Comparison of inhibition capability of scutellarein and scutellarin towards important liver UDP-glucuronosyltransferase (UGT) isoforms.

PubMed

Ma, Guang-You; Cao, Yun-Feng; Hu, Cui-Min; Fang, Zhong-Ze; Sun, Xiao-Yu; Hong, Mo; Zhu, Zhi-Tu

2014-03-01

Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand-Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP-glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (Ki ) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the Ki values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro-in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components. Copyright © 2013 John Wiley & Sons, Ltd.

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

PubMed

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  < 0.1, low possibility; 0.1 < [I]/Ki  < 1, medium possibility; [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. Copyright © 2016 John Wiley & Sons, Ltd.

Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups

PubMed Central

Murphy, Sharon E.; Park, Sung-Shim L.; Thompson, Elizabeth F.; Wilkens, Lynne R.; Patel, Yesha; Stram, Daniel O.; Le Marchand, Loic

2014-01-01

Nicotine metabolism influences smoking behavior and differences in metabolism probably contribute to ethnic variability in lung cancer risk. We report here on the proportion of nicotine metabolism by cytochrome P450 2A6-catalyzed C-oxidation, UDP-glucuronosyl transferase 2B10 (UGT2B10)-catalyzed N-glucuronidation and flavin monooxygenase 3-catalyzed N-oxidation in five ethnic/racial groups and the role of UGT2B10 genotype on the metabolic patterns observed. Nicotine and its metabolites were quantified in urine from African American (AA, n = 364), Native Hawaiian (NH, n = 311), White (n = 437), Latino (LA, n = 453) and Japanese American (JA, n = 674) smokers. Total nicotine equivalents, the sum of nicotine and six metabolites, and nicotine metabolism phenotypes were calculated. The relationship of UGT2B10 genotype to nicotine metabolic pathways was determined for each group; geometric means were computed and adjusted for age, sex, creatinine, and body mass index. Nicotine metabolism patterns were unique across the groups, C-oxidation was lowest in JA and NH (P < 0.0001), and N-glucuronidation lowest in AA (P < 0.0001). There was no difference in C-oxidation among Whites and AA and LA. Nicotine and cotinine glucuronide ratios were 2- and 3-fold lower in AA compared with Whites. Two UGT variants, a missense mutation (Asp67Tyr, rs61750900) and a splice variant (rs116294140) accounted for 33% of the variation in glucuronidation. In AA, the splice variant accounted for the majority of the reduced nicotine glucuronidation. UGT2B10 variant allele carriers had increased levels of C-oxidation (P = 0.0099). Our data indicate that the relative importance of nicotine metabolic pathways varies by ethnicity, and all pathways should be considered when characterizing the role of nicotine metabolism on smoking behavior and cancer risk. PMID:25233931

Prolonged neutropenia after irinotecan-based chemotherapy in a child with polymorphisms of UGT1A1 and SLCO1B1.

PubMed

Sakaguchi, S; Garcia-Bournissen, F; Kim, R; Schwarz, U I; Nathan, P C; Ito, S

2009-12-01

Genetic polymorphisms of uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), and SLCO1B1 coding organic anion-transporter polypeptide 1B1, are independent risk factors known to increase irinotecan toxicity in adults. Although combined occurrence of polymorphisms in these 2 genes is likely to influence susceptibility to irinotecan toxicity, data are scarce, especially in children. We report an 11-year-old female with severe and prolonged neutropenia after irinotecan-based chemotherapy. The patient's genotyping revealed polymorphisms in both UGT1A1 and SLCO1B1. To our knowledge, this is the first case report of combined genotyping of both UGT1A1 and SLCO1B1 in a child with severe irinotecan toxicity.

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

PubMed

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

2013-09-01

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

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

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

Grant, D; Hall, I J; Eastmond, D

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 genotypemore » 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

Bisphenol-A glucuronidation in human liver and breast: identification of UDP-glucuronosyltransferases (UGTs) and influence of genetic polymorphisms.

