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Sample records for detoxifying udp glucuronosyltransferases

  1. Polymorphisms of UDP-glucuronosyltransferase 1A7 are not involved in pancreatic diseases.

    NARCIS (Netherlands)

    Verlaan, M.; Drenth, J.P.H.; Truninger, K.; Koudova, M.; Schulz, H.U.; Bargetzi, M.; Kunzli, B.; Friess, H.; Cerny, M.; Kage, A.; Landt, O.; Morsche, R.H.M. te; Rosendahl, J.; Luck, W.; Nickel, R.; Halangk, J.; Macek Jr, M.; Jansen, J.B.M.J.; Witt, H.

    2005-01-01

    BACKGROUND: Xenobiotic mediated cellular injury is thought to play a major role in the pathogenesis of pancreatic diseases. Genetic variations that reduce the expression or activity of detoxifying phase II biotransformation enzymes such as the UDP-glucuronosyltransferases might be important in this

  2. Variability of human hepatic UDP-glucuronosyltransferase activity

    NARCIS (Netherlands)

    Little, JM; Lester, R; Kuipers, F; Vonk, R; Mackenzie, PI; Drake, RR; Frame, L; Radominska-Pandya, A

    1999-01-01

    The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases usi

  3. Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase.

    Science.gov (United States)

    Roy Chowdhury, J; Roy Chowdhury, N; Falany, C N; Tephly, T R; Arias, I M

    1986-01-01

    UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining. Images Fig. 2. Fig. 3. Fig. 5. PMID:3085655

  4. Interaction of periodate-oxidized UDP-glucuronic acid with recombinant human liver UDP-glucuronosyltransferase 1A6.

    Science.gov (United States)

    Battaglia, E; Terrier, N; Mizeracka, M; Senay, C; Magdalou, J; Fournel-Gigleux, S; Radominska-Pandya, A

    1998-08-01

    Sodium periodate reacts with UDP-glucuronic acid (UDP-GlcUA) to generate a reactive derivative [periodate-oxidized UDP-GlcUA (o-UDP-GlcUA)]. The ability of this analog of UDP-GlcUA to inactivate and label the human recombinant UDP-glucuronosyltransferase (UGT) UGT1A6 via the UDP-GlcUA binding site was investigated. At an o-UDP-GlcUA concentration of 20 mM, the enzymatic activity of UGT1A6 was totally inactivated after 30 min of incubation at pH 7.4. Inhibition was irreversible, time-dependent, and concentration-dependent and exhibited pseudo-first order kinetics (kinact = 4.0 M-1.min-1). Cosubstrate protection with UDP-GlcUA was biphasic, with no protection in the first phase and almost total protection in the second phase, suggesting that at least 65% of the cross-linking occurs at the cosubstrate binding site. Partial inactivation by o-UDP-GlcUA led to a decrease in Vmax, suggesting that o-UDP-GlcUA can act as an active site-directed inhibitor. Furthermore, proteins, including the UGTs, from membrane fractions of a recombinant V79 cell line expressing the UGT1A6 enzyme and from rat liver microsomes were cross-linked by in situ periodate oxidation of [beta-32P]UDP-GlcUA. The present results suggest that periodate-oxidized UDP-GlcUA, which inactivates UGT1A6 by the possible formation of a Schiff base adduct with active site lysyl residues, can be used as a new affinity label for the UDP-GlcUA binding site.

  5. IMMUNOAFFINITY PURIFICATION AND RECONSTITUTION OF THE HUMAN BILIRUBIN PHENOL UDP-GLUCURONOSYLTRANSFERASE FAMILY

    NARCIS (Netherlands)

    SEPPEN, J; JANSEN, PLM; ELFERINK, RPJO

    1995-01-01

    When membrane proteins are solubilized and subjected to purification procedures, the loss of lipids surrounding the protein often results in irreversible inactivation. We describe a procedure for the immunoaffinity purification of the membrane protein UDP-glucuronosyltransferase from human liver. Th

  6. Effects of Dietary Components on Testosterone Metabolism via UDP-Glucuronosyltransferase

    OpenAIRE

    Carl eJenkinson; Andrea ePetroczi; Declan P. Naughton

    2013-01-01

    The potential interference in testosterone metabolism through ingested substances has ramifications for: (i) a range of pathologies such as prostate cancer, (ii) medication contra-indications, (iii) disruption to the endocrine system, and (iv) potential confounding effects on doping tests. Conjugation of anabolic steroids during phase II metabolism, mainly driven by UDP-glucuronosyltransferase (UGT) 2B7, 2B15, and 2B17, has been shown to be impaired in vitro by a range of compounds including ...

  7. Isolation and purification of rat liver morphine UDP-glucuronosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Puig, J.F.; Tephly, T.R.

    1986-03-05

    The enhancement of rat liver microsomal morphine (M) and 4-hydroxybiphenyl (4-HBP) UDP-glucuronyltransferase (UDPGT) activities by phenobarbital treatment has been proposed to represent increased activity of a single enzyme form, GT-2. They have separated M and 4-HBP UDPGT activities from Emulgen 911-solubilized microsomes obtained from livers of phenobarbital-treated Wistar rats. A sensitive assay procedure was developed to quantify M-UDPGT and 4-HBP-UDPGT activities using /sup 14/C-UDP-glucuronic acid (UDPGA) and reversed phase C-18 minicolumns whereby the radioactive glucuronides were differentially eluted from labeled UDPGA. Trisacryl DEAE, and chromatofocusing procedures were employed to separate M-UDPGT and 4-HBP-UDPGT in the presence of exogenous phosphatidylcholine (PC). The PC is necessary to stabilize UDPGT activities. M-UDPGT was isolated to apparent homogeneity and displayed a monomeric molecular weight of 56,000 daltons on SDS-PAGE. It reacted with M but not with 4-HBP, bilirubin, p-nitrophenol, testosterone, androsterone, estrone, 4-aminobiphenyl or ..cap alpha..-naphthylamine. 4-HBP-UDPGT did not react with M. Therefore, M and 4-HBP glucuronidations are catalyzed by separate enzymes in rat liver microsomes.

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

    OpenAIRE

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

    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 wit...

  9. Regulation of Sulfotransferase and UDP-Glucuronosyltransferase Gene Expression by the PPARs

    Directory of Open Access Journals (Sweden)

    Melissa Runge-Morris

    2009-01-01

    Full Text Available During phase II metabolism, a substrate is rendered more hydrophilic through the covalent attachment of an endogenous molecule. The cytosolic sulfotransferase (SULT and UDP-glucuronosyltransferase (UGT families of enzymes account for the majority of phase II metabolism in humans and animals. In general, phase II metabolism is considered to be a detoxication process, as sulfate and glucuronide conjugates are more amenable to excretion and elimination than are the parent substrates. However, certain products of phase II metabolism (e.g., unstable sulfate conjugates are genotoxic. Members of the nuclear receptor superfamily are particularly important regulators of SULT and UGT gene transcription. In metabolically active tissues, increasing evidence supports a major role for lipid-sensing transcription factors, such as peroxisome proliferator-activated receptors (PPARs, in the regulation of rodent and human SULT and UGT gene expression. This review summarizes current information regarding the regulation of these two major classes of phase II metabolizing enzyme by PPARs.

  10. Purification and characterization of a 4-hydroxybiphenyl UDP-glucuronosyltransferase from rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Styczynski, B.; Green, M.; Coffman, B.; Puig, J.; Tephly, T. (Univ. of Iowa, Iowa City (United States))

    1991-03-11

    A phenobarbital-inducible rat liver microsomal UDP-glucuronosyltransferase (4-HBP UDPGT) which catalyzes the glucuronidation of 4-hydroxybiphenyl has been purified to homogeneity. The apparent subunit molecular weight of this protein is 52,500 as determined by SDS-PAGE. 4-HBP UDPGT was shown to react with 4-hydroxybiphenyl, p-nitrophenol and 4-methylumbelliferone, but did not react with morphine, testosteron or chloramphenicol. Upon treatment with Endoglycosidase H, the 4-HBP UDPGT underwent about a 2,000 dalton decrease in subunit molecular weight, suggesting that this protein in N-glycosylated. Western blot analysis has revealed immunorecognition of 4-HBP UDPGT by antibodies raised in rabbit against rat 3{alpha}- and 17{beta}-hydroxysteroid UDPGTs. Additionally, the authors have obtained the N-terminal amino acid sequence of 4-HBP UDPGT. These data provide evidence which suggests that this protein is different from another UDPGT previously shown to react with 4-hydroxybiphenyl, testosterone and chloramphenicol.

  11. In vitro characterization of glucuronidation of vanillin: identification of human UDP-glucuronosyltransferases and species differences.

    Science.gov (United States)

    Yu, Jian; Han, Jing-Chun; Hua, Li-Min; Gao, Ya-Jie

    2013-09-01

    Vanillin is a food flavoring agent widely utilized in foods, beverages, drugs, and perfumes and has been demonstrated to exhibit multiple pharmacological activities. Given the importance of glucuronidation in the metabolism of vanillin, the UDP-glucuronosyltransferase conjugation pathway of vanillin was investigated in this study. Vanillin glucuronide was identified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and a hydrolysis reaction catalyzed by β-glucuronidase. The kinetic study showed that vanillin glucuronidation by HLMs and HIMs followed Michaelis-Menten kinetics and the kinetic parameters were as follows: 134.9 ± 13.5 μM and 81.3 ± 11.3 μM for K(m) of HLMs and HIMs, 63.8 ± 2.0 nmol/min/mg pro and 13.4 ±2.0 nmol/min/mg pro for Vmax of HLMs and HIMs. All UDP-glucuronosyltransferase (UGT) isoforms except UGT1A4, 1A9, and 2B7 showed the capability to glucuronidate vanillin, and UGT1A6 exerted the higher V(max)/K(m) values than other UGT isoforms for the glucuronidation of vanillin when assuming expression of isoforms is similar in recombinant UGTs. Kinetic analysis using liver microsomes from six studied speices indicated that vanillin had highest affinity for the monkey liver microsomes enzyme (K(m)  = 25.6 ± 3.2 μM) and the lowest affinity for the mice liver microsomes enzyme (K(m)  = 149.1 ± 18.4 μM), and intrinsic clearance was in the following order: monkey > dog > minipig > mice > rat ~ human. These data collectively provided important information for understanding glucuronidation of vanillin.

  12. Phenobarbital induction and chemical synergism demonstrate the role of UDP-glucuronosyltransferases in detoxification of naphthalophos by Haemonchus contortus larvae.

    Science.gov (United States)

    Kotze, Andrew C; Ruffell, Angela P; Ingham, Aaron B

    2014-12-01

    We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Altered UDP-Glucuronosyltransferase and Sulfotransferase Expression and Function during Progressive Stages of Human Nonalcoholic Fatty Liver Disease

    OpenAIRE

    Hardwick, Rhiannon N.; Ferreira, Daniel W.; More, Vijay R.; Lake, April D.; Lu, Zhenqiang; Manautou, Jose E.; Slitt, Angela L.; Cherrington, Nathan J

    2013-01-01

    The UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) represent major phase II drug-metabolizing enzymes that are also responsible for maintaining cellular homeostasis by metabolism of several endogenous molecules. Perturbations in the expression or function of these enzymes can lead to metabolic disorders and improper management of xenobiotics and endobiotics. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver damage ranging from steatosis to nonalcoholic...

  14. Structure–inhibition relationship of ginsenosides towards UDP-glucuronosyltransferases (UGTs)

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Zhong-Ze [The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); 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 (China); Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892 (United States); Cao, Yun-Feng [Key Laboratory of Contraceptives and Devices Research(NPFPC),Shanghai Engineer and Technology Research Center of Reproductive Health Drug and Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032 (China); 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 (China); Hu, Cui-Min [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892 (United States); Hong, Mo; Sun, Xiao-Yu [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 (China); Ge, Guang-Bo; Liu, Yong; Zhang, Yan-Yan; Yang, Ling [Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 116023 Dalian (China); Sun, Hong-Zhi, E-mail: zzfang228@gmail.com [The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China)

    2013-03-01

    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 employed 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

  15. Development of UDP-glucuronosyltransferase activity toward digitoxigenin-monodigitoxoside in neonatal rats.

    Science.gov (United States)

    Watkins, J B; Klaassen, C D

    1985-01-01

    Glucuronidation is low or undetectable in embryonic and early fetal tissues and changes to adult levels at rates depending on the acceptor, tissue, and species. Because other data indicate there may be a specific UDP-glucuronosyltransferase (GT) in the liver of adult rats that glucuronidates digitoxigenin-monodigitoxoside (DIG), the development of GT activity in neonatal rats toward DIG was compared with that of other acceptors. Conjugation of p-nitrophenol and 1-naphthol was higher at birth and decreased to adult levels by 20 days of age. Glucuronidation of chloramphenicol, morphine, valproic acid, and bilirubin increased from birth to adult activity by 20 days of age. Conjugation of phenolphthalein, estrone, and diethylstilbestrol was low in 1-day-old rats and higher than adult in 20-day-old animals. In contrast, glucuronidation of DIG was barely detectable (9% of adult) in 20-day-old rats. The concentration of UDP-glucuronic acid was 50% of adult levels at birth and increased to adult values by 10 days of age. Administration of 3-methylcholanthrene on days 6 to 9 after birth significantly stimulated GT activity toward 1-naphthol, p-nitrophenol, and morphine, whereas phenobarbital precociously increased conjugation of chloramphenicol, valproic acid, morphine, and diethylstilbestrol. Pregnenolone-16 alpha-carbonitrile enhanced the development of GT activity toward morphine, chloramphenicol, valproic acid, bilirubin, diethylstilbestrol, and estrone. Glucuronidation of DIG was not increased after 3-methylcholanthrene or phenobarbital, but could be induced after pregnenolone-16 alpha-carbonitrile to 7% of adult values in 10-day-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam

    Energy Technology Data Exchange (ETDEWEB)

    Thomassin, J.; Tephly, T.R. (Univ. of Iowa, Iowa City (USA))

    1990-09-01

    Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of (3H)flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with (3H) flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000).

  17. Interaction between oblongifolin C and UDP-glucuronosyltransferase isoforms in human liver and intestine microsomes.

    Science.gov (United States)

    Gao, Cui; Shi, Rong; Wang, Tianming; Tan, Hongsheng; Xu, Hongxi; Ma, Yueming

    2015-01-01

    1. Oblongifolin C (OC) is a potential natural anticancer candidate, and its metabolic profile has not yet been established. 2. One major OC glucuronidation metabolite (OCG) has been identified in a pool of human liver microsomes (HLMs). Chemical inhibition experiments suggested that OCG was mainly formed by UGT1A. A screen of recombinant UDP-glucuronosyltransferase isoforms (UGTs) indicated that UGT1A1 primarily mediates OC conjugation, with minor contributions from UGT1A3 and UGT1A8. Enzyme kinetic studies showed that UGT1A1 was the main UGT isoform involved in OCG in HLMs. 3. Further investigation suggested that OC is a broad inhibitor of UGTs. Additionally, OC competitively inhibited UGT1A6 with a Ki value of 3.49 ± 0.57 μM, whereas non-competitively inhibited UGT1A10 with a Ki value of 2.12 ± 0.18 μM. 4. Understanding the interaction between OC and UGTs will greatly contribute to future investigations regarding the inter-individual differences in OC metabolism in clinical trials and potential drug-drug interactions.

  18. Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes.

    Science.gov (United States)

    Tan, Bo; Cai, Weimin; Zhang, Jinlian; Zhou, Ning; Ma, Guo; Yang, Ping; Zhu, Qing; Zhu, Yizhun

    2014-09-01

    Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far. Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one. Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CLint) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities. Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.

  19. Chirality Influence of Zaltoprofen Towards UDP-Glucuronosyltransferases (UGTs) Inhibition Potential.

    Science.gov (United States)

    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.

  20. Glucuronidation of drugs and drug-induced toxicity in humanized UDP-glucuronosyltransferase 1 mice.

    Science.gov (United States)

    Kutsuno, Yuki; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2014-07-01

    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.

  1. Glucose induces intestinal human UDP-glucuronosyltransferase (UGT) 1A1 to prevent neonatal hyperbilirubinemia.

    Science.gov (United States)

    Aoshima, Naoya; Fujie, Yoshiko; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2014-09-11

    Inadequate calorie intake or starvation has been suggested as a cause of neonatal jaundice, which can further cause permanent brain damage, kernicterus. This study experimentally investigated whether additional glucose treatments induce the bilirubin-metabolizing enzyme--UDP-glucuronosyltransferase (UGT) 1A1--to prevent the onset of neonatal hyperbilirubinemia. Neonatal humanized UGT1 (hUGT1) mice physiologically develop jaundice. In this study, UGT1A1 expression levels were determined in the liver and small intestine of neonatal hUGT1 mice that were orally treated with glucose. In the hUGT1 mice, glucose induced UGT1A1 in the small intestine, while it did not affect the expression of UGT1A1 in the liver. UGT1A1 was also induced in the human intestinal Caco-2 cells when the cells were cultured in the presence of glucose. Luciferase assays demonstrated that not only the proximal region (-1300/-7) of the UGT1A1 promoter, but also distal region (-6500/-4050) were responsible for the induction of UGT1A1 in the intestinal cells. Adequate calorie intake would lead to the sufficient expression of UGT1A1 in the small intestine to reduce serum bilirubin levels. Supplemental treatment of newborns with glucose solution can be a convenient and efficient method to treat neonatal jaundice while allowing continuous breastfeeding.

  2. New insights into the risk of phthalates: Inhibition of UDP-glucuronosyltransferases.

    Science.gov (United States)

    Liu, Xin; Cao, Yun-Feng; Ran, Rui-Xue; Dong, Pei-Pei; Gonzalez, Frank J; Wu, Xue; Huang, Ting; Chen, Jian-Xin; Fu, Zhi-Wei; Li, Rong-Shan; Liu, Yong-Zhe; Sun, Hong-Zhi; Fang, Zhong-Ze

    2016-02-01

    Wide utilization of phthalates-containing products results in the significant exposure of humans to these compounds. Many adverse effects of phthalates have been documented in rodent models, but their effects in humans exposed to these chemicals remain unclear until more mechanistic studies on phthalate toxicities can be carried out. To provide new insights to predict the potential adverse effects of phthalates in humans, the recent study investigated the inhibition of representative phthalates di-n-octyl ortho-phthalate (DNOP) and diphenyl phthalate (DPhP) towards the important xenobiotic and endobiotic-metabolizing UDP-glucuronosyltransferases (UGTs). An in vitro UGTs incubation system was employed to study the inhibition of DNOP and DPhP towards UGT isoforms. DPhP and DNOP weakly inhibited the activities of UGT1A1, UGT1A7, and UGT1A8. 100 µM of DNOP inhibited the activities of UGT1A3, UGT1A9, and UGT2B7 by 41.8% (p [I]/Ki > 0.1, medium possibility; [I]/Ki > 1, high possibility), these studies predicted in vivo drug-drug interaction might occur when the plasma concentration of DPhP was above 0.089 µM. Taken together, this study reveales the potential for adverse effects of phthalates DNOP and DPhP as a result of UGT inhibition.

  3. High throughput screening assay for UDP-glucuronosyltransferase 1A1 glucuronidation profiling.

    Science.gov (United States)

    Trubetskoy, O V; Finel, M; Kurkela, M; Fitzgerald, M; Peters, N R; Hoffman, F M; Trubetskoy, V S

    2007-06-01

    Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.

  4. The First Aspartic Acid of the DQxD Motif for Human UDP-Glucuronosyltransferase 1A10 Interacts with UDP-Glucuronic Acid during Catalysis

    Science.gov (United States)

    Xiong, Yan; Patana, Anne-Sisko; Miley, Michael J.; Zielinska, Agnieszka K.; Bratton, Stacie M.; Miller, Grover P.; Goldman, Adrian; Finel, Moshe; Redinbo, Matt R.; Radominska-Pandya, Anna

    2008-01-01

    All UDP-glucuronosyltransferase enzymes (UGTs) share a common cofactor, UDP-glucuronic acid (UDP-GlcUA). The binding site for UDP-GlcUA is localized to the C-terminal domain of UGTs on the basis of amino acid sequence homology analysis and crystal structures of glycosyltransferases, including the C-terminal domain of human UGT2B7. We hypothesized that the 393DQMD-NAK399 region of human UGT1A10 interacts with the glucuronic acid moiety of UDP-GlcUA. Using site-directed mutagenesis and enzymatic analysis, we demonstrated that the D393A mutation abolished the glucuronidation activity of UGT1A10 toward all substrates. The effects of the alanine mutation at Q394, D396, and K399 on glucuronidation activities were substrate-dependent. Previously, we examined the importance of these residues in UGT2B7. Although D393 (D398 in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q394 (Q399 in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate binding may explain these differences between UGT1A10 and UGT2B7. We thus postulate that D393 is critical for the binding of glucuronic acid and that proximal residues, e.g., Q394 (Q399 in UGT2B7), play a subtle role in cosubstrate binding in UGT1A10 and UGT2B7. Hence, this study provides important new information needed for the identification and understanding of the binding sites of UGTs, a major step forward in elucidating their molecular mechanism. PMID:18048489

  5. Induction of UDP-glucuronosyltransferase activities in Gunn, heterozygous, and Wistar rat livers by pregnenolone-16 alpha-carbonitrile.

    Science.gov (United States)

    Watkins, J B; Klaassen, C D

    1982-01-01

    The effect of pregnenolone-16 alpha-carbonitrile (PCN) on UDP-glucuronosyltransferase (UDP-GT) activity was comprehensively examined in Wistar (JJ), heterozygous (Jj) and Gunn (jj) rats with eleven different acceptors for glucuronic acid. UDP-GT activity after 3-methylcholanthrene (3-MC) and phenobarbital (PB) treatment was studied in additional rats for comparative purposes. Conjugation of group-1 aglycones (1-naphthol and p-nitrophenol) was much lower in Gunn than in Wistar rats. PCN did not alter UDP-GT conjugation of these acceptors. UDP-GT activity toward group-1 aglycones was increased by 3-MC in Wistar and heterozygous rats but was not enhanced in Gunn rats by any inducer. Activity toward group-2 aglycones (morphine, chloramphenicol, valproic acid) was similar in control rats of all genotypes. PCN increased chloramphenicol conjugation, whereas PB enhanced the glucuronidation of all group-2 aglycones in Wistar, heterozygous, and Gunn rats. Conjugation of group-3 acceptors (bilirubin and digitoxigenin monodigitoxoside, DIG) was deficient in Gunn rats and was not inducible. PCN increased glucuronidation of bilirubin and DIG in Wistar and heterozygous rats. The concentration of UDP-glucuronic acid (UDPGA) in liver was similar in control animals of all genotypes and was increased in rats treated with 3-MC. The other inducers did not affect hepatic UDPGA levels. Thus, 3-MC, PB, and PCN induce UDP-GT activities toward different groups of acceptors of glucuronic acid. The results support the hypothesis that PCN induces a form of UDP-GT that preferentially conjugates the group-3 acceptors, bilirubin and DIG.

  6. Inhibition of UDP-Glucuronosyltransferase (UGT) Isoforms by Arctiin and Arctigenin.

    Science.gov (United States)

    Zhang, Hui; Zhao, Zhenying; Wang, Tao; Wang, Yijia; Cui, Xiao; Zhang, Huijuan; Fang, Zhong-Ze

    2016-07-01

    Arctiin is the major pharmacological ingredient of Fructus Arctii, and arctigenin is the metabolite of arctiin formed via the catalysis of human intestinal bacteria. The present study aims to investigate the inhibition profile of arctiin and arctigenin on important phase II drug-metabolizing enzymes UDP-glucuronosyltransferases (UGTs), indicating the possible herb-drug interaction. In vitro screening experiment showed that 100 μM of arctiin and arctigenin inhibited the activity of UGT1A3, 1A9, 2B7, and 2B15. Homology modeling-based in silico docking of arctiin and arctigenin into the activity cavity of UGT2B15 showed that hydrogen bonds and hydrophobic interactions contributed to the strong binding free energy of arctiin (-8.14 kcal/mol) and arctigenin (-8.43 kcal/mol) with UGT2B15. Inhibition kinetics study showed that arctiin and arctigenin exerted competitive and noncompetitive inhibition toward UGT2B15, respectively. The inhibition kinetic parameters (Ki ) were calculated to be 16.0 and 76.7 μM for the inhibition of UGT2B15 by arctiin and arctigenin, respectively. Based on the plasma concentration of arctiin and arctigenin after administration of 100 mg/kg of arctiin, the [I]/Ki values were calculated to be 0.3 and 0.007 for arctiin and arctigenin, respectively. Based on the inhibition evaluation standard ([I]/Ki   1, high possibility), arctiin might induce drug-drug interaction with medium possibility. Based on these results, clinical monitoring the utilization of Fructus Arctii is very important and necessary. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Genetic factors affecting gene transcription and catalytic activity of UDP-glucuronosyltransferases in human liver.

    Science.gov (United States)

    Liu, Wanqing; Ramírez, Jacqueline; Gamazon, Eric R; Mirkov, Snezana; Chen, Peixian; Wu, Kehua; Sun, Chang; Cox, Nancy J; Cook, Edwin; Das, Soma; Ratain, Mark J

    2014-10-15

    The aim of this study was to discover cis- and trans-acting factors significantly affecting mRNA expression and catalytic activity of human hepatic UDP-glucuronosyltransferases (UGTs). Transcription levels of five major hepatic UGT1A (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) and five UGT2B (UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) genes were quantified in human liver tissue samples (n = 125) using real-time PCR. Glucuronidation activities of 14 substrates were measured in 47 livers. We genotyped 167 tagSNPs (single-nucleotide polymorphisms) in UGT1A (n = 43) and UGT2B (n = 124), as well as the known functional UGT1A1*28 and UGT2B17 CNV (copy number variation) polymorphisms. Transcription levels of 15 transcription factors (TFs) known to regulate these UGTs were quantified. We found that UGT expression and activity were highly variable among the livers (median and range of coefficient of variations: 135%, 74-217% and 52%, 39-105%, respectively). CAR, PXR and ESR1 were found to be the most important trans-regulators of UGT transcription (median and range of correlation coefficients: 46%, 6-58%; 47%, 9-58%; and 52%, 24-75%, respectively). Hepatic UGT activities were mainly determined by UGT gene transcription levels. Twenty-one polymorphisms were significantly (FDR-adjusted P transcription and testosterone glucuronidation rate, in addition to that attributable to the UGT2B17 CNV. Our study discovered novel pharmacogenetic markers and provided detailed insight into the genetic network regulating hepatic UGTs.

  8. Sequential metabolism of sesamin by cytochrome P450 and UDP-glucuronosyltransferase in human liver.

    Science.gov (United States)

    Yasuda, Kaori; Ikushiro, Shinichi; Kamakura, Masaki; Munetsuna, Eiji; Ohta, Miho; Sakaki, Toshiyuki

    2011-09-01

    Our previous study revealed that CYP2C9 played a central role in sesamin monocatecholization. In this study, we focused on the metabolism of sesamin monocatechol that was further converted into the dicatechol form by cytochrome P450 (P450) or the glucuronide by UDP-glucuronosyltransferase (UGT). Catecholization of sesamin monocatechol enhances its antioxidant activity, whereas glucuronidation strongly reduces its antioxidant activity. In human liver microsomes, the glucuronidation activity was much higher than the catecholization activity toward sesamin monocatechol. In contrast, in rat liver microsomes, catecholization is predominant over glucuronidation. In addition, rat liver produced two isomers of the glucuronide, whereas human liver produced only one glucuronide. These results suggest a significant species-based difference in the metabolism of sesamin between humans and rats. Kinetic studies using recombinant human UGT isoforms identified UGT2B7 as the most important UGT isoform for glucuronidation of sesamin monocatechol. In addition, a good correlation was observed between the glucuronidation activity and UGT2B7-specific activity in in vitro studies using 10 individual human liver microsomes. These results strongly suggest that UGT2B7 plays an important role in glucuronidation of sesamin monocatechol. Interindividual difference among the 10 human liver microsomes is approximately 2-fold. These results, together with our previous results on the metabolism of sesamin by human P450, suggest a small interindividual difference in sesamin metabolism. We observed the methylation activity toward sesamin monocatechol by catechol O-methyl transferase (COMT) in human liver cytosol. On the basis of these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in the human liver.

  9. Inhibition of UDP-Glucuronosyltransferases (UGTs) Activity by constituents of Schisandra chinensis.

    Science.gov (United States)

    Song, Jin-Hui; Cui, Li; An, Li-Bin; Li, Wen-Tao; Fang, Zhong-Ze; Zhang, Yan-Yan; Dong, Pei-Pei; Wu, Xue; Wang, Li-Xuan; Gonzalez, Frank J; Sun, Xiao-Yu; Zhao, De-Wei

    2015-10-01

    Structure-activity relationship for the inhibition of Schisandra chinensis's ingredients toward (Uridine-Diphosphate) UDP-glucuronosyltransferases (UGTs) activity was performed in the present study. In vitro incubation system was employed to screen the inhibition capability of S. chinensis's ingredients, and in silico molecular docking method was carried out to explain possible mechanisms. At 100 μM of compounds, the activity of UGTs was inhibited by less than 90% by schisandrol A, schisandrol B, schisandrin, schisandrin C, schisantherin A, gomisin D, and gomisin G. Schisandrin A exerted strong inhibition toward UGT1A1 and UGT1A3, with the residual activity to be 7.9% and 0% of control activity. Schisanhenol exhibited strong inhibition toward UGT2B7, with the residual activity to be 7.9% of control activity. Gomisin J of 100 μM inhibited 91.8% and 93.1% of activity of UGT1A1 and UGT1A9, respectively. Molecular docking prediction indicated different hydrogen bonds interaction resulted in the different inhibition potential induced by subtle structure alteration among schisandrin A, schisandrin, and schisandrin C toward UGT1A1 and UGT1A3: schisandrin A > schisandrin > schisandrin C. The detailed inhibition kinetic evaluation showed the strong inhibition of gomisin J toward UGT1A9 with the inhibition kinetic parameter (Ki ) to be 0.7 μM. Based on the concentrations of gomisin J in the plasma of the rats given with S. chinensis, high herb-drug interaction existed between S. chinensis and drugs mainly undergoing UGT1A9-mediated metabolism. In conclusion, in silico-in vitro method was used to give the inhibition information and possible inhibition mechanism for S. chinensis's components toward UGTs, which guide the clinical application of S. chinensis.

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

    Science.gov (United States)

    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

  11. Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus.

    Science.gov (United States)

    Fujiwara, Ryoichi; Nguyen, Nghia; Chen, Shujuan; Tukey, Robert H

    2010-03-16

    High levels of unconjugated bilirubin (UCB) in newborn children is associated with a reduction in hepatic UDP glucuronosyltransferase (UGT) 1A1 activity that can lead to CNS toxicity, brain damage, and even death. Little is known regarding those events that lead to UCB accumulation in brain tissue, and therefore, we sought to duplicate this condition in mice. The human UGT1 locus, encoding all 9-UGT1A genes including UGT1A1, was expressed in Ugt1(-/-) mice. Because the most common clinical condition associated with jaundice in adults is Gilbert's syndrome, which is characterized by an allelic polymorphism in the UGT1A1 promoter, hyperbilirubinemia was monitored in humanized UGT1 mice that expressed either the Gilbert's UGT1A1*28 allele [Tg(UGT1(A1*28))Ugt1(-/-) mice] or the normal UGT1A1*1 allele [Tg(UGT1(A1*1))Ugt1(-/-) mice]. Adult Tg(UGT1(A1*28))Ugt1(-/-) mice expressed elevated levels of total bilirubin (TB) compared with Tg(UGT1(A1*1))Ugt1(-/-) mice, confirming that the promoter polymorphism associated with the UGT1A1*28 allele contributes to hyperbilirubinemia in mice. However, TB accumulated to near toxic levels during neonatal development, a finding that is independent of the Gilbert's UGT1A1*28 promoter polymorphism. Whereas serum TB levels eventually returned to adult levels, TB clearance in neonatal mice was not associated with hepatic UGT1A1 expression. In approximately 10% of the humanized UGT1 mice, peak TB levels culminated in seizures followed by death. UCB deposition in brain tissue and the ensuing seizures were associated with developmental milestones and can be prevented by enhancing regulation of the UGT1A1 gene in neonatal mice.

  12. Inactivation by UDP-glucuronosyltransferase enzymes: the end of androgen signaling.

    Science.gov (United States)

    Chouinard, Sarah; Yueh, Mei-Fei; Tukey, Robert H; Giton, Frank; Fiet, Jean; Pelletier, Georges; Barbier, Olivier; Bélanger, Alain

    2008-04-01

    Conjugation by UDP-Glucuronosyltransferase (UGT) is the major pathway of androgen metabolism and elimination in the human. High concentrations of glucuronide conjugates of androsterone (ADT) and androstane-3alpha,17beta-diol (3alpha-diol) are present in circulation and several studies over the last 30 years have concluded that the serum levels of these metabolites might reflect the androgen metabolism in several tissues, including the liver and androgen target tissues. Three UGT2B enzymes are responsible for the conjugation of DHT and its metabolites ADT and 3alpha-diol: UGT2B7, B15 and B17. UGT2B7 is expressed in the liver and skin whereas UGT2B15 and B17 were found in the liver, prostate and skin. Very specific antibodies against each UGT2B enzyme have been obtained and used for immunohistochemical studies in the human prostate. It was shown that UGT2B17 is expressed in basal cells whereas UGT2B15 is only localized in luminal cells, where it inactivates DHT. By using LNCaP cells, we have also demonstrated that the expression and activity of UGT2B15 and B17 are modulated by several endogenous prostate factors including androgen. Finally, to study the physiological role of UGT2B enzymes, transgenic mice bearing the human UGT2B15 gene were recently obtained. A decrease in reproductive tissue weight from transgenic animals compared to those from control animals was observed. In conclusion, the conjugation by UGT2B7, B15 and B17, which represents a non-reversible step in androgen metabolism, is an important means by which androgens are regulated locally. It is also postulated that UGT enzymes protect the tissue from deleteriously high concentrations of active androgen.

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

    Science.gov (United States)

    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.

  14. New insights for the risk of bisphenol A: inhibition of UDP-glucuronosyltransferases (UGTs).

    Science.gov (United States)

    Jiang, Hua-Mao; Fang, Zhong-Ze; Cao, Yun-Feng; Hu, Cui-Min; Sun, Xiao-Yu; Hong, Mo; Yang, Ling; Ge, Guang-Bo; Liu, Yong; Zhang, Yan-Yan; Dong, Qiang; Liu, Ren-Jie

    2013-10-01

    Bisphenol A (BPA), the important endocrine-disrupting chemical (EDC), has been reported to be able to induce various toxicity. The present study aims to understand the toxicity behavior of bisphenol A through evaluating the inhibition profile of bisphenol A towards UDP-glucuronosyltransferase (UGT) isoforms. In vitro recombinant UGTs-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employed as probe reaction for all the tested UGT isoforms. The results showed that bisphenol A exerted stronger inhibition towards UGT2B isoforms than UGT1A isoforms. Furthermore, the inhibition kinetic type and parameters (K(i)) were determined for the inhibition of bisphenol A towards UGT2B4, 2B7, 2B15, and 2B17. Bisphenol A exhibited the competitive inhibition towards UGT2B4, and noncompetitive inhibition towards UGT2B7, 2B15 and 2B17. The inhibition kinetic parameters (K(i)) were calculated to be 1.1, 32.6, 5.6, and 19.9 μM for UGT2B4, 2B7, 2B15 and 2B17, respectively. In combination with the in vivo concentration of bisphenol A, the elevation of exposure dose was predicted to increase by 29.1%, 1%, 5.7%, and 1.6% for UGT2B4, 2B7, 2B15, and 2B17, indicating the high influence of bisphenol A towards the in vivo UGT2B isofroms-mediated metabolism of xenobiotics and endogenous substances. All these data provide the supporting information for deeper understanding of toxicology of bisphenol A.

  15. Endogenous Protein Interactome of Human UDP-Glucuronosyltransferases Exposed by Untargeted Proteomics

    Science.gov (United States)

    Rouleau, Michèle; Audet-Delage, Yannick; Desjardins, Sylvie; Rouleau, Mélanie; Girard-Bock, Camille; Guillemette, Chantal

    2017-01-01

    The conjugative metabolism mediated by UDP-glucuronosyltransferase enzymes (UGTs) significantly influences the bioavailability and biological responses of endogenous molecule substrates and xenobiotics including drugs. UGTs participate in the regulation of cellular homeostasis by limiting stress induced by toxic molecules, and by controlling hormonal signaling networks. Glucuronidation is highly regulated at genomic, transcriptional, post-transcriptional and post-translational levels. However, the UGT protein interaction network, which is likely to influence glucuronidation, has received little attention. We investigated the endogenous protein interactome of human UGT1A enzymes in main drug metabolizing non-malignant tissues where UGT expression is most prevalent, using an unbiased proteomics approach. Mass spectrometry analysis of affinity-purified UGT1A enzymes and associated protein complexes in liver, kidney and intestine tissues revealed an intricate interactome linking UGT1A enzymes to multiple metabolic pathways. Several proteins of pharmacological importance such as transferases (including UGT2 enzymes), transporters and dehydrogenases were identified, upholding a potential coordinated cellular response to small lipophilic molecules and drugs. Furthermore, a significant cluster of functionally related enzymes involved in fatty acid β-oxidation, as well as in the glycolysis and glycogenolysis pathways were enriched in UGT1A enzymes complexes. Several partnerships were confirmed by co-immunoprecipitations and co-localization by confocal microscopy. An enhanced accumulation of lipid droplets in a kidney cell model overexpressing the UGT1A9 enzyme supported the presence of a functional interplay. Our work provides unprecedented evidence for a functional interaction between glucuronidation and bioenergetic metabolism. PMID:28217095

  16. Expression and inducibility of UDP-glucuronosyltransferase 1As in MCF-7 human breast carcinoma cells.

    Science.gov (United States)

    Hanioka, Nobumitsu; Iwabu, Hiroyuki; Hanafusa, Hiroyuki; Nakada, Shintaro; Narimatsu, Shizuo

    2012-03-01

    UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms broadly expressed in hepatic and extrahepatic tissues and examined the expression and inducibility of UGT1As (UGT1A1 and UGT1A3-1A10) in MCF-7 cells (human breast carcinoma cell line). Reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated that UGT1A1, UGT1A6 and UGT1A9 mRNAs as well as the mRNAs of transcriptional regulators (AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; ERα, oestrogen receptor α; ERβ, oestrogen receptor β; and GR, glucocorticoid receptor) are expressed in MCF-7 cells. UGT1A6 mRNA level in MCF-7 cells was significantly increased to 1.9 times by β-naphthoflavone (BNF), whereas UGT1A1 and UGT1A9 mRNA levels were not affected by BNF. There were no significant changes in the mRNAs of UGT1A1, UGT1A6 and UGT1A9 in MCF-7 cells by treatment with phenobarbital (PB) and dexamethasone (DEX) in MCF-7 cells. The kinetics of 7-ethyl-10-hydroxycamptothecin (SN-38), 5-hydroxytryptamine (5-HT) and 4-methylumbelliferone (4-MU) glucuronidation by microsomes from control and BNF-treated MCF-7 cells fitted with the Michaelis-Menten model, and the V(max) and CL(int) values were significantly increased to 7.5-8.5 times and 5.9-10.4 times by BNF treatment, respectively. These findings suggest that BNF induces UGT1A6 in MCF-7 cells and that the increase may be mediated by AhR but not pregnane X receptor (PXR)/constitutive androstane receptor (CAR). The information gained in this study should help predict and assess the toxicity of environmental chemicals.

  17. Identification and preliminary characterization of UDP-glucuronosyltransferases catalyzing formation of ethyl glucuronide.

    Science.gov (United States)

    Schwab, Nicole; Skopp, Gisela

    2014-04-01

    Ethyl glucuronide (EtG), a minor metabolite of ethanol, is used as a marker of alcohol consumption in a variety of clinical and forensic settings. At present there are very few studies of UDP-glucuronosyltransferases (UGT), responsible for catalyzing EtG formation, and the possible effect of nutritional components, e.g. flavonoids, which are extensively glucuronidated, on EtG formation has not been addressed at all. The following incubation conditions were optimized with regard to previously published conditions: buffer, substrate concentration, and incubation time. Isolation of EtG from the incubation mixture was also optimized. Recombinant UGT enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15) were screened for their activity towards ethanol, and kinetic data were then established for all enzymes. It was decided to study the effect of the flavonoids quercetin and kaempferol on glucuronidation of ethanol. Isolation was by solid-phase extraction (SPE) to minimize matrix effects. Analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS-MS), with EtG-d5 as the internal standard. SPE was vital to avoid severe ion suppression after direct injection of the incubation solution. EtG formation was observed for all enzymes under investigation; their kinetics followed the Michaelis-Menten model, meaning the maximum reaction rate achieved at saturating substrate concentrations (V(max)) and the substrate concentration at which the reaction rate is half of V(max) (Michaelis-Menten constant, K(m)) could be calculated. The highest rate of glucuronidation was observed with UGT1A9 and 2B7. After co-incubation with both flavonoids, formation of EtG was significantly reduced for all enzymes except for UGT2B15, whose activity did not seem to be affected. Results reveal that multiple UGT isoforms are capable of catalyzing glucuronidation of ethanol; nevertheless, the effect of UGT polymorphism on glucuronidation of ethanol needs further study. Formation of Et

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

    Science.gov (United States)

    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...

  19. Effect of UDP-glucuronosyltransferase 1A8 polymorphism on raloxifene glucuronidation.

    Science.gov (United States)

    Kokawa, Yuki; Kishi, Naoki; Jinno, Hideto; Tanaka-Kagawa, Toshiko; Narimatsu, Shizuo; Hanioka, Nobumitsu

    2013-05-13

    Raloxifene is an antiestrogen marketed for the treatment of osteoporosis. The major metabolic pathway of raloxifene is glucuronidation at 6- and/or 4'-positions, which is mainly catalyzed by UDP-glucuronosyltransferase 1A8 (UGT1A8) expressed in extrahepatic tissues such as the small intestine and colon. Two non-synonymous allelic variants, termed UGT1A8*2 (518C>G, A173G) and UGT1A8*3 (830G>A, C277Y), have been found in Caucasian, African-American and Asian populations. In this study, the effect of amino acid substitutions in UGT1A8 on raloxifene glucuronidation was studied using recombinant UGT1A8 enzymes of wild-type (UGT1A8.1) and variant UGT1A8 (UGT1A8.2 and UGT1A8.3) expressed in Sf9 cells. Raloxifene 6- and 4'-glucuronidation by UGT1A8.1 exhibited negative allosteric kinetics. The Km and Vmax values of UGT1A8.1 were 15.0 μM and 111 pmol/min/mg protein for 6-glucuronidation, and 9.35 μM and 232 pmol/min/mg protein for 4'-glucuronidation, respectively. The kinetics of raloxifene 6-glucuronidation by UGT1A8.2 was positive allosteric, whereas the kinetics of raloxifene 4'-glucuronidation was negative allosteric. The S50 value of raloxifene 6-glucuronidation was markedly low (1.2%) compared with the Km value of UGT1A8.1, and the Km value for raloxifene 4'-glucuronidation was 29% that of UGT1A8.1. The Vmax value for raloxifene 6-glucuronidation by UGT1A8.2 was comparable to that of UGT1A8.1, whereas the Vmax value for raloxifene 4'-glucuronidation was significantly lower (54%) than that of UGT1A8.1. The activities of raloxifene 6- and 4'-glucuronidation in UGT1A8.3 were markedly lower than those of UGT1A8.1. In mycophenolic acid glucuronidation, the kinetics by wild-type and variant UGT1A8s fitted the Michaelis-Menten model. The Km and Vmax values of UGT1A8.1 were 123 μM and 4820 pmol/min/mg protein, respectively. The Km and Vmax values of UGT1A8.2 were comparable to those of UGT1A8.1. The Km value of UGT1A8.3 was similar to that of UGT1A8.1, whereas the Vmax

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

    Science.gov (United States)

    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

  1. Unravelling the transcriptomic landscape of the major phase II UDP-glucuronosyltransferase drug metabolizing pathway using targeted RNA sequencing.

    Science.gov (United States)

    Tourancheau, A; Margaillan, G; Rouleau, M; Gilbert, I; Villeneuve, L; Lévesque, E; Droit, A; Guillemette, C

    2016-02-01

    A comprehensive view of the human UDP-glucuronosyltransferase (UGT) transcriptome is a prerequisite to the establishment of an individual's UGT metabolic glucuronidation signature. Here, we uncover the transcriptome landscape of the 10 human UGT gene loci in normal and tumoral metabolic tissues by targeted RNA next-generation sequencing. Alignment on the human hg19 reference genome identifies 234 novel exon-exon junctions. We recover all previously known UGT1 and UGT2 enzyme-coding transcripts and identify over 130 structurally and functionally diverse novel UGT variants. We further expose a revised genomic structure of UGT loci and provide a comprehensive repertoire of transcripts for each UGT gene. Data also uncover a remodelling of the UGT transcriptome occurring in a tissue- and tumor-specific manner. The complex alternative splicing program regulating UGT expression and protein functions is likely critical in determining detoxification capacity of an organ and stress-related responses, with significant impact on drug responses and diseases.

  2. Role of cytochrome P450 and UDP-glucuronosyltransferases in metabolic pathway of homoegonol in human liver microsomes.

    Science.gov (United States)

    Kwon, Soon Sang; Kim, Ju Hyun; Jeong, Hyeon-Uk; Ahn, Kyung-Seop; Oh, Sei-Ryang; Lee, Hye Suk

    2015-08-01

    Homoegonol is being evaluated for the development of a new antiasthmatic drug. Based on a pharmacokinetic study of homoegonol in rats, homoegonol is almost completely eliminated via metabolism, but no study on its metabolism has been reported in animals and humans. Incubation of homoegonol in human liver microsomes in the presence of the reduced form of nicotinamide adenine dinucleotide phosphate and UDP-glucuronic acid resulted in the formation of five metabolites: 4-O-demethylhomoegonol (M1), hydroxyhomoegonol (M2 and M3), 4-O-demethylhomoegonol glucuronide (M4), and homoegonol glucuronide (M5). We characterized the cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for homoegonol metabolism using human liver microsomes, and cDNA-expressed CYP and UGT enzymes. CYP1A2 played a more prominent role than CYP3A4 and CYP2D6 in the 4-O-demethylation of homoegonol to M1. CYP3A4 was responsible for the hydroxylation of homoegonol to M2. The hydroxylation of homoegonol to M3 was insufficient to characterize CYP enzymes. Glucuronidation of homoegonol to M5 was mediated by UGT1A1, UGT1A3, UGT1A4, and UGT2B7 enzymes, whereas M4 was formed from 4-O-demethylhomoegonol by UGT1A1, UGT1A8, UGT1A10, and UGT2B15 enzymes.

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

    Directory of Open Access Journals (Sweden)

    Mohamed eOuzzine

    2014-10-01

    Full Text Available UDP-glucuronosyltransferases (UGTs form a multigenic family of membrane-bound enzymes expressed in various tissues, including brain. They catalyze the formation of β-Dglucuronides from structurally unrelated substances (drugs, other xenobiotics, as well as endogenous compounds by the linkage of glucuronic acid from the high energy donor, UDP-αD-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. They are also associated to brain interfaces devoid of blood-brain barrier, 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 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.

  4. Traditional herbal formulas to as treatments for musculoskeletal disorders: Their inhibitory effects on the activities of human microsomal cytochrome p450s and udp-glucuronosyltransferases

    Directory of Open Access Journals (Sweden)

    Seong Eun Jin

    2016-01-01

    Abbreviation used: BPTSS: Bangpungtongseong-san, OJS: Ojeok-san, OYSGS: Oyaksungi-san, CYP450s: cytochrome P450s, UGTs: UDP-glucuronosyltransferases, MSDs: Musculoskeletal disorders, NSAIDs: nonsteroidal anti-inflammatory drugs, EOMCC: 7-ethoxy-methyloxy-3-cyanocoumarin, DBOMF: di(benzyloxymethoxyfluorescein, BOMCC: 7-benzyloxy-4-trifluoromethylcoumarin, HPLC: High-performance liquid chromatography, PDA: photo diode array, SEM: standard error of the mean, UDPGA: uridine 5'-diphosphoglucuronic acid.

  5. UGT酶的遗传药理学研究进展%Progress in the pharmacogenomics of UDP-glucuronosyltransferase

    Institute of Scientific and Technical Information of China (English)

    郭栋; 庞良芳; 周宏灏

    2011-01-01

    尿苷二磷酸葡萄糖醛酸基转移酶(UDP-glucuronosyltransferase,UGT),是人体内重要的催化Ⅱ相结合反应的酶.UGT广泛分布于人体的肝、肾、胃肠道以及各种腺体组织,因其结构和细胞内分布的特殊性,对其研究落后于CYP450酶.近20年来随着实验技术的发展,越来越多的研究开始关注UGT.随着研究进展,UGT的底物在不断地扩展,包括有内源性激素、药物以及毒物.个体间、种族间葡萄糖醛酸化活性存在明显差异,UGT基因多态性是此现象的重要原因之一.对UGT基因多态性的研究日益增多,已经成立命名委员会.UGT按照序列相似性分两个大的亚家族,本文分别对其中主要参与药物代谢的UGT1 A和UGT2家族的某些成员的遗传药理学研究进展进行综述.%UDP-glucuronosyltransferase is an important enzyme involved in the phase II conjugation reaction. UGT is wildly expressed in human liver, kidney, stomach intestine tract and glandular organs. Because of the specificity of its structure and intra-cellular distribution, the study in UGT is lagged behind of the CYP450. Recent two decades, following the progress of the experiment technology, more and more research were focused on the UGT. Following these, the probe drug of UGT are enlarging, it involved in the metabolism of endogenous hormone, drugs and some kinds of toxicity. The polymorphisms of UGT gene were one of the most important reasons lead to the large inter-individual and inter-racial variability of the glucuronodiation activity. There are two subgroups, UGT1A and UGT2, depending on the similarity of the amino acid sequence. This paper reviews the current concept and new advances in the pharmacogenomics of UGT genes which have played an important role in drug metabolism.

  6. Effect of Honokiol on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Yong Yeon Cho

    2013-09-01

    Full Text Available Honokiol is a bioactive component isolated from the medicinal herbs Magnolia officinalis and Magnolia grandiflora that has antioxidative, anti-inflammatory, antithrombotic, and antitumor activities. The inhibitory potentials of honokiol on eight major human cytochrome P450 (CYP enzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, and four UDP-glucuronosyltransferases (UGTs 1A1, 1A4, 1A9, and 2B7 in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Honokiol strongly inhibited CYP1A2-mediated phenacetin O-deethylation, CYP2C8-mediated amodiaquine N-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4-hydroxylation, and UGT1A9-mediated propofol glucuronidation with Ki values of 1.2, 4.9, 0.54, 0.57, and 0.3 μM, respectively. Honokiol also moderately inhibited CYP2B6-mediated bupropion hydroxylation and CYP2D6-mediated bufuralol 1'-hydroxylation with Ki values of 17.5 and 12.0 μM, respectively. These in vitro results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9.

  7. UDP-glucuronosyltransferase 2B7 C802T (His268Tyr) polymorphism in bladder cancer cases.

    Science.gov (United States)

    Zimmermann, Anna; Blaszkewicz, Meinolf; Roth, Gerhard; Seidel, Thilo; Dietrich, Holger; Schutschkow, Olga; Bolt, Hermann M; Golka, Klaus

    2008-01-01

    A study of Chinese benzidine workers indicated elevated levels of UDP-glucuronosyltransferase (UGT) 2B7 T/T activity in carriers for development of bladder cancer. The present study was designed to investigate the possible impact of the presence of UGT2B7 genotype on bladder cancer risk in Caucasians. UGT2B7 polymorphism at locus C(802)T (His(268)Tyr) was detected using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based procedure. The study group consisted of 211 bladder cancer cases and 210 controls suffering from different urological diseases, but without any history of cancer. Both groups were recruited from a Department of Urology located in a center of former chemical and rubber industries in Germany. Furthermore, 171 bladder cancer cases with a history of occupational exposure to aromatic amines surveyed for compensation due to an occupational disease were investigated. T/T genotype frequencies in bladder cancer cases, urological controls, and exposed patients appeared similar (27 vs. 35 vs. 25%). This study indicated that there were ethnic differences between Caucasian and Chinese general populations with respect to the UGT2B7 genotype. Furthermore, in contrast to an earlier investigation in benzidine-exposed Chinese bladder cancer patients, no relevant differences between bladder cancer patients and urological hospital controls were observed in Germany.

  8. Multiple UDP- Glucuronosyltransferases in Human Liver Microsomes Glucuronidate Both R- and S-7-Hydroxywarfarin into Two Metabolites

    Science.gov (United States)

    Pugh, C. Preston; Pouncey, Dakota L; Hartman, Jessica H.; Nshimiyimana, Robert; Desrochers, Linda P.; Goodwin, Thomas E.; Boysen, Gunnar; Miller, Grover P.

    2014-01-01

    The widely used anticoagulant Coumadin (R/S-warfarin) undergoes oxidation by cytochromes P450 into hydroxywarfarins that subsequently become conjugated for excretion in urine. Hydroxywarfarins may modulate warfarin metabolism transcriptionally or through direct inhibition of cytochromes P450 and thus, UGT action toward hydroxywarfarin elimination may impact levels of the parent drugs and patient responses. Nevertheless, relatively little is known about conjugation by UDP-glucuronosyltransferases in warfarin metabolism. Herein, we identified probable conjugation sites, kinetic mechanisms and hepatic UGT isoforms involved in microsomal glucuronidation of R- and S-7-hydroxywarfarin. Both compounds underwent glucuronidation at C4 and C7 hydroxyl groups based on elution properties and spectral characteristics. Their formation demonstrated regio- and enantioselectivity by UGTs and resulted in either Michaelis-Menten or substrate inhibition kinetics. Glucuronidation at the C7 hydroxyl group occurred more readily than at the C4 group, and the reaction was overall more efficient for R-7-hydroxywarfarin due to higher affinity and rates of turnover. The use of these mechanisms and parameters to model in vivo clearance demonstrated that contributions of substrate inhibition would lead to underestimation of metabolic clearance than that predicted by Michaelis-Menten kinetics. Lastly, these processes were driven by multiple UGTs indicating redundancy in glucuronidation pathways and ultimately metabolic clearance of R- and S-7-hydroxywarfarin. PMID:25447818

  9. Impact of fatty acids on human UDP-glucuronosyltransferase 1A1 activity and its expression in neonatal hyperbilirubinemia.

    Science.gov (United States)

    Shibuya, Ayako; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2013-10-09

    While breast milk has been known as a cause of neonatal hyperbilirubinemia, the underlying mechanism of breast milk-induced jaundice has not been clarified. Here, the impact of fatty acids on human UDP-glucuronosyltransferase (UGT) 1A1--the sole enzyme that can metabolize bilirubin--were examined. Oleic acid, linoleic acid, and docosahexaenoic acid (DHA) strongly inhibited UGT1A1 activity. Forty-eight hours after a treatment with a lower concentration of DHA (10 mg/kg), total bilirubin significantly increased in neonatal hUGT1 mice, which are human neonatal jaundice models. In contrast, treatments with higher concentrations of fatty acids (0.1-10 g/kg) resulted in a decrease in serum bilirubin in hUGT1 mice. It was further demonstrated that the treatment with higher concentrations of fatty acids induced UGT1A1, possibly by activation of peroxisome proliferator-activated receptors. Our data indicates that activation of peroxisome proliferator-activated receptors would increase UGT1A1 expression, resulting in reduction of serum bilirubin levels in human infants.

  10. Induction of the UDP-Glucuronosyltransferase 1A1 during the Perinatal Period Can Cause Neurodevelopmental Toxicity.

    Science.gov (United States)

    Hirashima, Rika; Michimae, Hirofumi; Takemoto, Hiroaki; Sasaki, Aya; Kobayashi, Yoshinori; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2016-09-01

    Anticonvulsants can increase the risk of developing neurotoxicity in infants; however, the underlying mechanism has not been elucidated to date. Thyroxine [3,5,3',5'-l-tetraiodothyronine (T4)] plays crucial roles in the development of the central nervous system. In this study, we hypothesized that induction of UDP-glucuronosyltransferase 1A1 (UGT1A1)-an enzyme involved in the metabolism of T4-by anticonvulsants would reduce serum T4 levels and cause neurodevelopmental toxicity. Exposure of mice to phenytoin during both the prenatal and postnatal periods significantly induced UGT1A1 and decreased serum T4 levels on postnatal day 14. In the phenytoin-treated mice, the mRNA levels of synaptophysin and synapsin I in the hippocampus were lower than those in the control mice. The thickness of the external granule cell layer was greater in phenytoin-treated mice, indicating that induction of UGT1A1 during the perinatal period caused neurodevelopmental disorders. Exposure to phenytoin during only the postnatal period also caused these neurodevelopmental disorders. A T4 replacement attenuated the increase in thickness of the external granule cell layer, indicating that the reduced T4 was specifically associated with the phenytoin-induced neurodevelopmental disorder. In addition, these neurodevelopmental disorders were also found in the carbamazepine- and pregnenolone-16-α-carbonitrile-treated mice. Our study is the first to indicate that UGT1A1 can control neurodevelopment by regulating serum T4 levels.

  11. Metabolic Disposition of Luteolin Is Mediated by the Interplay of UDP-Glucuronosyltransferases and Catechol-O-Methyltransferases in Rats.

    Science.gov (United States)

    Wang, Liping; Chen, Qingwei; Zhu, Lijun; Li, Qiang; Zeng, Xuejun; Lu, Linlin; Hu, Ming; Wang, Xinchun; Liu, Zhongqiu

    2017-03-01

    Luteolin partially exerts its biologic effects via its metabolites catalyzed by UDP-glucuronosyltransferases (UGTs) and catechol-O-methyltransferases (COMTs). However, the interplay of UGTs and COMTs in mediating luteolin disposition has not been well clarified. In this study, we investigated the glucuronidation and methylation pathways of luteolin mediated by the interplay of UGTs and COMTs in vivo and in vitro. A total of nine luteolin metabolites was detected in rat plasma and bile by liquid chromatography-tandem mass spectrometry, namely, three glucuronides, two methylated metabolites, and four methylated glucuronides. Luteolin-3'-glucuronide (Lut-3'-G) exhibited the highest systemic exposure among these metabolites. Kinetics studies in rat liver S9 fractions suggested two pathways, as follows: 1) Luteolin was glucuronidated to luteolin-7-glucuronide, luteolin-4'-glucuronide, and Lut-3'-G by UGTs, and then Lut-7-G was methylated to chrysoeriol-7-glucuronide and diosmetin-7-glucuronide by COMTs. 2) Alternatively, luteolin was methylated to chrysoeriol and diosmetin by COMTs, and then chrysoeriol and diosmetin were glucuronidated by UGTs to their respective glucuronides. The methylation rate of luteolin was significantly increased by the absence of glucuronidation, whereas the glucuronidation rate was increased by the absence of methylation, but to a lesser extent. In conclusion, two pathways mediated by the interplay of UGTs and COMTs are probably involved in the metabolic disposition of luteolin. The glucuronidation and methylation of luteolin compensate for each other, although glucuronidation is the predominant pathway.

  12. Induction of digitoxigenin monodigitoxoside UDP-glucuronosyltransferase activity by glucocorticoids and other inducers of cytochrome P-450p in primary monolayer cultures of adult rat hepatocytes and in human liver.

    Science.gov (United States)

    Schuetz, E G; Hazelton, G A; Hall, J; Watkins, P B; Klaassen, C D; Guzelian, P S

    1986-06-25

    We have recently proposed that glucocorticoids induce cytochrome P-450p, a liver microsomal hemoprotein originally isolated from rats treated with the antiglucocorticoid pregnenolone 16 alpha-carbonitrile (PCN), through a mechanism that involves a stereospecific recognition system clearly distinguishable from the classic glucocorticoid receptor (Schuetz, E. G., Wrighton, S. A., Barwick, J. L., and Guzelian, P. S. (1984) J. Biol. Chem. 259, 1999-2012). We now report that digitoxigenin monodigitoxoside UDP-glucuronosyltransferase (DIG UDP-glucuronosyltransferase), a liver microsomal enzyme activity induced by PCN in rats, is also inducible, as is P-450p, in primary monolayer cultures of adult rat hepatocytes. DIG UDP-glucuronosyltransferase activity closely resembled reported characteristics of induction of P-450p in its time course of induction, concentration-response relationships, exclusivity of induction by steroids with glucocorticoid properties, unusual rank order of potency of glucocorticoid agonists, unusually high ED50 for induction by glucocorticoids, enhanced induction rather than inhibition by anti-glucocorticoids in the presence of glucocorticoids, and finally, induction by nonsteroidal inducers of P-450p. DIG UDP-glucuronosyltransferase activity was also readily detected in human liver microsomes and was elevated in two patients who had received inducers of P-450p. We conclude that the liver enzymes controlled by the postulated PCN recognition system include not only P-450p but also one or more UDP-glucuronosyltransferases.

  13. The donor substrate specificity of the human beta 1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues.

    Science.gov (United States)

    Ouzzine, Mohamed; Gulberti, Sandrine; Levoin, Nicolas; Netter, Patrick; Magdalou, Jacques; Fournel-Gigleux, Sylvie

    2002-07-12

    The human beta1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galbeta1,3Galbeta1,4Xylbeta-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His(308) and Arg(277) residues as essential determinants for the donor substrate (UDP-glucuronic acid) selectivity of the human GlcAT-I. Analysis of the UDP-glucuronic acid-binding site by computational modeling in conjunction with site-directed mutagenesis indicated that both residues interact with glucuronic acid. Substitution of His(308) by arginine induced major changes in the donor substrate specificity of GlcAT-I. Interestingly, the H308R mutant was able to efficiently utilize nucleotide sugars UDP-glucose, UDP-mannose, and UDP-N-acetylglucosamine, which are not naturally accepted by the wild-type enzyme, as co-substrate in the transfer reaction. To gain insight into the role of Arg(277), site-directed mutagenesis in combination with chemical modification was carried out. Substitution of Arg(277) with alanine abrogated the activity of GlcAT-I. Furthermore, the arginine-directed reagent 2,3-butanedione irreversibly inhibited GlcAT-I, which was effectively protected against inactivation by UDP-glucuronic acid but not by UDP-glucose. It is noteworthy that the activity of the H308R mutant toward UDP-glucose was unaffected by the arginine-directed reagent. Our results are consistent with crucial interactions between the His(308) and Arg(277) residues and the glucuronic acid moiety that governs the specificity of GlcAT-I toward the nucleotide sugar donor substrate.

  14. Gene therapy with bilirubin-UDP-glucuronosyltransferase in the Gunn rat model of Crigler-Najjar syndrome type 1.

    Science.gov (United States)

    Li, Q; Murphree, S S; Willer, S S; Bolli, R; French, B A

    1998-03-01

    Crigler-Najjar syndrome type 1 (CN type 1) is an autosomal recessive disorder characterized by nonhemolytic jaundice resulting from mutations to the gene encoding bilirubin-UDP-glucuronosyltransferase (UDPGT). The Gunn rat is an accurate animal model of this disease because the bilirubin-UDPGT gene in this strain carries a premature stop codon. The primary objective of this study was to complement this deficiency in vivo using liver-directed gene therapy. The efficiency of adenovirus type 5 (Ad5)-mediated gene transfer to the neonatal rat liver was first assessed by intravenous (i.v.) injection of an Ad5 vector carrying a nuclear-localized LacZ gene. An Ad5 vector expressing the cDNA encoding human bilirubin-UDPGT (Ad5/CMV/hUG-Br1) was then generated and injected i.v. into neonatal Gunn rats. Plasma samples were collected and bilirubin levels were determined at regular intervals. Although the mean level of bilirubin in homozygous Gunn rats 1-2 days after birth was already 14.5-fold higher than that of heterozygous siblings, treatment with Ad5/CMV/hUG-Br1 reduced plasma bilirubin to normal levels within 1 week. Plasma bilirubin in the treated homozygous rats remained normal for 4 weeks before gradually climbing to intermediate levels that were approximately half that of untreated homozygotes by 12 weeks. Administration of Ad5-mediated gene therapy to neonatal Gunn rats effectively complemented the deficiency in bilirubin-UDPGT, resulting in substantial reductions in plasma bilirubin over a 3-month period. The efficacy of Ad5-mediated gene therapy in neonates suggests that this approach might be effective against other hepatic disorders, including autosomal recessive deficiencies in lipid metabolism and vascular homeostasis.

  15. Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells.

    Science.gov (United States)

    Nishiyama, Takahito; Izawa, Tadashi; Usami, Mami; Ohnuma, Tomokazu; Ogura, Kenichiro; Hiratsuka, Akira

    2010-04-01

    Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process.

  16. Identification of the human UDP-glucuronosyltransferase isoforms involved in the glucuronidation of the phytochemical ferulic acid.

    Science.gov (United States)

    Li, Xiaojun; Shang, Liang; Wu, Yaohua; Abbas, Suzanne; Li, Dong; Netter, Patrick; Ouzzine, Mohamed; Wang, Hui; Magdalou, Jacques

    2011-01-01

    Ferulic acid (FA), a member of the hydroxycinnamate family, is an abundant dietary antioxidant that may offer beneficial effects against cancer, cardiovascular disease, diabetes, osteoarthritis and Alzheimer's disease. In this study, evidence for sulfation and glucuronidation of FA was investigated upon incubation with human liver microsomes and cytosol. Two main glucuronides, M1 (ether O-glucuronide) and M2 (ester acylglucuronide), were formed with a similar affinity (apparent K(m) 3.53 and 5.15 mM, respectively). A phenol sulfoconjugate was also formed with a higher affinity (K(m) 0.53 mM). Identification of the UDP-glucuronosyltransferase (UGT) isoforms involved in FA glucuronidation was investigated with 12 human recombinant enzymes. FA was mainly glucuronidated by UGT1A isoforms and by UGT2B7. UGT1A4, 2B4, 2B15 and 2B17 failed to glucuronidate the substance. Examination of the kinetic constants revealed that FA was mainly glucuronidated by UGT1A1 at the two nucleophilic groups. UGT1A3 was able to glucuronidate these two positions with the same, but low, efficiency. UGT1A6 and 1A8 were involved in the formation of the ether glucuronide only, whereas UGT1A7, 1A10 and 2B7 preferentially glucuronidated the carboxyl group. Moreover, octyl gallate, a marker substrate of UGT1A1, competitively inhibited FA glucuronidation mediated by this isoform. Altogether, the results suggest that FA glucuronidation is primarily mediated by UGT1A1.

  17. Effects of variant UDP-glucuronosyltransferase 1A1 gene, glucose-6-phosphate dehydrogenase deficiency and thalassemia on cholelithiasis

    Science.gov (United States)

    Huang, Yang-Yang; Huang, Ching-Shui; Yang, Sien-Sing; Lin, Min-Shung; Huang, May-Jen; Huang, Ching-Shan

    2005-01-01

    AIM: To test the hypothesis that the variant UDP-glucuronosyltransferase 1A1 (UGT1A1) gene, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and thalassemia influence bilirubin metabolism and play a role in the development of cholelithiasis. METHODS: A total of 372 Taiwan Chinese with cholelithiasis who had undergone cholecystectomy and 293 healthy individuals were divided into case and control groups, respectively. PCR and restriction fragment length polymorphism were used to analyze the promoter area and nucleotides 211, 686, 1 091, and 1 456 of the UGT1A1 gene for all subjects and the gene variants for thalassemia and G6PD deficiency. RESULTS: Variation frequencies for the cholelithiasis patients were 16.1%, 25.8%, 5.4%, and 4.3% for A(TA)6 TAA/A(TA)7TAA (6/7), heterozygosity within the coding region, compound heterozygosity, and homozygosity of the UGT1A1 gene, respectively. Comparing the case and control groups, a statistically significant difference in frequency was demonstrated for the homozygous variation of the UGT1A1 gene (P = 0.012, χ2 test), but not for the other variations. Further, no difference was demonstrated in a between-group comparison of the incidence of G6PD deficiency and thalassemia (2.7% vs 2.4% and 5.1% vs 5.1%, respectively). The bilirubin levels for the cholelithiasis patients with the homozygous variant-UGT1A1 gene were significantly different from the control analog (18.0 ± 6.5 and 12.7 ± 2.9 μmol/L, respectively; Pcholelithiasis in Taiwan Chinese. PMID:16237771

  18. Importance of UDP-glucuronosyltransferase 1A1 expression in skin and its induction by UVB in neonatal hyperbilirubinemia.

