP3/IP3R/SERCA Ca2+signaling pathway to stimulate 5-HT release; Ca2+influx is inhibitory. UTP-induced Vm-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca2+oscillations or Ica/VOCC). UTP-gated signaling pathways triggered by activation of P2Y4R stimulate 5-HT release. PMID:28751862

UTP - Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells.

PubMed

Liñán-Rico, Andromeda; Ochoa-Cortes, Fernando; Zuleta-Alarcon, Alix; Alhaj, Mazin; Tili, Esmerina; Enneking, Josh; Harzman, Alan; Grants, Iveta; Bergese, Sergio; Christofi, Fievos L

2017-01-01

Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca 2+ -dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca 2+ imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca 2+ oscillations in BON. UTP evoked a biphasic concentration-dependent Ca 2+ response. Cells responded in the order of UTP, ATP > UTPγS > UDP > MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y 4 , 50% cells), UDP (P2Y 6 , 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca 2+ responses were blocked with inhibitors of PLC, IP3R, SERCA Ca 2+ pump, La 3+ sensitive Ca 2+ channels or chelation of intracellular free Ca 2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca 2+ pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca 2+ currents (I Ca ), V m -depolarization and inhibited I K (not I A ) currents. An I Kv 7.2/7.3 K + channel blocker XE-991 mimicked UTP-induced V m -depolarization and blocked UTP-responses. XE-991 blocked I K and UTP caused further reduction. La 3+ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca 2+ buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y 4 /P2Y 6 R. Zero-Ca 2+ buffer augmented Ca 2+ responses and 5-HT release. Conclusion: UTP activates a predominant P2Y 4 R pathway to trigger Ca 2+ oscillations via internal Ca 2+ mobilization through a PLC/IP 3 /IP3R/SERCA Ca 2+ signaling pathway to stimulate 5-HT release; Ca 2+ influx is inhibitory. UTP-induced V m -depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca 2+ oscillations or I ca /VOCC). UTP-gated signaling pathways triggered by activation of P2Y 4 R stimulate 5-HT release.

Thio-Linked UDP–Peptide Conjugates as O-GlcNAc Transferase Inhibitors

PubMed Central

2018-01-01

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay. PMID:29723473

A kinetic study on the chemical cleavage of nucleoside diphosphate sugars.

PubMed

Huhta, Eija; Parjanen, Atte; Mikkola, Satu

2010-03-30

Nucleoside diphosphate sugars serve in essential roles in metabolic processes. They have, therefore, been used in mechanistic studies on glycosylation reactions, and their analogues have been synthesised as enzyme and receptor inhibitors. Despite extensive biochemical research, little is known about their chemical reactions. In the present work the chemical cleavage of two different types of nucleoside diphosphate sugars has been studied. UDP-Glc is phosphorylated at the anomeric carbon, whereas in ADP-Rib C-1 is unsubstituted, allowing hence the equilibrium between cyclic hemiacetal and acyclic carbonyl forms. Due to the structural difference, these substrates react via different pathways under slightly alkaline conditions: while UDP-Glc reacts exclusively by a nucleophilic attack of a glucose hydroxyl group on the diphosphate moiety, ADP-Rib undergoes a complex reaction sequence that involves isomerisation processes of the acyclic ribose sugar and results in a release of ADP. Copyright 2009 Elsevier Ltd. All rights reserved.

Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

PubMed

Nordström, Viola; Willershäuser, Monja; Herzer, Silke; Rozman, Jan; von Bohlen Und Halbach, Oliver; Meldner, Sascha; Rothermel, Ulrike; Kaden, Sylvia; Roth, Fabian C; Waldeck, Clemens; Gretz, Norbert; de Angelis, Martin Hrabě; Draguhn, Andreas; Klingenspor, Martin; Gröne, Hermann-Josef; Jennemann, Richard

2013-01-01

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.

Identification of Human UDP-Glucuronosyltransferase 1A4 as the Major Isozyme Responsible for the Glucuronidation of 20(S)-Protopanaxadiol in Human Liver Microsomes

PubMed Central

Li, Jia; He, Chunyong; Fang, Lianxiang; Yang, Li; Wang, Zhengtao

2016-01-01

20(S)-protopanaxadiol (PPD), one of the representative aglycones of ginsenosides, has a broad spectrum of pharmacological activities. Although phase I metabolism has been investigated extensively, information regarding phase II metabolism of this compound remains to be elucidated. Here, a glucuronidated metabolite of PPD in human liver microsomes (HLMs) and rat liver microsomes (RLMs) was unambiguously identified as PPD-3-O-β-d-glucuronide by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. The chemical inhibition and recombinant human UDP-Glucuronosyltransferase (UGT) isoforms assay showed that the PPD glucuronidation was mainly catalyzed by UGT1A4 in HLM, whereas UGT1A3 showed weak catalytic activity. In conclusion, PPD-3-O-β-d-glucuronide was first identified as the principal glucuronidation metabolite of PPD in HLMs, which was catalyzed by UGT1A4. PMID:27005621

Understanding Substrate Selectivity of Human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes

PubMed Central

Dong, Dong; Ako, Roland; Hu, Ming; Wu, Baojian

2015-01-01

The UDP-glucuronosyltransferase (UGT) enzyme catalyzes the glucuronidation reaction which is a major metabolic and detoxification pathway in humans. Understanding the mechanisms for substrate recognition by UGT assumes great importance in an attempt to predict its contribution to xenobiotic/drug disposition in vivo. Spurred on by this interest, 2D/3D-quantitative structure activity relationships (QSAR) and pharmacophore models have been established in the absence of a complete mammalian UGT crystal structure. This review discusses the recent progress in modeling human UGT substrates including those with multiple sites of glucuronidation. A better understanding of UGT active site contributing to substrate selectivity (and regioselectivity) from the homologous enzymes (i.e., plant and bacterial UGTs, all belong to family 1 of glycosyltransferase (GT1)) is also highlighted, as these enzymes share a common catalytic mechanism and/or overlapping substrate selectivity. PMID:22385482

P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport

PubMed Central

Köttgen, Michael; Löffler, Thomas; Jacobi, Christoph; Nitschke, Roland; Pavenstädt, Hermann; Schreiber, Rainer; Frische, Sebastian; Nielsen, Søren; Leipziger, Jens

2003-01-01

Extracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl– secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K+ channels. We therefore speculated that a basolateral P2Y6 receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y6 receptor using crypt [Ca2+]i measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y6elevates cAMP, we coexpressed the P2Y1 or P2Y6 receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl– currents. In oocytes expressing the P2Y1 receptor, ATP transiently activated the endogenous Ca2+-activated Cl– current, but not CFTR. In contrast, in oocytes expressing the P2Y6receptor, UDP transiently activated the Ca2+-activated Cl– current and subsequently CFTR. CFTR Cl– currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y6 receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca2+]i and cAMP. In support of these data P2Y6 receptor stimulation differentially activates CFTR in Xenopus oocytes. PMID:12569163

Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum

PubMed Central

1995-01-01

It has been proposed that the UDP-Glc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked Glc1Man7-9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin- like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP- Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides. PMID:7642696

Characterization of ppGalNAc-T18, a member of the vertebrate-specific Y subfamily of UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases.

PubMed

Li, Xing; Wang, Jing; Li, Wei; Xu, Yingjiao; Shao, Dong; Xie, Yinyin; Xie, Wenxian; Kubota, Tomomi; Narimatsu, Hisashi; Zhang, Yan

2012-05-01

The first step of mucin-type O-glycosylation is catalyzed by members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T; EC 2.4.1.41) family. Each member of this family has unique substrate specificity and expression profiles. In this report, we describe a new subfamily of ppGalNAc-Ts, designated the Y subfamily. The Y subfamily consists of four members, ppGalNAc-T8, -T9, -T17 and -T18, in which the conserved YDX(5)WGGENXE sequence in the Gal/GalNAc-T motif of ppGalNAc-Ts is mutated to LDX(5)YGGENXE. Phylogenetic analysis revealed that the Y subfamily members only exist in vertebrates. All four Y subfamily members lack in vitro GalNAc-transferase activity toward classical substrates possibly because of the UDP-GalNAc-binding pocket mutants. However, ppGalNAc-T18, the newly identified defining member, was localized in the endoplasmic reticulum rather than the Golgi apparatus in lung carcinoma cells. The knockdown of ppGalNAc-T18 altered cell morphology, proliferation potential and changed cell O-glycosylation. ppGalNAc-T18 can also modulate the in vitro GalNAc-transferase activity of ppGalNAc-T2 and -T10, suggesting that it may be a chaperone-like protein. These findings suggest that the new Y subfamily of ppGalNAc-Ts plays an important role in protein glycosylation; characterizing their functions will provide new insight into the role of ppGalNAc-Ts.

The development of response surface pathway design to reduce animal numbers in toxicity studies

PubMed Central

2014-01-01

Background This study describes the development of Response Surface Pathway (RSP) design, assesses its performance and effectiveness in estimating LD50, and compares RSP with Up and Down Procedures (UDPs) and Random Walk (RW) design. Methods A basic 4-level RSP design was used on 36 male ICR mice given intraperitoneal doses of Yessotoxin. Simulations were performed to optimise the design. A k-adjustment factor was introduced to ensure coverage of the dose window and calculate the dose steps. Instead of using equal numbers of mice on all levels, the number of mice was increased at each design level. Additionally, the binomial outcome variable was changed to multinomial. The performance of the RSP designs and a comparison of UDPs and RW were assessed by simulations. The optimised 4-level RSP design was used on 24 female NMRI mice given Azaspiracid-1 intraperitoneally. Results The in vivo experiment with basic 4-level RSP design estimated the LD50 of Yessotoxin to be 463 μg/kgBW (95% CI: 383–535). By inclusion of the k-adjustment factor with equal or increasing numbers of mice on increasing dose levels, the estimate changed to 481 μg/kgBW (95% CI: 362–566) and 447 μg/kgBW (95% CI: 378–504 μg/kgBW), respectively. The optimised 4-level RSP estimated the LD50 to be 473 μg/kgBW (95% CI: 442–517). A similar increase in power was demonstrated using the optimised RSP design on real Azaspiracid-1 data. The simulations showed that the inclusion of the k-adjustment factor, reduction in sample size by increasing the number of mice on higher design levels and incorporation of a multinomial outcome gave estimates of the LD50 that were as good as those with the basic RSP design. Furthermore, optimised RSP design performed on just three levels reduced the number of animals from 36 to 15 without loss of information, when compared with the 4-level designs. Simulated comparison of the RSP design with UDPs and RW design demonstrated the superiority of RSP. Conclusion Optimised RSP design reduces the number of animals needed. The design converges rapidly on the area of interest and is at least as efficient as both the UDPs and RW design. PMID:24661560

Effect of drainage on CO2, CH4, and N2O fluxes from aquaculture ponds during winter in a subtropical estuary of China.

PubMed

Yang, Ping; Lai, Derrick Y F; Huang, Jia F; Tong, Chuan

2018-03-01

Aquaculture ponds are dominant features of the landscape in the coastal zone of China. Generally, aquaculture ponds are drained during the non-culture period in winter. However, the effects of such drainage on the production and flux of greenhouse gases (GHGs) from aquaculture ponds are largely unknown. In the present study, field-based research was performed to compare the GHG fluxes between one drained pond (DP, with a water depth of 0.05m) and one undrained pond (UDP, with a water depth of 1.16m) during one winter in the Min River estuary of southeast China. Over the entire study period, the mean CO 2 flux in the DP was (0.75±0.12) mmol/(m 2 ·hr), which was significantly higher than that in the UDP of (-0.49±0.09) mmol/(m 2 ·hr) (p<0.01). This indicates that drainage drastically transforms aquaculture ponds from a net sink to a net source of CO 2 in winter. Mean CH 4 and N 2 O emissions were significantly higher in the DP compared to those in the UDP (CH 4 =(0.66±0.31) vs. (0.07±0.06) mmol/(m 2 ·hr) and N 2 O=(19.54±2.08) vs. (0.01±0.04) µmol/(m 2 ·hr)) (p<0.01), suggesting that drainage would also significantly enhance CH 4 and N 2 O emissions. Changes in environmental variables (including sediment temperature, pH, salinity, redox status, and water depth) contributed significantly to the enhanced GHG emissions following pond drainage. Furthermore, analysis of the sustained-flux global warming and cooling potentials indicated that the combined global warming potentials of the GHG fluxes were significantly higher in the DP than in the UDP (p<0.01), with values of 739.18 and 26.46mgCO 2 -eq/(m 2 ·hr), respectively. Our findings suggested that drainage of aquaculture ponds can increase the emissions of potent GHGs from the coastal zone of China to the atmosphere during winter, further aggravating the problem of global warming. Copyright © 2017. Published by Elsevier B.V.

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae*

PubMed Central

Wesener, Darryl A.; Levengood, Matthew R.

2017-01-01

The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis. In each species, the galactan is constructed from uridine 5′-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope. PMID:28039359

Diabetes mellitus reduces activity of human UDP-glucuronosyltransferase 2B7 in liver and kidney leading to decreased formation of mycophenolic acid acyl-glucuronide metabolite.

PubMed

Dostalek, Miroslav; Court, Michael H; Hazarika, Suwagmani; Akhlaghi, Fatemeh

2011-03-01

Mycophenolic acid (MPA) is an immunosuppressive agent commonly used after organ transplantation. Altered concentrations of MPA metabolites have been reported in diabetic kidney transplant recipients, although the reason for this difference is unknown. We aimed to compare MPA biotransformation and UDP-glucuronosyltransferase (UGT) expression and activity between liver (n = 16) and kidney (n = 8) from diabetic and nondiabetic donors. Glucuronidation of MPA, as well as the expression and probe substrate activity of UGTs primarily responsible for MPA phenol glucuronide (MPAG) formation (UGT1A1 and UGT1A9), and MPA acyl glucuronide (AcMPAG) formation (UGT2B7), was characterized. We have found that both diabetic and nondiabetic human liver microsomes and kidney microsomes formed MPAG with similar efficiency; however, AcMPAG formation was significantly lower in diabetic samples. This finding is supported by markedly lower glucuronidation of the UGT2B7 probe zidovudine, UGT2B7 protein, and UGT2B7 mRNA in diabetic tissues. UGT genetic polymorphism did not explain this difference because UGT2B7*2 or *1c genotype were not associated with altered microsomal UGT2B7 protein levels or AcMPAG formation. Furthermore, mRNA expression and probe activities for UGT1A1 or UGT1A9, both forming MPAG but not AcMPAG, were comparable between diabetic and nondiabetic tissues, suggesting the effect may be specific to UGT2B7-mediated AcMPAG formation. These findings suggest that diabetes mellitus is associated with significantly reduced UGT2B7 mRNA expression, protein level, and enzymatic activity of human liver and kidney, explaining in part the relatively low circulating concentrations of AcMPAG in diabetic patients.

Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

PubMed Central

Rabausch, U.; Juergensen, J.; Ilmberger, N.; Böhnke, S.; Fischer, S.; Schubach, B.; Schulte, M.

2013-01-01

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. PMID:23686272

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

PubMed

Mann, Paul A; Müller, Anna; Wolff, Kerstin A; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J; Pinho, Mariana G; Schneider, Tanja; Roemer, Terry

2016-05-01

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

Mur Ligase Inhibitors as Anti-bacterials: A Comprehensive Review.

PubMed

Sangshetti, Jaiprakash N; Joshi, Suyog S; Patil, Rajendra H; Moloney, Mark G; Shinde, Devanand B

2017-01-01

Exploring a new target for antibacterial drug discovery has gained much attention because of the emergence of Multidrug Resistance (MDR) strains of bacteria. To overcome this problem the development of novel antibacterial was considered as highest priority task and was one of the biggest challenge since multiple factors were involved. The bacterial peptidoglycan biosynthetic pathway has been well documented in the last few years and has been found to be imperative source for the development of novel antibacterial agents with high target specificity as they are essential for bacterial survival and have no homologs in humans. We have therefore reviewed the process of peptidoglycan biosynthesis which involves various steps like formation of UDP-Nacetylglucosamine (GlcNAc), UDP-N-acetylmuramic acid (MurNAc) and lipid intermediates (Lipid I and Lipid II) which are controlled by various enzymes like GlmS, GlmM, GlmU enzyme, followed by Mur Ligases (MurAMurF) and finally by MraY and MurG respectively. These four amide ligases MurC-MurF can be used as the source for the development of novel multi-target antibacterial agents as they shared and conserved amino acid regions, catalytic mechanisms and structural features. This review begins with the need for novel antibacterial agents and challenges in their development even after the development of bacterial genomic studies. An overview of the peptidoglycan monomer formation, as a source of disparity in this process is presented, followed by detailed discussion of structural and functional aspects of all Mur enzymes and different chemical classes of their inhibitors along with their SAR studies and inhibitory potential. This review finally emphasizes on different patents and novel Mur inhibitors in the development phase. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mann, Paul A.; Müller, Anna; Wolff, Kerstin A.

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA servesmore » as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.« less

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. 1

PubMed Central

Funk, Christoph; Brodelius, Peter E.

1990-01-01

Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5-trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [O-14C-methyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: trans-cinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia. PMID:16667674

Crystal structure of Helicobacter pylori pseudaminic acid biosynthesis N-acetyltransferase PseH: implications for substrate specificity and catalysis.

PubMed

Ud-Din, Abu I; Liu, Yu C; Roujeinikova, Anna

2015-01-01

Helicobacter pylori infection is the common cause of gastroduodenal diseases linked to a higher risk of the development of gastric cancer. Persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. It belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. The crystal structure of the PseH complex with cofactor acetyl-CoA has been determined at 2.3 Å resolution. This is the first crystal structure of the GNAT superfamily member with specificity to UDP-4-amino-4,6-dideoxy-β-L-AltNAc. PseH is a homodimer in the crystal, each subunit of which has a central twisted β-sheet flanked by five α-helices and is structurally homologous to those of other GNAT superfamily enzymes. Interestingly, PseH is more similar to the GNAT enzymes that utilize amino acid sulfamoyl adenosine or protein as a substrate than a different GNAT-superfamily bacterial nucleotide-sugar N-acetyltransferase of the known structure, WecD. Analysis of the complex of PseH with acetyl-CoA revealed the location of the cofactor-binding site between the splayed strands β4 and β5. The structure of PseH, together with the conservation of the active-site general acid among GNAT superfamily transferases, are consistent with a common catalytic mechanism for this enzyme that involves direct acetyl transfer from AcCoA without an acetylated enzyme intermediate. Based on structural homology with microcin C7 acetyltransferase MccE and WecD, the Michaelis complex can be modeled. The model suggests that the nucleotide- and 4-amino-4,6-dideoxy-β-L-AltNAc-binding pockets form extensive interactions with the substrate and are thus the most significant determinants of substrate specificity. A hydrophobic pocket accommodating the 6'-methyl group of the altrose dictates preference to the methyl over the hydroxyl group and thus to contributes to substrate specificity of PseH.

IN VITRO METABOLISM OF THYROID HORMONES BY RECOMBINANT HUMAN UDP-GLUCORONOSYLTRANSFERASES AND SULFOTRANSFERASES

EPA Science Inventory

Endocrine disruptors can decrease thyroid hormone levels via the induction of hepatic uridinediphosphate-glucoronosyltransferases (UGTs) and sulfotransferases (SULTs). Due to their ability to catalyze glucuronidation and sulfation of hormones and xenobiotics, UGTs and SULTs play ...

Transcriptional Analysis of the vanC Cluster from Enterococcus gallinarum Strains with Constitutive and Inducible Vancomycin Resistance

PubMed Central

Panesso, Diana; Abadía-Patiño, Lorena; Vanegas, Natasha; Reynolds, Peter E.; Courvalin, Patrice; Arias, Cesar A.