PubMed

Street, Christina M; Zhu, Zhaohui; Finel, Moshe; Court, Michael H

2017-01-01

1. Bisphenol-A is a ubiquitous environmental contaminant that is primarily metabolized by glucuronidation and associated with various human diseases including breast cancer. Here we identified UDP-glucuronosyltransferases (UGTs) and genetic polymorphisms responsible for interindividual variability in bisphenol-A glucuronidation in human liver and breast. 2. Hepatic UGTs showing the highest bisphenol-A glucuronidation activity included UGT2B15 and UGT1A9. Relative activity factor normalization indicated that UGT2B15 contributes >80% of activity at bisphenol-A concentrations under 5 μM, while UGT1A9 contributes up to 50% of activity at higher concentrations. 3. Bisphenol-A glucuronidation by liver microsomes (46 donors) ranged from 0.25 to 4.3 nmoles/min/mg protein. Two-fold higher glucuronidation (p = 0.018) was observed in UGT1A9 *22/*22 livers compared with *1/*1 and *1/*22 livers. However, no associations were observed for UGT2B15*2 or UGT1A1*28 genotypes. 4. Bisphenol-A glucuronidation by breast microsomes (15 donors) ranged from <0.2 to 56 fmoles/min/mg protein. Breast mRNA expression of UGTs capable of glucuronidating bisphenol-A was highest for UGT1A1, followed by UGT2B4, UGT1A9, UGT1A10, UGT2B7 and UGT2B15. Bisphenol-A glucuronidation was over 10-fold lower in breast tissues with the UGT1A1*28 allele compared with tissues without this allele (p = 0.006). 5. UGT2B15 and UGT1A9 contribute to glucuronidation variability in liver, while UGT1A1 is important in breast.

UGT-29 protein expression and localization during bacterial infection in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Wong, Rui-Rui; Lee, Song-Hua; Nathan, Sheila

2014-09-01

The nematode Caenorhabditis elegans is routinely used as an animal model to delineate complex molecular mechanisms involved in the host response to pathogen infection. Following up on an earlier study on host-pathogen interaction, we constructed a ugt-29::GFP transcriptional fusion transgenic worm strain to examine UGT-29 protein expression and localization upon bacterial infection. UGT-29 orthologs can be found in higher organisms including humans and is proposed as a member of the UDP-Glucoronosyl Transferase family of proteins which are involved in phase II detoxification of compounds detrimental to the host organism. Under uninfected conditions, UGT-29::GFP fusion protein was highly expressed in the C. elegans anterior pharynx and intestine, two major organs involved in detoxification. We further evaluated the localization of the enzyme in worms infected with the bacterial pathogen, Burkholderia pseudomallei. The infected ugt-29::GFP transgenic strain exhibited increased fluorescence in the pharynx and intestine with pronounced fluorescence also extending to body wall muscle. This transcriptional fusion GFP transgenic worm is a convenient and direct tool to provide information on UGT detoxification enzyme gene expression and could be a useful tool for a number of diverse applications.

Mapping the UDP-Glucuronic Acid Binding Site in UDP-Glucuronosyltransferase-1 A10 by Homology-based Modeling: Confirmation with Biochemical Evidence†

PubMed Central

Banerjee, Rajat; Pennington, Matthew W.; Garza, Amanda; Owens, Ida S.

2008-01-01

The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP-moiety in substrates, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315 and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E /G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of affinity-ligand, 5-azido-uridine-[β-32P]-diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high and low affinity binding sites. 2-Nitro 5-thiocyanobenzoic acid-digested UGT1A10-His bound with radiolabeled affinity-ligand revealed an 11.3- and 14.3-kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R- and 1A10-HisK404E showed 1.3-fold greater- and 50% less-label in the 14.3-kDa peptide, respectively, compared to 1A10-His without affecting the 11.3-kDa peptide. Scatchard analysis of binding data of affinity-ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in Kd, respectively. Our results indicate: K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high affinity sites and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321

Over-expression of UDP-glycosyltransferase gene UGT2B17 is involved in chlorantraniliprole resistance in Plutella xylostella (L.).

PubMed

Li, Xiuxia; Zhu, Bin; Gao, Xiwu; Liang, Pei

2017-07-01

UDP-glycosyltransferases (UGTs) are phase II detoxification enzymes widely distributed within living organisms. Their involvement in the biotransformation of various lipophilic endogenous compounds and phytoalexins in insects has been documented. However, the roles of this enzyme family in insecticide resistance have rarely been reported. Here, the functions of UGTs in chlorantraniliprole resistance in Plutella xylostella were investigated. Treatment with sulfinpyrazone and 5-nitrouracil (both inhibitors of UGT enzymes) significantly increased the toxicity of chlorantraniliprole against the third instar larvae of P. xylostella. Among the 23 UGT transcripts examined, only UGT2B17 was found to be over-expressed (with a range from 30.7- to 77.3-fold) in all four chlorantraniliprole-resistant populations compared to the susceptible one (CHS). The knock-down of UGT2B17 by RNA interference (RNAi) dramatically increased the toxicity of chlorantraniliprole by 27.4% and 29.8% in the CHS and CHR (resistant) populations, respectively. In contrast, exposure to phenobarbital significantly increased the relative expression of UGT2B17 while decreasing the toxicity of chlorantraniliprole to the larvae by 14.0%. UGT2B17 is involved in the detoxification of chlorantraniliprole, and its over-expression may play an important role in chlorantraniliprole resistance in P. xylostella. These results shed some light upon and further our understanding of the mechanisms of diamide insecticide resistance in insects. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