    Science.gov (United States)

    Sumida, Kyohei; Kawana, Makiko; Kouno, Emi; Itoh, Tomoo; Takano, Shuhei; Narawa, Tomoya; Tukey, Robert H; Fujiwara, Ryoichi

    2013-11-01

    UDP-glucuronosyltransferase (UGT) 1A1 is the sole enzyme that can metabolize bilirubin. Human infants physiologically develop hyperbilirubinemia as the result of inadequate expression of UGT1A1 in the liver. Although phototherapy using blue light is effective in preventing jaundice, sunlight has also been suggested, but without conclusive evidence, to reduce serum bilirubin levels. We investigated the mRNA expression pattern of human UGT1A1 in human skin, human skin keratinocyte (HaCaT) cells, and skin of humanized UGT1 mice. The effects of UVB irradiation on the expression of UGT1A1 in the HaCaT cells were also examined. Multiple UGT1A isoforms, including UGT1A1, were expressed in human skin and HaCaT cells. When HaCaT cells were treated with UVB-exposed tryptophan, UGT1A1 mRNA and activity were significantly induced. Treatment of the HaCaT cells with 6-formylindolo[3,2-b]carbazole, which is one of the tryptophan derivatives formed by UVB, resulted in an induction of UGT1A1 mRNA and activity. In neonates, the expression of UGT1A1 was greater in the skin; in adults, UGT1A1 was expressed mainly in the liver. Treatment of humanized UGT1 mice with UVB resulted in a reduction of serum bilirubin levels, along with increased UGT1A1 expression and activity in the skin. Our data revealed a protective role of UGT1A1 expressed in the skin against neonatal hyperbilirubinemia. Sunlight, a natural and free source of light, makes it possible to treat neonatal jaundice while allowing mothers to breast-feed neonates.

  19. Altered UDP-glucuronosyltransferase and sulfotransferase expression and function during progressive stages of human nonalcoholic fatty liver disease.

    Science.gov (United States)

    Hardwick, Rhiannon N; Ferreira, Daniel W; More, Vijay R; Lake, April D; Lu, Zhenqiang; Manautou, Jose E; Slitt, Angela L; Cherrington, Nathan J

    2013-03-01

    The UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) represent major phase II drug-metabolizing enzymes that are also responsible for maintaining cellular homeostasis by metabolism of several endogenous molecules. Perturbations in the expression or function of these enzymes can lead to metabolic disorders and improper management of xenobiotics and endobiotics. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Because the liver plays a central role in the metabolism of xenobiotics, the purpose of the current study was to determine the effect of human NAFLD progression on the expression and function of UGTs and SULTs in normal, steatosis, NASH (fatty), and NASH (not fatty/cirrhosis) samples. We identified upregulation of UGT1A9, 2B10, and 3A1 and SULT1C4 mRNA in both stages of NASH, whereas UGT2A3, 2B15, and 2B28 and SULT1A1, 2B1, and 4A1 as well as 3'-phosphoadenosine-5'-phosphosulfate synthase 1 were increased in NASH (not fatty/cirrhosis) only. UGT1A9 and 1A6 and SULT1A1 and 2A1 protein levels were decreased in NASH; however, SULT1C4 was increased. Measurement of the glucuronidation and sulfonation of acetaminophen (APAP) revealed no alterations in glucuronidation; however, SULT activity was increased in steatosis compared with normal samples, but then decreased in NASH compared with steatosis. In conclusion, the expression of specific UGT and SULT isoforms appears to be differentially regulated, whereas sulfonation of APAP is disrupted during progression of NAFLD.

  20. Hepatic UDP-glucuronosyltransferase enzyme activity decreased during salt acclimation of sea lamprey juveniles from Minho river basin

    Directory of Open Access Journals (Sweden)

    Marta Candeias

    2015-11-01

    Full Text Available Sea lamprey (Petromyzon marinus L., 1758 is an anadromous species which migrates twice during its life cycle between freshwater and seawater. During downstream migration, the juveniles are subject to salinity changes ranging between 0 and 35. The aim of this study was to evaluate the influence of salinity in hepatic biomarkers of stress and biotransformation of juveniles from Minho river basin, Portugal, during trophic migration. Sampled juveniles (macrophthalmia were transported alive to the laboratory and maintained in 200 L tanks with LSS life support system. The specimens were separated in three groups of five pools (#8: i macrophthalmia, salinity 0 for 7 days; ii macrophthalmia, salinity 0 for 30 days and, iii macrophthalmia subjected to a salt gradient up to salinity 35, for 30 days. At final of the experiments the weight and the length of specimens were determined. Microsomes and cytosol obtained by centrifugation of liver homogenates were used for fluorimetric quantification of glutathione (GSH, glutathione disulphide (GSSG and malondialdehyde (MDA and, spectrophotometric determination of catalase (CTT1 and UDP-glucuronosyltransferase (UDPGT activities. Stats include ANOVA I and Duncan test. The results showed that salt exposure of the animals caused a decrease in the body weight and condition factor (K without changes the hepatic somatic index (p <0.05. It was also observed a decrease in the GSH/GSSG ratio and UDPGT activity, markers of oxidative stress and loss of biotransformation capacity of liver macrophthalmia (p <0.05. This disturbances may jeopardize the success of sea lamprey trophic migration, if occur a permanent or accidental exposure of juveniles to organic pollutants on the path to the sea. In addition, the salt exposure did not change the liver cytosolic MDA content (p <0.05. The significant increase in CTT1 activity may have contributed to prevent hepatic oxidative damages in the sea lamprey juveniles.

  1. Quantitative Atlas of Cytochrome P450, UDP-Glucuronosyltransferase, and Transporter Proteins in Jejunum of Morbidly Obese Subjects.

    Science.gov (United States)

    Miyauchi, Eisuke; Tachikawa, Masanori; Declèves, Xavier; Uchida, Yasuo; Bouillot, Jean-Luc; Poitou, Christine; Oppert, Jean-Michel; Mouly, Stéphane; Bergmann, Jean-François; Terasaki, Tetsuya; Scherrmann, Jean-Michel; Lloret-Linares, Célia

    2016-08-01

    Protein expression levels of drug-metabolizing enzymes and transporters in human jejunal tissues excised from morbidly obese subjects during gastric bypass surgery were evaluated using quantitative targeted absolute proteomics. Protein expression levels of 15 cytochrome P450 (CYP) enzymes, 10 UDP-glucuronosyltransferase (UGT) enzymes, and NADPH-P450 reductase (P450R) in microsomal fractions from 28 subjects and 49 transporters in plasma membrane fractions from 24 of the same subjects were determined using liquid chromatography-tandem mass spectrometry. Based on average values, UGT1A1, UGT2B15, UGT2B17, SGLT1, and GLUT2 exhibited high expression levels (over 10 fmol/μg protein), though UGT2B15 expression was detected at a high level in only one subject. CYP2C9, CYP2D6, CYP3A5, UGT1A6, P450R, ABCG2, GLUT5, PEPT1, MCT1, 4F2 cell-surface antigen heavy chain (4F2hc), LAT2, OSTα, and OSTβ showed intermediate levels (1-10 fmol/μg protein), and CYP1A1, CYP1A2, CYP1B1, CYP2C18, CYP2C19, CYP2J2, CYP3A7, CYP4A11, CYP51A1, UGT1A3, UGT1A4, UGT1A8, UGT2B4, ABCC1, ABCC4, ABCC5, ABCC6, ABCG8, TAUT, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OCTN1, CNT2, PCFT, MCT4, GLUT4, and SLC22A18 showed low levels (less than 1 fmol/μg protein). The greatest interindividual difference (364-fold) was detected for UGT2B17. However, differences in expression levels of other quantified UGTs (except UGT2B15 and UGT2B17), CYPs (except CYP1A1 and CYP3A5), and P450R, and all quantified transporters, were within 10-fold. Expression levels of CYP1A2 and GLUT4 were significantly correlated with body-mass index. The levels of 4F2hc showed significant gender differences. Smokers showed increased levels of UGT1A1 and UGT1A3. These findings provide a basis for understanding the changes in molecular mechanisms of jejunal metabolism and transport, as well as their interindividual variability, in morbidly obese patients.

  2. Identification of Human UDP-Glucuronosyltransferase 1A4 as the Major Isozyme Responsible for the Glucuronidation of 20(S)-Protopanaxadiol in Human Liver Microsomes.

    Science.gov (United States)

    Li, Jia; He, Chunyong; Fang, Lianxiang; Yang, Li; Wang, Zhengtao

    2016-03-09

    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.

  3. Oligomerization of the UDP-glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation.

    Science.gov (United States)

    Operaña, Theresa N; Tukey, Robert H

    2007-02-16

    UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of beta-d-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.

  4. Glucuronidation of zearalenone, zeranol and four metabolites in vitro: formation of glucuronides by various microsomes and human UDP-glucuronosyltransferase isoforms.

    Science.gov (United States)

    Pfeiffer, Erika; Hildebrand, Andreas; Mikula, Hannes; Metzler, Manfred

    2010-10-01

    Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α-zearalanol (α-ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α-ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP-glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14-hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α-ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.

  5. Effect of dietary eugenol on xenobiotic metabolism and mediation of UDP-glucuronosyltransferase and cytochrome P450 1A1 expression in rat liver.

    Science.gov (United States)

    Iwano, Hidetomo; Ujita, Wakako; Nishikawa, Miyu; Ishii, Satomi; Inoue, Hiroki; Yokota, Hiroshi

    2014-03-01

    Xenobiotic-metabolizing enzymes (XMEs) play an important role in the elimination and detoxification of xenobiotics and drugs. A variety of natural dietary agents are known to protect against cancer by inducing XME. To elucidate the molecular mechanism of XME induction, we examined the effect of dietary eugenol (4-allyl-1-hydroxy-2-methoxybenzene) on xenobiotic metabolism. In this study, rats were administered dietary eugenol for 4 weeks to investigate the various effects of UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) expression. In rats administered dietary eugenol, expression levels of hepatic CYP1A 1 were reduced to 40% than of the controls, while expression of hepatic UGT1A6, UGT1A7 and UGT2B1 increased to 2-3 times than observed in the controls. Hepatic protein levels of UGT1A6 and 2B1 were also elevated in the eugenol-treated rats. These results suggest that the natural compound eugenol improves the xenobiotic-metabolizing systems that suppress and induce the expression of CYP1A1 and UGT, respectively.

  6. Glucuronidation of drugs in humanized UDP-glucuronosyltransferase 1 mice: Similarity with glucuronidation in human liver microsomes.

    Science.gov (United States)

    Kutsuno, Yuki; Sumida, Kyohei; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2013-10-01

    Uridine 5'-diphosphate-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various endogenous and exogenous substrates. Among 19 functional human UGTs, UGT1A family enzymes largely contribute to the metabolism of clinically used drugs. While the UGT1A locus is conserved in mammals such as humans, mice, and rats, species differences in drug glucuronidation have been reported. Recently, humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) have been developed. To evaluate the usefulness of hUGT1 mice to predict human glucuronidation of drugs, UGT activities, and inhibitory effects on UGTs were examined in liver microsomes of hUGT1 mice as well as in those of wild-type mice and humans. Furosemide acyl-glucuronidation was sigmoidal and best fitted to the Hill equation in hUGT1 mice and human liver microsomes, while it was fitted to the substrate inhibition equation in mouse liver microsomes. Kinetic parameters of furosemide glucuronidation were very similar between hUGT1 mice and human liver microsomes. The kinetics of S-naproxen acyl-glucuronidation and inhibitory effects of compounds on furosemide glucuronidation in hUGT1 liver microsomes were also slightly, but similar to those in human liver microsomes, rather than in wild-type mice. While wild-type mice lack imipramine and trifluoperazine N-glucuronidation potential, hUGT1 mice showed comparable N-glucuronidation activity to that of humans. Our data indicate that hUGT1 mice are promising tools to predict not only in vivo human drug glucuronidation but also potential drug-drug interactions.

  7. Coupling of UDP-glucuronosyltransferases and multidrug resistance-associated proteins is responsible for the intestinal disposition and poor bioavailability of emodin

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Wei; Feng, Qian; Li, Ye; Ye, Ling [Department of Pharmaceutics, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong (China); Hu, Ming, E-mail: mhu@uh.edu [Department of Pharmaceutics, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong (China); Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 1441 Moursund Street, Houston, TX 77030 (United States); Liu, Zhongqiu, E-mail: liuzq@smu.edu.cn [Department of Pharmaceutics, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong (China)

    2012-12-15

    Emodin is a poorly bioavailable but promising plant-derived anticancer drug candidate. The low oral bioavailability of emodin is due to its extensive glucuronidation in the intestine and liver. Caco-2 cell culture model was used to investigate the interplay between UDP-glucuronosyltransferases (UGTs) and efflux transporters in the intestinal disposition of emodin. Bidirectional transport assays of emodin at different concentrations were performed in the Caco-2 monolayers with or without multidrug resistance-associated protein (MRP) and breast cancer resistance protein (BCRP) efflux transporter chemical inhibitors. The bidirectional permeability of emodin and its glucuronide in the Caco-2 monolayers was determined. Emodin was rapidly metabolized to emodin glucuronide in Caco-2 cells. LTC4, a potent inhibitor of MRP2, decreased the efflux of emodin glucuronide and also substantially increased the intracellular glucuronide level in the basolateral-to-apical (B–A) direction. MK-571, chemical inhibitor of MRP2, MRP3, and MRP4, significantly reduced the efflux of glucuronide in the apical-to-basolateral (A–B) and B–A directions in a dose-dependent manner. However, dipyridamole, a BCRP chemical inhibitor demonstrated no effect on formation and efflux of emodin glucuronide in Caco-2 cells. In conclusion, UGT is a main metabolic pathway for emodin in the intestine, and the MRP family is composed of major efflux transporters responsible for the excretion of emodin glucuronide in the intestine. The coupling of UGTs and MRP efflux transporters causes the extensive metabolism, excretion, and low bioavailability of emodin. -- Highlights: ► Glucuronidation is the main reason for the poor oral bioavailability of emodin. ► Efflux transporters are involved in the excretion of emodin glucuronide. ► The intestine is the main organ for metabolism of emodin.

  8. Herb–drug interaction prediction based on the high specific inhibition of andrographolide derivatives towards UDP-glucuronosyltransferase (UGT) 2B7

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Hai-Ying, E-mail: cmu4h-mhy@126.com [The Fourth Affiliated Hospital of China Medical University, Shenyang 110032 (China); Sun, Dong-Xue [School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016 (China); Cao, Yun-Feng [The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); Joint Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Zhongshan Road, Dalian 116023 (China); Ai, Chun-Zhi [Joint Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Zhongshan Road, Dalian 116023 (China); Qu, Yan-Qing [Thyroid Surgery, Yantaishan Hospital, Yantai, Shandong (China); Hu, Cui-Min [Joint Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Zhongshan Road, Dalian 116023 (China); Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057 (United States); Jiang, Changtao [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892 (United States); Dong, Pei-Pei [Academy of Integrative Medicine, Dalian Medical University, Dalian 116044 (China); Sun, Xiao-Yu; Hong, Mo [Joint Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Zhongshan Road, Dalian 116023 (China); Tanaka, Naoki; Gonzalez, Frank J. [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892 (United States); and others

    2014-05-15

    Herb–drug interaction strongly limits the clinical application of herbs and drugs, and the inhibition of herbal components towards important drug-metabolizing enzymes (DMEs) has been regarded as one of the most important reasons. The present study aims to investigate the inhibition potential of andrographolide derivatives towards one of the most important phase II DMEs UDP-glucuronosyltransferases (UGTs). Recombinant UGT isoforms (except UGT1A4)-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation were employed to firstly screen the andrographolide derivatives' inhibition potential. High specific inhibition of andrographolide derivatives towards UGT2B7 was observed. The inhibition type and parameters (K{sub i}) were determined for the compounds exhibiting strong inhibition capability towards UGT2B7, and human liver microsome (HLMs)-catalyzed zidovudine (AZT) glucuronidation probe reaction was used to furtherly confirm the inhibition behavior. In combination of inhibition parameters (K{sub i}) and in vivo concentration of andrographolide and dehydroandrographolide, the potential in vivo inhibition magnitude was predicted. Additionally, both the in vitro inhibition data and computational modeling results provide important information for the modification of andrographolide derivatives as selective inhibitors of UGT2B7. Taken together, data obtained from the present study indicated the potential herb–drug interaction between Andrographis paniculata and the drugs mainly undergoing UGT2B7-catalyzed metabolic elimination, and the andrographolide derivatives as potential candidates for the selective inhibitors of UGT2B7. - Highlights: • Specific inhibition of andrographolide derivatives towards UGT2B7. • Herb-drug interaction related withAndrographis paniculata. • Guidance for design of UGT2B7 specific inhibitors.

  9. The glucuronidation of R- and S-lorazepam: human liver microsomal kinetics, UDP-glucuronosyltransferase enzyme selectivity, and inhibition by drugs.

    Science.gov (United States)

    Uchaipichat, Verawan; Suthisisang, Chuthamanee; Miners, John O

    2013-06-01

    The widely used hypnosedative-anxiolytic agent R,S-lorazepam is cleared predominantly by conjugation with glucuronic acid in humans, but the enantioselective glucuronidation of lorazepam has received little attention. The present study characterized the kinetics of the separate R and S enantiomers of lorazepam by human liver microsomes (HLMs) and by a panel of recombinant human UDP-glucuronosyltransferase (UGT) enzymes. Respective mean K(m) and V(max) values for R- and S-lorazepam glucuronidation by HLM were 29 ± 8.9 and 36 ± 10 µM, and 7.4 ± 1.9 and 10 ± 3.8 pmol/min ⋅ mg. Microsomal intrinsic clearances were not significantly different, suggesting the in vivo clearances of R- and S-lorazepam are likely to be similar. Both R- and S-lorazepam were glucuronidated by UGT2B4, 2B7, and 2B15, whereas R-lorazepam was additionally metabolized by the extrahepatic enzymes UGT1A7 and 1A10. Based on in vitro clearances and consideration of available in vivo and in vitro data, UGT2B15 is likely to play an important role in the glucuronidation of R- and S-lorazepam. However, the possible contribution of other enzymes and the low activities observed in vitro indicate that the lorazepam enantiomers are of limited use as substrate probes for UGT2B15. To identify potential drug-drug interactions, codeine, fluconazole, ketamine, ketoconazole, methadone, morphine, valproic acid, and zidovudine were screened as inhibitors of R- and S-lorazepam glucuronidation by HLM. In vitro-in vivo extrapolation suggested that, of these drugs, only ketoconazole had the potential to inhibit lorazepam clearance to a clinically significant extent.

  10. Accurate identification of UDP-glucuronosyltransferase 1A1 (UGT1A1) inhibitors using UGT1A1-overexpressing HeLa cells.

    Science.gov (United States)

    Sun, Hua; Zhou, Xiaotong; Wu, Baojian

    2015-01-01

    1. UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an irreplaceable role in detoxification of bilirubin and many drugs (e.g., SN-38). Here we aimed to explore the potential of UGT1A1-overexpressing HeLa cells (or HeLa1A1 cells) as a tool to accurately identify UGT1A1 inhibitors. 2. Determination of glucuronidation rates (β-estradiol and SN-38 as the substrates) was performed using HeLa1A1 cells and uridine diphosphoglucuronic acid (UDPGA)-supplemented cDNA expressed UGT1A1 enzyme (or microsomes). The inhibitory effects (IC50 values) of 20 structurally diverse compounds on the UGT1A1 activity were determined using HeLa1A1 cells and microsomal incubations. 3. In HeLa1A1 cells, the IC50 values for inhibition of β-estradiol glucuronidation by the tested compounds ranged from 0.33 to 94.6 µM. In the microsomal incubations, the IC50 values ranged from 0.47 to 155 µM. It was found that the IC50 values of all test compounds derived from the cells were well consistent with those from the microsomes (deviated by less than two-fold). Further, the IC50 values from the cells were strongly correlated with those from microsomes (r = 0.944, p HeLa cells were an appropriate tool to accurately depict the inhibition profiles of chemicals against UGT1A1.

  11. Developmental onset of bilirubin-induced neurotoxicity involves Toll-like receptor 2-dependent signaling in humanized UDP-glucuronosyltransferase1 mice.

    Science.gov (United States)

    Yueh, Mei-Fei; Chen, Shujuan; Nguyen, Nghia; Tukey, Robert H

    2014-02-21

    Biological and signaling events that connect developmentally induced hyperbilirubinemia to bilirubin-induced neurological dysfunction (BIND) and CNS toxicity in humans are poorly understood. In mammals, UDP-glucuronosyltransferase 1A1 (UGT1A1) is the sole enzyme responsible for bilirubin glucuronidation, a rate-limiting step necessary for bilirubin metabolism and clearance. Humanized mice that express the entire UGT1 locus (hUGT1) and the UGT1A1 gene, develop neonatal hyperbilirubinemia, with 8-10% of hUGT1 mice succumbing to CNS damage, a phenotype that is presented by uncontrollable seizures. We demonstrate that neuroinflammation and reactive gliosis are prominent features of bilirubin brain toxicity, and a disturbed redox status resulting from activation of NADPH oxidase is an important contributing mechanism found in BIND. Using knock-out mice and primary brain cells, we connect a key pattern recognition receptor, Toll-like receptor 2 (TLR2), to hyperbilirubinemia-induced signaling. We illustrate a requirement for TLR2 signaling in regulating gliosis, proinflammatory mediators, and oxidative stress when neonatal mice encounter severe hyperbilirubinemia. TLR2-mediated gliosis strongly correlates with pronounced neuroinflammation in the CNS with up-regulation of TNFα, IL-1β, and IL-6, creating a pro-inflammatory CNS environment. Gene expression and immunohistochemistry staining show that hUGT1/Tlr2(-/-) mice fail to activate glial cells, proinflammatory cytokines, and stress response genes. In addition, bilirubin-induced apoptosis was significantly enhanced by blocking TLR2 signaling indicating its anti-apoptotic property. Consequently, a higher neonatal death rate (57.1%) in hUGT1/Tlr2(-/-) mice was observed when compared with hUGT1 mice (8.7%). These results suggest that TLR2 signaling and microglia neuroinflammation are linked to a repair and/or protection mode against BIND.

  12. 3D-QSAR studies on UDP-glucuronosyltransferase 2B7 substrates using the pharmacophore and VolSurf approaches.

    Science.gov (United States)

    Ako, Roland; Dong, Dong; Wu, Baojian

    2012-09-01

    UDP-glucuronosyltransferase 2B7 (UGT2B7) is an important enzyme responsible for clearance of many drugs. Here, we report two 3D quantitative structure-activity relationship (QSAR) models for UGT2B7 using the pharmacophore and VolSurf approach, respectively. The dataset included 53 structurally diverse UGT2B7 substrates, 36 of which were used for the training set and 17 of which for the external test set. Pharmacophore-based 3D-QSAR model (or hypothesis) was developed using the Discovery Studio program. A user-defined "glucuronidation site" feature was forcefully included in a pharmacophore hypothesis. VolSurf-based 3D-QSAR model was generated using the VolSurf program. This involves calculation of VolSurf descriptors, variable selection with the FFD algorithm, and partial least squares (PLS) analyses. The best pharmacophore model (r(2) = 0.736) consists of one glucuronidation site, one hydrogen bond acceptor, and three hydrophobic regions. Using this model, K(m) values for 14 of 17 test substrates were predicted within one log unit. The yielded VolSurf (PLS) model with two components shows statistical significance in both fitting and internal predicting (r(2) = 0.866, q(2) = 0.728). Further, the K(m) values for all test substrates were predicted within one log unit. In addition, the VolSurf model reveals an overlay of chemical features influencing the enzyme-substrate binding. Those include molecular size and shape, integy moments, capacity factors, best volumes of DRY probe, H-bonding, and log P. In conclusion, the pharmacophore and VolSurf approaches are successfully utilized to establish predictive models for UGT2B7. The derived models should be an efficient tool for high throughput prediction of UGT2B7 metabolism.

  13. Effects of aqueous extract of Ruta graveolens and its ingredients on cytochrome P450, uridine diphosphate (UDP-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate (NAD(PH-quinone oxidoreductase in mice

    Directory of Open Access Journals (Sweden)

    Yune-Fang Ueng

    2015-09-01

    Full Text Available Ruta graveolens (the common rue has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes, effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP, uridine diphosphate (UDP-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate (NAD(PH:quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(PH:quinone oxidoreductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (Fmix increased Cyp2b activities in males. The mixture of rutin and Fmix increased Cyp1a and Cyp2b activities. These results revealed that rutin and Fmix contributed at least in part to the P450 induction by rue.

  14. Protein kinase Cα and Src kinase support human prostate-distributed dihydrotestosterone-metabolizing UDP-glucuronosyltransferase 2B15 activity.

    Science.gov (United States)

    Chakraborty, Sunit K; Basu, Nikhil K; Jana, Sirsendu; Basu, Mousumi; Raychoudhuri, Amit; Owens, Ida S

    2012-07-13

    Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17β-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70-100 and 80-90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130-140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D(3) enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-(33)P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55-58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated

  15. Characterization of niflumic acid as a selective inhibitor of human liver microsomal UDP-glucuronosyltransferase 1A9: application to the reaction phenotyping of acetaminophen glucuronidation.

    Science.gov (United States)

    Miners, John O; Bowalgaha, Kushari; Elliot, David J; Baranczewski, Pawel; Knights, Kathleen M

    2011-04-01

    Enzyme selective inhibitors represent the most valuable experimental tool for reaction phenotyping. However, only a limited number of UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors have been identified to date. This study characterized the UGT enzyme selectivity of niflumic acid (NFA). It was demonstrated that 2.5 μM NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentrations (50-100 μM) inhibited UGT1A1 and UGT2B15 but had little effect on the activities of UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B17. NFA inhibited 4-methylumbelliferone and propofol (PRO) glucuronidation by recombinant UGT1A9 and PRO glucuronidation by human liver microsomes (HLM) according to a mixed (competitive-noncompetitive) mechanism, with K(i) values ranging from 0.10 to 0.40 μM. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver microsomal UGT1A1 (K(i) range 14-18 μM), whereas competitive inhibition (K(i) 62 μM) was observed with UGT2B15. NFA was subsequently applied to the reaction phenotyping of human liver microsomal acetaminophen (APAP) glucuronidation. Consistent with previous reports, APAP was glucuronidated by recombinant UGT1A1, UGT1A6, UGT1A9, and UGT2B15. NFA concentrations in the range of 2.5 to 100 μM inhibited APAP glucuronidation by UGT1A1, UGT1A9, and UGT2B15 but not by UGT1A6. The mean V(max) for APAP glucuronidation by HLM was reduced by 20, 35, and 40%, respectively, in the presence of 2.5, 50, and 100 μM NFA. Mean K(m) values decreased in parallel with V(max), although the magnitude of the decrease was smaller. Taken together, the NFA inhibition data suggest that UGT1A6 is the major enzyme involved in APAP glucuronidation.

  16. Isoform selectivity and kinetics of morphine 3- and 6-glucuronidation by human udp-glucuronosyltransferases: evidence for atypical glucuronidation kinetics by UGT2B7.

    Science.gov (United States)

    Stone, Andrew N; Mackenzie, Peter I; Galetin, Aleksandra; Houston, J Brian; Miners, John O

    2003-09-01

    Morphine elimination involves UDP-glucuronosyltransferase (UGT) catalyzed conjugation with glucuronic acid to form morphine 3- and 6-glucuronides (M3G and M6G, respectively). It has been proposed that UGT2B7 is the major enzyme involved in these reactions, but there is evidence to suggest that other isoforms also catalyze morphine glucuronidation in man. Thus, we have characterized the selectivity and kinetics of M3G and M6G formation by recombinant human UGTs. UGT 1A1, 1A3, 1A6, 1A8, 1A9, 1A10, and 2B7 all catalyzed M3G formation, but only UGT2B7 formed M6G. The kinetics of M3G formation by the UGT1A family isoforms was consistent with a single enzyme Michaelis-Menten model, with apparent Km values ranging from 2.6 to 37.4 mM. In contrast, M3G and M6G formation by UGT2B7 exhibited atypical kinetics. The atypical kinetics may be described by a model with high- and low-affinity Km values (0.42 and 8.3 mM for M3G, and 0.97 and 7.4 mM for M6G) from fitting to a biphasic Michaelis-Menten model. However, a multisite model with an interaction between two identical binding sites in a negative cooperative manner provides a more realistic approach to modeling these data. According to this model, the respective binding affinities (Ks) for M3G and M6G were 1.76 and 1.41 mM, respectively. These data suggest that M6G formation may be used as a selective probe for UGT2B7 activity, and morphine glucuronidation by UGT2B7 appears to involve the simultaneous binding of two substrate molecules, highlighting the need for careful analysis of morphine glucuronidation kinetics in vitro.

  17. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    Science.gov (United States)

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined.

  18. INTRODUCTION OF AN N-GLYCOSYLATION SITE INTO UDP-GLUCURONOSYLTRANSFERASE 2B3 ALTERS ITS SENSITIVITY TO CYTOCHROME P450 3A1-DEPENDENT MODULATION

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    Tatsuro Nakamura

    2016-11-01

    Full Text Available Our previous studies have demonstrated functional protein-protein interactions between cytochrome P450 (CYP 3A and UDP-glucuronosyltransferase (UGT. However, the role of carbohydrate chains of UGTs in the interaction with CYP is not well understood. To address this issue, we examined whether CYP3A1 modulates the function of UGT2B3 which lacks potential glycosylation sites. We also examined whether the introduction of N-glycosylation to UGT2B3 affects CYP3A-dependent modulation of UGT function. To introduce a potential glycosylation site into UGT2B3, Ser 316 of UGT2B3 was substituted with Asn by site-directed mutagenesis. A baculovirus-Sf-9 cell system for expressing CYP3A1 and UGT2B3/UGT2B3(S316N was established using a Bac-to-Bac system. Glycosylation of UGT2B3(S316N was demonstrated in this expression system. The microsomal activity of recombinant UGT was determined using 4-methylumbelliferone as a substrate. The effect of CYP3A1 co-expression on UGT function was examined by comparing the kinetic profiles between single (UGT alone and double expression (UGT plus CYP systems. The kinetics of the two expression systems fitted a Michaelis-Menten equation. When the 4-MU concentration was varied, co-expression of CYP3A1 lowered the Vmax of UGT2B3-mediated conjugation. Conversely, for UGT2B3(S316N, the Vmax in the dual expression system was higher than that in the single expression system. The data obtained demonstrate that the introduction of N-glycosylation to UGT2B3 alters its sensitivity to CYP3A1-dependent modulation while CYP3A1 enhanced UGT2B3(S316N activity, and wild-type UGT2B3 was suppressed by CYP3A1. These data suggest that N-glycosylation of UGT is one of the determinants regulating the interaction between CYP3A and UGT.

  19. 尿苷二磷酸葡醛酸转移酶的研究进展%Progress of UDP-glucuronosyltransferases

    Institute of Scientific and Technical Information of China (English)

    崔冬雪

    2013-01-01

    UDP-glucuronosyltransferases (UGT) are the most important phase Ⅱ drug metabolizing enzyme, which can metabolize not only various endogenous substances, such as bilirubin, steroid hormones, thyroid hormones, bile acids and fat-soluble vitamins, but also many drugs, such as opioids, analgesics, NSAID and anticonvulsants. UGT plays an important role in drug absorbtion, metabolism, distribution and excretion. Furthermore, the inhibition or induction of UGT could not only result in serious drug-drug interactions, but also induce metabolic disorders of endogenous substances , for which evaluation of the inhibitory or induction effects of compounds on UGT is very important in clinic. This paper reviewed UGT in terms of its classification, tissue distribution, effect on drug absorption, gene polymorphism and related drug-drug interactions.%尿苷二磷酸葡醛酸转移酶(UGT)是体内最重要的Ⅱ相代谢酶,它可以参与许多内源性物质如胆红素、甾体激素、甲状腺激素、胆汁酸和脂溶性维生素等的代谢,在许多药物如阿片类药物、镇痛药、非甾体抗炎药和抗惊厥药等的代谢中也发挥着重要的作用.UGT在药物的吸收、分布、代谢和排泄中发挥重要作用.研究UGT特别是其基因多态性及其介导的药物-药物相互作用不仅可以指导临床用药,也可以揭示内源性物质代谢紊乱的机制.本文就UGT的分类、组织分布、对药物吸收的影响、基因多态性及其所介导的药物-药物相互作用进行综述.

  20. Bilirubin UDP-Glucuronosyltransferase 1A1 (UGT1A1) Gene Promoter Polymorphisms and HPRT, Glycophorin A, and Micronuclei Mutant Frequencies in Human Blood

    Energy Technology Data Exchange (ETDEWEB)

    Grant, D; Hall, I J; Eastmond, D; Jones, I M; Bell, D A

    2004-10-06

    A dinucleotide repeat polymorphism (5-, 6-, 7-, or 8-TA units) has been identified within the promoter region of UDP-glucuronosyltransferase 1A1 gene (UGT1A1). The 7-TA repeat allele has been associated with elevated serum bilirubin levels that cause a mild hyperbilirubinemia (Gilbert's syndrome). Studies suggest that promoter transcriptional activity of UGT1A1 is inversely related to the number of TA repeats and that unconjugated bilirubin concentration increases directly with the number of TA repeat elements. Because bilirubin is a known antioxidant, we hypothesized that UGT1A1 repeats associated with higher bilirubin may be protective against oxidative damage. We examined the effect of UGT1A1 genotype on somatic mutant frequency in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene in human lymphocytes and the glycophorin A (GPA) gene of red blood cells (both N0, NN mutants), and the frequency of lymphocyte micronuclei (both kinetochore (K) positive or micronuclei K negative) in 101 healthy smoking and nonsmoking individuals. As hypothesized, genotypes containing 7-TA and 8-TA displayed marginally lower GPA{_}NN mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). In contrast, our analysis showed that lower expressing UGT1A1 alleles (7-TA and 8-TA) were associated with modestly increased HPRT mutation frequency (p<0.05) while the same low expression genotypes were not significantly associated with micronuclei frequencies (K-positive or K-negative) when compared to high expression genotypes (5-TA and 6-TA). We found weak evidence that UGT1A1 genotypes containing 7-TA and 8-TA were associated with increased GPA{_}N0 mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). These data suggest that UGT1A1 genotype may modulate somatic mutation of some types, in some cell lineages, by a mechanism not involving bilirubin antioxidant activity. More detailed studies examining UGT1A1 promoter variation, oxidant/antioxidant balance and

  1. Glucuronidation of edaravone by human liver and kidney microsomes: biphasic kinetics and identification of UGT1A9 as the major UDP-glucuronosyltransferase isoform.

    Science.gov (United States)

    Ma, Liping; Sun, Jianguo; Peng, Ying; Zhang, Rong; Shao, Feng; Hu, Xiaoling; Zhu, Jianping; Wang, Xiaojin; Cheng, Xuefang; Zhu, Yinci; Wan, Ping; Feng, Dong; Wu, Hui; Wang, Guangji

    2012-04-01

    Edaravone was launched in Japan in 2001 and was the first neuroprotectant developed for the treatment of acute cerebral infarction. Edaravone is mainly eliminated as glucuronide conjugate in human urine (approximately 70%), but the mechanism involved in the elimination pathway remains unidentified. We investigated the glucuronidation of edaravone in human liver microsomes (HLM) and human kidney microsomes (HKM) and identified the major hepatic and renal UDP-glucuronosyltransferases (UGTs) involved. As we observed, edaravone glucuronidation in HLM and HKM exhibited biphasic kinetics. The intrinsic clearance of glucuronidation at high-affinity phase (CL(int1)) and low-affinity phase (CL(int2)) were 8.4 ± 3.3 and 1.3 ± 0.2 μl · min(-1) · mg(-1), respectively, for HLM and were 45.3 ± 8.2 and 1.8 ± 0.1 μl · min(-1) · mg(-1), respectively, for HKM. However, in microsomal incubations contained with 2% bovine serum albumin, CL(int1) and CL(int2) were 16.4 ± 1.2 and 3.7 ± 0.3 μl · min(-1) · mg(-1), respectively, for HLM and were 78.5 ± 3.9 and 3.6 ± 0.5 μl · min(-1) · mg(-1), respectively, for HKM. Screening with 12 recombinant UGTs indicated that eight UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B17) produced a significant amount of glucuronide metabolite. Thus, six UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A9, UGT2B7, and UGT2B17) expressed in human liver or kidney were selected for kinetic studies. Among them, UGT1A9 exhibited the highest activity (CL(int1) = 42.4 ± 9.5 μl · min(-1) · mg(-1)), followed by UGT2B17 (CL(int) = 3.3 ± 0.4 μl · min(-1) · mg(-1)) and UGT1A7 (CL(int) = 1.7 ± 0.2 μl · min(-1) · mg(-1)). Inhibition study found that inhibitor of UGT1A9 (propofol) attenuated edaravone glucuronidation in HLM and HKM. In addition, edaravone glucuronidation in a panel of seven HLM was significantly correlated (r = 0.9340, p = 0.0021) with propofol glucuronidation. Results indicated that UGT1A9 was the main UGT isoform

  2. UDP-glucuronosyltransferase-mediated metabolic activation of the tobacco carcinogen 2-amino-9H-pyrido[2,3-b]indole.

    Science.gov (United States)

    Tang, Yijin; LeMaster, David M; Nauwelaërs, Gwendoline; Gu, Dan; Langouët, Sophie; Turesky, Robert J

    2012-04-27

    2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by (1)H NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation by UGTs to form, respectively, N(2)-(β-D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N(2)-Gl) and N(2)-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON(2)-Gl). HONH-AαC also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN(2)-O-Gl). AαC-HN(2)-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-AαC adduct at 20-50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (V(max)/K(m)) at forming AαC-N(2)-Gl (0.67 μl·min(-1)·mg of protein(-1)), and UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 μl·min(-1)·mg of protein(-1)), whereas UGT1A1 was most efficient at forming AαC-HON(2)-Gl (5.0 μl·min(-1)·mg of protein(-1)). Human hepatocytes produced AαC-N(2)-Gl and AαC-HN(2)-O-Gl in abundant quantities, but AαC-HON(2)-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a

  3. Human UDP-Glucuronosyltransferase 1A1 is the Primary Enzyme Responsible for the N-glucuronidation of N-hydroxy-PhIP in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Malfatti, M A; Felton, J S

    2004-04-06

    UDP-Glucuronosyltransferase 1A proteins (UGT1A) catalyze the glucuronidation of many endogenous and xenobiotic compounds including heterocyclic amines and their hydroxylated metabolites (the main topic of this study). Studies have shown that in humans UGT1A mediated glucuronidation is an important pathway in the detoxification of food-borne carcinogenic heterocyclic amines. The biotransformation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant heterocyclic amine found in cooked meats, is highly dependent on cytochrome P4501A2 hydroxylation followed by UGT catalyzed glucuronidation of the N-hydroxy-PhIP reactive intermediate. To determine which UGT1A proteins are involved in the glucuronidation of N-hydroxy-PhIP, microsomal preparations from baculovirus infected insect cells that express all of the known functional human UGT1A isozymes (UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8, -1A9, -1A10) were exposed to N-hydroxy-PhIP and the reaction products were isolated by HPLC. All UGT1A proteins except UGT1A6 showed some degree of activity towards N-hydroxy-PhIP. The formation of both N-hydroxy-PhIP-N{sup 2}-glucuronide and N-hydroxy-PhIP-N3-glucuronide was both time and substrate concentration dependent in all the microsomal incubations that showed appreciable activity. UGT1A1 was the most efficient in converting N-hydroxy-PhIP to both conjugates producing 5 times more of the N{sup 2}-conjugate than UGT1A4, the next active UGT, and 286 times more than UGT1A7, the least active UGT. With an apparent Km of 52 {micro}M and a K{sub cat} of 114 min-1, UGT1A1 was also the most catalytically efficient in forming N-hydroxy-PhIP-N{sup 2}-glucuronide. Catalytic constants for UGT1A4, UGT1A8 and UGT1A9 were 52 min-1, 35 min{sup -1} and 3.7 min{sup -1}, respectively. The catalytic efficiency for N-hydroxy-PhIP-N3-glucuronide formation was 8, 10, and 6 times lower for UGT1A1, -1A4, and -1A8, respectively, when compared to the k{sub cat} values for N

  4. Morphine glucuronidation and glucosidation represent complementary metabolic pathways that are both catalyzed by UDP-glucuronosyltransferase 2B7: kinetic, inhibition, and molecular modeling studies.

    Science.gov (United States)

    Chau, Nuy; Elliot, David J; Lewis, Benjamin C; Burns, Kushari; Johnston, Martin R; Mackenzie, Peter I; Miners, John O

    2014-04-01

    Morphine 3-β-D-glucuronide (M3G) and morphine 6-β-D-glucuronide (M6G) are the major metabolites of morphine in humans. More recently, morphine-3-β-d-glucoside (M-3-glucoside) was identified in the urine of patients treated with morphine. Kinetic and inhibition studies using human liver microsomes (HLM) and recombinant UGTs as enzyme sources along with molecular modeling were used here to characterize the relationship between morphine glucuronidation and glucosidation. The M3G to M6G intrinsic clearance (C(Lint)) ratio (∼5.5) from HLM supplemented with UDP-glucuronic acid (UDP-GlcUA) alone was consistent with the relative formation of these metabolites in humans. The mean C(Lint) values observed for M-3-glucoside by incubations of HLM with UDP-glucose (UDP-Glc) as cofactor were approximately twice those for M6G formation. However, although the M3G-to-M6G C(Lint) ratio remained close to 5.5 when human liver microsomal kinetic studies were performed in the presence of a 1:1 mixture of cofactors, the mean C(Lint) value for M-3-glucoside formation was less than that of M6G. Studies with UGT enzyme-selective inhibitors and recombinant UGT enzymes, along with effects of BSA on morphine glycosidation kinetics, were consistent with a major role of UGT2B7 in both morphine glucuronidation and glucosidation. Molecular modeling identified key amino acids involved in the binding of UDP-GlcUA and UDP-Glc to UGT2B7. Mutagenesis of these residues abolished morphine glucuronidation and glucosidation. Overall, the data indicate that morphine glucuronidation and glucosidation occur as complementary metabolic pathways catalyzed by a common enzyme (UGT2B7). Glucuronidation is the dominant metabolic pathway because the binding affinity of UDP-GlcUA to UGT2B7 is higher than that of UDP-Glc.

  5. The Effect of UDP-glucuronosyltransferase 1A1 Expression on the Mutagenicity and Metabolism of the Cooked-Food Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4-5,b]pyridine in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Malfatti, M A; Wu, R W; Felton, J S

    2004-08-13

    UDP-glucuronosyltransferase proteins (UGT) catalyze the glucuronidation of both endogenous and xenobiotic compounds. In previous studies UGT1A1 has been implicated in the detoxification of certain food-borne-carcinogenic-heterocyclic amines. To determine the importance of UDP-glucuronosyltransferase 1A1 (UGT1A1) in the biotransformation of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), genetically modified CHO cells that are nucleotide excision repair-deficient, and express cytochrome P4501A2 (UV5P3 cell line) were transfected with a cDNA plasmid of human UGT1A1 to establish the UDPglucuronosyltransferase 1A1 expressing 5P3hUGT1A1 cell line. Expression of the UGT1A1 gene was verified by screening neogene expressing clonal isolates (G-418 resistant) for their sensitivity to cell killing from PhIP exposure. Five of eleven clones were chosen for further analysis due to their resistance to cell killing. Western blot analysis was used to confirm the presence of the UGT1A1 and CYP1A2 proteins. All five clones displayed a 52 kDa protein band, which corresponded to a UGT1A1 control protein. Only four of the clones had a protein band that corresponded to the CYP1A2 control protein. Correct fragment size of the cDNAs in the remaining 4 clones was confirmed by RT-PCR and quantification of the mRNA product was accomplished by real-time RT-PCR. Expression of UGT1A1 in the transfected cells was 10{sup 4}-10{sup 5} fold higher relative to the UV5P3 parental cells. One clone (No.14) had a 10 fold higher increase in expression at 1.47 x 10{sup 5} over the other three clones. This clone was also the most active in converting N-hydroxy-PhIP to N-hydroxy-PhIP glucuronide conjugates in microsomal metabolism assays. Based on the D{sub 50} values, the cytotoxic effect of PhIP was decreased {approx}350 fold in the 5P3hUGT1A1 cells compared to the UV5P3 control cells. In addition no significant increase in mutation frequency was observed in the

  6. A long-standing mystery solved: the formation of 3-hydroxydesloratadine is catalyzed by CYP2C8 but prior glucuronidation of desloratadine by UDP-glucuronosyltransferase 2B10 is an obligatory requirement.

    Science.gov (United States)

    Kazmi, Faraz; Barbara, Joanna E; Yerino, Phyllis; Parkinson, Andrew

    2015-04-01

    Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating long-lasting antihistamine that is widely used for the treatment of allergic rhinitis and chronic idiopathic urticaria. For over 20 years, it has remained a mystery as to which enzymes are responsible for the formation of 3-hydroxydesloratadine, the major active human metabolite, largely due to the inability of any in vitro system tested thus far to generate this metabolite. In this study, we demonstrated that cryopreserved human hepatocytes (CHHs) form 3-hydroxydesloratadine and its corresponding O-glucuronide. CHHs catalyzed the formation of 3-hydroxydesloratadine with a Km of 1.6 μM and a Vmax of 1.3 pmol/min per million cells. Chemical inhibition of cytochrome P450 (P450) enzymes in CHHs demonstrated that gemfibrozil glucuronide (CYP2C8 inhibitor) and 1-aminobenzotriazole (general P450 inhibitor) inhibited 3-hydroxydesloratadine formation by 91% and 98%, respectively. Other inhibitors of CYP2C8 (gemfibrozil, montelukast, clopidogrel glucuronide, repaglinide, and cerivastatin) also caused extensive inhibition of 3-hydroxydesloratadine formation (73%-100%). Assessment of desloratadine, amodiaquine, and paclitaxel metabolism by a panel of individual CHHs demonstrated that CYP2C8 marker activity robustly correlated with 3-hydroxydesloratadine formation (r(2) of 0.70-0.90). Detailed mechanistic studies with sonicated or saponin-treated CHHs, human liver microsomes, and S9 fractions showed that both NADPH and UDP-glucuronic acid are required for 3-hydroxydesloratadine formation, and studies with recombinant UDP-glucuronosyltransferase (UGT) and P450 enzymes implicated the specific involvement of UGT2B10 in addition to CYP2C8. Overall, our results demonstrate for the first time that desloratadine glucuronidation by UGT2B10 followed by CYP2C8 oxidation and a deconjugation event are responsible for the formation of 3-hydroxydesloratadine. Copyright © 2015 by The American

  7. In vitro inhibitory effects of non-steroidal anti-inflammatory drugs on 4-methylumbelliferone glucuronidation in recombinant human UDP-glucuronosyltransferase 1A9--potent inhibition by niflumic acid.

    Science.gov (United States)

    Mano, Yuji; Usui, Takashi; Kamimura, Hidetaka

    2006-01-01

    The inhibitory potencies of non-steroidal anti-inflammatory drugs (NSAIDs) on UDP-glucuronosyltransferase (UGT) 1A9 activity were investigated in recombinant human UGT1A9 using 4-methylumbelliferone (4-MU) as a substrate for glucuronidation. 4-MU glucuronidation (4-MUG) showed Michaelis-Menten kinetics with a Km value of 6.7 microM. The inhibitory effects of the following seven NSAIDs were investigated: acetaminophen, diclofenac, diflunisal, indomethacin, ketoprofen, naproxen and niflumic acid. Niflumic acid had the most potent inhibitory effect on 4-MUG with an IC50 value of 0.0341 microM. The IC50 values of diflunisal, diclofenac and indomethacin were 1.31, 24.2, and 34.1 microM, respectively, while acetaminophen, ketoprofen and naproxen showed less potent inhibition. Niflumic acid, diflunisal, diclofenac and indomethacin inhibited 4-MUG competitively with Ki values of 0.0275, 0.710, 53.3 and 69.9 microM, respectively, being similar to each IC50 value. In conclusion, of the seven NSAIDs investigated, niflumic acid was the most potent inhibitor of recombinant UGT1A9 via 4-MUG in a competitive manner.

  8. Effect of a New Prokinetic Agent DA-9701 Formulated with Corydalis Tuber and Pharbitidis Semen on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Hye Young Ji

    2012-01-01

    Full Text Available DA-9701 is a new botanical drug composed of the extracts of Corydalis tuber and Pharbitidis semen, and it is used as an oral therapy for the treatment of functional dyspepsia in Korea. The inhibitory potentials of DA-9701 and its component herbs, Corydalis tuber and Pharbitidis semen, on the activities of seven major human cytochrome P450 (CYP enzymes and four UDP-glucuronosyltransferase (UGT enzymes in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. DA-9701 and Corydalis tuber extract slightly inhibited UGT1A1-mediated etoposide glucuronidation, with 50% inhibitory concentration (IC50 values of 188 and 290 μg/mL, respectively. DA-9701 inhibited CYP2D6-catalyzed bufuralol 1′-hydroxylation with an inhibition constant (Ki value of 6.3 μg/mL in a noncompetitive manner. Corydalis tuber extract competitively inhibited CYP2D6-mediated bufuralol 1′-hydroxylation, with a Ki value of 3.7 μg/mL, whereas Pharbitidis semen extract showed no inhibition. The volume in which the dose could be diluted to generate an IC50 equivalent concentration (volume per dose index value of DA-9701 for inhibition of CYP2D6 activity was 1.16 L/dose, indicating that DA-9701 may not be a potent CYP2D6 inhibitor. Further clinical studies are warranted to evaluate the in vivo extent of the observed in vitro interactions.

  9. In vitro glucuronidation of the primary metabolite of 10-chloromethyl-11-demethyl-12-oxo-calanolide A by human liver microsomes and its interactions with UDP-glucuronosyltransferase substrates.

    Science.gov (United States)

    Liu, Xin; Sheng, Li; Zhao, Manman; Mi, Jiaqi; Liu, Zhihao; Li, Yan

    2015-02-01

    F18 (10-chloromethyl-11-demethyl-12-oxo-calanolide), an analog of (+)-Calanolide A, is a novel small-molecule nonnucleoside reverse transcriptase inhibitor for the therapy of human immunodeficiency virus (HIV) infection. M3, the most abundant primary metabolite of F18 in human liver microsomes (HLMs) and rat liver microsomes (RLMs), is mainly excreted in bile as a glucuronide conjugate in rats after oral administration. The aim of this study was to identify the UDP glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of M3 by HLMs and recombinant human UGTs and investigate the metabolic interactions of M3 with the substrates of UGTs in HLMs. As a result, UGT1A1 was the major isozyme responsible for the glucuronidation of M3, followed by UGT1A4, UGT1A9 and UGT2B7. M3 exhibited significant inhibition against UGT1A9 and UGT2B7 in both HLMs and recombinant human UGTs. In addition, M3 inhibited UGT1A9 catalyzed mycophenolic acid (MPA) glucuronidation with Ki of 0.39 μM, and M3 also inhibited the glucuronidation of 3'-azido-3'-deoxythymidine (AZT) by a "mixed-type" mechanism with Ki of 16.8 μM. The results suggest that UGT1A1 provides the major contribution to M3 glucuronidation in vitro and M3 has the potential to interact with xenobiotics and endogenous chemicals that are UGT1A9 and UGT 2B7 substrates.

  10. Estrogen receptor alpha, fos-related antigen-2, and c-Jun coordinately regulate human UDP glucuronosyltransferase 2B15 and 2B17 expression in response to 17beta-estradiol in MCF-7 cells.

    Science.gov (United States)

    Hu, Dong Gui; Mackenzie, Peter I

    2009-08-01

    UDP-glucuronosyltransferase 2B15 and 2B17 expression is up-regulated by 17beta-estradiol in MCF-7 breast cancer cells, as assessed by quantitative real-time polymerase chain reaction. Using 5'-deletion mapping and site-directed mutagenesis, we demonstrate that 17beta-estradiol activation of UGT2B15 gene transcription is mediated by a 282-base pair fragment positioned -454 to -172 nucleotides from the translation start site. This region contains two putative activator protein-1 (AP-1) elements, one imperfect estrogen response element (ERE), and two consensus ERE half-sites. We propose that these five sites act as an estrogen response unit (ERU), because mutation in any site reduces activation of the UGT2B15 promoter by 17beta-estradiol. Despite the presence of two AP-1 elements, the UGT2B15 promoter is not responsive to the AP-1 activator phorbol 12-myristate 13-acetate. Although electrophoretic mobility shift assays (EMSA) indicate that the AP-1 proteins c-Jun and Fos-related antigen 2 (Fra-2) bound to the distal AP-1 site, binding of Jun or Fos family members to the proximal AP-1 site was not detected by EMSA. Chromatin immunoprecipitation assays showed a 17beta-estradiol-induced recruitment of estrogen receptor (ER) alpha, c-Jun, and Fra-2 to the 282-bp ERU. The involvement of these three transcription factors in the stimulation of UGT2B15 gene expression by 17beta-estradiol was confirmed by siRNA silencing experiments. Mutagenesis and siRNA experiments indicate that UGT2B17 expression is also regulated by 17beta-estradiol via the ERU, which is fully conserved in both promoters. Because UGT2B15 and UGT2B17 inactivate steroid hormones by glucuronidation, the regulation of their genes by 17beta-estradiol may maintain steroid hormone homeostasis and prevent excessive estrogen signaling activity.

  11. Gilbert's syndrome: High frequency of the (TA)7 TAA allele in India and its interaction with a novel CAT insertion in promoter of the gene for bilirubin UDP-glucuronosyltransferase 1 gene

    Institute of Scientific and Technical Information of China (English)

    Shabana Farheen; Sanghamitra Sengupta; Amal Santra; Suparna Pal; Gopal Krishna Dhali; Meenakshi Chakravorty; Partha P Majumder; Abhijit Chowdhury

    2006-01-01

    AIM: To identify the variants in UDP-glucuronosyltransferase 1 (UGT1A1) gene in Gilbert's syndrome (GS) and to estimate the association between homozygosity for TA insertion and GS in India, as well as the frequency of TA insertion and its impact among normal controls in India.METHODS: Ninety-five GS cases and 95 normal controls were selected. Liver function and other tests were done. The promoter and all 5 exons of UGT1A1 gene were resequenced. Functional assessment of a novel trinucleotide insertion was done byin silico analysis and by estimating UGT1A1 promoter activity carried out by luciferase reporter assay of appropriate constructs in Hep G2 cell line.RESULTS: Among the GS patients, 80% were homozygous for the TA insertion, which was several-fold higher than reports from other ethnic groups. The mean UCB level was elevated among individuals with only one copy of this insertion, which was not significantly different from those with two copies. Many new DNA variants in UGT1A1 gene were discovered, including a trinucleotide (CAT) insertion in the promoter found in a subset (10%) of GS patients, but not among normal controls. In-silico analysis showed marked changes in the DNA-folding of the promoter and functional analysis showed a 20-fold reduction in transcription efficiency of UGT1A1 gene resulting from this insertion, thereby significantly elevating the UCB level.CONCLUSION: The genetic epidemiology of GS is variable across ethnic groups and the epistatic interactions among UGT1A1 promoter variants modulate bilirubin glucuronidation.

  12. Glucuronidation of odorant molecules in the rat olfactory system: activity, expression and age-linked modifications of UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT2A1, and relation to mitral cell activity.

    Science.gov (United States)

    Leclerc, Séverine; Heydel, Jean-Marie; Amossé, Valérie; Gradinaru, Daniela; Cattarelli, Martine; Artur, Yves; Goudonnet, Hervé; Magdalou, Jacques; Netter, Patrick; Pelczar, Hélène; Minn, Alain

    2002-11-15

    The aim of the present study was to examine the glucuronidation of a series of odorant molecules by homogenates prepared either with rat olfactory mucosa, olfactory bulb or brain. Most of the odorant molecules tested were efficiently conjugated by olfactory mucosa, whereas olfactory bulb and brain homogenates displayed lower activities and glucuronidated only a few molecules. Important age-related changes in glucuronidation efficiency were observed in olfactory mucosa and bulb. Therefore, we studied changes in expression of two UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT2A1, in 1-day, 1- and 2-week-, 3-, 12- and 24-month-old rats. UGT1A6 was expressed at the same transcriptional level in the olfactory mucosa, bulb and brain, throughout the life period studied. UGT2A1 mRNA was expressed in both olfactory mucosa and olfactory bulb, in accordance with previous results [Mol. Brain Res. 90 (2001) 83], but UGT2A1 transcriptional level was 400-4000 times higher than that of UGT1A6. Moreover, age-dependent variations in UGT2A1 mRNA expression were observed. As it has been suggested that drug metabolizing enzymes could participate in olfactory function, mitral cell electrical activity was recorded during exposure to different odorant molecules in young, adult and old animals. Age-related changes in the amplitude of response after stimulation with several odorant molecules were observed, and the highest responses were obtained with molecules that were not efficiently glucuronidated by olfactory mucosa. In conclusion, the present work presents new evidence of the involvement of UGT activity in some steps of the olfactory process.

  13. In vitro glucuronidation of Armillarisin A: UDP-glucuronosyltransferase 1A9 acts as a major contributor and significant species differences.

    Science.gov (United States)

    Sun, Dongxue; Zhu, Liangliang; Xiao, Ling; Xia, Yangliu; Ge, Guangbo; Cao, Yunfeng; Wu, Yan; Yin, Jun; Yang, Ling

    2014-11-01

    1. This study is performed to investigate liver microsomal glucuronidation of Armillarisin A (A.A), an effective cholagogue drug, aiming at characterizing the involved UDP-glucuronosyltranferases (UGT) and revealing potential species differences. 2. A.A glucuronidation in human liver microsomes (HLM) generates one metabolite (M2) glucuronidated at the phenol hydroxyl group, obeying Michaelis-Menten kinetic model. Multiple isoforms including UGT1A1, 1A7, 1A9 and 2B15 can catalyze A.A glucuronidation. Kinetic assays and chemical inhibition studies both demonstrate that UGT1A9 is responsible for A.A glucuronidation in HLM. A.A glucuronidation in Cynomolgus monkey microsomes (CyLM) also follows Michaelis-Menten model, but can additionally catalyze the traced glucuronosyl substitution at the alcohol hydroxyl group (M1). The reactions in liver microsomes from Sprague-Dawley rats (RLM), ICR/CD-1 mouse (MLM), Beagle dog (DLM) all display biphasic kinetics and only M2 is detected. HLM, RLM and CyLM exhibit very similar catalytic activities towards A.A glucuronidation, with the intrinsic clearance values of respective 38, 37 and 37 μL/min/mg, which are much higher than MLM and DLM. 3. This in vitro study indicates that UGT1A9 acts as a major contributor to A.A glucuronidation in human liver, and the reaction displays large species differences.

  14. Androgen-stimulated UDP-glucose dehydrogenase expression limits prostate androgen availability without impacting hyaluronan levels

    Science.gov (United States)

    Wei, Qin; Galbenus, Robert; Raza, Ashraf; Cerny, Ronald L.; Simpson, Melanie A.

    2009-01-01

    UDP-glucose dehydrogenase (UGDH) oxidizes UDP-glucose to UDP-glucuronate, an essential precursor for production of hyaluronan (HA), proteoglycans, and xenobiotic glucuronides. High levels of HA turnover in prostate cancer are correlated with aggressive progression. UGDH expression is high in the normal prostate even though HA accumulation is virtually undetectable. Thus, its normal role in the prostate may be to provide precursors for glucuronosyltransferase enzymes, which inactivate and solubilize androgens by glucuronidation. In this report, we quantified androgen dependence of UGDH, glucuronosyltransferase, and HA synthase expression. Androgen dependent and independent human prostate cancer cell lines were used to test the effects of UGDH manipulation on tumor cell growth, HA production and androgen glucuronidation. Dihydrotestosterone (DHT) increased UGDH expression ≈2.5-fold in androgen dependent cells. However, upregulation of UGDH did not affect HA synthase expression or enhance HA production. Mass spectrometric analysis showed that DHT was converted to a glucuronide, DHT-G, at a six-fold higher level in androgen dependent cells relative to androgen independent cells. The increased solubilization and elimination of DHT corresponded to slower cellular growth kinetics, which could be reversed in androgen dependent cells by treatment with a UDP-glucuronate scavenger. Collectively, these results suggest that dysregulated expression of UGDH could promote the development of androgen independent tumor cell growth by increasing available levels of intracellular androgen. PMID:19244115

  15. Correlation of Polymorphisms of UDP-Glucuronosyltransferase 1A7 Gene to Genetic Susceptibility of Lung Cancer%尿苷二磷酸葡糖醛酸转移酶1A7基因多态与肺癌遗传易感性的关系

    Institute of Scientific and Technical Information of China (English)

    冯飞跃; 周彤; 潘凯枫; 张联; 林东昕; 梁刚; 吕文富; 缪小平; 于春媛; 谭文; 周翊峰; 孙瞳; 康滨

    2005-01-01

    背景与目的:代谢酶尿苷二磷酸葡糖醛酸转移酶1A7(UDP-glucuronosyltcansferase,UGT1A7)可催化香烟中的致癌物质苯并[α]芘、亚硝胺NNK和杂环胺PhIP与葡糖醛酸结合使之失活,在解毒机制中起重要作用.本实验旨在研究UGT1A7基因多态与肺癌易感性的关系.方法:以聚合酶链反应-变性高效液相色谱(DHPLC)技术和聚合酶链反应-限制性片段长度多态性方法,分析317例正常对照和312例肺癌患者外周血淋巴细胞基因组DNA,UGT1A7 129~131和208位点多态基因型分布,及其与肺癌风险的关系.结果:与携带UGT1A7*1/*1基因型个体比较,携带UGT1A7*3/*1基因型的个体患肺腺癌的风险增高1.80倍(校正的OR为1.80;95%CI 1.03~3.12),携带UGT1A7*3基因型的个体患肺腺癌的风险增高1.59倍(校正的OR为1.59,95%CI 0.96~2.63).UGT1A7多态与肺鳞癌风险不相关.结论:UGT1A7基因多态可能是中国人肺癌遗传易感性因素.

  16. Divergent Expression and Metabolic Functions of Human Glucuronosyltransferases through Alternative Splicing.

    Science.gov (United States)

    Rouleau, Michèle; Tourancheau, Alan; Girard-Bock, Camille; Villeneuve, Lyne; Vaucher, Jonathan; Duperré, Anne-Marie; Audet-Delage, Yannick; Gilbert, Isabelle; Popa, Ion; Droit, Arnaud; Guillemette, Chantal

    2016-09-27

    Maintenance of cellular homeostasis and xenobiotic detoxification is mediated by 19 human UDP-glucuronosyltransferase enzymes (UGTs) encoded by ten genes that comprise the glucuronidation pathway. Deep RNA sequencing of major metabolic organs exposes a substantial expansion of the UGT transcriptome by alternative splicing, with variants representing 20% to 60% of canonical transcript expression. Nearly a fifth of expressed variants comprise in-frame sequences that may create distinct structural and functional features. Follow-up cell-based assays reveal biological functions for these alternative UGT proteins. Some isoforms were found to inhibit or induce inactivation of drugs and steroids in addition to perturbing global cell metabolism (energy, amino acids, nucleotides), cell adhesion, and proliferation. This work highlights the biological relevance of alternative UGT expression, which we propose increases protein diversity through the evolution of metabolic regulators from specific enzymes.

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

    Data.gov (United States)

    U.S. Environmental Protection Agency — 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 activity....

  18. Susceptibility to Exposure to Heterocyclic Amines from Cooked Food: Role of UDP-glucuronosyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Malfatti, M A; Felton, J S

    2005-08-22

    A number of carcinogenic heterocyclic amines (PhIP, MeIQx, and DiMeIQx) are produced from the condensation of creatinine, hexoses and amino acids during the cooking of meat (1). There are many variables that impact the production and subsequent ingestion of these compounds in our diet. Temperature, type of meat product, cooking method, doneness, and other factors affect the quantity of these carcinogens consumed by humans. Estimates of ingestion of these carcinogens are 1-20 ng/kg body weight per day (2). Human case control studies that correlate meat consumption from well-done cooking practices with cancer incidence indicate excess tumors for breast, colon, stomach, esophagus, and possibly prostate (3-5).

  19. Effects of dietary anticarcinogens and nonsteroidal anti-inflammatory drugs on rat gastrointestinal UDP-glucuronosyltransferases.

    NARCIS (Netherlands)

    Logt, E.M.J. van der; Roelofs, H.M.J.; Lieshout, E.M.M. van; Nagengast, F.M.; Peters, W.H.M.

    2004-01-01

    BACKGROUND: Dietary compounds or nonsteroidal anti-inflammatory drugs (NSAIDs) may reduce cancer rates. Elevation of phase II detoxification enzymes might be one of the mechanisms leading to cancer prevention. We investigated the effects of dietary anticarcinogens and NSAIDs on rat gastrointestinal

  20. Effects of dietary anticarcinogens and nonsteroidal anti-inflammatory drugs on rat gastrointestinal UDP-glucuronosyltransferases.

    NARCIS (Netherlands)

    Logt, E.M.J. van der; Roelofs, H.M.J.; Lieshout, E.M.M. van; Nagengast, F.M.; Peters, W.H.M.

    2004-01-01

    BACKGROUND: Dietary compounds or nonsteroidal anti-inflammatory drugs (NSAIDs) may reduce cancer rates. Elevation of phase II detoxification enzymes might be one of the mechanisms leading to cancer prevention. We investigated the effects of dietary anticarcinogens and NSAIDs on rat gastrointestinal

  1. Polymorphic expression of UDP-glucuronosyltransferase UGTlA gene in human colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Min Wang

    Full Text Available BACKGROUND: Polymorphism of genes encoding drug-metabolizing enzymes is known to play an important role in increased susceptibility of colorectal cancer. UGT1A gene locus has been suggested to define tissue-specific glucuronidation activity. Reduced capacity of glucuronidation is correlated with the development of colorectal cancer. Therefore, we sought to explore polymorphism of UGTlA gene in human colorectal cancer. METHODS: Cancerous and healthy tissues were obtained from selectedpatients. Blood samples were collected and UGTlA mRNA transcriptions were analyzed. Genomic DNA was prepared and UGTlA8 exon-1 sequences were amplified, visualized and purified. The extracted DNA was subcloned and sequenced. Two-tailed Fisher's exact test, Odds ratios (ORs, confidence interval (CIs and Logistics Regression Analysis were used for statistical analysis. RESULTS: UGTlA mRNA expression was reduced in cancerous tissues compared with healthy tissues from the same patient . The UGTlA mRNA expression of healthy tissue in study patients was lower than control . The mRNA expression of cancerous tissue was down-regulated in UGTlAl, 1A3, 1A4, lA6, 1A9 and up-regulated in UGTlA8 and UGTlAl0 UGT1A5 and UGT1A7 were not expressed in colonic tissue of either group. The allele frequency of WT UGTlA8*1 was higher (p = 0.000, frequency of UGTlA8*3 was lowered in control group (p = 0.000. The expression of homozygous UGTlA8*1 was higher in control group (p = 0.000. Higher frequency of both heterozygous UGTlA8*1/*3 and UGTlA8*2/*3 were found in study group (p = 0.000; p = 0.000. The occurrence of colorectal cancer was mainly related to the presence of polymorphic UGTlA8*3 alleles (p = 0.000. CONCLUSION: Regulation of human UGT1A genes is tissue-specific. Individual variation in polymorphic expressions of UGTlA gene locus was noted in all types of colonic tissue tested, whereas hepatic tissue expression was uniform. The high incidence of UGTlA8 polymorphism exists in colorectal cancer patients. UGTlA8*1 allele is a protective factor and UGTlA8*3 allele is a risk factor.

  2. The functional glycosyltransferase signature sequence of the human beta 1,3-glucuronosyltransferase is a XDD motif.

    Science.gov (United States)

    Gulberti, Sandrine; Fournel-Gigleux, Sylvie; Mulliert, Guillermo; Aubry, André; Netter, Patrick; Magdalou, Jacques; Ouzzine, Mohamed

    2003-08-22

    The human beta 1,3-glucuronosyltransferase I (GlcAT-I) is the key enzyme responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide of proteoglycans (GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta 1-O-serine). We have investigated the role of aspartate residues Asp194-Asp195-Asp196 corresponding to the glycosyltransferase DXD signature motif, in GlcAT-I function by UDP binding experiments, kinetic analyses, and site-directed mutagenesis. We presented the first evidence that Mn2+ is not only essential for GlcAT-I activity but is also required for cosubstrate binding. In agreement, kinetic studies were consistent with a metal-activated enzyme model whereby activation probably occurs via binding of a Mn2+.UDP-GlcA complex to the enzyme. Mutational analysis showed that the Asp194-Asp195-Asp196 motif is a major element of the UDP/Mn2+ binding site. Furthermore, determination of the individual role of each aspartate showed that substitution of Asp195 as well as Asp196 to alanine strongly impaired GlcAT-I activity, whereas Asp194 replacement produced only a moderate alteration of the enzyme activity. These findings along with molecular modeling and three-dimensional structure comparison of the GlcAT-I catalytic center with that of the Bacillus subtilis glycosyltransferase SpsA provided evidence that the interactions of Asp195 with the ribose moiety of UDP and of Asp196 with the metal cation Mn2+ were crucial for GlcAT-I function. Altogether, these results indicated that, similarly to the SpsA enzyme, the nucleotide binding site of GlcAT-I contains a XDD motif rather than a DXD motif.