2005-01-01

The vanC glycopeptide resistance gene cluster encodes enzymes required for synthesis of peptidoglycan precursors ending in d-Ala-d-Ser. Enterococcus gallinarum BM4174 and SC1 are constitutively and inducibly resistant to vancomycin, respectively. Analysis of peptidoglycan precursors in both strains indicated that UDP-MurNAc-tetrapeptide and UDP-MurNAc-pentapeptide[d-Ser] were synthesized in E. gallinarum SC1 only in the presence of vancomycin (4 μg/ml), whereas the “resistance” precursors accumulated in the cytoplasm of BM4174 cells under both inducing and noninducing conditions. Northern hybridization and reverse transcription-PCR experiments revealed that all the genes from the cluster, vanC-1, vanXYC, vanT, vanRC, and vanSC, were transcribed from a single promoter. In the inducible SC1 isolate, transcriptional regulation appeared to be responsible for inducible expression of resistance. Promoter mapping in E. gallinarum BM4174 revealed that the transcriptional start site was located 30 nucleotides upstream from vanC-1 and that the −10 promoter consensus sequence had high identity with that of the vanA cluster. Comparison of the deduced sequence of the vanSC genes from isolates with constitutive and inducible resistance revealed several amino acid substitutions located in the X box (R200L) and in the region between the F and G2 boxes (D312N, D312A, and G320S) of the putative sensor kinase proteins from isolates with constitutive resistance. PMID:15728903

Species differences in drug glucuronidation: Humanized UDP-glucuronosyltransferase 1 mice and their application for predicting drug glucuronidation and drug-induced toxicity in humans.

PubMed

Fujiwara, Ryoichi; Yoda, Emiko; Tukey, Robert H

2018-02-01

More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

40 CFR 721.2076 - D-Glucuronic acid, polymer with 6-deoxy-L-mannose and D-glucose, acetate, calcium magnesium...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false D-Glucuronic acid, polymer with 6...-Glucuronic acid, polymer with 6-deoxy-L-mannose and D-glucose, acetate, calcium magnesium potassium sodium... identified as D-Glucuronic acid, polymer with 6-deoxy-L-mannose and D-glucose, acetate, calcium magnesium...

A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3)

PubMed Central

van Wijk, Xander M.; Lawrence, Roger; Thijssen, Victor L.; van den Broek, Sebastiaan A.; Troost, Ran; van Scherpenzeel, Monique; Naidu, Natasha; Oosterhof, Arie; Griffioen, Arjan W.; Lefeber, Dirk J.; van Delft, Floris L.; van Kuppevelt, Toin H.

2015-01-01

Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 μM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50–60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5′-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.—Van Wijk, X. M., Lawrence, R., Thijssen, V. L., van den Broek, S. A., Troost, R., van Scherpenzeel, M., Naidu, N., Oosterhof, A., Griffioen, A. W., Lefeber, D. J., van Delft, F. L., van Kuppevelt, T. H. A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3). PMID:25868729

Expression of the human UDP-galactose transporter gene hUGT1 in tobacco plants' enhanced plant hardness.

PubMed

Abedi, Tayebeh; Khalil, Mohamed Farouk Mohamed; Koike, Kanae; Hagura, Yoshio; Tazoe, Yuma; Ishida, Nobuhiro; Kitamura, Kenji; Tanaka, Nobukazu

2018-04-09

We reported previously that tobacco plants transformed with the human UDP-galactose transporter 1 gene (hUGT1) had enhanced growth, displayed characteristic traits, and had an increased proportion of galactose (hyper-galactosylation) in the cell wall matrix polysaccharides. Here, we report that hUGT1-transgenic plants have an enhanced hardness. As determined by breaking and bending tests, the leaves and stems of hUGT1-transgenic plants were harder than those of control plants. Transmission electron microscopy revealed that the cell walls of palisade cells in leaves, and those of cortex cells and xylem fibers in stems of hUGT1-transgenic plants, were thicker than those of control plants. The increased amounts of total cell wall materials extracted from the leaves and stems of hUGT1-transgenic plants supported the increased cell wall thickness. In addition, the cell walls of the hUGT1-transgenic plants showed an increased lignin contents, which was supported by the up-regulation of lignin biosynthetic genes. Thus, the heterologous expression of hUGT1 enhanced the accumulation of cell wall materials, which was accompanied by the increased lignin content, resulting in the increased hardness of the leaves and stems of hUGT1-trangenic plants. The enhanced accumulation of cell wall materials might be related to the hyper-galactosylation of cell wall matrix polysaccharides, most notably arabinogalactan, because of the enhanced UDP-galactose transport from the cytosol to the Golgi apparatus by hUGT1, as suggested in our previous report. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Developmentally regulated expression of ectonucleotidases NTPDase5 and NTPDase6 and UDP-responsive P2Y receptors in the rat cochlea.

PubMed

O'Keeffe, Mary G; Thorne, Peter R; Housley, Gary D; Robson, Simon C; Vlajkovic, Srdjan M

2010-04-01

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate complex extracellular P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from this family (NTPDase1-8) are expressed in the adult rat cochlea. This study reports the changes in expression of NTPDase5 and NTPDase6 in the developing rat cochlea. These two intracellular members of the E-NTPDase family can be released in a soluble form and show preference for nucleoside 5'-diphosphates, such as UDP and GDP. Here, we demonstrate differential spatial and temporal patterns for NTPDase5 and NTPDase6 expression during cochlear development, which are indicative of both cytosolic and extracellular action via pyrimidines. NTPDase5 is noted during the early postnatal period in developing sensory hair cells and supporting Deiters' cells of the organ of Corti, and primary auditory neurons located in the spiral ganglion. In contrast, NTPDase6 is confined to the embryonic and early postnatal hair cell bundles. NTPDase6 immunolocalisation in the developing cochlea underpins its putative role in hair cell bundle development, probably via cytosolic action, whilst NTPDase5 may have a broader extracellular role in the development of sensory and neural tissues in the rat cochlea. Both NTPDase5 and NTPDase6 colocalize with UDP-preferring P2Y(4), P2Y(6) and P2Y(14) receptors during cochlear development, but this strong association was lost in the adult cochlea. Spatiotemporal topographic expression of NTPDase5 and NTPDase6 and P2Y receptors in adult and developing cochlear tissues provide strong support for the role of pyrimidinergic signalling in cochlear development.

Gene stacking of multiple traits for high yield of fermentable sugars in plant biomass

DOE PAGES

Aznar, Aude; Chalvin, Camille; Shih, Patrick M.; ...

2018-01-09

Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. Here, we have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase andmore » the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. In conclusion, the results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.« less

Gene stacking of multiple traits for high yield of fermentable sugars in plant biomass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aznar, Aude; Chalvin, Camille; Shih, Patrick M.

Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. Here, we have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase andmore » the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. In conclusion, the results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.« less

Thiamethoxam Resistance in Aphis gossypii Glover Relies on Multiple UDP-Glucuronosyltransferases

PubMed Central

Pan, Yiou; Tian, Fayi; Wei, Xiang; Wu, Yongqiang; Gao, Xiwu; Xi, Jinghui; Shang, Qingli

2018-01-01

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II enzymes that conjugate a variety of small lipophilic molecules with UDP sugars and alter them into more water-soluble metabolites. Therefore, glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. In this study, two inhibitors of UGT enzymes, sulfinpyrazone and 5-nitrouracil, significantly increased the toxicity of thiamethoxam against the resistant strain of Aphis gossypii, which indicates that UGTs are involved in thiamethoxam resistance in the cotton aphid. Based on transcriptome data, 31 A. gossypii UGTs belonging to 11 families (UGT329, UGT330, UGT341, UGT342, UGT343, UGT344, UGT345, UGT348, UGT349, UGT350, and UGT351) were identified. Compared with the thiamethoxam-susceptible strain, the transcripts of 23 UGTs were elevated, and the transcripts of 13 UGTs (UGT344J2, UGT348A2, UGT344D4, UGT341A4, UGT343B2, UGT342B2, UGT350C3, UGT344N2, UGT344A14, UGT344B4, UGT351A4, UGT344A11, and UGT349A2) were increased by approximately 2.0-fold in the resistant cotton aphid. The suppression of selected UGTs significantly increased the insensitivity of resistant aphids to thiamethoxam, suggesting that the up-regulated UGTs might be associated with thiamethoxam tolerance. This study provides an overall view of the possible metabolic factor UGTs that are relevant to the development of insecticide resistance. The results might facilitate further work to validate the roles of these UGTs in thiamethoxam resistance. PMID:29670540

[Powder modification technology used for the preparation of the hydrophilic decoction pieces of indigo naturalis and the modification principle].

PubMed

Zhang, Ding-Kun; Lin, Jun-Zhi; Liu, Jian-Yun; Qin, Chun-Feng; Guo, Zhi-Ping; Han, Li; Yang, Ming

2013-07-01

The hydrophilicity of the normal decoction pieces (NDP) of Indigo Naturalis is not good, therefore, it is not suit for decoctions. In this paper, powder modification technology is used and some NDP and alcohol are ground together in the vibromill to prepare the hydrophilic decoction pieces (HDP) of Indigo Naturalis. Initially, the properties of NDP, ultrafine decoction pieces (UDP) and HDP are compared, the hydrophilicity of UDP was promoted slightly, that of HDP is promoted dramatically. Then, three batches of Indigo Naturalis are prepared to HDP separately, but there is no obvious difference in the contact angle. Furthermore, the size distribution, surface area and micro-shape of HDP are bigger than that of UDP and smaller than NDP. The contents of indigo and indirubin in three decoction pieces are the same, as well as the species of inorganic substance, although there is a little difference in the proportion of five inorganic substances. The fact suggests the change of physical state and the qualitative and quantitative change of organism and inorganic substances are not the main factors to influence the hydrophilicity. In addition, hydroxyl, methylene and methyl can be identified at the wavenumber of 3 356 cm(-1) and 1 461 cm(-1) in infrared spectrum; the content of alcohol in HDP is 0.67% measured by gas chromatogram. The stability of HDP in the heating condition is studied, the fact suggests the hydrophilic effect of HDP at 40 degrees C is relatively stable. All above research suggests that the alcohol is the main factor to influence the hydrophilicity and maybe the intermolecular force which fixed alcohol molecule on the surface of Indigo Naturalis is the basic principle to produce the hydrophilicity.

Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa.

PubMed

Wetterhorn, Karl M; Gabardi, Kaitlyn; Michlmayr, Herbert; Malachova, Alexandra; Busman, Mark; McCormick, Susan P; Berthiller, Franz; Adam, Gerhard; Rayment, Ivan

2017-12-19

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

Functionalized Anodic Aluminum Oxide Membrane–Electrode System for Enzyme Immobilization

PubMed Central

2015-01-01

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min–1 on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min–1. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process. PMID:25025628

Molecular Recognition at Purine and Pyrimidine Nucleotide (P2) Receptors

PubMed Central

Jacobson, Kenneth A.; Constanzi, Stefano; Ohno, Michihiro; Joshi, Bhalchandra V.; Besada, Pedro; Xu, Bin; Tchilibon, Susanna

2015-01-01

In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X1 (derivatives of pyridoxal phosphates and suramin), P2X3 (A-317491), P2X7 (derivatives of the isoquinoline KN-62), P2Y1 (nucleotide analogues MRS 2179 and MRS 2279), P2Y2 (thiouracil derivatives such as AR-C126313), and P2Y12 (nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y1 receptors. The dinucleotide INS 37217 potently activates the P2Y2 receptor. UTP-γ-S and UDP-β-S are selective agonists for P2Y2/P2Y4 and P2Y6 receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y1 receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7. PMID:15078212

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.

PubMed

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-10-29

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis

PubMed Central

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-01-01

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200–300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP. PMID:24127606

Isolation, purification, and characterization of AgUCGalT1, a galactosyltransferase involved in anthocyanin galactosylation in purple celery (Apium graveolens L.).

PubMed

Feng, Kai; Xu, Zhi-Sheng; Liu, Jie-Xia; Li, Jing-Wen; Wang, Feng; Xiong, Ai-Sheng

2018-06-01

This study showed that a galactosyltransferase, AgUCGalT1, is involved in anthocyanin galactosylation in purple celery. Celery is a well-known vegetable because of its rich nutrients, low calories, and medicinal values. Its petioles and leaf blades are the main organs acting as nutrient sources. UDP-galactose: cyanidin 3-O-galactosyltransferase can transfer the galactosyl moiety from UDP-galactose to the 3-O-position of cyanidin through glycosylation. This process enhances the stability and water solubility of anthocyanins. In the present study, LC-MS data indicated that abundant cyanidin-based anthocyanins accumulated in the petioles of purple celery ('Nanxuan liuhe purple celery'). A gene encoding UDP-galactose: cyanidin 3-O-galactosyltransferase, namely AgUCGalT1, was isolated from purple celery and expressed in Escherichia coli BL21 (DE3). Sequence alignments revealed that the AgUCGalT1 protein contained a highly conserved putative secondary plant glycosyltransferase (PSPG) motif. The glycosylation product catalyzed by AgUCGalT1 was detected using UPLC equipment. The recombinant AgUCGalT1 had an optimal enzyme activity at 35 °C and pH 8.0, and showed highest enzyme activity toward cyanidin among the enzyme activities involving other substances, namely, peonidin, quercetin, and kaempferol. The expression levels of AgUCGalT1 were positively correlated with the total anthocyanin contents in purple and non-purple celery varieties. Crude enzymes extracted from purple celery exhibited glycosylation ability, whereas crude enzymes obtained from non-purple celery did not have this ability. This work provided evidence as a basis for investigations on the function of AgUCGalT1 in anthocyanin glycosylation in purple celery.

The Lifetime of UDP-galactose:Ceramide Galactosyltransferase Is Controlled by a Distinct Endoplasmic Reticulum-associated Degradation (ERAD) Regulated by Sigma-1 Receptor Chaperones*

PubMed Central

Hayashi, Teruo; Hayashi, Eri; Fujimoto, Michiko; Sprong, Hein; Su, Tsung-Ping

2012-01-01

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER. PMID:23105111

Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle

2012-11-01

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitormore » design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 {angstrom} movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k{sub cat}. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.« less

Enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7.

PubMed

Fang, Zhong-Ze; Wang, Haina; Cao, Yun-Feng; Sun, Dong-Xue; Wang, Li-Xuan; Hong, Mo; Huang, Ting; Chen, Jian-Xing; Zeng, Jia

2015-03-01

UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)-carprofen and (S)-carprofen towards multiple UGT isoforms. The results showed that (S)-carprofen exhibited stronger inhibition potential than (R)-carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)-carprofen and (S)-carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)-carprofen and (R)-carprofen towards UGT2B7. A Lineweaver-Burk plot showed that both (S)-carprofen and (R)-carprofen exhibited competitive inhibition towards UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameter (Ki ) was calculated to be 7.0 μM and 31.1 μM for (S)-carprofen and (R)-carprofen, respectively. Based on the standard for drug-drug interaction, the threshold for (S)-carprofen and (R)-carprofen to induce a drug-drug interaction is 0.7 μM and 3.1 μM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)-carprofen and (R)-carprofen to possibly induce the drug-drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)-carprofen is more important than (R)-carprofen to avoid a possible drug-drug interaction between carprofen and the drugs mainly undergoing UGT2B7-catalyzed metabolism. © 2014 Wiley Periodicals, Inc.

Downregulation of putative UDP-glucose: flavonoid 3-O-glucosyltransferase gene alters flower coloring in Phalaenopsis.

PubMed

Chen, Wen-Huei; Hsu, Chi-Yin; Cheng, Hao-Yun; Chang, Hsiang; Chen, Hong-Hwa; Ger, Mang-Jye

2011-06-01

Anthocyanin is the primary pigment contributing to red, violet, and blue flower color formation. The solubility of anthocyanins is enhanced by UDP glucose: flavonoid 3-O-glucosyltransferase (UFGT) through transfer of the glucosyl moiety from UDP-glucose to 3-hydroxyl group to produce the first stable pigments. To assess the possibility that UFGT is involved in the flower color formation in Phalaenopsis, the transcriptional activities of PeUFGT3, and other flower color-related genes in developing red or white flower buds were examined using RT-PCR analysis. In contrast with chalcone synthase, chalcone isomerase, and anthocyanidin synthase genes, PeUFGT3 transcriptional activity was higher expressed in the red color of Phalaenopsis cultivars. In the red labellum of Phalaenopsis 'Luchia Lady', PeUFGT3 also showed higher expression levels than that in the white perianth. PeUFGT3 was predominantly expressed in the red region of flower among various Phalaenopsis cultivars. To investigate the role of PeUFGT3 in red flower color formation, PeUFGT3 was specifically knocked down using RNA interference technology via virus inducing gene silencing in Phalaenopsis. The PeUFGT3-suppressed Phalaenopsis exhibited various levels of flower color fading that was well correlated with the extent of reduced level of PeUFGT3 transcriptional activity. Furthermore, there was a significant decrease in anthocyanin content in the PeUFGT3-suppressed Phalaenopsis flowers. The decrease of anthocyanin content due to PeUFGT3 gene silencing possibly caused the faded flower color in PeUFGT3-suppressed Phalaenopsis. Consequently, these results suggested that the glycosylation-related gene PeUFGT3 plays a critical role in red color formation in Phalaenopsis.

Crystal Structure of the Catalytic Domain of Drosophila [beta]1,4-Galactosyltransferase-7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramakrishnan, Boopathy; Qasba, Pradman K.

2010-11-03

The {beta}1,4-galactosyltransferase-7 ({beta}4Gal-T7) enzyme, one of seven members of the {beta}4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member {beta}4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of {beta}4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 {angstrom} resolution. In the crystalmore » structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of {beta}4Gal-T7 and {beta}4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH{sub 2}OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the {beta}4Gal-T7 crystal structure shows that the aromatic side chain of Tyr{sup 177} interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.« less

New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies*

PubMed Central

Liu, Yao; Rodrigues, João P. G. L. M.; Bonvin, Alexandre M. J. J.; Zaal, Esther A.; Berkers, Celia R.; Heger, Michal; Gawarecka, Katarzyna; Swiezewska, Ewa; Breukink, Eefjan; Egmond, Maarten R.

2016-01-01

Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-MurNAc-pentapeptide and heptaprenyl phosphate (C35-P, short-chain homolog of undecaprenyl phosphate, the endogenous substrate of MraY) as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDP-MurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I and UMP. We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue in MraY (His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide. PMID:27226570

Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

PubMed

Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

2017-07-01

Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum*

PubMed Central

Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luis

2013-01-01

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions. PMID:23615908

Biosynthesis of GDP-fucose and other sugar nucleotides in the blood stages of Plasmodium falciparum.

PubMed

Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luis

2013-06-07

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

Acute exposure to offshore produced water has an effect on stress- and secondary stress responses in three-spined stickleback Gasterosteus aculeatus.

PubMed

Knag, Anne Christine; Taugbøl, Annette

2013-09-01

Pollution is one of today's greatest problems, and the release of contaminants into the environment can cause adverse changes in vitally important biological pathways. In this study, we exposed three-spined stickleback Gasterosteus aculeatus to produced water (PW), i.e. wastewater from offshore petroleum production. PW contains substances such as alkylphenols (APs) and aromatic hydrocarbons (PAHs) known to induce toxicant stress and endocrine disruption in a variety of organisms. Following exposure to PW, a standardized confinement treatment was applied as a second stressor (PW-stress), testing how fish already under stress from the pollutant would respond to an additional stressor. The endpoint for analysis was a combination of blood levels of cortisol and glucose, in addition to transcribed levels of a set of genes related to toxicant stress, endocrine disruption and general stress. The findings of this study indicate that low doses of PW do not induce vitellogenin in immature female stickleback, but do cause an upregulation of cytochrome (CYP1A) and UDP-glucuronsyltransferase (UDP-GT), two biomarkers related to toxicant stress. However, when the second stressor was applied, both genes were downregulated, indicating that the confinement exposure had a suppressive effect on the expression of toxicant biomarkers (CYP1A and UDP-GT). Further, two of the stress related genes, heat shock protein 90 (HSP90) and stress-induced phosphoprotein (STIP), were upregulated in both PW- and PW-stress-treatment, but not in the water control confinement treatment, indicating that PW posed as a larger stress-factor than confinement for these genes. The confinement stressor caused an increased level of glucose in both control and PW-treated fish, indicating hyperglycemia, a commonly reported stress response in fish. © 2013.

Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

PubMed Central

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J.

2012-01-01

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3-Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45 % identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10–50, primarily by decreasing kcat. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens. PMID:22646091

Crystal structures of Trypanosoma cruzi UDP-galactopyranose mutase implicate flexibility of the histidine loop in enzyme activation.

PubMed

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J

2012-06-19

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k(cat). Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

PHENOBARBITAL AFFECTS THYROID HISTOLOGY AND LARVAL DEVELOPMENT IN THE AFRICAN CLAWED FROG XENOPUS LAEVIS

EPA Science Inventory

The abstract highlights our recent study to explore endocrine disrupting effects of phenobarbital in the African clawed frog, Xenopus laevis. In mammals, this chemical is known to induce the biotransforming enzyme UDP-glucuronosyltransferase (UDPGT) resulting in increased thyroid...

Probing the Catalytic Promiscuity of a Regio- and Stereospecific C-Glycosyltransferase from Mangifera indica.

PubMed

Chen, Dawei; Chen, Ridao; Wang, Ruishan; Li, Jianhua; Xie, Kebo; Bian, Chuancai; Sun, Lili; Zhang, Xiaolin; Liu, Jimei; Yang, Lin; Ye, Fei; Yu, Xiaoming; Dai, Jungui

2015-10-19

The catalytic promiscuity of the novel benzophenone C-glycosyltransferase, MiCGT, which is involved in the biosynthesis of mangiferin from Mangifera indica, was explored. MiCGT exhibited a robust capability to regio- and stereospecific C-glycosylation of 35 structurally diverse druglike scaffolds and simple phenolics with UDP-glucose, and also formed O- and N-glycosides. Moreover, MiCGT was able to generate C-xylosides with UDP-xylose. The OGT-reversibility of MiCGT was also exploited to generate C-glucosides with simple sugar donor. Three aryl-C-glycosides exhibited potent SGLT2 inhibitory activities with IC50  values of 2.6×, 7.6×, and 7.6×10(-7)  M, respectively. These findings demonstrate for the first time the significant potential of an enzymatic approach to diversification through C-glycosidation of bioactive natural and unnatural products in drug discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improved Polysaccharide Production by Homologous Co-overexpression of Phosphoglucomutase and UDP Glucose Pyrophosphorylase Genes in the Mushroom Coprinopsis cinerea.

PubMed

Zhou, Jiangsheng; Bai, Yang; Dai, Rujuan; Guo, Xiaoli; Liu, Zhong-Hua; Yuan, Sheng

2018-05-09

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions.

Scalable SCPPM Decoder

NASA Technical Reports Server (NTRS)

Quir, Kevin J.; Gin, Jonathan W.; Nguyen, Danh H.; Nguyen, Huy; Nakashima, Michael A.; Moision, Bruce E.

2012-01-01

A decoder was developed that decodes a serial concatenated pulse position modulation (SCPPM) encoded information sequence. The decoder takes as input a sequence of four bit log-likelihood ratios (LLR) for each PPM slot in a codeword via a XAUI 10-Gb/s quad optical fiber interface. If the decoder is unavailable, it passes the LLRs on to the next decoder via a XAUI 10-Gb/s quad optical fiber interface. Otherwise, it decodes the sequence and outputs information bits through a 1-GB/s Ethernet UDP/IP (User Datagram Protocol/Internet Protocol) interface. The throughput for a single decoder unit is 150-Mb/s at an average of four decoding iterations; by connecting a number of decoder units in series, a decoding rate equal to that of the aggregate rate is achieved. The unit is controlled through a 1-GB/s Ethernet UDP/IP interface. This ground station decoder was developed to demonstrate a deep space optical communication link capability, and is unique in the scalable design to achieve real-time SCPP decoding at the aggregate data rate.

Role of extrahepatic UDP-glucuronosyltransferase 1A1: Advances in understanding breast milk-induced neonatal hyperbilirubinemia.

PubMed

Fujiwara, Ryoichi; Maruo, Yoshihiro; Chen, Shujuan; Tukey, Robert H

2015-11-15

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepatic tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression. Copyright © 2015 Elsevier Inc. All rights reserved.

Studies on the genetic linkage of bilirubin and androsterone UDP-glucuronyltransferases by cross-breeding of two mutant rat strains.

PubMed Central

Nagai, F; Homma, H; Tanase, H; Matsui, M

1988-01-01

Gunn rats, which have defects in bilirubin and 4-nitrophenol UDP-glucuronyltransferases (GT), were crossed with LA Wistar rats with a defect in androsterone GT. The F1 hybrids showed normal GT activities towards androsterone, bilirubin and 4-nitrophenol, demonstrating that Gunn and LA ('low activity') Wistar rats inherit a homozygous dominant trait for androsterone GT and bilirubin GT respectively. The F2 progeny showed four different combinations of bilirubin and androsterone GT activities: defects in both GT activities, a single defect in bilirubin GT activity, a single defect in androsterone GT activity and two normal GT activities. They were segregated in the approximate ratio of 1:3:3:9, which is compatible with Mendel's Principle of Independent Assortment. These results provide evidence that androsterone GT and bilirubin GT are located on different chromosomes. In the F2 generation, defective bilirubin and 4-nitrophenol GT activities were not segregated, indicating that these two mutant genes are closely linked on the same chromosome. PMID:3138978

The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng

2014-10-02

Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) hasmore » been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.« less

Differences in response of glucuronide and glutathione conjugating enzymes to aflatoxin B/sub 1/ and N-acetylaminofluorene in underfed rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajpurohit, R.; Krishnaswamy, K.

Changes in the hepatic drug/xenobiotic-metabolizing enzymes in underfed rats exposed to aflatoxin B/sub 1/ and N-acetylaminofluorene were investigated. Neither carcinogen, fed at the level of 10 ..mu..g and 0.667 mg per 100 g body weight, respectively, over a period of 3 wk, had any significant influence on cytochrome P-450 and aryl hydrocarbon hydroxylase in the undernourished rats. Significantly low activities of UDP-glucuronyltransferase and glutathione S-transferase were observed in food-restricted animals fed on aflatoxin B/sub 1/. N-acetylaminofluorene, on the other hand stimulated both the enzyme activities in the underfed group, to as much observed in the respective well-fed treated group. UDP-Glucuronyltransferasemore » and glutathione S-transferase in undernutrition seem to respond differently to aflatoxin B/sub 1/ and N-acetylaminofluorene. Further studies are needed to assess the possible consequences of such alterations.« less

Expression of UDP-glucuronosyltransferase 1A4 in human placenta at term

PubMed Central

Østby, Lene; Stuen, Ina; Sundby, Eirik

2010-01-01

The placenta contains a large variety of metabolizing enzymes, among them UDP-glucuronosyltransferase (UGT). Several UGT2B isozymes have so far been detected in human placenta, but little is known on placental expression of UGT1A isozymes. The antiepileptic drug lamotrigine (LTG) is a UGT1A4-substrate, and its serum concentration falls by over 50% during pregnancy, leading to impaired seizure control. The placenta may be involved in this. Microsomes from term placentas of 4 LTG-users and 10 healthy control subjects were prepared. Western blot analysis detected UGT1A proteins in all placentas. The presence of UGT1A4 in placenta from LTG users was confirmed with UGT1A4 commercial standard and a specific UGT1A4 primary antibody. Since LTG is primarily metabolized by UGT1A4 and this isozyme is shown to be present in placenta at term, it may be hypothesized that the placenta is involved in the fall of LTG serum concentrations during pregnancy. PMID:21302032

Rapid prediction of chemical metabolism by human UDP-glucuronosyltransferase isoforms using quantum chemical descriptors derived with the electronegativity equalization method.

PubMed

Sorich, Michael J; McKinnon, Ross A; Miners, John O; Winkler, David A; Smith, Paul A

2004-10-07

This study aimed to evaluate in silico models based on quantum chemical (QC) descriptors derived using the electronegativity equalization method (EEM) and to assess the use of QC properties to predict chemical metabolism by human UDP-glucuronosyltransferase (UGT) isoforms. Various EEM-derived QC molecular descriptors were calculated for known UGT substrates and nonsubstrates. Classification models were developed using support vector machine and partial least squares discriminant analysis. In general, the most predictive models were generated with the support vector machine. Combining QC and 2D descriptors (from previous work) using a consensus approach resulted in a statistically significant improvement in predictivity (to 84%) over both the QC and 2D models and the other methods of combining the descriptors. EEM-derived QC descriptors were shown to be both highly predictive and computationally efficient. It is likely that EEM-derived QC properties will be generally useful for predicting ADMET and physicochemical properties during drug discovery.

Role of extrahepatic UDP-glucuronosyltransferase 1A1: advances in understanding breast milk-induced neonatal hyperbilirubinemia

PubMed Central

Fujiwara, Ryoichi; Maruo, Yoshihiro; Chen, Shujuan; Tukey, Robert H.

2015-01-01

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepatic tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression. PMID:26342858

β-Glucoside Activators of Mung Bean UDP-Glucose: β-Glucan Synthase 1

PubMed Central

Callaghan, Theresa; Ross, Peter; Weinberger-Ohana, Patricia; Garden, Gwenn; Benziman, Moshe

1988-01-01

Heat-stable activators of membranous β-glucan synthase have been isolated from the supernatant fraction of crude mung bean (Vigna radiata) extracts by DEAE-cellulose and silica-gel chromatography. One of the activators has been partially purified and characterized on the basis of susceptibility to various enzymes and by analysis of the products formed upon total acid hydrolysis, alkaline-methanolysis, and β-glucosidase digestion. This activator has the characteristics of a 1,2-dioleoyl diglyceride containing β-linked glucose residue(s) at the C-3 position. When expressed per mole of glucosyl residues, the maximal Ka value of the activator is estimated to be 25 micromolar. Both the intact glucosyl and fatty acid moiety are essential to the stimulatory effect of the activator. PMID:16666038

Milk metabolome relates enteric methane emission to milk synthesis and energy metabolism pathways.

PubMed

Antunes-Fernandes, E C; van Gastelen, S; Dijkstra, J; Hettinga, K A; Vervoort, J

2016-08-01

Methane (CH4) emission of dairy cows contributes significantly to the carbon footprint of the dairy chain; therefore, a better understanding of CH4 formation is urgently needed. The present study explored the milk metabolome by gas chromatography-mass spectrometry (milk volatile metabolites) and nuclear magnetic resonance (milk nonvolatile metabolites) to better understand the biological pathways involved in CH4 emission in dairy cattle. Data were used from a randomized block design experiment with 32 multiparous Holstein-Friesian cows and 4 diets. All diets had a roughage:concentrate ratio of 80:20 (dry matter basis) and the roughage was grass silage (GS), corn silage (CS), or a mixture of both (67% GS, 33% CS; 33% GS, 67% CS). Methane emission was measured in climate respiration chambers and expressed as CH4 yield (per unit of dry matter intake) and CH4 intensity (per unit of fat- and protein-corrected milk; FPCM). No volatile or nonvolatile metabolite was positively related to CH4 yield, and acetone (measured as a volatile and as a nonvolatile metabolite) was negatively related to CH4 yield. The volatile metabolites 1-heptanol-decanol, 3-nonanone, ethanol, and tetrahydrofuran were positively related to CH4 intensity. None of the volatile metabolites was negatively related to CH4 intensity. The nonvolatile metabolites acetoacetate, creatinine, ethanol, formate, methylmalonate, and N-acetylsugar A were positively related to CH4 intensity, and uridine diphosphate (UDP)-hexose B and citrate were negatively related to CH4 intensity. Several volatile and nonvolatile metabolites that were correlated with CH4 intensity also were correlated with FPCM and not significantly related to CH4 intensity anymore when FPCM was included as covariate. This suggests that changes in these milk metabolites may be related to changes in milk yield or metabolic processes involved in milk synthesis. The UDP-hexose B was correlated with FPCM, whereas citrate was not. Both metabolites were still related to CH4 intensity when FPCM was included as covariate. The UDP-hexose B is an intermediate of lactose metabolism, and citrate is an important intermediate of Krebs cycle-related energy processes. Therefore, the negative correlation of UDP-hexose B and citrate with CH4 intensity may reflect a decrease in metabolic activity in the mammary gland. Our results suggest that an integrative approach including milk yield and composition, and dietary and animal traits will help to explain the biological metabolism of dairy cows in relation to methane CH4 emission. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Activation by intracellular GDP, metabolic inhibition and pinacidil of a glibenclamide-sensitive K-channel in smooth muscle cells of rat mesenteric artery.

PubMed Central

Zhang, H; Bolton, T B

1995-01-01

1. Single-channel recordings were made from cell-attached and isolated patches, and whole-cell currents were recorded under voltage clamp from single smooth muscle cells obtained by enzymic digestion of a small branch of the rat mesenteric artery. 2. In single voltage-clamped cells 1 mM uridine diphosphate (UDP) or guanidine diphosphate (GDP) added to the pipette solution, or pinacidil (100 microM) a K-channel opener (KCO) applied in the bathing solution, evoked an outward current of up to 100pA which was blocked by glibenclamide (10 microM). In single cells from which recordings were made by the 'perforated patch' (nystatin pipette) technique, metabolic inhibition by 1 mM NaCN and 10 mM 2-deoxy-glucose also evoked a similar glibenclamide-sensitive current. 3. Single K-channel activity was observed in cell-attached patches only infrequently unless the metabolism of the cell was inhibited, whereupon channel activity blocked by glibenclamide was seen; pinacidil applied to the cell evoked similar glibenclamide-sensitive channel activity. If the patch was pulled off the cell to form an isolated inside-out patch, similar glibenclamide-sensitive single-channel currents were observed in the presence of UDP and/or pinacidil to those seen in cell-attached mode; channel conductance was 20 pS (60:130 K-gradient) and openings showed no voltage-dependence and noisy inward currents, typical of the nucleoside diphosphate (NDP) activated K-channel (KNDP) seen previously in rabbit portal vein. 4. Formation of an isolated inside-out patch into an ATP-free solution did not increase the probability of channel opening which declined with time even when some single-channel activity had occurred in the cell-attached mode before detachment. However, application of 1 mM UDP or GDP, but not ATP, to inside-out patches evoked single-channel activity. Application of ATP-free solution to isolated patches, previously exposed to ATP and in which channel activity had been seen, did not evoke channel activity. 5. It is concluded that small conductance K-channels (KNDP) open in smooth muscle cells from this small artery in response to UDP or GDP acting from the inside, or pinacidil acting from the outside; the same channels open during inhibition of metabolism presumably mainly due to the rise in nucleoside diphosphates, but a fall in the ATP concentration on the inside of the channel did not by itself evoke channel activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7735693

Soil CO2 CH4 and N2O fluxes from an afforested lowland raised peatbog in Scotland: implications for drainage and restoration

NASA Astrophysics Data System (ADS)

Yamulki, S.; Anderson, R.; Peace, A.; Morison, J. I. L.

2013-02-01

The effect of tree (lodgepole pine) planting with and without intensive drainage on soil greenhouse gas (GHG) fluxes was assessed after 45 yr at a raised peatbog in West Flanders Moss, central Scotland. Fluxes of CO2 CH4 and N2O from the soil were monitored over a 2-yr period every 2 to 4 weeks using the static opaque chamber method in a randomised experimental block trial with the following treatments: drained and planted (DP), undrained and planted (uDP), undrained and unplanted (uDuP) and for reference also from an adjoining near-pristine area of bog at East Flanders Moss (n-pris). There was a strong seasonal pattern in both CO2 and CH4 effluxes which were significantly higher in late spring and summer months because of warmer temperatures. Effluxes of N2O were low and no significant differences were observed between the treatments. Annual CH4 emissions increased with the proximity of the water table to the soil surface across treatments in the order: DP < uDP < uDuP < n-pris with mean annual effluxes over the 2-yr monitoring period of 0.15, 0.64, 7.70 and 22.63 g CH4 m-2 yr-1, respectively. For CO2, effluxes increased in the order uDP < DP< n-pris < uDuP, with mean annual effluxes of 1.23, 1.66, 1.82 and 2.55 kg CO2 m-2 yr-1, respectively. CO2 effluxes dominated the total net GHG emission, calculated using the global warming potential (GWP) of the three GHGs for each treatment (76-98%), and only in the n-pris site was CH4 a substantial contribution (23%). Based on soil effluxes only, the near pristine (n-pris) peatbog had 43% higher total net GHG emission compared with the DP treatment because of high CH4 effluxes and the DP treatment had 33% higher total net emission compared with the uDP because drainage increased CO2 effluxes. Restoration is likely to increase CH4 emissions, but reduce CO2 effluxes. Our study suggests that if estimates of CO2 uptake by vegetation from similar peatbog sites were included, the total net GHG emission of restored peatbog would still be higher than that of the peatbog with trees.

A tandem array of UDP-glycosyltransferases from the UGT73C subfamily glycosylate sapogenins, forming a spectrum of mono- and bisdesmosidic saponins.

PubMed

Erthmann, Pernille Østerbye; Agerbirk, Niels; Bak, Søren

2018-05-01

This study identifies six UGT73Cs all able to glucosylate sapogenins at positions 3 and/or 28 which demonstrates that B. vulgaris has a much richer arsenal of UGTs involved in saponin biosynthesis than initially anticipated. The wild cruciferous plant Barbarea vulgaris is resistant to some insects due to accumulation of two monodesmosidic triterpenoid saponins, oleanolic acid 3-O-β-cellobioside and hederagenin 3-O-β-cellobioside. Insect resistance depends on the structure of the sapogenin aglycone and the glycosylation pattern. The B. vulgaris saponin profile is complex with at least 49 saponin-like metabolites, derived from eight sapogenins and including up to five monosaccharide units. Two B. vulgaris UDP-glycosyltransferases, UGT73C11 and UGT73C13, O-glucosylate sapogenins at positions 3 and 28, forming mainly 3-O-β-D-glucosides. The aim of this study was to identify UGTs responsible for the diverse saponin oligoglycoside moieties observed in B. vulgaris. Twenty UGT genes from the insect resistant genotype were selected and heterologously expressed in Nicotiana benthamiana and/or Escherichia coli. The extracts were screened for their ability to glycosylate sapogenins (oleanolic acid, hederagenin), the hormone 24-epibrassinolide and sapogenin monoglucosides (hederagenin and oleanolic acid 3-O-β-D-glucosides). Six UGTs from the UGT73C subfamily were able to glucosylate both sapogenins and both monoglucosides at positions 3 and/or 28. Some UGTs formed bisdesmosidic saponins efficiently. At least four UGT73C genes were localized in a tandem array with UGT73C11 and possibly UGT73C13. This organization most likely reflects duplication events followed by sub- and neofunctionalization. Indeed, signs of positive selection on several amino acid sites were identified and modelled to be localized on the UGT protein surface. This tandem array is proposed to initiate higher order bisdesmosidic glycosylation of B. vulgaris saponins, leading to the recently discovered saponin structural diversity, however, not directly to known cellobiosidic saponins.

Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli.