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

PubMed

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

2017-08-01

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

Glucuronidation of Drugs and Drug-Induced Toxicity in Humanized UDP-Glucuronosyltransferase 1 Mice

PubMed Central

Kutsuno, Yuki; Itoh, Tomoo; Tukey, Robert H.

2014-01-01

UDP-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various drugs. Although experimental rodents are used in preclinical studies to predict glucuronidation and toxicity of drugs in humans, species differences in glucuronidation and drug-induced toxicity have been reported. Humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) were recently developed. In this study, acyl-glucuronidations of etodolac, diclofenac, and ibuprofen in liver microsomes of hUGT1 mice were examined and compared with those of humans and regular mice. The kinetics of etodolac, diclofenac, and ibuprofen acyl-glucuronidation in hUGT1 mice were almost comparable to those in humans, rather than in mice. We further investigated the hepatotoxicity of ibuprofen in hUGT1 mice and regular mice by measuring serum alanine amino transferase (ALT) levels. Because ALT levels were increased at 6 hours after dosing in hUGT1 mice and at 24 hours after dosing in regular mice, the onset pattern of ibuprofen-induced liver toxicity in hUGT1 mice was different from that in regular mice. These data suggest that hUGT1 mice can be valuable tools for understanding glucuronidations of drugs and drug-induced toxicity in humans. PMID:24764149

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

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

Ma, Hai-Ying, E-mail: cmu4h-mhy@126.com; Sun, Dong-Xue; Cao, Yun-Feng

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 themore » 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.« less

Role of UDP-Glucuronosyltransferase (UGT) 2B2 in Metabolism of Triiodothyronine: Effect of Microsomal Enzyme Inducers in Sprague Dawley and UGT2B2-Deficient Fischer 344 Rats

PubMed Central

Richardson, Terrilyn A.; Klaassen, Curtis D.

2010-01-01

Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) can impact thyroid hormone homeostasis in rodents. Increased glucuronidation can result in reduction of serum thyroid hormone and a concomitant increase in thyroid-stimulating hormone (TSH). UGT2B2 is thought to glucuronidate triiodothyronine (T3). The purposes of this study were to determine the role of UGT2B2 in T3 glucuronidation and whether increased T3 glucuronidation mediates the increased TSH observed after MEI treatment. Sprague Dawley (SD) and UGT2B2-deficient Fischer 344 (F344) rats were fed a control diet or diet containing pregnenolone-16α-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum thyroxine (T4), T3, and TSH concentrations, hepatic androsterone/T4/T3 glucuronidation, and thyroid follicular cell proliferation were determined. In both SD and F344 rats, MEI treatments decreased serum T4, whereas serum T3 was maintained (except with PCB treatment). Hepatic T4 glucuronidation increased significantly after MEI in both rat strains. Compared with the other MEI, only PCN treatment significantly increased T3 glucuronidation (281 and 497%) in both SD and UGT2B2-deficient F344 rats, respectively, and increased both serum TSH and thyroid follicular cell proliferation. These data demonstrate an association among increases in T3 glucuronidation, TSH, and follicular cell proliferation after PCN treatment, suggesting that T3 is glucuronidated by other PCN-inducible UGTs in addition to UGT2B2. These data also suggest that PCN (rather than 3-MC or PCB) promotes thyroid tumors through excessive TSH stimulation of the thyroid gland. PMID:20421340

Effect of the β-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases (UGTs)

PubMed Central

Oleson, Lauren; Court, Michael H.