  3. UDP-sulfoquinovose formation by Sulfolobus acidocaldarius.

    Science.gov (United States)

    Zolghadr, Behnam; Gasselhuber, Bernhard; Windwarder, Markus; Pabst, Martin; Kracher, Daniel; Kerndl, Martina; Zayni, Sonja; Hofinger-Horvath, Andreas; Ludwig, Roland; Haltrich, Dietmar; Oostenbrink, Chris; Obinger, Christian; Kosma, Paul; Messner, Paul; Schäffer, Christina

    2015-03-01

    The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-D-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD(+)-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-D-glucose is converted via UDP-4-keto-D-glucose to UDP-D-glucose-5,6-ene, the structure of which was determined by (1)H and (13)C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-D-glucose and NAD(+) could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-D-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested.

  4. Effects of dietary components on testosterone metabolism via UDP‐glucuronosyltransferase (UGT

    Directory of Open Access Journals (Sweden)

    Carl eJenkinson

    2013-07-01

    Full Text Available The potential interference in testosterone metabolism through ingested substances has ramifications for: i a range of pathologies such as prostate cancer, ii medication contra-indications, iii disruption to the endocrine system, and iv potential confounding effects on doping tests. Conjugation of anabolic steroids during phase II metabolism, mainly driven by UDP-glucuronosyltransferase (UGT 2B7, 2B15 and 2B17, has been shown to be impaired in vitro by a range of compounds including xenobiotics and pharmaceuticals. Following early reports on the effects of a range of xenobiotics on UGT activity in vitro, the work was extended to reveal similar effects with common non-steroidal anti-inflammatory drugs. Notably, recent studies have evidenced inhibitory effects of the common foodstuffs green tea and red wine, along with their constituent flavonoids and catechins. This review amalgamates the existing evidence for the inhibitory effects of various pharmaceutical and dietary substances on the rate of UGT glucuronidation of testosterone; and evaluates the potential consequences for health linked to steroid levels, interaction with treatment drugs metabolised by the UGT enzyme and steroid abuse in sport.

  5. Arabidopsis thaliana glucuronosyltransferase in family GT14

    DEFF Research Database (Denmark)

    Dilokpimol, Adiphol; Geshi, Naomi

    2014-01-01

    of glucuronic acid residues to β-1,3- and β-1,6-linked galactans of arabinogalactan (Knoch et al. 2013). The knockout mutant of this gene resulted in the enhanced growth rate of hypocotyls and roots of seedlings, suggesting an involvement of AtGlcAT 14A in cell elongation. AtGlcAt14A belongs to the family GT14...... in the Carbohydrate Active Enzyme database (CAZy; www.cazy.org), in which a total of 11 proteins, including AtGLCAT 14A, are classified from Arabidopsis thaliana. In this paper, we report the enzyme activities for the rest of the Arabidopsis GT14 isoforms, analyzed in the same way as for AtGlcAT 14A. Evidently, two...... other Arabidopsis GT14 isoforms, At5g15050 and At2g37585, also possess the glucuronosyltransferase activity adding glucuronic acid residues to β-1,3- and β-1,6-linked galactans. Therefore, we named At5g15050 and At2g37585 as AtGlcAT 14B and AtGlcAT 14C, respectively. © 2014 Landes Bioscience....

  6. UDP-galactose 4'-epimerase activities toward UDP-Gal and UDP-GalNAc play different roles in the development of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Jennifer M I Daenzer

    Full Text Available In both humans and Drosophila melanogaster, UDP-galactose 4'-epimerase (GALE catalyzes two distinct reactions, interconverting UDP-galactose (UDP-gal and UDP-glucose (UDP-glc in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine (UDP-galNAc and UDP-N-acetylglucosamine (UDP-glcNAc. All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans.

  7. Association of the golgi UDP-galactose transporter with UDP-galactose: ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum

    NARCIS (Netherlands)

    Sprong, H.; Degroote, S.; Nilsson, T.; Kawakita, M.; Ishida, N.; van der Sluijs, P.; van Meer, G.

    2003-01-01

    UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose: ceramide galactosyltransferase. It is not known how UDP-g

  8. Molecular cloning, chromosomal mapping, and characterization of the mouse UDP-galactose: Ceramide galactosyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Coetzee, T.; Fujita, N.; Marcus, J. [Univ. of North Carolina, Chapel Hill, NC (United States)] [and others

    1996-07-01

    UDP-galactose:ceramide galactosyltransferase (CGT) (EC 2.11.62) catalyzes the final step in the synthesis of galactocerebroside, a glycosphingolipid characteristically abundant in myelin. In this report, we describe the isolation of genomic clones spanning the mouse CGT gene. The mouse CGT gene consists of six exons that span a minimum of 70 kb of DNA and that encode a 541 amino acid translation product with extensive sequence similarity to the rat CGT enzyme and to UDP-glucuronosyltransferases (UGT). The 5{prime}-untranslated region of the mouse CGT gene is encoded by a separate exon located approximately 25 kb upstream of the first protein-encoding exon. Furthermore, the genomic organization of the five encoding region exons of the mouse CGT gene resembles that of the human UGT1 and rat UGT2B1 genes. Finally, analysis of somatic cell hybrids by PCR and fluorescence in situ hybridization to metaphase chromosomes has localized the mouse CGT gene to chromosome 3, bands E3-F1. 26 refs., 5 figs., 1 tab.

  9. Effect of the beta-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases.

    Science.gov (United States)

    Oleson, Lauren; Court, Michael H

    2008-09-01

    Glucuronidation studies using microsomes and recombinant uridine diphosphoglucuronosyltransferases (UGTs) can be complicated by the presence of endogenous beta-glucuronidases, leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used beta-glucuronidase inhibitor, although it is not clear whether this reagent should be added routinely to glucuronidation incubations. Here we have determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and recombinant UGTs (rUGTs). Despite the use of buffered incubation solutions, it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM did not enhance any of the glucuronidation activities evaluated that could be considered consistent with inhibition of beta-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for glucuronidation of 5-hydroxytryptamine and estradiol by pHLMs, with a 35% decrease at 20 mM saccharolactone concentration. Endogenous beta-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes and insect-cell expressed rUGTs, but not for kidney, intestinal or human embryonic kidney HEK293 microsomes. However, the extent of hydrolysis was relatively small, representing only 9-19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations. If saccharolactone is used, concentrations should be titrated to achieve activity enhancement without inhibition.

  10. Identification and characterization of human UDP-glucuronosyltransferases responsible for the in-vitro glucuronidation of arctigenin.

    Science.gov (United States)

    Xin, Hong; Xia, Yang-Liu; Hou, Jie; Wang, Ping; He, Wei; Yang, Ling; Ge, Guang-Bo; Xu, Wei

    2015-12-01

    This study aimed to characterize the glucuronidation pathway of arctigenin (AR) in human liver microsomes (HLM) and human intestine microsomes (HIM). HLM and HIM incubation systems were employed to catalyse the formation of AR glucuronide. The glucuronidation activity of commercially recombinant UGT isoforms towards AR was screened. A combination of chemical inhibition assay and kinetic analysis was used to determine the UGT isoforms involved in the glucuronidation of AR in HLM and HIM. AR could be extensively metabolized to one mono-glucuronide in HLM and HIM. The mono-glucuronide was biosynthesized and characterized as 4'-O-glucuronide. UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7 and 2B17 participated in the formation of 4'-O-G, while UGT2B17 demonstrated the highest catalytic activity in this biotransformation. Both kinetic analysis and chemical inhibition assays demonstrated that UGT1A9, UGT2B7 and UGT2B17 played important roles in AR-4'-O-glucuronidation in HLM. Furthermore, HIM demonstrated moderate efficiency for AR-4'-O-glucuronidation, implying that AR may undergo a first-pass metabolism during the absorption process. UGT1A9, UGT2B7 and UGT2B17 were the major isoforms responsible for the 4'-O-glucuronidation of AR in HLM, while UGT2B7 and UGT2B17 were the major contributors to this biotransformation in HIM. © 2015 Royal Pharmaceutical Society.

  11. Effects of variant UDP-glucuronosyltransferase 1A1 gene,glucose-6-phosphate dehydrogenase deficiency and thalassemia on cholelithiasis

    Institute of Scientific and Technical Information of China (English)

    Yang-Yang Huang; Ching-Shui Huang; Sien-Sing Yang; Min-Shung Lin; May-Jen Huang; Ching-Shan Huang

    2005-01-01

    AIM: To test the hypothesis that the variant UDPglucuronosyltransferase 1A1 (UGT1A1) gene, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and thalassemia influence bilirubin metabolism and play a role in the development of cholelithiasis.METHODS: A total of 372 Taiwan Chinese with cholelithiasis who had undergone cholecystectomy and 293 healthy individuals were divided into case and control groups,respectively. PCR and restriction fragment length polymorphism were used to analyze the promoter area and nucleotides 211, 686, 1 091, and 1 456 of the UGT1A1 gene for all subjects and the gene variants for thalassemia and G6PD deficiency.RESULTS: Variation frequencies for the cholelithiasis patients were 16.1%, 25.8%, 5.4%, and 4.3% for A(TA)6TAA/A(TA)7TAA (6/7), heterozygosity within the coding region, compound heterozygosity, and homozygosity of the UGT1A1 gene, respectively. Comparing the case and control groups, a statistically significant difference in frequency was demonstrated for the homozygous variation of the UGT1A1 gene (P = 0.012, χ2 test), but not for the other variations. Further, no difference was demonstrated in a between-group comparison of the incidence of G6PD deficiency and thalassemia (2.7% vs 2.4% and 5.1% vs 5.1%, respectively). The bilirubin levels for the cholelithiasis patients with the homozygous variant-UGT1A1 gene were significantly different from the control analog (18.0±6.5 and 12.7±2.9 μmol/L, respectively; P<0.001, Student's ttest).CONCLUSION: Our results show that the homozygous variation in the UGT1A1 gene is a risk factor for the development of cholelithiasis in Taiwan Chinese.

  12. Effects of a novel organophosphorus pesticide (RPR-V) on extra hepatic detoxifying enzymes after repeated oral doses in rats.

    Science.gov (United States)

    Mahboob, Mohammed; Kaleem, Mohammed; Siddiqui, Javed

    2004-10-01

    The effects of a novel organophosphorous pesticide, 2-butenoic acid-3-(diethoxy phosphinothionyl) ethyl ester (RPR-V) on glutathione S-transferases (GST), UDP-glucuronyl transferases (UDPGT) and the level of glutathione (GSH) were evaluated in rats after repeated oral administration at 33 microg kg(-1)day(-1) (low), 66 microg kg(-1)day(-1) (mid) and 99 microg kg(-1)day(-1) (high) for 90 days and at 28 days (withdrawal) post-treatment. GSH level and GST in kidney; GSH level in brain decreased significantly at mid and high doses on 45th and 90th day (P RPR-V has the potential to modulate the extrahepatic detoxifying enzymes and thereby interact with other physiological processes in the exposed organisms.

  13. A novel glucuronosyltransferase has an unprecedented ability to catalyse continuous two-step glucuronosylation of glycyrrhetinic acid to yield glycyrrhizin.

    Science.gov (United States)

    Xu, Guojie; Cai, Wei; Gao, Wei; Liu, Chunsheng

    2016-10-01

    Glycyrrhizin is an important bioactive compound that is used clinically to treat chronic hepatitis and is also used as a sweetener world-wide. However, the key UDP-dependent glucuronosyltransferases (UGATs) involved in the biosynthesis of glycyrrhizin remain unknown. To discover unknown UGATs, we fully annotated potential UGATs from Glycyrrhiza uralensis using deep transcriptome sequencing. The catalytic functions of candidate UGATs were determined by an in vitro enzyme assay. Systematically screening 434 potential UGATs, we unexpectedly found one unique GuUGAT that was able to catalyse the glucuronosylation of glycyrrhetinic acid to directly yield glycyrrhizin via continuous two-step glucuronosylation. Expression analysis further confirmed the key role of GuUGAT in the biosynthesis of glycyrrhizin. Site-directed mutagenesis revealed that Gln-352 may be important for the initial step of glucuronosylation, and His-22, Trp-370, Glu-375 and Gln-392 may be important residues for the second step of glucuronosylation. Notably, the ability of GuUGAT to catalyse a continuous two-step glucuronosylation reaction was determined to be unprecedented among known glycosyltransferases of bioactive plant natural products. Our findings increase the understanding of traditional glycosyltransferases and pave the way for the complete biosynthesis of glycyrrhizin.

  14. Bilirubin diglucuronide synthesis by a UDP-glucuronic acid-dependent enzyme system in rat liver microsomes.

    Science.gov (United States)

    Blanckaert, N; Gollan, J; Schmid, R

    1979-01-01

    Incubation of rat liver homogenate or microsomal preparations with bilirubin or bilirubin monoglucuronide with (BMG) resulted in formation of bilirubin diglucuronide (BDG). Both synthesis of BMG and its conversion to BDG were critically dependent on the presence of UDP-glucuronic acid. Pretreatment of the animals with phenobarbital stimulated both reactions. When 33 microM bilirubin was incubated with microsomal preparations from phenobarbital-treated rats, 80-90% of the substrate was converted to bilirubin glucuronides; the reaction products consisted of almost equal amounts of BMG and BDG. When phenobarbital pretreatment was omitted or when the substrate concentration was increased to 164 microM bilirubin, proportionally more BMG and less BDG were formed. Homogenate and microsomes from homozygous Gunn rats neither synthesized BMG nor converted BMG to BDG. These findings in vitro suggest an explanation for the observations in vivo that, in conditions of excess bilirubin load or of genetically decreased bilirubin UDP glucuronosyltransferase (EC 2.4.1.17) activity, proportionally more BMG and less BDG are excreted in bile. PMID:109837

  15. Chemically-induced alteration of UDP-glucuronic acid concentration in rat liver.

    Science.gov (United States)

    Watkins, J B; Klaassen, C D

    1983-01-01

    Since many xenobiotics alter hepatic UDP-glucuronosyltransferase activity, their effect on UDPGA concentration was determined. Rats were pretreated with: 1) microsomal enzyme inducers (7,8-benzoflavone, benzo(a)pyrene, butylated hydroxyanisole, isosafrole, 3-methylcholanthrene, phenobarbital, pregnenolone-16 alpha-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin, trans-stilbene oxide); 2) inhibitors of microsomal enzymes (cobaltous chloride, piperonyl butoxide, SKF 525-A, borneol, galactosamine); 3) hepatotoxins (allyl alcohol, aflatoxin B1, alpha-naphthylisothiocyanate, bromobenzene, cadmium chloride, carbon tetrachloride, 1,1-dichloroethylene), and 4) commonly used anesthetics (pentobarbital, urethane, diethyl ether, halothane, enflurane, methoxyflurane). Rats were decapitated before removal of the liver. All inducers except PCN and isosafrole increased UDPGA 36-85% above control. Mixed-function oxidase inhibitors had no effect whereas borneol and galactosamine reduced UDPGA 85-90%. Aflatoxin B1 and cadmium produced decreases of 59 and 25%, respectively. Hepatic UDPGA content was diminished 70-95% after exposure to the inhalation anesthetics, whereas the other anesthetics reduced UDPGA about 25%. Thus, numerous xenobiotics alter the concentration of UDPGA in rat liver, which may influence the rate of glucoronidation.

  16. The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis.

    Science.gov (United States)

    Rautengarten, Carsten; Ebert, Berit; Moreno, Ignacio; Temple, Henry; Herter, Thomas; Link, Bruce; Doñas-Cofré, Daniela; Moreno, Adrián; Saéz-Aguayo, Susana; Blanco, Francisca; Mortimer, Jennifer C; Schultink, Alex; Reiter, Wolf-Dieter; Dupree, Paul; Pauly, Markus; Heazlewood, Joshua L; Scheller, Henrik V; Orellana, Ariel

    2014-08-05

    Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP-l-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP-l-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP-l-Rha/UDP-d-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP-l-Rha and UDP-d-Gal for matrix polysaccharide biosynthesis.

  17. Strong Inhibition of Celastrol Towards UDP-Glucuronosyl Transferase (UGT 1A6 and 2B7 Indicating Potential Risk of UGT-Based Herb-Drug Interaction

    Directory of Open Access Journals (Sweden)

    Qi Wang

    2012-06-01

    Full Text Available Celastrol, a quinone methide triterpene isolated from Tripterygium wilfordii Hook F., has various biochemical and pharmacological activities, and is now being developed as a promising anti-tumor agent. Inhibitory activity of compounds towards UDP-glucuronosyltransferase (UGT is an important cause of clinical drug-drug interactions and herb-drug interactions. The aim of the present study is to investigate the inhibition of celastrol towards two important UDP-glucuronosyltransferase (UGT isoforms UGT1A6 and UGT2B7. Recombinant UGT isoforms and non-specific substrate 4-methylumbelliferone (4-MU were used. The results showed that celastrol strongly inhibited the UGT1A6 and 2B7-mediated 4-MU glucuronidation reaction, with 0.9 ± 0.1% and 1.8 ± 0.2% residual 4-MU glucuronidation activity at 100 μM of celastrol, respectively. Furthermore, inhibition kinetic study (Dixon plot and Lineweaver-Burk plot demonstrated that celastrol noncompetitively inhibited the UGT1A1-mediated 4-MU glucuronidation, and competitively inhibited UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameters (Ki were calculated to be 0.49 μM and 0.045 μM for UGT1A6 and UGT2B7, respectively. At the therapeutic concentration of celastrol for anti-tumor utilization, the possibility of celastrol-drug interaction and celastrol-containing herbs-drug interaction were strongly indicated. However, given the complicated nature of herbs, these results should be viewed with more caution.

  18. Portable UDP port forwarding in user space

    Directory of Open Access Journals (Sweden)

    Vancea Florin

    2009-05-01

    Full Text Available Port forwarding is frequently used to selectively expose services available on remote machines to clients running on the local machine or on machines connected to the local network. Services running over TCP are easy to forward using SSH, at least as long as the details of the transported protocol are transparent. Tools are available for other classes of tunneling, including UDP tunneling. However, thesetools are not very portable and may be limited to root/admin usage or may simply not be available. We are presenting a Java client/server pair that may be successfully used in almost any environment to forward a UDP dialog over an existing forwarded TCPconnection. The tool is tested for traffic like SNMP with multiple clients and multiple targets.

  19. Use of UDP for efficient imagery dissemination

    Science.gov (United States)

    Prandolini, Robert; Au, T. A.; Lui, Andrew K.; Owen, Michael J.; Grigg, Mark W.

    2000-05-01

    In the defence organization, imagery represents an important information source for users in the tactical, operational and strategic environments. Its wider dissemination may rely on deployed communication networks that are often unstable with high bit error rates and outages. This paper presents efficient techniques for imagery dissemination using the user datagram protocol (UDP). The use of UDP is compared with the popular transmission control protocol and shown to be superior in performance for error prone IP networks. We have employed a wavelet based coder producing an embedded bit-stream. The packetization of the bit-stream is investigated and we show that it is better to tile an image into independent embedded bit-streams when the network performance is poor. Variable-size tiling is compared with, and shown to be superior to, a fixed-size tiling approach. Selective re-transmission of lost packets is implemented for efficient imagery dissemination using UDP. The selective re- transmission scheme is a function of network bandwidth, delay times, error rates and the significance of the packet.

  20. A TCP-Over-UDP Test Harness

    Energy Technology Data Exchange (ETDEWEB)

    Dunigan, TH

    2002-10-31

    This report describes an implementation of a TCP-like protocol that runs over UDP. This TCP-like implementation, which does not require kernel modifications, provides a test harness for evaluating variations of the TCP transport protocol over the Internet. The test harness provides a tunable and instrumented version of TCP that supports Reno, NewReno, and SACK/FACK. The test harness can also be used to characterize the TCP-performance of a network path. Several case studies illustrate how one can tune the transport protocol to improve performance.

  1. Degrading and Detoxifying Industrial Waste Water using Bioremediation Approach

    OpenAIRE

    Agrawal, P K; Sangeet Prabha; Shalu Mittal

    2014-01-01

    Bioremediation uses various microorganisms to detoxify or degrade various harmful substances in the nature, particularly soil and water. In the proposed work, five species of micro-organisms were used to analyse their impact on various physico-chemical parameters of water. In the first attempt the actual physico chemical parameters of the collected sample water were noted down (Fresh sample parameters). Then the sample water was treated with micro-organisms (one at a time). The growth of micr...

  2. Ligand Binding and Substrate Discrimination by UDP-Galactopyranose Mutase

    Energy Technology Data Exchange (ETDEWEB)

    Gruber, Todd D.; Borrok, M. Jack; Westler, William M.; Forest, Katrina T.; Kiessling, Laura L.; (UW)

    2009-07-31

    Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5{prime}-diphosphate (UDP) Galf, the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 {angstrom} resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the

  3. The interconversion of UDP-arabinopyranose and UDP-arabinofuranose is indispensable for plant development in Arabidopsis.

    Science.gov (United States)

    Rautengarten, Carsten; Ebert, Berit; Herter, Thomas; Petzold, Christopher J; Ishii, Tadashi; Mukhopadhyay, Aindrila; Usadel, Björn; Scheller, Henrik Vibe

    2011-04-01

    L-Ara, an important constituent of plant cell walls, is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. Nucleotide sugar mutases have been demonstrated to interconvert UDP-Larabinopyranose (UDP-Arap) and UDP-L-arabinofuranose (UDP-Araf) in rice (Oryza sativa). These enzymes belong to a small gene family encoding the previously named Reversibly Glycosylated Proteins (RGPs). RGPs are plant-specific cytosolic proteins that tend to associate with the endomembrane system. In Arabidopsis thaliana, the RGP protein family consists of five closely related members. We characterized all five RGPs regarding their expression pattern and subcellular localizations in transgenic Arabidopsis plants. Enzymatic activity assays of recombinant proteins expressed in Escherichia coli identified three of the Arabidopsis RGP protein family members as UDP-L-Ara mutases that catalyze the formation of UDP-Araf from UDP-Arap. Coimmunoprecipitation and subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry analysis revealed a distinct interaction network between RGPs in different Arabidopsis organs. Examination of cell wall polysaccharide preparations from RGP1 and RGP2 knockout mutants showed a significant reduction in total L-Ara content (12–31%) compared with wild-type plants. Concomitant downregulation of RGP1 and RGP2 expression results in plants almost completely deficient in cell wall–derived L-Ara and exhibiting severe developmental defects.

  4. Capillary electrophoretic study on UDP-sugars in cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Glucose is an important regulator of cell growth and metabolism. Uridine diphosphate sugars (UDP-sugars), as the intermediate products of metabolism, play pivotal roles as precursors in the synthesis of complex carbohydrates and glycolipids as well as lectose. It is very important to study their metabolism in cells in clinical biochemistry. A capillary electrophoretic method has been developed for the analysis of UDP-sugars and nucleotides. By using an uncoated capillary (70cm×50 μm) and 20 mmol/L borax buffer (pH 9), 4 important UDP-sugars can be analyzed in 15 min at 22 kV with satisfactory precision and sensitivity. The developed method has been applied to analyze UDP-sugars concentrations in lymphocytes, fibroblasts and mesangial cells, and the results show it not only is much better than HPLC method, but also can be used to measure the energy charge of cells.

  5. Capillary electrophoretic study on UDP-sugars in cells

    Institute of Scientific and Technical Information of China (English)

    许国旺; R.Lehmann; 路鑫; H.M.Liebich; 张玉奎; 卢佩章

    2000-01-01

    Glucose is an important regulator of cell growth and metabolism. Uridine diphosphate sugars (UDP-sugars), as the intermediate products of metabolism, play pivotal roles as precursors in the synthesis of complex carbohydrates and glycolipids as well as lectose. It is very important to study their metabolism in cells in clinical biochemistry. A capillary electrophoretic method has been developed for the analysis of UDP-sugars and nucleotides. By using an uncoated capillary (70cm×50μm) and 20 mmol/L borax buffer (pH 9), 4 important UDP-sugars can be analyzed in 15 min at 22 kV with satisfactory precision and sensitivity. The developed method has been applied to analyze UDP-sugars concentrations in lymphocytes, fibroblasts and mesangial cells, and the results show it not only is much better than HPLC method, but also can be used to measure the energy charge of cells.

  6. Use of Giardia, which appears to have a single nucleotide-sugar transporter for UDP-GlcNAc, to identify the UDP-Glc transporter of Entamoeba.

    Science.gov (United States)

    Banerjee, Sulagna; Cui, Jike; Robbins, Phillips W; Samuelson, John

    2008-05-01

    Nucleotide-sugar transporters (NSTs) transport activated sugars (e.g. UDP-GlcNAc) from the cytosol to the lumen of the endoplasmic reticulum or Golgi apparatus where they are used to make glycoproteins and glycolipids. UDP-Glc is an important component of the N-glycan-dependent quality control (QC) system for protein folding. Because Entamoeba has this QC system while Giardia does not, we hypothesized that transfected Giardia might be used to identify the UDP-Glc transporter of Entamoeba. Here we show Giardia membranes transport UDP-GlcNAc and have apyrases, which hydrolyze nucleoside-diphosphates to make the antiporter nucleoside-monophosphate. The only NST of Giardia (GlNst), which we could identify, transports UDP-GlcNAc in transfected Saccharomyces and is present in perinuclear and peripheral vesicles and increases in expression during encystation. Entamoeba membranes transport three nucleotide-sugars (UDP-Gal, UDP-GlcNAc, and UDP-Glc), and Entamoeba has three NSTs, one of which has been shown previously to transport UDP-Gal (EhNst1). Here we show recombinant EhNst2 transports UDP-Glc in transfected Giardia, while recombinant EhNst3 transports UDP-GlcNAc in transfected Saccharomyces. In summary, all three NSTs of Entamoeba and the single NST of Giardia have been molecularly characterized, and transfected Giardia provides a new system for testing heterologous UDP-Glc transporters.

  7. A saponin-detoxifying enzyme mediates suppression of plant defences

    Science.gov (United States)

    Bouarab, K.; Melton, R.; Peart, J.; Baulcombe, D.; Osbourn, A.

    2002-08-01

    Plant disease resistance can be conferred by constitutive features such as structural barriers or preformed antimicrobial secondary metabolites. Additional defence mechanisms are activated in response to pathogen attack and include localized cell death (the hypersensitive response). Pathogens use different strategies to counter constitutive and induced plant defences, including degradation of preformed antimicrobial compounds and the production of molecules that suppress induced plant defences. Here we present evidence for a two-component process in which a fungal pathogen subverts the preformed antimicrobial compounds of its host and uses them to interfere with induced defence responses. Antimicrobial saponins are first hydrolysed by a fungal saponin-detoxifying enzyme. The degradation product of this hydrolysis then suppresses induced defence responses by interfering with fundamental signal transduction processes leading to disease resistance.

  8. Target Proteins in Human Autoimmunity: Cytochromes P450 and Udp-Glycoronosyltransferases

    Directory of Open Access Journals (Sweden)

    Petra Obermayer-Straub

    2000-01-01

    Full Text Available Cytochromes P450 (CYPs and UDP-glucuronosyltransferases (UGTs are targets of autoantibodies in several hepatic and extrahepatic autoimmune diseases. Autoantibodies directed against hepatic CYPs and UGTs were first detected by indirect immunofluorescence as antiliver and/or kidney microsomal antibodies. In autoimmune hepatitis (AIH type 2, liver and/or kidney microsomal (LKM type 1 autoantibodies are detected and are directed against CYP2D6. About 10% of AIH-2 sera further contain LKM-3 autoantibodies directed against family 1 UGTs. Chronic infections by hepatitis C virus and hepatitis delta virus may induce several autoimmune phenomena, and multiple autoantibodies are detected. Anti-CYP2D6 autoantibodies are detected in up to 4% of patients with chronic hepatitis C, and anti-CYP2A6 autoantibodies are detected in about 2% of these patients. In contrast, 14% of patients with chronic hepatitis delta virus infections generate anti-UGT autoantibodies. In a small minority of patients, certain drugs are known to induce immune-mediated, idiosyncratic drug reactions, also known as ’drug-induced hepatitis’. Drug-induced hepatitis is often associated with autoantibodies directed against hepatic CYPs or other hepatic proteins. Typical examples are tienilic acid-induced hepatitis with anti-CYP2C9, dihydralazine hepatitis with anti-CYP1A2, halothane hepatitis with anti-CYP2E1 and anticonvulsant hepatitis with anti-CYP3A. Recent data suggest that alcoholic liver disease may be induced by mechanisms similar to those that are active in drug-induced hepatitis. Autoantibodies directed against several CYPs are further detected in sera from patients with the autoimmune polyglandular syndrome type 1. Patients with autoimmune polyglandular syndrome type 1 with hepatitis often develop anti-CYP1A2; patients with adrenal failure develop anti-CYP21, anti- CYP11A1 or CYP17; and patients with gonadal failure develop anti-CYP11A1 or CYP17. In idiopathic Addison disease

  9. Role of UDP-Glucuronosyltransferase 1A1 in the Metabolism and Pharmacokinetics of Silymarin Flavonolignans in Patients with HCV and NAFLD

    Directory of Open Access Journals (Sweden)

    Ying Xie

    2017-01-01

    Full Text Available Silymarin is the most commonly used herbal medicine by patients with chronic liver disease. Silymarin flavonolignans undergo rapid first-pass metabolism primarily by glucuronidation. The aims of this investigation were: (1 to determine the association of UGT1A1*28 polymorphism with the area under the plasma concentration-time curves (AUCs for silybin A (SA and silybin B (SB; (2 to evaluate the effect of UGT1A1*28 polymorphism on the profile of flavonolignan glucuronide conjugates found in the plasma; and (3 to investigate the role of UGT1A1 enzyme kinetics on the pharmacokinetics of SA and SB. AUCs and metabolic ratios for thirty-three patients with chronic liver disease administered oral doses of silymarin were compared between different UGT1A1*28 genotypes. The AUCs, metabolic ratios, and the profiles of major SA and SB glucuronides did not differ significantly among the three UGT1A1 genotypes. In contrast, an increase in the proportion of sulfated flavonolignan conjugates in plasma was observed in subjects with UGT1A1*28/*28 genotype compared to subjects carrying wild type alleles. Differences in SA and SB in vitro intrinsic clearance estimates for UGTIA1 correlated inversely with SA and SB exposures observed in vivo indicating a major role for UGT1A1 in silymarin metabolism. In addition, a significant difference in the metabolic ratio observed between patients with NAFLD and HCV suggests that any effect of UGT1A1 polymorphism may be obscured by a greater effect of liver disease on the pharmacokinetics of silymarin. Taken together, these results suggest the presence of the UGT1A1*28 allele does not contribute significantly to a large inter-subject variability in the pharmacokinetics of silybin A and silybin B which may obscure the ability to detect beneficial effects of silymarin in patients with liver disease.

  10. Induction of UDP-glucuronosyltransferase 2B15 gene expression by the major active metabolites of tamoxifen, 4-hydroxytamoxifen and endoxifen, in breast cancer cells.

    Science.gov (United States)

    Chanawong, Apichaya; Hu, Dong Gui; Meech, Robyn; Mackenzie, Peter I; McKinnon, Ross A

    2015-06-01

    We previously reported upregulation of UGT2B15 by 17β-estradiol in breast cancer MCF7 cells via binding of the estrogen receptor α (ERα) to an estrogen response unit (ERU) in the proximal UGT2B15 promoter. In the present study, we show that this ERα-mediated upregulation was significantly reduced by two ER antagonists (fulvestrant and raloxifene) but was not affected by a third ER antagonist, 4-hydroxytamoxifen (4-OHTAM), a major active tamoxifen (TAM) metabolite. Furthermore, we found that, similar to 17β-estradiol, 4-OHTAM and endoxifen (another major active TAM metabolite) elevated UGT2B15 mRNA levels, and that this stimulation was significantly abrogated by fulvestrant. Further experiments using 4-OHTAM revealed a critical role for ERα in this regulation. Specifically; knockdown of ERα expression by anti-ERα small interfering RNA reduced the 4-OHTAM-mediated induction of UGT2B15 expression; 4-OHTAM activated the wild-type but not the ERU-mutated UGT2B15 promoter; and chromatin immunoprecipitation assays showed increased ERα occupancy at the UGT2B15 ERU in MCF7 cells upon exposure to 4-OHTAM. Together, these data indicate that both 17β-estradiol and the antiestrogen 4-OHTAM upregulate UGT2B15 in MCF7 cells via the same ERα-signaling pathway. This is consistent with previous observations that both 17β-estradiol and TAM upregulate a common set of genes in MCF7 cells via the ER-signaling pathway. As 4-OHTAM is a UGT2B15 substrate, the upregulation of UGT2B15 by 4-OHTAM in target breast cancer cells is likely to enhance local metabolism and inactivation of 4-OHTAM within the tumor. This represents a potential mechanism that may reduce TAM therapeutic efficacy or even contribute to the development of acquired TAM resistance.

  11. A G protein-coupled receptor for UDP-glucose.

    Science.gov (United States)

    Chambers, J K; Macdonald, L E; Sarau, H M; Ames, R S; Freeman, K; Foley, J J; Zhu, Y; McLaughlin, M M; Murdock, P; McMillan, L; Trill, J; Swift, A; Aiyar, N; Taylor, P; Vawter, L; Naheed, S; Szekeres, P; Hervieu, G; Scott, C; Watson, J M; Murphy, A J; Duzic, E; Klein, C; Bergsma, D J; Wilson, S; Livi, G P

    2000-04-14

    Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

  12. Maleic acid treatment of biologically detoxified corn stover liquor.

    Science.gov (United States)

    Kim, Daehwan; Ximenes, Eduardo A; Nichols, Nancy N; Cao, Guangli; Frazer, Sarah E; Ladisch, Michael R

    2016-09-01

    Elimination of microbial and enzyme inhibitors from pretreated lignocellulose is critical for effective cellulose conversion and yeast fermentation of liquid hot water (LHW) pretreated corn stover. In this study, xylan oligomers were hydrolyzed using either maleic acid or hemicellulases, and other soluble inhibitors were eliminated by biological detoxification. Corn stover at 20% (w/v) solids was LHW pretreated LHW (severity factor: 4.3). The 20% solids (w/v) pretreated corn stover derived liquor was recovered and biologically detoxified using the fungus Coniochaeta ligniaria NRRL30616. After maleic acid treatment, and using 5 filter paper units of cellulase/g glucan (8.3mg protein/g glucan), 73% higher cellulose conversion from corn stover was obtained for biodetoxified samples compared to undetoxified samples. This corresponded to 87% cellulose to glucose conversion. Ethanol production by yeast of pretreated corn stover solids hydrolysate was 1.4 times higher than undetoxified samples, with a reduction of 3h in the fermentation lag phase.

  13. Enzymatic Strategies to Detoxify Gluten: Implications for Celiac Disease

    Directory of Open Access Journals (Sweden)

    Ivana Caputo

    2010-01-01

    Full Text Available Celiac disease is a permanent intolerance to the gliadin fraction of wheat gluten and to similar barley and rye proteins that occurs in genetically susceptible subjects. After ingestion, degraded gluten proteins reach the small intestine and trigger an inappropriate T cell-mediated immune response, which can result in intestinal mucosal inflammation and extraintestinal manifestations. To date, no pharmacological treatment is available to gluten-intolerant patients, and a strict, life-long gluten-free diet is the only safe and efficient treatment available. Inevitably, this may produce considerable psychological, emotional, and economic stress. Therefore, the scientific community is very interested in establishing alternative or adjunctive treatments. Attractive and novel forms of therapy include strategies to eliminate detrimental gluten peptides from the celiac diet so that the immunogenic effect of the gluten epitopes can be neutralized, as well as strategies to block the gluten-induced inflammatory response. In the present paper, we review recent developments in the use of enzymes as additives or as processing aids in the food biotechnology industry to detoxify gluten.

  14. Degrading and Detoxifying Industrial Waste Water using Bioremediation Approach

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    P. K. Agrawal

    2014-06-01

    Full Text Available Bioremediation uses various microorganisms to detoxify or degrade various harmful substances in the nature, particularly soil and water. In the proposed work, five species of micro-organisms were used to analyse their impact on various physico-chemical parameters of water. In the first attempt the actual physico chemical parameters of the collected sample water were noted down (Fresh sample parameters. Then the sample water was treated with micro-organisms (one at a time. The growth of microbes was noted carefully over 96 hours after inoculation. The physico chemical parameters were recorded again and were compared with the fresh sample parameters. The results were analysed for any change and on this basis an impact factor was developed. The study reveals all the selected microbes have a great capacity of degrading and simplifying the complex molecules into simpler ones. Bioremediative treatment further enhances this capacity and therefore this approach can be utilized on large scale to minimize pollution of water bodies.

  15. Acrolein-detoxifying isozymes of glutathione transferase in plants.

    Science.gov (United States)

    Mano, Jun'ichi; Ishibashi, Asami; Muneuchi, Hitoshi; Morita, Chihiro; Sakai, Hiroki; Biswas, Md Sanaullah; Koeduka, Takao; Kitajima, Sakihito

    2017-02-01

    Acrolein is a lipid-derived highly reactive aldehyde, mediating oxidative signal and damage in plants. We found acrolein-scavenging glutathione transferase activity in plants and purified a low K M isozyme from spinach. Various environmental stressors on plants cause the generation of acrolein, a highly toxic aldehyde produced from lipid peroxides, via the promotion of the formation of reactive oxygen species, which oxidize membrane lipids. In mammals, acrolein is scavenged by glutathione transferase (GST; EC 2.5.1.18) isozymes of Alpha, Pi, and Mu classes, but plants lack these GST classes. We detected the acrolein-scavenging GST activity in four species of plants, and purified an isozyme showing this activity from spinach (Spinacia oleracea L.) leaves. The isozyme (GST-Acr), obtained after an affinity chromatography and two ion exchange chromatography steps, showed the K M value for acrolein 93 μM, the smallest value known for acrolein-detoxifying enzymes in plants. Peptide sequence homology search revealed that GST-Acr belongs to the GST Tau, a plant-specific class. The Arabidopsis thaliana GST Tau19, which has the closest sequence similar to spinach GST-Acr, also showed a high catalytic efficiency for acrolein. These results suggest that GST plays as a scavenger for acrolein in plants.

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

    Science.gov (United States)

    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.

  17. Detoxification of Abrus precatorius L. seeds by Ayurvedic Shodhana process and anti-inflammatory potential of the detoxified extract

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    Sagar B Dhoble

    2014-01-01

    Full Text Available Background: Abrus precatorius seeds traditionally used for the treatment of sciatica and alopecia contains the toxic protein, abrin, a Type II Ribosome Inactivating Protein. Ayurveda recommends the use of Abrus seeds after the Shodhana process (detoxification. Objective: The current study was aimed at performing the Shodhana process, swedana (boiling of Abrus precatorius seeds using water as a medium and to evaluate the anti-inflammatory potential of seed extract post detoxification. Materials and Methods: Non-detoxified and detoxified extracts were prepared and subsequently subjected to various in vitro and in vivo assays. In hemagglutination assay, the non-detoxified extract shows higher agglutination of RBCs than detoxified extract indicating riddance of toxic hemagglutinating proteins by Shodhana. This was confirmed by the SDSPAGE analysis of detoxified extract revealing the absence of abrin band in detoxified extract when compared to non-detoxified extract. Results: The cytotoxicity assay in HeLa cell line expresses a higher reduction in growth percentage of the cells with non-detoxified extract as compared to detoxified extract indicating successful detoxification. Brine shrimp lethality test indicated the reduction in toxicity index of detoxified extract as compared to non-detoxified extract. Further, the whole body apoptosis assay in zebrafish revealed that percentage of viable cells were greater for detoxified extract than non-detoxified extract. The anti-inflammatory studies using carrageenan induced paw edema model in rats was carried out on the extracts with doses of 100 mg/kg and 200 mg/kg, per oral, where the detoxified extract exhibited significant inhibition of rat paw edema at both the doses comparable to that of Diclofenac sodium. Conclusion: Absence of toxicity and the retention of the anti-inflammatory activity of detoxified Abrus seed extract confirmed that the Swedana process is effective in carrying out the detoxification

  18. Ingestive behavior of finishing sheep fed detoxified castor bean meal

    Directory of Open Access Journals (Sweden)

    Daniel Cézar da Silva

    2016-06-01

    Full Text Available ABSTRACT Castor bean crops stand out in the Northeastern Brazil for oil production, producing coproducts with potential for animal diets. Thus, this work evaluated the effect on ingestive behavior when 0, 33, 67 and 100% of detoxified castor bean meal (DCBM were included to substitute soy bean meal in diets for sheep. The randomized blocks design was used with five sheep in each treatment. Dry matter intake and neutral detergent fiber intake were not affected (P > .05 by the inclusion of DCBM in the diet, with means of 1362.6 and 582.98 g/animal/day, respectively. Substitution of soybean meal by DCBM did not affect (P > .05 times of rumination, idle and total chewing, with averages of 181.33, 347.04 and 366.24 minute/12 h, respectively. A quadratic effect (P < .05 was found for feeding time, with minimum of 164.56 min/12 h, when 60% of DCBM was included in the diet. A quadratic effect (P < .05 was verified for eating efficiency with maximum of 4.43 g DM/minute and 2.08 g NDF/minute. Rumination efficiency in g DM and NDF/minute were not affected (P < .05, with means of 4.31 and 1.84, respectively. The substitution of soybean meal by DCBM decreases feeding time when 60% of it was used but does not influence the intake of DM and NDF, time spent in ruminating and idle, and total chewing time. The use of 60% of DCBM increases feeding efficiency of DM and NDF, and does not compromise the efficiency of rumination.

  19. Efficacy of T-2 toxin detoxifying agent in broiler chickens

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    Nešić V.

    2012-01-01

    Full Text Available This investigation was conducted in order to investigate the efficacy of the detoxifying agent Mycofix® Plus (MP in the prevention and/or alleviation in vivo adverse effects of T-2 toxin in broilers. In addition, the adsorbing potential of MP was estimated in vitro. Mean degradation levels of T-2 toxin with MP in vitro, as measured by HPTLC, varied from 26.06 to 31.02 % and the adsorption ability was elevated in acidic environment (pH 3. In vivo trial was performed on 160 one day old "Ross" broiler chicks and lasted for 21 days. Birds were divided into 4 equal groups as follows: Group 1 - negative control; Group 2 - positive control - 2 ppm T-2 toxin; Group 3 - 2 ppm T-2 toxin+2 kg/t MP; Group 4 - 2 kg/t MP. Broilers fed the diet containing 2 mg/kg of T-2 toxin without MP developed typical T-2 toxicosis. Birds that were fed the diet containing both T-2 and MP had better performances and no oral ulcerations as the dominant sign of T-2 toxicosis were observed. Histopathological examination of tissues originating from birds fed the diet containing T-2 toxin revealed degenerative changes in the oral and small intestine mucosa, necroses of enterocytes and hepatocytes, as well as depletion of lymphocytes in the bursa Fabricii. Immunohistochemical examination also revealed negative effects of T-2 toxin on cells proliferation in intestineal and bile duct mucosa, as well as on lymphocytes from bursa Fabricii. The macroscopic and microscopic structure of the liver, intestine and bursa Fabricii of broilers fed a diet containing T-2 toxin and MP was mostly preserved. Cutaneous basophile hypersensitivity reaction was weaker in broilers fed mixtures containing 2 mg/kg T-2 toxin.

  20. Crystal structures of Mycobacteria tuberculosis and Klebsiella pneumoniae UDP-galactopyranose mutase in the oxidised state and Klebsiella pneumoniae UDP-galactopyranose mutase in the (active) reduced state

    OpenAIRE

    2005-01-01

    Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the D-galactofuranose sugar found in bacterial and parasitic cell walls, including those of many pathogens. UDP-Galf is made from UDP-galactopyranose by the enzyme UDP-galactopyranose mutase. The enzyme requires the reduced FADH− co-factor for activity. The structure of the Mycobacterium tuberculosis mutase with FAD has been determined to 2.25Å. The structures of Klebsiella pneumoniae mutase with FAD and with FADH− bound have bee...

  1. Structural basis of cooperativity in human UDP-glucose dehydrogenase.

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    Venkatachalam Rajakannan

    Full Text Available BACKGROUND: UDP-glucose dehydrogenase (UGDH is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics. METHODOLOGY: Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle. CONCLUSION: In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

  2. Synthesis of gamma-chaconine and gamma-solanine are catalyzed in potato by two separate glycosyltransferases: UDP-glucose:solanidine glucosyltransferase and UDP-galactose:solanidine galactosyltransferase.

    Science.gov (United States)

    Zimowski, J

    1997-01-01

    UDP-glucose:solanidine glucosyltransferase and UDP-galactose:solanidine galactosyltransferase from cytosol of potato sprouts were partially separated by Sephadex G-100 and Q-Sepharose chromatographies, proving the existence of different glycosylation systems in biosynthesis of alpha-chaconine and alpha-solanine.

  3. UDP-glucosyltransferase activity toward exogenous substrates in Drosophila melanogaster.

    Science.gov (United States)

    Real, M D; Ferré, J; Chapa, F J

    1991-05-01

    To investigate the capacity of Drosophila extracts to glucosylate exogenous substrates we have developed a fast and sensitive method for the detection of UDP-glucosyltransferase activity using 4-nitrophenol, 1-naphthol, or 2-naphthol as substrates. High-performance liquid chromatography was used to separate and quantitate the reaction products, allowing detection of activities that produced as little as 1 pmol of 2-naphthol glucoside (fluorescence detection) or 16 pmol of 4-nitrophenol glucoside (absorbance detection). Optimal activity was found at 43 degrees C and alkaline pH. The affinity of the Drosophila enzyme was 250-fold higher for 1-naphthol or 2-naphthol (Km approximately 4 microM) than for 4-nitrophenol and UDP-glucose (Km approximately 1 mM).

  4. UDP Large-Payload Capability Detection for DNSSEC

    Science.gov (United States)

    Rikitake, Kenji; Nakao, Koji; Shimojo, Shinji; Nogawa, Hiroki

    Domain Name System (DNS) is a major target for the network security attacks due to the weak authentication. A security extension DNSSEC has been proposed to introduce the public-key authentication, but it is still on the deployment phase. DNSSEC assumes IP fragmentation allowance for exchange of its messages over UDP large payloads. IP fragments are often blocked on network packet filters for administrative reasons, and the blockage may prevent fast exchange of DNSSEC messages. In this paper, we propose a scheme to detect the UDP large-payload transfer capability between two DNSSEC hosts. The proposed detection scheme does not require new protocol elements of DNS and DNSSEC, so it is applicable by solely modifying the application software and configuration. The scheme allows faster capability detection to probe the end-to-end communication capability between two DNS hosts by transferring a large UDP DNS message. The DNS software can choose the maximum trans-mission unit (MTU) on the application level using the probed detection results. Implementation test results show that the proposed scheme shortens the detection and transition time on fragment-blocked transports.

  5. Purification and properties of an unusual UDP-glucose dehydrogenase, NADPH-dependent, from Xanthomonas albilineans.

    Science.gov (United States)

    Blanch, María; Legaz, María-Estrella; Vicente, C

    2008-01-01

    Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxydation of NADPH is linearly dependent on the amount of oxygen supplied. The enzyme has been purified at homogeneity. The value of pI of the purified enzyme is 8.98 and its molecular mass has been estimated as about 14 kDa. The enzyme shows a michaelian kinetics for UDP-glucose concentrations. The value of K(m) for UDP-glucose is 0.87 mM and 0.26 mM for NADPH, although the enzyme has three different sites to interact with NADPH. The enzyme is inhibited by UDP-glucose concentrations higher than 1.3 mM. N-Terminal sequence has been determined as IQPYNH.

  6. Metabolizable energy and nutrient digestibility of detoxified castor meal and castor cake for poultry

    Directory of Open Access Journals (Sweden)

    João Batista Matos Júnior

    2011-11-01

    Full Text Available This experiment aimed to determine the metabolizable energy and the digestibility of detoxified castor meal and castor cake nutrients for broilers. A total of 180 Cobb-500TM broilers from 21 to 32 days of age was randomly distributed in three treatments with six replicates of 10 broilers - 5 males and 5 females. The experimental treatments were a reference-diet and two test-diets composed of 80% of reference-diet and 20% of detoxified castor cake or castor meal. The adaptation period ranged from 21 to 27 days of age and the total excreta collection period ranged from 28 to 32 days of age. The detoxified castor meal presented apparent metabolizable energy of 2,032 kcal/kg of natural matter, apparent metabolizable energy corrected for nitrogen balance of 1,829 kcal/kg of natural matter and the digestibility coefficients of 57.8% dry matter, 68.4% crude protein, 10.5% crude fiber, 35.9% nitrogen free extract, 80.0% ether extract and 26.6% ash. In the detoxified castor cake, the apparent metabolizable energy content was 2,550 kcal/kg of natural matter, the nitrogen corrected apparent metabolizable energy balance was of 2,320 kcal/kg of natural matter and the digestibility coefficients of 60.6% for dry matter, 77.2% for crude protein, 14.0% for crude fiber, 45.7% for nitrogen free extract, 85.4% for ether extract and 28.2% for ash. The metabolizable energy and digestibility of nutrients in detoxified castor cake are higher than in the detoxified castor meal.

  7. Toxicity of crude and detoxified Tityus serrulatus venom in anti-venom-producing sheep

    Science.gov (United States)

    Ferreira, Marina G.; Duarte, Clara G.; Oliveira, Maira S.; Castro, Karen L. P.; Teixeira, Maílson S.; Reis, Lílian P. G.; Zambrano, José A.; Kalapothakis, Evanguedes; Michel, Ana Flávia R. M.; Soto-Blanco, Benito; Chávez-Olórtegui, Carlos

    2016-01-01

    Specific anti-venom used to treat scorpion envenomation is usually obtained from horses after hyperimmunization with crude scorpion venom. However, immunized animals often become ill because of the toxic effects of the immunogens used. This study was conducted to evaluate the toxic and immunogenic activities of crude and detoxified Tityus serrulatus (Ts) venom in sheep during the production of anti-scorpionic anti-venom. Sheep were categorized into three groups: G1, control, immunized with buffer only; G2, immunized with crude Ts venom; and G3, immunized with glutaraldehyde-detoxified Ts venom. All animals were subjected to clinical exams and supplementary tests. G2 sheep showed mild clinical changes, but the other groups tolerated the immunization program well. Specific antibodies generated in animals immunized with either Ts crude venom or glutaraldehyde-detoxified Ts venom recognized the crude Ts venom in both assays. To evaluate the lethality neutralization potential of the produced sera, individual serum samples were pre-incubated with Ts crude venom, then subcutaneously injected into mice. Efficient immune protection of 56.3% and 43.8% against Ts crude venom was observed in G2 and G3, respectively. Overall, the results of this study support the use of sheep and glutaraldehyde-detoxified Ts venom for alternative production of specific anti-venom. PMID:27297422

  8. Apparent digestibility of nutrients, energy, and amino acid of nontoxic and detoxified physic nut cakes for Nile tilapia

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    Hamilton Hisano

    2015-09-01

    Full Text Available Abstract:The objective of this work was to evaluate the apparent digestibility coefficients of nutrients, energy, and amino acids of nontoxic and detoxified physic nut cakes treated with solvent plus posterior extrusion, for Nile tilapia. The apparent digestibility coefficients of crude protein and gross energy were higher for detoxified than for nontoxic physic nut cake. However, the apparent digestibility coefficient of ether extract of the nontoxic physic nut cake was higher than that of the detoxified one. The apparent digestibility coefficient of amino acids of both feed ingredients was superior to 80%, except for glycine, for the nontoxic psychic nut cake, and for threonine, for the detoxified one. Nontoxic and detoxified physic nut cakes show apparent digestibility coefficient values equivalent to those of the other evaluated oilseeds and potential for inclusion in Nile tilapia diets.

  9. Tomato UDP-Glucose Sterol Glycosyltransferases: A Family of Developmental and Stress Regulated Genes that Encode Cytosolic and Membrane-Associated Forms of the Enzyme

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    Karla Ramirez-Estrada

    2017-06-01

    Full Text Available Sterol glycosyltransferases (SGTs catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic

  10. TLR-Activated Gap Junction Channels Protect Mice against Bacterial Infection through Extracellular UDP Release.

    Science.gov (United States)

    Qin, Juliang; Zhang, Guangxu; Zhang, Xiaoyu; Tan, Binghe; Lv, Zhangsheng; Liu, Mingyao; Ren, Hua; Qian, Min; Du, Bing

    2016-02-15

    Extracellular UDP (eUDP), released as a danger signal by stressed or apoptotic cells, plays an important role in a series of physiological processes. Although the mechanism of eUDP release in apoptotic cells has been well defined, how the eUDP is released in innate immune responses remains unknown. In this study, we demonstrated that UDP was released in both Escherichia coli-infected mice and LPS- or Pam3CSK4-treated macrophages. Also, LPS-induced UDP release could be significantly blocked by selective TLR4 inhibitor Atractylenolide I and selective gap junction inhibitors carbenoxolone and flufenamic acid (FFA), suggesting the key role of TLR signaling and gap junction channels in this process. Meanwhile, eUDP protected mice from peritonitis by reducing invaded bacteria that could be rescued by MRS2578 (selective P2Y6 receptor inhibitor) and FFA. Then, connexin 43, as one of the gap junction proteins, was found to be clearly increased by LPS in a dose- and time-dependent manner. Furthermore, if we blocked LPS-induced ERK signaling by U0126, the expression of connexin 43 and UDP release was also inhibited dramatically. In addition, UDP-induced MCP-1 secretion was significantly reduced by MRS2578, FFA, and P2Y6 mutation. Accordingly, pretreating mice with U0126 and Gap26 increased invaded bacteria and aggravated mice death. Taken together, our study reveals an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of gap junction channel-mediated UDP release in infectious diseases. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis.

    Science.gov (United States)

    Ebert, Berit; Rautengarten, Carsten; Guo, Xiaoyuan; Xiong, Guangyan; Stonebloom, Solomon; Smith-Moritz, Andreia M; Herter, Thomas; Chan, Leanne Jade G; Adams, Paul D; Petzold, Christopher J; Pauly, Markus; Willats, William G T; Heazlewood, Joshua L; Scheller, Henrik Vibe

    2015-04-01

    Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.

  12. A UDP-X diphosphatase from Streptococcus pneumoniae hydrolyzes precursors of peptidoglycan biosynthesis.

    Directory of Open Access Journals (Sweden)

    Krisna C Duong-Ly

    Full Text Available The gene for a Nudix enzyme (SP_1669 was found to code for a UDP-X diphosphatase. The SP_1669 gene is localized among genes encoding proteins that participate in cell division in Streptococcus pneumoniae. One of these genes, MurF, encodes an enzyme that catalyzes the last step of the Mur pathway of peptidoglycan biosynthesis. Mur pathway substrates are all derived from UDP-glucosamine and all are potential Nudix substrates. We showed that UDP-X diphosphatase can hydrolyze the Mur pathway substrates UDP-N-acetylmuramic acid and UDP-N-acetylmuramoyl-L-alanine. The 1.39 Å resolution crystal structure of this enzyme shows that it folds as an asymmetric homodimer with two distinct active sites, each containing elements of the conserved Nudix box sequence. In addition to its Nudix catalytic activity, the enzyme has a 3'5' RNA exonuclease activity. We propose that the structural asymmetry in UDP-X diphosphatase facilitates the recognition of these two distinct classes of substrates, Nudix substrates and RNA. UDP-X diphosphatase is a prototype of a new family of Nudix enzymes with unique structural characteristics: two monomers, each consisting of an N-terminal helix bundle domain and a C-terminal Nudix domain, form an asymmetric dimer with two distinct active sites. These enzymes function to hydrolyze bacterial cell wall precursors and degrade RNA.

  13. Structures of complexes of a metal-independent glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-acetylgalactosamine (UDP-GalNAc) and its hydrolysis products.

    Science.gov (United States)

    Pham, Tram T K; Stinson, Brittany; Thiyagarajan, Nethaji; Lizotte-Waniewski, Michelle; Brew, Keith; Acharya, K Ravi

    2014-03-21

    Mammalian members of glycosyltransferase family 6 (GT6) of the CAZy database have a GT-A fold containing a conserved Asp-X-Asp (DXD) sequence that binds an essential metal cofactor. Bacteroides ovatus GT6a represents a GT6 clade found in more than 30 Gram-negative bacteria that is similar in sequence to the catalytic domains of mammalian GT6, but has an Asn(95)-Ala-Asn(97) (NXN) sequence substituted for the DXD motif and metal-independent catalytic activity. Co-crystals of a low activity mutant of BoGT6a (E192Q) with UDP-GalNAc contained protein complexes with intact UDP-GalNAc and two forms with hydrolysis products (UDP plus GalNAc) representing an initial closed complex and later open form primed for product release. Two cationic residues near the C terminus of BoGT6a, Lys(231) and Arg(243), interact with the diphosphate moiety of UDP-GalNAc, but only Lys(231) interacts with the UDP product and may function in leaving group stabilization. The amide group of Asn(95), the first Asn of the NXN motif, interacts with the ribose moiety of the substrate. This metal-independent GT6 resembles its metal-dependent homologs in undergoing conformational changes on binding UDP-GalNAc that arise from structuring the C terminus to cover this substrate. It appears that in the GT6 family, the metal cofactor functions specifically in binding the UDP moiety in the donor substrate and transition state, actions that can be efficiently performed by components of the polypeptide chain.

  14. A structural basis for the allosteric regulation of non-hydrolysing UDP-GlcNAc 2-epimerases

    Science.gov (United States)

    Velloso, Lucas M; Bhaskaran, Shyam S; Schuch, Raymond; Fischetti, Vincent A; Stebbins, C Erec

    2008-01-01

    The non-hydrolysing bacterial UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) catalyses the conversion of UDP-GlcNAc into UDP-N-acetylmannosamine, an intermediate in the biosynthesis of several cell-surface polysaccharides. This enzyme is allosterically regulated by its substrate UDP-GlcNAc. The structure of the ternary complex between the Bacillus anthracis UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, showed direct interactions between UDP and its substrate, and between the complex and highly conserved enzyme residues, identifying the allosteric site of the enzyme. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme. Kinetic data and mutagenesis of the highly conserved UDP-GlcNAc-interacting residues confirm their importance in the substrate binding and catalysis of the enzyme. This constitutes the first example to our knowledge, of an enzymatic allosteric activation by direct interaction between the substrate and the allosteric activator. PMID:18188181

  15. A Structural Basis for the Allosteric Regulatin of Non-Hydrolysing UDP-G1cNAc 2-Epimerases

    Energy Technology Data Exchange (ETDEWEB)

    Velloso,L.; Bhaskaran, S.; Schuch, R.; Fischetti, V.; Stebbins, C.

    2008-01-01

    The non-hydrolysing bacterial UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) catalyses the conversion of UDP-GlcNAc into UDP-N-acetylmannosamine, an intermediate in the biosynthesis of several cell-surface polysaccharides. This enzyme is allosterically regulated by its substrate UDP-GlcNAc. The structure of the ternary complex between the Bacillus anthracis UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, showed direct interactions between UDP and its substrate, and between the complex and highly conserved enzyme residues, identifying the allosteric site of the enzyme. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme. Kinetic data and mutagenesis of the highly conserved UDP-GlcNAc-interacting residues confirm their importance in the substrate binding and catalysis of the enzyme. This constitutes the first example to our knowledge, of an enzymatic allosteric activation by direct interaction between the substrate and the allosteric activator.

  16. Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Mathapati, Santosh; Bishi, Dillip Kumar [Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai (India); Frontier Lifeline Pvt Ltd. and Dr. K. M. Cherian Heart Foundation, Mogappair, Chennai (India); Healthcare and Energy Materials Laboratory, NUSNNI, Faculty of Engineering, National University of Singapore (Singapore); Guhathakurta, Soma [Departmet of Engineering Design, Indian Institute of Technology Madras, Chennai (India); Cherian, Kotturathu Mammen [Frontier Lifeline Pvt Ltd. and Dr. K. M. Cherian Heart Foundation, Mogappair, Chennai (India); Venugopal, Jayarama Reddy; Ramakrishna, Seeram [Healthcare and Energy Materials Laboratory, NUSNNI, Faculty of Engineering, National University of Singapore (Singapore); Verma, Rama Shanker, E-mail: vermars@iitm.ac.in [Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai (India)

    2013-04-01

    Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p > 0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP–G–CA–ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP–G–CA–ET). DBP–G–CA–ET exhibited a significant (p > 0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (p < 0.05). Lyophilized acellular detoxified GLUT cross-linked bovine pericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions. - Highlights: ► We improved the quality of patch biomaterial for cardiovascular surgical procedures. ► Bovine pericardium was

  17. Wheat bran feruloyl oligosaccharides modulate the phase II detoxifying/antioxidant enzymes via Nrf2 signaling.

    Science.gov (United States)

    Zhang, Huijuan; Wang, Jing; Liu, Yingli; Sun, Baoguo

    2015-03-01

    The antioxidant activities of wheat bran feruloyl oligosaccharides (FOs) were determined in rats by determining the activities and mRNA expression levels of phase II detoxifying/antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and heme oxygenase-1 (HO-1) in rat organs. FOs was given by gavage at doses of 0.25, 0.5, and 0.75 mmol/kg body weight every day for 15 days. Compared with the control group, the activities of SOD, CAT, and GSH-Px in FOs treatment groups significantly (Plevels of SOD, CAT, and HO-1 in organs. Moreover, the immunoblot analysis revealed increased nuclear factor-E2-related factor (Nrf2) protein expression levels in organs and there were positive correlations between the mRNA expression of phase II detoxifying/antioxidant enzymes and the expressions of Nrf2 protein, which demonstrated FOs treatment could modulate the detoxifying/antioxidant enzymes via Nrf2 signaling. Copyright © 2014. Published by Elsevier B.V.

  18. Aldo-keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants.

    Science.gov (United States)

    Vemanna, Ramu S; Vennapusa, Amaranatha Reddy; Easwaran, Murugesh; Chandrashekar, Babitha K; Rao, Hanumantha; Ghanti, Kirankumar; Sudhakar, Chinta; Mysore, Kirankumar S; Makarla, Udayakumar

    2017-07-01

    In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  19. Fermented wheat powder induces the antioxidant and detoxifying system in primary rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Margherita La Marca

    Full Text Available Many plants exhibit antioxidant properties which may be useful in the prevention of oxidative stress reactions, such as those mediated by the formation of free radical species in different pathological situations. In recent years a number of studies have shown that whole grain products in particular have strong antioxidant activity. Primary cultures of rat hepatocytes were used to investigate whether and how a fermented powder of wheat (Lisosan G is able to modulate antioxidant and detoxifying enzymes, and whether or not it can activate Nrf2 transcription factor or inhibit NF-kB activation. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by 0.7 mg/ml Lisosan G treatment. In particular,quinone oxidoreductase and heme oxygenase-1 were induced, although to different degrees, at the transcriptional, protein and/or activity levels by the treatment. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus after 1 h of Lisosan G treatment was revealed by immunoblotting. Lisosan G was also observed to decrease H2O2-induced toxicity Taken together, these results show that this powder of wheat is an effective inducer of ARE/Nrf2-regulated antioxidant and detoxifying genes and has the potential to inhibit the translocation of NF-kB into the nucleus.

  20. Sistem Pencegahan UDP DNS Flood Dengan Filter Firewall Pada Router Mikrotik

    Directory of Open Access Journals (Sweden)

    Doni Aprilianto

    2017-05-01

    Full Text Available Serangan terhadap server jaringan dapat terjadi kapan saja,  jenis serangan yang dapat menyebabkan efek yang signifikan pada sebuah router adalah UDP-Flooding. UDP (User Datagram Protocol-Flooding adalah jenis serangan yang memanfaatkan protokol UDP dengan mengurangi sambungan (connectionless untuk menyerang target. Dalam analisis ini menggunakan metode penelitian deskriptif untuk memperoleh data secara langsung dengan melakukan teknik flooding serta teknik pencegahannya terhadap server yang telah dirancang. Dengan menggunakan Filter Rules yang telah dibuat, packet yang melalui port DNS selain IP Address yang telah di allow jika mencoba melakukan request atau flood DNS ke IP Public ISP pada router mikrotik, maka packet tersebut akan langsung di drop oleh pengaturan rules tersebut. kesimpulan yang dapat diambil yaitu penerapan filter firewall pada router mikrotik dapat mengurangi jumlah paket data UDP yang dikirimkan oleh attacker melalui port DNS sebanyak 60% dari jumlah paket yang masuk jika tanpa firewall.

  1. The Biosynthesis of UDP-d-QuiNAc in Bacillus cereus ATCC 14579

    Science.gov (United States)

    Hwang, Soyoun; Aronov, Avi; Bar-Peled, Maor

    2015-01-01

    N-acetylquinovosamine (2-acetamido-2,6-di-deoxy-d-glucose, QuiNAc) is a relatively rare amino sugar residue found in glycans of few pathogenic gram-negative bacteria where it can play a role in infection. However, little is known about QuiNAc-related polysaccharides in gram-positive bacteria. In a routine screen for bacillus glycan grown at defined medium, it was surprising to identify a QuiNAc residue in polysaccharides isolated from this gram-positive bacterium. To gain insight into the biosynthesis of these glycans, we report the identification of an operon in Bacillus cereus ATCC 14579 that contains two genes encoding activities not previously described in gram-positive bacteria. One gene encodes a UDP-N-acetylglucosamine C4,6-dehydratase, (abbreviated Pdeg) that converts UDP-GlcNAc to UDP-4-keto-4,6-d-deoxy-GlcNAc (UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose); and the second encodes a UDP-4-reductase (abbr. Preq) that converts UDP-4-keto-4,6-d-deoxy-GlcNAc to UDP-N-acetyl-quinovosamine in the presence of NADPH. Biochemical studies established that the sequential Pdeg and Preq reaction product is UDP-d-QuiNAc as determined by mass spectrometry and one- and two-dimensional NMR experiments. Also, unambiguous evidence for the conversions of the dehydratase product, UDP-α-d-4-keto-4,6-deoxy-GlcNAc, to UDP-α-d-QuiNAc was obtained using real-time 1H-NMR spectroscopy and mass spectrometry. The two genes overlap by 4 nucleotides and similar operon organization and identical gene sequences were also identified in a few other Bacillus species suggesting they may have similar roles in the lifecycle of this class of bacteria important to human health. Our results provide new information about the ability of Bacilli to form UDP-QuiNAc and will provide insight to evaluate their role in the biology of Bacillus. PMID:26207987

  2. The Biosynthesis of UDP-D-QuiNAc in Bacillus cereus ATCC 14579.

    Directory of Open Access Journals (Sweden)

    Soyoun Hwang

    Full Text Available N-acetylquinovosamine (2-acetamido-2,6-di-deoxy-D-glucose, QuiNAc is a relatively rare amino sugar residue found in glycans of few pathogenic gram-negative bacteria where it can play a role in infection. However, little is known about QuiNAc-related polysaccharides in gram-positive bacteria. In a routine screen for bacillus glycan grown at defined medium, it was surprising to identify a QuiNAc residue in polysaccharides isolated from this gram-positive bacterium. To gain insight into the biosynthesis of these glycans, we report the identification of an operon in Bacillus cereus ATCC 14579 that contains two genes encoding activities not previously described in gram-positive bacteria. One gene encodes a UDP-N-acetylglucosamine C4,6-dehydratase, (abbreviated Pdeg that converts UDP-GlcNAc to UDP-4-keto-4,6-D-deoxy-GlcNAc (UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose; and the second encodes a UDP-4-reductase (abbr. Preq that converts UDP-4-keto-4,6-D-deoxy-GlcNAc to UDP-N-acetyl-quinovosamine in the presence of NADPH. Biochemical studies established that the sequential Pdeg and Preq reaction product is UDP-D-QuiNAc as determined by mass spectrometry and one- and two-dimensional NMR experiments. Also, unambiguous evidence for the conversions of the dehydratase product, UDP-α-D-4-keto-4,6-deoxy-GlcNAc, to UDP-α-D-QuiNAc was obtained using real-time 1H-NMR spectroscopy and mass spectrometry. The two genes overlap by 4 nucleotides and similar operon organization and identical gene sequences were also identified in a few other Bacillus species suggesting they may have similar roles in the lifecycle of this class of bacteria important to human health. Our results provide new information about the ability of Bacilli to form UDP-QuiNAc and will provide insight to evaluate their role in the biology of Bacillus.