PubMed

Bouhss, A; Mengin-Lecreulx, D; Blanot, D; van Heijenoort, J; Parquet, C

1997-09-30

The comparison of the amino acid sequences of 20 cytoplasmic peptidoglycan synthetases (MurC, MurD, MurE, MurF, and Mpl) from various bacterial organisms has allowed us to detect common invariants: seven amino acids and the ATP-binding consensus sequence GXXGKT/S all at the same position in the alignment. The Mur synthetases thus appeared as a well-defined class of closely functionally related proteins. The conservation of a constant backbone length between certain invariants suggested common structural motifs. Among the other enzymes catalyzing a peptide bond formation driven by ATP hydrolysis to ADP and Pi, only folylpoly-gamma-l-glutamate synthetases presented the same common conserved amino acid residues, except for the most N-terminal invariant D50. Site-directed mutageneses were carried out to replace the K130, E174, H199, N293, N296, R327, and D351 residues by alanine in the MurC protein from Escherichia coli taken as model. For this purpose, plasmid pAM1005 was used as template, MurC being highly overproduced in this genetic setting. Analysis of the Vmax values of the mutated proteins suggested that residues K130, E174, and D351 are essential for the catalytic process whereas residues H199, N293, N296, and R327 were not. Mutations K130A, H199A, N293A, N296A, and R327A led to important variations of the Km values for one or more substrates, thereby indicating that these residues are involved in the structure of the active site and suggesting that the binding order of the substrates could be ATP, UDP-MurNAc, and alanine. The various mutated murC plasmids were tested for their effects on the growth, cell morphology, and peptidoglycan cell content of a murC thermosensitive strain at 42 degrees C. The observed effects (complementation, altered morphology, and reduced peptidoglycan content) paralleled more or less the decreased values of the MurC activity of each mutant.

A barley UDP-glucosyltransferase inactivates nivalenol and provides Fusarium head blight resistance in transgenic wheat

USDA-ARS?s Scientific Manuscript database

Fusarium Head Blight is a disease of cereal crops that causes severe yield losses and mycotoxin contamination of grain. The main causal pathogen, Fusarium graminearum, produces the trichothecene toxins deoxynivalenol or nivalenol as virulence factors. Nivalenol-producing isolates are most prevalent ...

Cell wall composition and digestibility alterations in Brachypodium distachyon achieved through reduced expression of the UDP-arabinopyranose mutase

USDA-ARS?s Scientific Manuscript database

Nucleotide-activated sugars are essential substrates for plant cell wall carbohydrate-polymer biosynthetic glycosyltransferase enzymes. The most prevalent sugars in grass cell walls include glucose (Glc), xylose (Xyl), and arabinose (Ara). These sugars are biosynthetically related via the uridine di...

Transgenic wheat expressing a barley UDP-glucosyltransferase detoxifies deoxynivalenol and provides high levels of resistance to Fusarium graminearum

USDA-ARS?s Scientific Manuscript database

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a devastating disease of wheat and barley that results in huge economic losses worldwide. During infection, F. graminearum produces trichothecene mycotoxins, such as deoxynivalenol (DON), that increase fungal virulence and decreas...

A QUANTITATIVE MODEL FOR XENOBIOTIC METABOLIZING ENZYME (XME) INDUCTION REGULATED BY THE PREGNANE X RECEPTOR (PXR)

EPA Science Inventory

The nuclear receptor, PXR, is an integral part of the regulation of hepatic metabolism. It has been shown to regulate specific CYPs (phase I drug-metabolizing enzymes) as well as certain phase II drug metabolism activities, including UDP-glucuronosyl transferase (UGT), sulfotran...

Identification and characterization of the ecdysteroid UDPglucosyltransferase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus

Treesearch

Christopher I. Riegel; Carita Lanner-Herrera; James M. Slavicek

1994-01-01

We have located, cloned, sequenced and characterized the ecdysteroid UDP-glucosyltransferase gene (egt) gene from the baculovirus Lymantria dispar multinucleocapsid nuclear polyhedrosis virus,(LdMNPV), which is specific for the gypsy moth (L. dispar). The egt gene from the related baculovirus Autographa californica...

Enzymatic synthesis of novel phloretin glucosides.

PubMed

Pandey, Ramesh Prasad; Li, Tai Feng; Kim, Eun-Hee; Yamaguchi, Tokutaro; Park, Yong Il; Kim, Joong Su; Sohng, Jae Kyung

2013-06-01

A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli.

Enzymatic Synthesis of Novel Phloretin Glucosides

PubMed Central

Pandey, Ramesh Prasad; Li, Tai Feng; Kim, Eun-Hee; Yamaguchi, Tokutaro; Park, Yong Il; Kim, Joong Su

2013-01-01

A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli. PMID:23542617

THE EFFECT OF BACULOVIRUS INFECTION ON ECDYSTEROID TITER IN GYPSY MOTH LARVAE (LYMANTRIA DISPAR).

EPA Science Inventory

Insect baculovirus carries a gene refered to as egt. This gene encodes an enzyme known as ecdysteroid UDP-glucosyl transferase which catalyzes the sugar conjugation of ecdysteroids. Using a gypsy moth embryonic cell line EGT activity of Lymantria dispar nuclear polyhedrosis virus...

Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

USDA-ARS?s Scientific Manuscript database

UDP-glucuronosyltransferase (UGT) activity toward the flavonoid quercetin and UGT protein were characterized in 3 equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats, n=8/age using villin to control for enterocyte content. SI microsomal intrinsic clearance of quercetin...

Success in the Urban Setting: Ohio's Urban Demonstration Projects.

ERIC Educational Resources Information Center

Ohio State Dept. of Education, Columbus.

The Urban Demonstration Projects (UDP) combined rehabilitative, preventive, and developmental services in a coordinated school and community effort to test the impact of a maximal educational program for disadvantaged students in Ohio's urban schools. This report, which was prepared by staff members from the various projects throughout the State,…

[Regulation of terpene metabolism]. [Mentha piperita, Mentha spicata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Croteau, R.

1989-01-01

Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes

USDA-ARS?s Scientific Manuscript database

The dietary bioavailability of the isoflavone genistein is decreased in older rats compared to young adults. Since flavonoids are metabolized extensively by the UDP-glucuronosyltransferases (UGTs), we hypothesized that UGT flavonoid conjugating activity changes with age. The effect of age on flavono...

Enabling IP Header Compression in COTS Routers via Frame Relay on a Simplex Link

NASA Technical Reports Server (NTRS)

Nguyen, Sam P.; Pang, Jackson; Clare, Loren P.; Cheng, Michael K.

2010-01-01

NASA is moving toward a networkcentric communications architecture and, in particular, is building toward use of Internet Protocol (IP) in space. The use of IP is motivated by its ubiquitous application in many communications networks and in available commercial off-the-shelf (COTS) technology. The Constellation Program intends to fit two or more voice (over IP) channels on both the forward link to, and the return link from, the Orion Crew Exploration Vehicle (CEV) during all mission phases. Efficient bandwidth utilization of the links is key for voice applications. In Voice over IP (VoIP), the IP packets are limited to small sizes to keep voice latency at a minimum. The common voice codec used in VoIP is G.729. This new algorithm produces voice audio at 8 kbps and in packets of 10-milliseconds duration. Constellation has designed the VoIP communications stack to use the combination of IP/UDP/RTP protocols where IP carries a 20-byte header, UDP (User Datagram Protocol) carries an 8-byte header, and RTP (Real Time Transport Protocol) carries a 12-byte header. The protocol headers total 40 bytes and are equal in length to a 40-byte G.729 payload, doubling the VoIP latency. Since much of the IP/UDP/RTP header information does not change from IP packet to IP packet, IP/UDP/RTP header compression can avoid transmission of much redundant data as well as reduce VoIP latency. The benefits of IP header compression are more pronounced at low data rate links such as the forward and return links during CEV launch. IP/UDP/RTP header compression codecs are well supported by many COTS routers. A common interface to the COTS routers is through frame relay. However, enabling IP header compression over frame relay, according to industry standard (Frame Relay IP Header Compression Agreement FRF.20), requires a duplex link and negotiations between the compressor router and the decompressor router. In Constellation, each forward to and return link from the CEV in space is treated independently as a simplex link. Without negotiation, the COTS routers are prevented from entering into the IP header compression mode, and no IP header compression would be performed. An algorithm is proposed to enable IP header compression in COTS routers on a simplex link with no negotiation or with a one-way messaging. In doing so, COTS routers can enter IP header compression mode without the need to handshake through a bidirectional link as required by FRF.20. This technique would spoof the routers locally and thereby allow the routers to enter into IP header compression mode without having the negotiations between routers actually occur. The spoofing function is conducted by a frame relay adapter (also COTS) with the capability to generate control messages according to the FRF.20 descriptions. Therefore, negotiation is actually performed between the FRF.20 adapter and the connecting COTS router locally and never occurs over the space link. Through understanding of the handshaking protocol described by FRF.20, the necessary FRF.20 negotiations messages can be generated to control the connecting router, not only to turn on IP header compression but also to adjust the compression parameters. The FRF.20 negotiation (or control) message is composed in the FRF.20 adapter by interpreting the incoming router request message. Many of the fields are simply transcribed from request to response while the control field indicating response and type are modified.

Effects of Andrographis paniculata and Orthosiphon stamineus extracts on the glucuronidation of 4-methylumbelliferone in human UGT isoforms.

PubMed

Ismail, Sabariah; Hanapi, Nur Aziah; Ab Halim, Mohd Rohaimi; Uchaipichat, Verawan; Mackenzie, Peter I

2010-05-14

The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU) by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA) as the cofactor, MgCl(2), cell lysate of respective isoform, and 4MU at the approximate apparent K(m) or S(50) value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 microg/mL and 0.01, 0.10, 1.0, 10 and 50 microg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC(50) 1.70 microg/mL for UGT1A3, 2.57 microg/mL for UGT1A8, 2.82 microg/mL for UGT2B7, 5.00 micorg/mL for UGT1A1, 5.66 microg/mL for UGT1A6, 9.88 microg/mL for UGT1A7 and 15.66 microg/mL for UGT1A10). Both extracts showed less than 70% inhibition of UGT2B15, so the IC(50) values were >50 microg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

Differences in metabolite-mediated toxicity of tamoxifen in rodents versus humans elucidated with DNA/microsome electro-optical arrays and nanoreactors.

PubMed

Zhao, Linlin; Krishnan, Sadagopan; Zhang, Yun; Schenkman, John B; Rusling, James F

2009-02-01

Tamoxifen, a therapeutic and chemopreventive breast cancer drug, was chosen as a model compound because of acknowledged species specific toxicity differences. Emerging approaches utilizing electro-optical arrays and nanoreactors based on DNA/microsome films were used to compare metabolite-mediated toxicity differences of tamoxifen in rodents versus humans. Hits triggered by liver enzyme metabolism were first provided by arrays utilizing a DNA damage end point. The arrays feature thin-film spots containing an electrochemiluminescent (ECL) ruthenium polymer ([Ru(bpy)(2)PVP(10)](2+); PVP, polyvinylpyridine), DNA, and liver microsomes. When DNA damage resulted from reactions with tamoxifen metabolites, it was detected by an increase in light from the oxidation of the damaged DNA by the ECL metallopolymer. The slope of ECL generation versus enzyme reaction time correlated with the rate of DNA damage. An approximate 2-fold greater ECL turnover rate was observed for spots with rat liver microsomes compared to that with human liver microsomes. These results were supported by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of reaction products using nanoreactors featuring analogous films on silica nanoparticles, allowing the direct measurement of the relative formation rate for alpha-(N(2)-deoxyguanosinyl)tamoxifen. We observed 2-5-fold more rapid formation rates for three major metabolites, i.e., alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and tamoxifen N-oxide, catalyzed by rat liver microsomes compared to human liver microsomes. Comparable formation rates were observed for N-desmethyl tamoxifen with rat and human liver microsomes. A better detoxifying capacity for human liver microsomes than rat liver microsomes was confirmed utilizing glucuronyltransferase in microsomes together with UDP-glucuronic acid. Taken together, lower genotoxicity and higher detoxication rates presented by human liver microsomes correlate with the lower risk of tamoxifen in causing liver carcinoma in humans, provided the glucuronidation pathway is active.

Selective and Catalyst-free Oxidation of D-Glucose to D-Glucuronic acid induced by High-Frequency Ultrasound

NASA Astrophysics Data System (ADS)

Amaniampong, Prince N.; Karam, Ayman; Trinh, Quang Thang; Xu, Kai; Hirao, Hajime; Jérôme, François; Chatel, Gregory

2017-01-01

This systematic experimental investigation reveals that high-frequency ultrasound irradiation (550 kHz) induced oxidation of D-glucose to glucuronic acid in excellent yield without assistance of any (bio)catalyst. Oxidation is induced thanks to the in situ production of radical species in water. Experiments show that the dissolved gases play an important role in governing the nature of generated radical species and thus the selectivity for glucuronic acid. Importantly, this process yields glucuronic acid instead of glucuronate salt typically obtained via conventional (bio)catalyst routes, which is of huge interest in respect of downstream processing. Investigations using disaccharides revealed that radicals generated by high frequency ultrasound were also capable of promoting tandem hydrolysis/oxidation reactions.

Cell wall composition and digestibility alterations in Brachypodium distachyon achieved through reduced expression of the UDP-arabinopyranose mutase

PubMed Central

Rancour, David M.; Hatfield, Ronald D.; Marita, Jane M.; Rohr, Nicholas A.; Schmitz, Robert J.

2015-01-01

Nucleotide-activated sugars are essential substrates for plant cell-wall carbohydrate-polymer biosynthesis. The most prevalent grass cell wall (CW) sugars are glucose (Glc), xylose (Xyl), and arabinose (Ara). These sugars are biosynthetically related via the UDP–sugar interconversion pathway. We sought to target and generate UDP–sugar interconversion pathway transgenic Brachypodium distachyon lines resulting in CW carbohydrate composition changes with improved digestibility and normal plant stature. Both RNAi-mediated gene-suppression and constitutive gene-expression approaches were performed. CWs from 336 T0 transgenic plants with normal appearance were screened for complete carbohydrate composition. RNAi mutants of BdRGP1, a UDP-arabinopyranose mutase, resulted in large alterations in CW carbohydrate composition with significant decreases in CW Ara content but with minimal change in plant stature. Five independent RNAi-RGP1 T1 plant lines were used for in-depth analysis of plant CWs. Real-time PCR analysis indicated that gene expression levels for BdRGP1, BdRGP2, and BdRGP3 were reduced in RNAi-RGP1 plants to 15–20% of controls. CW Ara content was reduced by 23–51% of control levels. No alterations in CW Xyl and Glc content were observed. Corresponding decreases in CW ferulic acid (FA) and ferulic acid-dimers (FA-dimers) were observed. Additionally, CW p-coumarates (pCA) were decreased. We demonstrate the CW pCA decrease corresponds to Ara-coupled pCA. Xylanase-mediated digestibility of RNAi-RGP1 Brachypodium CWs resulted in a near twofold increase of released total carbohydrate. However, cellulolytic hydrolysis of CW material was inhibited in leaves of RNAi-RGP1 mutants. Our results indicate that targeted manipulation of UDP–sugar biosynthesis can result in biomass with substantially altered compositions and highlights the complex effect CW composition has on digestibility. PMID:26136761

PROTEOMIC ANALYSIS OPTIMIZATION: SELECTIVE PROTEIN SAMPLE ON-COLUMN RETENTION IN REVERSE-PHASE LIQUID CHROMATOGRAPHY

EPA Science Inventory

Why work was done?

To be able to identify, on a proteomic level, cytochromes P450 (CYP) and UDP-glucuronosyltransferases (UGT) in mouse liver microsomes for the conazole exposure study IRP # NHEERL-ECD-SCN-CZ-2002-01-R1_Addendum 1. The new enrichment method was necessary beca...

Development of Real Time System for Data Communication Based on SCO UNIX

NASA Astrophysics Data System (ADS)

Hua, Ying-Min

2002-01-01

The real time system based on SCO UNIX has the multiple tasks properties as on other UNIX system. The costs is lower than other UNIX system. In this paper the usage of multiple serial communication and UDP communication is mainly introduced. The data housekeeping and system monitor are described.

Gene regulation of UDP-galactose synthesis and transport: Potential rate limiting processes in initiation of milk production in humans

USDA-ARS?s Scientific Manuscript database

Lactose synthesis is believed to be rate-limiting for milk production. However, understanding the molecular events controlling lactose synthesis in humans is still rudimentary. We have utilized our established model of the RNA isolated from breast milk fat globule from 7 healthy exclusively breastfe...

[Regulation of terpene metabolism]. Annual progress report, March 15, 1988--March 14, 1989

DOE Office of Scientific and Technical Information (OSTI.GOV)

Croteau, R.

1989-12-31

Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

Age-related increases in F344 rat intestine microsomal quercetin glucuronidation

USDA-ARS?s Scientific Manuscript database

The objective of this study was to establish the extent age modifies intestinal quercetin glucuronidation capacity. Pooled microsomal fractions of three equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats (n=8/group) were employed to model the enzyme kinetics of UDP-gl...

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

PubMed Central

Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

2003-01-01

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase. PMID:12429023

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

PubMed

Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

2003-03-01

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.

Agm1/Pgm3-Mediated Sugar Nucleotide Synthesis Is Essential for Hematopoiesis and Development▿

PubMed Central

Greig, Kylie T.; Antonchuk, Jennifer; Metcalf, Donald; Morgan, Phillip O.; Krebs, Danielle L.; Zhang, Jian-Guo; Hacking, Douglas F.; Bode, Lars; Robb, Lorraine; Kranz, Christian; de Graaf, Carolyn; Bahlo, Melanie; Nicola, Nicos A.; Nutt, Stephen L.; Freeze, Hudson H.; Alexander, Warren S.; Hilton, Douglas J.; Kile, Benjamin T.

2007-01-01

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity. PMID:17548465

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.

PubMed

Lim, Michelle Yi-Xiu; LaMonte, Gregory; Lee, Marcus C S; Reimer, Christin; Tan, Bee Huat; Corey, Victoria; Tjahjadi, Bianca F; Chua, Adeline; Nachon, Marie; Wintjens, René; Gedeck, Peter; Malleret, Benoit; Renia, Laurent; Bonamy, Ghislain M C; Ho, Paul Chi-Lui; Yeung, Bryan K S; Chow, Eric D; Lim, Liting; Fidock, David A; Diagana, Thierry T; Winzeler, Elizabeth A; Bifani, Pablo

2016-09-19

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

Crystal Structure of a UDP-glucose-specific Glycosyltransferase from a Mycobacterium Species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fulton, Zara; McAlister, Adrian; Wilce, Matthew C.J.

2008-10-24

Glycosyltransferases (GTs) are a large and ubiquitous family of enzymes that specifically transfer sugar moieties to a range of substrates. Mycobacterium tuberculosis contains a large number of GTs, many of which are implicated in cell wall synthesis, yet the majority of these GTs remain poorly characterized. Here, we report the high resolution crystal structures of an essential GT (MAP2569c) from Mycobacterium avium subsp. paratuberculosis (a close homologue of Rv1208 from M. tuberculosis) in its apo- and ligand-bound forms. The structure adopted the GT-A fold and possessed the characteristic DXD motif that coordinated an Mn{sup 2+} ion. Atypical of most GTsmore » characterized to date, MAP2569c exhibited specificity toward the donor substrate, UDP-glucose. The structure of this ligated complex revealed an induced fit binding mechanism and provided a basis for this unique specificity. Collectively, the structural features suggested that MAP2569c may adopt a 'retaining' enzymatic mechanism, which has implications for the classification of other GTs in this large superfamily.« less

Synthesis of FUDP-N-acetylglucosamine and FUDP-glucose in Saccharomyces cerevisiae cells treated with 5-fluorouracil.

PubMed

Günther Sillero, María A; Pérez-Zúñiga, Francisco; Gomes, Joana; de Carvalho, Ana Isabel; Martins, Susana; Silles, Eduardo; Sillero, Antonio

2008-03-01

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.

Radioimmune assay of ganglioside GM/sub 1/ synthase using cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Honke, K.; Taniguchi, N.; Makita, A.