2009-01-01

Glucuronidation studies using microsomes and recombinant UDP-glucuronosyltransferases (rUGTs) can be complicated by the presence of endogenous β-glucuronidases leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used β-glucuronidase inhibitor, although as of yet it is not clear whether this reagent should be routinely added to glucuronidation incubations. Here we determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and 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 failed to show enhancement of any of the glucuronidation activities evaluated that could be considered consistent with inhibition of β-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for 5-hydroxytryptamine and estradiol glucuronidation by pHLMs with 35% decrease at 20 mM saccharolactone concentration. Endogenous β-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 or intestinal microsomes, or HEK293 microsomes. However, the extent of hydrolysis was relatively small representing only 9 to 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 and, if used, saccharolactone concentrations should be titrated to achieve activity enhancement without inhibition. PMID:18718121

A tandem array of UDP-glycosyltransferases from the UGT73C subfamily glycosylate sapogenins, forming a spectrum of mono- and bisdesmosidic saponins.

PubMed

Erthmann, Pernille Østerbye; Agerbirk, Niels; Bak, Søren

2018-05-01

This study identifies six UGT73Cs all able to glucosylate sapogenins at positions 3 and/or 28 which demonstrates that B. vulgaris has a much richer arsenal of UGTs involved in saponin biosynthesis than initially anticipated. The wild cruciferous plant Barbarea vulgaris is resistant to some insects due to accumulation of two monodesmosidic triterpenoid saponins, oleanolic acid 3-O-β-cellobioside and hederagenin 3-O-β-cellobioside. Insect resistance depends on the structure of the sapogenin aglycone and the glycosylation pattern. The B. vulgaris saponin profile is complex with at least 49 saponin-like metabolites, derived from eight sapogenins and including up to five monosaccharide units. Two B. vulgaris UDP-glycosyltransferases, UGT73C11 and UGT73C13, O-glucosylate sapogenins at positions 3 and 28, forming mainly 3-O-β-D-glucosides. The aim of this study was to identify UGTs responsible for the diverse saponin oligoglycoside moieties observed in B. vulgaris. Twenty UGT genes from the insect resistant genotype were selected and heterologously expressed in Nicotiana benthamiana and/or Escherichia coli. The extracts were screened for their ability to glycosylate sapogenins (oleanolic acid, hederagenin), the hormone 24-epibrassinolide and sapogenin monoglucosides (hederagenin and oleanolic acid 3-O-β-D-glucosides). Six UGTs from the UGT73C subfamily were able to glucosylate both sapogenins and both monoglucosides at positions 3 and/or 28. Some UGTs formed bisdesmosidic saponins efficiently. At least four UGT73C genes were localized in a tandem array with UGT73C11 and possibly UGT73C13. This organization most likely reflects duplication events followed by sub- and neofunctionalization. Indeed, signs of positive selection on several amino acid sites were identified and modelled to be localized on the UGT protein surface. This tandem array is proposed to initiate higher order bisdesmosidic glycosylation of B. vulgaris saponins, leading to the recently discovered

Single Nucleotide Polymorphisms in B-Genome Specific UDP-Glucosyl Transferases Associated with Fusarium Head Blight Resistance and Reduced Deoxynivalenol Accumulation in Wheat Grain.

PubMed

Sharma, Pallavi; Gangola, Manu P; Huang, Chen; Kutcher, H Randy; Ganeshan, Seedhabadee; Chibbar, Ravindra N

2018-01-01

An in vitro spike culture method was optimized to evaluate Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) and used to screen a population of ethyl methane sulfonate treated spike culture-derived variants (SCDV). Of the 134 SCDV evaluated, the disease severity score of 47 of the variants was ≤30%. Single nucleotide polymorphisms (SNP) in the UDP-glucosyltransferase (UGT) genes, TaUGT-2B, TaUGT-3B, and TaUGT-EST, differed between AC Nanda (an FHB-susceptible wheat variety) and Sumai-3 (an FHB-resistant wheat cultivar). SNP at 450 and 1,558 bp from the translation initiation site in TaUGT-2B and TaUGT-3B, respectively were negatively correlated with FHB severity in the SCDV population, whereas the SNP in TaUGT-EST was not associated with FHB severity. Fusarium graminearum strain M7-07-1 induced early expression of TaUGT-2B and TaUGT-3B in FHB-resistant SCDV lines, which were associated with deoxynivalenol accumulation and reduced FHB disease progression. At 8 days after inoculation, deoxynivalenol concentration varied from 767 ppm in FHB-resistant variants to 2,576 ppm in FHB-susceptible variants. The FHB-resistant SCDV identified can be used as new sources of FHB resistance in wheat improvement programs.