  3. Arabidopsis thaliana AtUTr7 Encodes a Golgi-Localized UDP-Glucose/UDP-Galactose Transporter that Affects Lateral Root Emergence

    Institute of Scientific and Technical Information of China (English)

    Michael Handford; Cecilia Rodríguez-Furlán; Lorena Marchant; Marcelo Segura; Daniela Gómez; Elena Alvarez-Buyll; Guang-Yan Xiong; Markus Pauly; Ariel Orellana

    2012-01-01

    Nucleotide sugar transporters (NSTs) are antiporters comprising a gene family that plays a fundamental role in the biosynthesis of complex cell wall polysaccharides and glycoproteins in plants.However,due to the limited number of related mutants that have observable phenotypes,the biological function(s) of most NSTs in cell wall biosynthesis and assembly have remained elusive.Here,we report the characterization of AtUTr7 from Arabidopsis (Arabidopsis thaliana (L.) Heynh.),which is homologous to multi-specific UDP-sugar transporters from Drosophila melanogaster,humans,and Caenorhabditis elegans.We show that AtUTr7 possesses the common structural characteristics conserved among NSTs.Using a green fluorescent protein (GFP) tagged version,we demonstrate that AtUTr7 is localized in the Golgi apparatus.We also show that AtUTr7 is widely expressed,especially in the roots and in specific floral organs.Additionally,the results of an in vitro nucleotide sugar transport assay carried out with a tobacco and a yeast expression system suggest that AtUTr7 is capable of transferring UDP-Gal and UDP-GIc,but not a range of other UDP-and GDP-sugars,into the Golgi lumen.Mutants lacking expression of AtUTr7 exhibited an early proliferation of lateral roots as well as distorted root hairs when cultivated at high sucrose concentrations.Furthermore,the distribution of homogalacturonan with a low degree of methyl esterification differed in lateral root tips of the mutant compared to wild-type plants,although additional analytical procedures revealed no further differences in the composition of the root cell walls.This evidence suggests that the transport of UDP-Gal and UDP-GIc into the Golgi under conditions of high root biomass production plays a role in lateral root and root hair development.

  4. The uridine diphosphate glucuronosyltransferases: quantitative structure-activity relationships for hydroxyl polychlorinated biphenyl substrates

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Degao [Dalian University of Technology, Department of Environmental Science and Technology, Dalian (China)

    2005-10-01

    Quantitative structure-activity relationships (QSARs), which relate the glucuronidation of hydroxyl polychlorinated biphenyls (OH-PCBs) - catalyzed by the uridine diphosphate glucuronosyltransferases (UGTs) - to their physicochemical properties and molecular structural parameters, can be used to predict the rate constants and interpret the mechanism of glucuronidation. In this study, QSARs have been developed that use 23 semi-empirical calculated quantum chemical descriptors to predict the logarithms of the constants 1/K{sub m} and V{sub max}, related to enzyme kinetics. A partial least squares regression method was used to select the optimal set of descriptors to minimize the multicollinearity between the descriptors, as well as to maximize the cross-validated coefficient (Q{sup 2} {sub cum}) values. The key descriptors affecting log(1/K{sub m}) were E{sub lumo}- E{sub homo} (the energy gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital) and q{sub C}{sup -} (the largest negative net atomic charge on a carbon atom), while the key descriptors affecting log V{sub max} were the polarizability {alpha}, the Connolly solvent-excluded volume (CSEV), and logP (the logarithm of the partition coefficient for octanol/water). From the results obtained it can be concluded that hydrophobic and electronic aspects of OH-PCBs are important in the glucuronidation of OH-PCBs. (orig.)

  5. Separation by FPLC chromatofocusing of UDP-glucosyltransferases from three developmental stages of Drosophila melanogaster.

    Science.gov (United States)

    Rausell, C; Llorca, J; Real, M D

    1997-01-01

    Variation of UDP-glucosyltransferase activity, during Drosophila melanogaster development, was analyzed. The endogenous metabolite xanthurenic acid and the xenobiotic compounds 1-naphthol and 2-naphthol were used as substrates. Developmentally regulated differences were observed for the three substrates, suggesting the presence of UDP-glucosyltransferase isoenzymes. This was further confirmed by FPLC chromatofocusing on a Mono P column: seven peaks of UDP-glucosyltransferase activity (pHs: > or = 6.3, 5.8, 5.5, 4.9, 4.5, 4.2, < or = 4.0) with either single or overlapping substrate specificity were detected. A single xanthurenic acid:UDP-glucosyltransferase activity (pl 5.8) was found throughout development. In contrast, a gradual increase in the number of 2-napthol:UDP-glucosyltransferase-isoenzymes (pl from 6.3 to 4.0) was observed during development, whereas no isoenzymes specific for 1-naphthol were resolved. Based on the distribution and substrate specificity of the eluted peaks in the three developmental stages analyzed, the presence of seven or possibly eight UDP-glucosyltransferase isoenzymes is proposed.

  6. Reproductive responses and productive characteristics in ewes supplemented with detoxified castor meal for a long period

    Directory of Open Access Journals (Sweden)

    Liliane Moreira Silva

    2014-08-01

    Full Text Available The objective of the present study was to evaluate the effects of supplementation with detoxified castor meal (DCM in the diet of ewes during pregnancy, partum, and post-partum on the weight development of their offspring and at slaughter. The study included 56 ewes with synchronized estrus that were naturally mated. At the beginning of pregnancy and in post-partum, hepatic and renal function-related parameters and progesterone levels were measured. At slaughter, the proximate composition and fatty acid profile were determined in the loin of ewes. There was no effect of diet on reproductive response after estrus synchronization. At the beginning of pregnancy, albumin and creatinine levels were lower in the DCM group. Supplementation with DCM did not alter the weight or body condition of ewes at partum. However, at weaning, the DCM group showed a higher loin-eye area (LEA in relation to the group fed diets without detoxified castor meal (WDCM. At partum, as well as at weaning, the offspring of the ewes supplemented with DCM had a larger LEA than the WDCM group. In post-partum, levels of glucose, urea, protein, and cholesterol were lower in the DCM group. The return to cyclicity was similar in both groups, with an average of 47 days after partum. At slaughter, neither anatomical and carcass components nor the results of the proximate analysis were affected by the type of diet, except for an increase in heptadecanoic acid in the DCM group. Supplementation with detoxified castor meal in the diet of ewes does not affect lambing, pregnancy, prolificacy, return to cyclicity, milk production, blood biochemical parameters, or carcass characteristics.

  7. Molecular cloning of a novel glucuronokinase/putative pyrophosphorylase from zebrafish acting in an UDP-glucuronic acid salvage pathway.

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    Roman Gangl

    Full Text Available In animals, the main precursor for glycosaminoglycan and furthermore proteoglycan biosynthesis, like hyaluronic acid, is UDP-glucuronic acid, which is synthesized via the nucleotide sugar oxidation pathway. Mutations in this pathway cause severe developmental defects (deficiency in the initiation of heart valve formation. In plants, UDP-glucuronic acid is synthesized via two independent pathways. Beside the nucleotide sugar oxidation pathway, a second minor route to UDP-glucuronic acid exist termed the myo-inositol oxygenation pathway. Within this myo-inositol is ring cleaved into glucuronic acid, which is subsequently converted to UDP-glucuronic acid by glucuronokinase and UDP-sugar pyrophosphorylase. Here we report on a similar, but bifunctional enzyme from zebrafish (Danio rerio which has glucuronokinase/putative pyrophosphorylase activity. The enzyme can convert glucuronic acid into UDP-glucuronic acid, required for completion of the alternative pathway to UDP-glucuronic acid via myo-inositol and thus establishes a so far unknown second route to UDP-glucuronic acid in animals. Glucuronokinase from zebrafish is a member of the GHMP-kinase superfamily having unique substrate specificity for glucuronic acid with a Km of 31 ± 8 µM and accepting ATP as the only phosphate donor (Km: 59 ± 9 µM. UDP-glucuronic acid pyrophosphorylase from zebrafish has homology to bacterial nucleotidyltransferases and requires UTP as nucleosid diphosphate donor. Genes for bifunctional glucuronokinase and putative UDP-glucuronic acid pyrophosphorylase are conserved among some groups of lower animals, including fishes, frogs, tunicates, and polychaeta, but are absent from mammals. The existence of a second pathway for UDP-glucuronic acid biosynthesis in zebrafish likely explains some previous contradictory finding in jekyll/ugdh zebrafish developmental mutants, which showed residual glycosaminoglycans and proteoglycans in knockout mutants of UDP

  8. Crystal structures of Mycobacteria tuberculosis and Klebsiella pneumoniae UDP-galactopyranose mutase in the oxidised state and Klebsiella pneumoniae UDP-galactopyranose mutase in the (active) reduced state.

    Science.gov (United States)

    Beis, Konstantinos; Srikannathasan, Velupillai; Liu, Huanting; Fullerton, Stephen W B; Bamford, Vicki A; Sanders, David A R; Whitfield, Chris; McNeil, Mike R; Naismith, James H

    2005-05-13

    Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the d-galactofuranose sugar found in bacterial and parasitic cell walls, including those of many pathogens. UDP-Galf is made from UDP-galactopyranose by the enzyme UDP-galactopyranose mutase. The enzyme requires the reduced FADH- co-factor for activity. The structure of the Mycobacterium tuberculosis mutase with FAD has been determined to 2.25 A. The structures of Klebsiella pneumoniae mutase with FAD and with FADH- bound have been determined to 2.2 A and 2.35 A resolution, respectively. This is the first report of the FADH(-)-containing structure. Two flavin-dependent mechanisms for the enzyme have been proposed, one, which involves a covalent adduct being formed at the flavin and the other based on electron transfer. Using our structural data, we have examined the two mechanisms. The electron transfer mechanism is consistent with the structural data, not surprisingly, since it makes fewer demands on the precise positioning of atoms. A model based on a covalent adduct FAD requires repositioning of the enzyme active site and would appear to require the isoalloxazine ring of FADH- to buckle in a particular way. However, the FADH- structure reveals that the isoalloxazine ring buckles in the opposite sense, this apparently requires the covalent adduct to trigger profound conformational changes in the protein or to buckle the FADH- opposite to that seen in the apo structure.

  9. A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

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    Matías D. Asención Diez

    2013-05-01

    Full Text Available In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase or unregulated UDP-Glc PPase (EC 2.7.7.9. In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.

  10. Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus.

    Science.gov (United States)

    Chen, Wendy Yiting; Marcellin, Esteban; Hung, Jacky; Nielsen, Lars Keld

    2009-07-03

    The molecular weight of hyaluronan is important for its rheological and biological function. The molecular mechanisms underlying chain termination and hence molecular weight control remain poorly understood, not only for hyaluronan synthases but also for other beta-polysaccharide synthases, e.g. cellulose, chitin, and 1,3-betaglucan synthases. In this work, we manipulated metabolite concentrations in the hyaluronan pathway by overexpressing the five genes of the hyaluronan synthesis operon in Streptococcus equi subsp. zooepidemicus. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased molecular weight, whereas overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased molecular weight. The highest molecular mass observed was at 3.4 +/- 0.1 MDa twice that observed in the wild-type strain, 1.8 +/- 0.1 MDa. The data indicate that (a) high molecular weight is achieved when an appropriate balance of UDP-N-acetylglucosamine and UDP-glucuronic acid is achieved, (b) UDP-N-acetylglucosamine exerts the dominant effect on molecular weight, and (c) the wild-type strain has suboptimal levels of UDP-N-acetylglucosamine. Consistent herewith molecular weight correlated strongly (rho = 0.84, p = 3 x 10(-5)) with the concentration of UDP-N-acetylglucosamine. Data presented in this paper represent the first model for hyaluronan molecular weight control based on the concentration of activated sugar precursors. These results can be used to engineer strains producing high molecular weight hyaluronan and may provide insight into similar polymerization mechanisms in other polysaccharides.

  11. Heterologous expression of active human uridine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells

    Institute of Scientific and Technical Information of China (English)

    Ya-Kun Chen; Xin Li; Shu-Qing Chen; Su Zeng

    2005-01-01

    AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR)using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418.The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.

  12. Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: a strategy to promote cartilage repair.

    Science.gov (United States)

    Venkatesan, N; Barré, L; Benani, A; Netter, P; Magdalou, J; Fournel-Gigleux, S; Ouzzine, M

    2004-12-28

    Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, beta1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1beta down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by (35)S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1beta-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1beta-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1beta. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.

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

    Science.gov (United States)

    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.

  14. Lightweight UDP Pervasive Protocol in Smart Home Environment Based on Labview

    Science.gov (United States)

    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.

  15. Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase.

    Science.gov (United States)

    Kim, Hun; Choi, Jongkeun; Kim, Truc; Lokanath, Neratur K; Ha, Sung Chul; Suh, Se Won; Hwang, Hye-Yeon; Kim, Kyeong Kyu

    2010-04-01

    UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg(2+)-bound forms at 2.9 A and 2.3 A resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg(2+) ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.

  16. Genetic and structural validation of Aspergillus fumigatus UDP-N-acetylglucosamine pyrophosphorylase as an antifungal target.

    Science.gov (United States)

    Fang, Wenxia; Du, Ting; Raimi, Olawale G; Hurtado-Guerrero, Ramon; Urbaniak, Michael D; Ibrahim, Adel F M; Ferguson, Michael A J; Jin, Cheng; van Aalten, Daan M F

    2013-08-01

    The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme.

  17. Glucuronidation of the oxidative cytochrome P450-mediated phenolic metabolites of the endocrine disruptor pesticide: methoxychlor by human hepatic UDP-glucuronosyl transferases.

    Science.gov (United States)

    Hazai, Eszter; Gagne, Peter V; Kupfer, David

    2004-07-01

    Methoxychlor, a currently used pesticide, is a proestrogen exhibiting estrogenic activity in mammals in vivo. Methoxychlor undergoes oxidative metabolism by cytochromes P450, yielding 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (bis-OH-M) as main metabolites. Since humans may be exposed to these estrogenic metabolites, which are potential substrates of UDP-glucuronosyltransferases (UGTs), their glucuronide conjugation was investigated with human liver preparations and individual UGTs. Incubation of both mono-OH-M and bis-OH-M with human liver microsomes formed monoglucuronides. The structures of the glucuronides were identified by liquid chromatography/tandem mass spectometry. Examination of cDNA-expressed recombinant human hepatic UGTs revealed that several catalyze glucuronidation of both compounds. Among the cDNA-expressed UGT1A enzymes, UGT1A9 seemed to be the main catalyst of formation of mono-OH-M-glucuronide, whereas UGT1A3 seemed to be the most active in bis-OH-M-glucuronide formation. Furthermore, the chiral selectivity of mono-OH-M glucuronidation was examined. The results of the incubation of single enantiomers generally agreed with the chiral analyses of mono-OH-M derived from the glucuronidase digestion of the glucuronides of the racemic mono-OH-M. There was a relatively slight but consistent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9, and UGT2B15 enzymes for glucuronidation of the S- over the R-mono-OH-M, whereas in human liver microsomes differences were observed among donors in generating the respective R/S-mono-OH-M ratio. Since it was previously shown that human liver microsomes demethylate methoxychlor mainly into S-mono-OH-M, the observation that UGT1A isoforms preferentially glucuronidate the S-mono-OH-M suggests a suitable mechanism for eliminating this major enantiomer. This enantiomeric preference, however, is not extended to all samples of

  18. Metabolic engineering of Lactobacillus casei for production of UDP-N-acetylglucosamine.

    Science.gov (United States)

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

    2012-07-01

    UDP-sugars are used as glycosyl donors in many enzymatic glycosylation processes. In bacteria UDP-N-acetylglucosamine (UDP-GlcNAc) is synthesized from fructose-6-phosphate 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). In this work several metabolic engineering strategies, aimed to increment UDP-GlcNAc biosynthesis, were applied in the probiotic bacterium Lactobacillus casei strain BL23. This strain does not produce exopolysaccharides, therefore it could be a suitable host for the production of oligosaccharides. The genes glmS, glmM, and glmU coding for GlmS, GlmM, and GlmU activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous over-expression. The recombinant L. casei strain over-expressing simultaneously the genes glmM and glmS showed a 3.47 times increase in GlmS activity and 6.43 times increase in GlmM activity with respect to the control strain. Remarkably, these incremented activities resulted in about fourfold increase of the UDP-GlcNAc pool. In L. casei BL23 wild type strain transcriptional analyses showed that glmM and glmU are constitutively transcribed. By contrast, glmS transcription is down-regulated with a 21-fold decrease of glmS mRNA in cells cultured with N-acetylglucosamine as the sole carbon source compared to cells cultured with glucose. Our results revealed for the first time that GlmS, GlmM, and GlmU are responsible for UDP-GlcNAc biosynthesis in lactobacilli. Copyright © 2012 Wiley Periodicals, Inc.

  19. Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120.

    Science.gov (United States)

    Kawano, Yusuke; Sekine, Midori; Ihara, Masaki

    2014-05-01

    Exopolysaccharides produced by photosynthetic cyanobacteria have received considerable attention in recent years for their potential applications in the production of renewable biofuels. Particularly, cyanobacterial cellulose is one of the most promising products because it is extracellularly secreted as a non-crystalline form, which can be easily harvested from the media and converted into glucose units. In cyanobacteria, the production of UDP-glucose, the cellulose precursor, is a key step in the cellulose synthesis pathway. UDP-glucose is synthesized from UTP and glucose-1-phosphate (Glc-1P) by UDP-glucose pyrophosphorylase (UGPase), but this pathway in cyanobacteria has not been well characterized. Therefore, to elucidate the overall cellulose biosynthesis pathway in cyanobacteria, we studied the putative UGPase All3274 and seven other putative NDP-sugar pyrophosphorylases (NSPases), All4645, Alr2825, Alr4491, Alr0188, Alr3400, Alr2361, and Alr3921 of Anabaena sp. PCC 7120. Assays using the purified recombinant proteins revealed that All3274 exhibited UGPase activity, All4645, Alr2825, Alr4491, Alr0188, and Alr3921 exhibited pyrophosphorylase activities on ADP-glucose, CDP-glucose, dTDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine, respectively. Further characterization of All3274 revealed that the kcat for UDP-glucose formation was one or two orders lower than those of other known UGPases. The activity and dimerization tendency of All3274 increased at higher enzyme concentrations, implying catalytic activation by dimerization. However, most interestingly, All3274 dimerization was inhibited by UTP and Glc-1P, but not by UDP-glucose. This study presents the first in vitro characterization of a cyanobacterial UGPase, and provides insights into biotechnological attempts to utilize the photosynthetic production of cellulose from cyanobacteria.

  20. Increased turnover of dopamine in caudate nucleus of detoxified alcoholic patients

    DEFF Research Database (Denmark)

    Kumakura, Yoshitaka; Gjedde, Albert; Caprioli, Daniele

    2013-01-01

    (k(loss)), and we also calculated the total distribution volume of decarboxylated metabolites and unmetabolized FDOPA as a steady-state index of the dopamine storage capacity (V(d)) in brain. The craving scores in the 12 alcoholics correlated positively with the rate of loss (k(loss)) in the left...... ventral striatum. We conclude that craving is most pronounced in the individuals with relatively rapid dopamine turnover in the left ventral striatum. The blood-brain clearance rate (K), corrected for subsequent loss of radiolabeled molecules from brain, was completely normal throughout the brain...... for rapid dopamine turnover in the ventral striatum subserves craving and reward-dependence, leading to an acquired state of increased dopamine turnover in the dorsal striatum of detoxified alcoholic patients....

  1. Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering.

    Science.gov (United States)

    Mathapati, Santosh; Bishi, Dillip Kumar; Guhathakurta, Soma; Cherian, Kotturathu Mammen; Venugopal, Jayarama Reddy; Ramakrishna, Seeram; Verma, Rama Shanker

    2013-04-01

    Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p>0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP-G-CA-ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP-G-CA-ET). DBP-G-CA-ET exhibited a significant (p>0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (ppericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Pampa, K.J., E-mail: sagarikakj@gmail.com [Department of Studies in Microbiology, University of Mysore, Mysore 570 006 (India); Lokanath, N.K. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Girish, T.U. [Department of General Surgery, JSS Medical College and Hospital, JSS University, Mysore 570 015 (India); Kunishima, N. [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148 (Japan); Rai, V.R. [Department of Studies in Microbiology, University of Mysore, Mysore 570 006 (India)

    2014-10-24

    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 by 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.

  3. Efflux Transport Characterization of Resveratrol Glucuronides in UDP-Glucuronosyltransferase 1A1 Transfected HeLa Cells: Application of a Cellular Pharmacokinetic Model to Decipher the Contribution of Multidrug Resistance-Associated Protein 4.

    Science.gov (United States)

    Wang, Shuai; Li, Feng; Quan, Enxi; Dong, Dong; Wu, Baojian

    2016-04-01

    Resveratrol undergoes extensive metabolism to form biologically active glucuronides in humans. However, the transport mechanisms for resveratrol glucuronides are not fully established. Here, we aimed to characterize the efflux transport of resveratrol glucuronides using UGT1A1-overexpressing HeLa cells (HeLa1A1 cells), and to determine the contribution of multidrug resistance-associated protein (MRP) 4 to cellular excretion of the glucuronides. Two glucuronide isomers [i.e., resveratrol 3-O-glucuronide (R3G) and resveratrol 4'-O-glucuronide (R4'G)] were excreted into the extracellular compartment after incubation of resveratrol (1-100 μM) with HeLa1A1 cells. The excretion rate was linearly related to the level of intracellular glucuronide, indicating that glucuronide efflux was a nonsaturable process. MK-571 (a dual inhibitor of UGT1A1 and MRPs) significantly decreased the excretion rates of R3G and R4'G while increasing their intracellular levels. Likewise, short-hairpin RNA (shRNA)-mediated silencing of MRP4 caused a significant reduction in glucuronide excretion but an elevation in glucuronide accumulation. Furthermore, β-glucuronidase expressed in the cells catalyzed the hydrolysis of the glucuronides back to the parent compound. A cellular pharmacokinetic model integrating resveratrol transport/metabolism with glucuronide hydrolysis/excretion was well fitted to the experimental data, allowing derivation of the efflux rate constant values in the absence or presence of shRNA targeting MRP4. It was found that a large percentage of glucuronide excretion (43%-46%) was attributed to MRP4. In conclusion, MRP4 participated in cellular excretion of R3G and R4'G. Integration of mechanistic pharmacokinetic modeling with transporter knockdown was a useful method to derive the contribution percentage of an exporter to overall glucuronide excretion.

  4. GLUCURONIDATION OF N-OH-2-AMINO-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE (PHIP) BY HUMAN MICROSOMAL UDP-GLUCURONOSYLTRANSFERASES: IDENTIFICATION OF SPECIFIC UGT1A FAMILY ISOFORMS INVOLVED. (R825280)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  5. Pen and Pal are nucleotide-sugar dehydratases that convert UDP-GlcNAc to UDP-6-deoxy-D-GlcNAc-5,6-ene and then to UDP-4-keto-6-deoxy-L-AltNAc for CMP-pseudaminic acid synthesis in Bacillus thuringiensis.

    Science.gov (United States)

    Li, Zi; Hwang, Soyoun; Ericson, Jaime; Bowler, Kyle; Bar-Peled, Maor

    2015-01-09

    CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-D-GlcNAc-5,6-ene. Pen contains strongly bound NADP(+) and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-D-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-L-AltNAc. Pal is NAD(+)-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-L-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.

  6. Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis[OPEN

    Science.gov (United States)

    Ebert, Berit; Rautengarten, Carsten; Guo, Xiaoyuan; Xiong, Guangyan; Stonebloom, Solomon; Smith-Moritz, Andreia M.; Herter, Thomas; Chan, Leanne Jade G.; Adams, Paul D.; Petzold, Christopher J.; Pauly, Markus; Willats, William G.T.; Heazlewood, Joshua L.; Scheller, Henrik Vibe

    2015-01-01

    Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis. PMID:25804536

  7. In vivo induction of phase II detoxifying enzymes, glutathione transferase and quinone reductase by citrus triterpenoids

    Directory of Open Access Journals (Sweden)

    Ahmad Hassan

    2010-09-01

    Full Text Available Abstract Background Several cell culture and animal studies demonstrated that citrus bioactive compounds have protective effects against certain types of cancer. Among several classes of citrus bioactive compounds, limonoids were reported to prevent different types of cancer. Furthermore, the structures of citrus limonoids were reported to influence the activity of phase II detoxifying enzymes. The purpose of the study was to evaluate how variations in the structures of citrus limonoids (namely nomilin, deacetyl nomilin, and isoobacunoic acid and a mixture of limonoids would influence phase II enzyme activity in excised tissues from a mouse model. Methods In the current study, defatted sour orange seed powder was extracted with ethyl acetate and subjected to silica gel chromatography. The HPLC, NMR and mass spectra were used to elucidate the purity and structure of compounds. Female A/J mice were treated with three limonoids and a mixture in order to evaluate their effect on phase II enzymes in four different tissues. Assays for glutathione S-transferase and NAD(PH: quinone reductase (QR were used to evaluate induction of phase II enzymatic activity. Results The highest induction of GST against 1-chloro-2,4-dinitrobenzene (CDNB was observed in stomach (whole, 58% by nomilin, followed by 25% isoobacunoic acid and 19% deacetyl nomilin. Deacetyl nomilin in intestine (small as well as liver significantly reduced GST activity against CDNB. Additionally isoobacunoic acid and the limonoid mixture in liver demonstrated a significant reduction of GST activity against CDNB. Nomilin significantly induced GST activity against 4-nitroquinoline 1-oxide (4NQO, intestine (280% and stomach (75% while deacetyl nomilin showed significant induction only in intestine (73%. Induction of GST activity was also observed in intestine (93% and stomach (45% treated with the limonoid mixture. Finally, a significant induction of NAD(PH: quinone reductase (QR activity was

  8. Detoxified Haemophilus ducreyi cytolethal distending toxin and induction of toxin specific antibodies in the genital tract.

    Science.gov (United States)

    Lundqvist, Annika; Fernandez-Rodrigues, Julia; Ahlman, Karin; Lagergård, Teresa

    2010-08-16

    Haemophilus ducreyi causes genital ulceration (chancroid), a sexually transmitted infection and still an important factor which contributes to the spread of HIV in developing countries. The bacterium produces a cytolethal distending toxin (HdCDT) causing cell cycle arrest and apoptosis/necrosis of human cells and contributes to the aggravation of ulcers. The aim of the study was to induce toxin-neutralizing antibodies in the genital tract of mice. Repeated subcutaneous (sc) immunisations with 5-10microg active HdCDT induced low levels of serum anti-HdCDT IgG without neutralizing capacity. High levels of specific IgG1 antibodies in serum and genital tract were generated after sc immunisations with 10microg formaldehyde detoxified HdCDT toxoid alone and the addition of aluminium salts or RIBI (based on the lipid A moiety) as adjuvant further increased the level of serum antibodies. A high correlation was found between elevated levels of anti-HdCDT IgG in sera, the level of neutralizing activity and the antibody level in genital tract (r=0.8). Thus, induction of high antibody levels specific to HdCDT in the genital tissue can be achieved by parenteral immunisation with the toxoid. The HdCDT toxoid can be considered as a candidate component in vaccine against chancroid.

  9. Chemical composition and fermentation characteristics of sugar cane silage enriched with detoxified castor bean meal

    Directory of Open Access Journals (Sweden)

    A.C. Oliveira

    2015-02-01

    Full Text Available This experiment was designed to evaluate the effects of the inclusion of detoxified castor bean meal (CM content on the chemical-bromatological composition, as well as the fermentation characteristics and dry matter losses of sugarcane silage. The treatments consisted of four levels (0, 7, 14 and 21% natural matter of addition of castor bean meal. The design was completely randomized, with five replicates. The material was ensiled in PVC silos of 50-cm in height and 10-cm in diameter, which were opened after 60 days. The density of fodder maintained in the silos was equal to 750kg of natural matter/m3. In order to quantify the gas and effluent losses, the silos were weighed at the beginning and at the end of the experiment. The addition of castor bean meal has increased the levels of DM and crude protein and reduced the levels of neutral detergent fiber, N-NH3, ethanol, and gas and effluent losses from silages, but did not affect pH values. During ensiling, alcoholic fermentation was controlled with the inclusion of the additive.

  10. Serotonergic function, substance craving, and psychopathology in detoxified alcohol-addicted males undergoing tryptophan depletion.

    Science.gov (United States)

    Wedekind, Dirk; Herchenhein, Thomas; Kirchhainer, Julia; Bandelow, Borwin; Falkai, Peter; Engel, Kirsten; Malchow, Berend; Havemann-Reinecke, Ursula

    2010-12-01

    Alcohol addiction is associated with alterations of central nervous dopaminergic and serotonergic functions. Acute tryptophan depletion has not yet been applied in detoxified alcohol-addicted patients in order to investigate its impact on psychopathology, psychoneuroendocrinology, and substance craving behaviour. 25 alcohol-addicted males randomly either received a tryptophan-free or tryptophan-containing amino acid drink and 7 days later the respective other drink. Anxiety, depression, and craving were assessed before and 5 h after the drink. Tryptophan, 5-HIAA, dopamine, norepinephrine, epinephrine, and HVA in serum were measured before and after both treatments. Nocturnal urinary cortisol measurements and genotyping for the HTTLPR polymorphism of the SLC6A4 gene were performed. Tryptophan depletion resulted in a significant reduction of total and free serum tryptophan while the tryptophan-rich drink increased serum levels. Both treatments caused a significant increase of serum serotonin levels, however, serum 5-HIAA was decreased after depletion but increased after sham depletion. Dopamine and norepinephrine were elevated after tryptophan depletion and sham. Depletion increased depression scores (MADRS), while the full amino acid drink improved state and trait anxiety ratings (STAI) and substance craving. Urinary cortisol excretion was not affected by both treatments. Patients with the ll genotype of the serotonin transporter gene displayed lower baseline tryptophan levels compared to patients with the heterozygous genotype. Results suggest an impaired serotonergic function in alcohol-addicted males.

  11. Detoxifying antitumoral drugs via nanoconjugation: the case of gold nanoparticles and cisplatin.

    Directory of Open Access Journals (Sweden)

    Joan Comenge

    Full Text Available Nanoparticles (NPs have emerged as a potential tool to improve cancer treatment. Among the proposed uses in imaging and therapy, their use as a drug delivery scaffold has been extensively highlighted. However, there are still some controversial points which need a deeper understanding before clinical application can occur. Here the use of gold nanoparticles (AuNPs to detoxify the antitumoral agent cisplatin, linked to a nanoparticle via a pH-sensitive coordination bond for endosomal release, is presented. The NP conjugate design has important effects on pharmacokinetics, conjugate evolution and biodistribution and results in an absence of observed toxicity. Besides, AuNPs present unique opportunities as drug delivery scaffolds due to their size and surface tunability. Here we show that cisplatin-induced toxicity is clearly reduced without affecting the therapeutic benefits in mice models. The NPs not only act as carriers, but also protect the drug from deactivation by plasma proteins until conjugates are internalized in cells and cisplatin is released. Additionally, the possibility to track the drug (Pt and vehicle (Au separately as a function of organ and time enables a better understanding of how nanocarriers are processed by the organism.

  12. Episodic memory in detoxified alcoholics: contribution of grey matter microstructure alteration.

    Directory of Open Access Journals (Sweden)

    Sandra Chanraud

    Full Text Available Even though uncomplicated alcoholics may likely have episodic memory deficits, discrepancies exist regarding to the integrity of brain regions that underlie this function in healthy subjects. Possible relationships between episodic memory and 1 brain microstructure assessed by magnetic resonance diffusion tensor imaging (DTI, 2 brain volumes assessed by voxel-based morphometry (VBM were investigated in uncomplicated, detoxified alcoholics.Diffusion and morphometric analyses were performed in 24 alcohol dependent men without neurological or somatic complications and in 24 healthy men. The mean apparent coefficient of diffusion (ADC and grey matter volumes were measured in the whole brain. Episodic memory performance was assessed using a French version of the Free and Cued Selective Reminding Test (FCSRT. Correlation analyses between verbal episodic memory, brain microstructure, and brain volumes were carried out using SPM2 software.In those with alcohol dependence, higher ADC was detected mainly in frontal, temporal and parahippocampal regions, and in the cerebellum. In alcoholics, regions with higher ADC typically also had lower grey matter volume. Low verbal episodic memory performance in alcoholism was associated with higher mean ADC in parahippocampal areas, in frontal cortex and in the left temporal cortex; no correlation was found between regional volumes and episodic memory scores. Regression analyses for the control group were not significant.These findings support the hypothesis that regional microstructural but no macrostructural alteration of the brain might be responsible, at least in part, for episodic memory deficits in alcohol dependence.

  13. Preclinical evaluation of a chemically detoxified pneumolysin as pneumococcal vaccine antigen

    Science.gov (United States)

    Hermand, Philippe; Vandercammen, Annick; Mertens, Emmanuel; Di Paolo, Emmanuel; Verlant, Vincent; Denoël, Philippe; Godfroid, Fabrice

    2017-01-01

    ABSTRACT The use of protein antigens able to protect against the majority of Streptococcus pneumoniae serotypes is envisaged as stand-alone and/or complement to the current capsular polysaccharide-based pneumococcal vaccines. Pneumolysin (Ply) is a key virulence factor that is highly conserved in amino acid sesec-typsecquence across pneumococcal serotypes, and therefore may be considered as a vaccine target. However, native Ply cannot be used in vaccines due to its intrinsic cytolytic activity. In the present work a completely, irreversibly detoxified pneumolysin (dPly) has been generated using an optimized formaldehyde treatment. Detoxi-fication was confirmed by dPly challenge in mice and histological analysis of the injection site in rats. Immunization with dPly elicited Ply-specific functional antibodies that were able to inhibit Ply activity in a hemolysis assay. In addition, immunization with dPly protected mice against lethal intranasal challenge with Ply, and intranasal immunization inhibited nasopharyngeal colonization after intranasal challenge with homologous or heterologous pneumococcal strain. Our findings supported dPly as a valid candidate antigen for further pneumococcal vaccine development. PMID:27768518

  14. The Antagonistic Action of Heat-Clearing and Detoxifying Chinese Drugs on Endotoxins

    Institute of Scientific and Technical Information of China (English)

    张霞; 崔乃杰; 王家泰

    2001-01-01

    @@In the recent decade, interest in treatment and prevention of many critical, severe and acute diseases caused by bacterial endotoxins has been aroused along with the advance of the knowledge on the nature of the endotoxin and the conditions involved. In abroad, attention has been mainly payed to raising antisera and monocolonal antibodies against the endotoxin and the induced mediators. However, the allergic reactions and the cost are still the problems. Till now, there is no drug that can antagonize endotoxin with high effectiveness and low toxicity. Clinical treatments are still confined in inhibiting or killing the pathogen, and correcting the internal environmental disturbance. Being less toxic and rich in resources with low cost and less side-effects, screening of effective Chinese drugs for antagonizing endotoxin is of important and practical significance. Endotoxin belongs to the category of toxic evils, or more precisely, the heat toxin in TCM. Therefore the application of heat-clearing and detoxifying Chinese drugs to antagonizing endotoxins is consistent with the theory of TCM. Some achievements in this field are reported as follows.

  15. Zero-valent iron pretreatment for detoxifying iodine in liquid crystal display (LCD) manufacturing wastewater.

    Science.gov (United States)

    Lee, J W; Cha, D K; Oh, Y K; Ko, K B; Song, J S

    2009-05-15

    This study investigated reductive transformation of iodine by zero-valent iron (ZVI), and the subsequent detoxification of iodine-laden wastewater. ZVI completely reduced aqueous iodine to non-toxic iodide. Respirometric bioassay illustrated that the presence of iodine increase the lag phase before the onset of oxygen consumption. The length of lag phase was proportional to increasing iodine dosage. The reduction products of iodine by ZVI did not exhibit any inhibitory effect on the biodegradation. The cumulative biological oxidation associated with iodine toxicity was closely fitted to Gompertz model. When iodine-laden wastewater was continuously fed to a bench-scale activated sludge unit, chemical oxygen demand (COD) removal efficiencies decreased from above 90% to below 80% along with a marked decrease in biomass concentration. On the other hand, the COD removal efficiency and biomass concentration remained constant in the integrated ZVI-activated sludge system. Respirometric bioassay with real iodine-laden LCD manufacturing wastewater demonstrated that ZVI was effective for detoxifying iodine and consequently enhancing biodegradability of wastewater. This result suggested that ZVI pretreatment may be a feasible option for the removal of iodine in LCD processing wastewater, instead of more costly processes such as adsorption and chemical oxidation, which are commonly in the iodine-laden LCD wastewater treatment facility.

  16. Potential chemoprevention activity of pterostilbene by enhancing the detoxifying enzymes in the HT-29 cell line.

    Science.gov (United States)

    Harun, Zaliha; Ghazali, Ahmad Rohi

    2012-01-01

    Detoxifying enzymes are present in most epithelial cells of the human gastrointestinal tract where they protect against xenobiotics which may cause cancer. Induction of examples such as glutathione S-transferase (GST) and its thiol conjugate, glutathione (GSH) as well as NAD(P)H: quinoneoxidoreductase (NQO1) facilitate the excretion of carcinogens and thus preventing colon carcinogenesis. Pterostilbene, an analogue of resveratrol, has demonstrated numerous pharmacological activities linked with chemoprevention. This study was conducted to investigate the potential of pterostilbene as a chemopreventive agent using the HT-29 colon cancer cell line to study the modulation of GST and NQO1 activities as well as the GSH level. Initially, our group, established the optimum dose of 24 hours pterostilbene treatment using MTT assays. Then, effects of pterostilbene (0-50 μM) on GST and NQO1 activity and GSH levels were determined using GST, NQO1 and Ellman assays, respectively. MTT assay of pterostilbene (0-100 μM) showed no cytotoxicity toward the HT-29 cell line. Treatment increased GST activity in the cell line significantly (p<0.05) at 12.5 and 25.0 μM. In addition, treatment at 50 μM increased the GSH level significantly (p<0.05). Pterostilbene also enhanced NQO1 activity significantly (p<0.05) at 12.5 μM and 50 μM. Hence, pterostilbene is a potential chemopreventive agent capable of modulation of detoxifiying enzyme levels in HT-29 cells.

  17. Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants

    Science.gov (United States)

    Gregori, Josep; Esteban, Juan I.; Cubero, María; Garcia-Cehic, Damir; Perales, Celia; Casillas, Rosario; Alvarez-Tejado, Miguel; Rodríguez-Frías, Francisco; Guardia, Jaume; Domingo, Esteban; Quer, Josep

    2013-01-01

    We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity. PMID:24391758

  18. Toxic Compound, Anti-Nutritional Factors and Functional Properties of Protein Isolated from Detoxified Jatropha curcas Seed Cake

    Directory of Open Access Journals (Sweden)

    Worapot Suntornsuk

    2010-12-01

    Full Text Available Jatropha curcas is a multipurpose tree, which has potential as an alternative source for biodiesel. All of its parts can also be used for human food, animal feed, fertilizer, fuel and traditional medicine. J. curcas seed cake is a low-value by-product obtained from biodiesel production. The seed cake, however, has a high amount of protein, with the presence of a main toxic compound: phorbol esters as well as anti-nutritional factors: trypsin inhibitors, phytic acid, lectin and saponin. The objective of this work was to detoxify J. curcas seed cake and study the toxin, anti-nutritional factors and also functional properties of the protein isolated from the detoxified seed cake. The yield of protein isolate was approximately 70.9%. The protein isolate was obtained without a detectable level of phorbol esters. The solubility of the protein isolate was maximal at pH 12.0 and minimal at pH 4.0. The water and oil binding capacities of the protein isolate were 1.76 g water/g protein and 1.07 mL oil/g protein, respectively. The foam capacity and stability, including emulsion activity and stability of protein isolate, had higher values in a range of basic pHs, while foam and emulsion stabilities decreased with increasing time. The results suggest that the detoxified J. curcas seed cake has potential to be exploited as a novel source of functional protein for food applications.

  19. Novel UDP-GalNAc Derivative Structures Provide Insight into the Donor Specificity of Human Blood Group Glycosyltransferase.

    Science.gov (United States)

    Wagner, Gerd K; Pesnot, Thomas; Palcic, Monica M; Jørgensen, Rene

    2015-12-25

    Two closely related glycosyltransferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the "tucked under" conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.

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

    CERN Document Server

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

    2015-01-01

    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.

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

    Science.gov (United States)

    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.

  2. An Intrusion Detection System Against UDP Flood Attack and Ping of Death Attack (DDOS in MANET

    Directory of Open Access Journals (Sweden)

    Ankur Ashok Acharya

    2016-04-01

    Full Text Available DDoS is one of the serious attacks in the ad hoc network. Among lot many DDoS attacks, UDP flood attack and Ping of death attack are considered to be important as these two attacks may cause severe damage to the network. To provide better security to the network, efficient intrusion detection (IDS system is required to monitor the network continuously, keeping track of malicious activities and policy violations and produce report to the network administrator. UDP flood attack and ping of death attack are given importance in this paper as they are not well addressed in the existing research works. Packet capture and packet decoder is used to identify the packets and retrieve the packet details. A threshold is set for each node that is connected to the network. If the packet flow into the node exceeds the threshold that is set then the administrator is notified about the same.

  3. Laccase enzyme detoxifies hydrolysates and improves biogas production from hemp straw and miscanthus.

    Science.gov (United States)

    Schroyen, Michel; Van Hulle, Stijn W H; Holemans, Sander; Vervaeren, Han; Raes, Katleen

    2017-07-27

    The impact of various phenolic compounds, vanillic acid, ferulic acid, p-coumaric acid and 4-hydroxybenzoic acid on anaerobic digestion of lignocellulosic biomass (hemp straw and miscanthus) was studied. Such phenolic compounds have been known to inhibit biogas production during anaerobic digestion. The different phenolic compounds were added in various concentrations: 0, 100, 500, 1000 and 2000mg/L. A difference in inhibition of biomethane production between the phenolic compounds was noted. Hydrolysis rate, during anaerobic digestion of miscanthus was inhibited up to 50% by vanillic acid, while vanillic acid had no influence on the initial rate of biogas production during the anaerobic digestion of hemp straw. Miscanthus has a higher lignin concentration (12-30g/100gDM) making it less accessible for degradation, and in combination with phenolic compounds released after harsh pretreatments, it can cause severe inhibition levels during the anaerobic digestion, lowering biogas production. To counter the inhibition, lignin degrading enzymes can be used to remove or degrade the inhibitory phenolic compounds. The interaction of laccase and versatile peroxidase individually with the different phenolic compounds was studied to have insight in the polymerization of inhibitory compounds or breakdown of lignocellulose. Hemp straw and miscanthus were incubated with 0, 100 and 500mg/L of the different phenolic compounds for 0, 6 and 24h and pretreated with the lignin degrading enzymes. A laccase pretreatment successfully detoxified the substrate, while versatile peroxidase however was inhibited by 100mg/L of each of the individual phenolic compounds. Finally a combination of enzymatic detoxification and subsequent biogas production showed that a decrease in phenolic compounds by laccase treatment can considerably lower the inhibition levels of the biogas production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Characterization of mouse UDP-glucose pyrophosphatase, a Nudix hydrolase encoded by the Nudt14 gene

    Energy Technology Data Exchange (ETDEWEB)

    Heyen, Candy A.; Tagliabracci, Vincent S.; Zhai, Lanmin [Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Roach, Peter J., E-mail: proach@iupui.edu [Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States)

    2009-12-25

    Recombinant mouse UDP-glucose pyrophosphatase (UGPPase), encoded by the Nudt14 gene, was produced in Escherichia coli and purified close to homogeneity. The enzyme catalyzed the conversion of [{beta}-{sup 32}P]UDP-glucose to [{sup 32}P]glucose-1-P and UMP, confirming that it hydrolyzed the pyrophosphate of the nucleoside diphosphate sugar to generate glucose-1-P and UMP. The enzyme was also active toward ADP-ribose. Activity is dependent on the presence of Mg{sup 2+} and was greatest at alkaline pH above 8. Kinetic analysis indicated a K{sub m} of {approx}4 mM for UDP-glucose and {approx}0.3 mM for ADP-ribose. Based on V{sub max}/K{sub m} values, the enzyme was {approx}20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of M{sub r} 54,000 from a monomer of 30,000 as judged by SDS-PAGE. The dimerization was not affected by the presence of glucose-1-P or UDP-glucose. Using antibodies raised against the recombinant protein, Western analysis indicated that UGPPase was widely expressed in mouse tissues, including skeletal muscle, liver, kidney, heart, lung, fat, heart and pancreas with a lower level in brain. It was generally present as a doublet when analyzed by SDS-PAGE, suggesting the occurrence of some form of post-translational modification. Efforts to interconvert the species by adding or inhibiting phosphatase activity were unsuccessful, leaving the nature of the modification unknown. Sequence alignments and database searches revealed related proteins in species as distant as Drosophila melanogaster and Caenorhabditis elegans.

  5. Activity and Crystal Structure of Arabidopsis thalianaUDP-N-Acetylglucosamine Acyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Sang Hoon; Chung, Hak Suk; Raetz, Christian R.H.; Garrett, Teresa A. (Vassar); (CUD- South Korea); (Duke)

    2012-08-31

    The UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase, encoded by lpxA, catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria, the (R)-3-hydroxyacyl-ACP-dependent acylation of the 3-OH group of UDP-GlcNAc. Recently, we demonstrated that the Arabidopsis thaliana orthologs of six enzymes of the bacterial lipid A pathway produce lipid A precursors with structures similar to those of Escherichia coli lipid A precursors [Li, C., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 11387-11392]. To build upon this finding, we have cloned, purified, and determined the crystal structure of the A. thaliana LpxA ortholog (AtLpxA) to 2.1 {angstrom} resolution. The overall structure of AtLpxA is very similar to that of E. coli LpxA (EcLpxA) with an {alpha}-helical-rich C-terminus and characteristic N-terminal left-handed parallel {beta}-helix (L{beta}H). All key catalytic and chain length-determining residues of EcLpxA are conserved in AtLpxA; however, AtLpxA has an additional coil and loop added to the L{beta}H not seen in EcLpxA. Consistent with the similarities between the two structures, purified AtLpxA catalyzes the same reaction as EcLpxA. In addition, A. thaliana lpxA complements an E. coli mutant lacking the chromosomal lpxA and promotes the synthesis of lipid A in vivo similar to the lipid A produced in the presence of E. coli lpxA. This work shows that AtLpxA is a functional UDP-GlcNAc acyltransferase that is able to catalyze the same reaction as EcLpxA and supports the hypothesis that lipid A molecules are biosynthesized in Arabidopsis and other plants.

  6. Human liver morphine UDP-glucuronyl transferase enantioselectivity and inhibition by opioid congeners and oxazepam.

    OpenAIRE

    Wahlström, A; Pacifici, G. M.; Lindström, B; Hammar, L.; Rane, A.

    1988-01-01

    1. Morphine uridine diphosphate glucuronyl transferase (UDP-GT) was studied in human liver microsomes. The (-)- and (+)-morphine enantiomers were used as substrates and inhibitors, such as oxazepam and various opioid congeners were employed to characterize the different glucuronidation pathways. The kinetics of the oxazepam inhibition were studied in the rat liver. 2. The overall glucuronidation of (+)-morphine was higher than that of (-)-morphine. The morphine congeners tested, potently inhi...

  7. Elucidation of substrate specificity in Aspergillus nidulans UDP-galactose-4-epimerase.

    Directory of Open Access Journals (Sweden)

    Sean A Dalrymple

    Full Text Available The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD(+ and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K m and k cat for the enzyme were determined to be 0.11 mM and 12.8 s(-1, respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.

  8. Potentiometric analysis of UDP-galactopyranose mutase: stabilization of the flavosemiquinone by substrate.

    Science.gov (United States)

    Fullerton, Stephen W B; Daff, Simon; Sanders, David A R; Ingledew, W John; Whitfield, Chris; Chapman, Stephen K; Naismith, James H

    2003-02-25

    UDP-galactopyranose mutase is a flavoprotein which catalyses the interconversion of UDP-galactopyranose and UDP-galactofuranose. The enzyme is of interest because it provides the activated biosynthetic precursor of galactofuranose, a key cell wall component of many bacterial pathogens. The reaction mechanism of this mutase is intriguing because the anomeric oxygen forms a glycosidic bond, which means that the reaction must proceed by a novel mechanism involving ring breakage and closure. The structure of the enzyme is known, but the mechanism, although speculated on, is not resolved. The overall reaction is electrically neutral but a crypto-redox reaction is suggested by the requirement that the flavin must adopt the reduced form for activity. Herein we report a thermodynamic analysis of the enzyme's flavin cofactor with the objective of defining the system and setting parameters for possible reaction schemes. The analysis shows that the neutral semiquinone (FADH(*)) is stabilized in the presence of substrate and the fully reduced flavin is the anionic FADH(-) rather than the neutral FADH(2). The anionic FADH(-) has the potential to act as a rapid 1-electron donor/acceptor without being slowed by a coupled proton transfer and is therefore an ideal crypto-redox cofactor.

  9. In vivo and in vitro analysis of the effect of various acid-secretion blockers on UDP-galactosyltransferase activities in rat gastric mucosa

    OpenAIRE

    2002-01-01

    Gastric mucus glycoprotein is important for the protection of gastric mucosa from acid. UDP-galactosyltransfer-ase (UDP-Gal-T) is a key enzyme for the synthesis of gastric mucus glycoprotein. In this study, we investigted the effects of five acid-secretion blockers, cimetidine, ranitidine, famotidine, roxatidine and omprazole on the UDP-Gal-T activity in rat gastric mucosa to clarify the interaction of the acid-secretion blocker and the gastric mucosal barrier. Intraperitoneal administration ...

  10. Separation and purification of uridine diphosphate-glucuronosyltransferases by chromatofocusing on a high-performance liquid chromatograph.

    Science.gov (United States)

    Takanashi, H; Homma, H; Matsui, M

    1989-06-01

    A rapid method for the separation and purification of uridine diphosphate-glucuronosyltransferases (GT) was developed with the use of chromatofocusing on a high-performance liquid chromatograph. GT isoenzymes solubilized from hepatic microsomes of Wistar rats were separated on a Mono P column, a pre-packed column for chromatofocusing. Using 4-nitrophenol, testosterone and androsterone as substrates, four fractions with different GT activities were separated in a pH gradient from 9.5 to 7.0. Two isoenzymes, testosterone GT and androsterone GT were purified to apparent homogeneity. They were eluted at pH 8.9 and 8.0 and had subunit molecular weight values of 50000 and 52000, respectively. Approximately 10 mg of solubilized microsomal proteins was applied and the elution was completed within 2 h. Addition of N-nitrodiethylamine, an in vitro activator of GT activity, enhanced the GT activity toward 4-nitrophenol in the three fractions. This chromatographic analysis confirmed the absence of androsterone GT isoenzyme in LA Wistar rats, a mutant strain in terms of androsterone glucuronidation.

  11. UDP-GalNAc :polypeptide N-acetylgalactosaminyltransferase 14%UDP-GalNAc:多肽N-乙酰氨基半乳糖转移酶-14

    Institute of Scientific and Technical Information of China (English)

    郭晓丹; 吴琛; 康现江

    2010-01-01

    UDP-GalNAc:多肽N-乙酰氨基半乳糖转移酶家族(简称GalNAc-T)是黏蛋白O-糖基化的起始酶,N-乙酰氨基半乳糖转移酶-14(GalNAc-T14)是该家族中最新发现的成员.近年来有人指出,O-糖基化可能与肿瘤的发生发展具有密切关系,因此对N-乙酰氨基半乳糖转移酶家族的研究也受到越来越多重视.本文主要综述了GalNAc-T14的命名、结构、分布、功能以及潜在的应用价值.

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

    Science.gov (United States)

    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/gcell dry weight ) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 gproduct /L, 86% yield (based on UDP), and a total turnover number of 103 gUDP-glc /gcell 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.

  13. The critical role of UDP-galactose-4-epimerase in osteoarthritis: modulating proteoglycans synthesis of the articular chondrocytes.

    Science.gov (United States)

    Wen, Yinxian; Qin, Jun; Deng, Yu; Wang, Hui; Magdalou, Jacques; Chen, Liaobin

    2014-10-03

    UDP-galactose-4-epimerase (GALE) is a key enzyme catalyzing the interconversion of UDP-glucose and UDP-galactose, as well as UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine, which are all precursors for the proteoglycans (PGs) synthesis. However, whether GALE is essential in cartilage homeostasis remains unknown. Therefore, we investigated the role of GALE in PGs synthesis of human articular chondrocytes, the GALE expression in OA, and the regulation of GALE expression by interleukin-1beta (IL-1β). Silencing GALE gene with specific siRNAs resulted in a markedly inhibition of PGs synthesis in human articular chondrocytes. GALE protein levels were also decreased in both human and rat OA cartilage, thus leading to losses of PGs contents. Moreover, GALE mRNA expression was stimulated by IL-1β in early phase, but suppressed in late phase, while the suppression of GALE expression induced by IL-1β was mainly mediated by stress-activated protein kinase/c-Jun N-terminal kinase pathway. These data indicated a critical role of GALE in maintaining cartilage homeostasis, and suggested that GALE inhibition might contribute to OA progress.

  14. Efficient and configurable transmission protocol based on UDP in grid computing

    Institute of Scientific and Technical Information of China (English)

    Jigang WANG; Guochang GU; Chunguang MA; Weidong ZHONG

    2009-01-01

    At present,mainstream data transfer protocols are not always a good match for the diverse demands of grid computing.Considering this situation,this article proposes an efficient and configurable data transfer protocol (ECUDP) for grid computing.The ECUDP is based on the standard user datagram protocol (UDP),but with a collection of optimizations that meet the challenge of providing configurability and reliability while main-taining performance that meets the communication requirements of demanding applications.Experimental results show that the ECUDP performs efficiently in various grid computing scenarios and the performance analysis model can provide a good estimation of its performance.

  15. A simplified radioactive assay for the enzyme UDP-glucose pyrophosphorylase

    Energy Technology Data Exchange (ETDEWEB)

    Evers, C.A.; Palatnik, M.

    1981-11-15

    A radioactive assay for the enzyme UDP-glucose pyrophosphorylase has been modified. The modifications allow the enzymes to be assayed in one simple step and eliminate two procedures (a boiling step and an alkaline phosphatase incubation) which lengthen the time required to do the assay, make it somewhat cumbersome, and which must be carefully controlled. Furthermore, the modifications allow the simultaneous measurement of large numbers of samples. The modified assay is sensitive enough to quantitate activity in a mutant of Dictyostelium discoideum which with previous spectrophotometric assays could not be accurately measured.

  16. UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y(6) receptors

    DEFF Research Database (Denmark)

    Hou, Mingyan; Harden, T Kendall; Kuhn, Cynthia M;

    2002-01-01

    Mitogenic effects of the extracellular nucleotides ATP and UTP are mediated by P2Y(1), P2Y(2), and P2Y(4) receptors. However, it has not been possible to examine the highly expressed UDP-sensitive P2Y(6) receptor because of the lack of stable, selective agonists. In rat aorta smooth muscle cells...... (vascular smooth muscle cells; VSMC), UDP and UTP stimulated (3)H-labeled thymidine incorporation with similar pEC(50) values (5.96 and 5.69). Addition of hexokinase did not reduce the mitogenic effect of UDP. In cells transfected with P2Y receptors the stable pyrimidine agonist uridine 5'-O-(2...

  17. An adaptive FEC to protect RoHC and UDP-Lite H.264 video critical data

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhi-kui

    2006-01-01

    This paper describes how to use an Adaptive Forward Error Correction (AFEC) algorithm to efficiently protect the critical data areas of compressed headers and UDP-Lite packets for data transport in a radio link layer of a wireless connection.Augmented with RoHC and UDP-Lite, for H.264 video transmissions over wireless channels in a heterogeneous wired-wireless environment, the erroneous packet payloads can be useful and better able to cope with lost packets (native UDP case), by adopting some of the erasure and error resilient modes in H.264. The context transfer during inter/intra handover is also discussed. Simulations demonstrated that the proposed scenario significantly improves the PSNR performance and video quality.

  18. Yield and nutritional content of Pleurotus sajor caju on wheat straw supplemented with raw and detoxified mahua cake.

    Science.gov (United States)

    Gupta, Aditi; Sharma, Satyawati; Saha, Supradip; Walia, Suresh

    2013-12-15

    The effect of supplementation of wheat straw (WS) with raw/detoxified mahua cake (MC) on yield and nutritional quality of Pleurotus sajor caju was studied. Raw cake significantly enhanced the yield compared to control and could be tolerated up to a 10% addition. Detoxification further improved the mushroom yield giving a maximum of 1024.7 g kg(-1) from WS supplemented with 20% saponin free detoxified mahua cake. Chemical analysis of fruit bodies revealed that they are rich in proteins (27.4-34.8%), soluble sugars (28.6-32.2%) and minerals. Glucose, trehalose and glutamic acid, alanine were the major sugars and amino acids detected by HPLC analysis, respectively. HPLC studies further confirmed the absence of saponins (characteristic toxins present in MC) in both fruit bodies and spent. Degradation of complex molecules in spent was monitored via FTIR. The study proved beneficial for effective management of agricultural wastes along with production of nutrient rich and saponin free fruit bodies/spent.

  19. Detoxifying enzyme studies on cotton leafhopper, Amrasca biguttula biguttula (Ishida, resistance to neonicotinoid insecticides in field populations in Karnataka, India

    Directory of Open Access Journals (Sweden)

    Halappa Banakar

    2016-12-01

    Full Text Available The cotton leafhopper (Amrasca biguttula biguttula Ishida is considered to be an alarming insect pest causing both quantitative and qualitative loss in cotton. In situ bioassay studies were done and the role of detoxifying enzymes in conferring resistance to neonicotinoid groups of insecticides in low (MUD, medium (DVG, high (HVR and very high (GLB pesticide usage areas of Karnataka were determined. Bioassay studies showed that imidacloprid, thiamethoxam, acetamiprid, thiacloprid and clothianidin registered varying levels of resistance for all the locations studied. The resistance ratio was high in imidacloprid (3.35, 8.57, 9.15 and 12.27 fold respectively and the lowest in dinoferuran (1.86, 5.13, 6.71 and 9.88 fold respectively. Furthermore, the enzyme activity ratio (glutathione-S-transferase was relatively greater, and corresponded to the higher LC50 values of neonicotinoids for very high, high, medium and low pesticide usage areas. Our study suggested that the higher activity of the detoxifying enzyme in the resistance population of cotton leafhopper apparently has a significant role in endowing resistance to neonicotinoid groups of insecticides. However, this study recommends using neonicotinoids in cotton growing areas with caution.

  20. High glucose recovery from direct enzymatic hydrolysis of bisulfite-pretreatment on non-detoxified furfural residues.

    Science.gov (United States)

    Xing, Yang; Bu, Lingxi; Sun, Dafeng; Liu, Zhiping; Liu, Shijie; Jiang, Jianxin

    2015-10-01

    This study reports four schemes to pretreat wet furfural residues (FRs) with sodium bisulfite for production of fermentable sugar. The results showed that non-detoxified FRs (pH 2-3) had great potential to lower the cost of bioconversion. The optimal process was that unwashed FRs were first pretreated with bisulfite, and the whole slurry was then directly used for enzymatic hydrolysis. A maximum glucose yield of 99.4% was achieved from substrates pretreated with 0.1 g NaHSO3/g dry substrate (DS), at a relatively low temperature of 100 °C for 3 h. Compared with raw material, enzymatic hydrolysis at a high-solid of 16.5% (w/w) specifically showed more excellent performance with bisulfite treated FRs. Direct bisulfite pretreatment improved the accessibility of substrates and the total glucose recovery. Lignosulfonate in the non-detoxified slurry decreased the non-productive adsorption of cellulase on the substrate, thus improving enzymatic hydrolysis.

  1. Increased deposition of chondroitin/dermatan sulfate glycosaminoglycan and upregulation of β1,3-glucuronosyltransferase I in pulmonary fibrosis.

    Science.gov (United States)

    Venkatesan, Narayanan; Ouzzine, Mohamed; Kolb, Martin; Netter, Patrick; Ludwig, Mara S

    2011-02-01

    Pulmonary fibrosis (PF) is characterized by increased deposition of proteoglycans (PGs), in particular core proteins. Glycosaminoglycans (GAGs) are key players in tissue repair and fibrosis, and we investigated whether PF is associated with changes in the expression and structure of GAGs as well as in the expression of β1,3-glucuronosyltransferase I (GlcAT-I), a rate-limiting enzyme in GAG synthesis. Lung biopsies from idiopathic pulmonary fibrosis (IPF) patients and lung tissue from a rat model of bleomycin (BLM)-induced PF were immunostained for chondroitin sulfated-GAGs and GlcAT-I expression. Alterations in disaccharide composition and sulfation of chondroitin/dermatan sulfate (CS/DS) were evaluated by fluorophore-assisted carbohydrate electrophoresis (FACE) in BLM rats. Lung fibroblasts isolated from control (saline-instilled) or BLM rat lungs were assessed for GAG structure and GlcAT-I expression. Disaccharide analysis showed that 4- and 6-sulfated disaccharides were increased in the lungs and lung fibroblasts obtained from fibrotic rats compared with controls. Fibrotic lung fibroblasts and transforming growth factor-β(1) (TGF-β(1))-treated normal lung fibroblasts expressed increased amounts of hyaluronan and 4- and 6-sulfated chondroitin, and neutralizing anti-TGF-β(1) antibody diminished the same. TGF-β(1) upregulated GlcAT-I and versican expression in lung fibroblasts, and signaling through TGF-β type I receptor/p38 MAPK was required for TGF-β(1)-mediated GlcAT-I and CS-GAG expression in fibroblasts. Our data show for the first time increased expression of CS-GAGs and GlcAT-I in IPF, fibrotic rat lungs, and fibrotic lung fibroblasts. These data suggest that alterations of sulfation isomers of CS/DS and upregulation of GlcAT-I contribute to the pathological PG-GAG accumulation in PF.

  2. AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Kim

    2017-03-01

    Full Text Available AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP or uridine 5′-diphospho-glucuronosyltransferase (UGT enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7 enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

  3. AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

    Science.gov (United States)

    Kim, Ju-Hyun; Kwon, Soon-Sang; Kong, Tae Yeon; Cheong, Jae Chul; Kim, Hee Seung; In, Moon Kyo; Lee, Hye Suk

    2017-03-10

    AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography-tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

  4. No association between cyclooxygenase-2 and uridine diphosphate glucuronosyltransferase 1A6 genetic polymorphisms and colon cancer risk

    Institute of Scientific and Technical Information of China (English)

    Cheryl L Thompson; Sarah J Plummer; Alona Merkulova; Iona Cheng; Thomas C Tucker; Graham Casey; Li Li

    2009-01-01

    AIM: To investigate the association of variations in the cyclooxygenase-2 (COX2) and uridine diphosphate glucuronosyltransferase 1A6 (UGT1A6) genes and nonsteroidal anti-inflammatory drugs (NSAIDs) use with risk of colon cancer. METHODS: NSAIDs, which are known to reduce the risk of colon cancer, act directly on COX2 and reduce its activity. Epidemiological studies have associated variations in the COX2 gene with colon cancer risk, but others were unable to replicate this finding. Similarly, enzymes in the UGT1A6 gene have been demonstrated to modify the therapeutic effect of NSAIDs on colon adenomas. Polymorphisms in the UGT1A6 gene have been statistically shown to interact with NSAID intake to influence risk of developing colon adenomas, but not colon cancer. Here we examined the association of tagging single nucleotide polymorphisms (SNPs) in the COX2 and UGT1A6 genes, and their interaction with NSAID consumption, on risk of colon cancer in a population of 422 colon cancer cases and 481 population controls. RESULTS: No SNP in either gene was individually statistically significantly associated with colon cancer, nor did they statistically significantly change the protective effect of NSAID consumption in our sample. Like others, we were unable to replicate the association of variants in the COX2 gene with colon cancer risk ( P > 0.05), and we did not observe that these variants modify the protective effect of NSAIDs ( P > 0.05). We were able to confirm the lack of association of variants in UGT1A6 with colon cancer risk, although further studies will have to be conducted to confirm the association of these variants with colon adenomas. CONCLUSION: Our study does not support a role of COX2 and UGT1A6 genetic variations in the development of colon cancer.

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

    Science.gov (United States)

    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.

  6. Using UDP Datagram to Realize a Distributed Control Mode at High-Speed Data Communication

    Science.gov (United States)

    Zhao, Lindi

    This article presents a distributed control mode at high-speed data communication by using UDP datagram based on a dual-port RAM, with the purpose of exploring an efficient, low CPU occupancy, high-speed, reliable and large amounts of data exchange mode. One port of the proposed dual-port RAM mode is connected to local CPU, which can be easily read and write data and the other port is connected to the reading and writing and communication control dedicated logic, which is responsible for organizing, sending, receiving and analyzing UDP datagram and also for completing the tasks of reading and writing dual-port RAM and sending interrupt request to the local processor. This model not only realizes efficient data exchange, but also provides real-time interrupt response mechanism. In this article, it discusses the functional modules required for these functions, describes the composition of the system and the advantages of technical performance. It also analyzes the feasibility of its development, and demonstrates that this model is an advanced, high performance distributed control communication program with great promising.

  7. Highly Scalable, UDP-Based Network Transport Protocols for Lambda Grids and 10 GE Routed Networks

    Energy Technology Data Exchange (ETDEWEB)

    PI: Robert Grossman Co-PI: Stephen Eick

    2009-08-04

    Summary of Report In work prior to this grant, NCDM developed a high performance data transport protocol called SABUL. During this grant, we refined SABUL’s functionality, and then extended both the capabilities and functionality and incorporated them into a new protocol called UDP-based Data transport Protocol, or UDT. We also began preliminary work on Composable UDT, a version of UDT that allows the user to choose among different congestion control algorithms and implement the algorithm of his choice at the time he compiles the code. Specifically, we: · Investigated the theoretical foundations of protocols similar to SABUL and UDT. · Performed design and development work of UDT, a protocol that uses UDP in both the data and control channels. · Began design and development work of Composable UDT, a protocol that supports the use of different congestion control algorithms by simply including the appropriate library when compiling the code. · Performed experimental studies using UDT and Composable UDT using real world applications such as the Sloan Digital Sky Survey (SDSS) astronomical data sets. · Released several versions of UDT and Composable, the most recent being v3.1.

  8. Extracellular UDP and P2Y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in IFN-β production.

    Science.gov (United States)

    Li, Ruimei; Tan, Binghe; Yan, Yan; Ma, Xiaobin; Zhang, Na; Zhang, Zhi; Liu, Mingyao; Qian, Min; Du, Bing

    2014-11-01

    Extracellular nucleotides that constitute a "danger signal" play an important role in the regulation of immune responses. However, the function and mechanism of extracellular UDP and P2Y6 in antiviral immunity remain unknown. In this study, we demonstrated the in vitro and in vivo protection of UDP/P2Y6 signaling in vesicular stomatitis virus (VSV) infection. First, we demonstrated that VSV-infected cells secrete UDP from the cytoplasm as a danger signal to arouse surrounding cells. Meanwhile, expression of the UDP-specific receptor P2Y6 also was enhanced by VSV. Consequently, UDP protects RAW 264.7 cells, murine embryonic fibroblasts, bone marrow-derived macrophages, and L929 cells from VSV and GFP lentivirus infection. This protection can be blocked by the P2Y6 selective antagonist MRS2578 or IFN-α/β receptor-blocking Ab. VSV-induced cell death and virus replication were both enhanced significantly by knocking down and knocking out P2Y6 in different cells. Mechanistically, UDP facilitates IFN-β secretion through the p38/JNK- and ATF-2/c-Jun-signaling pathways, which are crucial in promoting antiviral immunity. Interestingly, UDP was released through a caspase-cleaved pannexin-1 channel in VSV-induced apoptotic cells and protected cells from infection through P2Y6 receptor in an autocrine or paracrine manner. Furthermore, UDP also protected mice from VSV infection through P2Y6 receptors in an acute neurotropic infection mouse model. Taken together, these results demonstrate the important role of extracellular UDP and P2Y6 as a danger signal in antiviral immune responses and suggest a potential therapeutic role for UDP/P2Y6 in preventing and controlling viral diseases.