1986-01-01

A radioimmune assay for uridine 5'-diphosphate-galactose (UDP-Gal):GM/sub 2/ galactosyltransferase, which synthesizes GM/sub 1/, has been developed utilizing cholera toxin. This assay is more sensitive and simpler than previously used assays. Radioactive nucleotide substrate and GM/sub 2/ were incubated with an enzyme sample, and a radiolabeled product, GM/sub 1/, was reacted with cholera toxin. The GM/sub 1/-cholera toxin complex was further reacted with anti-cholera toxin and Staphylococcus aureus cell suspension. The resulting complex was transferred onto a nitrocellulose membrane and quantitated by liquid scintillation counting. This assay was found to be sensitive for the detection of 100 pmol of the reactionmore » product, GM/sub 1/. With this assay method, some properties of the crude enzyme extracts from rat liver were studied. The enzyme had a pH optimum of 6.5-7.0 and required Mn/sup 2 +/. The K/sub m/ values for UDP-Gal and GM/sub 2/ were 0.12 mM and 6 ..mu..M, respectively.« less

Base-modified GDP-mannose derivatives and their substrate activity towards a yeast mannosyltransferase.

PubMed

Collier, Alice; Wagner, Gerd K

2017-11-27

We have previously developed a new class of inhibitors and chemical probes for glycosyltransferases through base-modification of the sugar-nucleotide donor. The key feature of these donor analogues is the presence of an additional substituent at the nucleobase. To date, the application of this general concept has been limited to UDP-sugars and UDP-sugar-dependent glycosyltransferases. Herein, we report for the first time the application of our approach to a GDP-mannose-dependent mannosyltransferase. We have prepared four GDP-mannose derivatives with an additional substituent at either position 6 or 8 of the nucleobase. These donor analogues were recognised as donor substrates by the mannosyltransferase Kre2p from yeast, albeit with significantly lower turnover rates than the natural donor GDP-mannose. The presence of the additional substituent also redirected enzyme activity from glycosyl transfer to donor hydrolysis. Taken together, our results suggest that modification of the donor nucleobase is, in principle, a viable strategy for probe and inhibitor development against GDP-mannose-dependent GTs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selective and Catalyst-free Oxidation of D-Glucose to D-Glucuronic acid induced by High-Frequency Ultrasound

PubMed Central

Amaniampong, Prince N.; Karam, Ayman; Trinh, Quang Thang; Xu, Kai; Hirao, Hajime; Jérôme, François; Chatel, Gregory

2017-01-01

This systematic experimental investigation reveals that high-frequency ultrasound irradiation (550 kHz) induced oxidation of D-glucose to glucuronic acid in excellent yield without assistance of any (bio)catalyst. Oxidation is induced thanks to the in situ production of radical species in water. Experiments show that the dissolved gases play an important role in governing the nature of generated radical species and thus the selectivity for glucuronic acid. Importantly, this process yields glucuronic acid instead of glucuronate salt typically obtained via conventional (bio)catalyst routes, which is of huge interest in respect of downstream processing. Investigations using disaccharides revealed that radicals generated by high frequency ultrasound were also capable of promoting tandem hydrolysis/oxidation reactions. PMID:28084448

Rare ginsenoside Ia synthesized from F1 by cloning and overexpression of the UDP-glycosyltransferase gene from Bacillus subtilis: synthesis, characterization, and in vitro melanogenesis inhibition activity in BL6B16 cells.

PubMed

Wang, Dan-Dan; Jin, Yan; Wang, Chao; Kim, Yeon-Ju; Perez, Zuly Elizabeth Jimenez; Baek, Nam In; Mathiyalagan, Ramya; Markus, Josua; Yang, Deok-Chun

2018-01-01

Ginsenoside F1 has been described to possess skin-whitening effects on humans. We aimed to synthesize a new ginsenoside derivative from F1 and investigate its cytotoxicity and melanogenesis inhibitory activity in B16BL6 cells using recombinant glycosyltransferase enzyme. Glycosylation has the advantage of synthesizing rare chemical compounds from common compounds with great ease. UDP-glycosyltransferase (BSGT1) gene from Bacillus subtilis was selected for cloning. The recombinant glycosyltransferase enzyme was purified, characterized, and utilized to enzymatically transform F1 into its derivative. The new product was characterized by NMR techniques and evaluated by MTT, melanin count, and tyrosinase inhibition assay. The new derivative was identified as (20 S )-3 β ,6 α ,12 β ,20-tetrahydroxydammar-24-ene-20- O - β -D-glucopyranosyl-3- O - β -D-glucopyranoside (ginsenoside Ia), which possesses an additional glucose linked into the C-3 position of substrate F1. Ia had been previously reported; however, no in vitro biological activity was further examined. This study focused on the mass production of arduous ginsenoside Ia from accessible F1 and its inhibitory effect of melanogenesis in B16BL6 cells. Ia showed greater inhibition of melanin and tyrosinase at 100 μmol/L than F1 and arbutin. These results suggested that Ia decreased cellular melanin synthesis in B16BL6 cells through downregulation of tyrosinase activity. To our knowledge, this is the first study to report on the mass production of rare ginsenoside Ia from F1 using recombinant UDP-glycosyltransferase isolated from B. subtillis and its superior melanogenesis inhibitory activity in B16BL6 cells as compared to its precursor. In brief, ginsenoside Ia can be applied for further study in cosmetics.

A diet containing the soy phytoestrogen genistein causes infertility in female rats partially deficient in UDP glucuronyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seppen, Jurgen, E-mail: j.seppen@amc.uva.nl

Soy beans contain genistein, a natural compound that has estrogenic effects because it binds the estrogen receptor with relatively high affinity. Genistein is therefore the most important environmental estrogen in the human diet. Detoxification of genistein is mediated through conjugation by UDP-glucuronyltransferase 1 and 2 (UGT1 and UGT2) isoenzymes. Gunn rats have a genetic deficiency in UGT1 activity, UGT2 activities are not affected. Because our Gunn rats stopped breeding after the animal chow was changed to a type with much higher soy content, we examined the mechanism behind this soy diet induced infertility. Gunn and control rats were fed dietsmore » with and without genistein. In these rats, plasma levels of genistein and metabolites, fertility and reproductive parameters were determined. Enzyme assays showed reduced genistein UGT activity in Gunn rats, as compared to wild type rats. Female Gunn rats were completely infertile on a genistein diet, wild type rats were fertile. Genistein diet caused a persistent estrus, lowered serum progesterone and inhibited development of corpora lutea in Gunn rats. Concentrations of total genistein in Gunn and control rat plasma were identical and within the range observed in humans after soy consumption. However, Gunn rat plasma contained 25% unconjugated genistein, compared to 3.6% in control rats. This study shows that, under conditions of reduced glucuronidation, dietary genistein exhibits a strongly increased estrogenic effect. Because polymorphisms that reduce UGT1 expression are prevalent in the human population, these results suggest a cautionary attitude towards the consumption of large amounts of soy or soy supplements. -- Highlights: ► Gunn rats are partially deficient in detoxification by UDP glucuronyltransferases. ► Female Gunn rats are infertile on a soy containing diet. ► Soy contains genistein, a potent phytoestrogen. ► Inefficient glucuronidation of genistein causes female infertility.« less

Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes.

PubMed

Kanabar, Varsha; Tedaldi, Lauren; Jiang, Jingqian; Nie, Xiaodan; Panina, Irina; Descroix, Karine; Man, Francis; Pitchford, Simon C; Page, Clive P; Wagner, Gerd K

2016-10-01

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell-cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1: ) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1: also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1: , which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development. © The Author 2016. Published by Oxford University Press.

Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes

PubMed Central

Kanabar, Varsha; Tedaldi, Lauren; Jiang, Jingqian; Nie, Xiaodan; Panina, Irina; Descroix, Karine; Man, Francis; Pitchford, Simon C; Page, Clive P; Wagner, Gerd K

2016-01-01

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell–cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1 also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1, which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development. PMID:27233805

Identification of O-GlcNAcylated proteins in Plasmodium falciparum.

PubMed

Kupferschmid, Mattis; Aquino-Gil, Moyira Osny; Shams-Eldin, Hosam; Schmidt, Jörg; Yamakawa, Nao; Krzewinski, Frédéric; Schwarz, Ralph T; Lefebvre, Tony

2017-11-29

Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans. The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS). While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins. This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.

[Human drug metabolizing enzymes. II. Conjugation enzymes].

PubMed

Vereczkey, L; Jemnitz, K; Gregus, Z

1998-09-01

In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia

PubMed Central

Wyroba, E.; Kwaśniak, P.; Miller, K.; Kobyłecki, K.; Osińska, M.

2016-01-01

Protein products of paralogous genes resulting from whole genome duplication may acquire new functions. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue (distinct from that of Rab7a directly involved in phagocytosis) was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala diminished the incorporation of [P32] by 37% and of [C14-]UDP-glucose by 24% into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells in contrast to non-mutagenized recombinant Rab7b correctly incorporated in the cytostome area. Using nano LC-MS/MS to compare the peptide map of Rab7b with that after deglycosylation with a mixture of five enzymes of different specificity we identified a peptide ion at m/z=677.63+ representing a glycan group attached to Thr200. Based on its mass and quantitative assays with [P32] and [C14]UDP-glucose, the suggested composition of the adduct attached to Thr200 is (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Paramecium octaurelia Rab7b is crucial for the proper localization/function of this protein. Moreover, the two Rab7 paralogues differ also in another PTM: substantially more phosphorylated amino acid residues are in Rab7b than in Rab7a. PMID:27349314

Structure of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC).

PubMed

Deva, Taru; Baker, Edward N; Squire, Christopher J; Smith, Clyde A

2006-12-01

The bacterial cell wall provides essential protection from the external environment and confers strength and rigidity to counteract internal osmotic pressure. Without this layer the cell would be easily ruptured and it is for this reason that biosynthetic pathways leading to the formation of peptidoglycan have for many years been a prime target for effective antibiotics. Central to this pathway are four similar ligase enzymes which add peptide groups to glycan moieties. As part of a program to better understand the structure-function relationships in these four enzymes, the crystal structure of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC) has been determined to 2.6 A resolution. The structure was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and refined to a crystallographic R factor of 0.212 (R(free) = 0.259). The enzyme has a modular multi-domain structure very similar to those of other members of the mur family of ATP-dependent amide-bond ligases. Detailed comparison of these four enzymes shows that considerable conformational changes are possible. These changes, together with the recruitment of two different N-terminal domains, allow this family of enzymes to bind a substrate which is identical at one end and at the other has the growing peptide tail which will ultimately become part of the rigid bacterial cell wall. Comparison of the E. coli and Haemophilus influenzae structures and analysis of the sequences of known MurC enzymes indicate the presence of a ;dimerization' motif in almost 50% of the MurC enzymes and points to a highly conserved loop in domain 3 that may play a key role in amino-acid ligand specificity.

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

PubMed

El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

2003-03-01

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

PubMed

Horynová, Milada; Takahashi, Kazuo; Hall, Stacy; Renfrow, Matthew B; Novak, Jan; Raška, Milan

2012-02-01

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system. Copyright © 2011 Elsevier Inc. All rights reserved.

Regional Heritability Mapping Provides Insights into Dry Matter Content in African White and Yellow Cassava Populations.

PubMed

Okeke, Uche Godfrey; Akdemir, Deniz; Rabbi, Ismail; Kulakow, Peter; Jannink, Jean-Luc

2018-03-01

The HarvestPlus program for cassava ( Crantz) fortifies cassava with β-carotene by breeding for carotene-rich tubers (yellow cassava). However, a negative correlation between yellowness and dry matter (DM) content has been identified. We investigated the genetic control of DM in white and yellow cassava. We used regional heritability mapping (RHM) to associate DM with genomic segments in both subpopulations. Significant segments were subjected to candidate gene analysis and candidates were validated with prediction accuracies. The RHM procedure was validated via a simulation approach and revealed significant hits for white cassava on chromosomes 1, 4, 5, 10, 17, and 18, whereas hits for the yellow were on chromosome 1. Candidate gene analysis revealed genes in the carbohydrate biosynthesis pathway including plant serine-threonine protein kinases (SnRKs), UDP (uridine diphosphate)-glycosyltransferases, UDP-sugar transporters, invertases, pectinases, and regulons. Validation using 1252 unique identifiers from the SnRK gene family genome-wide recovered 50% of the predictive accuracy of whole-genome single nucleotide polymorphisms for DM, whereas validation using 53 likely genes (extracted from the literature) from significant segments recovered 32%. Genes including an acid invertase, a neutral or alkaline invertase, and a glucose-6-phosphate isomerase were validated on the basis of an a priori list for the cassava starch pathway, and also a fructose-biphosphate aldolase from the Calvin cycle pathway. The power of the RHM procedure was estimated as 47% when the causal quantitative trait loci generated 10% of the phenotypic variance (sample size = 451). Cassava DM genetics are complex and RHM may be useful for complex traits. Copyright © 2018 Crop Science Society of America.

Solubilization of an Arabinan Arabinosyltransferase Activity from Mung Bean Hypocotyls1

PubMed Central

Nunan, Kylie Joy; Scheller, Henrik Vibe

2003-01-01

The biosynthesis of polysaccharides destined for the plant cell wall and the subsequent assembly of the cell wall are poorly understood processes that are currently the focus of much research. Arabinan, a component of the pectic polysaccharide rhamnogalacturonan I, is composed of arabinosyl residues connected via various glycosidic linkages, and therefore, the biosynthesis of arabinan is likely to involve more than one arabinosyltransferase. We have studied the transfer of [14C]arabinose (Ara) from UDP-l-arabinopyranose onto polysaccharides using microsomal membranes isolated from mung bean (Vigna radiata) hypocotyls. [14C]arabinosyl and [14C]xylosyl residues were incorporated into endogenous products due to the presence of UDP-Xyl-4-epimerase activity. Enzymatic digestion of endogenous products with endo-arabinanase released very little radiolabeled sugars, whereas digestion with arabinofuranosidase released some [14C]Ara. Microsomal membranes solubilized with the detergent octyl glucoside were able to add a single [14C]Ara residue onto (1→5)-linked α-l-arabino-oligosaccharide acceptors. The reaction had a pH optimum of 6.5 and a requirement for manganese ions. However, enzymatic digestion of the radiolabeled oligosaccharides with endo-arabinanase and arabinofuranosidases could not fully release the radiolabeled Ara residue, indicating that the [14C]Ara residue was not a (1→2)-, (1→3)-, or (1→5)-linked α-l-arabinofuranosyl residue. Rather, mild acid treatment of the product suggested that the radiolabeled Ara residue was in a pyranose conformation, and this result was confirmed by thin-layer chromatography of radiolabeled partially methylated sugars. Using microsomal membranes separated on a discontinuous sucrose gradient, the arabinosyltransferase activity appears to be mainly localized to Golgi membranes. PMID:12746538

Crystal structure of Os79 (Os04g0206600) from Oryza sativa: a UDP-glucosyltransferase involved in the detoxification of deoxynivalenol

USDA-ARS?s Scientific Manuscript database

Fusarium head blight is a plant disease with significant agricultural and health impact which affects cereal crops such as wheat, barley, and maize and is characterized by reduced grain yield and the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). Studies have identified trich...

77 FR 43089 - Evaluation of an Up-and-Down Procedure for Acute Dermal Systemic Toxicity Testing: Request for...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-23

... Systemic Toxicity Testing: Request for Nominations for an Independent Expert Panel and Submission of... systemic toxicity testing. NICEATM requests nominations of scientific experts who can be considered for the...) Test Guideline 425 in 2001 (OECD, 2001). The oral UDP reduces animal use by up to 70% compared to the...

Tools and Techniques for Simplifying the Analysis of Captured Packet Data

ERIC Educational Resources Information Center

Cavaiani, Thomas P.

2008-01-01

Students acquire an understanding of the differences between TCP and UDP (connection-oriented vs. connection-less) data transfers as they analyze network packet data collected during one of a series of labs designed for an introductory network essentials course taught at Boise State University. The learning emphasis of the lab is not on the…

Improving Operational Effectiveness of Tactical Long Endurance Unmanned Aerial Systems (TALEUAS) by Utilizing Solar Power

DTIC Science & Technology

2014-06-01

Speed xiii TEK Total Energy Compensated TSP traveling salesman problem UAV unmanned aerial vehicle UDP user datagram protocol UKF unscented...discretized map, and use the map to optimally solve the navigation task. The optimal navigation solution utilizes the well-known “ travelling salesman problem ...2 C. FORMULATION OF THE PROBLEM .................................................. 3 D

Inhibition of Glucuronokinase by Substrate Analogs 1

PubMed Central

Gillard, Douglas F.; Dickinson, David B.

1978-01-01

Glucuronokinase from Lilium longiflorum pollen was purified 30- to 40- fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and all were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a Ki value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mm ATP, while the rates for 10 mm GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl α-glucuronoside, methyl β-glucuronoside, β-glucuronic acid-1-phosphate, and 4-O-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The β-glucuronic acid-1-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depending on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective. PMID:16660589

Transgene silencing of sucrose synthase in alfalfa stem vascular tissue by a truncated phosphoenolpyruvate carboxylase: sucrose synthase construct

USDA-ARS?s Scientific Manuscript database

An important role of sucrose synthase (SUS, EC 2.4.1.13) in plants is to provide UDP-glucose needed for cellulose synthesis in cell walls. We examined if over-expressing SUS in alfalfa (Medicago sativa L.) would increase cellulose content of stem cell walls. Alfalfa plants were transformed with two ...

2-acylamido analogues of N-acetylglucosamine prime formation of chitin oligosaccharides by yeast chitin synthase 2

USDA-ARS?s Scientific Manuscript database

Chitin, a polymer of beta-1,4-linked N-acetylglucosamine (GlcNAc), is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases (CSs) transfer GlcNAc from UDP-GlcNAc to pre-existing chitin chains in reactions that are typically stimulated by free GlcNAc. The eff...

User Data Package (UDP) for Packaged Cogeneration Systems (PCS)

DTIC Science & Technology

1990-05-01

Standards for PURPA Compliance ............ ...................... 10 1.3 Selected Commercial, Institutional, and Multi-unit Technically Feasible...percent. The Federal Energy Regulatory Commission (FERC), in accordance with Section 201 of the Public Utility Regulatory Policies Act ( PURPA ) of 1978...percent of the time the engine was running, or if 57 percent of the recovered engine heat were stored. Additional requirements for PURPA efficiency that

DEPENDENCE OF ECDYSTEROID METABOLISM AND DEVELOPMENT IN HOST LARVAE ON THE TIME OF BACULOVIRUS INFECTION AND THE ACTIVITY OF THE UDP-GLUCOSYL TRANSFERASE GENE.

EPA Science Inventory

Infection of fourth-instar gypsy moth (Lymantria dispar, Lepidoptera: Lymantriidae) larvae with the wild-type (Wt) gypsy moth baculovirus, LdNPV on the first day post-molt, or infection of fifth instars on the fifth day post-molt, results in elevated ecdysteroid levels in both he...

The Effects of Cognitive Jamming on Wireless Sensor Networks Used for Geolocation

DTIC Science & Technology

2012-03-01

continuously sends out random bits to the channel without following any MAC-layer etiquette [31]. Normally, the underlying MAC protocol allows...23 UDP User Datagram Protocol . . . . . . . . . . . . . . . . . . . 30 MIMO Multiple Input Multiple Output . . . . . . . . . . . . . . . 70...information is packaged and distributed on the network layer, only the physical measurements are considered. This protocol is used to detect faulty nodes

On-target labeling of intracellular metabolites combined with chemical mapping of individual hyphae revealing cytoplasmic relocation of isotopologues.