Thiamethoxam Resistance in Aphis gossypii Glover Relies on Multiple UDP-Glucuronosyltransferases

PubMed Central

Pan, Yiou; Tian, Fayi; Wei, Xiang; Wu, Yongqiang; Gao, Xiwu; Xi, Jinghui; Shang, Qingli

2018-01-01

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II enzymes that conjugate a variety of small lipophilic molecules with UDP sugars and alter them into more water-soluble metabolites. Therefore, glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. In this study, two inhibitors of UGT enzymes, sulfinpyrazone and 5-nitrouracil, significantly increased the toxicity of thiamethoxam against the resistant strain of Aphis gossypii, which indicates that UGTs are involved in thiamethoxam resistance in the cotton aphid. Based on transcriptome data, 31 A. gossypii UGTs belonging to 11 families (UGT329, UGT330, UGT341, UGT342, UGT343, UGT344, UGT345, UGT348, UGT349, UGT350, and UGT351) were identified. Compared with the thiamethoxam-susceptible strain, the transcripts of 23 UGTs were elevated, and the transcripts of 13 UGTs (UGT344J2, UGT348A2, UGT344D4, UGT341A4, UGT343B2, UGT342B2, UGT350C3, UGT344N2, UGT344A14, UGT344B4, UGT351A4, UGT344A11, and UGT349A2) were increased by approximately 2.0-fold in the resistant cotton aphid. The suppression of selected UGTs significantly increased the insensitivity of resistant aphids to thiamethoxam, suggesting that the up-regulated UGTs might be associated with thiamethoxam tolerance. This study provides an overall view of the possible metabolic factor UGTs that are relevant to the development of insecticide resistance. The results might facilitate further work to validate the roles of these UGTs in thiamethoxam resistance. PMID:29670540

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

PubMed

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. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Identification of UGT2B9*2 and UGT2B33 isolated from female rhesus monkey liver.

PubMed

Dean, Brian; Arison, Byron; Chang, Steve; Thomas, Paul E; King, Christopher

2004-06-01

Two UDP-glucuronosyltransferases (UGT2B9(*)2 and UGT2B33) have been isolated from female rhesus monkey liver. Microsomal preparations of the cell lines expressing the UGTs catalyzed the glucuronidation of the general substrate 7-hydroxy-4-(trifluoromethyl)coumarin in addition to selected estrogens (beta-estradiol and estriol) and opioids (morphine, naloxone, and naltrexone). UGT2B9(*)2 displayed highest efficiency for beta-estradiol-17-glucuronide production and did not catalyze the glucuronidation of naltrexone. UGT2B33 displayed highest efficiency for estriol and did not catalyze the glucuronidation of beta-estradiol. UGT2B9(*)2 was found also to catalyze the glucuronidation of 4-hydroxyestrone, 16-epiestriol, and hyodeoxycholic acid, while UGT2B33 was capable of conjugating 4-hydroxyestrone, androsterone, diclofenac, and hyodeoxycholic acid. Three glucocorticoids (cortisone, cortisol, and corticosterone) were not substrates for glucuronidation by liver or kidney microsomes or any expressed UGTs. Our current data suggest the use of beta-estradiol-3-glucuronidation, beta-estradiol-17-glucuronidation, and estriol-17-glucuronidation to assay UGT1A01, UGT2B9(*)2, and UGT2B33 activity in rhesus liver microsomes, respectively.

Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes.

PubMed

Gufford, Brandon T; Robarge, Jason D; Eadon, Michael T; Gao, Hongyu; Lin, Hai; Liu, Yunlong; Desta, Zeruesenay; Skaar, Todd C

2018-04-01

Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes ( P  <   .05). Rifampin-induced miRNA expression changes correlated with mRNA changes and miRNAs were identified that may modulate conjugating enzyme expression. NAT2 gene expression was most strongly repressed (1.3-fold) by rifampin while UGT1A4 and UGT1A1 genes were most strongly induced (7.9- and 4.8-fold, respectively). Physiologically based pharmacokinetic modeling (PBPK) was used to simulate the clinical consequences of rifampin induction of CYP3A4- and UGT1A4-mediated midazolam metabolism. Simulations evaluating isolated UGT1A4 induction predicted increased midazolam N-glucuronide exposure (~4-fold) with minimal reductions in parent midazolam exposure (~10%). Simulations accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements.

Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function.