  9. Rational design of organophosphorus hydrolase with high catalytic efficiency for detoxifying a V-type nerve agent.

    Science.gov (United States)

    Jeong, Young-Su; Choi, Jung Min; Kyeong, Hyun-Ho; Choi, Jae-Youl; Kim, Eui-Joong; Kim, Hak-Sung

    2014-07-01

    V-type nerve agents, known as VX, are organophosphate (OP) compounds, and show extremely toxic effects on human and animals by causing cholinergic overstimulation of synapses. The bacterial organophosphorus hydrolase (OPH) has attracted much attention for detoxifying V-type agents through hydrolysis of the P-S bond. However, low catalytic efficiency of OPH has limited the practical use of the enzyme. Here we present rational design of OPH with high catalytic efficiency for a V-type nerve agent. Based on the model structure of the enzyme and substrate docking simulation, we predicted the key residues that appear to enhance the access of the substrate to the active site of the enzyme, and constructed numerous OPH mutants. Of them, double mutant, L271/Y309A, was shown to exhibit a 150-fold higher catalytic efficiency for VX than the wild-type.

  10. Relationship Between the Development of Methamidophos Resistance and the Activities of Three Detoxifying Enzymes in Brown Planthopper,Nilaparvata lugens

    Institute of Scientific and Technical Information of China (English)

    LIuZe-wen; HANZhao-jun; ZHANGLing-chun

    2004-01-01

    Methamidophos resistance of brown planthopper(BPH), Nilaparvata lugens was selected in laboratory for 19 generations (F1 to F19). The resistance development in BPH was approximatively shaped as the letter "S" : resistance change was small before the fifth generation and after the fifteenth generation, and the changing pattern was sharp between the fifth and the fifteenth generation. Esterase might play an important role in the resistance development, because the esterase activity and the number of individuals with high activities increased along with the resistance development. The esterase activities of insecticide-sensitive population S, field population F0, its selective generations F, F10 and F15 were highly correlated with the resistance ratios of these generations, and the coefficient was 0.9899. Mixed-function oxidases and giutathione S-transferase also might play some roles in the resistance development, but the big change in the activities of the two detoxifying enzymes both took place before the tenth generation.

  11. Relationship Between the Development of Methamidophos Resistance and the Activities of Three Detoxifying Enzymes in Brown Planthopper, Nilaparvata lugens

    Institute of Scientific and Technical Information of China (English)

    LIU Ze-wen; HAN Zhao-jun; ZHANG Ling-chun

    2004-01-01

    Methamidophos resistance of brown planthopper(BPH), Nilaparvata lugens was selected in laboratory for 19 generations (F1 to F19). The resistance development in BPH was approximatively shaped as the letter "S" : resistance change was small before the fifth generation and after the fifteenth generation, and the changing pattern was sharp between the fifth and the fifteenth generation. Esterase might play an important role in the resistance development, because the esterase activity and the number of individuals with high activities increased along with the resistance development. The esterase activities of insecticide-sensitive population S, field population F0, its selective generations Fs, F10 and F15 were highly correlated with the resistance ratios of these generations, and the coefficient was 0.9899. Mixed-function oxidases and glutathione S-transferase also might play some roles in the resistance development, but the big change in the activities of the two detoxifying enzymes both took place before the tenth generation.

  12. Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tsung-Pao; Pan, Yun-Ru; Fu, Chien-Yu; Chang, Hwan-You, E-mail: hychang@life.nthu.edu.tw

    2010-10-15

    UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellular spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.

  13. Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease.

    Directory of Open Access Journals (Sweden)

    Michelle Oppenheimer

    Full Text Available UDP-galactopyranose mutase (UGM is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf. Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ∼1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.

  14. Aeromonas surface glucan attached through the O-antigen ligase represents a new way to obtain UDP-glucose.

    Science.gov (United States)

    Merino, Susana; Bouamama, Lamiaa; Knirel, Yuriy A; Senchenkova, Sofya N; Regué, Miguel; Tomás, Juan M

    2012-01-01

    We previously reported that A. hydrophila GalU mutants were still able to produce UDP-glucose introduced as a glucose residue in their lipopolysaccharide core. In this study, we found the unique origin of this UDP-glucose from a branched α-glucan surface polysaccharide. This glucan, surface attached through the O-antigen ligase (WaaL), is common to the mesophilic Aeromonas strains tested. The Aeromonas glucan is produced by the action of the glycogen synthase (GlgA) and the UDP-Glc pyrophosphorylase (GlgC), the latter wrongly indicated as an ADP-Glc pyrophosphorylase in the Aeromonas genomes available. The Aeromonas glycogen synthase is able to react with UDP or ADP-glucose, which is not the case of E. coli glycogen synthase only reacting with ADP-glucose. The Aeromonas surface glucan has a role enhancing biofilm formation. Finally, for the first time to our knowledge, a clear preference on behalf of bacterial survival and pathogenesis is observed when choosing to produce one or other surface saccharide molecules to produce (lipopolysaccharide core or glucan).

  15. The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities

    DEFF Research Database (Denmark)

    Hassan, H; Reis, C A; Bennett, E P;

    2000-01-01

    The initiation step of mucin-type O-glycosylation is controlled by a large family of homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Differences in kinetic properties, substrate specificities, and expression patterns of these isoenzymes provide for diff...

  16. Aeromonas surface glucan attached through the O-antigen ligase represents a new way to obtain UDP-glucose.

    Directory of Open Access Journals (Sweden)

    Susana Merino

    Full Text Available We previously reported that A. hydrophila GalU mutants were still able to produce UDP-glucose introduced as a glucose residue in their lipopolysaccharide core. In this study, we found the unique origin of this UDP-glucose from a branched α-glucan surface polysaccharide. This glucan, surface attached through the O-antigen ligase (WaaL, is common to the mesophilic Aeromonas strains tested. The Aeromonas glucan is produced by the action of the glycogen synthase (GlgA and the UDP-Glc pyrophosphorylase (GlgC, the latter wrongly indicated as an ADP-Glc pyrophosphorylase in the Aeromonas genomes available. The Aeromonas glycogen synthase is able to react with UDP or ADP-glucose, which is not the case of E. coli glycogen synthase only reacting with ADP-glucose. The Aeromonas surface glucan has a role enhancing biofilm formation. Finally, for the first time to our knowledge, a clear preference on behalf of bacterial survival and pathogenesis is observed when choosing to produce one or other surface saccharide molecules to produce (lipopolysaccharide core or glucan.

  17. In Bacillus subtilis, the SatA (formerly YyaR) acetyltransferase detoxifies streptothricin via lysine acetylation.

    Science.gov (United States)

    Burckhardt, Rachel M; Escalante-Semerena, Jorge C

    2017-08-25

    Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA(+) restored streptothricin resistance to B. subtilis satA strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-CoA. A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA(+) in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil.Importance Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The alluded enzyme is a member of a family of proteins that is broadly distributed in all domains of life, but poorly studied in B. subtilis and B. anthracis. The initial characterization of the enzyme provides insights into its mechanism of catalysis. Copyright © 2017 American Society for

  18. Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution.

    Science.gov (United States)

    Smith, James; Yang, Yiwen; Levy, Shahar; Adelusi, Oluwatoyin Oluwayemi; Hahn, Michael G; O'Neill, Malcolm A; Bar-Peled, Maor

    2016-10-07

    Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDP-apiose as the donor. UDP-apiose (UDP-Api) together with UDP-xylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans.

  19. Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato.

    Science.gov (United States)

    Moehs, C P; Allen, P V; Friedman, M; Belknap, W R

    1997-02-01

    A cDNA encoding solanidine glucosyltransferase (SGT) was isolated from potato. The cDNA was selected from a yeast expression library using a positive selection based on the higher toxicity of steroidal alkaloid aglycons relative to their associated glycosylated forms. The cDNA contained an open reading frame encoding a 56 kDa polypeptide with regions of similarity to previously characterized UDP-glucosyltransferases. The enzyme activity and reaction products of recombinant SGT in yeast were consistent with those observed for the endogenous enzyme from potato. SGT mRNA and protein accumulated in tubers in response to wounding. The time course for SGT mRNA accumulation paralleled that of 3-hydroxy-3-methylglutaryl-coenzymeA isoform 1 (hmg1) mRNA. Steady-state SGT mRNA levels also increased transiently upon wounding of leaves.

  20. Characterization and Evaluation of TCP and UDP-based Transport on Real Networks

    Energy Technology Data Exchange (ETDEWEB)

    Cottrell, L.

    2005-01-25

    Standard TCP (Reno TCP) does not perform well on fast long distance networks, due to its AIMD congestion control algorithm. In this paper we consider the effectiveness of various alternatives, in particular with respect to their applicability to a production environment. We then characterize and evaluate the achievable throughput, stability and intra-protocol fairness of different TCP stacks (Scalable, HSTCP, HTCP, Fast TCP, Reno, BICTCP, HSTCP-LP and LTCP) and a UDP based application level transport protocol (UDTv2) on both production and testbed networks. The characterization is made with respect to both the transient traffic (entry and exit of different streams) and the steady state traffic on production Academic and Research networks, using paths with RTTs differing by a factor of 10. We also report on measurements made with 10Gbits/sec NICs with and without TCP Offload Engines, on 10Gbits/s dedicated paths set up for SC2004.

  1. [UDP-glucuronyltransferases in detoxification and activation metabolism of endogenous compounds and xenobiotics].

    Science.gov (United States)

    Fedejko, Barbara; Mazerska, Zofia

    2011-01-01

    Glucuronidation is a crucial pathway of metabolism and excretion of endogenous compounds and xenobiotics. UDP-glucuronyltransferases, UGT, catalyse transformations of bilirubine, steroids and thyroid hormones, bile acids as well as exogenous compounds, including drugs, carcinogens, environmental pollutants and nutrient components. From therapeutic point of view, the participation of UGTs in drug metabolism is of particular significance. Polymorphism of UGT1A and UGT2B genes resulted in various susceptibility of substrates to conjugation with glucuronic acid. Deactivation of xenobiotics and the following excretion of hydrophilic conjugates is a common task of glucuronidation, which should lead to detoxification. However, a lot of glucuronides were known, which expressed the comparable or even higher reactivity than that of the native compound. There are, among others, acyl glucuronides of carboxylic acids, morphine 6-O-glucuronide or retinoid glucuronides. They are able to bind cellular macromolecules with low or high strength and, as a consequence, their toxicity is saved or even increased, respectively.

  2. Manila clams from Hg polluted sediments of Marano and Grado lagoons (Italy) harbor detoxifying Hg resistant bacteria in soft tissues

    Energy Technology Data Exchange (ETDEWEB)

    Baldi, Franco, E-mail: baldi@unive.it [Dipartimento di Scienze Molecolari e Nanosistemi, Cà Foscari University of Venice, Dorsoduro 2137, 30123 Venice (Italy); Gallo, Michele; Marchetto, Davide [Dipartimento di Scienze Molecolari e Nanosistemi, Cà Foscari University of Venice, Dorsoduro 2137, 30123 Venice (Italy); Faleri, Claudia [Department of Environmental Science ‘G. Sarfatti’, University of Siena, 53100 Siena (Italy); Maida, Isabel; Fani, Renato [Dipartimento di Biologia Evoluzionistica, Via Romana, 17, University of Florence, 50125 Florence (Italy)

    2013-08-15

    A mechanism of mercury detoxification has been suggested by a previous study on Hg bioaccumulation in Manila clams (Ruditapes philippinarum) in the polluted Marano and Grado lagoons and in this study we demonstrate that this event could be partly related to the detoxifying activities of Hg-resistant bacteria (MRB) harbored in clam soft tissues. Therefore, natural clams were collected in six stations during two different periods (winter and spring) from Marano and Grado Lagoons. Siphons, gills and hepatopancreas from acclimatized clams were sterile dissected to isolate MRB. These anatomical parts were glass homogenized or used for whole, and they were lying on a solid medium containing 5 mg l{sup −1} HgCl{sub 2} and incubated at 30 °C. A total of fourteen bacterial strains were isolated and were identified by 16S rDNA sequencing and analysis, revealing that strains were representative of eight bacterial genera, four of which were Gram-positive (Enterococcus, Bacillus, Jeotgalicoccus and Staphylococcus) and other four were Gram-negative (Stenotrophomonas, Vibrio, Raoultella and Enterobacter). Plasmids and merA genes were found and their sequences determined. Fluorescence in situ hybridization (FISH) technique shows the presence of Firmicutes, Actinobacteria and Gammaproteobacteria by using different molecular probes in siphon and gills. Bacterial clumps inside clam flesh were observed and even a Gram-negative endosymbiont was disclosed by transmission electronic microscope inside clam cells. Bacteria harbored in cavities of soft tissue have mercury detoxifying activity. This feature was confirmed by the determination of mercuric reductase in glass-homogenized siphons and gills. -- Highlights: ► We isolated Gram-positive and Gram-negative Hg resistant strains from soft tissues of Ruditapes philippinarum. ► We identify 14 mercury resistant strains by 16S rRNA gene sequences. ► Bacteria in siphon and gill tissues of clams were observed by TEM and identified

  3. UDP-glucose pyrophosphorylase influences polysaccharide synthesis, cell wall components, and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose.

    Science.gov (United States)

    Li, Mengjiao; Chen, Tianxi; Gao, Tan; Miao, Zhigang; Jiang, Ailiang; Shi, Liang; Ren, Ang; Zhao, Mingwen

    2015-09-01

    UDP-glucose pyrophosphorylase (UGP) is a key enzyme involved in carbohydrate metabolism, but there are few studies on the functions of this enzyme in fungi. The ugp gene of Ganoderma lucidum was cloned, and enzyme kinetic parameters of the UGP recombinant protein were determined in vitro, revealing that this protein was functional and catalyzed the reversible conversion between Glc-1-P and UDP-Glc. ugp silencing by RNA interference resulted in changes in the levels of the intermediate metabolites Glc-1-P and UDP-Glc. The compounds and structure of the cell wall in the silenced strains were also altered compared with those in the wild-type strains. Moreover, the number of hyphal branches was also changed in the silenced strains. To verify the role of UGP in hyphal branching, a ugp-overexpressing strain was constructed. The results showed that the number of hyphal branches was influenced by UGP. The mechanism underlying hyphal branching was further investigated by adding exogenous Glc-1-P. Our results showed that hyphal branching was regulated by a change in the cytosolic Ca(2+) concentration, which was affected by the level of the intermediate metabolite Glc-1-P, in G. lucidum. Our findings indicate the existence of an interaction between carbon metabolism and Ca(2+) signaling in this fungus.

  4. Decomposition of 2-chloroethylethylsulfide on copper oxides to detoxify polymer-based spherical activated carbons from chemical warfare agents.

    Science.gov (United States)

    Fichtner, S; Hofmann, J; Möller, A; Schrage, C; Giebelhausen, J M; Böhringer, B; Gläser, R

    2013-11-15

    For the decomposition of chemical warfare agents, a hybrid material concept was applied. This consists of a copper oxide-containing phase as a component with reactive functionality supported on polymer-based spherical activated carbon (PBSAC) as a component with adsorptive functionality. A corresponding hybrid material was prepared by impregnation of PBSAC with copper(II)nitrate and subsequent calcination at 673K. The copper phase exists predominantly as copper(I)oxide which is homogeneously distributed over the PBSAC particles. The hybrid material containing 16 wt.% copper on PBSAC is capable of self-detoxifying the mustard gas surrogate 2-chloroethylethylsulfide (CEES) at room temperature. The decomposition is related to the breakthrough behavior of the reactant CEES, which displaces the reaction product ethylvinylsulfide (EVS). This leads to a combined breakthrough of CEES and EVS. The decomposition of CEES is shown to occur catalytically over the copper-containing PBSAC material. Thus, the hybrid material can even be considered to be self-cleaning.

  5. Effect of dietary acids on the formation of aflatoxin B2a as a means to detoxify aflatoxin B1.

    Science.gov (United States)

    Rushing, Blake R; Selim, Mustafa I

    2016-09-01

    Aflatoxin B1 (AFB1) is a class 1 carcinogen and a common food contaminant worldwide with widely uncontrolled human exposure. The ability of organic acids to transform AFB1 into a known detoxified form, aflatoxin B2a (AFB2a), was investigated using high performance liquid chromatography-electrospray ionisation-time of flight mass spectrometry (HPLC/ESI/TOF/MS). The identity of the transformation product was confirmed by accurate mass measurement, chromatographic separation from other aflatoxins, H(1)-nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. Of the weak acids tested, citric acid was found to be the most effective for AFB2a formation. At room temperature, 1 M citric acid was able to convert > 97% of AFB1 to AFB2a over 96 h of treatment. Up to 98% transformation was achieved by boiling AFB1 in the presence of citric acid for 20 min. AFB1 hydration after ingestion was explored by spiking AFB1 into simulated gastric fluid containing citric acid. Under these conditions, > 71% of AFB1 was hydrated to AFB2a and did not show any reversion to the parent compound after being transferred to a neutral solution. These results provide a basis for a practical and effective method for detoxification of AFB1 in contaminated foods.

  6. Decolorizing and detoxifying textile wastewater, containing both soluble and insoluble dyes, in a full scale combined anaerobic/aerobic system.

    Science.gov (United States)

    Frijters, C T M J; Vos, R H; Scheffer, G; Mulder, R

    2006-03-01

    The wastewater originating from the bleaching and dyeing processes in the textile factory Ten Cate Protect in Nijverdal (the Netherlands) was successfully treated in a sequential anaerobic/aerobic system. In the system, a combination of an anaerobic 70-m3 fluidized bed reactor and a 450-m3 aerobic basin with integrated tilted plate settlers, 80-95% of the color was removed. The color was largely removed in the preacidification basin and the anaerobic reactor. Color, deriving from both reactive as well as disperse, was anaerobically removed, indicating that these type of dyes were reduced to colorless products. Interestingly, the vat dyes, the anthraquinones and indigoids, which were thought to be removed mainly aerobically, were largely anaerobically decolorized. Apparently the anaerobic system is capable of effectively removing the color of both soluble as insoluble dyes. The treated effluent of the sequential anaerobic/aerobic treatment showed no toxicity towards the bioluminescent bacterium Vibrio fisheri (EC20 (95%) > 45%). Partially bypassing the anaerobic stage resulted in increased toxicity (EC20 (95%) of 9% and 14%) in the effluent of the aerobic treatment and caused significant decrease of color removal. The results of this study show a main contribution of anaerobic treatment in decolorizing and detoxifying the textile wastewater in the sequential anaerobic/aerobic system.

  7. The role of detoxifying enzymes in the resistance of the cowpea aphid (Aphis craccivora Koch to thiamethoxam

    Directory of Open Access Journals (Sweden)

    Abdallah Ibrahim Saleh

    2016-01-01

    Full Text Available The cowpea aphid (Aphis craccivora Koch is considered a serious insect pest attacking several crops. We carried out biochemical studies to elucidate the role of the metabolising enzymes in conferring resistance to thiamethoxam, in two strains (resistant and susceptible of the cowpea aphid. Bioassay experiments showed that the thiamethoxam selected strain developed a 48 fold resistance after consecutive selection with thiamethoxam for 12 generations. This resistant strain also exhibited cross-resistance to the tested carbamates; pirimicarb and carbosulfan, organophosphorus (malathion, fenitrothion, and chlorpyrifos-methyl, and the neonicotinoid (acetamiprid. Synergism studies have indicated that S,S,S-tributyl phosphorotrithioate (DEF, a known inhibitor for esterases, increased thiamethoxam toxicity 5.58 times in the resistant strain compared with the susceptible strain. Moreover, the biochemical determination revealed that carboxylestersae activity was 30 times greater in the resistant strain than in the susceptible strain. In addition, the enzyme activity of glutathione S-transferase (GST and mixed function oxidases (mfo increased only in the resistant strain 3.7 and 2.7 times, respectively, in relation to the susceptible (the control. Generally, our results suggest that the higher activity of the detoxifying enzymes, particularly carboxylesterase, in the resistant strain of the cowpea aphid, apparently have a significant role in endowing resistance to thiamethoxam, although additional mechanisms may contribute.

  8. The Anopheles gambiae oxidation resistance 1 (OXR1 gene regulates expression of enzymes that detoxify reactive oxygen species.

    Directory of Open Access Journals (Sweden)

    Giovanna Jaramillo-Gutierrez

    Full Text Available BACKGROUND: OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage. METHODOLOGY/PRINCIPAL FINDINGS: OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT and glutathione peroxidase (Gpx expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response. CONCLUSION: The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection.

  9. Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells

    OpenAIRE

    Taguchi, G; Imura, H.; Maeda, Y.; Kodaira, R; Hayashida, N.; Shimosaka, M; Okazaki, M.

    2000-01-01

    The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%. Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, ...

  10. Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus.

    Science.gov (United States)

    Patel, Kinnari B; Toh, Evelyn; Fernandez, Ximena B; Hanuszkiewicz, Anna; Hardy, Gail G; Brun, Yves V; Bernards, Mark A; Valvano, Miguel A

    2012-05-01

    Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.

  11. Functional consequence of covalent reaction of phosphoenolpyruvate with UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA).

    Science.gov (United States)

    Zhu, Jin-Yi; Yang, Yan; Han, Huijong; Betzi, Stephane; Olesen, Sanne H; Marsilio, Frank; Schönbrunn, Ernst

    2012-04-13

    The enzyme MurA has been an established antibiotic target since the discovery of fosfomycin, which specifically inhibits MurA by covalent modification of the active site residue Cys-115. Early biochemical studies established that Cys-115 also covalently reacts with substrate phosphoenolpyruvate (PEP) to yield a phospholactoyl adduct, but the structural and functional consequences of this reaction remained obscure. We captured and depicted the Cys-115-PEP adduct of Enterobacter cloacae MurA in various reaction states by X-ray crystallography. The data suggest that cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM), the product of the MurB reaction, with PEP covalently attached to Cys-115. The uniqueness and rigidity of this "dormant" complex was previously not recognized and presumably accounts for the failure of drug discovery efforts toward the identification of novel and effective MurA inhibitors. We demonstrate that recently published crystal structures of MurA from various organisms determined by different laboratories were indeed misinterpreted and actually contain UNAM and covalently bound PEP. The Cys-115-PEP adduct was also captured in vitro during the reaction of free MurA and substrate UDP-N-acetylglucosamine or isomer UDP-N-acetylgalactosamine. The now available series of crystal structures allows a comprehensive view of the reaction cycle of MurA. It appears that the covalent reaction of MurA with PEP fulfills dual functions by tightening the complex with UNAM for the efficient feedback regulation of murein biosynthesis and by priming the PEP molecule for instantaneous reaction with substrate UDP-N-acetylglucosamine.

  12. The UDP-glucuronate decarboxylase gene family in Populus: structure, expression, and association genetics.

    Directory of Open Access Journals (Sweden)

    Qingzhang Du

    Full Text Available In woody crop plants, the oligosaccharide components of the cell wall are essential for important traits such as bioenergy content, growth, and structural wood properties. UDP-glucuronate decarboxylase (UXS is a key enzyme in the synthesis of UDP-xylose for the formation of xylans during cell wall biosynthesis. Here, we isolated a multigene family of seven members (PtUXS1-7 encoding UXS from Populus tomentosa, the first investigation of UXSs in a tree species. Analysis of gene structure and phylogeny showed that the PtUXS family could be divided into three groups (PtUXS1/4, PtUXS2/5, and PtUXS3/6/7, consistent with the tissue-specific expression patterns of each PtUXS. We further evaluated the functional consequences of nucleotide polymorphisms in PtUXS1. In total, 243 single-nucleotide polymorphisms (SNPs were identified, with a high frequency of SNPs (1/18 bp and nucleotide diversity (πT = 0.01033, θw = 0.01280. Linkage disequilibrium (LD analysis showed that LD did not extend over the entire gene (r (2<0.1, P<0.001, within 700 bp. SNP- and haplotype-based association analysis showed that nine SNPs (Q <0.10 and 12 haplotypes (P<0.05 were significantly associated with growth and wood property traits in the association population (426 individuals, with 2.70% to 12.37% of the phenotypic variation explained. Four significant single-marker associations (Q <0.10 were validated in a linkage mapping population of 1200 individuals. Also, RNA transcript accumulation varies among genotypic classes of SNP10 was further confirmed in the association population. This is the first comprehensive study of the UXS gene family in woody plants, and lays the foundation for genetic improvements of wood properties and growth in trees using genetic engineering or marker-assisted breeding.

  13. Structures of Bacteroides fragilis uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA).

    Science.gov (United States)

    Ngo, Alice; Fong, Kai T; Cox, Daniel L; Chen, Xi; Fisher, Andrew J

    2015-05-01

    Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes a reversible reaction for adding an O-acyl group to the GlcNAc in UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A constitutes a major component of lipopolysaccharides, also referred to as endotoxins, which form the outer monolayer of the outer membrane of Gram-negative bacteria. Ligand-free and UDP-GlcNAc-bound crystal structures of LpxA from Bacteroides fragilis NCTC 9343, the most common pathogenic bacteria found in abdominal abscesses, have been determined and are presented here. The enzyme crystallizes in a cubic space group, with the crystallographic threefold axis generating the biological functional homotrimer and with each monomer forming a nine-rung left-handed β-helical (LβH) fold in the N-terminus followed by an α-helical motif in the C-terminus. The structure is highly similar to LpxA from other organisms. Yet, despite sharing a similar LβH structure with LpxAs from Escherichia coli and others, previously unseen calcium ions are observed on the threefold axis in B. fragilis LpxA to help stabilize the trimeric assembly.

  14. Interaction of arylsulfatase A with UDP-N-acetylglucosamine:Lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase.

    Science.gov (United States)

    Schierau, A; Dietz, F; Lange, H; Schestag, F; Parastar, A; Gieselmann, V

    1999-02-05

    The critical step in lysosomal targeting of soluble lysosomal enzymes is the recognition by an UDP-N-acetylglucosamine:lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase. The structure of the determinant common to all lysosomal enzymes for proper recognition by the phosphotransferase is not completely understood. Our current knowledge is largely based on the introduction of targeted amino acid substitutions into lysosomal enzymes and analysis of their effects on phosphotransferase recognition. We have investigated the effect of eight anti-arylsulfatase A monoclonal antibodies on the interaction of arylsulfatase A with the lysosomal enzyme phosphotransferase in vitro. We also show that a lysine-rich surface area of arylsulfatases A and B is essential for proper recognition by the phosphotransferase. Monoclonal antibodies bind to at least six different epitopes at different locations on the surface of arylsulfatase A. All antibodies bind outside the lysine-rich recognition area, but nevertheless Fab fragments of these antibodies prevent interaction of arylsulfatase A with the phosphotransferase. Our data support a model in which binding of arylsulfatase A to the phosphotransferase is not restricted to a limited surface area but involves the simultaneous recognition of large parts of arylsulfatase A.

  15. UDP-glucosyltransferase71c5, a major glucosyltransferase, mediates abscisic acid homeostasis in Arabidopsis.

    Science.gov (United States)

    Liu, Zhen; Yan, Jin-Ping; Li, De-Kuan; Luo, Qin; Yan, Qiujie; Liu, Zhi-Bin; Ye, Li-Ming; Wang, Jian-Mei; Li, Xu-Feng; Yang, Yi

    2015-04-01

    Abscisic acid (ABA) plays a key role in plant growth and development. The effect of ABA in plants mainly depends on its concentration, which is determined by a balance between biosynthesis and catabolism of ABA. In this study, we characterize a unique UDP-glucosyltransferase (UGT), UGT71C5, which plays an important role in ABA homeostasis by glucosylating ABA to abscisic acid -: glucose ester (GE) in Arabidopsis (Arabidopsis thaliana). Biochemical analyses show that UGT71C5 glucosylates ABA in vitro and in vivo. Mutation of UGT71C5 and down-expression of UGT71C5 in Arabidopsis cause delay in seed germination and enhanced drought tolerance. In contrast, overexpression of UGT71C5 accelerates seed germination and reduces drought tolerance. Determination of the content of ABA and ABA-GE in Arabidopsis revealed that mutation in UGT71C5 and down-expression of UGT71C5 resulted in increased level of ABA and reduced level of ABA-GE, whereas overexpression of UGT71C5 resulted in reduced level of ABA and increased level of ABA-GE. Furthermore, altered levels of ABA in plants lead to changes in transcript abundance of ABA-responsive genes, correlating with the concentration of ABA regulated by UGT71C5 in Arabidopsis. Our work shows that UGT71C5 plays a major role in ABA glucosylation for ABA homeostasis.

  16. UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.

    Science.gov (United States)

    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

    2016-01-01

    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.

  17. Overexpression of UDP-glucose pyrophosphorylase gene could increase cellulose content in Jute (Corchorus capsularis L.).

    Science.gov (United States)

    Zhang, Gaoyang; Qi, Jianmin; Xu, Jiantang; Niu, Xiaoping; Zhang, Yujia; Tao, Aifen; Zhang, Liwu; Fang, Pingping; Lin, Lihui

    2013-12-13

    In this study, the full-length cDNA of the UDP-glucose pyrophosphorylase gene was isolated from jute by homologous cloning (primers were designed according to the sequence of UGPase gene of other plants) and modified RACE techniques; the cloned gene was designated CcUGPase. Using bioinformatic analysis, the gene was identified as a member of the UGPase gene family. Real-time PCR analysis revealed differential spatial and temporal expression of the CcUGPase gene, with the highest expression levels at 40 and 120d. PCR and Southern hybridization results indicate that the gene was integrated into the jute genome. Overexpression of CcUGPase gene in jute revealed increased height and cellulose content compared with control lines, although the lignin content remained unchanged. The results indicate that the jute UGPase gene participates in cellulose biosynthesis. These data provide an important basis for the application of the CcUGPase gene in the improvement of jute fiber quality. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. A synchronous Gigabit Ethernet protocol stack for high-throughput UDP/IP applications

    CERN Document Server

    Födisch, P; Sandmann, J; Büchner, A; Enghardt, W; Kaever, P

    2015-01-01

    State of the art detector readout electronics require high-throughput data acquisition (DAQ) systems. In many applications, e. g. for medical imaging, the front-end electronics are set up as separate modules in a distributed DAQ. A standardized interface between the modules and a central data unit is essential. The requirements on such an interface are varied, but demand almost always a high throughput of data. Beyond this challenge, a Gigabit Ethernet interface is predestined for the broad requirements of Systems-on-a-Chip (SoC) up to large-scale DAQ systems. We have implemented an embedded protocol stack for a Field Programmable Gate Array (FPGA) capable of high-throughput data transmission and clock synchronization. A versatile stack architecture for the User Datagram Protocol (UDP) and Internet Control Message Protocol (ICMP) over Internet Protocol (IP) such as Address Resolution Protocol (ARP) as well as Precision Time Protocol (PTP) is presented. With a point-to-point connection to a host in a MicroTCA ...

  19. Performance of TCP/IP/ UDP adaptive header compression algorithm for wireless network

    Directory of Open Access Journals (Sweden)

    Prof.N.Penchalaiah

    2010-08-01

    Full Text Available The convergence of mobile technologies will characterize the future telecommunication systems based on TCP/IP protocols. These Protocols can be used to build both wired and wireless parts on same platform. But TCP/IP headers overheads are large, so it utilizes more bandwidth even small services, whose payload is small. So it definitely needheader compression to save bandwidth, as we know bandwidth is the most expensive and scared resource in wireless systems.There are many approaches used to compress the headers overloads. Most commonly used approaches are, an adaptive robust TCP/IP header compression lgorithm for 3G wireless networks and an adaptive robust header compression algorithm based on UDP-RTS/CTS handshake for real-timestreams in wireless networks such a 3G platforms. But these approaches are not efficient in adaptive nature. So In this paper we are proposing one approach to solve the problem efficiently even in adaptive nature. The aim of this algorithm is to adjust the dimension of Variable Sliding Window (VSW in W-LSB encoding with the accurate estimation of wireless channel state to achieve the good balance of compression ratio and error-resistant robustness for the adaptive use in wireless link. We present simulation results that demonstrate theeffectiveness of this adaptive algorithm over wireless link and comparative study of existing approaches

  20. Identification of UDP-linked murein precursors as contaminants in recombinant proteins of low molecular weight.

    Science.gov (United States)

    Ram, M K; Andrade, L J; Phillips, T B; van Schravendijk, M R

    1999-11-01

    The A(280)/A(260) ratio of a purified protein is frequently used as an indication of the purity of the preparation with respect to nucleic acids. We show here that for low-molecular-weight recombinant proteins purified from Escherichia coli, a low A(280)/A(260) ratio can also result from contamination with UDP-linked murein precursors derived from bacterial cell wall metabolism. Although these precursors are small molecules of molecular weight 1000-1200, they comigrate in gel filtration with recombinant human FKBP (MW 11,820). This gel filtration behavior, which is distinct from that of unmodified mononucleotides, does not reflect binding interactions with FKBP, but is an intrinsic property of these precursors. Therefore, these molecules would be expected to copurify with other low-molecular-weight proteins, especially in the abbreviated purification protocols made possible by freeze-thaw release of recombinant proteins from E. coli (Johnson, B. H., and Hecht, M. H. (1994) BioTechnology 12, 1357-1360). Several alternative strategies are discussed for integrating these findings into the design of improved purification procedures for low-molecular-weight recombinant proteins.

  1. UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides.

    Science.gov (United States)

    Mohamed, Amal A A; Ceunen, Stijn; Geuns, Jan M C; Van den Ende, Wim; De Ley, Marc

    2011-07-01

    A short-term experiment was designed to measure the transcript levels of downstream genes contributing to the biosynthesis of steviol glycosides. Stevia rebaudiana plants were subjected to long- and short-day conditions for different time intervals. Samples from both lower and upper leaves were collected. Using quantitative real-time polymerase chain reaction, the transcript levels of three UDP-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The results were compared with the steviol glycoside contents measured in the leaves, which were quantified by reversed phase HPLC. In the same daylength condition, steviol glycoside concentration and the transcript levels of the three UGT genes were higher in upper leaves than in lower leaves. Steviol glycosides accumulated more in plants under short-day conditions. Under these conditions, a highly significant correlation was found between UGT85C2 transcription and total steviol glycoside accumulation in the upper leaves. This suggests that the glycosylation of steviol to form steviolmonoside is the rate-limiting step in the glycosylation pathway of steviol glycosides. In these upper leaves, a relatively high accumulation of rebaudioside A compared to stevioside was also observed, however, without correlation with the transcription of UGT76G1.

  2. The detoxifying effects of structural elements of persimmon tannin on Chinese cobra phospholipase A2 correlated with their structural disturbing effects well

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    2017-07-01

    Full Text Available The effects of persimmon tannin (PT characteristic structural elements on Naja atra phospholipase A2 (PLA2-induced lethality, myotoxicity, and hemolysis in mice models were determined. In addition, methods including surface plasmon resonance, dynamic light scattering, and Fourier transform infrared spectroscopy were explored to uncover the possible detoxifying mechanisms of PT on snake venom PLA2. Our results revealed that PT characteristic elements (EGCG, ECG, A-type EGCG dimer, and A-type ECG dimer could neutralize the lethality, myotoxicity, and hemolysis of PLA2. Moreover, the detoxifying effects of the four structural elements correlated with their structural disturbing effects well. Our results proved that A-type EGCG dimer and A-type ECG dimer may be structural requirements for the detoxifying effects of PT. We propose that the high affinity of A-type EGCG dimer and A-type ECG dimer for PLA2 and the considerable spatial structural disturbance of PLA2 induced by the dimers may be responsible for their antilethality, antimyotoxicity, and antihemolysis on Chinese cobra PLA2 in vivo.

  3. Long-term effects of a novel phosphorothionate (RPR-II) on detoxifying enzymes in brain, lung, and kidney rats.

    Science.gov (United States)

    Mahboob, M; Siddiqui, M K J

    2002-11-01

    The effects of a phosphorothionate, 2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester (RPR-II), on the activities of glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) and the level of glutathione (GSH) were evaluated in rats after administration of RPR-II at 0.014 (low), 0.028 (medium), and 0.042 (high) mgkg(-1)day(-1) for 90 days and also at 28 days (withdrawal) after stopping treatment. Brain GST activity and GSH level decreased significantly at the high dose on the 45th and 90th days of treatment. Dose- and time-dependent decreases in GST activity and GSH was level were observed in lung at medium and high doses and in kidneys at all three doses on both the 45th and 90th days. UDPGT activity increased significantly in kidneys at the medium and high doses at 45 and 90 days. Brain and lung did not display any significant variations in UDPGT activity when compared with the control. Interestingly, the withdrawal study revealed that the effect was reversible within 28 days of cessation of treatment, when enzyme activity reverted to levels close to those of controls. The study revealed that RPR-II affected the GSH- and GST-dependent detoxification system of the treated tissues of rat and its potential to modulate the enzymes is in the order kidneys>lung>brain. The present subacute study suggests that RPR-II may bring about physiological upsets by altering GSH- and GST-dependent events in different tissues of exposed organisms.

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

    Science.gov (United States)

    McCorvie, Thomas J; Timson, David J

    2013-07-25

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

  5. Evaluación de ataques UDP Flood utilizando escenarios virtuales como plataforma experimental

    Directory of Open Access Journals (Sweden)

    Deyci Toscano

    2011-07-01

    Full Text Available Los ataques por denegación de servicio (DoS tienen como propósito imposibilitar el acceso a los servicios de una organización durante un periodo indefinido; por lo general, están dirigidos a los servidores de una empresa, para que no puedan ser accedidos por usuarios autorizados. El presente trabajo se enfoca en la evaluación de ataques DoS tipo UDP Flood, utilizando como plataforma de experimentación un entorno virtual de red que permite identificar cómo actúan dichos ataques en la saturación del ancho de banda; para llevarlo a cabo se diseñó e implementó una red híbrida con segmentación WAN, LAN y DMZ que inhabilita el acceso interno y externo a un servicio Web expuesto. Las herramientas evaluadas fueron UPD Unicorn, Longcat Flooder y UDPl.pl Script de Perl; las dos primeras instaladas sobre Windows, y la última, sobre Linux. Para validar está investigación se desarrolló un mecanismo de detección y mitigación de los ataques a nivel del firewall e IDS/IPS, evitando de este modo la saturación de la red. Finalmente, se evaluó el consumo dememoria, CPUy ancho de banda durante el ataque, la detección y la evasión, con el fin de determinar cuál genera mayor impacto. Los resultados demuestran que el mecanismo detecta, controla y mitiga los ataques.

  6. Evaluación de ataques UDP Flood utilizando escenarios virtuales como plataforma experimental

    Directory of Open Access Journals (Sweden)

    Walter Fuertes

    2012-05-01

    Full Text Available Los ataques por denegación de servicio (DoS tienen como propósito imposibilitar el acceso a los servicios de una organización durante un periodo indefinido; por lo general, están dirigidos a los servidores de una empresa, para que no puedan ser accedidos por usuarios autorizados. El presente trabajo se enfoca en la evaluación de ataques DoS tipo UDP Flood, utilizando como plataforma de experimentación un entorno virtual de red que permite identificar cómo actúan dichos ataques en la saturación del ancho de banda; para llevarlo a cabo se diseñó e implementó una red híbrida con segmentación WAN, LAN y DMZ que inhabilita el acceso interno y externo a un servicio Web expuesto. Las herramientas evaluadas fueron UPD Unicorn, Longcat Flooder y UDPl.pl Script de Perl; las dos primeras instaladas sobre Windows, y la última, sobre Linux. Para validar está investigación se desarrolló un mecanismo de detección y mitigación de los ataques a nivel del firewall e IDS/IPS, evitando de este modo la saturación de la red. Finalmente, se evaluó el consumo dememoria, CPUy ancho de banda durante el ataque, la detección y la evasión, con el fin de determinar cuál genera mayor impacto. Los resultados demuestran que el mecanismo detecta, controla y mitiga los ataques. 

  7. Up-regulation of sucrose synthase and UDP-glucose pyrophosphorylase impacts plant growth and metabolism.

    Science.gov (United States)

    Coleman, Heather D; Ellis, Dave D; Gilbert, Margarita; Mansfield, Shawn D

    2006-01-01

    The effects of the overexpression of sucrose synthase (SuSy) and UDP-glucose pyrophosphorylase (UGPase) on plant growth and metabolism were evaluated in tobacco (Nicotiana tabacum cv. Xanthi). T(1) transgenic plants expressing either gene under the control of a tandem repeat cauliflower mosaic virus 35S promoter (2x35S) or a xylem-localized 4CL promoter (4-coumarate:CoA ligase; 4CL) were generated, and reciprocally crossed to generate plants expressing both genes. Transcript levels, enzyme activity, growth parameters, fibre properties and carbohydrate content of stem tissue were quantified. The expression profiles of both genes confirmed the expression pattern of the promoters: 2x35S expressed more strongly in leaves, while 4CL expression was highest in stem tissue. In-depth plant characterization revealed that the single-transgene lines showed significant increases in the height growth compared with corresponding control lines. The double-transgene plants demonstrated an additive effect, proving to be even taller than the single-transgene parents. Several of these lines had associated increases in soluble sugar content. Although partitioning of storage carbohydrates into starch or cellulose was not observed, the increased height growth and increases in soluble carbohydrates suggest a role for SuSy as a marker in sink strength and lend credit to the function of UGPase in a similar role. The up-regulation of these two genes, although not increasing the percentage cellulose content, was effective in increasing the total biomass, and thus the overall cellulose yield, from a given plant.

  8. Octamerization is essential for enzymatic function of human UDP-glucose pyrophosphorylase.

    Science.gov (United States)

    Führing, Jana; Damerow, Sebastian; Fedorov, Roman; Schneider, Julia; Münster-Kühnel, Anja-Katharina; Gerardy-Schahn, Rita

    2013-04-01

    Uridine diphosphate-glucose pyrophosphorylase (UGP) occupies a central position in carbohydrate metabolism in all kingdoms of life, since its product uridine diphosphate-glucose (UDP-glucose) is essential in a number of anabolic and catabolic pathways and is a precursor for other sugar nucleotides. Its significance as a virulence factor in protists and bacteria has given momentum to the search for species-specific inhibitors. These attempts are, however, hampered by high structural conservation of the active site architecture. A feature that discriminates UGPs of different species is the quaternary organization. While UGPs in protists are monomers, di- and tetrameric forms exist in bacteria, and crystal structures obtained for the enzyme from yeast and human identified octameric UGPs. These octamers are formed by contacts between highly conserved amino acids in the C-terminal β-helix. Still under debate is the question whether octamerization is required for the functionality of the human enzyme. Here, we used single amino acid replacements in the C-terminal β-helix to interrogate the impact of highly conserved residues on octamer formation and functional activity of human UGP (hUGP). Replacements were guided by the sequence of Arabidopsis thaliana UGP, known to be active as a monomer. Correlating the data obtained in blue native PAGE, size exclusion chromatography and enzymatic activity testing, we prove that the octamer is the active enzyme form. This new insight into structure-function relationships in hUGP does not only improve the understanding of the catalysis of this important enzyme, but in addition broadens the basis for studies aimed at designing drugs that selectively inhibit UGPs from pathogens.

  9. Cloning and characterization of a novel UDP-glycosyltransferase gene induced by DON from wheat

    Institute of Scientific and Technical Information of China (English)

    MA Xin; DU Xu-ye; LIU Guo-juan; YANG Zai-dong; HOU Wen-qian; WANG Hong-wei; FENG De-shun; LI An-fei; KONG Ling-rang

    2015-01-01

    Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is a destructive disease of wheat throughout the world. However, the mechanisms of host resistance to FHB are stil largely unclear. Deoxynivalenol (DON) produced by F. graminearum which enhances the pathogen to spread could be converted into inactive form D3G by UDP-glycosyltrans-ferases (UGTs). A DON responsive UGT gene, designated as TaUGT4, was ifrst cloned from wheat in this study. The putative open reading frame (ORF) of TaUGT4 was 1 386 bp, encoding 461 amino acids protein. TaUGT4 was placed on chromosome 2D using a set of nul i-tetrasomic lines of wheat cultivar Chinese Spring (CS). When fused with eGFP at C terminal, TaUGT4 was shown to localize in cytoplasm of the transformed tobacco cel s. The transcriptional analysis revealed that TaUGT4 was strongly induced by F. graminearum or DON in both of FHB-resistant cultivar Sumai 3 and susceptible cultivar Kenong 199, especial y in Sumai 3 under DON treatment. Similar increase of TaUGT4 expression was observed in Sumai 3 and Kenong 199 in response to salicylic acid (SA) treatment. But interestingly, the transcripts level of TaUGT4 in Sumai 3 showed signiifcantly higher than that in Kenong 199 after treated with methyl jasmonate (MeJA). According to the expression patterns, TaUGT4 might lead to different effects between FHB-resistant genotype and susceptible genotype in the process against F. graminearum inoculation. It had also been discussed in this paper that JA signaling pathway might play a signiifcant role in the resistance against F. graminearum compared to SA signaling pathway.

  10. Isolation and characterization of a cDNA clone of UDP-galactose: flavonoid 3-O-galactosyltransferase (UF3GaT) expressed in Vigna mungo seedlings.

    Science.gov (United States)

    Mato, M; Ozeki, Y; Itoh, Y; Higeta, D; Yoshitama, K; Teramoto, S; Aida, R; Ishikura, N; Shibata, M

    1998-11-01

    Four cDNA clones were isolated from Vigna mungo seedlings by the screening with cDNA encoding UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) of Antirrhinum majus as a probe; the product of the gene corresponding to one cDNA was more highly expressed in the first simple leaves than in stems. Nucleotide sequence analysis revealed 1,691 bp (including 326 bp non-reading) containing an open reading frame of 455 amino acids. The deduced amino acid sequence showed 42% and 23% identity with those of A. majus UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petunia hybrida UDP-rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase (RT), respectively. One region of the cDNA (amino acids 325 to 387) showed similarity to ceramide UDP-galactosyltransferases of mice, rats and humans. A crude extract from Escherichia coli, in which the protein was expressed from the cDNA, showed high UF3GaT activity but low UF3GT activity, and was similar in K(m), optimal pH and substrate specificity to UF3GaT from V. mungo. We conclude that we have obtained UDP-galactose:flavonoid 3-O-galactosyltransferase (UF3GaT) cDNA from V. mungo.

  11. Neurophysiological correlates of response inhibition predict relapse in detoxified alcoholic patients: some preliminary evidence from event-related potentials

    Directory of Open Access Journals (Sweden)

    Petit G

    2014-06-01

    Full Text Available Géraldine Petit, Agnieszka Cimochowska, Charles Kornreich, Catherine Hanak, Paul Verbanck, Salvatore CampanellaLaboratory of Psychological Medicine and Addictology, ULB Neuroscience Institute (UNI, Université Libre de Bruxelles (ULB, Brussels, BelgiumBackground: Alcohol dependence is a chronic relapsing disease. The impairment of response inhibition and alcohol-cue reactivity are the main cognitive mechanisms that trigger relapse. Despite the interaction suggested between the two processes, they have long been investigated as two different lines of research. The present study aimed to investigate the interaction between response inhibition and alcohol-cue reactivity and their potential link with relapse.Materials and methods: Event-related potentials were recorded during a variant of a “go/no-go” task. Frequent and rare stimuli (to be inhibited were superimposed on neutral, nonalcohol-related, and alcohol-related contexts. The task was administered following a 3-week detoxification course. Relapse outcome was measured after 3 months, using self-reported abstinence. There were 27 controls (seven females and 27 patients (seven females, among whom 13 relapsed during the 3-month follow-up period. The no-go N2, no-go P3, and the “difference” wave (P3d were examined with the aim of linking neural correlates of response inhibition on alcohol-related contexts to the observed relapse rate.Results: Results showed that 1 at the behavioral level, alcohol-dependent patients made significantly more commission errors than controls (P<0.001, independently of context; 2 through the subtraction no-go P3 minus go P3, this inhibition deficit was neurophysiologically indexed in patients with greater P3d amplitudes (P=0.034; and 3 within the patient group, increased P3d amplitude enabled us to differentiate between future relapsers and nonrelapsers (P=0.026.Conclusion: Our findings suggest that recently detoxified alcoholics are characterized by poorer

  12. Structural and Functional Analysis of Campylobacter jejuni PseG: a Udp-sugarhydrolase from the Pseudaminic Acid Biosynthetic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    E Rangarajan; A Proteau; Q Cui; S Logan; Z Potetinova; D Whitfield; E Purisima; M Cygler; A Matte; et al.

    2011-12-31

    Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-{beta}-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 {angstrom} resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His{sup 17}-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His{sup 17} functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C-O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.

  13. UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Identification and separation of two distinct transferase activities

    DEFF Research Database (Denmark)

    Sørensen, T; White, T; Wandall, H H;

    1995-01-01

    Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) (White, T., Bennett, E.P., Takio, K., Sørensen, T., Bonding, N......., and Clausen, H. (1995) J. Biol. Chem. 270, 24156-24165). Here we report detailed studies of the acceptor substrate specificity of GalNAc-transferase purified by this scheme as well as the Gal-NAc-transferase activity, which, upon repeated affinity chromatography, evaded purification by this affinity ligand...

  14. Intestinal detoxifi cation time of hand-foot-and-mouth disease in children with EV71 infection and the related factors

    Institute of Scientific and Technical Information of China (English)

    Shu Teng; Yi Wei; Shi-Yong Zhao; Xian-Yao Lin; Qi-Min Shao; Juan Wang

    2015-01-01

    Background: Hand-foot-and-mouth disease (HFMD) is a common pediatric infectious disease caused by a variety of intestinal viruses. Enterovirus 71 (EV71) is the primary pathogen that might cause severe symptoms and even death in children with HFMD. This study aimed to investigate the intestinal detoxification time of HFMD children with EV71 infection and its related factors. Methods: Sixty-five HFMD children with EV71 infection were followed up. Their stool samples were collected once every 4 to 7 days. Viral nucleic acids were detected byfl uorescent polymerase chain reaction until the results became negative. The positive rates of viral nucleic acids were analyzed by the Kaplan-Meier method. The Log-rank test and Cox-Mantel test were used to analyze factors affecting the HFMD children with EV71 infection. Results: On the 2nd, 4th, 6th and 10th week, the positive rates of viral nucleic acids in stool samples of the 65 children were 94.6%, 48.1%, 17.2% and 0, respectively. Univariate analysis showed that the intestinal detoxifi cation time of the children were related to gender, pre-admission disease course, severity of disease, and use of steroids or gamma globulin (P<0.05). Multivariate analysis showed that the severity of disease was an independent factor affecting the intestinal detoxification time (P<0.05), with a relative risk of 2.418. Conclusions: The longest intestinal detoxifi cation time of HFMD children with EV71 infection was 10 weeks. The severity of disease was an important factor affecting the intestinal detoxification time of HFMD children with EV71 infection. Severe HFMD children with EV71 infection had a longer intestinal detoxifi cation time.

  15. Adjuvant effects elicited by novel oligosaccharide variants of detoxified meningococcal lipopolysaccharides on Neisseria meningitidis recombinant PorA protein: a comparison in mice.

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    Ojas H Mehta

    Full Text Available Neisseria meningitidis lipopolysaccharide (LPS has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS, and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus were immunized with a recombinant PorA P1.7-2,4 (rPorA protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16, each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines.

  16. A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

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    Belshoff Alex C

    2011-05-01

    Full Text Available Abstract Background Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes. Results We utilized a stable isotope resolved metabolomics (SIRM approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits

  17. Downregulation of the UDP-arabinomutase gene in switchgrass (Panicum virgatum L. results in increased cell wall lignin while reducing arabinose-glycans

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    Jonathan Duran Willis

    2016-10-01

    Full Text Available Switchgrass (Panicum virgatum L. is a C4 perennial prairie grass and a lignocellulosic biofuels feedstock. 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 crosslink other cell wall polymers. Grasses have predominately 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/reversible glycosylated polypeptides (UAM/RGPs catalyze the interconversion between UDP-arabinopyranose (UDP-Arap and UDP-Araf. In switchgrass we knocked down expression of the endogenous PvUAM1 gene 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. 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 morphologically similar to non-transgenics. There was decreased cell wall-associated arabinose in leaves and stems by over 50%, but there was an increase in cellulose in these organs. 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, but had increased glucose in cell walls. The increased glucose detected in stems and leaves indicates that attenuation of PvUAM1 expression might have downstream effects on starch

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

    Science.gov (United States)

    Willis, Jonathan D.; Smith, James A.; Mazarei, Mitra; Zhang, Ji-Yi; Turner, Geoffrey B.; Decker, Stephen R.; Sykes, Robert W.; Poovaiah, Charleson R.; Baxter, Holly L.; Mann, David G. J.; Davis, Mark F.; Udvardi, Michael K.; Peña, Maria J.; Backe, Jason; Bar-Peled, Maor; Stewart, C. N.

    2016-01-01

    Background: Switchgrass (Panicum virgatum L.) is a C4 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. Results: The expression of 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. Conclusion: Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell

  19. Molecular and functional analysis of UDP-N-acetylglucosamine Pyrophosphorylases from the Migratory Locust, Locusta migratoria.

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    Xiaojian Liu

    Full Text Available UDP-N-acetylglucosamine pyrophosphorylases (UAP function in the formation of extracellular matrix by producing N-acetylglucosamine (GlcNAc residues needed for chitin biosynthesis and protein glycosylation. Herein, we report two UAP cDNA's derived from two different genes (LmUAP1 and LmUAP2 in the migratory locust Locusta migratoria. Both the cDNA and their deduced amino acid sequences showed about 70% identities between the two genes. Phylogenetic analysis suggests that LmUAP1 and LmUAP2 derive from a relatively recent gene duplication event. Both LmUAP1 and LmUAP2 were widely expressed in all the major tissues besides chitin-containing tissues. However, the two genes exhibited different developmental expression patterns. High expression of LmUAP1 was detected during early embryogenesis, then decreased greatly, and slowly increased before egg hatch. During nymphal development, the highest expression of LmUAP1 appeared just after molting but declined in each inter-molting period and then increased before molting to the next stage, whereas LmUAP2 was more consistently expressed throughout all these stages. When the early second- and fifth-instar nymphs (1-day-old were injected with LmUAP1 double-stranded RNA (dsRNA, 100% mortality was observed 2 days after the injection. When the middle second- and fifth-instar nymphs (3- to 4-day-old were injected with LmUAP1 dsRNA, 100% mortality was observed during their next molting process. In contrast, when the insects at the same stages were injected with LmUAP2 dsRNA, these insects were able to develop normally and molt to the next stage successfully. It is presumed that the lethality caused by RNAi of LmUAP1 is due to reduced chitin biosynthesis of the integument and midgut, whereas LmUAP2 is not essential for locust development at least in nymph stage. This study is expected to help better understand different functions of UAP1 and UAP2 in the locust and other insect species.

  20. Molecular and functional analysis of UDP-N-acetylglucosamine Pyrophosphorylases from the Migratory Locust, Locusta migratoria.

    Science.gov (United States)

    Liu, Xiaojian; Li, Feng; Li, Daqi; Ma, Enbo; Zhang, Wenqing; Zhu, Kun Yan; Zhang, Jianzhen

    2013-01-01

    UDP-N-acetylglucosamine pyrophosphorylases (UAP) function in the formation of extracellular matrix by producing N-acetylglucosamine (GlcNAc) residues needed for chitin biosynthesis and protein glycosylation. Herein, we report two UAP cDNA's derived from two different genes (LmUAP1 and LmUAP2) in the migratory locust Locusta migratoria. Both the cDNA and their deduced amino acid sequences showed about 70% identities between the two genes. Phylogenetic analysis suggests that LmUAP1 and LmUAP2 derive from a relatively recent gene duplication event. Both LmUAP1 and LmUAP2 were widely expressed in all the major tissues besides chitin-containing tissues. However, the two genes exhibited different developmental expression patterns. High expression of LmUAP1 was detected during early embryogenesis, then decreased greatly, and slowly increased before egg hatch. During nymphal development, the highest expression of LmUAP1 appeared just after molting but declined in each inter-molting period and then increased before molting to the next stage, whereas LmUAP2 was more consistently expressed throughout all these stages. When the early second- and fifth-instar nymphs (1-day-old) were injected with LmUAP1 double-stranded RNA (dsRNA), 100% mortality was observed 2 days after the injection. When the middle second- and fifth-instar nymphs (3- to 4-day-old) were injected with LmUAP1 dsRNA, 100% mortality was observed during their next molting process. In contrast, when the insects at the same stages were injected with LmUAP2 dsRNA, these insects were able to develop normally and molt to the next stage successfully. It is presumed that the lethality caused by RNAi of LmUAP1 is due to reduced chitin biosynthesis of the integument and midgut, whereas LmUAP2 is not essential for locust development at least in nymph stage. This study is expected to help better understand different functions of UAP1 and UAP2 in the locust and other insect species.

  1. Species and Gender Differences Affect the Metabolism of Baicalein via Glucuronidation%黄芩素在人及不同种属肝微粒体中的UDP-glucuronosyltransferase代谢差异

    Institute of Scientific and Technical Information of China (English)

    吴媛; 董瑞华; 高欣; 刘泽源

    2013-01-01

    . Besides, our data also show that there are great differences in gender in some species, BG, BG’and BGG in SD rats and BG in dogs have remarkable differences. The species and gender metabolic differences we observed between animals and human liver provide key informa-tion for delineating Baicalein pharmacokinetics needed for human health risk assessment. In conclusion, species and gender affect Baicalein metabolism to a different degree, and experimental animals are expected to be useful to predict the Baicalein glucuronidation in humans.%目的:研究黄芩素在不同种属肝微粒体中的UDP-葡萄糖醛酸转移酶(UDP-glucuronosyltransferase, UDPGA)代谢差异特性。方法使用肝微粒体体外代谢孵育法、HPLC-UV分析方法,选用不同种属的肝微粒体进行黄芩素UDPGA体外代谢研究。结果黄芩素在人肝微粒体及不同种属的肝微粒中,加入UDPGA进行37℃恒温孵育,孵育结束后离心,取上清液,经HPLC-UV分离检测得到3个代谢产物,分别是:黄芩素-7-O-β-葡萄糖醛酸结合物、黄芩素-6-O-β-葡萄糖醛酸结合物和黄芩素-6-O-葡萄糖醛酸结合物-7-O-β-葡萄糖醛酸结合物;通过与标准品对照确定黄芩素的三个代谢产物都是葡萄糖醛酸化的代谢产物。同时,不同种属间UGT代谢物的活性表现出较大差异,黄芩素-7-O-β-葡萄糖醛酸结合物在人肝微粒体中的代谢活性最强,Km=1.61,Vmax=0.77(BG在人肝微粒体中的代谢活性是SD雌鼠的25.2倍);黄芩素-6-O-β-葡萄糖醛酸结合物在比格犬肝微粒体中代谢活性最强,Km=3.05,Vmax=3.51(雄性比格犬肝微粒体的活性是雄性恒河猴肝微粒体的2.6倍);黄芩素-6-O-葡萄糖醛酸-7-O-β-葡萄糖醛酸结合物在猪肝微粒体中代谢活性最强,Km=5.38, Vmax=0.17(猪肝微粒体的活性是人肝微粒体的13.6倍),其他依次是犬、恒河猴、鼠和人。结论黄芩素在人及不同种属肝微

  2. AglM and VNG1048G, Two Haloarchaeal UDP-Glucose Dehydrogenases, Show Different Salt-Related Behaviors

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    Lina Kandiba

    2016-08-01

    Full Text Available Haloferax volcanii AglM and Halobacterium salinarum VNG1048G are UDP-glucose dehydrogenases involved in N-glycosylation in each species. Despite sharing >60% sequence identity and the ability of VNG1048G to functionally replace AglM in vivo, these proteins behaved differently as salinity changed. Whereas AglM was active in 2–4 M NaCl, VNG1048G lost much of its activity when salinity dropped below 3 M NaCl. To understand the molecular basis of this phenomenon, each protein was examined by size exclusion chromatrography in 2 M NaCl. Whereas AglM appeared as a dodecamer, VNG1048G was essentially detected as a dodecamer and a dimer. The specific activity of the VNG1048G dodecamer was only a sixth of that of AglM, while the dimer was inactive. As such, not only was the oligomeric status of VNG1048G affected by lowered salinity, so was the behavior of the individual dodecamer subunits. Analyzing surface-exposed residues in homology models of the two UDP-glucose dehydrogenases revealed the more acidic and less basic VNG1048G surface, further explaining the greater salt-dependence of the Hbt. salinarum enzyme.

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

    Science.gov (United States)

    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.

  4. Molecular and biochemical characterization of the UDP-glucose: Anthocyanin 5-O-glucosyltransferase from Vitis amurensis.

    Science.gov (United States)

    He, Fei; Chen, Wei-Kai; Yu, Ke-Ji; Ji, Xiang-Nan; Duan, Chang-Qing; Reeves, Malcolm J; Wang, Jun

    2015-09-01

    Generally, red Vitis vinifera grapes only contain monoglucosidic anthocyanins, whereas most non-vinifera red grapes of the Vitis genus have both monoglucosidic and bis-glucosidic anthocyanins, the latter of which are believed to be more hydrophilic and more stable. Although previous studies have established the biosynthetic mechanism for formation of monoglucosidic anthocyanins, less attention has been paid to that of bis-glucosidic anthocyanins. In the present research, the full-length cDNA of UDP-glucose: anthocyanin 5-O-glucosyltransferase from Vitis amurensis Rupr. cv. 'Zuoshanyi' grape (Va5GT) was cloned. After acquisition and purification of recombinant Va5GT, its enzymatic parameters were systematically analyzed in vitro. Recombinant Va5GT used malvidin-3-O-glucoside as its optimum glycosidic acceptor when UDP-glucose was used as the glycosidic donor. Va5GT-GFP was found to be located in the cytoplasm by analyzing its subcellular localization with a laser-scanning confocal fluorescence microscope, and this result was coincident with its metabolic function of modifying anthocyanins in grape cells. Furthermore, the relationship between the transcriptional expression of Va5GT and the accumulation of anthocyanidin bis-glucosides during berry development suggested that Va5GT is a key enzyme in the biosynthesis of bis-glucosidic anthocyanins in V. amurensis grape berries.

  5. Development of Client-Server Application by Using UDP Socket Programming for Remotely Monitoring CNC Machine Environment in Fixture Process

    Directory of Open Access Journals (Sweden)

    Darmawan Darmawan

    2016-08-01

    Full Text Available The use of computer technology in manufacturing industries can improve manufacturing flexibility significantly, especially in manufacturing processes; many software applications have been utilized to improve machining performance. However, none of them has discussed the abilities to perform direct machining. In this paper, an integrated system for remote operation and monitoring of Computer Numerical Control (CNC machines is put into consideration. The integrated system includes computerization, network technology, and improved holding mechanism. The work proposed by this research is mainly on the software development for such integrated system. It uses Java three-dimensional (3D programming and Virtual Reality Modeling Language (VRML at the client side for visualization of machining environment. This research is aimed at developing a control system to remotely operate and monitor a self-reconfiguration fixture mechanism of a CNC milling machine through internet connection and integration of Personal Computer (PC-based CNC controller, a server side, a client side and CNC milling. The performance of the developed system was evaluated by testing with one type of common protocols particularly User Datagram Protocol (UDP.  Using UDP, the developed system requires 3.9 seconds to complete the close clamping, less than 1 second to release the clamping and it can deliver 463 KiloByte.

  6. Implementation and Design of UDP Flow Measurement System%一种流方式的UDP测量系统的设计与实现

    Institute of Scientific and Technical Information of China (English)

    晋国青; 郭海峰

    2011-01-01

    近年来迅猛发展的P2P、流媒体、网络游戏等UDP新应用占总流量的比重越来越大,这些UDP数据没有TCP数据本身的流的标识,也没有流开始的握手和流结束时候的Reset包.面向高速网络环境,提出一种流方式的UPD系统(FlowUDP)的思路.该系统考虑骨干网络中UDP流的单向特性,使用了winpcap捕包技术.系统包括网络数据包捕获模块、数据包重放模块、数据包拼流模块、UDP分析模块,同时采用stl中的map模板类,通过自定义高效的比较函数,提高了UDP数据包性能,从而提高系统运行效率.针对复杂网络环境下的数据特点,系统采用分步骤小工具设计方式,方便系统扩充新的分析功能.通过一个骨干网数据集和两个DARPA1999数据集,可以看出,UDP数据流的总体发展是占网络带宽越来越多,而且小的UDP流比较多,网络带宽发展越来越快,UDP流的平均时同越来越短.%In recent years, the rapid development of P2P, streaming media, network games and new application of UDP play an important role in the current Internet. These UDP data have not TCP flow identification itself, nor the flow started handshake and the reset packets of the flow over. The UDP flow measurement system is studied. A FlowUDP idea is proposed for high-speed network environment. The system considered the one-way characteristic of UDP flow in backbone network, used the winpeap packet capture technology. The system included network packet capture module, packet replay module, packet spell flow module and UDP analysis module, and adopted the map template classes in stl to improve the performance of UDP data packet through a comparison function of efficient custom, so as to improve the efficiency of the system. According to the data characteristic of complex network environment, the system adopted the step small tools design way to expand a new analysis function. Three data sets (a backbone data sets and two DARPA1999 data sets

  7. The relative merits of dolomitic and calcitic limestone in detoxifying and revegetating acidic, nickel- and copper-contaminated soils in the Sudbury mining and smelting region of Canada

    Energy Technology Data Exchange (ETDEWEB)

    McHale, D.; Winterhalder, K. [Laurentian Univ., Sudbury, Ontario (Canada)

    1996-12-31

    Soils in the Sudbury mining and smelting region have been rendered phytotoxic and barren by acidification and Particulate metal contamination, but can be detoxified revegetated by the surface application of an growth is better on soil treated ground limestone. On certain barren sites, plant growth is better on soil treated with dolomitic limestone than with calcitic limestone and greenhouse experiments using mung beans (Vigna radiata) have shown superior root and shoot growth on certain contaminated soils when the limestone is dolomitic rather than calcitic. Results of experiments with species used in revegetation (Agrostis gigantea and Lotus corniculatus) suggest that leguminous species are more sensitive to Ca:Mg ratio than grasses, that the plant response to this ratio is greater at lowering liming levels, and that the response is more marked on more toxic soils. The effects of calcium:magnesium ratio of the limestone used in revegetating acidic, metal-contaminated soils are clearly complex, interactive and difficult to interpret. Further studies are needed, but meanwhile it is recommended that the practice of using dolomitic limestone to detoxify barren Sudbury soils be continued, since there is a risk of induced magnesium deficiency at certain sites when calcitic limestone is used.

  8. Inhibitory Effects of Aschantin on Cytochrome P450 and Uridine 5′-diphospho-glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Soon-Sang Kwon

    2016-04-01

    Full Text Available Aschantin is a bioactive neolignan found in Magnolia flos with antiplasmodial, Ca2+-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP and uridine 5′-diphospho-glucuronosyltransferase (UGT enzymes of human liver microsomes to determine if mechanistic aschantin–enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1′-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.

  9. Down-regulation of UDP-glucuronic Acid Biosynthesis Leads to Swollen Plant Cell Walls and Severe Developmental Defects Associated with Changes in Pectic Polysaccharides*

    Science.gov (United States)

    Reboul, Rebecca; Geserick, Claudia; Pabst, Martin; Frey, Beat; Wittmann, Doris; Lütz-Meindl, Ursula; Léonard, Renaud; Tenhaken, Raimund

    2011-01-01

    UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation. PMID:21949134

  10. Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides.

    Science.gov (United States)

    Reboul, Rebecca; Geserick, Claudia; Pabst, Martin; Frey, Beat; Wittmann, Doris; Lütz-Meindl, Ursula; Léonard, Renaud; Tenhaken, Raimund

    2011-11-18

    UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

  11. Evidence of Kinetic Control of Ligand Binding and Staged Product Release in MurA (enolpyruvyl UDP-GlcNAc synthase)-catalyzed Reactions

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, S.; Zhang, F; Chindemi, P; Junop, M; Berti, P

    2009-01-01

    MurA (enolpyruvyl UDP-GlcNAc synthase) catalyzes the first committed step in peptidoglycan biosynthesis. In this study, MurA-catalyzed breakdown of its tetrahedral intermediate (THI), with a k{sub cat}/K{sub M} of 520 M{sup -1} s{sup -1}, was far slower than the normal reaction, and 3 x 10{sup 5}-fold slower than the homologous enzyme, AroA, reacting with its THI. This provided kinetic evidence of slow binding and a conformationally constrained active site. The MurA cocrystal structure with UDP-N-acetylmuramic acid (UDP-MurNAc), a potent inhibitor, and phosphite revealed a new 'staged' MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site. The closed-to-staged transition involved breaking eight MurA {center_dot} ligand ion pairs, and three intraprotein hydrogen bonds helping hold the active site loop closed. These were replaced with only two MurA {center_dot} UDP-MurNAc ion pairs, two with phosphite, and seven new intraprotein ion pairs or hydrogen bonds. Cys115 appears to have an important role in forming the staged conformation. The staged conformation appears to be one step in a complex choreography of release of the product from MurA.