PubMed

Hu, Jie-Bi; Chen, Yu-Chie; Urban, Pawel L

2012-06-05

A microscale analytical platform integrating microbial cell culture, isotopic labeling, along with visual and mass spectrometric imaging with single-cell resolution has been developed and applied in the monitoring of cellular metabolism in fungal mycelium. The method implements open chips with a two-dimensional surface pattern composed of hydrophobic and hydrophilic zones. Two hydrophilic islands are used as medium reservoirs, while the hydrophobic area constitutes the support for the growing aerial hyphae, which do not have direct contact with the medium. The first island, containing (12)C(6)-glucose medium, was initially inoculated with the mycelium (Neurospora crassa), and following the initial incubation period, the hyphae progressed toward the second medium island, containing an isotopically labeled substrate ((13)C(6)-glucose). The (13)C atoms were gradually incorporated into cellular metabolites, which was revealed by MALDI-MS. The fate of the chitin-biosynthesis precursor, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), was monitored by recording mass spectra with characteristic isotopic patterns, which indicated the presence of various (12)C/(13)C isotopologues. The method enabled mapping the (13)C-labeled UDP-GlcNAc in fungal mycelium and recording its redistribution in hyphae, directly on the chip.

Radio astronomy interferometer network testing for a Malaysia-China real-time e-VLBI

NASA Astrophysics Data System (ADS)

Abidin, Zamri Zainal; Hashim, Shaiful Jahari; Wei, Lim Yang; Zhong, Chen; Rosli, Zulfazli

2018-01-01

The uv-coverage of the current VLBI network between Australia northern Asia will be significantly enhanced with an existence of a middle baseline VLBI station located in Malaysia. This paper investigated the connecting route of the first half of the Asia-Oceania VLBI network i.e. from Malaysia to China. The investigation of transmission network characteristics between Malaysia and China was carried out in order to perform a real-time and reliable data transfer within the e-VLBI network for future eVLBI observations. MyREN (Malaysia) and CSTNET (China) high-speed research networks were utilized for this proposed e-VLBI connection. Preliminary network test was performed by ping, traceroute, and iperf prior to data transfer tests, which were evaluated with three types of protocols namely FTP, Tsunami-UDT and UDT. The results showed that, on average, there were eighteen hops between Malaysia and China networks with 98 ms round trip time (RTT) delay. Overall UDP protocol has a better throughput compared to TCP protocol. UDP can reach a maximum rate of 90 Mbps with 0% packet loss. In this feasibility test, the VLBI test data was successfully transferred between Malaysia and China by utilizing the three types of data transfer protocols.

Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

PubMed

Gurung, Rit Bahadur; Gong, So Youn; Dhakal, Dipesh; Le, Tuoi Thi; Jung, Na Rae; Jung, Hye Jin; Oh, Tae Jin; Sohng, Jae Kyung

2017-09-28

Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, antiinflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP- α-D-glucose or UDP-α-D-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'- O -β- glucoside, curcumin 4',4''-di- O -β-glucoside, curcumin 4'- O -β-2-deoxyglucoside, and curcumin 4',4''-di- O -β-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'- O -β-glucoside and curcumin 4'- O -β-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

Species-specific glucosylation of DIMBOA in larvae of the rice Armyworm.

PubMed

Sasai, Hiroaki; Ishida, Masahiro; Murakami, Kenjiro; Tadokoro, Naoko; Ishihara, Atsushi; Nishida, Ritsuo; Mori, Naoki

2009-06-01

DIMBOA [2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] is a benzoxazinoid (Bx), part of the chemical defense system of graminaceous plants such as maize, wheat, and rye. When Bombyx mori larvae were fed artificial diets containing DIMBOA, they died in three days. In contrast, Mythimna separata larvae, a serious pest of rice, maize, sorghum, wheat etc., grew well on the same diets. Three kinds of glucosides [1-(2-hydroxy-4-methoxyphenylamino)-1-deoxy-beta-glucopyranoside-1,2-carbamate (methoxy glucoside carbamate), 2-O-beta-glucopyranosyl-4-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA-2-O-Glc), and 2-O-beta-glucopyranosyl-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (HMBOA-2-O-Glc)] were identified by LC-MS and NMR analyses from the frass of M. separata that had been fed on a DIMBOA-containing diet. Furthermore, the incubation of DIMBOA with a midgut tissue suspension of M. separata in the presence of UDP-D-glucose generated DIMBOA-2-O-Glc. These findings strongly suggest that glucosylation by UDP-glucosyltransferase(s) was important for detoxification to circumvent the defenses of host plants against M. separata larvae.

Decapitation improves the efficiency of Cd phytoextraction by Celosia argentea Linn.

PubMed

Liu, Jie; Zhang, Xuehong; Mo, Lingyun; Yao, Shiyin; Wang, Yixuan

2017-08-01

The effect of decapitation on enhancing plant growth and Cd accumulation in Celosia argentea Linn. was evaluated using a pot experiment. Decapitation significantly enhanced the growth of C. argentea. The numbers of branch and leaf in the decapitated plants (DP) were significantly higher than those in undecapitated plants (UDP, p < 0.05). Decapitation increased the biomass by 75%-105% for roots, 108%-152% for stems, and 80%-107% for leaves. Although the transpiration and photosynthesis rates were not significantly different between DP and UPD, decapitation significantly increased the total leaf area and total transpiration per plant (p < 0.05). The higher total transpiration per plant resulted in a higher leaf Cd concentration in DP. DP accumulated Cd in shoots (197, 275, and 425 μg plant -1 ) that were 2.5-2.8 times higher than UDP (78, 108, and 152 μg plant -1 ), with the soils containing 1, 5, and 10 mg kg -1 Cd. Results suggested that decapitation is a novel and convenient method to improve the phytoextraction efficiency of C. argentea in Cd contaminated soils. Copyright © 2017 Elsevier Ltd. All rights reserved.

Suppression of abnormal morphology and extracytoplasmic function sigma activity in Bacillus subtilis ugtP mutant cells by expression of heterologous glucolipid synthases from Acholeplasma laidlawii.

PubMed

Matsuoka, Satoshi; Seki, Takahiro; Matsumoto, Kouji; Hara, Hiroshi

2016-12-01

Glucolipids in Bacillus subtilis are synthesized by UgtP processively transferring glucose from UDP-glucose to diacylglycerol. Here we conclude that the abnormal morphology of a ugtP mutant is caused by lack of glucolipids, since the same morphology arises after abolition of glucolipid production by disruption of pgcA and gtaB, which are involved in UDP-glucose synthesis. Conversely, expression of a monoglucosyldiacylglycerol (MGlcDG) produced by 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii (alMGS) almost completely suppressed the ugtP disruptant phenotype. Activation of extracytoplasmic function (ECF) sigmas (SigM, SigV, and SigX) in the ugtP mutant was decreased by alMGS expression, and was suppressed to low levels by MgSO 4 addition. When alMGS and alDGS (A. laidlawii 1,2-diacylglycerol-3-glucose (1-2)-glucosyltransferase producing diglucosyldiacylglycerol (DGlcDG)) were simultaneously expressed, SigX activation was repressed to wild type level. These observations suggest that MGlcDG molecules are required for maintenance of B. subtilis cell shape and regulation of ECF sigmas, and DGlcDG regulates SigX activity.

Genome-wide identification of glycosyltransferases converting phloretin to phloridzin in Malus species.

PubMed

Zhou, Kun; Hu, Lingyu; Li, Pengmin; Gong, Xiaoqing; Ma, Fengwang

2017-12-01

Phloridzin (phloretin 2'-O-glucoside) is the most abundant phenolic compound in Malus species, accounting for up to 18% of the dry weight in leaves. Glycosylation of phloretin at the 2' position is the last and key step in phloridzin biosynthesis. It is catalyzed by a uridine diphosphate (UDP)-glucose:phloretin 2'-O-glucosyltransferase (P2'GT), which directly determines the concentration of phloridzin. However, this process is poorly understood. We conducted a large-scale investigation of phloridzin accumulations in leaves from 64 Malus species and cultivars. To identify the responsible P2'GT, we performed a genome-wide analysis of the expression patterns of UDP-dependent glycosyltransferase genes (UGTs). Two candidates were screened preliminarily in Malus spp. cv. Adams (North American Begonia). Results from further qRT-PCR analyses of the genotypes showed a divergence in phloridzin production. Our assays of enzyme activity also suggested that MdUGT88F4 and MdUGT88F1 regulate the conversion of phloretin to phloridzin in Malus plants. Finally, when they were silenced in 'GL-3' ('Royal Gala'), the concentrations of phloridzin and phloretin (and trilobatin) were significantly reduced and increased, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

Biotransformation of anthelmintics and the activity of drug-metabolizing enzymes in the tapeworm Moniezia expansa.

PubMed

Prchal, Lukáš; Bártíková, Hana; Bečanová, Aneta; Jirásko, Robert; Vokřál, Ivan; Stuchlíková, Lucie; Skálová, Lenka; Kubíček, Vladimír; Lamka, Jiří; Trejtnar, František; Szotáková, Barbora

2015-04-01

The sheep tapeworm Moniezia expansa is very common parasite, which affects ruminants such as sheep, goats as well as other species. The benzimidazole anthelmintics albendazole (ABZ), flubendazole (FLU) and mebendazole (MBZ) are often used to treat the infection. The drug-metabolizing enzymes of helminths may alter the potency of anthelmintic treatment. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (ABZ, MBZ and FLU) in M. expansa. Activities of biotransformation enzymes were determined in subcellular fractions. Metabolites of the anthelmintics were detected and identified using high performance liquid chromatography/ultra-violet/VIS/fluorescence or ultra-high performance liquid chromatography/mass spectrometry. Reduction of MBZ, FLU and oxidation of ABZ were proved as well as activities of various metabolizing enzymes. Despite the fact that the conjugation enzymes glutathione S-transferase, UDP-glucuronosyl transferase and UDP-glucosyl transferase were active in vitro, no conjugated metabolites of anthelmintics were identified either ex vivo or in vitro. The obtained results indicate that sheep tapeworm is able to deactivate the administered anthelmintics, and thus protects itself against their action.

Quantum Chemical and Docking Insights into Bioavailability Enhancement of Curcumin by Piperine in Pepper.

PubMed

Patil, Vaishali M; Das, Sukanya; Balasubramanian, Krishnan

2016-05-26

We combine quantum chemical and molecular docking techniques to provide new insights into how piperine molecule in various forms of pepper enhances bioavailability of a number of drugs including curcumin in turmeric for which it increases its bioavailability by a 20-fold. We have carried out docking studies of quantum chemically optimized piperine structure binding to curcumin, CYP3A4 in cytochrome P450, p-Glycoprotein and UDP-glucuronosyltransferase (UGT), the enzyme responsible for glucuronosylation, which increases the solubility of curcumin. All of these studies establish that piperine binds to multiple sites on the enzymes and also intercalates with curcumin forming a hydrogen bonded complex with curcumin. The conjugated network of double bonds and the presence of multiple charge centers of piperine offer optimal binding sites for piperine to bind to enzymes such as UDP-GDH, UGT, and CYP3A4. Piperine competes for curcumin's intermolecular hydrogen bonding and its stacking propensity by hydrogen bonding with enolic proton of curcumin. This facilitates its metabolic transport, thereby increasing its bioavailability both through intercalation into curcumin layers through intermolecular hydrogen bonding, and by inhibiting enzymes that cause glucuronosylation of curcumin.

TAPBPR bridges UDP-glucose:glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway

PubMed Central

Neerincx, Andreas; Hermann, Clemens; Antrobus, Robin; van Hateren, Andy; Cao, Huan; Trautwein, Nico; Stevanović, Stefan; Elliott, Tim; Deane, Janet E; Boyle, Louise H

2017-01-01

Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex. DOI: http://dx.doi.org/10.7554/eLife.23049.001 PMID:28425917

Profiling serum bile acid glucuronides in humans: gender divergences, genetic determinants and response to fenofibrate

PubMed Central

Trottier, Jocelyn; Perreault, Martin; Rudkowska, Iwona; Levy, Cynthia; Dallaire-Theroux, Amélie; Verreault, Mélanie; Caron, Patrick; Staels, Bart; Vohl, Marie-Claude; Straka, Robert J.; Barbier, Olivier

2014-01-01

Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes detoxifies cholestatic bile acids (BAs). We aimed at i) characterizing the circulating BA-glucuronide (-G) pool composition in humans, ii) evaluating how sex and UGT polymorphisms influence this composition, and iii) analyzing the effects of lipid-lowering drug fenofibrate on the circulating BA-G profile in 300 volunteers and 5 cholestatic patients. Eleven BA-Gs were determined in pre- and post-fenofibrate samples. Men exhibited higher BA-G concentrations, and various genotype/BA-G associations were discovered in relevant UGT genes. The chenodeoxycholic acid-3G concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA-3G formation. Fenofibrate exposure increased the serum levels of 5 BA-G species, including CDCA-3G, and up-regulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrates that fenofibrate stimulates BA glucuronidation in humans, and thus reduces bile acid toxicity in the liver. PMID:23756370

User Data Package (UDP) for Packaged Cogeneration Systems (PCS)

DTIC Science & Technology

1990-05-01

4 1.2 Operating Efficiency Standards for PURPA Compliance ......... ..................... 10 1.3 Selected Commercial, Institutional, and Multi...or 81 percent. The Federal Energy Regulatory Commission (FERC), in accordance with Section 201 of the Public Utility Regulatory Policies Act ( PURPA ...least 57 percent of the time the engine was running, or if 57 percent of the recovered engine heat were stored. Additional requirements for PURPA

High-Level Fosfomycin Resistance in Vancomycin-Resistant Enterococcus faecium

PubMed Central

Guo, Yan; Tomich, Adam D.; McElheny, Christi L.; Cooper, Vaughn S.; Tait-Kamradt, Amelia; Wang, Minggui; Hu, Fupin; Rice, Louis B.; Sluis-Cremer, Nicolas

2017-01-01

Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 μg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays. PMID:29048285

Overexpression of poplar xylem sucrose synthase in tobacco leads to a thickened cell wall and increased height.

PubMed

Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong

2015-01-01

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.

Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

PubMed Central

Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong

2015-01-01

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue. PMID:25807295

BDE 47 AND PCB 153 INCREASE THYROXINE CATABOLISM IN PRIMARY RAT AND HUMAN HEPATOCYTES: THE UTILITY OF HEPATOCYTES AS SCREENING TOOLS FOR POTENTIAL THYROID HORMONE DISRUPTORS

EPA Science Inventory

Studies demonstrate that exposure to 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) and 2,2',4,4',5,5'· hexachlorobiphenyl (PCB 153) decrease serum thyroxine (T4)levels in laboratory animals 1,2,3. The T4 decrease in rodents is thought to occur through the induction of UDP-glucurono...

Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor

PubMed Central

Van Roy, F. P.; Heirwegh, K. P. M.

1968-01-01

1. Conjugated bilirubin is conveniently determined by coupling with the diazonium salt of ethyl anthranilate. 2. This method has been used in the development of assays for UDP-glucuronyltransferase (EC 2.4.1.17), with bilirubin as substrate, in rat liver homogenates, microsomal preparations and partly purified fractions. 3. Chromatographic analysis suggests that bilirubin monoglucuronide is the product of the enzyme systems studied. PMID:5660631

Information Sharing for Medical Triage Tasking During Mass Casualty/Humanitarian Operations

DTIC Science & Technology

2009-12-01

military patrol units or surreptitious " cloak and dagger " fact gathering missions to gain photographic/video graphic data for dissemination to the...fractured command and control organization and retarded deployment of resources. Tragedies such as Hurricane Katrina in 2005, the September 11 attacks of...with PKI certificates and HMAC protection from replay attacks and UDP flooding [17]. 3. Triage Graphical User Interface (GUI) Currently the GUI for

Naval Open Architecture Machinery Control Systems for Next Generation Integrated Power Systems

DTIC Science & Technology

2012-05-01

PORTABLE) OS / RTOS ADAPTATION MIDDLEWARE (FOR OS PORTABILITY) MACHINERY CONTROLLER FRAMEWORK MACHINERY CONTROL SYSTEM SERVICES POWER CONTROL SYSTEM...SERVICES SHIP SYSTEM SERVICES TTY 0 TTY N … OPERATING SYSTEM ( OS / RTOS ) COMPUTER HARDWARE UDP IP TCP RAW DEV 0 DEV N … POWER MANAGEMENT CONTROLLER...operating systems (DOS, Windows, Linux, OS /2, QNX, SCO Unix ...) COMPUTERS: ISA compatible motherboards, workstations and portables (Compaq, Dell

Exploring reaction pathways for O-GlcNAc transferase catalysis. A string method study.

PubMed

Kumari, Manju; Kozmon, Stanislav; Kulhánek, Petr; Štepán, Jakub; Tvaroška, Igor; Koča, Jaroslav

2015-03-26

The inverting O-GlcNAc glycosyltransferase (OGT) is an important post-translation enzyme, which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to the hydroxyl group of the Ser/Thr of cytoplasmic, nuclear, and mitochondrial proteins. In the past, three different catalytic bases were proposed for the reaction: His498, α-phosphate, and Asp554. In this study, we used hybrid quantum mechanics/molecular mechanics (QM/MM) Car-Parrinello molecular dynamics to investigate reaction paths using α-phosphate and Asp554 as the catalytic bases. The string method was used to calculate the free-energy reaction profiles of the tested mechanisms. During the investigations, an additional mechanism was observed. In this mechanism, a proton is transferred to α-phosphate via a water molecule. Our calculations show that the mechanism with α-phosphate acting as the base is favorable. This reaction has a rate-limiting free-energy barrier of 23.5 kcal/mol, whereas reactions utilizing Asp554 and water-assisted α-phosphate have barriers of 41.7 and 40.9 kcal/mol, respectively. Our simulations provide a new insight into the catalysis of OGT and may thus guide rational drug design of transition-state analogue inhibitors with potential therapeutic use.

Transgenic Wheat Expressing a Barley UDP-Glucosyltransferase Detoxifies Deoxynivalenol and Provides High Levels of Resistance to Fusarium graminearum.

PubMed

Li, Xin; Shin, Sanghyun; Heinen, Shane; Dill-Macky, Ruth; Berthiller, Franz; Nersesian, Natalya; Clemente, Thomas; McCormick, Susan; Muehlbauer, Gary J

2015-11-01

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a devastating disease of wheat that results in economic losses worldwide. During infection, F. graminearum produces trichothecene mycotoxins, including deoxynivalenol (DON), that increase fungal virulence and reduce grain quality. Transgenic wheat expressing a barley UDP-glucosyltransferase (HvUGT13248) were developed and evaluated for FHB resistance, DON accumulation, and the ability to metabolize DON to the less toxic DON-3-O-glucoside (D3G). Point-inoculation tests in the greenhouse showed that transgenic wheat carrying HvUGT13248 exhibited significantly higher resistance to disease spread in the spike (type II resistance) compared with nontransformed controls. Two transgenic events displayed complete suppression of disease spread in the spikes. Expression of HvUGT13248 in transgenic wheat rapidly and efficiently conjugated DON to D3G, suggesting that the enzymatic rate of DON detoxification translates to type II resistance. Under field conditions, FHB severity was variable; nonetheless, transgenic events showed significantly less-severe disease phenotypes compared with the nontransformed controls. In addition, a seedling assay demonstrated that the transformed plants had a higher tolerance to DON-inhibited root growth than nontransformed plants. These results demonstrate the utility of detoxifying DON as a FHB control strategy in wheat.

Regioselective formation of quercetin 5-O-glucoside from orally administered quercetin in the silkworm, Bombyx mori.

PubMed

Hirayama, Chikara; Ono, Hiroshi; Tamura, Yasumori; Konno, Kotaro; Nakamura, Masatoshi

2008-03-01

The cocoons of some races of the silkworm, Bombyx mori, have been shown to contain 5-O-glucosylated flavonoids, which do not occur naturally in the leaves of their host plant, mulberry (Morus alba). Thus, dietary flavonoids could be biotransformed in this insect. In this study, we found that after feeding silkworms a diet rich in the flavonol quercetin, quercetin 5-O-glucoside was the predominant metabolite in the midgut tissue, while quercetin 5,4'-di-O-glucoside was the major constituent in the hemolymph and silk glands. UDP-glucosyltransferase (UGT) in the midgut could transfer glucose to each of the hydroxyl groups of quercetin, with a preference for formation of 5-O-glucoside, while quercetin 5,4'-di-O-glucoside was predominantly produced if the enzyme extracts of either the fat body or silk glands were incubated with quercetin 5-O-glucoside and UDP-glucose. These results suggest that dietary quercetin was glucosylated at the 5-O position in the midgut as the first-pass metabolite of quercetin after oral absorption, then glucosylated at the 4'-O position in the fat body or silk glands. The 5-O-glucosylated flavonoids retained biological activity in the insect, since the total free radical scavenging capacity of several tissues increased after oral administration of quercetin.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salminen, S.O.; Streeter, J.G.

Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with /sup 14/C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O/sub 2/ (2% in the gas phase). Uptake and conversion of /sup 14/C to CO/sub 2/ were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO/sub 2/, and fructose was respired at a rate of at least double that of glucose. Sucrose and glucose were converted to CO/sub 2/ at a very low but measurable rate (<0.1more » nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO/sub 2/ at a rate 30 times greater than the conversion of carbon Number 6 to CO/sub 2/, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase.« less

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase in nuclei and rimmed vacuoles of muscle fibers in DMRV (distal myopathy with rimmed vacuoles).

PubMed

Ishihara, Shoichiro; Tomimitsu, Hiroyuki; Fujigasaki, Hiroto; Saito, Fumiaki; Mizusawa, Hidehiro

2008-03-01

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key molecule in the pathogenesis of distal myopathy with rimmed vacuoles (DMRV) and hereditary inclusion body myopathy (HIBM) and almost all such patients have some mutations in GNE. However, subcellular localization of GNE and the mechanism of muscular damage have not been clarified. A rabbit polyclonal antibody for GNE was prepared. Immunohistochemistry was performed using anti-GNE and anti-nuclear protein antibodies. Western blotting with subcellular fractionated proteins was performed to determine subcellular localization of GNE. The sizes of myonuclei were quantified in muscle biopsies from patients with DMRV and amyotrophic lateral sclerosis (ALS). In DMRV muscles, immunohistochemistry identified GNE in sarcoplasm and specifically in myonuclei and rimmed vacuoles (RV). Nuclear proteins were also found in RVs. Immunohistochemistry showed colocalization of GNE and emerin in C2C12 cells. Western blotting revealed the presence of GNE in nuclear fractions of human embryonic kidney (HEK) 293T cells. The mean size of myonuclei of DMRV was significantly larger than that of ALS. GNE is present in myonuclei near nuclear membrane. Our results suggest that myonuclei are involved in RV formation in DMRV, and that mutant GNE in myonuclei seems to play some role in this process.

Chirality Influence of Zaltoprofen Towards UDP-Glucuronosyltransferases (UGTs) Inhibition Potential.

PubMed

Jia, Lin; Hu, Cuimin; Wang, Haina; Liu, Yongzhe; Liu, Xin; Zhang, Yan-Yan; Li, Wei; Wang, Li-Xuan; Cao, Yun-Feng; Fang, Zhong-Ze

2015-06-01

Zaltoprofen (ZLT) is a nonsteroidal antiinflammation drug, and has been clinically employed to treat rheumatoid arthritis, osteoarthritis, and other chronic inflammatory pain conditions. The present study aims to investigate the chirality influence of zaltoprofen towards the inhibition potential towards UDP-glucuronosyltransferases (UGTs) isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation system was employed to investigate the inhibition of (R)-zaltoprofen and (S)-zaltoprofen towards UGT isoforms. The inhibition difference capability was observed for the inhibition of (R)-zaltoprofen and (S)-zaltoprofen towards UGT1A8 and UGT2B7, but not for other tested UGT isoforms. (R)-zaltoprofen exhibited noncompetitive inhibition towards UGT1A8 and competitive inhibition towards UGT2B7. The inhibition kinetic parameters were calculated to be 35.3 μM and 19.2 μM for UGT1A8 and UGT2B7. (R)-zaltoprofen and (S)-zaltoprofen exhibited a different inhibition type towards UGT1A7. Based on the reported maximum plasma concentration of (R)-zaltoprofen in vivo, a high drug-drug interaction between (R)-zaltoprofen and the drugs mainly undergoing UGT1A7, UGT1A8, and UGT2B7-catalyzed glucuronidation was indicated. © 2015 Wiley Periodicals, Inc.

Glucuronidation of Drugs and Drug-Induced Toxicity in Humanized UDP-Glucuronosyltransferase 1 Mice

PubMed Central

Kutsuno, Yuki; Itoh, Tomoo; Tukey, Robert H.

2014-01-01

UDP-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various drugs. Although experimental rodents are used in preclinical studies to predict glucuronidation and toxicity of drugs in humans, species differences in glucuronidation and drug-induced toxicity have been reported. Humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) were recently developed. In this study, acyl-glucuronidations of etodolac, diclofenac, and ibuprofen in liver microsomes of hUGT1 mice were examined and compared with those of humans and regular mice. The kinetics of etodolac, diclofenac, and ibuprofen acyl-glucuronidation in hUGT1 mice were almost comparable to those in humans, rather than in mice. We further investigated the hepatotoxicity of ibuprofen in hUGT1 mice and regular mice by measuring serum alanine amino transferase (ALT) levels. Because ALT levels were increased at 6 hours after dosing in hUGT1 mice and at 24 hours after dosing in regular mice, the onset pattern of ibuprofen-induced liver toxicity in hUGT1 mice was different from that in regular mice. These data suggest that hUGT1 mice can be valuable tools for understanding glucuronidations of drugs and drug-induced toxicity in humans. PMID:24764149

UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum

PubMed Central

Ferris, Sean P.; Jaber, Nikita S.; Molinari, Maurizio; Arvan, Peter; Kaufman, Randal J.

2013-01-01

Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER. PMID:23864712

Endogenous purinergic signaling is required for osmotic volume regulation of retinal glial cells.

PubMed

Wurm, Antje; Lipp, Stephan; Pannicke, Thomas; Linnertz, Regina; Krügel, Ute; Schulz, Angela; Färber, Katrin; Zahn, Dirk; Grosse, Johannes; Wiedemann, Peter; Chen, Ju; Schöneberg, Torsten; Illes, Peter; Reichenbach, Andreas; Bringmann, Andreas

2010-03-01

Intense neuronal activity in the sensory retina is associated with a volume increase of neuronal cells (Uckermann et al., J. Neurosci. 2004, 24:10149) and a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., Vis. Neurosci. 1999, 16:1157). Here, we show the existence of an endogenous purinergic mechanism that prevents hypoosmotic swelling of retinal glial (Müller) cells in mice. In contrast to the cells from wild-type mice, hypoosmotic stress induced rapid swelling of glial cell somata in retinal slices from mice deficient in P2Y(1), adenosine A(1) receptors, or ecto-5'-nucleotidase (CD73). Consistently, glial cell bodies in retinal slices from wild-type mice displayed osmotic swelling when P2Y(1) or A(1) receptors, or CD73, were pharmacologically blocked. Exogenous ATP, UTP, and UDP inhibited glial swelling in retinal slices, while the swelling of isolated glial cells was prevented by ATP but not by UTP or UDP, suggesting that uracil nucleotides indirectly regulate the glial cell volume via activation of neuronal P2Y(4/6) and neuron-to-glia signaling. It is suggested that autocrine/paracrine activation of purinergic receptors and enzymes is crucially involved in the regulation of the glial cell volume.

Comparison of inhibition capability of scutellarein and scutellarin towards important liver UDP-glucuronosyltransferase (UGT) isoforms.

PubMed

Ma, Guang-You; Cao, Yun-Feng; Hu, Cui-Min; Fang, Zhong-Ze; Sun, Xiao-Yu; Hong, Mo; Zhu, Zhi-Tu

2014-03-01

Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand-Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP-glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (Ki ) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the Ki values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro-in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components. Copyright © 2013 John Wiley & Sons, Ltd.

Soybean greatly reduces valproic acid plasma concentrations: A food–drug interaction study

PubMed Central

Marahatta, Anu; Bhandary, Bidur; Jeong, Seul-Ki; Kim, Hyung-Ryong; Chae, Han-Jung

2014-01-01

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents. PMID:24618639

Soybean greatly reduces valproic acid plasma concentrations: a food-drug interaction study.

PubMed

Marahatta, Anu; Bhandary, Bidur; Jeong, Seul-Ki; Kim, Hyung-Ryong; Chae, Han-Jung

2014-03-12

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.

Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus.

PubMed

Ishibashi, Mihoko; Kurokawa, Kenji; Nishida, Satoshi; Ueno, Kohji; Matsuo, Miki; Sekimizu, Kazuhisa

2007-09-01

Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.

Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

PubMed Central

Talhaoui, Ibtissam; Bui, Catherine; Oriol, Rafael; Mulliert, Guillermo; Gulberti, Sandrine; Netter, Patrick; Coughtrie, Michael W. H.; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie

2010-01-01

Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, 163DVD165 and 221FWGWGREDDE230, are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp224 as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp228 acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics. PMID:20843813

Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

PubMed

Wyroba, E; Kwaśniak, P; Miller, K; Kobyłecki, K; Osińska, M

2016-04-11

Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

Identification of the Molecular and Genetic Basis of PX2, a Glycosphingolipid Blood Group Antigen Lacking on Globoside-deficient Erythrocytes*

PubMed Central

Westman, Julia S.; Benktander, John; Storry, Jill R.; Peyrard, Thierry; Hult, Annika K.; Hellberg, Åsa; Teneberg, Susann; Olsson, Martin L.

2015-01-01

The x2 glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/Pk-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-β-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcβ3Gal, as x2. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P1k phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x2 and sialylated forms of x2, whereas x2 is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x2. Knockdown experiments with siRNA against B3GALNT1 diminished x2 levels. We conclude that x2 fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x2 joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P1k or P2k erythrocyte units are preferentially selected for transfusion to Pk patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels. PMID:26055721

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis*

PubMed Central

Basu, Debarati; Liang, Yan; Liu, Xiao; Himmeldirk, Klaus; Faik, Ahmed; Kieliszewski, Marcia; Held, Michael; Showalter, Allan M.

2013-01-01

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [14C]Gal from UDP-[14C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)7 and anhydrous hydrogen fluoride-deglycosylated d(AO)51. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [14C]Gal to (AO)7, and the resulting product co-eluted with (AO)7 by reverse-phase HPLC. Acid hydrolysis of the [14C]Gal-(AO)7 product released 14C-radiolabel as Gal only. Base hydrolysis of the [14C]Gal-(AO)7 product released a 14C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg2+ or Mn2+ for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function. PMID:23430255

Identification of human UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin.

PubMed

Sadeque, Abu J M; Usmani, Khawja A; Palamar, Safet; Cerny, Matthew A; Chen, Weichao G

2012-04-01

Lorcaserin, a selective serotonin 5-HT(2C) receptor agonist, is a weight management agent in clinical development. Lorcaserin N-carbamoyl glucuronidation governs the predominant excretory pathway of lorcaserin in humans. Human UDP-glucuronosyltransferases (UGTs) responsible for lorcaserin N-carbamoyl glucuronidation are identified herein. Lorcaserin N-carbamoyl glucuronide formation was characterized by the following approaches: metabolic screening using human tissues (liver, kidney, intestine, and lung) and recombinant enzymes, kinetic analyses, and inhibition studies. Whereas microsomes from all human tissues studied herein were found to be catalytically active for lorcaserin N-carbamoyl glucuronidation, liver microsomes were the most efficient. With recombinant UGT enzymes, lorcaserin N-carbamoyl glucuronidation was predominantly catalyzed by three UGT2Bs (UGT2B7, UGT2B15, and UGT2B17), whereas two UGT1As (UGT1A6 and UGT1A9) played a minor role. UGT2B15 was most efficient, with an apparent K(m) value of 51.6 ± 1.9 μM and V(max) value of 237.4 ± 2.8 pmol/mg protein/min. The rank order of catalytic efficiency of human UGT enzymes for lorcaserin N-carbamoyl glucuronidation was UGT2B15 > UGT2B7 > UGT2B17 > UGT1A9 > UGT1A6. Inhibition of lorcaserin N-carbamoyl glucuronidation activities of UGT2B7, UGT2B15, and UGT2B17 in human liver microsomes by mefenamic acid, bisphenol A, and eugenol further substantiated the involvement of these UGT2B isoforms. In conclusion, multiple human UGT enzymes catalyze N-carbamoyl glucuronidation of lorcaserin; therefore, it is unlikely that inhibition of any one of these UGT activities will lead to significant inhibition of the lorcaserin N-carbamoyl glucuronidation pathway. Thus, the potential for drug-drug interaction by concomitant administration of a drug(s) that is metabolized by any of these UGTs is remote.

Molecular mechanisms and cell signaling of 20-hydroxyeicosatetraenoic acid in vascular pathophysiology

PubMed Central

Fan, Fan; Ge, Ying; Lv, Wenshan; Elliott, Matthew R.; Muroya, Yoshikazu; Hirata, Takashi; Booz, George W.; Roman, Richard J.

2016-01-01

Cytochrome P450s enzymes catalyze the metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatetraenoic acid and hydroxyeicosatetraeonic acid (HETEs). 20-HETE is a vasoconstrictor that depolarizes vascular smooth muscle cells by blocking K+ channels. EETs serve as endothelial derived hyperpolarizing factors. Inhibition of the formation of 20-HETE impairs the myogenic response and autoregulation of renal and cerebral blood flow. Changes in the formation of EETs and 20-HETE have been reported in hypertension and drugs that target these pathways alter blood pressure in animal models. Sequence variants in CYP4A11 and CYP4F2 that produce 20-HETE, UDP-glucuronosyl transferase involved in the biotransformation of 20-HETE and soluble epoxide hydrolase that inactivates EETs are associated with hypertension in human studies. 20-HETE contributes to the regulation of vascular hypertrophy, restenosis, angiogenesis and inflammation. It also promotes endothelial dysfunction and contributes to cerebral vasospasm and ischemia-reperfusion injury in the brain, kidney and heart. This review will focus on the role of 20-HETE in vascular dysfunction, inflammation, ischemic and hemorrhagic stroke and cardiac and renal ischemia reperfusion injury. PMID:27100515

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme.

PubMed

Gallage, Nethaji J; Hansen, Esben H; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

2014-06-19

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme

PubMed Central

Gallage, Nethaji J.; Hansen, Esben H.; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

2014-01-01

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco. PMID:24941968

Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carver, M.A.; Turco, S.J.

1991-06-15

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-({sup 3}H)galactose or GDP-({sup 14}C)mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The ({sup 3}H)galactose- or ({sup 14}C)mannose-labeled product was (1) cleaved by phosphatidylinositol-specific phospholipase C; (2) deaminated by nitrous acid; and (3) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of themore » two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.« less

Glucuronic acid and the ethanol metabolite ethyl-glucuronide cause Toll-like receptor 4 activation and enhanced pain

PubMed Central

Lewis, Susannah S.; Hutchinson, Mark R.; Zhang, Yingning; Hund, Dana K.; Maier, Steven F.; Rice, Kenner C.; Watkins, Linda R.

2013-01-01

We have previously observed that the non-opioid morphine metabolite, morphine-3-glucuronide, enhances pain via a toll-like receptor 4 (TLR4) dependent mechanism. The present studies were undertaken to determine whether TLR4-dependent pain enhancement generalizes to other classes of glucuronide metabolites. In silico modeling predicted that glucuronic acid alone and ethyl glucuronide, a minor but long-lasting ethanol metabolite, would dock to the same MD-2 portion of the TLR4 receptor complex previously characterized as the docking site for morphine-3-glucuronide. Glucuronic acid, ethyl glucuronide and ethanol all caused an increase in TLR4-dependent reporter protein expression in a cell line transfected with TLR4 and associated co-signaling molecules. Glucuronic acid-, ethyl glucuronide-, and ethanol-induced increases in TLR4 signaling were blocked by the TLR4 antagonists LPS-RS and (+)-naloxone. Glucuronic acid and ethyl glucuronide both caused allodynia following intrathecal injection in rats, which was blocked by intrathecal co-administration of the TLR4 antagonist LPS-RS. The finding that ethyl glucuronide can cause TLR4-dependent pain could have implications for human conditions such as hangover headache and alcohol withdrawal hyperalgesia, as well as suggesting that other classes of glucuronide metabolites could have similar effects. PMID:23348028

Detecting the Gender Dimension of the Choice of the Teaching Profession Prior to the Economic Crisis and IMF (International Monetary Fund) Memorandum in Greece--A Case Study

ERIC Educational Resources Information Center

Asimaki, Anna; Vergidis, Dimitris K.

2013-01-01

The purpose of this research paper is the investigation of, and the sociological approach to, and interpretation of the attitudes of male and female students in the University Department of Primary Education (U.D.P.E.) at the University of Patras in Greece, before the enforcement of the IMF Memorandum, concerning the choice of the teaching…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brand, B.; Boos, W.

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose. This reaction was exploited to preparatively synthesize ({sup 14}C)trehalose from exogenous ({sup 14}C)glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.

Method and Apparatus for Processing UDP Data Packets

NASA Technical Reports Server (NTRS)

Murphy, Brandon M. (Inventor)

2017-01-01

A method and apparatus for processing a plurality of data packets. A data packet is received. A determination is made as to whether a portion of the data packet follows a selected digital recorder standard protocol based on a header of the data packet. Raw data in the data packet is converted into human-readable information in response to a determination that the portion of the data packet follows the selected digital recorder standard protocol.

Exploiting Spatial Channel Occupancy Information in WLANs

DTIC Science & Technology

2014-05-15

transmit signal UDP user datagram protocol WLAN wireless local area network ix Acknowledgements I owe a great debt of gratitude to my advisor, Professor...information. Unlike in wired networks , each node in a wireless network observes a different medium depending on its location. As a result, standard local... wireless LANs [15, 23, 29]. In [23], Li et. al. model the throughput of an 802.11 network using full spatial information. Their approach is from a

Long term change in atmospheric dust absorption, dust scattering and black carbon aerosols scattering coefficient parameters over western Indian locations

NASA Astrophysics Data System (ADS)

Satoliya, Anil Kumar; Vyas, B. M.; Shekhawat, M. S.