PubMed

Kundu, Rakesh; Dasgupta, Suman; Biswas, Anindita; Bhattacharya, Anirban; Pal, Bikas C; Bandyopadhyay, Debashis; Bhattacharya, Shelley; Bhattacharya, Samir

2008-08-13

Cajanus cajan Linn. (Leguminosae) is a nontoxic edible herb, widely used in Indian folk medicine for the prevention of various liver disorders. In the present study we have demonstrated that methanol-aqueous fraction (MAF2) of Cajanus cajan leaf extract could prevent the chronically treated alcohol induced rat liver damage. Chronic doses of alcohol (3.7 g/ kg) orally administered to rats for 28 days and liver function marker enzymes such as GPT, GOT, ALP and anti-oxidant enzyme activities were determined. Effect of MAF2 at a dose of 50mg/kg body weight on alcohol treated rats was noted. Alcohol effected significant increase in liver marker enzyme activities and reduced the activities of anti-oxidant enzymes. Co-administration of MAF2 reversed the liver damage due to alcohol; it decreased the activities of liver marker enzymes and augmented antioxidant enzyme activities. We also demonstrate significant decrease of the phase II detoxifying enzyme, UDP-glucuronosyl transferase (UGT) activity along with a three- and two-fold decrease of UGT2B gene and protein expression respectively. MAF2 co-administration normalized UGT activity and revived the expression of UGT2B with a concomitant expression and nuclear translocation of Nrf2, a transcription factor that regulates the expression of many cytoprotective genes. Cajanus cajan extract therefore shows a promise in therapeutic use in alcohol induced liver dysfunction.

Quantum Chemical and Docking Insights into Bioavailability Enhancement of Curcumin by Piperine in Pepper.

PubMed

Patil, Vaishali M; Das, Sukanya; Balasubramanian, Krishnan

2016-05-26

We combine quantum chemical and molecular docking techniques to provide new insights into how piperine molecule in various forms of pepper enhances bioavailability of a number of drugs including curcumin in turmeric for which it increases its bioavailability by a 20-fold. We have carried out docking studies of quantum chemically optimized piperine structure binding to curcumin, CYP3A4 in cytochrome P450, p-Glycoprotein and UDP-glucuronosyltransferase (UGT), the enzyme responsible for glucuronosylation, which increases the solubility of curcumin. All of these studies establish that piperine binds to multiple sites on the enzymes and also intercalates with curcumin forming a hydrogen bonded complex with curcumin. The conjugated network of double bonds and the presence of multiple charge centers of piperine offer optimal binding sites for piperine to bind to enzymes such as UDP-GDH, UGT, and CYP3A4. Piperine competes for curcumin's intermolecular hydrogen bonding and its stacking propensity by hydrogen bonding with enolic proton of curcumin. This facilitates its metabolic transport, thereby increasing its bioavailability both through intercalation into curcumin layers through intermolecular hydrogen bonding, and by inhibiting enzymes that cause glucuronosylation of curcumin.

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

PubMed

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

2016-09-10

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

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed Central

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

2016-01-01

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

Identification of Diet-Derived Constituents as Potent Inhibitors of Intestinal Glucuronidation

PubMed Central

Gufford, Brandon T.; Chen, Gang; Lazarus, Philip; Graf, Tyler N.; Oberlies, Nicholas H.

2014-01-01

Drug-metabolizing enzymes within enterocytes constitute a key barrier to xenobiotic entry into the systemic circulation. Furanocoumarins in grapefruit juice are cornerstone examples of diet-derived xenobiotics that perpetrate interactions with drugs via mechanism-based inhibition of intestinal CYP3A4. Relative to intestinal CYP3A4-mediated inhibition, alternate mechanisms underlying dietary substance–drug interactions remain understudied. A working systematic framework was applied to a panel of structurally diverse diet-derived constituents/extracts (n = 15) as inhibitors of intestinal UDP-glucuronosyl transferases (UGTs) to identify and characterize additional perpetrators of dietary substance–drug interactions. Using a screening assay involving the nonspecific UGT probe substrate 4-methylumbelliferone, human intestinal microsomes, and human embryonic kidney cell lysates overexpressing gut-relevant UGT1A isoforms, 14 diet-derived constituents/extracts inhibited UGT activity by >50% in at least one enzyme source, prompting IC50 determination. The IC50 values of 13 constituents/extracts (≤10 μM with at least one enzyme source) were well below intestinal tissue concentrations or concentrations in relevant juices, suggesting that these diet-derived substances can inhibit intestinal UGTs at clinically achievable concentrations. Evaluation of the effect of inhibitor depletion on IC50 determination demonstrated substantial impact (up to 2.8-fold shift) using silybin A and silybin B, two key flavonolignans from milk thistle (Silybum marianum) as exemplar inhibitors, highlighting an important consideration for interpretation of UGT inhibition in vitro. Results from this work will help refine a working systematic framework to identify dietary substance–drug interactions that warrant advanced modeling and simulation to inform clinical assessment. PMID:25008344

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product–Drug Interaction

PubMed Central

Gufford, Brandon T.; Chen, Gang; Vergara, Ana G.; Lazarus, Philip; Oberlies, Nicholas H.