  12. Effects of thyroid status and thyrostatic drugs on hepatic glucuronidation of lodothyronines and other substrates in rats - Induction of phenol UDP-glucuronyltransferase by methimazole

    NARCIS (Netherlands)

    T.J. Visser (Theo); E. Kaptein (Ellen); A.L. Gijzel (Anthonie); W.W. de Herder (Wouter); M.L. Cannon (Mark); F. Bonthuis (Fred); W.J. de Greef (W.)

    1996-01-01

    textabstractGlucuronidation of iodothyronines in rat liver is catalyzed by at least three UDP-glucuronyltransferases (UGTs): bilirubin UGT, phenol UGT, and androsterone UGT. Bilirubin and phenol UGT activities are regulated by thyroid hormone, but the effect of thyroid status on hepatic glucuronidat

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

    Science.gov (United States)

    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.

  14. GneZ, a UDP-GlcNAc 2-epimerase, is required for S-layer assembly and vegetative growth of Bacillus anthracis.

    Science.gov (United States)

    Wang, Ya-Ting; Missiakas, Dominique; Schneewind, Olaf

    2014-08-15

    Bacillus anthracis, the causative agent of anthrax, forms an S-layer atop its peptidoglycan envelope and displays S-layer proteins and Bacillus S-layer-associated (BSL) proteins with specific functions to support cell separation of vegetative bacilli and growth in infected mammalian hosts. S-layer and BSL proteins bind via the S-layer homology (SLH) domain to the pyruvylated secondary cell wall polysaccharide (SCWP) with the repeat structure [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→]n, where α-GlcNAc and β-GlcNAc are substituted with two and one galactosyl residues, respectively. B. anthracis gneY (BAS5048) and gneZ (BAS5117) encode nearly identical UDP-GlcNAc 2-epimerase enzymes that catalyze the reversible conversion of UDP-GlcNAc and UDP-ManNAc. UDP-GlcNAc 2-epimerase enzymes have been shown to be required for the attachment of the phage lysin PlyG with the bacterial envelope and for bacterial growth. Here, we asked whether gneY and gneZ are required for the synthesis of the pyruvylated SCWP and for S-layer assembly. We show that gneZ, but not gneY, is required for B. anthracis vegetative growth, rod cell shape, S-layer assembly, and synthesis of pyruvylated SCWP. Nevertheless, inducible expression of gneY alleviated all the defects associated with the gneZ mutant. In contrast to vegetative growth, neither germination of B. anthracis spores nor the formation of spores in mother cells required UDP-GlcNAc 2-epimerase activity.

  15. UDP-N-Acetyl glucosamine pyrophosphorylase as novel target for controlling Aedes aegypti – molecular modeling, docking and simulation studies

    Directory of Open Access Journals (Sweden)

    Bhagath Kumar Palaka

    2014-12-01

    Full Text Available Aedes aegypti is a vector that transmits diseases like dengue fever, chikungunya, and yellow fever. It is distributed in all tropical and subtropical regions of the world. According to WHO reports, 40% of the world’s population is currently at risk for dengue fever. As vaccines are not available for such diseases, controlling mosquito population becomes necessary. Hence, this study aims at UDP-N-acetyl glucosamine pyrophosphorylase of Aedes aegypti (AaUAP, an essential enzyme for chitin metabolim in insects, as a drug target. Structure of AaUAP was predicted and validated using in-silico approach. Further, docking studies were performed using a set of 10 inhibitors out of which NAG9 was found to have good docking score, which was further supported by simulation studies. Hence, we propose that NAG9 can be considered as a potential hit in designing new inhibitors to control Aedes aegypti.

  16. UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors.

    Science.gov (United States)

    Zinsser, Veronika L; Lindert, Steffen; Banford, Samantha; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J

    2015-03-01

    Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

  17. A diet containing the soy phytoestrogen genistein causes infertility in female rats partially deficient in UDP glucuronyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Seppen, Jurgen, E-mail: j.seppen@amc.uva.nl

    2012-11-01

    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 diets 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.

  18. Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

    Energy Technology Data Exchange (ETDEWEB)

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J. (Virginia Tech); (UMC)

    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 inhibitor 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.

  19. Mechanisms of Nrf2/Keap1-Dependent Phase II Cytoprotective and Detoxifying Gene Expression and Potential Cellular Targets of Chemopreventive Isothiocyanates

    Directory of Open Access Journals (Sweden)

    Biswa Nath Das

    2013-01-01

    Full Text Available Isothiocyanates (ITCs are abundantly found in cruciferous vegetables. Epidemiological studies suggest that chronic consumption of cruciferous vegetables can lower the overall risk of cancer. Natural ITCs are key chemopreventive ingredients of cruciferous vegetables, and one of the prime chemopreventive mechanisms of natural isothiocyanates is the induction of Nrf2/ARE-dependent gene expression that plays a critical role in cellular defense against electrophiles and reactive oxygen species. In the present review, we first discuss the underlying mechanisms how natural ITCs affect the intracellular signaling kinase cascades to regulate the Keap1/Nrf2 activities, thereby inducing phase II cytoprotective and detoxifying enzymes. We also discuss the potential cellular protein targets to which natural ITCs are directly conjugated and how these events aid in the chemopreventive effects of natural ITCs. Finally, we discuss the posttranslational modifications of Keap1 and nucleocytoplasmic trafficking of Nrf2 in response to electrophiles and oxidants.

  20. A live attenuated Salmonella Enteritidis secreting detoxified heat labile toxin enhances mucosal immunity and confers protection against wild-type challenge in chickens.

    Science.gov (United States)

    Lalsiamthara, Jonathan; Kamble, Nitin Machindra; Lee, John Hwa

    2016-06-04

    A live attenuated Salmonella Enteritidis (SE) capable of constitutively secreting detoxified double mutant Escherichia coli heat labile toxin (dmLT) was developed. The biologically adjuvanted strain was generated via transformation of a highly immunogenic SE JOL1087 with a plasmid encoding dmLT gene cassette; the resultant strain was designated JOL1641. A balanced-lethal host-vector system stably maintained the plasmid via auxotrophic host complementation with a plasmid encoded aspartate semialdehyde dehydrogenase (asd) gene. Characterization by western blot assay revealed the dmLT subunit proteins in culture supernatants of JOL1641. For the investigation of adjuvanticity and protective efficacy, chickens were immunized via oral or intramuscular routes with PBS, JOL1087 and JOL1641. Birds immunized with JOL1641 showed significant (P ≤ 0.05) increases in intestinal SIgA production at the 1(st) and 2(nd) weeks post-immunization via oral and intramuscular routes, respectively. Interestingly, while both strains showed significant splenic protection via intramuscular immunization, JOL1641 outperformed JOL1087 upon oral immunization. Oral immunization of birds with JOL1641 significantly reduced splenic bacterial counts. The reduction in bacterial counts may be correlated with an adjuvant effect of dmLT that increases SIgA secretion in the intestines of immunized birds. The inclusion of detoxified dmLT in the strain did not cause adverse reactions to birds, nor did it extend the period of bacterial fecal shedding. In conclusion, we report here that dmLT could be biologically incorporated in the secretion system of a live attenuated Salmonella-based vaccine, and that this construction is safe and could enhance mucosal immunity, and protect immunized birds against wild-type challenge.

  1. PHENOBARBITAL AFFECTS THYROID HISTOLOGY AND LARVAL DEVELOPMENT IN THE AFRICAN CLAWED FROG XENOPUS LAEVIS

    Science.gov (United States)

    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...

  2. Cloning a Full-length cDNA Encoding UDP-glucose Pyrophosphorylase from Amorpha fruticosa by PCR-based Methods

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A method based on degenerate Oligo-primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase (UGP), a key enzyme producing UDP-glucose in the synthesis of sucrose and cell ulose, is cloned by using this method. We design 5' RACE primers based on UGP A1 fragment, which obtains from degenerate PCR. Inverse PCR and nested PCR enable cloning of the remainder 5' and 3' end fragments of the gene. The deduced amino acid sequence exhibits significant homology with the other UGP genes cloned. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes.

  3. Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance

    DEFF Research Database (Denmark)

    Lacour, S.; Bechet, E.; Cozzone, A.J.

    2008-01-01

    -kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been...... shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides. Methodology....../Principal Findings: Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc...

  4. A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2.

    Science.gov (United States)

    Gómez, Hansel; Rojas, Raúl; Patel, Divya; Tabak, Lawrence A; Lluch, José M; Masgrau, Laura

    2014-05-01

    It is estimated that >50% of proteins are glycosylated with sugar tags that can modulate protein activity through what has been called the sugar code. Here we present the first QM/MM calculations of human GalNAc-T2, a retaining glycosyltransferase, which initiates the biosynthesis of mucin-type O-glycans. Importantly, we have characterized a hydrogen bond between the β-phosphate of UDP and the backbone amide group from the Thr7 of the sugar acceptor (EA2 peptide) that promotes catalysis and that we propose could be a general catalytic strategy used in peptide O-glycosylation by retaining glycosyltransferases. Additional important substrate-substrate interactions have been identified, for example, between the β-phosphate of UDP with the attacking hydroxyl group from the acceptor substrate and with the substituent at the C2' position of the transferred sugar. Our results support a front-side attack mechanism for this enzyme, with a barrier height of ~20 kcal mol(-1) at the QM(M05-2X/TZVP//BP86/SVP)/CHARMM22 level, in reasonable agreement with the experimental kinetic data. Experimental and in silico mutations show that transferase activity is very sensitive to changes in residues Glu334, Asn335 and Arg362. Additionally, our calculations for different donor substrates suggest that human GalNAc-T2 would be inactive if 2'-deoxy-Gal or 2'-oxymethyl-Gal were used, while UDP-Gal is confirmed as a valid sugar donor. Finally, the analysis herein presented highlights that both the substrate-substrate and the enzyme-substrate interactions are mainly concentrated on stabilizing the negative charge developing at the UDP leaving group as the transition state is approached, identifying this as a key aspect of retaining glycosyltransferases catalysis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Karr, Dale B.; Nix, Jay C.; Sobrado, Pablo; Tanner, John J. (LBNL); (Missouri); (VPI)

    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 has 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.

  6. A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2†

    Science.gov (United States)

    Gómez, Hansel; Rojas, Raúl; Patel, Divya; Lluch, José M.

    2016-01-01

    It is estimated that >50% of proteins are glycosylated with sugar tags that can modulate protein activity through what has been called the sugar code. Here we present the first QM/MM calculations of human GalNAc-T2, a retaining glycosyltransferase, which initiates the biosynthesis of mucin-type O-glycans. Importantly, we have characterized a hydrogen bond between the β-phosphate of UDP and the backbone amide group from the Thr7 of the sugar acceptor (EA2 peptide) that promotes catalysis and that we propose could be a general catalytic strategy used in peptide O-glycosylation by retaining glycosyltransferases. Additional important substrate–substrate interactions have been identified, for example, between the β-phosphate of UDP with the attacking hydroxyl group from the acceptor substrate and with the substituent at the C2′ position of the transferred sugar. Our results support a front-side attack mechanism for this enzyme, with a barrier height of ~20 kcal mol−1 at the QM(M05-2X/TZVP//BP86/SVP)/CHARMM22 level, in reasonable agreement with the experimental kinetic data. Experimental and in silico mutations show that transferase activity is very sensitive to changes in residues Glu334, Asn335 and Arg362. Additionally, our calculations for different donor substrates suggest that human GalNAc-T2 would be inactive if 2′-deoxy-Gal or 2′-oxymethyl-Gal were used, while UDP-Gal is confirmed as a valid sugar donor. Finally, the analysis herein presented highlights that both the substrate–substrate and the enzyme–substrate interactions are mainly concentrated on stabilizing the negative charge developing at the UDP leaving group as the transition state is approached, identifying this as a key aspect of retaining glycosyltransferases catalysis. PMID:24643241

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

    Directory of Open Access Journals (Sweden)

    Barvkar Vitthal T

    2012-05-01

    Full Text Available Abstract 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

  8. The metastability of human UDP-galactose 4'-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding.

    Science.gov (United States)

    Pey, Angel L; Padín-Gonzalez, Esperanza; Mesa-Torres, Noel; Timson, David J

    2014-11-15

    Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization.

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

    Science.gov (United States)

    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.

  10. Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.

    Science.gov (United States)

    Wohlers, T M; Christacos, N C; Harreman, M T; Fridovich-Keil, J L

    1999-02-01

    Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency. The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine. In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance. G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized. Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes.

  11. Study and Analysis of the Behavior of UDP Encapsulated IPV4 Packet With ESP Protection in IPV4-IPV6 Translation

    Directory of Open Access Journals (Sweden)

    Geocey Shejy

    2015-01-01

    Full Text Available Internet address is now in a transition phase from IPV4 address to IPV6 address; the reason behind this is the lack of address in IPV4.But sudden transition to IPV6 is not possible and hence transition strategies are devised by IETF. One of the transition strategy ie. Header Translation is analysed in this paper. The study is conducted on NAT-PT, one of the IPV4-IPV6 translation gateway which translates IPV4 header in to IPV6 header and vice versa. The IPV4 packet encapsulated with IPSec is injected in to an IPV4- IPV6 network. But IPSec and NAT exhibit incompatibilities so that an IPSec protected IP packet cannot cross NAT-PT. The paper thoroughly analyze one of the IPSec protocol i.e. ESP behavior in NAT-PT It is observed that when ESP encapsulated packet cannot crosses NAT-PT.UDP encapsulation of the packet is done. The analysis of the IPSec protected IP packet encapsulated in UDP shows successful traversal of the packet with IPSec security. Lesser overhead without affecting the efficiency is observed as an advantage of UDP Encapsulation.

  12. Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control.

    Science.gov (United States)

    Zuber, C; Fan, J Y; Guhl, B; Parodi, A; Fessler, J H; Parker, C; Roth, J

    2001-09-11

    The UDP-glucose:glycoprotein glucosyltransferase (GT) is a protein folding sensor and glycosyltransferase that constitutes an important component of the protein quality control machinery. With the use of quantitative immunogold electron microscopy, we established the subcellular distribution of GT in rat liver and pancreas and Drosophila melanogaster salivary gland as well as cell lines and correlated it with that of glucosidase II, calreticulin, and pre-Golgi intermediate markers. Labeling for GT, as well as for glucosidase II and calreticulin, was found in the endoplasmic reticulum (ER), including nuclear envelope and pre-Golgi intermediates located between ER and Golgi apparatus, and in the cell periphery. In the rough ER, labeling for GT was inhomogeneous, with variously sized labeled and unlabeled cisternal regions alternating, indicative of a meshwork of quality control checkpoints. Notably, labeling intensity for GT was highest in pre-Golgi intermediates, corresponding to twice that of rough ER, whereas the Golgi apparatus exhibited no specific labeling. These results suggest that protein quality control is not restricted to the ER and that the pre-Golgi intermediates, by virtue of the presence of GT, glucosidase II, and calreticulin, are involved in this fundamental cellular process.

  13. Genome-Wide Identification, Expression Patterns, and Functional Analysis of UDP Glycosyltransferase Family in Peach (Prunus persica L. Batsch)

    Science.gov (United States)

    Wu, Boping; Gao, Liuxiao; Gao, Jie; Xu, Yaying; Liu, Hongru; Cao, Xiangmei; Zhang, Bo; Chen, Kunsong

    2017-01-01

    Peach (Prunus persica L. Batsch) is a commercial grown fruit trees, important because of its essential nutrients and flavor promoting secondary metabolites. The glycosylation processes mediated by UDP-glycosyltransferases (UGTs) play an important role in regulating secondary metabolites availability. Identification and characterization of peach UGTs is therefore a research priority. A total of 168 peach UGT genes that distributed unevenly across chromosomes were identified based on their conserved PSPG motifs. Phylogenetic analysis of these genes with plant UGTs clustered them into 16 groups (A–P). Comparison of the patterns of intron–extron and their positions within genes revealed one highly conserved intron insertion event in peach UGTs. Tissue specificity, temporal expression patterns in peach fruit during development and ripening, and in response to abiotic stress UV-B irradiation was investigated using RNA-seq strategy. The relationship between UGTs transcript levels and concentrations of glycosylated volatiles was examined to select candidates for functional analysis. Heterologous expressing these candidate genes in Escherichia coli identified UGTs that were involved in the in vitro volatile glycosylation. Our results provide an important source for the identification of functional UGT genes to potential manipulate secondary biosynthesis in peach. PMID:28382047

  14. Evaluation of Saccharomyces cerevisiae Y5 for ethanol production from enzymatic hydrolysate of non-detoxified steam-exploded corn stover.

    Science.gov (United States)

    Li, Yun; Gao, Kai; Tian, Shen; Zhang, Sijin; Yang, Xiushan

    2011-11-01

    Saccharomyces cerevisiae Y5 was used to produce ethanol from enzymatic hydrolysate of non-detoxified steam-exploded corn stover, with and without a nitrogen source, and decreasing inoculum size. The results indicated that the ethanol concentration of 44.55 g/L, corresponding to 94.5% of the theoretical yield was obtained after 24 h, with an inoculum size of 10% (v/v) and nitrogen source (corn steep liquor, CSL) of 40 mL/L. With the same inoculum size, and without CSL, the ethanol concentration was 43.21 g/L, corresponding to 91.7% of the theoretical value after 60 h. With a decreased inoculum size of 5% (v/v), and without CSL, the ethanol concentration was 40.00 g/L, corresponding to 85.8% of the theoretical value after 72 h. The strain offers the potential to improve the economy of cellulosic ethanol production by simplifying the production process and reducing the costs associated with the process such as water, capital equipment and nutrient supplementation.

  15. Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Linlin [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001 (China); Liu, Ziwen [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Department of Surgery, Peking Union Medical College Hospital, Beijing 100730 (China); Yan, Ruilan [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Johnson, Stephen [Carbon Dynamics Institute, LLC, 2835 via Verde Drive, Springfield, IL 62703-4325 (United States); Zhao, Yupei [Department of Surgery, Peking Union Medical College Hospital, Beijing 100730 (China); Fang, Xiubin [Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001 (China); Cao, Deliang, E-mail: dcao@siumed.edu [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States)

    2009-09-18

    Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

  16. Enhancing Nutritional Contents of Lentinus sajor-caju Using Residual Biogas Slurry Waste of Detoxified Mahua Cake Mixed with Wheat Straw

    Science.gov (United States)

    Gupta, Aditi; Sharma, Satyawati; Kumar, Ashwani; Alam, Pravej; Ahmad, Parvaiz

    2016-01-01

    Residual biogas slurries (BGS) of detoxified mahua cake and cow dung were used as supplements to enhance the yield and nutritional quality of Lentinus sajor-caju on wheat straw (WS). Supplementation with 20% BGS gave a maximum yield of 1155 gkg-1 fruit bodies, furnishing an increase of 95.1% over WS control. Significant increase (p ≤ 0.05) in protein content (29.6-38.9%), sugars (29.1-32.3%) and minerals (N, P, K, Fe, Zn) was observed in the fruit bodies. Principle component analysis (PCA) was performed to see the pattern of correlation within a set of observed variables and how these different variables varied in different treatments. PC1 and PC2 represented 90% of total variation in the observed variables. Moisture (%), lignin (%), celluloses (%), and C/N ratio were closely correlated in comparison to Fe, N, and saponins. PCA of amino acids revealed that, PC1 and PC2 represented 74% of total variation in the data set. HPLC confirmed the absence of any saponin residues (characteristic toxins of mahua cake) in fruit bodies and mushroom spent. FTIR studies showed significant degradation of celluloses (22.2-32.4%), hemicelluloses (14.1-23.1%) and lignin (27.4-39.23%) in the spent, along with an increase in nutrition content. The study provided a simple, cost effective approach to improve the yield and nutritional quality of L. sajor-caju by resourceful utilization of BGS.

  17. Enhancing nutritional contents of Lentinus sajor-caju using residual biogas slurry waste of detoxified mahua cake mixed with wheat straw

    Directory of Open Access Journals (Sweden)

    Aditi Gupta

    2016-10-01

    Full Text Available Residual biogas slurries (BGS of detoxified mahua cake (DMC and cow dung (CD were used as supplements to enhance the yield and nutritional quality of Lentinus sajor-caju on wheat straw (WS. Supplementation with 20% BGS gave a maximum yield of 1155 gkg-1 fruit bodies, furnishing an increase of 95.1% over WS control. Significant increase (p≤0.05 in protein content (29.6-38.9%, sugars (29.1-32.3% and minerals (N, P, K, Fe, Zn was observed in the fruit bodies. Principle component analysis (PCA was performed to see the pattern of correlation within a set of observed variables and how these different variables varied in different treatments. PC1 and PC2 represented 90% of total variation in the observed variables. Moisture (%, lignin (%, celluloses (% and C/N ratio were closely correlated in comparison to Fe, N and saponins. PCA of amino acids revealed that, PC1 and PC2 represented 74% of total variation in the data set. HPLC confirmed the absence of any saponin residues (characteristic toxins of mahua cake in fruit bodies and mushroom spent. FTIR studies showed significant degradation of celluloses (22.2-32.4%, hemicelluloses (14.1-23.1% and lignin (27.4-39.23% in the spent, along with an increase in nutrition content. The study provided a simple, cost effective approach to improve the yield and nutritional quality of Lentinus sajor-caju by resourceful utilization of BGS.

  18. Different Levels in Orexin Concentrations and Risk Factors Associated with Higher Orexin Levels: Comparison between Detoxified Opiate and Methamphetamine Addicts in 5 Chinese Cities

    Directory of Open Access Journals (Sweden)

    Haoran Zhang

    2013-01-01

    Full Text Available This study sought to explore the degree of orexin levels in Chinese opiate and methamphetamine addicts and the differences between them. The cross-sectional study was conducted among detoxified drug addicts from Mandatory Detoxification Center (MDC in five Chinese cities. Orexin levels were assayed with radioimmunoassay (RIA. Mann-Whitney U test and Kruskal-Wallis test were used to detect differences across groups, and logistic regression was used to explore the association between orexin levels and characteristics of demographic and drug abuse. Between November 2009 and January 2011, 285 opiates addicts, 112 methamphetamine addicts, and 79 healthy controls were enrolled. At drug withdrawal period, both opiate and methamphetamine addicts had lower median orexin levels than controls, and median orexin levels in opiate addicts were higher than those in methamphetamine addicts (all above P<0.05. Adjusted odds of the above median concentration of orexin were higher for injection than “chasing the dragon” (AOR = 3.1, 95% CI = 1.2–7.9. No significant factors associated with orexin levels of methamphetamine addicts were found. Development of intervention method on orexin system by different administration routes especially for injected opiate addicts at detoxification phase may be significant and was welcome.

  19. Una variedad genética de la UDP-glucuronosil transferasa asociada a toxicidad gastrointestinal por irinotecan A prevalent genetic variety of UDP-glycuronosyl transferase predicts high risk of irinotecan toxicity

    Directory of Open Access Journals (Sweden)

    Matías Valsecchi

    2007-02-01

    Full Text Available Los avances en genética y biología molecular han impulsado la aparición de nuevas áreas de estudio en la medicina, como la farmacogenómica, la cual intenta predecir la respuesta y toxicidad a drogas en función de la variabilidad genética de cada individuo, constituyendo los llamados síndromes fármacogenómicos. La oncología podría beneficiarse debido a la gran toxicidad de sus fármacos. Hay varios loci genéticos que se están analizando por su potencial valor predictivo y hasta ahora sólo tres de ellos demostraron cierto grado de utilidad clínica. En especial, el estudio del número de repeticiones del dinucleótido timina-adenina (TA en el promotor de la enzima UDP-glucuronosil-transferasa (UGT mostró ser capaz de predecir neutropenia severa en pacientes expuestos a dosis intermedias y altas de irinotecan. Comunicamos el caso de una paciente con cáncer de pulmón de células pequeñas que padeció toxicidad hematológica y gastrointestinal grave tras haber sido tratada con dosis relativamente bajas (65 mg/m² de irinotecan, y en quien un análisis del ADN leucocitario mostró la presencia de homocigosis para siete repeticiones de TA. Este caso es un ejemplo de aplicabilidad clínica del test, se discute su utilidad como predictor de toxicidad y la conducta a tomar frente a pacientes con estas características.The advances in genetics and molecular biology have raised new areas in medicine, such as pharmacogenomics, which tries to predict drug responses and toxicities based on the individual genetic variability, describing the so called: pharmacogenomic syndromes. Oncology would find this development extremely useful because of the severe toxicity of chemotherapy. There are a lot of genetic loci under investigation for their potential in predicting drug toxicity, but only three of them have showed clinical usefulness up to now. In particular, quantification of the number of thymine-adenine (TA dinucleotics in the promoter region

  20. Genome-Wide Identification and Functional Characterization of UDP-Glucosyltransferase Genes Involved in Flavonoid Biosynthesis in Glycine max.

    Science.gov (United States)

    Yin, Qinggang; Shen, Guoan; Di, Shaokang; Fan, Cunying; Chang, Zhenzhan; Pang, Yongzhen

    2017-09-01

    Flavonoids, natural products abundant in the model legume Glycine max, confer benefits to plants and to animal health. Flavonoids are present in soybean mainly as glycoconjugates. However, the mechanisms of biosynthesis of flavonoid glycosides are largely unknown in G. max. In the present study, 212 putative UDP-glycosyltransferase (UGT) genes were identified in G. max by genome-wide searching. The GmUGT genes were distributed differentially among the 20 chromosomes, and they were expressed in various tissues with distinct expression profiles. We further analyzed the enzymatic activities of 11 GmUGTs that are potentially involved in flavonoid glycosylation, and found that six of them (UGT72X4, UGT72Z3, UGT73C20, UGT88A13, UGT88E19 and UGT92G4) exhibited activity toward flavonol, isoflavone, flavone and flavanol aglycones with different kinetic properties. Among them, UGT72X4, UGT72Z3 and UGT92G4 are flavonol-specific UGTs, and UGT73C20 and UGT88E19 exhibited activity toward both flavonol and isoflavone aglycones. In particular, UGT88A13 exhibited activity toward epicatechin, but not for the flavonol aglycones kaempferol and quercetin. Overexpression of these six GmUGT genes significantly increased the contents of isoflavone and flavonol glucosides in soybean hairy roots. In addition, overexpression of these six GmUGT genes also affected flavonol glycoside contents differently in seedlings and seeds of transgenic Arabidopsis thaliana. We provide valuable information on the identification of all UGT genes in soybean, and candidate GmUGT genes for potential metabolic engineering of flavonoid compounds in both Escherichia coli and plants. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. UDP-Xylose-stimulated glucuronyltransferase activity in wheat microsomal membranes: characterization and role in glucurono(arabino)xylan biosynthesis.

    Science.gov (United States)

    Zeng, Wei; Chatterjee, Mohor; Faik, Ahmed

    2008-05-01

    Microsomal membranes from etiolated wheat (Triticum aestivum) seedlings cooperatively incorporated xylose (Xyl), arabinose, and glucuronic acid residues from their corresponding uridine 5'-diphosphosugars into an ethanol-insoluble glucurono(arabino)xylan (GAX)-like product. A glucuronyltransferase activity that is enhanced by the presence of UDP-Xyl was also identified in these microsomes. Wheat glucuronyltransferase activity was optimal at pH 7 and required manganese ions, and several lines of evidence suggest its involvement in GAX-like biosynthesis. The GAX characteristics of the 14C-product were confirmed by digestion with a purified endo-xylanase from Aspergillus awamori (endo-xylanase III) and by total acid hydrolysis, resulting in a Xyl:arabinose:glucuronic acid molar ratio of approximately 105:34:1. Endo-xylanase III released only three types of oligosaccharides in addition to free Xyl. No radiolabel was released as xylobiose, xylotriose, or xylotetraose, indicating the absence of long stretches of unbranched Xyl residues in the nascent GAX-like product. High-pH anion exchange chromatography analysis of the resulting oligosaccharides along with known arabinoxylan oligosaccharide standards suggests that a portion of the nascent GAX-like product has a relatively regular structure. The other portion of the [14C]GAX-like polymer was resistant to proteinase K, endo-polygalacturonase, and endo-xylanase III (GH11 family) but was degraded by Driselase, supporting the hypothesis that the xylan backbone in this portion of the product is most likely highly substituted. Size exclusion chromatography indicated that the nascent GAX-like polymer had an apparent molecular mass of approximately 10 to 15 kD; however, mature GAXs from wheat cell walls had larger apparent molecular masses (>66 kD).

  2. Establishing the role of detoxifying enzymes in field-evolved resistance to various insecticides in the brown planthopper (Nilaparvata lugens) in South India.

    Science.gov (United States)

    Malathi, Vijayakumar Maheshwari; Jalali, Sushil K; Gowda, Dandinashivara K Sidde; Mohan, Muthugounder; Venkatesan, Thiruvengadam

    2017-02-01

    The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the major pests of rice throughout Asia. Extensive use of insecticides for suppressing N. lugens has resulted in the development of insecticide resistance leading to frequent control failures in the field. The aim of the present study was to evaluate resistance in the field populations of N. lugens from major rice growing states of South India to various insecticides. We also determined the activity of detoxifying enzymes (esterases [ESTs], glutathione S-transferases [GSTs], and mixed-function oxidases [MFOs]). Moderate levels of resistance were detected in the field populations to acephate, thiamethoxam and buprofezin (resistance factors 1.05-20.92 fold, 4.52-14.99 fold, and 1.00-18.09 fold, respectively) as compared with susceptible strain while there were low levels of resistance to imidacloprid (resistance factor 1.23-6.70 fold) and complete sensitivity to etofenoprox (resistance factor 1.05-1.66 fold). EST activities in the field populations were 1.06 to 3.09 times higher than the susceptible strain while for GST and MFO the ratios varied from 1.29 to 3.41 and 1.03 to 1.76, respectively. The EST activity was found to be correlated to acephate resistance (r = 0.999, P ≥ 0.001). The high selection pressure of organophosphate, neonicotinoid, and insect growth regulator (IGR) in the field is likely to be contributing for resistance in BPH to multiple insecticides, leading to control failures. The results obtained will be beneficial to IPM recommendations for the use of effective insecticides against BPH. © 2015 Institute of Zoology, Chinese Academy of Sciences.

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

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Jr., Richard M.; Polizzi, Samuel J.; Kadirvelraj, Renuka; Howard, Wesley W.; Wood, Zachary A. [Georgia

    2015-03-17

    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 show 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.

  4. A conserved UDP-glucose dehydrogenase encoded outside the hasABC operon contributes to capsule biogenesis in group A Streptococcus.

    Science.gov (United States)

    Cole, Jason N; Aziz, Ramy K; Kuipers, Kirsten; Timmer, Anjuli M; Nizet, Victor; van Sorge, Nina M

    2012-11-01

    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.

  5. Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.

    OpenAIRE

    Wohlers, T M; Christacos, N. C.; Harreman, M T; Fridovich-Keil, J L

    1999-01-01

    Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation ...

  6. Intake and digestibility of diets containing castor bean meal detoxified to finish of sheep Consumo e digestibilidade de dietas contendo farelo de mamona destoxificado para ovinos em terminação

    Directory of Open Access Journals (Sweden)

    Marcônio Martins Rodrigues

    2011-03-01

    Full Text Available The effect of including detoxified castor bean meal to substitute soy bean meal was evaluated to sheep finishing diets on intake, digestibility and energy value of the diets. A positive linear effect was verified for ether extract intake and acid detergent fiber with an intake raise of 0.015 g/BW0.75 in ether extract and 0.090 g/BW0.75 in acid detergent fiber per each unit percentage of detoxified castor bean meal added to the diet. There was maximun value in hemicellulose intake when included 39.55% of detoxified castor bean meal to the diet. Negative linear effect was verified for dry matter, organic matter, crude protein, total carbohydrates digestibility, respectively, with 0.0536, 0.0507, 0.0705 and 0.0572% decreases per unit percentage of detoxified castor bean meal added. Positive quadratic effect was verified for neutral detergent fiber and hemicellulose digestibility with 54.93 and 64.53% maximum in levels of the 38.6 and 31.4, respectively. Detoxified castor bean meal inclusion does not influence dry matter and nutrients intake, attending these animal class nutritional requirements. Including detoxified castor bean meal decreases dry mater, organic matter, crude protein, total carbohydrates digestibility, being in this situation recommended the inclusion of to 33% in the diet by allowing high neutral detergent fiber and hemicellulose digestibility. Despite these effects, the energy value of the diets is not influenced by the inclusion of detoxified castor bean meal.Avaliou-se a substituição do farelo de soja pelo farelo de mamona destoxificado em dietas para ovinos em terminação. O consumo de extrato etéreo e fibra em detergente ácido foi linear positivo, com aumento de 0,015 e 0,090g/UTM por unidade percentual de inclusão de farelo de mamona destoxificado, respectivamente. O consumo máximo de hemicelulose ocorreu para dietas que continham 39,55% de farelo de mamona destoxificado. A digestibilidade da matéria seca, mat

  7. Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius.

    Science.gov (United States)

    Lu, Chao; Zhao, Shoujing; Wei, Guanning; Zhao, Huijuan; Qu, Qingling

    2017-02-01

    Panax ginseng (Asian ginseng) and Panax quinquefolius (American ginseng) have been used as medicinal and functional herbal remedies worldwide. Different properties of P. ginseng and P. quinquefolius were confirmed not only in clinical findings, but also at cellular and molecular levels. The major pharmacological ingredients of P. ginseng and P. quinquefolius are the triterpene saponins known as ginsenosides. The P. ginseng roots contain a higher ratio of ginsenoside Rg1:Rb1 than that in P. quinquefolius. In ginseng plants, various ginsenosides are synthesized via three key reactions: cyclization, hydroxylation and glycosylation. To date, several genes including dammarenediol synthase (DS), protopanaxadiol synthase and protopanaxatriol synthase have been isolated in P. ginseng and P. quinquefolius. Although some glycosyltransferase genes have been isolated and identified association with ginsenoside synthesis in P. ginseng, little is known about the glycosylation mechanism in P. quinquefolius. In this paper, we cloned and identified a UDP-glycosyltransferase gene named Pq3-O-UGT2 from P. quinquefolius (GenBank accession No. KR106207). In vitro enzymatic activity experiments biochemically confirmed that Pq3-O-UGT2 catalyzed the glycosylation of Rh2 and F2 to produce Rg3 and Rd, and the chemical structure of the products were confirmed susing high performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS). High sequence similarity between Pq3-O-UGT2 and PgUGT94Q2 indicated a close evolutionary relationship between P. ginseng and P. quinquefolius. Moreover, we established both P. ginseng and P. quinquefolius RNAi transgenic roots lines. RNA interference of Pq3-O-UGT2 and PgUGT94Q2 led to reduce levels of ginsenoside Rd, protopanaxadiol-type and total ginsenosides. Expression of key genes including protopanaxadiol and protopanaxatriol synthases was up-regulated in RNAi lines, while expression of dammarenediol synthase gene

  8. Effect of single or combined application of UDP-glucose, GDNF and memantine on improvement of white matter injury in neonatal rats assessed with light and electron microscopy pathologically%UDP-糖、GDNF和美金胺改善新生大鼠缺血性脑白质病变的光电镜病理评估

    Institute of Scientific and Technical Information of China (English)

    李文娟; 毛凤霞; 陈惠金; 钱龙华

    2012-01-01

    Objective To evaluate pathologically the effect of the single or combined application of UDP-glucose, GDNF and memantine on the improvement of white matter injury in neonatal rats with periventricular leukomalacia (PVL) under light and electron microscopy. Methods A five-day-old neonatal ral model for PVL was esiabJished by ligation of the lateral common carotid artery following 120-minute hypoxia. Rats were randomly divided into six groups (30 rats in each group); sham-operated, PVL, LJDP-glucuse (UDP-glucose 2000 mg/kg jntraperi tone ally after PVL), GDNF (GDNF 100 μg/kg intracerebrally afler PVL) , imemantine ( memantine 20 mg/kg intraperitoneally after PVL) , and a combination administration of three drugs ( UDP-glucose, GDNF and memantine). The rats were sacrificed 7 or 21 days after PVL for assessment of pathological changes in the white matter under both light and electron microscopy. The number and thickness of the myelin sheath in the white matter were measured under electron microscopy, and both of pathological grading and scoring were undertaken under light microscopy. Results There was care and sparse myelinogenesis with a loose arrangement of nerve fibers in the white matter under electron microscopy in the PVL group at 7 and 21 days after PVL. The number and thickness of the myelin sheath in the PVL group were significantly less than in the sham-operated, UDP-glucose, GDNF, memantine and combination administration groups (P<0- 01). The results of pathological grading of white matter under light microscopy showed that all rats in the PVL group manifested either mild injury (38% -50% ) or severe injury (50%-62%) at 7 and 21 days after PVL. The majority of rats (50%-88%) in the four drug administration groups had normal white matter at 7 and 21 days after PVL. The pathological scores at 7 and 21 days after PVL in the PVL group were the highest, and they were significantly higher than in the other five groups (P < 0. 05). Conclusions The single or

  9. Downregulation of a pathogen-responsive tobacco UDP-Glc:phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation, enhances oxidative stress, and weakens virus resistance.

    Science.gov (United States)

    Chong, Julie; Baltz, Rachel; Schmitt, Corinne; Beffa, Roland; Fritig, Bernard; Saindrenan, Patrick

    2002-05-01

    Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid- and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.

  10. Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10).

    Science.gov (United States)

    Kubota, Tomomi; Shiba, Tomoo; Sugioka, Shigemi; Furukawa, Sanae; Sawaki, Hiromichi; Kato, Ryuich; Wakatsuki, Soichi; Narimatsu, Hisashi

    2006-06-01

    Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isozyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin beta sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isozymes to produce a wide range of O-glycans.

  11. Sinorhizobium meliloti low molecular mass phosphotyrosine phosphatase SMc02309 modifies activity of the UDP-glucose pyrophosphorylase ExoN involved in succinoglycan biosynthesis.

    Science.gov (United States)

    Medeot, Daniela B; Romina Rivero, María; Cendoya, Eugenia; Contreras-Moreira, Bruno; Rossi, Fernando A; Fischer, Sonia E; Becker, Anke; Jofré, Edgardo

    2016-03-01

    In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role in the control of exopolysaccharide (EPS) production. This study demonstrated that the chromosomal ORF SMc02309 from Sinorhizobium meliloti 2011 encodes a protein with significant sequence similarity to low molecular mass protein-tyrosine phosphatases (LMW-PTPs), such as the Escherichia coli Wzb. Unlike other well-characterized EPS biosynthesis gene clusters, which contain neighbouring LMW-PTPs and kinase, the S. meliloti succinoglycan (EPS I) gene cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical assays revealed that the SMc02309 protein hydrolyses p-nitrophenyl phosphate (p-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore, we show evidence that SMc02309 is not the LMW-PTP of the bacterial tyrosine-kinase (BY-kinase) ExoP. Nevertheless, ExoN, a UDP-glucose pyrophosphorylase involved in the first stages of EPS I biosynthesis, is phosphorylated at tyrosine residues and constitutes an endogenous substrate of the SMc02309 protein. Additionally, we show that the UDP-glucose pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I production and delays nodulation on Medicago sativa roots.

  12. Hepatic cytochrome P450 and UDP-glucuronosyl transferase are affected by five sources of dietary fiber in germ-free rats.

    Science.gov (United States)

    Nugon-Baudon, L; Roland, N; Flinois, J P; Beaune, P

    1996-02-01

    The influence of dietary fiber on xenobiotic-metabolizing enzymes (XME) was assessed using germ-free rats fed inulin and other sources of fiber (wheat bran, carrot, cocoa and oat). The consumption of cocoa fiber greatly modified the hepatic cytochrome P450 isoenzymatic profile, causing a strong enhancement of 1A2 and 2B1/B2 forms, concomitant with a significant decrease of the constitutive form 2C11, compared with all of the other types of fiber. Moreover, rats fed the cocoa fiber diet had a higher specific activity of hepatic UDP-glucuronosyl transferase than their carrot fiber- and wheat bran-fed counterparts. Intestinal UDP-glucuronosyl transferase was unaffected by the type of ingested fiber. Diet composition also did not alter the specific activity of glutathione-S-transferase in the liver, small intestine, or colon. Using earlier results obtained in heteroxenic rats, we show that intestinal microflora plays a key role in some of the effects of fiber on XME, although this is not a necessary prerequisite for all of the liver alterations.

  13. Impact of hormonal therapy on the detection of promoter hypermethylation of the detoxifying glutathione-S-transferase P1 gene (GSTP1 in prostate cancer

    Directory of Open Access Journals (Sweden)

    Krause Hans

    2006-06-01

    Full Text Available Abstract Background In spite of excellent cure rates for prostate cancer patients with favorable tumor characteristics, patients with unfavorable characteristics after radical prostatectomy are still at a significantly increased risk of tumor progression. Early adjuvant hormonal therapy (AHT has been shown to be of prognostic benefit in these patients. Unfortunately initiation and duration of early AHT in the individual patient is based on statistic data. PSA, as the standard prostate marker is neither able to reliably indicate minimal residual tumor disease in the early postoperative phase, nor can it be used for therapy monitoring due to the suppressive effect of hormonal therapy on PSA production. Promoter hypermethylation of the detoxifying glutathione-S-transferase P1 gene (GSTP1-HM has been shown to be the most common DNA alteration of primary prostatic carcinoma which, when used as a marker, is supposed to be able to overcome some of the disadvantages of PSA. However until now information on the impact of hormonal therapy on the detection of GSTP1-HM is lacking. The purpose of our study was to assess the impact of endocrine therapy on the detection of GSTP1-HM by methylation-specific PCR (MSP in prostate cancer. Methods Paraffin embedded tumor samples from the radical prostatectomy (RP specimens from 15 patients after hormonal therapy (HT (mean 8 months were assessed by MSP. In 8 of the patients the GSTP-1 status of the tumors before HT was assessed on the corresponding initial diagnostic biopsies. Results Following HT MSP showed GSTP1-HM in 13/15 of the RP specimens. In two patients analysis of the RP specimens failed to show GSTP1-HM. All initial tumor samples (8/8 biopsy specimens showed GSTP1-HM, including both patients negative for GSTP1 HM in the corresponding RP specimen. Conclusion In most cases hormonal therapy appears to not alter GSTP1 HM detection. However the change from a positive to a negative GSTP1 HM status in a subset of

  14. Effects of potential detoxifying agents on growth performance and deoxynivalenol (DON) urinary balance characteristics of nursery pigs fed DON-contaminated wheat.

    Science.gov (United States)

    Frobose, H L; Stephenson, E W; Tokach, M D; DeRouchey, J M; Woodworth, J C; Dritz, S S; Goodband, R D

    2017-01-01

    Two experiments were conducted to evaluate potential detoxifying agents on growth of nursery pigs fed deoxynivalenol (DON)-contaminated diets. Naturally DON-contaminated wheat (6 mg/kg) was used to achieve desired DON levels. In a 21-d study, 238 pigs (13.4 ± 1.8 kg BW) were used in a completely randomized design with a 2 × 2 + 1 factorial arrangement. Diets were: 1) Positive control (PC; SMB; Samirian Chemicals, Campbell, CA). There were 6 or 7 replicate pens/treatment and 7 pigs/pen. Analyzed DON was decreased by 92% when pelleted with SMB, but otherwise matched formulated levels. Overall, a DON × Product V interaction was observed for ADG ( 0.05) with a tendency for an interaction for ADFI ( 0.10). As anticipated, DON reduced ( 0.001) ADG and ADFI, but the interaction was driven by even poorer growth when Product V was added to NC diets. Pigs fed NC diets had 10% poorer G:F ( 0.001) than PC-fed pigs. Reductions in ADG due to DON were most distinct (50%) during the initial period. Adding SMB to NC diets improved ( 0.01) ADG, ADFI, and G:F, and improved ( 0.02) ADG and G:F compared to the PC diet. A urinary balance study was conducted using diets 3 to 5 from Exp. 1 to evaluate Product V and SMB on DON urinary metabolism. A 10 d adaptation was followed by a 7 d collection using 24 barrows in a randomized complete block design. Pigs fed NC + SMB diet had greater urinary DON output ( 0.05) than pigs fed NC + Product V, with NC pigs intermediate. Daily DON excretion was lowest ( 0.05) in the NC + SMB pigs. However, degradation of DON-sulfonate back to the parent DON molecule was observed as pigs fed NC + SMB excreted more DON than they consumed (164% of daily DON intake), greater ( 0.001) than pigs fed the NC (59%) or NC + Product V (48%). Overall, Product V did not alleviate DON effects on growth nor did it reduce DON absorption and excretion. However, hydrothermally processing DON-contaminated diets with 1.0% SMB restored ADFI and improved G:F. Even so, the

  15. 清热解毒酸奶加工工艺的研究%Research on the Processing Technology of Heat-clearing and Detoxifying Yoghurt

    Institute of Scientific and Technical Information of China (English)

    贾鲁彦; 李志成; 鲁周民

    2015-01-01

    Heat-clearing and Detoxifying Yoghurt was taken from fresh milk as main raw material , with mung bean juice, wild chrysanthemum juice, honeysuckle juice and sugar used as auxiliary materials. Orthogonal test results showed that 4 kinds of supplementary materials dosage effect on fermentation results in order of mung bean juice > wild chrysanthemum juice > sucrose > honeysuckle juice. The best formula was mung bean juice 20 %, the wild chrysanthemum juice 8%, honeysuckle juice 4%and 8%of sucrose added. Sterilization cooling access accounted for 4%of the total volume of fermentation ,after fermentation and ripening the acidity of yogurt products in about 80 °T, the viable count≥107 CFU/mL. By taking this procedure, the yoghurt would have refreshing taste and pleasant flavor. Also , the procedure was qualified with national standards , the yoghurt would be able to produce in factory.%以鲜牛乳为主料,绿豆浆、野菊花汁、金银花汁、蔗糖为辅料,研究了新型清热解毒酸奶加工工艺.正交优化试验结果表明4种辅料添加量对发酵结果的影响顺序依次为绿豆浆>野菊花汁>蔗糖>金银花汁. 辅料最优添加量(占原料总量)分别为绿豆浆20%、野菊花汁8%、金银花汁4%、蔗糖8%,灭菌冷却后接入占总体积4%的发酵剂,经发酵及后熟所得酸奶产品酸度在80 °T左右,活菌数≥107 CFU/mL.采用此工艺加工的酸奶口感清爽、风味良好,符合国家酸奶产品标准,可用于工厂化生产.

  16. Evidence of calcium-dependent pathway in the regulation of human beta1,3-glucuronosyltransferase-1 (GlcAT-I) gene expression: a key enzyme in proteoglycan synthesis.

    Science.gov (United States)

    Barré, Lydia; Venkatesan, Narayanan; Magdalou, Jacques; Netter, Patrick; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2006-08-01

    The importance of heparan- and chondroitin-sulfate proteoglycans in physiological and pathological processes led to the investigation of the regulation of beta1,3-glucuronosyltransferase I (GlcAT-I), responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide, a key step prior to polymerization of chondroitin- and heparan-sulfate chains. We have cloned and functionally characterized GlcAT-I 5'-flanking regulatory region. Mutation analysis and electrophoretic mobility shift assays demonstrated the importance of Sp1 motif located at -65/-56 position in promoter activity. Furthermore, we found that elevation of intracellular calcium concentration by the calcium ionophore ionomycin stimulated GlcAT-I gene expression as well as glycosaminoglycan chain synthesis in HeLa cells. Bisanthracycline, an anti-Sp1 compound, inhibited GlcAT-I basal promoter activity and suppressed ionomycin induction, suggesting the importance of Sp1 in calcium induction of GlcAT-I gene expression. Nuclear protein extracts from ionomycin-induced cells exhibited an increased DNA binding of Sp1 factor to the consensus sequence at position -65/-56. Signaling pathway analysis and MEK inhibition studies revealed the important role of p42/p44 MAPK in the stimulation of GlcAT-I promoter activity by ionomycin. The present study identifies, for the first time, GlcAT-I as a target of calcium-dependent signaling pathway and evidences the critical role of Sp1 transcription factor in the activation of GlcAT-I expression.

  17. In vitro metabolism of canagliflozin in human liver, kidney, intestine microsomes, and recombinant uridine diphosphate glucuronosyltransferases (UGT) and the effect of genetic variability of UGT enzymes on the pharmacokinetics of canagliflozin in humans.

    Science.gov (United States)

    Francke, Stephan; Mamidi, Rao N V S; Solanki, Bhavna; Scheers, Ellen; Jadwin, Andrew; Favis, Reyna; Devineni, Damayanthi

    2015-09-01

    O-glucuronidation is the major metabolic elimination pathway for canagliflozin. The objective was to identify enzymes and tissues involved in the formation of 2 major glucuronidated metabolites (M7 and M5) of canagliflozin and subsequently to assess the impact of genetic variations in these uridine diphosphate glucuronosyltransferases (UGTs) on in vivo pharmacokinetics in humans. In vitro incubations with recombinant UGTs revealed involvement of UGT1A9 and UGT2B4 in the formation of M7 and M5, respectively. Although M7 and M5 were formed in liver microsomes, only M7 was formed in kidney microsomes. Participants from 7 phase 1 studies were pooled for pharmacogenomic analyses. A total of 134 participants (mean age, 41 years; men, 63%; white, 84%) were included in the analysis. In UGT1A9*3 carriers, exposure of plasma canagliflozin (Cmax,ss , 11%; AUCτ,ss , 45%) increased relative to the wild type. An increase in exposure of plasma canagliflozin (Cmax,ss , 21%; AUCt,ss , 18%) was observed in participants with UGT2B4*2 genotype compared with UGT2B4*2 noncarriers. Metabolites further delineate the role of both enzymes. The pharmacokinetic findings in participants carrying the UGT1A9*3 and UGT2B4*2 allele implicate that UGT1A9 and UGT2B4 are involved in the metabolism of canagliflozin to M7 and M5, respectively. © 2015, The American College of Clinical Pharmacology.

  18. TEST FOR UDP

    Institute of Scientific and Technical Information of China (English)

    Jambalaya

    2004-01-01

    不久前看到一篇关于UPD论坛安全性的文章,因为闲来无事就从网上弄了一个下来,准备随便看看。我听很多人和我说CGI不安全,PHP安全,其实没有一种语言具备先天安全性,安全性是存在于编写代码人的思想中的,尽管PHP从刚开始的对magic_gpc_quote的限制到PHP4.O中对register_gtobals的禁止都没有阻拦住php各种论坛和系统的漏洞层出不穷,任何代码编写思想的不严谨和对漏洞成因的不了解都会直接或间接导致漏洞,如果你不信,那我们就来一起来看看吧!

  19. Identification of a novel UDP-GalNAc:GlcNAcβ-R β1-4 N-acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Mulder, H.; Spronk, B.A.; Schachter, H.; Neeleman, A.P.; Eijnden, D.H. van den; Jong-Brink, M. de; Kamerling, J.P.

    1995-01-01

    Both the albumen gland, one of the female accessory sex glands, and connective tissue of the freshwater snail Lymnaea stagnalis contain N-acetylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP-GalNAc in beta1-4 linkage to the terminal GlcNAc residue of GlcNAcbeta-R. The al

  20. 基于 UDP协议的利率衍生产品报价系统设计与实现%Design and Implement of the Interest Rate Derivatives Biding System Based on the UDP Protocol

    Institute of Scientific and Technical Information of China (English)

    姚毓才

    2014-01-01

    结合UDP协议、DES加密等技术,论述了一个基于UDP协议的利率衍生产品报价系统的设计思想和整体架构,着重剖析了基于UDP协议的通讯报文设计、报文内容DES加密算法,并针对UDP通讯中存在的可靠性问题,提供了一种类似于TCP协议中滑动窗口机制的措施。%It was discussed of a protocol in this paper based on UDP interest rate derivative products quo -tation system design ideas and the overall framework .Combining the UDP protocol , the DES encryption technology ,it analyzed the communication messages design , UDP protocol message content based on the DES encryption algorithm ,and in view of the reliability problem in UDP communication ,a similar measure was provided to the TCP protocol in the sliding window mechanism .

  1. Butanol production from detoxified Salix hydrolysate%预处理沙柳酶解液脱毒及其丁醇发酵

    Institute of Scientific and Technical Information of China (English)

    林增祥; 李荣秀; 刘莉; 沈兆兵; 史吉平

    2015-01-01

    为了提高沙柳原料的丁醇发酵效果,考察沙柳原料经过蒸爆、超微粉碎+稀酸和超微粉碎+稀碱预处理后补料酶解的效果,优化了沙柳酶解液活性炭脱毒工艺参数,并对经过脱毒处理的酶解液进行了丁醇发酵研究,结果表明:预处理沙柳原料酶解底物质量浓度为200 g/mL时,3种预处理方法中蒸爆处理法水解效果最好,每克底物的滤纸酶酶加量15 U,酶解96 h后,酶解液总糖质量浓度达到57 g/L。活性炭脱毒处理的最优条件:pH 4�8,碳加量4%(质量分数)、温度70℃、1 h,该条件下的沙柳水解液脱色率达到97�4%、糖损失率3�1%。3种预处理沙柳原料的酶解液经活性炭脱毒后都可以被丁醇梭菌正常利用发酵产丁醇,发酵液总溶剂( ABE)质量浓度约为14 g/L。%In this study, we studied methods to improve butanol fermentation of Salix hydrolysates after pretreatment and enzymatic hydrolysis. Salix samples were pretreated by steam explosion, ultrafine grind with dilute acid and ultrafine grind with dilute alkali, respectively. Before acetone⁃butanol⁃ethanol ( ABE) fermentation, we optimized the feeding enzyme hydrolysis and activated carbon detoxification. Steam explosion pretreated Salix samples had the highest sugar yield ( 57 g/L ) after enzymatic hydrolysis, with 200 g/mL substrate consistency, 15 FPU enzyme loading for 96 hours. The optimal condition of activated carbon detoxification was observed with 4% activated carbon loading at pH 4�8 and 70 ℃ for 1 hour. Under that condition, hydrolysate was decoloarated by 97�4% with 3�1% sugar loss. The detoxified Salix hydrolysates of three pretreatment methods could be fermented by Clostridium acetobutylicums, and the total product solvent ( ABE) was about 14 g/L.

  2. Structure of the Escherichia coli ArnA N-formyltransferase domain in complex with N(5) -formyltetrahydrofolate and UDP-Ara4N.

    Science.gov (United States)

    Genthe, Nicholas A; Thoden, James B; Holden, Hazel M

    2016-08-01

    ArnA from Escherichia coli is a key enzyme involved in the formation of 4-amino-4-deoxy-l-arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram-negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Indeed, it is thought that such modifications may be responsible for the repeated infections of cystic fibrosis patients with Pseudomonas aeruginosa. ArnA is a bifunctional enzyme with the N- and C-terminal domains catalyzing formylation and oxidative decarboxylation reactions, respectively. The catalytically competent cofactor for the formylation reaction is N(10) -formyltetrahydrofolate. Here we describe the structure of the isolated N-terminal domain of ArnA in complex with its UDP-sugar substrate and N(5) -formyltetrahydrofolate. The model presented herein may prove valuable in the development of new antimicrobial therapeutics.

  3. Epigenetic reprogramming of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) in HIV-1-infected CEM T cells.

    Science.gov (United States)

    Giordanengo, Valerie; Ollier, Laurence; Lanteri, Marion; Lesimple, Josette; March, Denise; Thyss, Sylvain; Lefebvre, Jean-Claude

    2004-12-01

    Sialylated glycoconjugates mediate several key lymphocyte functions. We previously reported that hyposialylation occurred in latently HIV-1-infected CEM T cells, despite the fully preserved catalytic activity of several sialyltransferases. We show now that these cells are affected by a down-regulation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), which leads to a dramatic decrease in the synthesis of CMP-sialic acid, the donor substrate of all sialyltransferases. The GNE gene promoter was found to be located in a CpG island with several regulatory motifs CREB, SP1, and AP-2. De novo hypermethylation of this promoter was observed in HIV-1-infected CEM cells. This phenomenon might explain some immunological disorders that persist in infected individuals despite long-term therapeutically controlled viral replication. Indeed, an overall decrease in sialic acid engraftment can affect glycoproteins, notably those in which the sialylation status is crucial to ensure homing, recirculation, and survival of lymphocytes.

  4. Vitis vinifera L. Single-Nucleotide Polymorphism Detection with High-Resolution Melting Analysis Based on the UDP-Glucose:Flavonoid 3-O-Glucosyltransferase Gene.

    Science.gov (United States)

    Pereira, Leonor; Martins-Lopes, Paula

    2015-10-21

    Vitis vinifera L. is a species with a large number of varieties, which differ in terms of anthocyanin content. The genes involved in the anthocyanin biosynthesis pathway have a direct effect in the anthocyanin profile of each variety, being potentially interesting for varietal identification. The current study aimed at the design of an assay suitable for the discrimination of the largest number of grapevine varieties. Two genes of the anthocyanin pathway, chalcone isomerase (CHI) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT), were sequenced in 22 grapevine varieties. The CHI gene presented 5 SNPs within the sequence. A total of 58 SNPs and 1 INDEL were found among the UFGT gene, allowing the discrimination of 18 different genotypes within the 22 grapevine varieties. A HRM assay designed for UFGT, containing 704 bp, produced differentiated melting curves for each of the 18 haplotypes. The developed HRM assay is efficient in grapevine varietal discrimination.

  5. Study of the 238U(d,p) surrogate reaction via the simultaneous measurement of gamma-decay and fission probabilities

    CERN Document Server

    Ducasse, Q; Aïche, M; Marini, P; Mathieu, L; Görgen, A; Guttormsen, M; Larsen, A C; Tornyi, T; Wilson, J N; Barreau, G; Boutoux, G; Czajkowski, S; Giacoppo, F; Gunsing, F; Hagen, T W; Lebois, M; Lei, J; Méot, V; Morillon, B; Moro, A; Renstrøm, T; Roig, O; Rose, S J; Sérot, O; Siem, S; Tsekhanovich, I; Tveten, G M; Wiedeking, M

    2015-01-01

    We investigated the 238U(d,p) reaction as a surrogate for the n + 238U reaction. For this purpose we measured for the first time the gamma-decay and fission probabilities of 239U* simultaneously and compared them to the corresponding neutron-induced data. We present the details of the procedure to infer the decay probabilities, as well as a thorough uncertainty analysis, including parameter correlations. Calculations based on the continuum-discretized coupled-channels and distorted-wave Born approximations were used to correct our data from detected protons originating from elastic and inelastic deuteron breakup. The corrected fission probability is in agreement with neutron-induced data, whereas the gamma-decay probability is much higher than the neutron-induced data. The performed statistical-model calculations are not able to explain these results.

  6. Biochemical characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE from Verrucomicrobium spinosum DSM 4136(T..

    Directory of Open Access Journals (Sweden)

    Sean E McGroty

    Full Text Available Verrucomicrobium spinosum is a Gram-negative bacterium that is related to bacteria from the genus Chlamydia. The bacterium is pathogenic towards Drosophila melanogaster and Caenorhabditis elegans, using a type III secretion system to facilitate pathogenicity. V. spinosum employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the V. spinosum genome provides evidence that the bacterium should be able to synthesize peptidoglycan de novo, since all of the necessary genes are present. The enzyme UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-2,6-diaminopimelate ligase (MurE (E.C. 6.3.2.15 catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The murE ortholog from V. spinosum (murE Vs was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:meso-2,6-diaminopimelate ligase activity in vivo using functional complementation. In vitro analysis using the purified recombinant enzyme demonstrated that MurEVs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. meso-Diaminopimelate was the preferred substrate with a K m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurEVs. Our kinetic analysis and structural model of MurEVs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that V. spinosum incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a V. spinosum culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.

  7. Research and realization of multimedia communication based on UDP%基于UDP的多媒体通信的研究与实现

    Institute of Scientific and Technical Information of China (English)

    高毅; 张曦煌; 王广翔

    2012-01-01

    针对直接使用UDP传输视频图像时的诸多问题,分析现有的基于UDP的数据传输协议的优缺点,在此基础上,提出一种改进的双重拥塞控制机制:在发送端添加基于反馈信息的被动拥塞控制算法,在接收端添加主动拥塞避免算法,有效地提高了传输效率.为进一步降低丢包率,提出缓冲区管理策略.实验结果表明,改进后的拥塞控制机制有效提高了数据传输的效率和可靠性.使用缓冲区管理策略后,可靠性还有待进一步提高.%Problems are always existing when using UDP to implement image transmission in the Internet. The paper analyses the advantages and disadvantages of the existing UDP_based data transfer protocols. On the basis of this, an improved dual congestion control mechanism is proposed. It adds a passive congestion control manner based on the feedback on the sender and an active congestion avoid manner on the receiver. After that the network performance is greatly improved. In order to reduce packet loss rate further, the paper gives a method to process the buffer. The experiment results show that reliability and efficiency have been improved a lot while using the improved congestion control mechanism. The reliability is improved better by adding the method of processing the buffer.

  8. Estudos do Mecanismo de Catálise da Pirofosforilase UDP-N-acetilglucosamina do Moniliophthora perniciosa por Métodos QM/MM

    Directory of Open Access Journals (Sweden)

    André Teles

    2015-09-01

    Full Text Available The fungus Moniliophthora perniciosa is the causal agent of the witches’ broom disease of cacao (Theobroma cacao. This fungus has a pyrophosphorylase enzyme to catalyze the formation of UDP-N-acetylglucosamine-1-phosphate, an intermediate  in the biosynthesis of the fungal cell wall. This enzyme was studied by QM/MM methods to understand its catalytic mechanism. Initially, the structure was refined and submitted to molecular dynamics simulations. Next, QM/MM calculations were used to scan the coordinates of the reaction from the reactants to products; an SN2 mechanism has been proposed of the aforementioned reaction. In this system, the nucleophile is the oxygen atom of the phosphate group from N-acetylglucosamine-1-phosphate. It attacks the phosphorus of the alfa-phosphate from the uridine triphosphate to form UDP-N-acetylglucosamine and pyrophosphate. The calculated reaction was exothermic (ΔH = -81.01 Kcal/mol. On the reaction pathway, the most energetic structure had the phosphorus atom attacked in a pentacoordinate configuration. This structure interacts with the catalytic site composed by the residues Gly112, Gly113, Arg116, Lys123 and Gly225 through hydrogen bonds. During the mechanism study, the geometry of the transition state was obtained. Thus, the generation of new compounds sharing a geometric similarity with this pentacoordinate transition state can lead to the development of more active compounds possessing antifungal proprieties against witch's broom. DOI: http://dx.doi.org/10.17807/orbital.v7i3.629 

  9. Cloning, expression and characterization of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA from Wolbachia endosymbiont of human lymphatic filarial parasite Brugia malayi.

    Directory of Open Access Journals (Sweden)

    Mohd Shahab

    Full Text Available 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 (Km 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.

  10. Molecular cloning and characterization of UDP-glucose: furaneol glucosyltransferase gene from grapevine cultivar Muscat Bailey A (Vitis labrusca × V. vinifera).

    Science.gov (United States)

    Sasaki, Kanako; Takase, Hideki; Kobayashi, Hironori; Matsuo, Hironori; Takata, Ryoji

    2015-10-01

    2,5-Dimethyl-4-hydroxy-3(2H)-furanone (furaneol) is an important aroma compound in fruits, such as pineapple and strawberry, and is reported to contribute to the strawberry-like note in some wines. Several grapevine species are used in winemaking, and furaneol is one of the characteristic aroma compounds in wines made from American grape (Vitis labrusca) and its hybrid grape. Furaneol glucoside was recently isolated as an important furaneol derivative from the hybrid grapevine cultivar, Muscat Bailey A (V. labrusca × V. vinifera), and this was followed by its isolation from some fruits such as strawberry and tomato. Furaneol glucoside is a significant 'aroma precursor of wine' because furaneol is liberated from it during alcoholic fermentation. In this study, a glucosyltransferase gene from Muscat Bailey A (UGT85K14), which is responsible for the glucosylation of furaneol was identified. UGT85K14 was expressed in the representative grape cultivars regardless of species, indicating that furaneol glucoside content is regulated by the biosynthesis of furaneol. On the other hand, furaneol glucoside content in Muscat Bailey A berry during maturation might be controlled by the expression of UGT85K14 along with the biosynthesis of furaneol. Recombinant UGT85K14 expressed in Escherichia coli is able to transfer a glucose moiety from UDP-glucose to the hydroxy group of furaneol, indicating that this gene might be UDP-glucose: furaneol glucosyltransferase in Muscat Bailey A. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Substrate specificities of three members of the human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3

    DEFF Research Database (Denmark)

    Wandall, H H; Hassan, H; Mirgorodskaya, E

    1997-01-01

    recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3......, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem...... repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results...

  12. Effect of galactosamine-induced hepatic UDP-glucuronic acid depletion on acetaminophen elimination in rats. Dispositional differences between hepatically and extrahepatically formed glucuronides of acetaminophen and other chemicals.

    Science.gov (United States)

    Gregus, Z; Madhu, C; Goon, D; Klaassen, C D

    1988-01-01

    Galactosamine (GAL) markedly depletes hepatic UDP-glucuronic acid (UDP-GA) whereas extrahepatic UDP-GA is minimally affected. This suggests that GAL predominantly inhibits hepatic glucuronidation. Therefore, the effect of GAL-induced hepatic UDP-GA depletion was examined in bile duct-cannulated rats to determine the role of hepatic glucuronidation in the disposition of acetaminophen (AA). GAL markedly altered the fate of AA-glucuronide but had little or no effect upon other AA metabolites. GAL decreased the biliary excretion of AA-glucuronide up to 92%, whereas reductions in blood levels and urinary excretion of AA-glucuronide did not exceed 50%. This suggests that AA-glucuronide excreted in bile is predominantly of hepatic origin whereas AA-glucuronide found in blood and urine is derived from both hepatic and extrahepatic tissues. Data in the present and previous studies [Gregus, Watkins, Thompson, Klaassen: J. Pharmacol. Exp. Ther. 225, 256, (1983)] indicate that GAL greatly reduced the biliary excretion of AA- and valproic acid-glucuronide whereas the biliary excretion of the glucuronides of phenolphthalein, iopanoic acid, bilirubin, and diethylstilbestrol was only partially decreased. This difference appears to be largely due to differential contributions by the liver and extrahepatic tissues in the glucuronidation of various compounds as well as the availability of glucuronides formed in extrahepatic tissues for biliary excretion. Specifically, the extrahepatically formed glucuronide conjugates of AA and valproic acid are not readily available for biliary excretion whereas the glucuronides of the other compounds are readily excreted into bile.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. High-Bandwidth Tactical-Network Data Analysis in a High-Performance-Computing (HPC) Environment: Transport Protocol (Transmission Control Protocol/User Datagram Protocol [TCP/UDP]) Analysis

    Science.gov (United States)

    2015-09-01

    Protocol [TCP/UDP]) Analysis 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kenneth D Renard and James R Adametz...guaranteed and ordered delivery of data from application to application. Internet Protocol ( IP ) does not guarantee either of these capabilities...that require reliable transport services, careful examination of TCP protocol performance is necessary. Data collected during test events includes IP

  14. TCP/UDP下一种网络透明传输服务编程接口实现方法%Implementation of a Service API on Transparent Network Communication Programming Based on TCP/UDP

    Institute of Scientific and Technical Information of China (English)

    何波玲; 隋菱歌

    2011-01-01

    To write network communication code based on TCP/UDP,you must understand some network communication concepts such as IP address,port,socket,TCP/UDP protocols etc.This paper implemented a service API(Application Programming Interface) that encapsulate all network communication concepts and make TCP programming and UDP programming with the same mode.The results show that the API can make network communication programming very easily with no network concepts and supposes real-time and reliable communication and multi-NIC(Network Interface Card) in same host.%使用在TCP/UDP协议进行网络通信,会涉及IP地址、端口号、套接字、TCP/UDP协议等网络通信概念,而且TCP传输和UDP传输的编程模式是不同的,对网络通信概念进行了封装,实现了一个网络通信透明传输服务系统,且TCP传输和UDP传输具有相同的编程模式.使用表明,用户无需了解网络通信概念,即可使用该系统进行通信编程,使用极为方便,实现网络通信的实时传输、可靠传输和同一主机的多网卡支持.