2018-05-01

The first time satellite space based measurement of atmospheric black carbon (BC) aerosols scattering coefficient at 550nm (BC SC at 550nm), dust aerosols scattering and dust aerosols extinction coefficient (DSC at 550nm and DEC at 550nm) parameters have been used to understand their long term trend of natural and anthropogenic aerosols behavior with its close association with ground based measured precipitation parameters such as Total Rain Fall (TRF), and Total Number of Rainy Days (TNRD) for the same period over western Indian regions concerned to the primary aerosols sources of natural activities. The basic objective of this study is an attempt to investigate the inter-correlation between dust and black carbon aerosols loading characteristics with a variation of rainfall pattern parameters as indirect aerosols induced effect i.e., aerosols-cloud interaction. The black carbon aerosols generated by diverse anthropogenic or human made activities are studied by choosing of measured atmospheric BC SC at 550nm parameter, whereas desert dust mineral aerosols primarily produced by varieties of natural activities pre-dominated of dust mineral desert aerosols mainly over Thar desert influenced area of hot climate and rural tropical site are investigated by selecting DSC at 550nm and DEC at 550nm of first semi-urban site i.e., Udaipur (UDP, 24.6°N, 73.35°E, 580m above surface level (asl)) situated in southern Rajasthan part as well as over other two Great Indian Thar desert locations i.e., Jaisalmer (JSM, 26.90°N, 69.90°E, 220m asl)) and Bikaner (BKN, 28.03°N, 73.30°E, 224m asl) located in the vicinity of the Thar desert region situated in Rajasthan state of the western Indian region. The source of the present study would be collection of longer period of monthly values of the above parameters of spanning 35 years i.e., 1980 to 2015. Such types of atmospheric aerosols-cloud monsoon interaction investigation is helpful in view of understanding their direct and indirect aerosols active role of optical absorption and scattering of solar light radiation at useful wavelength 550nm as well as heating of clouds over least explored region, i.e., the Thar desert region and also away from less dust dominated influenced provinces for longer period. The analysis of the above the result would also give a clear scientific evidence of alteration in enhancement in DSC at 550nm and DEC at 550nm and BC SC at 550nm variables with simultaneous corresponding reduction in the five yearly mean precipitation activity parameters such as TRF and TNRD. It is quite evident that anthropogenic BC aerosols activity are showing the significant increasing trend at all three locations, but it is more prominent over central Thar Desert influenced regime, i.e., JSM and BKN relative to semi-urban region i.e., UDP. The systematic increasing pattern of average monthly mean value of DSC at 550nm and DEC at 550nm or increasing aerosol loading have been revealed from acquiring their lowest value in January month and the highest values in July and retained with the broad peak values in pre-monsoon months. Subsequently, their respective values reduce sharply downward from August to December onwards. The mountain value of dust aerosols parameters, i.e., DSC at 550nm and DEC at 550nm are systematically enhanced toward from UDP to BKN and then maximized at JSM. It is clearly obvious fact that the following ascending order of desert aerosols loading influenced activity in different areas has been recorded, i.e., JSM> BKN>UDP. Several other interesting features of the earth-climate change implication in reference to the altering nature of reduction of precipitation parameter pattern with simultaneous observed elevated dust aerosol and BC aerosol loading have been also noticed in the course of present investigation. Overall reduction in rainfall pattern effect with increasing of dust aerosols loading or vice versa are seen more pronounced over JSM and lees prevalence over UDP. The more detailed investigations about other interesting results of Aerosols-Indian monsoon over western Indian locations are also discussed thoroughly in this paper.

Arabidopsis GUX Proteins Are Glucuronyltransferases Responsible for the Addition of Glucuronic Acid Side Chains onto Xylan

EPA Science Inventory

Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of β-(1,4)-linked xylosyl residues that are often substituted with sugar side chains, such as glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). It has recently been shown that muta...

Structure–inhibition relationship of ginsenosides towards UDP-glucuronosyltransferases (UGTs)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Zhong-Ze; Joint Center for Translational Medicine, Dalian Institute of Chemical Physics Chinese Academy of Sciences and The first Affiliated Hospital of Liaoning Medical University, No.457, Zhongshan Road, Dalian 116023; Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892

The wide utilization of ginseng provides the high risk of herb–drug interaction (HDI) with many clinical drugs. The inhibition of ginsenosides towards drug-metabolizing enzymes (DMEs) has been regarded as an important reason for herb–drug interaction (HDI). Compared with the deep studies on the ginsenosides' inhibition towards cytochrome P450 (CYP), the inhibition of ginsenosides towards the important phase II enzymes UDP-glucuronosyltransferases (UGTs) remains to be unclear. The present study aims to evaluate the inhibition behavior of ginsenosides towards important UGT isoforms located in the liver and intestine using in vitro methods. The recombinant UGT isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employedmore » as in vitro probe reaction. The results showed that structure-dependent inhibition existed for the inhibition of ginsenosides towards UGT isoforms. To clarify the possibility of in vivo herb–drug interaction induced by this kind of inhibition, the ginsenoside Rg{sub 3} was selected as an example, and the inhibition kinetic type and parameters (K{sub i}) were determined. Rg{sub 3} competitively inhibited UGT1A7, 2B7 and 2B15-catalyzed 4-MU glucuronidation reaction, and exerted noncompetitive inhibition towards UGT1A8-catalyzed 4-MU glucuronidation. The inhibition parameters (K{sub i} values) were calculated to be 22.6, 7.9, 1.9, and 2.0 μM for UGT1A7, 1A8, 2B7 and 2B15. Using human maximum plasma concentration of Rg{sub 3} (400 ng/ml (0.5 μM)) after intramuscular injection of 60 mg Rg{sub 3}, the area under the plasma concentration-time curve (AUC) was extrapolated to increase by 2.2%, 6.3%, 26.3%, and 25% for the co-administered drugs completely undergoing the metabolism catalyzed by UGT1A7, 1A8, 2B7 and 2B15, respectively. All these results indicated that the ginsenosides' inhibition towards UGT isoforms might be an important reason for ginseng–drug interaction. - Highlights: ► Structure-dependent inhibition of ginsenoside towards UDP-glucuronosyltransferases. ► Rg{sub 3}′ inhibition towards UGT isoforms can induce in vivo drug–drug interaction. ► Broadening knowledge on ginsenosides' inhibition towards drug-metabolizing enzymes.« less

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish ( Raphanus sativus L.) seedlings.

PubMed

Kato, Hideaki; Takeuchi, Yoshimi; Tsumuraya, Yoichi; Hashimoto, Yohichi; Nakano, Hirofumi; Kovác, Pavol

2003-06-01

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14)C]Gal into beta-(1-->4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.

The genes and enzymes of sucrose metabolism in moderately thermophilic methanotroph Methylocaldum szegediense O12.

PubMed

But, Sergey Y; Solntseva, Natalia P; Egorova, Svetlana V; Mustakhimov, Ildar I; Khmelenina, Valentina N; Reshetnikov, Alexander; Trotsenko, Yuri A

2018-05-01

Four enzymes involved in sucrose metabolism: sucrose phosphate synthase (Sps), sucrose phosphate phosphatase (Spp), sucrose synthase (Sus) and fructokinase (FruK), were obtained as his-tagged proteins from the moderately thermophilic methanotroph Methylocaldum szegediense O12. Sps, Spp, FruK and Sus demonstrated biochemical properties similar to those of other bacterial counterparts, but the translated amino acid sequences of Sps and Spp displayed high divergence from the respective microbial enzymes. The Sus of M. szegediense O12 catalyzed the reversible reaction of sucrose cleavage in the presence of ADP or UDP and preferred ADP as a substrate, thus implying a connection between sucrose and glycogen metabolism. Sus-like genes were found only in a few methanotrophs, whereas amylosucrase was generally used in sucrose cleavage in this group of bacteria. Like other microbial fructokinases, FruK of M. szegediense O12 showed a high specificity to fructose.

High-Bandwidth Tactical-Network Data Analysis in a High-Performance-Computing (HPC) Environment: Transport Protocol (Transmission Control Protocol/User Datagram Protocol [TCP/UDP]) Analysis

DTIC Science & Technology

2015-09-01

the network Mac8 Medium Access Control ( Mac ) (Ethernet) address observed as destination for outgoing packets subsessionid8 Zero-based index of...15. SUBJECT TERMS tactical networks, data reduction, high-performance computing, data analysis, big data 16. SECURITY CLASSIFICATION OF: 17...Integer index of row cts_deid Device (instrument) Identifier where observation took place cts_collpt Collection point or logical observation point on

Leveraging an SNMP Agent in Terminal Equipment for Network Monitoring of U.S. Navy SATCOM

DTIC Science & Technology

2011-09-01

Network Topology TWT Traveling-wave Tube TX Transmitter UCD Uplink Channel Descriptor UDP User Datagram Protocol UFO UHF Follow-On UHF Ultra High...through DSCS III, UFO , and Milstar” (Martin, n.d.a).  “Capabilities have grown dramatically with the development of satellite and electronics...Communication Systems (DSCS) II and III and the Global Broadcast Service (GBS) payload on the UHF Follow-On ( UFO ) satellite  In 1971, the DSCS II

Disturbance of Mammary UDP-Glucuronosyltransferase Represses Estrogen Metabolism and Exacerbates Experimental Breast Cancer.

PubMed

Zhou, Xueyan; Zheng, Ziqiang; Xu, Chang; Wang, Juan; Min, Mengjun; Zhao, Yun; Wang, Xi; Gong, Yinhan; Yin, Jiale; Guo, Meng; Guo, Dong; Zheng, Junnian; Zhang, Bei; Yin, Xiaoxing

2017-08-01

The progression of breast cancer is closely related to the levels of estrogens within the body. UDP-glucuronosyltransferase (UGT) is an important class of phase II metabolizing enzymes, playing a pivotal role in detoxifying steroid hormone. In the present study, we aim at uncovering the potential dysregulation pattern of UGT and its role in estrogen metabolism and in the pathogenesis of breast cancer. Female Sprague-Dawley rats were treated with 100 mg/kg dimethylbenz(a)anthracene (DMBA) to induce breast cancer. Our results showed that the expression and activity of UGT in mammary tissues were downregulated significantly in DMBA rats. Consistent with this, levels of estradiol, 4-hydroxylated estradiol, and 2-hydroxylated estradiol were increased in both mammary tissues and serum, supporting a notable accumulation of toxic estrogen species in the target tissue of breast cancer. In addition, we also observed the decreased cell migration, cell proliferation, and DNA damage in UGT-transfected MCF-7 cells, suggesting a protective role of UGT against estrogen-induced mammary carcinogenesis. Taken together, these results indicated that accumulation of estrogens induced by UGT deficiency is a critical factor to induce the development of breast cancer. UGT contributes to estrogen elimination, and its glucuronidation capacity influences the estrogen signaling pathway and the pathogenesis of breast cancer. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers

DOE PAGES

Olek, Anna T.; Rayon, Catherine; Makowski, Lee; ...

2014-07-10

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice ( Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain,more » elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. As a result, the arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.« less

A deletion affecting an LRR-RLK gene co-segregates with the fruit flat shape trait in peach.

PubMed

López-Girona, Elena; Zhang, Yu; Eduardo, Iban; Mora, José Ramón Hernández; Alexiou, Konstantinos G; Arús, Pere; Aranzana, María José

2017-07-27

In peach, the flat phenotype is caused by a partially dominant allele in heterozygosis (Ss), fruits from homozygous trees (SS) abort a few weeks after fruit setting. Previous research has identified a SSR marker (UDP98-412) highly associated with the trait, found suitable for marker assisted selection (MAS). Here we report a ∼10 Kb deletion affecting the gene PRUPE.6G281100, 400 Kb upstream of UDP98-412, co-segregating with the trait. This gene is a leucine-rich repeat receptor-like kinase (LRR-RLK) orthologous to the Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) group. PCR markers suitable for MAS confirmed its strong association with the trait in a collection of 246 cultivars. They were used to evaluate the DNA from a round fruit derived from a somatic mutation of the flat variety 'UFO-4', revealing that the mutation affected the flat associated allele (S). Protein BLAST alignment identified significant hits with genes involved in different biological processes. Best protein hit occurred with AtRLP12, which may functionally complement CLAVATA2, a key regulator that controls the stem cell population size. RT-PCR analysis revealed the absence of transcription of the partially deleted allele. The data support PRUPE.6G281100 as a candidate gene for flat shape in peach.

De Novo Assembly and Analysis of Polygonatum sibiricum Transcriptome and Identification of Genes Involved in Polysaccharide Biosynthesis.

PubMed

Wang, Shiqiang; Wang, Bin; Hua, Wenping; Niu, Junfeng; Dang, Kaikai; Qiang, Yi; Wang, Zhezhi

2017-09-12

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of β-fructofuranosidase ( sacA ), fructokinase ( scrK ), UDP-glucose 4-epimerase ( GALE ), Mannose-1-phosphate guanylyltransferase ( GMPP ), and UDP-glucose 6-dehydrogenase ( UGDH ), but negatively correlated with the expression of Hexokinase ( HK ). Through qRT-PCR validation and comprehensive analysis, we determined that sacA , HK , and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions.

Computational evaluation of exome sequence data using human and model organism phenotypes improves diagnostic efficiency

PubMed Central

Bone, William P.; Washington, Nicole L.; Buske, Orion J.; Adams, David R.; Davis, Joie; Draper, David; Flynn, Elise D.; Girdea, Marta; Godfrey, Rena; Golas, Gretchen; Groden, Catherine; Jacobsen, Julius; Köhler, Sebastian; Lee, Elizabeth M. J.; Links, Amanda E.; Markello, Thomas C.; Mungall, Christopher J.; Nehrebecky, Michele; Robinson, Peter N.; Sincan, Murat; Soldatos, Ariane G.; Tifft, Cynthia J.; Toro, Camilo; Trang, Heather; Valkanas, Elise; Vasilevsky, Nicole; Wahl, Colleen; Wolfe, Lynne A.; Boerkoel, Cornelius F.; Brudno, Michael; Haendel, Melissa A.; Gahl, William A.; Smedley, Damian

2016-01-01

Purpose: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. Genet Med 18 6, 608–617. Methods: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease–gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein–protein association neighbors. Genet Med 18 6, 608–617. Results: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease–gene associations and ranked the correct seeded variant in up to 87% when detectable disease–gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. Genet Med 18 6, 608–617. Conclusion: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders. Genet Med 18 6, 608–617. PMID:26562225

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers.

PubMed

Olek, Anna T; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; Himmel, Michael E; Bolin, Jeffrey T; Carpita, Nicholas C

2014-07-01

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize. © 2014 American Society of Plant Biologists. All rights reserved.

A new communication protocol family for a distributed spacecraft control system

NASA Technical Reports Server (NTRS)

Baldi, Andrea; Pace, Marco

1994-01-01

In this paper we describe the concepts behind and architecture of a communication protocol family, which was designed to fulfill the communication requirements of ESOC's new distributed spacecraft control system SCOS 2. A distributed spacecraft control system needs a data delivery subsystem to be used for telemetry (TLM) distribution, telecommand (TLC) dispatch and inter-application communication, characterized by the following properties: reliability, so that any operational workstation is guaranteed to receive the data it needs to accomplish its role; efficiency, so that the telemetry distribution, even for missions with high telemetry rates, does not cause a degradation of the overall control system performance; scalability, so that the network is not the bottleneck both in terms of bandwidth and reconfiguration; flexibility, so that it can be efficiently used in many different situations. The new protocol family which satisfies the above requirements is built on top of widely used communication protocols (UDP and TCP), provides reliable point-to-point and broadcast communication (UDP+) and is implemented in C++. Reliability is achieved using a retransmission mechanism based on a sequence numbering scheme. Such a scheme allows to have cost-effective performances compared to the traditional protocols, because retransmission is only triggered by applications which explicitly need reliability. This flexibility enables applications with different profiles to take advantage of the available protocols, so that the best rate between sped and reliability can be achieved case by case.

Single Nucleotide Polymorphisms in B-Genome Specific UDP-Glucosyl Transferases Associated with Fusarium Head Blight Resistance and Reduced Deoxynivalenol Accumulation in Wheat Grain.

PubMed

Sharma, Pallavi; Gangola, Manu P; Huang, Chen; Kutcher, H Randy; Ganeshan, Seedhabadee; Chibbar, Ravindra N

2018-01-01

An in vitro spike culture method was optimized to evaluate Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) and used to screen a population of ethyl methane sulfonate treated spike culture-derived variants (SCDV). Of the 134 SCDV evaluated, the disease severity score of 47 of the variants was ≤30%. Single nucleotide polymorphisms (SNP) in the UDP-glucosyltransferase (UGT) genes, TaUGT-2B, TaUGT-3B, and TaUGT-EST, differed between AC Nanda (an FHB-susceptible wheat variety) and Sumai-3 (an FHB-resistant wheat cultivar). SNP at 450 and 1,558 bp from the translation initiation site in TaUGT-2B and TaUGT-3B, respectively were negatively correlated with FHB severity in the SCDV population, whereas the SNP in TaUGT-EST was not associated with FHB severity. Fusarium graminearum strain M7-07-1 induced early expression of TaUGT-2B and TaUGT-3B in FHB-resistant SCDV lines, which were associated with deoxynivalenol accumulation and reduced FHB disease progression. At 8 days after inoculation, deoxynivalenol concentration varied from 767 ppm in FHB-resistant variants to 2,576 ppm in FHB-susceptible variants. The FHB-resistant SCDV identified can be used as new sources of FHB resistance in wheat improvement programs.

De Novo Assembly and Analysis of Polygonatum sibiricum Transcriptome and Identification of Genes Involved in Polysaccharide Biosynthesis

PubMed Central

Wang, Shiqiang; Wang, Bin; Hua, Wenping; Niu, Junfeng; Dang, Kaikai; Qiang, Yi; Wang, Zhezhi

2017-01-01

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of β-fructofuranosidase (sacA), fructokinase (scrK), UDP-glucose 4-epimerase (GALE), Mannose-1-phosphate guanylyltransferase (GMPP), and UDP-glucose 6-dehydrogenase (UGDH), but negatively correlated with the expression of Hexokinase (HK). Through qRT-PCR validation and comprehensive analysis, we determined that sacA, HK, and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions. PMID:28895881

Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice*

PubMed Central

Kasaikina, Marina V.; Fomenko, Dmitri E.; Labunskyy, Vyacheslav M.; Lachke, Salil A.; Qiu, Wenya; Moncaster, Juliet A.; Zhang, Jie; Wojnarowicz, Mark W.; Natarajan, Sathish Kumar; Malinouski, Mikalai; Schweizer, Ulrich; Tsuji, Petra A.; Carlson, Bradley A.; Maas, Richard L.; Lou, Marjorie F.; Goldstein, Lee E.; Hatfield, Dolph L.; Gladyshev, Vadim N.

2011-01-01

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency. PMID:21768092

Frog brain uridine diphosphate galactose–N-acetylgalactosaminyl-N-acetylneuraminylgalactosylglucosylceramide galactosyltransferase

PubMed Central

Yip, Morris C. M.; Dain, Joel A.

1970-01-01

1. The enzyme that catalyses the transfer of galactose from UDP-galactose to N-acetylgalactosaminyl-(1→4)-N-acetylneuraminyl-(2→3)-galactosyl-(1→4)-glucosylceramide (GM2) was found mainly in the heavy- and light-microsomal fractions of the adult frog brain. 2. The subcellular distribution of the enzyme, UDP-galactose–GM2 galactosyltransferase, parallels that of gangliosides in adult frog brain. 3. The enzymic activity was first detected at late gastrulation (Shumway stage 11½) and increased until the completion of the operculum (Shumway stage 25) and then decreased in the tadpoles. 4. In adult frog brain, the enzyme exhibited a pH optimum of 7.2–7.3 in both cacodylate and tris buffers. The enzyme required 10mm-Mn2+ for maximal activity and the Km for Mn2+ was determined as 2.2mm. The half-maximal velocity was obtained at a GM2 concentration of 0.18mm. Inhibition of the enzymic reaction was found when the GM2 concentration was greater than 1.38mm. 5. The enzymic activity was also inhibited by the products in the pathway of ganglioside synthesis, i.e. either by a mixture of gangliosides or by individual ganglioside components. The most active inhibitor was disialoganglioside. The degree of inhibition is a function of the individual ganglioside concentration. 6. A product-inhibition mechanism for the regulation of ganglioside biosynthesis is discussed. PMID:5484669

Characterization of an acidic polysaccharide isolated from the leaves of Corchorus olitorius (Moroheiya).

PubMed

Ohtani, K; Okai, K; Yamashita, U; Yuasa, I; Misaki, A

1995-03-01

An acidic polysaccharide was isolated from the water-soluble mucilage extracted from dried leaves of Corchorus olitorius, known as Moroheiya in Japan (3.0 g per 100 g). This polysaccharide showed a single peak in a Sepharose CL-6B column, and the specific rotation in H2O at 25 degrees C was +250 degrees. The polysaccharide was rich in uronic acid (65%), and consisted of rhamnose, glucose, galacturonic acid, and glucuronic acid in a molar ratio of 1.0:0.2:0.2:0.9:1.7, in addition to 3.7% of the acetyl group. A methylation analysis, Smith degradation study and fragmentation analysis suggested that this polysaccharide mainly consisted of O-4 substituted galacturonic acid and glucuronic acid, and O-2 substituted rhamnose residues, and that most of the (1-->4)-linked uronic acid residues were substituted at the O-3 position with glucuronic acid residues. This polysaccharide showed proliferative activity toward the murine splenocyte.