2015-01-01

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4′- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27–66 µM; UGT1A1, 3.2–8.3 µM; UGT1A8, 19–73 µM; and UGT1A10, 65–120 µM) encompassed reported intestinal tissue concentrations (20–310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product–drug interactions in the context of cancer prevention. PMID:26070840

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product-Drug Interaction.

PubMed

Gufford, Brandon T; Chen, Gang; Vergara, Ana G; Lazarus, Philip; Oberlies, Nicholas H; Paine, Mary F

2015-09-01

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4'- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27-66 µM; UGT1A1, 3.2-8.3 µM; UGT1A8, 19-73 µM; and UGT1A10, 65-120 µM) encompassed reported intestinal tissue concentrations (20-310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product-drug interactions in the context of cancer prevention. Copyright © 2015 by The American Society for Pharmacology and Experimental

Association of UGT1A1 variants and hyperbilirubinemia in breast-fed full-term Chinese infants.

PubMed

Zhou, Youyou; Wang, San-nan; Li, Hong; Zha, Weifeng; Wang, Xuli; Liu, Yuanyuan; Sun, Jian; Peng, Qianqian; Li, Shilin; Chen, Ying; Jin, Li

2014-01-01

A retrospective case control study of breast-fed full-term infants was carried out to determine whether variants in Uridine Diphosphate Glucuronosyl Transferase 1A1 (UGT1A1) and Heme Oxygenase-1 (HMOX1) were associated with neonatal hyperbilirubinemia. Eight genetic variants of UGT1A1 and 3 genetic variants of HMOX1 were genotyped in 170 hyperbilirubinemic newborns and 779 controls. Five significant associations with breast-fed hyperbilirubinemia were detected after adjusting for gender, birth season, birth weight, delivery mode, gestational age and False Discovery Rate (FDR) correction: the dominant effect of rs887829 (c-364t) (Odds Ratio (OR): 0.55; 95% Confidence Interval (CI): 0.34-0.89; p = 0.014), the additive effect of (TA)n repeat (OR: 0.59; 95%CI: 0.38-0.91; p = 0.017), the dominant effect of rs4148323 (Gly71Arg, G211A) (OR: 2.02; 95%CI: 1.44-2.85; p = 5.0×10-5), the recessive effect of rs6717546 (g+914a) (OR: 0.30; 95%CI: 0.11-0.83; p = 0.021) and rs6719561 (t+2558c) (OR: 0.38; 95%CI: 0.20-0.75; p = 0.005). Neonates carrying the minor allele of rs887829 (TA)n repeat had significantly lower peak bilirubin than wild types, while the minor allele carriers of rs4148323 had significantly higher peak bilirubin than wild types. No association was found in HMOX1. Our findings added to the understanding of the significance of UGT1A1 in association with neonatal hyperbilirubinemia in East Asian population. Additional studies were required to investigate the mechanisms of the protective effects.

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

PubMed

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

2018-08-01

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

Isothiocyanates induce UGT1A1 in humanized UGT1 mice in a CAR dependent fashion that is highly dependent upon oxidative stress.

PubMed

Yoda, Emiko; Paszek, Miles; Konopnicki, Camille; Fujiwara, Ryoichi; Chen, Shujuan; Tukey, Robert H

2017-04-19

Isothiocyanates, such as phenethyl isothiocyanate (PEITC), are formed following the consumption of cruciferous vegetables and generate reactive oxygen species (ROS) that lead to the induction of cytoprotective genes such as the UDP-glucuronosyltransferases (UGTs). The induction of ROS activates the Nrf2-Keap 1 pathway leading to the induction of genes through antioxidant response elements (AREs). UGT1A1, the sole enzyme responsible for the metabolism of bilirubin, can be induced following activation of Nrf2. When neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB) because of a developmental delay in expression of the UGT1A1 gene, were treated with PEITC, TSB levels were reduced. Liver and intestinal UGT1A1 were induced, along with murine CYP2B10, a consensus CAR target gene. In both neonatal and adult hUGT1/Car -/- mice, PEITC was unable to induce CYP2B10. A similar result was observed following analysis of UGT1A1 expression in liver. However, TSB levels were still reduced in hUGT1/Car -/- neonatal mice because of ROS induction of intestinal UGT1A1. When oxidative stress was blocked by exposing mice to N-acetylcysteine, induction of liver UGT1A1 and CYP2B10 by PEITC was prevented. Thus, new findings in this report link an important role in CAR activation that is dependent upon oxidative stress.