  15. Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5′-diphospho-glucuronosyltransferase isoforms

    Directory of Open Access Journals (Sweden)

    Munirah Haron

    2015-01-01

    Full Text Available Background: Glucuronidation catalyzed by uridine 5′- diphospho-glucuronosyltransferase (UGT is a major phase II drug metabolism reaction which facilitates drug elimination. Inhibition of UGT activity can cause drug-drug interaction. Therefore, it is important to determine the inhibitory potentials of drugs on glucuronidation. Objective: The objective was to evaluate the inhibitory potentials of mitragynine, 7-hydroxymitragynine, ketamine and buprenorphine, respectively on 4-methylumbelliferone (4-MU glucuronidation in rat liver microsomes, human liver microsomes and recombinant human UGT1A1 and UGT2B7 isoforms. Materials and Methods: The effects of the above four compounds on the formation of 4-MU glucuronide from 4-MU by rat liver microsomes, human liver microsomes, recombinant human UGT1A1 and UGT2B7 isoforms were determined using high-performance liquid chromatography with ultraviolet detection. Results: For rat liver microsomes, ketamine strongly inhibited 4-MU glucuronidation with an IC 50 value of 6.21 ± 1.51 mM followed by buprenorphine with an IC 50 value of 73.22 ± 1.63 mM. For human liver microsomes, buprenorphine strongly inhibited 4-MU glucuronidation with an IC 50 value of 6.32 ± 1.39 mM. For human UGT1A1 isoform, 7-hydroxymitragynine strongly inhibited 4-MU glucuronidation with an IC 50 value of 7.13 ± 1.16 mM. For human UGT2B7 isoform, buprenorphine strongly inhibited 4-MU glucuronidation followed by 7-hydroxymitragynine and ketamine with respective IC 50 values of 5.14 ± 1.30, 26.44 ± 1.31, and 27.28 ± 1.18 mM. Conclusions: These data indicate the possibility of drug-drug interaction if 7-hydroxymitragynine, ketamine, and buprenorphine are co-administered with drugs that are UGT2B7 substrates since these three compounds showed significant inhibition on UGT2B7 activity. In addition, if 7-hydroxymitragynine is to be taken with other drugs that are highly metabolized by UGT1A1, there is a possibility of drug

  16. Interaction of FAM5C with UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1): Implication of N-glycosylation in FAM5C secretion.

    Science.gov (United States)

    Terao, Yuya; Fujita, Hidenobu; Horibe, Sayo; Sato, Junya; Minami, Satomi; Kobayashi, Miwako; Matsuoka, Ichiro; Sasaki, Naoto; Satomi-Kobayashi, Seimi; Hirata, Ken-Ichi; Rikitake, Yoshiyuki

    2017-03-27

    N-glycosylation of proteins is important for protein folding and function. We have recently reported that FAM5C/BRINP3 contributes to the tumor necrosis factor-α-induced expression of leukocyte adhesion molecules in vascular endothelial cells (ECs). However, regulatory mechanism of the FAM5C biosynthesis is poorly understood. Co-immunoprecipitation assay revealed the interaction of FAM5C with UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), a glycoprotein folding-sensor enzyme. FAM5C ectopically expressed in HEK293 cells was localized to the endoplasmic reticulum and co-localized with endogenously expressed UGGT1. Molecular size of FAM5C was reduced by treatment with N-glycosidase F and in FAM5C-expressing cells cultured in the presence of the N-glycosylation inhibitor tunicamycin. FAM5C was secreted by the cells and the secretion of FAM5C was blocked by tunicamycin. Among six potential N-glycosylation sites, the potential site at Asn(168) was not N-glycosylated, and Asn(337), Asn(456), Asn(562), Asn(609), and Asn(641) mutants were poorly secreted by the cells. These results demonstrated that FAM5C is an N-glycosylated protein and N-glycosylation is necessary for the secretion of FAM5C.

  17. A new case of UDP-galactose transporter deficiency (SLC35A2-CDG): molecular basis, clinical phenotype, and therapeutic approach.

    Science.gov (United States)

    Dörre, K; Olczak, M; Wada, Y; Sosicka, P; Grüneberg, M; Reunert, J; Kurlemann, G; Fiedler, B; Biskup, S; Hörtnagel, K; Rust, S; Marquardt, T

    2015-09-01

    Congenital disorders of glycosylation (CDG) are a group of hereditary metabolic diseases characterized by abnormal glycosylation of proteins and lipids. Often, multisystem disorders with central nervous system involvement and a large variety of clinical symptoms occur. The main characteristics are developmental delay, seizures, and ataxia. In this paper we report the clinical and biochemical characteristics of a 5-year-old girl with a defective galactosylation of N-glycans, resulting in developmental delay, muscular hypotonia, epileptic seizures, inverted nipples, and visual impairment. Next generation sequencing revealed a de novo mutation (c.797G > T, p.G266V) in the X-chromosomal gene SLC35A2 (solute carrier family 35, UDP-galactose transporter, member A2; MIM 300896). While this mutation was found heterozygous, random X-inactivation of the normal allele will lead to loss of normal SLC35A2 activity in respective cells. The functional relevance of the mutation was demonstrated by complementation of UGT-deficient MDCK-RCA(r) and CHO-Lec8 cells by normal UGT-expression construct but not by the mutant version. The effect of dietary galactose supplementation on glycosylation was investigated, showing a nearly complete normalization of transferrin glycosylation.

  18. Genome-wide identification and tissue-specific expression analysis of UDP-glycosyltransferases genes confirm their abundance in Cicer arietinum (Chickpea) genome.

    Science.gov (United States)

    Sharma, Ranu; Rawat, Vimal; Suresh, C G

    2014-01-01

    UDP-glycosyltransferases (EC 2.4.1.x; UGTs) are enzymes coded by an important gene family of higher plants. They are involved in the modification of secondary metabolites, phytohormones, and xenobiotics by transfer of sugar moieties from an activated nucleotide molecule to a wide range of acceptors. This modification regulates various functions like detoxification of xenobiotics, hormone homeostasis, and biosynthesis of secondary metabolites. Here, we describe the identification of 96 UGT genes in Cicer arietinum (CaUGT) and report their tissue-specific differential expression based on publically available RNA-seq and expressed sequence tag data. This analysis has established medium to high expression of 84 CaUGTs and low expression of 12 CaUGTs. We identified several closely related orthologs of CaUGTs in other genomes and compared their exon-intron arrangement. An attempt was made to assign functional specificity to chickpea UGTs by comparing substrate binding sites with experimentally determined specificity. These findings will assist in precise selection of candidate genes for various applications and understanding functional genomics of chickpea.

  19. 一种串口与UDP协议实现透明转换的方法%Designing for the serial and UDP protocol conversion interface

    Institute of Scientific and Technical Information of China (English)

    周慎

    2013-01-01

    针对物联网技术Internet的接入要求,应用AVR单片机和以太网控制芯片设计开发了串口(RS-232)与UDP协议数据格式双向透明转换通用网关,为智能仪表或传感器的以太网接入和IP网络的统一管理提供了途径。通过测试验证了本方法的有效性、实时性和可靠性。%Demand on access the internet of Things, designed a serial and UDP protocol conversion interface which base on the AVR mcu and chip of ethernet control, the smart meter or sensor can connected and managed by IP network with this interface. Test result shown this interface has effective, real-time and reliable performance.

  20. Up-scaled Teer-UDP850/4 Unbalanced Magnetron Deposition System Used for Mass-Production of CrTiAlN Hard Coatings

    Institute of Scientific and Technical Information of China (English)

    ZHANGGuo-jun; YANGShi-cai; JIANGBai-ling; BAILi-jing; CHENDi-chum; WENXiao-bin; TEERD.G.

    2004-01-01

    Up-sca]ed deposition process of Teer-UDP850/4 has been established and used for massive production of CrTiAlN hard coatings in applications of anti-wear, cutting and forming tools. This deposition system uses four magnetrons that are arranged by unbalanced magnets to fomt closed magnetic field enabling the system running in high current density. Elemental metals of Cr, Ti and Al are used as the target materials which are co-deposited with nitrogen forming nlultialloy nitride, nanoscale multi-layer or superlattice hard coatings. The stthstrate turntable is designed as planet rotation mechanism with three folds so that components or tools with complicate geometry can be uniformly coated onto all their surfaces and cutting edges. The pawer units for the magnetrons are straight dc whilst the substrate is biased by pulsed de. Two solid heaters are installed in the system to enable running a wide range of deposition temperature from 200℃ to 500℃. The pumping system is powerful that incorporated with a polycold to pump the system to a good vacuum in a very shori time. A front door and a movable substrate table are available to benefit easily loading and unloading. Deposition procedure. properties and performance of the coatings is also presented in this paper.

  1. Up-scaled Teer-UDP850/4 Unbalanced Magnetron Deposition System Used for Mass-Production of CrTiAlN Hard Coatings

    Institute of Scientific and Technical Information of China (English)

    ZHANG Guo-jun; YANG Shi-cai; JIANG Bai-ling; BAI Li-jing; CHEN Di-chun; WEN Xiao-bin; TEER D.G.

    2004-01-01

    Up-scaled deposition process of Teer-UDP850/4 has been established and used for massive production of CrTiAlN hard coatings in applications of anti-wear, cutting and forming tools. This deposition system uses four magnetrons that are arranged by unbalanced magnets to form closed magnetic field enabling the system running in high current density.Elemental metals of Cr, Ti and Al are used as the target materials which are co-deposited with nitrogen forming multialloy nitride, nanoscale multi-layer or superlattice hard coatings. The substrate turntable is designed as planet rotation mechanism with three folds so that components or tools with complicate geometry can be uniformly coated onto all their surfaces and cutting edges. The power units for the magnetrons are straight dc whilst the substrate is biased by pulsed dc. Two solid heaters are installed in the system to enable running a wide range of deposition temperature from 200℃ to 500℃. The pumping system is powerful that incorporated with a polycold to pump the system to a good vacuum in a very short time. A front door and a movable substrate table are available to benefit easily loading and unloading. Deposition procedure,properties and performance of the coatings is also presented in this paper.

  2. Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

    Science.gov (United States)

    Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

    2012-05-01

    The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

  3. Differences in the glucuronidation of bisphenols F and S between two homologous human UGT enzymes, 1A9 and 1A10.

    Science.gov (United States)

    Gramec Skledar, Darja; Troberg, Johanna; Lavdas, Jason; Peterlin Mašič, Lucija; Finel, Moshe

    2015-01-01

    1. Bisphenol S (BPS) and bisphenol F (BPF) are bisphenol A (BPA) analogues commonly used in the manufacturing of industrial and consumer products. 2. Bisphenols are often detoxified through conjugation with glucuronic acid or sulfate. In this work, we have examined the glucuronidation of BPS and BPF by recombinant human UDP-glucuronosyltransferase (UGT) enzymes. In addition, we have reexamined BPA glucuronidation, using extra-hepatic UGTs that were not tested previously. 3. The results revealed that UGT1A9, primarily a hepatic enzyme, is mainly responsible for BPS glucuronidation, whereas UGT1A10, an intestine enzyme that is highly homologous to UGT1A9 at the protein level, is by far the most active UGT in BPF glucuronidation. In contrast to the latter two UGTs that display significant specificity in the glucuronidation of BPS and BPF, UGT2A1 that is mainly expressed in the airways, exhibited high activity toward all the tested bisphenols, BPS, BPF and BPA. UGT1A10 exhibited somewhat higher BPA glucuronidation activity than UGT1A9, but it was lower than UGT2A1 and UGT2B15. 4. The new findings demonstrate interesting differences in the glucuronidation patterns of bisphenols and provide new insights into the role of extra-hepatic tissues in their detoxification.

  4. Profiling serum bile acid glucuronides in humans: gender divergences, genetic determinants and response to fenofibrate

    Science.gov (United States)

    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

  5. Dietary moderately oxidized oil activates the Nrf2 signaling pathway in the liver of pigs

    Directory of Open Access Journals (Sweden)

    Varady Juliane

    2012-02-01

    Full Text Available Abstract Background Previous studies have shown that administration of oxidized oils increases gene expression and activities of various enzymes involved in xenobiotic metabolism and stress response in the liver of rats and guinea pigs. As these genes are controlled by nuclear factor erythroid-derived 2-like 2 (Nrf2, we investigated the hypothesis that feeding of oxidized fats causes an activation of that transcription factor in the liver which in turn activates the expression of antioxidant, cytoprotective and detoxifying genes. Methods Twenty four crossbred pigs were allocated to two groups of 12 pigs each and fed nutritionally adequate diets with either fresh rapeseed oil (fresh fat group or oxidized rapeseed oil prepared by heating at a temperature of 175°C for 72 h (oxidized fat group. Results After 29 days of feeding, pigs of the oxidized fat group had a markedly increased nuclear concentration of the transcription factor Nrf2 and a higher activity of cellular superoxide dismutase and T4-UDP glucuronosyltransferase in liver than the fresh fat group (P P Conclusion The present study shows for the first time that administration of an oxidized fat activates the Nrf2 in the liver of pigs which likely reflects an adaptive mechanism to prevent cellular oxidative damage. Activation of the NF-κB pathway might also contribute to this effect of oxidized fat.

  6. The positive effects of high-frequency right dorsolateral prefrontal cortex repetitive transcranial magnetic stimulation on memory, correlated with increases in brain metabolites detected by proton magnetic resonance spectroscopy in recently detoxified alcohol-dependent patients

    Directory of Open Access Journals (Sweden)

    Qiao J

    2016-09-01

    Full Text Available Jun Qiao,1,2 Guixing Jin,1,2 Licun Lei,3 Lan Wang,1,2 Yaqiang Du,3 Xueyi Wang1,2 1Institute of Mental Health, The First Hospital of Hebei Medical University, 2Brain Ageing and Cognitive Neuroscience Laboratory, Hebei Medical University, 3Department of Radiology, The First Hospital of Hebei Medical University, Hebei, People’s Republic of China Objective: To explore the effect of right dorsolateral prefrontal cortex (DLPFC repetitive transcranial magnetic stimulation (rTMS on memory, and its correlation with levels of hippocampal brain metabolites detected by proton magnetic resonance spectroscopy (1H-MRS in recently detoxified alcohol-dependent patients. Materials and methods: In this randomized, double-blind sham-controlled trial, alcohol-dependent patients were enrolled and randomized into two groups: the experimental group (rTMS, 10 Hz, on right DLPFC, 20 sessions and the control group (sham stimulation. Memory function was assessed using Hopkins Verbal Learning Test-Revised (HVLT-R and Brief Visuospatial Memory Test-Revised (BVMT-R before and after treatment. 1H-MRS was used to detect the levels of N-acetyl aspartic acid (NAA, choline (Cho, and creatine (Cr in bilateral hippocampi before and after treatment. Results: Thirty-eight patients (18 in the experimental group and 20 in the control group were included in the analyses. The experimental group showed significantly greater changes in HVLT-R, BVMT-R, NAA/Cr, and Cho/Cr after rTMS from baseline than the control group. The percentage change in BVMT-R and HVLT-R correlated with the percentage change in NAA/Cr and Cho/Cr in the right brain. Conclusion: High-frequency right DLPFC rTMS was associated with improvement in memory dysfunction, which is correlated with levels of hippocampal brain metabolites detected by 1H-MRS in recently detoxified alcohol-dependent patients. Keywords: alcohol dependence, memory, repetitive transcranial magnetic stimulation, MR spectroscopy

  7. Inhibitory Role of Greatwall-Like Protein Kinase Rim15p in Alcoholic Fermentation via Upregulating the UDP-Glucose Synthesis Pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Watanabe, Daisuke; Zhou, Yan; Hirata, Aiko; Sugimoto, Yukiko; Takagi, Kenichi; Akao, Takeshi; Ohya, Yoshikazu; Takagi, Hiroshi; Shimoi, Hitoshi

    2015-10-23

    The high fermentation rate of Saccharomyces cerevisiae sake yeast strains is attributable to a loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-family protein kinase that is conserved among eukaryotes. In the present study, we performed intracellular metabolic profiling analysis and revealed that deletion of the RIM15 gene in a laboratory strain impaired glucose-anabolic pathways through the synthesis of UDP-glucose (UDPG). Although Rim15p is required for the synthesis of trehalose and glycogen from UDPG upon entry of cells into the quiescent state, we found that Rim15p is also essential for the accumulation of cell wall β-glucans, which are also anabolic products of UDPG. Furthermore, the impairment of UDPG or 1,3-β-glucan synthesis contributed to an increase in the fermentation rate. Transcriptional induction of PGM2 (phosphoglucomutase) and UGP1 (UDPG pyrophosphorylase) was impaired in Rim15p-deficient cells in the early stage of fermentation. These findings demonstrate that the decreased anabolism of glucose into UDPG and 1,3-β-glucan triggered by a defect in the Rim15p-mediated upregulation of PGM2 and UGP1 redirects the glucose flux into glycolysis. Consistent with this, sake yeast strains with defective Rim15p exhibited impaired expression of PGM2 and UGP1 and decreased levels of β-glucans, trehalose, and glycogen during sake fermentation. We also identified a sake yeast-specific mutation in the glycogen synthesis-associated glycogenin gene GLG2, supporting the conclusion that the glucose-anabolic pathway is impaired in sake yeast. These findings demonstrate that downregulation of the UDPG synthesis pathway is a key mechanism accelerating alcoholic fermentation in industrially utilized S. cerevisiae sake strains.

  8. Glycosylation of α-dystroglycan: O-mannosylation influences the subsequent addition of GalNAc by UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases.

    Science.gov (United States)

    Tran, Duy T; Lim, Jae-Min; Liu, Mian; Stalnaker, Stephanie H; Wells, Lance; Ten Hagen, Kelly G; Live, David

    2012-06-15

    O-Linked glycosylation is a functionally and structurally diverse type of protein modification present in many tissues and across many species. α-Dystroglycan (α-DG), a protein linked to the extracellular matrix, whose glycosylation status is associated with human muscular dystrophies, displays two predominant types of O-glycosylation, O-linked mannose (O-Man) and O-linked N-acetylgalactosamine (O-GalNAc), in its highly conserved mucin-like domain. The O-Man is installed by an enzyme complex present in the endoplasmic reticulum. O-GalNAc modifications are initiated subsequently in the Golgi apparatus by the UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) enzymes. How the presence and position of O-Man influences the action of the ppGalNAc-Ts on α-DG and the distribution of the two forms of glycosylation in this domain is not known. Here, we investigated the interplay between O-Man and the addition of O-GalNAc by examining the activity of the ppGalNAc-Ts on peptides and O-Man-containing glycopeptides mimicking those found in native α-DG. These synthetic glycopeptides emulate intermediate structures, not otherwise readily available from natural sources. Through enzymatic and mass spectrometric methods, we demonstrate that the presence and specific location of O-Man can impact either the regional exclusion or the site of O-GalNAc addition on α-DG, elucidating the factors contributing to the glycosylation patterns observed in vivo. These results provide evidence that one form of glycosylation can influence another form of glycosylation in α-DG and suggest that in the absence of proper O-mannosylation, as is associated with certain forms of muscular dystrophy, aberrant O-GalNAc modifications may occur and could play a role in disease presentation.

  9. Systematic determination of the peptide acceptor preferences for the human UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase).

    Science.gov (United States)

    Perrine, Cynthia; Ju, Tongzhong; Cummings, Richard D; Gerken, Thomas A

    2009-03-01

    Mucin-type protein O-glycosylation is initiated by the addition of alpha-GalNAc to Ser/Thr residues of a polypeptide chain. The addition of beta-Gal to GalNAc by the UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase), forming the Core 1 structure (beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr), is a common and biologically significant subsequent step in O-glycan biosynthesis. What dictates the sites of Core 1 glycosylation is poorly understood; however, the peptide sequence and neighboring glycosylation effects have been implicated. To systematically address the role of the peptide sequence on the specificity of T-synthase, we used the oriented random glycopeptide: GAGAXXXX(T-O-GalNAc)XXXXAGAG (where X = G, A, P, V, I, F, Y, S, N, D, E, H, R, and K) as a substrate. The Core 1 glycosylated product was isolated on immobilized PNA (Arachis hypogaea) lectin and its composition determined by Edman amino acid sequencing for comparison with the initial substrate composition, from which transferase preferences were obtained. From these studies, elevated preferences for Gly at the +1 position with moderately high preferences for Phe and Tyr in the +3 position relative to the acceptor Thr-O-GalNAc were found. A number of smaller Pro enhancements were also observed. Basic residues, i.e., Lys, Arg, and His, in any position were disfavored, suggesting electrostatic interactions as an additional important component modulating transferase specificity. This work suggests that there are indeed subtle specific and nonspecific protein-targeting sequence motifs for this transferase.

  10. Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose: a simple analysis by 13C NMR of urinary menthol glucuronide.

    Science.gov (United States)

    Mendes, Ana C; Caldeira, M Madalena; Silva, Claudia; Burgess, Shawn C; Merritt, Matthew E; Gomes, Filipe; Barosa, Cristina; Delgado, Teresa C; Franco, Fatima; Monteiro, Pedro; Providencia, Luis; Jones, John G

    2006-11-01

    Menthol glucuronide was isolated from the urine of a healthy 70-kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U-(13)C]glucose. Glucuronide (13)C-excess enrichment levels were 4-6% and thus provided high signal-to-noise ratios (SNRs) for confident assignment of (13)C-(13)C spin-coupled multiplet components within each (13)C resonance by (13)C NMR. The [U-(13)C]glucuronide isotopomer derived via direct pathway conversion of [U-(13)C]glucose to [U-(13)C]UDP-glucose was resolved from [1,2,3-(13)C(3)]- and [1,2-(13)C(2)]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U-(13)C]glucose. In a second study, a group of four overnight-fasted patients (63 +/- 10 kg) with severe heart failure were given peppermint oil and infused with [U-(13)C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady-state plasma [U-(13)C]glucose enrichment of 4.6% +/- 0.6%. Menthol glucuronide was harvested and glucuronide (13)C-isotopomers were analyzed by (13)C NMR. [U-(13)C]glucuronide enrichment was 0.6% +/- 0.1%, and the sum of [1,2,3-(13)C(3)] and [1,2-(13)C(2)]glucuronide enrichments was 0.9% +/- 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% +/- 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% +/- 10% of GP. (c) 2006 Wiley-Liss, Inc.

  11. Use of a cell-free system to determine UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase activities in human hereditary inclusion body myopathy.

    Science.gov (United States)

    Sparks, Susan E; Ciccone, Carla; Lalor, Molly; Orvisky, Eduard; Klootwijk, Riko; Savelkoul, Paul J; Dalakas, Marinos C; Krasnewich, Donna M; Gahl, William A; Huizing, Marjan

    2005-11-01

    Hereditary inclusion body myopathy (HIBM) is an autosomal recessive neuromuscular disorder associated with mutations in uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine (ManNAc) kinase (MNK), the bifunctional and rate-limiting enzyme of sialic acid biosynthesis. We developed individual GNE and MNK enzymatic assays and determined reduced activities in cultured fibroblasts of patients, with HIBM harboring missense mutations in either or both the GNE and MNK enzymatic domains. To assess the effects of individual mutations on enzyme activity, normal and mutated GNE/MNK enzymatic domains were synthesized in a cell-free in vitro transcription-translation system and subjected to the GNE and MNK enzymatic assays. This cell-free system was validated for both GNE and MNK activities, and it revealed that mutations in one enzymatic domain (in GNE, G135V, V216A, and R246W; in MNK, A631V, M712T) affected not only that domain's enzyme activity, but also the activity of the other domain. Moreover, studies of the residual enzyme activity associated with specific mutations revealed a discrepancy between the fibroblasts and the cell-free systems. Fibroblasts exhibited higher residual activities of both GNE and MNK than the cell-free system. These findings add complexity to the tightly regulated system of sialic acid biosynthesis. This cell-free approach can be applied to other glycosylation pathway enzymes that are difficult to evaluate in whole cells because their substrate specificities overlap with those of ancillary enzymes.

  12. UDP-Xylose-Stimulated Glucuronyltransferase Activity in Wheat Microsomal Membranes: Characterization and Role in Glucurono(arabino)xylan Biosynthesis1[C

    Science.gov (United States)

    Zeng, Wei; Chatterjee, Mohor; Faik, Ahmed

    2008-01-01

    Microsomal membranes from etiolated wheat (Triticum aestivum) seedlings cooperatively incorporated xylose (Xyl), arabinose, and glucuronic acid residues from their corresponding uridine 5′-diphosphosugars into an ethanol-insoluble glucurono(arabino)xylan (GAX)-like product. A glucuronyltransferase activity that is enhanced by the presence of UDP-Xyl was also identified in these microsomes. Wheat glucuronyltransferase activity was optimal at pH 7 and required manganese ions, and several lines of evidence suggest its involvement in GAX-like biosynthesis. The GAX characteristics of the 14C-product were confirmed by digestion with a purified endo-xylanase from Aspergillus awamori (endo-xylanase III) and by total acid hydrolysis, resulting in a Xyl:arabinose:glucuronic acid molar ratio of approximately 105:34:1. Endo-xylanase III released only three types of oligosaccharides in addition to free Xyl. No radiolabel was released as xylobiose, xylotriose, or xylotetraose, indicating the absence of long stretches of unbranched Xyl residues in the nascent GAX-like product. High-pH anion exchange chromatography analysis of the resulting oligosaccharides along with known arabinoxylan oligosaccharide standards suggests that a portion of the nascent GAX-like product has a relatively regular structure. The other portion of the [14C]GAX-like polymer was resistant to proteinase K, endo-polygalacturonase, and endo-xylanase III (GH11 family) but was degraded by Driselase, supporting the hypothesis that the xylan backbone in this portion of the product is most likely highly substituted. Size exclusion chromatography indicated that the nascent GAX-like polymer had an apparent molecular mass of approximately 10 to 15 kD; however, mature GAXs from wheat cell walls had larger apparent molecular masses (>66 kD). PMID:18359844

  13. Effect of ecdysteroid UDP-glucosyltransferase gene deletion on efficacy of a baculovirus against Heliothis virescens and Trichoplusia ni (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Treacy, M F; All, J N; Ghidiu, G M

    1997-10-01

    Laboratory, greenhouse, and field studies were conducted to characterize the biological activity of a genetically altered form of Autographa californica (Speyer) nucleopolyhedrosis virus (AcNPV). The altered baculovirus (vEGTDEL) had a deletion in the ecdysteroid UDP-glucosyltransferase gene. Results from bioassays conducted with neonate and 3rd-instar tobacco budworm, Heliothis virescens (F.), as well as with 3rd-instar cabbage looper, Trichoplusia ni (Hübner), showed vEGTDEL caused larval death slightly, but significantly, quicker than AcNPV. Based on supposition (LT50 values were not calculated), it appeared that larval mortality occurred 0.5-1.0 d faster following exposure to vEGTDEL versus AcNPV. Greenhouse studies conducted against H. virescens on cotton showed that hastened virulence exhibited by vEGTDEL led to improved plant protection versus AcNPV. For example, following 5 weekly sessions of foliar application and H. virescens artificial infestation, cotton treated with wettable powder formulations of vEGTDEL or AcNPV at 2.5 x 10(12) OB/ha averaged 25.7 and 61.8% damaged flower buds, respectively. Although vEGTDEL tended to provide more consistent control of T. ni than AcNPV in greenhouse and field trials conducted on leafy vegetables, differences in efficacy between the 2 baculoviruses were marginal and usually not statistically significant. Generally, results from these studies suggest that genetic modification of NPVs to hasten their lethal effect may be a promising strategy for improving the insecticidal properties of the insect-specific pathogens.

  14. UDP-glucose pyrophosphorylase2 (OsUgp2),a pollen-preferential gene in rice, plays a critical role in starch accumulation during pollen maturation

    Institute of Scientific and Technical Information of China (English)

    MU Hong; KE JianHao; LIU Wei; ZHUANG ChuXiong; YIP WingKin

    2009-01-01

    UDP-glucose pyrophosphorylase (UGPase) is predominantly present and plays significant role in car-bohydrate metabolism in plants. Two homologous UGPase genes, OsUgp1 and OsUgp2, exist in rice genome. OsUgp1 has recently been reported to be essential for callose deposition during pollen mother cell and meiosis stages as well as for seed carbohydrate metabolism. In this study, a full-length cDNA of OsUgp2 was isolated from rice anther. Northern blot and RNA in situ hybridization indicated that the expression of OsUgp2 was preferentially in pollen and developmentally regulated. No tran-scripts were found in leaf, stem, lemma/palea, ripening grain and florets before the uninucleate micro-spore developmental stage, but a large quantity of OsUgp2 mRNA was found in pollen at the binucleate and mature stages. The immunolocalizaUon of OsUgp2 showed a similar expression pattern to that by RNA in situ hybridization. The function of OsUgp2 was investigated by dsRNA-mediated transcriptional gene silencing. The pollen fertility of 16 independent transgenic rice plants was found between 25%and 90%, which was correlated with the amount of OsUgp2 mRNA. The results of morphological changes and starch variation during pollen development in transgenic rice showed that the abnormal feature of pollen development appeared after the uninucleate microspore stage. Starch failed to accu-mulate in pollen and thus led to sterile pollens. These results demonstrated that OsUgp2 is a pol-len-preferential "late gone" and plays a key role during pollen maturation, especially for starch accu-mulation. OsUgp2 complements OsUgpl to fulfill the UGPase's functions necessary for the full processof pollen development.

  15. Varietal Yield Test on the Cultivation of Detoxified Potato with Pine Needle Mulch%松针土栽培脱毒马铃薯品种比较试验

    Institute of Scientific and Technical Information of China (English)

    周平; 付业春; 王朝海; 王朝贵; 顾尚敬

    2011-01-01

    With detoxified potato seeds of Eshu series as tested materials, the yields, proportions of big, middle and small potatos, aerial characteristics of plant of all the varieties were determined and the varieties suitable for sowing in pine needle mulch were selected. The results indicated that the fresh yields and single - hole weights of Eshu 8, 7 and 3 were higher and their growth period of plant was shorter. They were suitable for sowing in pine needle mulch in Bijie area.%以鄂薯系列脱毒种薯为试验材料,测定各品种的产量和大、中、小薯率及植株地上部特征特性,选择适宜在松针土中播种的品种.结果表明,鄂薯8号、鄂薯7号和鄂薯3号的鲜产、单窝重较高,植株生育期较短,适宜在毕节地区松针土中播种.

  16. Disease: H00208 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H00208 Hyperbilirubinemia; Crigler-Najjar syndrome, type I (CN1); Crigler-Najjar sy...dromes result in unconjugated hyperbilirubinemia caused by deficiency of bilirubin-UDP-glucuronosyltransfera...se which is involved in the detoxification of bilirubin by conjugation with glucu...ronic acid. Gilbert disease is a benign familial disorder characterized by low-grade chronic hyperbilirubine...sta E Hematologically important mutations: bilirubin UDP-glucuronosyltransferase gene mutations in Gilbert a

  17. Unexpected tolerance of glycosylation by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase revealed by electron capture dissociation mass spectrometry: carbohydrate as potential protective groups.

    Science.gov (United States)

    Yoshimura, Yayoi; Matsushita, Takahiko; Fujitani, Naoki; Takegawa, Yasuhiro; Fujihira, Haruhiko; Naruchi, Kentarou; Gao, Xiao-Dong; Manri, Naomi; Sakamoto, Takeshi; Kato, Kentaro; Hinou, Hiroshi; Nishimura, Shin-Ichiro

    2010-07-20

    UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs, EC 2.4.1.41), a family of key enzymes that initiate posttranslational modification with O-glycans in mucin synthesis by introduction of alpha-GalNAc residues, are structurally composed of a catalytic domain and a lectin domain. It has been known that multiple Ser/Thr residues are assigned in common mucin glycoproteins as potential O-glycosylation sites and more than 20 distinct isoforms of this enzyme family contribute to produce densely O-glycosylated mucin glycoproteins. However, it seems that the functional role of the lectin domain of ppGalNAcTs remains unclear. We considered that electron capture dissociation mass spectrometry (ECD-MS), a promising method for highly selective fragmentation at peptide linkages of glycopeptides to generate unique c and z series of ions, should allow for precise structural characterization to uncover the mechanism in O-glycosylation of mucin peptides by ppGalNAcTs. In the present study, it was demonstrated that a system composed of an electrospray source, a linear RFQ ion trap that isolates precursor ions, the ECD device, and a TOF mass spectrometer is a nice tool to identify the preferential O-glycosylation sites without any decomposition of the carbohydrate moiety. It should be noted that electrons used for ECD are accelerated within a range from 1.75 to 9.75 eV depending on the structures of glycopeptides of interest. We revealed for the first time that additional installation of a alpha-GalNAc residue at potential glycosylation sites by ppGalNAcT2 proceeds smoothly in various unnatural glycopeptides having alpha-Man, alpha-Fuc, and beta-Gal residues as well as alpha-GalNAc residues. The results may suggest that ppGalNAcT2 did not differentiate totally presubstituted sugar residues in terms of configuration of functional groups, d-, l-configuration, and even alpha-, beta-stereochemistry at an anomeric carbon atom when relatively short synthetic

  18. 多杀菌素对黄褐天幕毛虫解毒酶及保护酶的影响%Effect of Spinosad on the Detoxifying and Protective Enzymes of Malacosoma neustria testacea

    Institute of Scientific and Technical Information of China (English)

    刘丹; 严善春; 曹传旺; 廖月枝

    2012-01-01

    为了研究多杀菌素对黄褐天幕毛虫的杀虫活性及作用机制,采用叶片药膜法测定多杀菌素对黄褐天幕毛虫幼虫的毒杀效力,并用分光光度法检测LC50剂量药剂处理后不同时间点幼虫体内各种解毒酶和保护酶活性的变化.结果表明:多杀菌素对4龄、5龄幼虫均表现出极高的杀虫活性,见效速度块;而且黄褐天幕毛虫4龄、5龄幼虫经过多杀菌素处理后,谷胱甘肽S-转移酶均表现为复杂的抑制-激活-再抑制作用,多功能氧化酶活性则均表现为先抑制后激活的作用.多杀菌素对4龄幼虫羧酸酯酶活性具有激活作用,对乙酰胆碱酯酶和过氧化物酶均具有先激活后抑制作用,对超氧化物歧化酶具有先激活再抑制后激活的作用,对过氧化氢酶则表现为复杂的先抑制再激活后抑制作用.对5龄幼虫羧酸酯酶、乙酰胆碱酯酶、超氧化物歧化酶及过氧化物酶活性具有明显的抑制作用,而对过氧化氢酶则表现为先抑制再激活后抑制的作用.因此,多杀菌素能有效干扰昆虫解毒和保护系统,扰乱其正常的生理代谢,从而起到较好的毒杀效果.%To study the insecticidal activity and toxicity mechanism of spinosad, we assayed bioactivity of spinosad by leaf membrane method and its effects on the activities of detoxifying and protective enzymes in Malacosoma neustria testacea larvae by spectrophotometry. The results showed that spinosad had an extremely high toxicity against the 4' and 5' instar larvae. The glutathione S-transfer (GST) activity in 4th and 5th instar larvae was firstly inhibited, then induced, and finally inhibited, while the mixed-functional oxidase ( MFO) activity was inhibited and then enhanced. The induced effect on carboxylesterase ( CarE) , the induced and inhibited effect on acetylcholinesterase ( AchE) and peroxidase ( POD) , and the complicated effects on superoxide dismutase ( SOD) and Catalase ( CAT) were determined in 4

  19. Combined chemical-enzymic synthesis of deoxygenated oligosaccharide analogs: transfer of deoxygenated D-GlcpNAc residues from their UDP-GlcpNAc derivatives using N-acetylglucosaminyltransferase I.

    Science.gov (United States)

    Srivastava, G; Alton, G; Hindsgaul, O

    1990-10-25

    The 3''-, 4''-, and 6''-deoxy analogs of UDP-GlcpNAc have been synthesized chemically and found to act as donor-substrates for N-acetylglucosaminyltransferase-I (GnT-I) from human milk. Incubation of UDP-GlcpNAc and these deoxy analogs with GnT-I in the presence of alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -O(CH2)8COOMe gave beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----3)-[alpha-D-Manp- (1----6)]- beta-D-Manp-O(CH2)8COOMe (6), and the deoxy analogs 12-14 where HO-3, HO-4, and HO-6, respectively, of the beta-D-GlcNAc residue were replaced by hydrogen. The tetrasaccharide glycosides 6 and 12-14 were characterized by 1H-n.m.r. spectroscopy and evaluated as acceptors for GnT-II, the next enzyme in the pathway of biosynthesis of Asn-linked oligosaccharides. Deoxygenation of the 3-position of the beta-D-GlcNAc residue of 6 completely abolished its acceptor activity, whereas removal of HO-4 or HO-6 caused only modest decreases in activity.

  20. The Impact of Accelerated Right Prefrontal High-Frequency Repetitive Transcranial Magnetic Stimulation (rTMS) on Cue-Reactivity: An fMRI Study on Craving in Recently Detoxified Alcohol-Dependent Patients.

    Science.gov (United States)

    Herremans, Sarah C; Van Schuerbeek, Peter; De Raedt, Rudi; Matthys, Frieda; Buyl, Ronald; De Mey, Johan; Baeken, Chris

    2015-01-01

    In alcohol-dependent patients craving is a difficult-to-treat phenomenon. It has been suggested that high-frequency (HF) repetitive transcranial magnetic stimulation (rTMS) may have beneficial effects. However, exactly how this application exerts its effect on the underlying craving neurocircuit is currently unclear. In an effort to induce alcohol craving and to maximize detection of HF-rTMS effects to cue-induced alcohol craving, patients were exposed to a block and event-related alcohol cue-reactivity paradigm while being scanned with fMRI. Hence, we assessed the effect of right dorsolateral prefrontal cortex (DLPFC) stimulation on cue-induced and general alcohol craving, and the related craving neurocircuit. Twenty-six recently detoxified alcohol-dependent patients were included. First, we evaluated the impact of one sham-controlled stimulation session. Second, we examined the effect of accelerated right DLPFC HF-rTMS treatment: here patients received 15 sessions in an open label accelerated design, spread over 4 consecutive days. General craving significantly decreased after 15 active HF-rTMS sessions. However, cue-induced alcohol craving was not altered. Our brain imaging results did not show that the cue-exposure affected the underlying craving neurocircuit after both one and fifteen active HF-rTMS sessions. Yet, brain activation changes after one and 15 HF-rTMS sessions, respectively, were observed in regions associated with the extended reward system and the default mode network, but only during the presentation of the event-related paradigm. Our findings indicate that accelerated HF-rTMS applied to the right DLPFC does not manifestly affect the craving neurocircuit during an alcohol-related cue-exposure, but instead it may influence the attentional network.

  1. 菜籽粕脱毒液中硫代葡萄糖苷提取工艺的研究%Study on Extracting Procedure of Glucosinolates from Rapeseed Meal Detoxifying Solution

    Institute of Scientific and Technical Information of China (English)

    谭世语; 周铭; 罗建富

    2009-01-01

    本文用两步萃取法从菜籽粕脱毒液中提取硫代葡萄糖苷,以3,5-二硝基水杨酸比色法测定硫代葡萄糖苷含量,采用单因素试验法,分别考察可能影响硫代葡萄糖苷提取效果的各种因素,确定了最佳工艺条件:第一步萃取剂为异辛烷,母液与异辛烷体积比为2:1,搅拌时间为30 min,异辛烷萃取次数为2次,异辛烷单次萃取率达71.2%.硫代葡萄糖苷收率为83.7%.%This paper discussed the extraction of glucosinolates from rapeseed meal detoxifying solution. The content of glucosinolates was determined by 3,5-dinitrosalicylic acid colorimiay and the various factors responsible for the effect of extraction of glucosinolates were investigated respectively by single factor method. The most suitable extraction conditions were as follows:in the first step,where the volume ratio of mother solution to extractant, which was isooctane,was 2: 1, extraction yield reached 71.2% in the first extraction by isooctane. In this step,there were two extractions, each of which lasted for 30 min. The total recovery of glucosinolates was 83.7%.

  2. Effect of High Fructose Syrup Diet Exposure on the Activities of Detoxifying Enzymes in Honey Bees Apis mellifera ligustica%饲喂果葡糖浆对意大利蜜蜂解毒酶的影响

    Institute of Scientific and Technical Information of China (English)

    孟丽峰; 靳三省; 刁青云

    2013-01-01

    为了探讨果葡糖浆饲喂蜜蜂的安全性,以意大利蜜蜂(Apis mellifera ligustica)为实验材料,蔗糖作为对照,饲喂果葡糖浆2个月后,检测意大利蜜蜂体内解毒酶的变化情况.结果表明:饲喂果葡糖浆后,意大利蜜蜂大幼虫体内细胞色素P450比活力、成年工蜂腹部谷胱甘肽-S-转移酶和羧酸酯酶比活力均与对照无显著差异.短期饲喂果葡糖浆对蜜蜂是安全的,长期影响还有待于继续研究.%The activities of Cytochrome-P450,glutathione S-transferase and carboxylesterase in the worker bees of Apis mellifera ligustica were investigated after the bees were fed orally with high fructose syrup in two consecutive months.The results showed that compared with sucrose diet,high fructose syrup diet did not significantly affect the activities of three detoxifying enzymes.Feeding with high fructose syrups is safe to Apis mellifera ligustica in short time and long-time effects need further research.The results can be used to assess the security of high fructose syrups used as bee feed.

  3. Hydrolysate detoxified from steam exploded corn cob and its fermentation producing butanol fuels%蒸汽爆破玉米芯水解液脱毒及其发酵生产燃料丁醇

    Institute of Scientific and Technical Information of China (English)

    王风芹; 仝银杏; 李传斌; 谢慧; 宋安东

    2016-01-01

    The development and utilization of biomass have been paid great attention by many countries with the depletion of fossil energy sources. Due to the recalcitrant and complex structure of lignocellulosic biomass, various pretreatment methods are adopted to help biomass carbohydrate’s hydrolysis. Pretreatment can increase the biomass digestibility for efficient fermentable sugar production, but many fermentation inhibitors are also produced during this process, such as furan derivatives (furfural and 5-hydroxymethylfurfural), mini-molecule organic acid (acetic acid and formic acid), and lignin degradation products (coumaric acid, vanillin and other phenolics). Therefore, the detoxification process which removes fermentation inhibitors from hydrolysates is essential for the production of biomass energy; at the same time, for different fermentations have different adaptability of initial sugar concentration in fermentation liquor, the studies about the optimal initial sugar fermentation concentration are also meaningful. In order to study the best technology of butanol fuel production from steam exploded corn cob, 4 detoxification methods, which were over liming, D301 resin, over liming combined with D301 resin, and over liming combined with NKAII macro porous resin, were used to detoxify the hydrolysate of steam exploded corn cob, and their effects on butanol production were also determined in this research. The results showed that the D301 resin detoxification was the most efficient way to detoxify the hydrolysate of steam exploded corn cob. The removal ratio of vanillin could reach 100%; the removal ratio of formic acid, acetic acid, total phenol, furfural and 5-hydroxymethylfurfural reached 60%, 46.04%, 56.31%, 82.95% and 87.52%, respectively. At the same time, total sugar lost ratio was only 4.38%. Finally, butanol and ABE (actone, butanol, ethanol) production were 5.2 and 7.5 g/L by fermentation withC. acetobutylicumCICC 8016, and the utilization rate of glucose

  4. Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia

    NARCIS (Netherlands)

    Kotal, P; VanderVeere, CN; Sinaasappel, M; Elferink, RO; Vitek, L; Brodanova, M; Jansen, PLM; Fevery, J

    1997-01-01

    Patients with Crigler-Najjar syndrome and Gunn rats cannot form bilirubin glucuronides owing to a lack of bilirubin UDP-glucuronosyltransferase activity. Because increased serum and tissue bilirubin levels remain constant, an alternative excretory route has to substitute for this deficiency. Gunn ra

  5. PROTEOMIC ANALYSIS OPTIMIZATION: SELECTIVE PROTEIN SAMPLE ON-COLUMN RETENTION IN REVERSE-PHASE LIQUID CHROMATOGRAPHY

    Science.gov (United States)

    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...

  6. Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia

    NARCIS (Netherlands)

    Kotal, P; VanderVeere, CN; Sinaasappel, M; Elferink, RO; Vitek, L; Brodanova, M; Jansen, PLM; Fevery, J

    1997-01-01

    Patients with Crigler-Najjar syndrome and Gunn rats cannot form bilirubin glucuronides owing to a lack of bilirubin UDP-glucuronosyltransferase activity. Because increased serum and tissue bilirubin levels remain constant, an alternative excretory route has to substitute for this deficiency. Gunn ra

  7. Comparing the glucuronidation capacity of the feline liver with substrate-specific glucuronidation in dogs

    NARCIS (Netherlands)

    van Beusekom, C D; Fink-Gremmels, J; Schrickx, J A

    2014-01-01

    This study aimed to assess the overall glucuronidation capacity of cats, using prototypic substrates identified for human UDP-glucuronosyltransferases (UGTs). To this end, Michaelis-Menten kinetics were established for the substrates using feline hepatic microsomal fractions, and results were compar

  8. Comparing the glucuronidation capacity of the feline liver with substrate-specific glucuronidation in dogs

    NARCIS (Netherlands)

    van Beusekom, C D|info:eu-repo/dai/nl/314836497; Fink-Gremmels, J|info:eu-repo/dai/nl/119949997; Schrickx, J A|info:eu-repo/dai/nl/30483114X

    This study aimed to assess the overall glucuronidation capacity of cats, using prototypic substrates identified for human UDP-glucuronosyltransferases (UGTs). To this end, Michaelis-Menten kinetics were established for the substrates using feline hepatic microsomal fractions, and results were

  9. The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes

    Science.gov (United States)

    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...

  10. Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

    Science.gov (United States)

    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...

  11. Polymorphisms in genes encoding acetylsalicylic acid metabolizing enzymes are unrelated to upper gastrointestinal health in cardiovascular patients on acetylsalicylic acid.

    NARCIS (Netherlands)

    Oijen, M.G.H. van; Huybers, S.; Peters, W.H.M.; Drenth, J.P.H.; Laheij, R.J.F.; Verheugt, F.W.A.; Jansen, J.B.M.J.

    2005-01-01

    BACKGROUND: As acetylsalicylic acid is metabolized by UDP-glucuronosyltransferase 1A6 (UGT1A6) and cytochrome P450 2C9 (CYP2C9), interindividual differences in activity of these enzymes may modulate the effects and side-effects of acetylsalicylic acid. The objective of this study was to assess wheth

  12. 尿苷-5′-二磷酸葡萄糖醛酸转移酶对常见植物药组分的催化代谢及其相互作用%Catalytic metabolism and interaction of herbs and their components mediated by uridine 5′-diphosphate glucuronosyltransferases

    Institute of Scientific and Technical Information of China (English)

    高瑞; 陈昱; 张文; 夏春华; 张红; 黄世博; 熊玉卿

    2014-01-01

    尿苷-5′-二磷酸葡萄糖醛酸转移酶( UGT)是药物II相代谢反应最主要的酶,通过催化葡萄糖醛酸与内源性或外源性化学物质发生葡萄糖醛酸化反应,进而影响药物的药代动力学特征。许多植物药及其有效成分是UGT的底物,由UGT介导其催化代谢。本文概述了UGT酶介导某些植物药及其组分的催化代谢和对UGT酶活性的影响。%Uridine 5′-diphosphate glucuronosyltransferases ( UGT ) are the most important phase II drug metabolizing enzymes.They catalyze the glucuronidation of endogenous or exogenous chemicals , which then affect the pharmacokinetic characteristics of co -administrated drugs.Many herbs as well as their active ingredients are the substrates of UGT and their catalytic metabolism are mediated by UGT.This paper summarizes the catalytic metabolism of some herbs and their components mediated by UGT and their influence on UGT activity.

  13. Protective effect of detoxifying and collateral-dredging Baoshen Capsule on the kidney of diabetic rats%解毒通络保肾胶囊对糖尿病大鼠肾脏的保护作用

    Institute of Scientific and Technical Information of China (English)

    赵贤俊; 李才; 南征; 邓悦

    2005-01-01

    紧张素转换酶和血管紧张素Ⅱ水平:阳性对照组肾组织肾素活性较其余3组明显升高(P<0.01).阳性对照组和实验组肾组织中血管紧张素转换酶活性和血管紧张素Ⅱ含量较糖尿病对照组明显下降(P<0.01),其中血管紧张素转换酶活性阳性对照组较实验组下降明显(P<0.05).③肾脏纤维连接蛋白、兔抗鼠Ⅳ型胶原蛋白表达水平:阳性对照组和实验组较糖尿病对照组免疫染色明显减弱(P<0.01),介于正常对照组和糖尿病对照组之间.④肾皮质转化生长因子β1mRNA表达:阳性对照组和实验组的表达水平较糖尿病对照组显著下降,分别为糖尿病对照组的66.28%,64.75%(P<0.05).⑤组织学观察:光镜下阳性对照组和实验组大鼠肾小球系膜增生、肾小球基底膜增厚均较轻,且系膜区扩大不明显,肾小球基底膜无明显增厚且厚度均匀,系膜细胞增多不显著,上皮细胞足突均匀分布.结论:解毒通络保肾胶囊对糖尿病肾脏病变有保护作用,其机制可能是通过抑制肾组织肾素活性和血管紧张素转换酶,减少肾组织中血管紧张素Ⅱ含量,下凋糖尿病大鼠肾皮质转化生长因子β1mRNA表达,减少细胞外基质的沉积.%BACKGROUND: It is found in clinical observation that traditional Chinese drug detoxifying and collateral-dredging Baoshen Capsule has a good effect in prevention and treatment of diabetic nephropathy, however the concrete way of effect has not been clear.OBJECTIVE: To investigate the effects of protective effect of detoxifying and collateral-dredging Baoshen Capsule on the kidney of diabetic rats,and its mechanism.DESIGN:A randomized controlled trial.SETTING:Institute of Regenerative Medical Science of Jilin University and Institute of Traditional Chinese Medicine (TCM) of Changchun College of TCM.MATERIALS:The experiment was conducted from May 2002 to January 2003 at Pathological Laboratory, Institute of Regenerative Medical

  14. 菜籽粕脱毒液中植酸的提取及其抗氧化活性%Extraction and Antioxidant Activity of Phytic Acid from Detoxified Rapeseed Meal Solution

    Institute of Scientific and Technical Information of China (English)

    兰文菊; 彭密军; 彭胜; 吕强

    2011-01-01

    Detoxified rapeseed meal solution was extracted by alkaline precipitation method to obtain phytic acid and the antioxidant activity of phytic acid obtained was evaluated through exploring its effect on acid value and peroxide value of lard using BHT as the positive control.Meanwhile,the antioxidant activity was also investigated through comparing its scavenging capacities against hydroxyl,superoxide anion and DPPH free radicals as well as hydrogen peroxide,determined by flow injection chemiluminescence and visible spectrophotometric methods,with those of VC.The results showed that the best condition for the extraction of phytic acid was pH 8.Under the optimal extraction condition,the yield of phytic acid was 92.83% and the purity was 45.96%.The extracted phytic acid could inhibit rancidity and peroxidation in lard with a better effect than that of BHT.The extract revealed the strongest scavenging effect against superoxide anion free radicals,followed by hydroxyl free radicals,hydrogen peroxide and DPPH free radicals.Moreover,it was superior to vitamin C in scavenging superoxide anion and hydroxyl free radicals.Therefore,the extract is a good natural antioxidant.%采用碱沉淀法从菜籽粕脱毒液中回收提取植酸并对该制备植酸的抗氧化活性进行研究。以BHT为阳性对照,考察植酸对猪油的酸价和过氧化值的影响;采用流动化学发光法和可见分光光度法,以VC为阳性对照,考察植酸清除羟自由基(.OH)、超氧阴离子自由基(O2.)、1,1-二苯基-2-苦苯肼自由基(DPPH自由基)及H2O2的能力。结果表明:回收提取植酸以NaOH调节溶液pH8为优,得率达92.83%,植酸含量为45.96%;植酸对猪油的酸败和过氧化有一定的抑制作用且优于BHT;制备植酸(实验制备)清除各种自由基的能力依次为:O2.〉.OH〉H2O2〉DPPH自由基,且除DPPH自由基外对其他自由基清除效果均优于VC。表明植酸是一种较好的天然抗氧化剂。

  15. AN UDPE ASSAY FOR RAPID AND SENSITIVE DETECTION OF B URKHOLDEIA PSE UDOMALLEI%类鼻疽伯克霍尔德氏菌UDPE检测方法的建立和应用

    Institute of Scientific and Technical Information of China (English)

    宋亚军; 王津; 张敏丽; 郭兆彪; 杨瑞馥

    2001-01-01

    目的 建立防止产物污染的UDPE(UDG-Duplex PCR-EIA)技术,用于检测类鼻疽伯克霍尔德氏菌。方法选择类鼻疽伯克霍尔德氏菌FUR(Ferric Uptake Regulator)基因为靶序列,设计两对引物进行PCR扩增,用UDG(尿嘧啶糖基化酶)防止PCR产物污染,并用微孔板杂交-酶联显色检测扩增的PCR产物片段;用不同的模板考察方法的特异性和灵敏度。结果该检测系统可以特异性地检测类鼻疽伯克霍尔德氏菌;对于纯DNA模板,检测灵敏度可以达到10fg/μl;对于系列稀释菌液提取的模板,灵敏度可达0.1个菌/μl;对于模拟污水标本、模拟组织标本和模拟土壤标本的检测灵敏度分别为10个菌/μl,100个菌/μl和100个菌/μl;检测系统可以防止109个PCR产物分子的污染;检测系统可以在37℃下稳定保存7d。结论本研究建立了稳定的,防止产物污染,灵敏度和特异性均较理想的类鼻疽伯克霍尔德氏茵UDPE检测系统。%Aim To develop and objective, rapid, specific and sensitive UDPE (UDG - Duplex PCR - EIA)assay to detect the pathogen Burkholderia pseudomallei. Method Two sets of primers, targeting FUR (Ferric Uptake Regulator)gene of Burkholderia pseudomallei, were chosen to amplify certain fragments, and an enzyme called UDG(Uracil DNA Glycosylase)was added into the PCR reaction mixture to avoid carry - over caused by PCR products. The PGR products were detected by microwell - hybriadation and Enzyme- Immunoassay(EIA). Different templates were employed to evaluate the specificity and sensitivity of this detection system. Results This system can specifically detect Burkholderia pseudomallei without false positive result in realted species. 0.1U of UDG in the PCR reaction mixture can effectively prevent carry - over raised by 109 PCR product molecules. The detection limit of pure DNA template is as low as 10fg/μl;when applied to serial dilution bacteria suspension, the detection limit

  16. 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.

    Science.gov (United States)

    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 (D100), IV (F220) and V (F264) 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.

  17. Epigenetic modifications of triterpenoid ursolic acid in activating Nrf2 and blocking cellular transformation of mouse epidermal cells.

    Science.gov (United States)

    Kim, Hyuck; Ramirez, Christina N; Su, Zheng-Yuan; Kong, Ah-Ng Tony

    2016-07-01

    Ursolic acid (UA), a well-known natural triterpenoid found in abundance in blueberries, cranberries and apple peels, has been reported to possess many beneficial health effects. These effects include anticancer activity in various cancers, such as skin cancer. Skin cancer is the most common cancer in the world. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of antioxidative stress response with anticarcinogenic activity against UV- and chemical-induced tumor formation in the skin. Recent studies show that epigenetic modifications of Nrf2 play an important role in cancer prevention. However, the epigenetic impact of UA on Nrf2 signaling remains poorly understood in skin cancer. In this study, we investigated the epigenetic effects of UA on mouse epidermal JB6 P+ cells. UA inhibited cellular transformation by 12-O-tetradecanoylphorbol-13-acetate at a concentration at which the cytotoxicity was no more than 25%. Under this condition, UA induced the expression of the Nrf2-mediated detoxifying/antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferase 1A1. DNA methylation analysis revealed that UA demethylated the first 15 CpG sites of the Nrf2 promoter region, which correlated with the reexpression of Nrf2. Furthermore, UA reduced the expression of epigenetic modifying enzymes, including the DNA methyltransferases DNMT1 and DNMT3a and the histone deacetylases (HDACs) HDAC1, HDAC2, HDAC3 and HDAC8 (Class I) and HDAC6 and HDAC7 (Class II), and HDAC activity. Taken together, these results suggest that the epigenetic effects of the triterpenoid UA could potentially contribute to its beneficial effects, including the prevention of skin cancer.

  18. Metabolic detoxification pathways for 5-methoxy-sterigmatocystin in primary tracheal epithelial cells.

    Science.gov (United States)

    Cabaret, Odile; Puel, Olivier; Botterel, Françoise; Delaforge, Marcel; Bretagne, Stéphane

    2014-01-01

    1.  The health effects of inhaled mycotoxins remain poorly documented despite their presence in bioaerosols. 5-methoxy-sterigmatocystin is produced in association with sterigmatocystin by some Aspergillus spp., sometimes in larger amounts than sterigmatocystin. Whereas sterigmatocystin can be metabolized through cytochromes P450 (CYP), UDP-glucuronosyltransferases and sulfotransferases in airway epithelial cells, little is known about 5-methoxy-sterigmatocystin. 2.  The 5-methoxy-sterigmatocystin metabolites were analyzed using human recombinant CYP and porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. The induction of xenobiotic-metabolizing enzymes was examined by real-time quantitative PCR for mRNA expression and 7-ethoxyresorufin O-deethylation activity. 3.  CYP1A1 metabolized 5-methoxy-sterigmatocystin into hydroxy-nor-methoxy-sterigmatocystin, nor-methoxy-sterigmatocystin and dihydroxy-methoxy-sterigmatocystin. CYP1A2 led to monohydroxy-methoxy-sterigmatocystin. In PTEC, 5-methoxy-sterigmatocystin metabolism resulted into a glucuroconjugate of 5-methoxy-sterigmatocystin, a sulfoconjugate and a glucuroconjugate of monohydroxy-methoxy-sterigmatocystin. The exposure of PTEC for 24 h to 1 µM 5-methoxy-sterigmatocystin induced a significant increase in the mRNA levels of CYP1A1, without significant induction of the 7-ethoxyresorufin O-deethylation activity. 4.  These data suggest that 5-methoxy-sterigmatocystin is mainly detoxified in airway cells through conjugation, as sterigmatocystin. However, while CYP produced a reactive metabolite of sterigmatocystin, no such metabolite was detected with 5-methoxy-sterigmatocystin. Nevertheless, 5-methoxy-sterigmatocystin increases the CYP1A1 mRNA levels. The long-term consequences remain unknown.

  19. Intestinal glucuronidation protects against chemotherapy-induced toxicity by irinotecan (CPT-11).

    Science.gov (United States)

    Chen, Shujuan; Yueh, Mei-Fei; Bigo, Cyril; Barbier, Olivier; Wang, Kepeng; Karin, Michael; Nguyen, Nghia; Tukey, Robert H

    2013-11-19

    Camptothecin (CPT)-11 (irinotecan) has been used widely for cancer treatment, particularly metastatic colorectal cancer. However, up to 40% of treated patients suffer from severe late diarrhea, which prevents CPT-11 dose intensification and efficacy. CPT-11 is a prodrug that is hydrolyzed by hepatic and intestinal carboxylesterase to form SN-38, which in turn is detoxified primarily through UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed glucuronidation. To better understand the mechanism associated with toxicity, we generated tissue-specific Ugt1 locus conditional knockout mouse models and examined the role of glucuronidation in protecting against irinotecan-induced toxicity. We targeted the deletion of the Ugt1 locus and the Ugt1a1 gene specifically in the liver (Ugt1(ΔHep)) and the intestine (Ugt1(ΔGI)). Control (Ugt1(F/F)), Ugt1(ΔHep), and Ugt1(ΔGI) adult male mice were treated with different concentrations of CPT-11 daily for four consecutive days. Toxicities were evaluated with regard to tissue glucuronidation potential. CPT-11-treated Ugt1(ΔHep) mice showed a similar lethality rate to the CPT-11-treated Ugt1(F/F) mice. However, Ugt1(ΔGI) mice were highly susceptible to CPT-11-induced diarrhea, developing severe and lethal mucositis at much lower CPT-11 doses, a result of the proliferative cell loss and inflammation in the intestinal tract. Comparative expression levels of UGT1A1 in intestinal tumors and normal surrounding tissue are dramatically different, providing for the opportunity to improve therapy by differential gene regulation. Intestinal expression of the UGT1A proteins is critical toward the detoxification of SN-38, whereas induction of the UGT1A1 gene may serve to limit toxicity and improve the efficacy associated with CPT-11 treatment.

  20. Mutations in MurE, the essential UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase of Corynebacterium glutamicum: effect on L-lysine formation and analysis of systemic consequences.

    Science.gov (United States)

    Hochheim, Jennifer; Kranz, Angela; Krumbach, Karin; Sokolowsky, Sascha; Eggeling, Lothar; Noack, Stephan; Bocola, Marco; Bott, Michael; Marienhagen, Jan

    2017-02-01

    To explore systemic effects of mutations in the UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase (MurE) of Corynebacterium glutamicum, that leads to extracellular L-lysine accumulation by this bacterium. The analysis of a mutant cohort of C. glutamicum strains carrying all possible 20 amino acids at position 81 of MurE revealed unexpected effects on cellular properties. With increasing L-lysine accumulation the growth rate of the producing strain is reduced. A dynamic flux balance analysis including the flux over MurE fully supports this finding and suggests that further reductions at this flux control point would enhance L-lysine accumulation even further. The strain carrying the best MurE variant MurE-G81K produces 37 mM L-lysine with a yield of 0.17 g/g (L-lysine·HCl/glucose·H2O), bearing no other genetic modification. Interestingly, among the strains with high L-lysine titers, strain variants occur which, despite possessing the desired amino acid substitutions in MurE, have regained close to normal growth and correspondingly lower L-lysine accumulation. Genome analyses of such variants revealed the transposition of mobile genetic elements which apparently annulled the favorable consequences of the MurE mutations on L-lysine formation. MurE is an attractive target to achieve high L-lysine accumulation, and product formation is inversely related to the specific growth rate. Moreover, single point mutations leading to elevated L-lysine titers may cause systemic effects on different levels comprising also major genome modifications. The latter caused by the activity of mobile genetic elements, most likely due to the stress conditions being characteristic for microbial metabolite producers.

  1. Genomic and biochemical analysis of the diaminopimelate and lysine biosynthesis pathway in Verrucomicrobium spinosum: Identification and partial characterization of L,L-diaminopimelate aminotransferase and UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-meso-diaminopimelate ligase

    Directory of Open Access Journals (Sweden)

    Victoria R. Nachar

    2012-05-01

    Full Text Available The Gram-negative bacterium Verrucomicrobium spinosum has attracted interest in recent years following the sequencing and annotation of its genome. Comparative genomic analysis of V. spinosum using diaminopimelate/lysine metabolic genes from Chlamydia trachomatis suggests that V. spinosum employs the L,L-diaminopimelate aminotransferase (DapL pathway for diaminopimelate/lysine biosynthesis. The open reading frame corresponding to the putative dapL ortholog was cloned and the recombinant enzyme was shown to possess L,L-diaminopimelate aminotransferase activity in vitro. In vivo analysis using functional complementation confirmed that the dapL ortholog was able to functionally complement an E. coli mutant that confers auxotrophy for diaminopimelate and lysine. In addition to its role in lysine biosynthesis, the intermediate diaminopimelate has an integral role in peptidoglycan biosynthesis. To this end, the UDP-N-acetylmuramoylalanyl-D-glutamyl-2, 6-meso-diaminopimelate ligase ortholog was also identified, cloned and was shown to possess meso-diaminopimelate ligase activity in vivo. The L,L-diaminopimelate aminotransferase pathway has been experimentally confirmed in several bacteria, some of which are deemed pathogenic to animals. Since animals, and particularly humans, lack the genetic machinery for the synthesis of diaminopimelate/lysine de novo, the enzymes involved in this pathway are attractive targets for development of antibiotics. Whether dapL is an essential gene in any bacteria is currently not known. V. spinosum is an excellent candidate to investigate the essentiality of dapL, since the bacterium employs the DapL pathway for lysine and cell wall biosynthesis, is non-pathogenic to humans, facile to grow and can be genetically manipulated.

  2. Analysis of mucolipidosis II/III GNPTAB missense mutations identifies domains of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase involved in catalytic function and lysosomal enzyme recognition.

    Science.gov (United States)

    Qian, Yi; van Meel, Eline; Flanagan-Steet, Heather; Yox, Alex; Steet, Richard; Kornfeld, Stuart

    2015-01-30

    UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits.

  3. Design and implementation of UDP-based terminal adaptive protocol%基于UDP的终端性能自适应协议的设计与实现

    Institute of Scientific and Technical Information of China (English)

    王斌; 陈红梅; 张保平

    2013-01-01

    Aiming at terminal performance bottleneck among current data transfer process, a UDP-based terminal adaptive protocol was proposed. After the analysis and the comparison of many factors which affected terminal performance, this protocol viewed both the previous packet loss ratio and the current one as congestion detection parameters. It employed various rate adaption methods such as finite loop counter and process scheduling function in order to balance performance differences in real-time and ensured reliable and fast data transfer. Compared with traditional idle Automatic Repeat reQuest (ARQ) method, the average delay is reduced by more than 25%. The experimental results show that the proposed algorithm has the features of strong real-time, quick response, and it is compatible with large amount of data transmission, especially suitable for small amount of data transmission in engineering applications.%针对目前数据传输当中接收端性能瓶颈问题,提出一种基于UDP的终端自适应协议设计方法.通过对影响终端性能的若干因素的分析与比较,协议采用前后两次丢包率作为拥塞检测参数,结合有限循环计数器以及进程调度函数等多种速率适配方法,实时有效地平衡收发双方性能差异,并确保数据可靠快速地传输.与传统的空闲ARQ方式相比,平均延迟降低了25%以上.实验结果证实:该协议实时性强,反应迅速,支持大量数据传输,且更适于数据量不大的工程应用.

  4. CDP-Alcohol Hydrolase, a Very Efficient Activity of the 5′-Nucleotidase/UDP-Sugar Hydrolase Encoded by the ushA Gene of Yersinia intermedia and Escherichia coli▿ †

    Science.gov (United States)

    Alves-Pereira, Isabel; Canales, José; Cabezas, Alicia; Martín Cordero, Paloma; Costas, María Jesús; Cameselle, José Carlos

    2008-01-01

    Nucleoside 5′-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25°C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5′-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshAYi) of the Y. intermedia ushA gene (ushAYi) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshAEc). Therefore, UshAEc was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshAEc and UshAYi behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with kcat/Km ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshAEc conformers and the respective homology models constructed for UshAYi, and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations. PMID:18641143

  5. 类纸屏幕课件分阅系统中的UDP广播问题研究%RESEARCH ON UDP BROADCASTING ISSUES IN PAPER-LIKE SCREEN COURSEWARE RESPECTIVE READING SYSTEM

    Institute of Scientific and Technical Information of China (English)

    马捷; 张金; 石彬; 陈昕宇; 陈维政; 李天龙; 王鹏

    2012-01-01

    利用E-Ink技术类纸显示、无辐射、功耗低等特点,设计并实现一种环保节能的基于类纸屏幕广播的课堂教学系统.针对TCP协议开销较大、耗时长的问题,设计一种以UDP广播为主,辅以反馈和TCP传输的无线传输方法,通过对屏幕的即时截图将教师演示的课件内容以图片的形式即时传输并显示在学生手中具有E-Ink屏幕的电子阅读器设备上,从而实现课件的同步分阅.最后通过采集实验数据对该系统进行了性能分析,包括终端接收成功率、广播次数、数据包大小等.结果表明采用该无线传输方法的系统具有较高可行性.%Taking advantage of E-Ink technologies such as paper-like display, no radiation and low power consumption, the paper designs and implements an environment protective and energy preservative, paper-like screen broadcasting based classroom teaching system. To overcome the disadvantages of TCP protocol such as high cost and time consuming, the paper designs a wireless transmission approach which depends mainly on UDP broadcasting while supplemented by feedbacks and TCP transmission, through instant screen snatch, transmits in the form of images, the courseware contents that the teacher demonstrates, and displays them on electronic readable devices, which are held in students' hands, each equipped with an E-lnk screen. As a result the synchronous respective-reading of courseware is realized. Finally by collecting experimental data the performance of the system is analyzed, including the success rate of terminal receipt, broadcasting frequency, packet size and so on. The result validates the high feasibility of the system on account of the wireless transmission approach.

  6. IN VITRO GLUCURONIDATION OF APREPITANT: A MODERATE INHIBITOR OF UGT2B7

    OpenAIRE

    House, Larry; Ramirez, Jacqueline; Seminerio, Michael; Mirkov, Snezana; Ratain, Mark J.

    2015-01-01

    Aprepitant, an oral antiemetic, commonly used in the prevention of chemotherapy-induced nausea and vomiting, is primarily metabolized by CYP3A4. Aprepitant glucuronidation has yet to be evaluated in humans. The contribution of human UDP-glucuronosyltransferase (UGT) isoforms to the metabolism of aprepitant was investigated by performing kinetic studies, inhibition studies, and correlation analyses. In addition, aprepitant was evaluated as an inhibitor of UGTs.Glucuronidation of aprepitant was...

  7. Pharmacogenetic Study of Deferasirox, an Iron Chelating Agent

    OpenAIRE

    Ji Won Lee; Hyoung Jin Kang; Ji-Yeob Choi; Nam Hee Kim; Mi Kyung Jang; Chang-Woo Yeo; Sang Seop Lee; Hyery Kim; June Dong Park; Kyung Duk Park; Hee Young Shin; Jae-Gook Shin; Hyo Seop Ahn

    2013-01-01

    Transfusion-associated iron overload induces systemic toxicity. Deferasirox, a convenient long acting oral agent, has recently been introduced in clinical practice with a promising efficacy. But there are some patients who experience drug-related toxicities and cannot tolerate it. To investigate effect of genetic variations on the toxicities and find optimal target population, we analyzed the genetic polymorphisms of UDP-glucuronosyltransferase 1A (UGT1A) subfamily, multi-drug resistance-asso...

  8. Enzymic transfer of 6-modified D-galactosyl residues: synthesis of biantennary penta- and hepta-saccharides having two 6-deoxy-D-galactose residues at the nonreducing end and evaluation of 6-deoxy-D-galactosyl transfer to glycoprotein using bovine beta-(1-->4)-galactosyltransferase and UDP-6-deoxy-D-galactose.

    Science.gov (United States)

    Kajihara, Y; Endo, T; Ogasawara, H; Kodama, H; Hashimoto, H

    1995-04-19

    UDP-6-Deoxy-D-galactose and UDP-6-deoxy-6-fluoro-D-galactose were synthesized and their transfer to 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) by beta-(1-->4)-galactosyltransferase was examined. The transfer rates of 6-deoxy-D-galactose and 6-deoxy-6-fluoro-D-galactose were 1.3 and 0.2% of that of D-galactosyl transfer, respectively. The 2-acetamido-4-O-(6-deoxy-beta-D-galactopyranosyl)-2-deoxy-D-glucopyranose (6'-deoxy-N-acetyllactosamine) and methyl 2-acetamido-4-O-(6-deoxy-6-fluoro-beta-D-galactopyranosyl)-2-deoxy-D- glucopyranoside (6'-deoxy-6'-fluoro-N-acetyllactosamine) were synthesized enzymatically in 30 and 59% yields, respectively. Further, 6-deoxy-D-galactose could be completely transferred to N-linked type biantennary oligosaccharides having two N-acetyl-D-glucosaminyl residues at the nonreducing end to give the corresponding penta- and hepta-saccharides in 55 and 57% yields, respectively. An assay of 6-deoxy-D-galactosyl transfer using asialo agalacto alpha 1-acid glycoprotein as an acceptor suggested that 6-deoxy-D-galactose was transferred to about 30% of the N-acetyl-D-glucosaminyl residues in the N-linked oligosaccharides of the glycoprotein.

  9. Statin Lactonization by Uridine 5'-Diphospho-glucuronosyltransferases (UGTs)

    NARCIS (Netherlands)

    Schirris, T.J.J.; Ritschel, T.; Bilos, A.; Smeitink, J.A.M.; Russel, F.G.M.

    2015-01-01

    Statins are cholesterol-lowering drugs that have proven to be effective in lowering the risk of major cardiovascular events. Although well tolerated, statin-induced myopathies are the most common side effects. Compared to their pharmacologically active acid form, statin lactones are more potent indu

  10. 甲氧虫酰肼对舞毒蛾幼虫解毒酶及其体内蛋白质表达的影响%Effect of Methoxyfenozide on Activities of Detoxifying Enzymes and Expression of Proteins in Lymantria dispar larvae

    Institute of Scientific and Technical Information of China (English)

    廖月枝; 严善春; 曹传旺; 刘丹

    2012-01-01

    In order to study the insecticidal activity of methoxyfenozide ( RH-2485 ) against the larvae of Lymantria dispar ,the pesticide bioactivity of the chemical against the different instar larvae of L. dispar and its effect on the activities of detoxifying enzymes of the insect were assayed using leaf film method, and the expression of proteins in different tissues of the larvae were detected with SDS-PAGE. The results showed that methoxyfenozide had a high toxicity against the larvae,especially 2nd instar and 3rd instar, and the toxicity was obviously different with different larva instars. The activities of detoxifying enzymes, such as carboxylesterase ( CarE ) , MFO O-demethylase ( MFOD ) and glutathione S-transfer (GST) , in the 2nd , 4th , 6th instar larvae were significantly induced or inhibited by the methoxyfenozide treatment. The impact of methoxyfenozide on these enzymes was significantly different at different treatment time. After the 4th instar larvae were fed with methoxyfenozide, the protein expression pattern in the hemolymph ,midgut and epidermis was different from that in the control. The effect of methoxyfenozide on proteins in the hemolymph and midgut was obvious in 12 h and 24 h, whereas the effect on protein expression in epidermis tissue was more significant in 48 h. These results indicated that methoxyfenozide as a non-steroidal ecdysone had a higher biological activity against L. dispar, and the major detoxifying enzymes in the insect body were significantly interfered, showing that methoxyfenozide had a high toxic effect against L. dispar. The specific proteins were produced in the hemolymph , midgut and epidermal tissue, which might interfere with the normal physiological metabolism of insects and epidermal formation.%为研究甲氧虫酰肼(RH-2485)对舞毒蛾幼虫的杀虫活性,采用叶片药膜法测定该药剂对舞毒蛾不同龄期幼虫的生物活性及对其体内解毒酶活性的影响,并通过SDS-PAGE对舞毒

  11. Maleic acid treatment of biologically detoxified corn stover liquor

    Science.gov (United States)

    Elimination of microbial and/or enzyme inhibitors from pretreated lignocellulose is critical for effective cellulose conversion and yeast fermentation of liquid hot-water (LHW) pretreated corn stover. In this study, xylan oligomers were hydrolyzed using either maleic acid or hemicellulases. Other so...

  12. Hacia un estilo disciplinario desintoxicado (TOWARDS A DETOXIFIED DISCIPLINARY STYLE

    Directory of Open Access Journals (Sweden)

    Murillo Aguilar Osvaldo

    2009-04-01

    Full Text Available Resumen: En este ensayo se analizan desde la Orientación, los principales estilos que presentan las docentes y los docentes para el manejo de la disciplina en las aulas y los centros educativos.Para dicho análisis se utilizaron los enfoques de intervención cognitivos, de la Terapia Racional Emotivo Conductual y el Cognitivo - Conductual con el fin de exponer cuáles podrían ser algunas de las ideas o creencias centrales más notables en la gestión de la disciplina. Dichas ideas son abordadas en el ensayo como “tóxicos cognitivos” que pueden ser debatidos y modificados a fin de conseguir un mejor y más sano estilo para el manejo de la disciplina y por lo tanto un clima escolar más armónico.Por último, para las profesionales y los profesionales en Orientación puede ser un criterio más para abordar el tema en sus instituciones con sus compañeras y compañeros docentes.Abstract: This essay analyzes from a Guidance perspective the main styles used by teachers to handle discipline in classrooms and schools.Cognitive intervention approaches were used with the purpose of showing what might be the major ideas or beliefs in the management of discipline. These ideas are conceived in the article as "toxic cognitions" that can be discussed and modified to learn a better style of discipline.Lastly, for guidance counselors, it can be a resource for addressing the issue in their institutions with their fellow teachers.

  13. Current Methods to Detoxify Fly Ash from Waste Incineration

    Energy Technology Data Exchange (ETDEWEB)

    Hallgren, Christine; Stroemberg, Birgitta [TPS Termiska Processer AB, Nykoeping (Sweden)

    2004-07-01

    Fly ash from waste incineration contains large amounts of heavy metals and dioxins, which will cause a significant disposal problem within the coming years. The amount of fly ash produced in Sweden is currently approximately 60,000 tons/y. New technological options for the decontamination and/or inertization of incinerator fly ash are being developed with the objective of rendering a product that can be reused or, at least, be deposited at standard landfill sites with no risk. Many of these technologies have been tested at industrial scale or in pilot projects. The proposed alternatives include: Thermal treatments; Immobilization/stabilization by cement based techniques; Wet chemical treatments (extractions, immobilizations); Microbiological treatments. Of these, thermal treatments are the most promising solution. Depending on the temperature thermal treatments are classified in two main types: 1) low temperature (below 600 deg C) thermal treatments and 2) high temperature (above 1200 deg C) thermal treatments (vitrification). Most dioxins can be successfully destroyed at temperatures up to 400 deg C under oxygen deficient conditions and at temperatures up to 600 deg C under oxidising conditions. However most heavy metals remain in the fly ash after low temperature treatment. At a temperature of 900 deg C most heavy metals can also be removed in a 10% HCl atmosphere by forming volatile metal chlorides (CT-Fluapur process). During vitrification processes the fly ash melts and forms an inert glassy slag. The product does not leach any significant amount of heavy metals and is free from dioxin. The volume of the fly ash is significantly reduced. The product can be land filled at low costs or used as construction material. The properties of the product depend on the cooling process and on additives such as sand, limestone or waste glass. A series of vitrification methods at industrial size or in pilot scale using different furnaces are studied. Among these, plasma vitrification, electric arc and melting in a secondary combustion chamber by adding coke as a heating source (coke bed melting furnaces) or residual carbon in the fly ash (Rotary surface melting furnace) are the most common methods. In general, vitrification processes require a high-energy input and are therefore relatively cost intensive. Locking the hazardous components into the matrix by a stabilization/solidification with cement is a common alternative to decontamination. Mixing the fly ash with cement or asphalt is widely used for the reuse of fly ash from coal incineration, but it requires careful attention to any leaching of heavy metals if applied to fly ash from waste incineration. Studies by mixing fly ash with cement at concentrations from 5 to 70 % showed, that in most cases an additional pretreatment step, e.g. washing in HNO{sub 3} solution, is necessary to receive acceptable leaching behaviour and required properties as building material. Related European regulations are currently pending. On the other hand, the use of fly ash as filler for asphalt does not require any pretreatment and is already commonly applied in some countries such as the Netherlands as a well-established method. Solvent extraction methods such as acidic extraction (3R-process) or combined basic and acidic extraction (MR-process) are also designed to remove the contaminants. The effectiveness of these methods is only moderate and a further thermal treatment is required to destroy the dioxins. These methods require relatively high amounts of chemicals and wastewater management. However, they are supposed to be relatively cost effective. Other treatment options that are being tested at laboratory scale such as microbiological treatment and supercritical extraction are optimistic but have no realistic practical relevance at this state.

  14. Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins

    Directory of Open Access Journals (Sweden)

    Ilenia Siciliano

    2016-04-01

    Full Text Available Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N2, 0.1% O2 and 1% O2, 21% O2, then power (400, 700, 1000, 1150 W and exposure time (1, 2, 4, and 12 min were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min, a reduction in the concentration of total aflatoxins and AFB1 of over 70% was obtained. Aflatoxins B1 and G1 were more sensitive to plasma treatments compared to aflatoxins B2 and G2, respectively. Under plasma treatment, aflatoxin B1 was more sensitive compared to aflatoxin G1. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics.

  15. Chemical and molecular regulation of enzymes that detoxify carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Prestera, T.; Holtzclaw, W.D.; Zhang, Y., Talalay, P. (John Hopkins Univ. School of Medicine, Baltimore, MD (United States))

    1993-04-01

    Inductions of detoxication (phase 2) enzymes, such as glutathione transferases and NAD(P)H:(quinone-acceptor) oxidoreductase, are a major mechanism for protecting animals and their cells against the toxic and neoplastic effects of carcinogens. These inductions result from enhances transcription, and they are evoked by diverse chemical agents: oxidizable diphenols and phenylenediamines; Michael reaction acceptors; organic isothiocyanates; other electrophiles-e.g., alkyl and aryl halides; metal ions-e.g., HgCl[sub 2] and CdCl[sub 2]; trivalent arsenic derivatives; vicinal dimercaptans; organic hydroperoxides and hydrogen peroxide; and 1,2-dithiole-3-thiones. The molecular mechanisms of these inductions were analyzed with the help of a construct containing a 41-bp enhancer element derived from the 5[prime] upstream region of the mouse liver glutathione transferase Ya subunit gene ligated to the 5[prime] end of the isolated promoter region of this gene, and inserted into a plasmid containing a human growth hormone reporter gene. When this construct was transfected into Hep G2 human hepatoma cells, the concentrations of 28 compounds (from the above classes) required to double growth hormone production, and the concentrations required to double quinone reductase specific activities in Hepa 1c1c7 cells, spanned a range of four orders of magnitude but were closely linearly correlated. Six compounds tested were inactive in both systems. A 26-bp subregion of the above enhancer oligonucleotide (containing the two tandem [open quotes]AP-1 like[close quotes] sites but lacking the preceding ETS protein binding sequence) was considerably less responsive to the same inducers. We conclude that the 41-bp enhancer element mediates most, if not all, of the phase 2 enzyme inducer activity of all of these widely different classes of compounds. 33 refs., 4 figs., 2 tabs.

  16. Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins.

    Science.gov (United States)

    Siciliano, Ilenia; Spadaro, Davide; Prelle, Ambra; Vallauri, Dario; Cavallero, Maria Chiara; Garibaldi, Angelo; Gullino, Maria Lodovica

    2016-04-26

    Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N₂, 0.1% O₂ and 1% O₂, 21% O₂), then power (400, 700, 1000, 1150 W) and exposure time (1, 2, 4, and 12 min) were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min), a reduction in the concentration of total aflatoxins and AFB₁ of over 70% was obtained. Aflatoxins B₁ and G₁ were more sensitive to plasma treatments compared to aflatoxins B₂ and G₂, respectively. Under plasma treatment, aflatoxin B₁ was more sensitive compared to aflatoxin G₁. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics.

  17. Time to detoxify medical literature from guideline overdose

    Institute of Scientific and Technical Information of China (English)

    Dinesh Vyas; Arpita K Vyas

    2012-01-01

    The current financial turmoil in the United States has been attributed to multiple reasons including healthcare expenditure.Health care spending has increased from 5.7 percent of the gross domestic product (GDP)in 1965 to 16 percent of the GDP in 2004.Healthcare is driven with a goal to provide best possible care available at that period of time.Guidelines are generally assumed to have the high level of certainty and security as conclusions generated by the conventional scientific method leading many clinicians to use guidelines as the final arbiters of care.To provide the standard of care,physicians follow guidelines,proposed by either groups of physicians or various medical societies or government organizations like National Comprehensive Cancer Network.This has lead to multiple tests for the patient and has not survived the test of time.This independence leads to lacunae in the standardization of guidelines,hence flooding of literature with multiple guidelines and confusion to patients and physicians and eventually overtreatment,inefficiency,and patient inconvenience.There is an urgent need to restrict articles with Guidelines and develop some strategy like have an intermediate stage of pre-guidelines and after 5-10 years of trials,a systematic launch of the Guidelines.There can be better ways than this for putting together guidelines as has been suggested by multiple authors and researchers.

  18. Synthesis of a tetrasaccharide acceptor for use in the assay of UDP-GlcpNAc:beta-D-Galp-(1----4)-beta-D-GlcpNAc (GlcNAc to Gal) beta(1----3)-N-acetylglucosaminyltransferase activity and the pentasaccharide product that would be formed by its enzymic glycosylation.

    Science.gov (United States)

    Srivastava, G; Hindsgaul, O

    1992-02-07

    The tetrasaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-alpha-D- Manp-(1----6)-beta-D-Manp-OR (2) and the pentasaccharide beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp-OR (3), where R = (CH2)8COOMe, have been prepared by using combined chemical and enzymic procedures. Structure 2 is a substrate for UDP-GlcpNAc:beta-D-Galp-(1----4)- beta-D-GlcpNAc (GlcNAc to Gal) beta(1----3)-N-acetylglucosaminyltransferase, and 3 would be the product of its action. Antibodies raised against 3 are intended for use in an ELISA assay that would quantitate the enzymic conversion of immobilized 2 into 3.

  19. 益气清瘟解毒颗粒剂治疗流行性感冒的临床疗效评价%Clinical Effect of Granules for Qi-Refreshing and Pestilence-Eliminating-Detoxifying in Treatment of Influenza

    Institute of Scientific and Technical Information of China (English)

    徐红日; 程淼; 殷人易; 徐丽丽; 于会勇; 王明哲; 姜良铎; 周平安; 王成祥; 刘清泉; 王兰; 张立山; 寇兰俊; 李雁; 刘涓; 苗青; 郭楠

    2012-01-01

    目的 评价益气清瘟解毒颗粒剂治疗流行性感冒的有效性和安全性.方法 将临床收集的123例表寒里热型流行性感冒患者随机分为中药治疗组与西药对照组,中药治疗组口服益气清瘟解毒颗粒剂,西药对照组服用抗病毒药治疗.结果 益气清瘟解毒颗粒剂的退热效果优于西药对照组,中药治疗组改善流感患者的症状积分优于西药抗病毒药物,其疾病疗效、中医证候疗效及主要症状疗效均优于西药对照组,且具有抑制免疫炎性损伤的趋势.结论 益气清瘟解毒颗粒剂治疗表寒里热型流行性感冒疗效显著,安全可靠.%Objective To estimate the effect and safety of Granules for Qi - Refreshing and Pestilence - Eliminating -Detoxifying ( GQPD ) in treatment of influenza Methods Totally 123 influenza patients with the syndrome of Exterior - Cold and Interior - Heat in traditional Chinese medicine ( TCM ) were randomly divided as TCM group receiving QRPE and control group receiving antiviral drug. Results Compared with the antiviral drug, GQPD was better in antipyretic effect and improvement of influenza symptom scores, being better in effects for disease, TCM syndrome, and cardinal symptoms of influenza. And it showed a tendency to inhibit the immune - inflammatory injury. Conclusion The GQPD is effective and safe in treatment of influenza with TCM syndrome of Exterior - Cold and Interior - Heat.

  20. Attenuated Escherichia coli strains expressing the colonization factor antigen I (CFA/I) and a detoxified heat-labile enterotoxin (LThK63) enhance clearance of ETEC from the lungs of mice and protect mice from intestinal ETEC colonization and LT-induced fluid accumulation.

    Science.gov (United States)

    Byrd, Wyatt; Boedeker, Edgar C

    2013-03-15

    Although enterotoxigenic Escherichia coli (ETEC) infections are important causes of infantile and traveler's diarrhea there is no licensed vaccine available for those at-risk. Our goal is to develop a safe, live attenuated ETEC vaccine. We used an attenuated E. coli strain (O157:H7, Δ-intimin, Stx1-neg, Stx2-neg) as a vector (ZCR533) to prepare two vaccine strains, one strain expressing colonization factor antigen I (ZCR533-CFA/I) and one strain expressing CFA/I and a detoxified heat-labile enterotoxin (ZCR533-CFA/I+LThK63) to deliver ETEC antigens to mucosal sites in BALB/c mice. Following intranasal and intragastric immunization with the vaccine strains, serum IgG and IgA antibodies were measured to the CFA/I antigen, however, only serum IgG antibodies were detected to the heat-labile enterotoxin. Intranasal administration of the vaccine strains induced respiratory and intestinal antibody responses to the CFA/I and LT antigens, while intragastric administration induced only intestinal antibody responses with no respiratory antibodies detected to the CFA/I and LT antigens. Mice immunized intranasally with the vaccine strains showed enhanced clearance of wild-type (wt) ETEC bacteria from the lungs. Mice immunized intranasally and intragastrically with the vaccine strains were protected from intestinal colonization following oral challenge with ETEC wt bacteria. Mice immunized intragastrically with the ZCR533-CFA/I+LThK63 vaccine strain had less fluid accumulate in their intestine following challenge with ETEC wt bacteria or with purified LT as compared to the sham mice indicating that the immunized mice were protected from LT-induced intestinal fluid accumulation. Thus, mice intragastrically immunized with the ZCR533-CFA/I+LThK63 vaccine strain were able to effectively neutralize the activity of the LT enterotoxin. However, no difference in intestinal fluid accumulation was detected in the mice immunized intranasally with the vaccine strain as compared to the sham

  1. The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis

    DEFF Research Database (Denmark)

    Rautengarten, Carsten; Ebert, Berit; Moreno, Ignacio

    2014-01-01

    Delivery of nucleotide sugar substrates into the Golgi apparatus and endoplasmic reticulum for processes such as cell wall biosynthesis and protein glycosylation is critical for plant growth and development. Plant genomes encode large families of uncharacterized nucleotide sugar transporters......-of-function and overexpression lines for two of these transporters identified biochemical alterations supporting their roles in the biosynthesis of Rha- and Gal-containing polysaccharides. Thus, cell wall polysaccharide biosynthesis in the Golgi apparatus of plants is likely also regulated by substrate transport mechanisms....

  2. 一种作用于花青素和甜菊醇的甜菊糖基转移酶的基因克隆和功能分析%MOLECULAR CLONING AND CHARACTERIZATION OF STEVIA REBA UDIANA UDP-GLUCOSYLTRANSFERASE

    Institute of Scientific and Technical Information of China (English)

    马凌波; 张大兵; 陈亮; 陈睦传

    2003-01-01

    We repor here the cloning and characterization of a UDP-glucose flavonoid glucosyltransferase(srUFGT) in Stevia rebaudiana. The isolated cDNA was 1419bp in length encoding 473 deduced amino acidswith a predicted molecular mass of 53.2 kDa. The products of in vitro translation from an expression vectorhad anthocyanidins and steviol glucosyltransferase activity. Comparison of the activity of the recombinant UDP-glucosyltransferase toward a range of acceptor substrates suggests that it may participate in the synthesis of ste-viol glycosides. The results support the hypothesis that the flavonoid glucosyltransferases, which have a broadsubstrate specificity,may be not only involved in flavonoid glucosylation but also play a role in producing thewater-soluble steviol-glycosides in S. rebaudiana.%本文对一种新的甜菊糖基转移酶进行了基因克隆和功能分析.获得的基因cDNA全长1419bp,编码473个氨基酸,蛋白质分子量约53.2K Da.与常见的糖基转移酶基因比较,相似性达44%以上,且具有糖基转移酶的保守序列.体外异源表达获得的融合蛋白,具有在花青素类和甜菊醇等糖基受体上转糖基的酶活性.在对一系列不同底物的酶活性进行比较后,推测这种糖基转移酶在体内参与了甜菊糖苷的合成.结果表明,具有广泛的底物活性的类黄酮类糖基转移酶,在甜菊体内不仅对类黄酮转糖基,而且在生成水溶性甜菊糖苷的过程中也扮演重要的角色.

  3. The Impact of Glucuronidation on the Bioactivation and DNA Adduction of the Cooked-Food Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Malfatti, M A; Ubick, E A; Felton, J S

    2005-03-31

    UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of many different chemicals. Glucuronidation is especially important for detoxifying reactive intermediates from metabolic reactions, which otherwise can be biotransformed into highly reactive cytotoxic or carcinogenic species. Detoxification of certain food-borne carcinogenic heterocyclic amines (HAs) is highly dependent on UGT1A-mediated glucuronidation. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant carcinogenic HA found in well-done cooked meat, is extensively glucuronidated by UGT1A proteins. In humans, CYP1A2 catalyzed N-hydroxylation and subsequent UGT1A-mediated glucuronidation is a dominant pathway in the metabolism of PhIP. Therefore, changes in glucuronidation rates could significantly alter PhIP metabolism. To determine the importance of UGT1A-mediated glucuronidation in the biotransformation of PhIP, UGT1A proficient Wistar and UGT1A deficient Gunn rats were exposed to a single 100 {micro}g/kg oral dose of [{sup 14}C]-PhIP. Urine was collected over 24 h and the PhIP urinary metabolite profiles were compared between the two strains. After the 24 h exposure, livers and colon were removed and analyzed for DNA adduct formation by accelerator mass spectrometry. Wistar rats produced several PhIP and N-hydroxy-PhIP glucuronides that accounted for {approx}25% of the total amount of recovered urinary metabolites. In the Gunn rats, PhIP and N-hydroxy-PhIP glucuronides were reduced by 68-92%, compared to the Wistar rats, and comprised only 4% of the total amount of recovered urinary metabolites. PhIP-DNA adduct analysis from the Gunn rats revealed a correlation between reduced PhIP and N-hydroxy-PhIP glucuronide levels in the urine and increased hepatic DNA adducts, compared to the Wistar rats. These results indicate that UGT1A-mediated glucuronidation of PhIP and N-hydroxy-PhIP is an important pathway for PhIP detoxification. Failure to form glucuronide conjugates

  4. 尿苷二磷酸葡聚糖转移酶1A8*2在云南健康佤族、白族和藏族基因多态性研究%Genetic polymorphism of UDP-glucan transferase 1A8 * 2 in Yunnan province healthy Wa, Bai and Tibetan nationalities

    Institute of Scientific and Technical Information of China (English)

    赖泳; 黄民; 李嘉丽; 董寿堂; 李辉

    2012-01-01

    OBJECTIVE To investigate the allele and genotype frequencies of UDP-glucan transferase 1A8 * 2 in Yunnan province healthy Wa.Bai and Tibetan nationalities, and compare with other ethnics. METHODS The number of the healthy volunteers from Yunnan province Wa, Bai and Tibetan nationalities were 144,115 and 252, respectively. The PCR-RFLP method, which was verified by direct sequencing, was applied to genotype UDP-glucan transf erase 1A8 * 2. Then the genotypes frequencies of the nationalities were calculated to test whether in Hardy-Weinberg equilibrium or not. The genotypes and alleles frequencies between Yunnan province Wa,Bai and Tibetan nationalities and other ethnic groups were compared by Pearson's Chi-Square test. RESULTS The frequencies of CC, CG and GG genotypes were 16.7%, 52.8%, 30. 5% and 35.7%, 50. 4%, 13. 9%and 8. 3%, 78. 2%, 13. 5%, respectively, in Yunnan province Wa,Bai and Tibetan population. Moreover, The frequencies of allele G in Yunnan province Wa,Bai and Tibetan were 56. 9%, 39. 1% and 52. 6%, that were higher than African-Americans and German Caucasions. Furthermore, the mutation rate of Yunnan province Bai was higher than Japanese and lower than Chinese Han nationality. There were significant differences between Yunnan province Bai and Wa or Tibetan nationality about the frequencies of allele G(P<0. 01), but no difference between Yunnan province Wa and Tibetan nationality. A-bove all genotypes distribution all met Hardy-Weinberg equilibrium. CONCLUSION The UDP-glucan transf erase 1A8 * 2 gene distribution in Yunnan nationality Wa,Bai and Tibetan nationality mutation occurred in the situation has its own characteristics, Genotyping of UDP-glucan transf erase 1A8 * 2 will be helpful in guiding rational and individualized medication for individuals from Yunnan province Wa.Bai and Tibetan nationality using drugs that are substrates of UGT.%目的:了解云南佤族、白族和藏族人群中尿苷二磷酸葡聚糖转移酶1A8*2的基因多态

  5. 水稻施用硅肥对稻纵卷叶螟幼虫保护酶和解毒酶活性的影响%Effects of providing additional silicon to host plants on the activity of protective and detoxifying enzymes in Cnaphalocrocis medinalis larvae

    Institute of Scientific and Technical Information of China (English)

    韩永强; 礼章; 侯茂林

    2016-01-01

    【目的】研究施硅水稻对稻纵卷叶螟 Cnaphalocrocis medinalis(Guenée)幼虫保护酶(过氧化氢酶 CAT、过氧化物酶 POD、超氧化物歧化酶 SOD)和解毒酶(谷胱甘肽-S-转移酶 GST、乙酰胆碱酯酶AChE)活性的影响,为探明硅增强水稻抗稻纵卷叶螟的机理和稻纵卷叶螟的综合治理提供依据。【方法】采用感虫水稻品种 TN1,设置2种施硅水平,即0.32 g Si/kg 土壤(Si+)和不施硅(Si-),测定取食水稻24、48、72、96 h 后3龄幼虫体内 CAT、POD、SOD、GST、AChE 活性的动态变化。【结果】幼虫取食 Si+水稻前期(24~48 h),CAT、POD、SOD 活性呈上升趋势,并在48 h 时达到最高值;取食后期(48~96 h), CAT、POD、SOD 活性下降并在96 h 时显著低于 Si-处理。在 Si-处理中,幼虫 CAT 和 POD 活性“先降低、再升高”,SOD 活性呈一直增大的趋势。取食 Si+水稻幼虫 GST 活性始终显著高于 Si-处理,而 AChE 活性均低于 Si-水稻,并在72 h 和96 h 时显著低于 Si-水稻。处理间幼虫存活率存在显著差异,Si+水稻上1龄和3龄幼虫的存活率显著低于 Si-水稻。这些结果说明,硅处理可能使稻纵卷叶螟幼虫产生应激反应引起保护酶活性在短时间内增大,但取食一段时间后保护酶活性下降。AChE 活性受到抑制,会引起昆虫神经传导的异常反应,造成昆虫死亡。【结论】硅可能通过参与植物的生理代谢过程,诱导植物增加次生代谢物的合成和积累,引起稻纵卷叶螟保护酶和解毒酶的活性变化,可能会影响稻纵卷叶螟的存活。%[Objectives] To explore the effects of providing additional silicon (Si) to host plants on the activities of protective and detoxifying enzymes in Cnaphalocrocis medinalis larvae. Such information may provide evidence of Si-mediated resistance and improve management of insect pests in rice crops. [Methods] The

  6. Desenvolvimento e caracterização de matrizes poliméricas como veículo de componentes ativos do extrato etanólico da película de amendoim

    OpenAIRE

    2015-01-01

    A película de amendoim é um resíduo da indústria de alimentos. Esse resíduo é rico em compostos fenólicos como resveratrol e procianidinas e apresenta elevada atividade antioxidante e atividade farmacológica. Apesar de suas atividades farmacológicas, compostos fenólicos apresentam baixa biodisponibilidade devido à glucuronidação catalisada pelas enzimas UDP-glucuronosyltransferases (UGTs), que acontece na primeira passagem no intestino e/ou fígado, dificultando a utilização dos compostos fenó...

  7. 家蚕触角富集UDP葡萄糖醛酸基转移酶基因的克隆与表达分析%Cloning and Expression Analysis of an Antennal-enriched UDP glucosyltransferase Gene from Silkworm, Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    黄飞飞; 张军; 黄茂华; 王婷婷; 彭丽娜; 潘敏慧; 鲁成

    2011-01-01

    糖基化作用在抑制和排除一系列内生和外源化合物的毒性方面起重要作用,一系列尿苷二磷酸-糖基转移酶(UDPglycosyltransferases,UGTs)参与了糖基化反应过程.为了解家蚕中的UDP葡萄糖醛酸基转移酶的生理作用,克隆和表达了家蚕触角富集UDP葡萄糖醛酸基转移酶基因BmUGT013829.BmUGT013829基因cDNA全长1 545 bp,编码蛋白含有514个氨基酸残基.Western bltting检测发现BmUGT013829在家蚕脂肪体、头部、体壁和中肠组织中表达,在中肠组织的表达量低,在头部高量表达.免疫组织化学试验结果显示,BmUGT013829在头部的触角中表达水平最高.研究结果表明,BmUGT013829是一种触角富集尿苷二磷酸葡萄糖醛酸基转移酶,推测其在嗅觉中扮演着重要角色.%Glucosidation plays a major role in the inactivation and excretion of a wide variety of endogenous and exogenous compounds. This process involves a class of UDP-glycosyltransferases (UGTs). We report here the molecular cloning and expression characterization of the silkworm antennal-enriched UGT BmUG T013829. We cloned a complete 1 545 bp cDNA that encodes a 514 amino acid protein. Western blotting revealed that BmUGT013829 was expressed in the fat body, head, integument and midgut, and the protein was lowly expressed in the midgut and highly expressed in head. Immunohistochemical assay found that BmUGT013829 expression level was the highest in antennae compared to other components of the head. These patterns suggest that BmUGT013829 is an antennal-enriched UGT and may play a role in olfaction.

  8. Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain.

    Science.gov (United States)

    Montiel, Maria-Dolores; Krzewinski-Recchi, Marie-Ange; Delannoy, Philippe; Harduin-Lepers, Anne

    2003-01-01

    The nucleotide sequence of the short and long transcripts of beta1,4- N -acetylgalactosaminyltransferase have been submitted to the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under accession nos AJ517770 and AJ517771 respectively. The human Sd(a) antigen is formed through the addition of an N -acetylgalactosamine residue via a beta1,4-linkage to a sub-terminal galactose residue substituted with an alpha2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4- N -acetylgalactosaminyltransferase (Sd(a) beta1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sd(a) beta1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1(S) and E1(L), leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to alpha2,3-sialylated acceptor

  9. User Data Package (UDP) for Packaged Cogeneration Systems (PCS)

    Science.gov (United States)

    1990-05-01

    a. - s- v -W - s- s- m -v -W - s- 6 5 u u u u 3 .2 - - . - . . *-2 - I-in~~~~~~~ ~~~ Wn in0 nini n44 4 4 4 44 4 4 4 4 44...V 6- oe. V 0 N n In O ’ ’ - 0 0 -1 NNNNNNNNNN𔃺Inu~inui .2 2w -t t . -t t 0 00 0 0 0 000,1 l1 I ’ In~~~~~~~~~~~~ InI n nI n nI n nInI nIn!2 ’ nI n nI...I nIwI nI I nI<I nInI nInI 0. 0n�𔃺O0’.00’ 0 0_j_ j_ _ j w t-N m V. Iu - w x w o i4R ~ ~ ~ L 4 L1 n2a mo 0 x <W nWoQ< W w- f!2 Dw z-x<- N IT a2t

  10. Method and Apparatus for Processing UDP Data Packets

    Science.gov (United States)

    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.

  11. Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana

    National Research Council Canada - National Science Library

    Martinez, Viviana; Ingwers, Miles; Smith, James; Glushka, John; Yang, Ting; Bar-Peled, Maor

    2012-01-01

    .... Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana...

  12. Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)[W][OA

    Science.gov (United States)

    Ono, Eiichiro; Homma, Yu; Horikawa, Manabu; Kunikane-Doi, Satoshi; Imai, Haruna; Takahashi, Seiji; Kawai, Yosuke; Ishiguro, Masaji; Fukui, Yuko; Nakayama, Toru

    2010-01-01

    We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine (Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-O-glucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5 for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates, illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities. PMID:20693356

  13. Heme degradation and human disease: diversity is the soul of life.

    Science.gov (United States)

    Shibahara, Shigeki; Kitamuro, Tomomi; Takahashi, Kazuhiro

    2002-08-01

    We all depend on molecular oxygen and heme for our life, as evident from the pigments in blood and daily wastes. About 80% of serum bilirubin is derived from hemoglobin of senescent erythrocytes, which have finished their mission of 120 days and have been phagocytized by macrophages in the reticuloendothelial system. Here we present an overview of the heme degradation processes and relevant disorders by focusing on heme oxygenase-1 (HO-1), a key enzyme in heme catabolism. HO-1 cleaves the porphyrin macrocycle of heme at the expense of molecular oxygen to release a linear tetrapyrrole biliverdin, carbon monoxide, and ferrous iron; biliverdin is rapidly reduced to bilirubin. Bilirubin is transported to the liver (hepatocytes), conjugated with glucuronic acid by bilirubin UDP-glucuronosyltransferase, and excreted into bile. Genetic diversity, a strategy in the host defense, is seen in the human ho-1 and UDP-glucuronosyltransferase genes. Moreover, striking interspecies variations are noted in the regulation of HO-1 expression by hypoxia, heat shock, or interferon-gamma, each of which mainly represses HO-1 expression in human cells. Implications of such a variety are discussed in relevance to the pathogenesis of severe malaria caused by Plasmodium falciparum, the most ancient foe of humans.

  14. 多肽:N-乙酰氨基半乳糖转移酶2在SGC7901细胞中同时定位于高尔基体顺面囊和反面囊%UDP-Ga1NAc: polypeptide α-N-acetygalactosaminyltransferase 2 Localized on Both cis and trans Side of Golgi Stacks in SGC7901 Cells

    Institute of Scientific and Technical Information of China (English)

    周迎会; 杭赛宇; 仇灏; 贾伟; 徐岚; 姜智; 吴士良

    2009-01-01

    多肽:N.乙酰氨基半乳糖转移酶(ppGalNAcT)在高尔基体中催化粘蛋白型O-糖基化的第一步.首先进行了人ppGalNAcT2多克隆抗体的制备和鉴定,进一步通过对分离的亚细胞结构进行蛋白质印迹分析,免疫细胞化学后共聚焦显微镜观察此抗体和两个高尔基体标记GS28(顺面高尔基体的分子标志)和TGN38(反面高尔基体的分子标志)来研究ppGalNAcT2在SGC7901细胞株中的亚细胞定位.结果表明:约有60%的ppGalNAcT2信号和Gs28共定位,大约36%的ppGalNAcT2信号和TGN38共定位.约有34%的TGN38和ppGalNAcT2信号重叠,而约38%的反面高尔基体标志和ppGalNAcT2重叠.结论是:在SGC7901中,ppGalNAcT2同时定位于高尔基体顺面囊和反面囊中,实验证实了在高尔基体中进行粘蛋白型O-糖基化的起始反应.%Uridine diphosphate (UDP)-GalNAc : polypeptide N-acetylgalactosaminyltransfemse (ppGalNAcT) catalyzes the initial step in mucin type O-glycosylation in the Golgi apparatus. Here generation and characterization of a polyclonal antibody to human ppGalNAcT2 were described. The subcellular location of ppGalNAeT2 in SGC7901 cell line was investigated using Western blot analysis of fractionated cell extracts and confocal microscopy with this antibody and two Golgi markers: Golgi SNARE (soluble N-ethylmalemide-sensifive factor attachment protein receptor) of 28 ku (GS28) and trans-Golgi network (TGN) 38, markers for the c/s- and trans-Golgi apparatus, respectively. Morphometric analyses indicated that ~60% of the ppGalNAcT2 signal colocalized with the GS28, while~36% of the c/s-Golgi marker colocalized with the ppGalNAeT2. Approximately 34% of the ppGalNAcT2 signal colocalized with the TGN38, whereas 38% of the trans-Golgi marker colocalized with the ppGalNAcT2. The results provide unequivocal evidence for the location ofppGalNAcT2 within the Golgi apparatus, and further highlight the importance of this organelle in the initiation of O

  15. Glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes.

    Science.gov (United States)

    Yamanaka, Hiroyuki; Nakajima, Miki; Katoh, Miki; Yokoi, Tsuyoshi

    2007-09-01

    Glucuronidation of thyroxine is a major metabolic pathway facilitating its excretion. In this study, we characterized the glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes, and identified human UDP-glucuronosyltransferase (UGT) isoforms involved in the activity. Human jejunum microsomes showed a lower K(m) value (24.2 microM) than human liver (85.9 microM) and kidney (53.3 microM) microsomes did. Human kidney microsomes showed a lower V(max) value (22.6 pmol/min/mg) than human liver (133.4 pmol/min/mg) and jejunum (184.6 pmol/min/mg) microsomes did. By scaling-up, the in vivo clearances in liver, intestine, and kidney were estimated to be 1440, 702, and 79 microl/min/kg body weight, respectively. Recombinant human UGT1A8 (108.7 pmol/min/unit), UGT1A3 (91.6 pmol/min/unit), and UGT1A10 (47.3 pmol/min/unit) showed high, and UGT1A1 (26.0 pmol/min/unit) showed moderate thyroxine glucuronosyltransferase activity. The thyroxine glucuronosyltransferase activity in microsomes from 12 human livers was significantly correlated with bilirubin O-glucuronosyltransferase (r = 0.855, p microsomes was mainly catalyzed by UGT1A8 and UGT1A10 and to a lesser extent by UGT1A1, and the activity in human kidney microsomes was mainly catalyzed by UGT1A7, UGT1A9, and UGT1A10. The changes of activities of these UGT1A isoforms via inhibition and induction by administered drugs as well as genetic polymorphisms may be a causal factor of interindividual differences in the plasma thyroxine concentration.

  16. Process for removing and detoxifying cadmium from scrap metal including mixed waste

    Energy Technology Data Exchange (ETDEWEB)

    Kronberg, J.W.

    1994-07-01

    Cadmium-bearing scrap from nuclear applications, such as neutron shielding and reactor control and safety rods, must usually be handled as mixed waste since it is radioactive and the cadmium in it is both leachable and highly toxic. Removing the cadmium from this scrap, and converting it to a nonleachable and minimally radioactive form, would greatly simplify disposal or recycling. A process now under development will do this by shredding the scrap; leaching it with reagents which selectively dissolve out the cadmium; reprecipitating the cadmium as its highly insoluble sulfide; then fusing the sulfide into a glassy matrix to bring its leachability below EPA limits before disposal. Alternatively, the cadmium may be recovered for reuse. A particular advantage of the process is that all reagents (except the glass frit) can easily be recovered and reused in a nearly closed cycle, minimizing the risk of radioactive release. The process does not harm common metals such as aluminum, iron and stainless steel, and is also applicable to non-nuclear cadmium-bearing scrap such as nickel-cadmium batteries.

  17. Modified natural clinoptilolite detoxifies small mammal's organism loaded with lead II: genetic, cell, and physiological effects.

    Science.gov (United States)

    Topashka-Ancheva, Margarita; Beltcheva, Michaela; Metcheva, Roumiana; Rojas, J Antonio Heredia; Rodriguez-De la Fuente, Abraham O; Gerasimova, Tsvetelina; Rodríguez-Flores, Laura E; Teodorova, Svetla E

    2012-06-01

    The detoxification capacity of the clinoptilolite modification KLS-10-MA used as food additive in small mammals, chronically lead-exposed, was proven for the first time. The modified clinoptilolite was prepared based on natural Bulgarian clinoptilolite deposits. As a powder, it was mechanically mixed at 12.5% concentration with the conventional forage for small rodents. Lead in the form of aqueous solution of Pb(NO(3))(2) was diluted in the drinking water. In the ecotoxicological experiment covering 90 days, imprinting control region laboratory mice were used. They were allocated into four groups: group 1, (control): animals fed with conventional food for small rodents and water; group 2: animals fed with conventional food + clinosorbent KLS-10-MA and water; group 3: animals fed with conventional food and water + Pb(NO(3))(2); and group 4: animals fed with conventional food + KLS-10-MA and water + Pb(NO(3))(2). A group of non-exposed healthy animals was fed with conventional forage mixed with KLS-10-MA to prove eventual toxicity of the sorbent and influence on growth performance. The changes in the chromosome structure, mitotic index, erythrocyte form, erythropoiesis, and body weight gain were recorded. On day 90, the following relations were established: Pb-exposed and clinoptilolite-supplemented mice exhibited 2.3-fold lower chromosome aberrations frequency, 2.5-fold higher mitotic index, and 1.5-fold higher percentage normal erythrocytes 1.3-fold higher body weight compared to Pb-exposed and unsupplemented animals. The obtained data showed that the sorbent is practically non-toxic. The results of the present study encourage a further elaboration of a reliable drug based on the tested substance in the cases of chronic lead intoxication.

  18. Ethanol production from the enzymatic hydrolysis of non-detoxified steam-exploded corn stalk.

    Science.gov (United States)

    Yang, Xiushan; Zhang, Sijin; Zuo, Zhuang; Men, Xun; Tian, Shen

    2011-09-01

    To reduce water consumption and equipment investment, and simplify the technological process, a Pichia stipitis-adapted strain with improved tolerance against inhibitors and ethanol was used in ethanol production. The steam-exploded corn stalk was directly enzymatically hydrolyzed without detoxification, and then the enzymatic hydrolysate was used as the fermentation substrate. Results from laboratory experiments in shake flasks and fermentation tanks indicated that, after fermentation for 48 h, ethanol concentration reached to 43.42 g/L; the ethanol yield was 0.47 g(p)/g(s), which was 92.16% of the theoretical ethanol yield. The results of the present research demonstrated that the application of this strain avoided detoxification of the steam-pretreated material through washing, thus simplifying the technological process. In addition, the application of the adapted strain reduced water consumption and lowered the equipment investment of ethanol production from corn stalk, which are important factors in further promotion of the development of ethanol production from straw.

  19. Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

    Directory of Open Access Journals (Sweden)

    S.G. Fonseca

    1997-07-01

    Full Text Available The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 µg of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 µg was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice

  20. Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

    OpenAIRE

    1997-01-01

    The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immuni...

  1. Dynamic evolution of the LPS-detoxifying enzyme intestinal alkaline phosphatase in zebrafish and other vertebrates

    Directory of Open Access Journals (Sweden)

    Ye eYang

    2012-10-01

    Full Text Available Alkaline phosphatases (Alps are well-studied enzymes that remove phosphates from a variety of substrates. Alps function in diverse biological processes, including modulating host-bacterial interactions by dephosphorylating the Gram-negative bacterial cell wall component lipopolysaccharide (LPS. In animals, Alps are encoded by multiple genes characterized by either ubiquitous expression (named Alpls, for their liver expression, or their tissue-specific expression, for example in the intestine (Alpi. We previously characterized a zebrafish alpi gene (renamed here alpi.1 that is regulated by Myd88-dependent innate immune signaling and that is required to prevent a host’s excessive inflammatory reactions to its resident microbiota. Here we report the characterization of two new alp genes in zebrafish, alpi.2 and alp3. To understand their origins, we investigated the phylogenetic history of Alp genes in animals. We find that vertebrate Alp genes are organized in three clades with one of these clades missing from the mammals. We present evidence that these three clades originated during the two vertebrate genome duplications. We show that in zebrafish alpl is ubiquitously expressed, as it is in mammals, whereas the other three alps are specific to the intestine. Our phylogenetic analysis reveals that in contrast to Alpl, which has been stably maintained as a single gene throughout the vertebrates, the Alpis have been lost and duplicated multiple times independently in vertebrate lineages, likely reflecting the rapid and dynamic evolution of vertebrate gut morphologies, driven by changes in bacterial associations and diet.

  2. Catalase and ascorbate peroxidase-representative H2O2-detoxifying heme enzymes in plants.

    Science.gov (United States)

    Anjum, Naser A; Sharma, Pallavi; Gill, Sarvajeet S; Hasanuzzaman, Mirza; Khan, Ekhlaque A; Kachhap, Kiran; Mohamed, Amal A; Thangavel, Palaniswamy; Devi, Gurumayum Devmanjuri; Vasudhevan, Palanisamy; Sofo, Adriano; Khan, Nafees A; Misra, Amarendra Narayan; Lukatkin, Alexander S; Singh, Harminder Pal; Pereira, Eduarda; Tuteja, Narendra

    2016-10-01

    Plants have to counteract unavoidable stress-caused anomalies such as oxidative stress to sustain their lives and serve heterotrophic organisms including humans. Among major enzymatic antioxidants, catalase (CAT; EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) are representative heme enzymes meant for metabolizing stress-provoked reactive oxygen species (ROS; such as H2O2) and controlling their potential impacts on cellular metabolism and functions. CAT mainly occurs in peroxisomes and catalyzes the dismutation reaction without requiring any reductant; whereas, APX has a higher affinity for H2O2 and utilizes ascorbate (AsA) as specific electron donor for the reduction of H2O2 into H2O in organelles including chloroplasts, cytosol, mitochondria, and peroxisomes. Literature is extensive on the glutathione-associated H2O2-metabolizing systems in plants. However, discussion is meager or scattered in the literature available on the biochemical and genomic characterization as well as techniques for the assays of CAT and APX and their modulation in plants under abiotic stresses. This paper aims (a) to introduce oxidative stress-causative factors and highlights their relationship with abiotic stresses in plants; (b) to overview structure, occurrence, and significance of CAT and APX in plants;

  3. Tolerant yeast in situ detoxifies major class of toxic chemicals while producing ethanol

    Science.gov (United States)

    Renewable lignocellulosic materials contain abundant sugar source and biofuels conversion including cellulosic ethanol production from lignocellulosic biomass provides a sustainable alternative energy resource for a cleaner environment. In order to release the biomass sugars from the complex cellulo...

  4. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

    Science.gov (United States)

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P N; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino; Ferlenghi, Ilaria; Bagnoli, Fabio

    2016-06-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.

  5. Arbuscular mycorrhiza detoxifying response against arsenic and pathogenic fungus in soybean.

    Science.gov (United States)

    Spagnoletti, Federico N; Balestrasse, Karina; Lavado, Raúl S; Giacometti, Romina

    2016-11-01

    Uptake of Arsenic (As) in plant tissues can affect metabolism, causing physiological disorders, even death. As toxicity, but also pathogen infections trigger a generalised stress response called oxidative stress; however knowledge on the response of soybean (Glycine max L.) under multiple stressors is limited so far. Arbuscular mycorrhizal fungi (AMF) enhance the tolerance of host plants to abiotic and biotic stress. Thus, we investigated the effects of the AMF Rhizophagus intraradices on soybean grown in As-contaminated soils as well as in the presence of the pathogen Macrophomina phaseolina (charcoal rot of the stem). Plant parameters and degree of mycorrhizal colonization under the different assessed treatments were analyzed. Content of As in roots and leaves was quantified. Increasing As level in the soil stopped plant growth, but promoted plant As uptake. Inoculation of soybean plants with M. phaseolina accentuated As effect at all physiological levels. In the presence of mycorrhizal symbiosis biomass dramatically increased, and significantly reduced the As concentration in plant tissues. Mycorrhization decreased oxidative damage in the presence of both As and the pathogen. Furthermore, transcription analysis revealed that the high-affinity phosphate transporter from R. intraradices RiPT and the gene encoding a putative arsenic efflux pump RiArsA were up-regulated under higher As doses. These results suggest that R. intraradices is most likely to get involved in the defense response against M. phaseolina, but also in the reduction of arsenate to arsenite as a possible detoxification mechanism in AMF associations in soybean. R. intraradices actively participates in the soybean antioxidant defense response against arsenic stress and M. phaseolina infection. Copyright © 2016. Published by Elsevier Inc.

  6. Isothiocyanates may chemically detoxify mutagenic amines formed in heat processed meat.

    Science.gov (United States)

    Lewandowska, Anna; Przychodzeń, Witold; Kusznierewicz, Barbara; Kołodziejski, Dominik; Namieśnik, Jacek; Bartoszek, Agnieszka

    2014-08-15

    Meat consumption represents a dietary risk factor increasing the incidence of common cancers, probably due to carcinogenic amines (HAAs) formed upon meat heating. Interestingly, cancers whose incidence is increased by meat consumption, are decreased in populations consuming brassica vegetables regularly. This inverse correlation is attributed to brassica anticarcinogenic components, especially isothiocyanates (ITCs) that stimulate detoxification of food carcinogens. However, ITC reactivity towards amines generating stable thioureas, may also decrease mutagenicity of processed meat. We confirmed here that combining meat with cabbage (fresh or lyophilized), in proportions found in culinary recipes, limited by 17-20% formation of HAAs and significantly lowered mutagenic activity of fried burgers. Moreover, MeIQx mutagenicity was lowered in the presence of ITCs, as well as for synthetic ITC-MeIQx conjugates. This suggests that formation of thioureas could lead to chemical detoxification of food carcinogens, reducing the cancer risk associated with meat consumption.

  7. Bioprospecting for TRI101 in Fusarium: Searching for a Better Enzyme to Detoxify Deoxynivalenol (DON)

    Science.gov (United States)

    The mycotoxin deoxynivalenol (DON) is a common contaminant of wheat and barley in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. Previous research has shown the value of an enzyme (TRI101) to modify DON and reduce its toxicity. Recent work by...

  8. Genetic polymorphisms of smoking-related carcinogen detoxifying enzymes and head and neck cancer susceptibility.

    NARCIS (Netherlands)

    Lacko, M.; Oude Ophuis, M.B.; Peters, W.H.M.; Manni, J.J.

    2009-01-01

    Smoking and the consumption of alcohol are the main risk factors for head and neck cancer. However, interindividual variation in the activity of enzymes involved in the detoxification of tobacco smoke (pro)carcinogens, such as microsomal epoxide hydrolase (mEH), glutathione-S-transferases (GSTs) and

  9. Intraspecific variation among Tetranychid mites for ability to detoxify and to induce plant defenses.

    Science.gov (United States)

    Ozawa, Rika; Endo, Hiroki; Iijima, Mei; Sugimoto, Koichi; Takabayashi, Junji; Gotoh, Tetsuo; Arimura, Gen-Ichiro

    2017-02-27

    Two genotypes coexist among Kanzawa spider mites, one of which causes red scars and the other of which causes white scars on leaves, and they elicit different defense responses in host plants. Based on RNA-Seq analysis, we revealed here that the expression levels of genes involved in the detoxification system were higher in Red strains than White strains. The corresponding enzyme activities as well as performances for acaricide resistance and host adaptation toward Laminaceae were also higher in Red strains than White strains, indicating that Red strains were superior in trait(s) of the detox system. In subsequent generations of strains that had survived exposure to fenpyroximate, both strains showed similar resistance to this acaricide, as well as similar detoxification activities. The endogenous levels of salicylic acid and jasmonic acid were increased similarly in bean leaves damaged by original Red strains and their subsequent generations that inherited high detox activity. Jasmonic acid levels were increased in leaves damaged by original White strains, but not by their subsequent generations that inherited high detox activity. Together, these data suggest the existence of intraspecific variation - at least within White strains - with respect to their capacity to withstand acaricides and host plant defenses.

  10. Photosensitive dyes and self-detoxifying textiles: Degradation products and dye durability

    Science.gov (United States)

    Brewer, S. A.; Artiles, C. Perdomo; Taylor, J. A.; Dennis, M.

    2010-01-01

    The photochemical destruction of 2-(phenylthio)ethanol, a benign model for the toxic chemical, sulphur mustard, was investigated in both aqueous solution, and on a textile substrate. In both cases the first formed product was the sulphoxide, 2-(phenylsulphinyl)ethanol. Increasing the concentration of sensitiser did not necessarily lead to an increase in the rate of destruction of sulphide; which is attributed to the self-quenching of the reaction in the presence of higher concentrations of Rose Bengal. The oxidation of sulphide was more efficient on nylon fabric that had been dyed with Rose Bengal, than in aqueous solution; however, a significant quantity of sulphone was also formed on the fabric. The dyed fabric could be used repeatedly to destroy the model sulphide, although the Rose Bengal itself was gradually destroyed, but at a much slower rate than the model sulphide. The ability for the fabric dyed with Rose Bengal to destroy a biological organism was also demonstrated.

  11. Characterization and Digestibility of Detoxified Castor Oil Meal for Japanese Quails

    Directory of Open Access Journals (Sweden)

    PA dos Santos

    2015-12-01

    Full Text Available ABSTRACT These experiments were performed to determine the chemical composition, coefficients of nutrient and energy metabolizability, amino acid composition, and cytotoxicity of different castor oil meals subjected to different detoxification processes and added to the diet of Japanese quails. In the trial, 180 46-d-old female Japanese quails were distributed according to a completely randomized design into five treatments and with replicates of six bird each. The treatments consisted of following detoxification methods of castor oil meal: Castor oil meal A (CMA - recovery in alcohol at 80 °C for 20 minutes and drying at 80 °C; castor oil meal B (CMB and C (CMC - recovery in alcohol at 80 °C for 6 minutes, neutralization with 5% NaOH, and drying under direct sunlight sun for two days (CMB or pelleted (CMC; castor oil meal D (CMD - recovery in alcohol at 110 °C for 15 minutes and drying at 110 °C. Castor oil meal was added replacing 20% of the reference diet. There was slight chemical composition variation (1.21% in crude protein, 6% in dry matter, 2.2% in ether extract and 64 kcal/kg in gross energy among the castor oil meals submitted to the different treatments. The castor oil meal submitted to treatment C showed the highest amino acid values. In the cytotoxicity test, treatment D presented lower ricin activity. Castor oil meals A, C, and D may be included in Japanese quail diets; however, castor oil meal D is recommended due to the simplicity its industrial process, its low toxicity, and metabolizability coefficients obtained.

  12. Characterization of an acetyltransferase that detoxifies aromatic chemicals in Legionella pneumophila

    DEFF Research Database (Denmark)

    Kubiak, Xavier Jean Philippe; Dervins-Ravault, Delphine; Pluvinage, Benjamin;

    2012-01-01

    Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires' disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive in these e......Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires' disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive...

  13. Localized proton magnetic resonance spectroscopy of the cerebellum in detoxifying alcoholics.

    Science.gov (United States)

    Seitz, D; Widmann, U; Seeger, U; Nägele, T; Klose, U; Mann, K; Grodd, W

    1999-01-01

    An increased daily alcohol consumption results in neurological symptoms and morphological central nervous system changes, e.g. shrinkage of the frontal lobes and the cerebellar vermis. Brain shrinkage can be due to neuronal loss, gliosis, or alterations of (cell) membrane constitutes/myelin. Neuronal, glial, and metabolic changes can be measured in vivo with proton magnetic resonance spectroscopy. A total of 11 alcoholics and 10 age-matched volunteers were examined by magnetic resonance imaging and localized magnetic resonance spectroscopy at an echo time of 135 and 5 msec. Peak integral values were calculated for N-acetylaspartate (NAA), choline (Cho), myo-inositol (ml), glutamate/glutamine (Glx), and normalized to phosphocreatine/creatine (Cr). Patients had a significant shrinkage of the cerebellar vermis. NAA/Cr and Cho/Cr ratios were reduced in both sequences, but the NAA/Cr reduction was only significant in long echo time, although the Cho/Cr reduction was significant in short echo time. The ml/Cr and Glx/Cr ratios did not show any significant difference between volunteers and patients. The decrease of NAA/Cr in alcohol dependent patients is consistent with neuronal loss. The Cho/Cr decrease and an unchanged ml/Cr may reflect cell membrane modification or myelin alterations in alcohol-dependent patients. These changes lead to brain shrinkage, although hydration effects and gliosis are less likely.

  14. Neuropsychological Impairment in Detoxified Alcohol-Dependent Subjects with Preserved Psychosocial Functioning

    Directory of Open Access Journals (Sweden)

    Catherine Martelli

    2017-09-01

    Full Text Available BackgroundChronic alcoholism and its related cognitive impairments are associated with increased social, relational, and professional deficits which have a variable overall impact on social integration. These impairments are known to have varying severities and have rarely been studied among healthy alcohol-dependent subjects with preserved psychosocial functioning. Thus, the objective of this study is to describe neuropsychological performance in this particular population.MethodTwenty-nine socially adjusted alcohol-dependent men, hospitalized for a first or second withdrawal and abstinent for 3 weeks minimum, were compared to 29 healthy non-alcoholic controls. All subjects underwent clinical and psychiatric examination, neuropsychological tests of memory (M, working memory (WM, and executive functions (EF. Comparisons were performed using Student’s t-tests or Mann–Whitney U tests.ResultsNo group differences were found on the Self-Reported Social Adjustment Scale (SAS-SR or in the Mini-Mental State Examination. Compared to controls, patients had greater episodic, spatial, and WM deficits as well as slightly altered executive functions. In contrast, their executive functions (spontaneous flexibility, criteria generation, rule maintenance, and inhibitory control were relatively preserved.ConclusionOur sample of socially and professionally integrated alcoholic patients shows fewer cognitive deficits than described in previous studies. Our results suggest that early on, alcohol-dependent subjects develop compensatory adaptation processes to preserve social function and adaptation. Minor cognitive impairments should be screened early in the disease to integrate cognitive interventions into the health-care plan to thus eventually prevent further socio-professional marginalization.

  15. Carbonic anhydrase: a key regulatory and detoxifying enzyme for Karst plants.

    Science.gov (United States)

    Müller, Werner E G; Qiang, Li; Schröder, Heinz C; Hönig, Natalie; Yuan, Daoxian; Grebenjuk, Vlad A; Mussino, Francesca; Giovine, Marco; Wang, Xiaohong

    2014-01-01

    Karstification is a rapid process during which calcidic stones/limestones undergo dissolution with the consequence of a desertification of karst regions. A slow-down of those dissolution processes of Ca-carbonate can be approached by a reforestation program using karst-resistant plants that can resist alkaline pH and higher bicarbonate (HCO₃⁻) concentrations in the soil. Carbonic anhydrases (CA) are enzymes that mediate a rapid and reversible interconversion of CO₂ and HCO₃⁻. In the present study, the steady-state expression of a CA gene, encoding for the plant carbonic anhydrase from the parsley Petroselinum crispum, is monitored. The studies were primarily been performed during germination of the seeds up to the 12/14-day-old embryos. The CA cDNA was cloned. Quantitative polymerase chain reaction (qPCR) analysis revealed that the gene expression level of the P. crispum CA is strongly and significantly affected at more alkaline pH in the growth medium (pH 8.3). This abolishing effect is counteracted both by addition of HCO₃⁻ and by addition of polyphosphate (polyP) to the culture medium. In response to polyP, the increased pH in the vacuoles of the growing plants is normalized. The effect of polyP let us to propose that this polymer acts as a buffer system that facilitates the adjustment of the pH in the cytoplasm. In addition, it is proposed that polyP has the potential to act, especially in the karst, as a fertilizer that allows the karstic plants to cope with the adverse pH and HCO₃⁻ condition in the soil.

  16. Decreased striatal and enhanced thalamic dopaminergic responsivity in detoxified cocaine abusers

    Energy Technology Data Exchange (ETDEWEB)

    Volkow, N.D.; Wang, G.J.; Fowler, J.S. [Brookhaven National Lab., Stony Brook, NY (United States)] [and others

    1997-05-01

    It has been hypothesized that cocaine addiction could result from decreased brain dopamine (DA) function. However, little is known about changes in (DA) neurotransmission in human cocaine addiction. We used PET and [C-11]raclopride, a DA D2 receptor ligand sensitive to competition with endogenous DA, to measure relative changes in extracellular DA induced by methylphenidate (MP) in 20 cocaine abusers (3-6 weeks after cocaine discontinuation) and 23 controls. MP did not affect the transport of [C-11]raclopride from blood to brain (K1); however it induced a significant reduction in DA D2 receptor availability (Bmax/Kd) in striatum. The magnitude of ND-induced changes in striatal [C-11]raclopride binding were significantly larger in controls (21 + 13% change from baseline) than in cocaine abusers (9 {+-} 13 %) (ANOVA p < 0.005). In cocaine abusers, but not in controls, MP also decreased Bmax/Kd values in thalamus (29 {+-} 35 %) (ANOVA p < 0.005). There were no differences in plasma MP concentration between the groups. In striatum MP-induced changes in Bmax/Kd were significantly correlated with MP-induced changes in self reports of restlessness (r = 0.49, df 42, p < 0.002). In thalamus MP-induced changes in Bmax/Kd were significantly correlated with ND-induced changes in self reports of cocaine craving (r = 0.57, df 42, p < 0.0001). These results are compatible with a decrease in striatal DA brain function in cocaine abusers. They also suggest a participation of thalamic DA pathways in cocaine addiction.

  17. Biotransformation in Egyptian spiny mouse Acomys cahirinus.

    Science.gov (United States)

    Watkins, J B; LaFollette, J W; Sanders, R A

    1995-01-01

    The activities of several representative biotransformation enzymes were determined in male and female spiny mouse tissues. Cytochrome P450 monooxygenase activity toward benzo(a)pyrene was significantly greater in female spiny mouse intestine than in males. Activity toward benzphetamine in both sexes was high in the liver, with little activity in the kidney and intestine. Sulfotransferase activity was high in kidney and intestine of female spiny mice but undetectable in the same tissues in males. Hepatic glutathione S-transferase activity towards 1-chloro-2,4-dinitrobenzene in females was significantly higher than in males. UDP-Glucuronosyltransferase activity toward 1-naphthol in both sexes in the kidney was significantly higher than hepatic and intestinal activity. Intestinal N-acetyltransferase activity towards 2-aminofluorene and beta-naphthylamine was significantly greater in females than males. No consistent relation appeared to exist between biotransformation activities in spiny mouse and those in other related rodent species.

  18. Comparison of metabolism of sesamin and episesamin by drug-metabolizing enzymes in human liver.

    Science.gov (United States)

    Yasuda, Kaori; Ikushiro, Shinichi; Wakayama, Shuto; Itoh, Toshimasa; Yamamoto, Keiko; Kamakura, Masaki; Munetsuna, Eiji; Ohta, Miho; Sakaki, Toshiyuki

    2012-10-01

    Sesamin and episesamin are two epimeric lignans that are found in refined sesame oil. Commercially available sesamin supplements contain both sesamin and episesamin at an approximate 1:1 ratio. Our previous study clarified the sequential metabolism of sesamin by cytochrome P450 (P450) and UDP-glucuronosyltransferase in human liver. In addition, we revealed that sesamin caused a mechanism-based inhibition (MBI) of CYP2C9, the P450 enzyme responsible for sesamin monocatecholization. In the present study, we compared the metabolism and the MBI of episesamin with those of sesamin. Episesamin was first metabolized to the two epimers of monocatechol, S- and R-monocatechols in human liver microsomes. The P450 enzymes responsible for S- and R-monocatechol formation were CYP2C9 and CYP1A2, respectively. The contribution of CYP2C9 was much larger than that of CYP1A2 in sesamin metabolism, whereas the contribution of CYP2C9 was almost equal to that of CYP1A2 in episesamin metabolism. Docking of episesamin to the active site of CYP1A2 explained the stereoselectivity in CYP1A2-dependent episesamin monocatecholization. Similar to sesamin, the episesamin S- and R-monocatechols were further metabolized to dicatechol, glucuronide, and methylate metabolites in human liver; however, the contribution of each reaction was significantly different between sesamin and episesamin. The liver microsomes from CYP2C19 ultra-rapid metabolizers showed a significant amount of episesamin dicatechol. In this study, we have revealed significantly different metabolism by P450, UDP-glucuronosyltransferase, and catechol-O-methyltransferase for sesamin and episesamin, resulting in different biological effects.

  19. Modified natural clinoptilolite detoxifies small mammal's organism loaded with lead I. Lead disposition and kinetic model for lead bioaccumulation.

    Science.gov (United States)

    Beltcheva, Michaela; Metcheva, Roumiana; Popov, Nikolay; Teodorova, Svetla E; Heredia-Rojas, J Antonio; Rodríguez-de la Fuente, Abraham O; Rodríguez-Flores, Laura E; Topashka-Ancheva, Margarita

    2012-06-01

    Zeolites, especially clinoptilolites, have wide application in removing heavy metals from different solutions and wastewater. The detoxification capacity of the clinoptilolite sorbent KLS-10-MA, a modified natural Bulgarian zeolite, applied as a food supplement in conditions of an ecotoxicological experiment with conventional food and lead was demonstrated for the first time. Laboratory mice, inbred imprinting control region strain, were used in a 90-day ecotoxicological experiment. Animals were divided into four experimental groups. Lead bioaccumulations in exposed and non-supplemented/supplemented with KLS-10-MA animals were compared. As additional control, healthy animals non-exposed to Pb were fed with conventional forage mixed with 12.5% KLS-10-MA. The dietary inclusion of the sorbent reduced Pb concentrations in exposed and supplemented mice by 84%, 89%, 91%, 77%, and 88% in carcass, liver, kidneys, bones, and feces, respectively. A mathematical model was proposed to outline the common trends of bone Pb bioaccumulation in exposed and non-supplemented/supplemented animals. Characteristic parameters of the kinetics of Pb concentrations were determined. Based on the model, the coefficient of absorption of Pb by gastrointestinal mucosa in the supplemented mice was found-η = 3.53% (versus η = 15% in non-supplemented ones). The present study clearly indicates that there is a realistic perspective to create a new drug based on modified natural clinoptilolites in cases of chronic heavy metal intoxication, without negatively affecting the environment.

  20. Mechanisms to Detoxify Selected Organic Contaminants in Higher Plants and Microbes, and Their Potential Use in Landscape Management

    Science.gov (United States)

    2004-10-01

    et al. 2001). Type I, present in animals, plants and a number of microorganisms (e.g. strains of Bacillus, Staphylococcus, Actinomycetes , Pseudomonas...of 260 mg kg-1 Kassel et al., 2002 Soil green alga Chlamidomonas reinhardtii Marine green alga Dunaliella tertiolecta Algae able to absorb and