Conditional immortalization of Gunn rat hepatocytes: an ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer.

PubMed

Fox, I J; Chowdhury, N R; Gupta, S; Kondapalli, R; Schilsky, M L; Stockert, R J; Chowdhury, J R

1995-03-01

Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGR)-mediated endocytosis are being developed to transfer genes for the correction of bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT) deficiency. Ex vivo evaluation of these gene transfer vectors would be facilitated by a cell system that lacks bilirubin-UGT, but expresses differentiated liver functions, including ASGR. We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58). At 33 degrees C, the immortalized hepatocyte clones expressed SV40 large T antigen, synthesized DNA, and doubled in number every 2 to 3 days. At this temperature, differentiated hepatocyte markers, e.g., albumin, ASGR, and androsterone-UGT, were expressed at 5% to 10% of the levels found in primary hepatocytes maintained in culture for 24 hours. Glutathione-S-transferase Yp (GST-Yp), an oncofetal protein, was expressed in these cells at 33 degrees C, but was undetectable in primary hepatocytes. In contrast, when the cells were cultured at 39 degrees C or 37 degrees C, the large T antigen was degraded, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were observed. The expression of albumin, ASGR, and androsterone-UGT, and their corresponding mRNAs, increased to 25% to 40% of the level in primary hepatocytes, whereas GST-Yp expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red-labeled asialoorosomucoid, and binding and degradation of 125I-asialoorosomucoid. After liposome-mediated transfer of a plasmid containing the coding region of human bilirubin-UGT1, driven by the SV40 large T promoter, active human bilirubin-UGT1 was expressed in these cells. The immortalized cells were not tumorigenic after transplantation into severe combined immunodeficiency mice. These conditionally immortalized cells will be useful

The UDP-Glucuronosyltransferase (UGT) 1A Polymorphism c.2042C>G (rs8330) Is Associated with Increased Human Liver Acetaminophen Glucuronidation, Increased UGT1A Exon 5a/5b Splice Variant mRNA Ratio, and Decreased Risk of Unintentional Acetaminophen-Induced Acute Liver FailureS⃞

PubMed Central

Freytsis, Marina; Wang, Xueding; Peter, Inga; Guillemette, Chantal; Hazarika, Suwagmani; Duan, Su X.; Greenblatt, David J.; Lee, William M.

2013-01-01

Acetaminophen is cleared primarily by hepatic glucuronidation. Polymorphisms in genes encoding the acetaminophen UDP-glucuronosyltransferase (UGT) enzymes could explain interindividual variability in acetaminophen glucuronidation and variable risk for liver injury after acetaminophen overdose. In this study, human liver bank samples were phenotyped for acetaminophen glucuronidation activity and genotyped for the major acetaminophen-glucuronidating enzymes (UGTs 1A1, 1A6, 1A9, and 2B15). Of these, only three linked single nucleotide polymorphisms (SNPs) located in the shared UGT1A-3′UTR region (rs10929303, rs1042640, rs8330) were associated with acetaminophen glucuronidation activity, with rs8330 consistently showing higher acetaminophen glucuronidation at all the tested concentrations of acetaminophen. Mechanistic studies using luciferase-UGT1A-3′UTR reporters indicated that these SNPs do not alter mRNA stability or translation efficiency. However, there was evidence for allelic imbalance and a gene-dose proportional increase in the amount of exon 5a versus exon 5b containing UGT1A mRNA spliced transcripts in livers with the rs8330 variant allele. Cotransfection studies demonstrated an inhibitory effect of exon 5b containing cDNAs on acetaminophen glucuronidation by UGT1A1 and UGT1A6 cDNAs containing exon 5a. In silico analysis predicted that rs8330 creates an exon splice enhancer site that could favor exon 5a (over exon 5b) utilization during splicing. Finally, the prevalence of rs8330 was significantly lower (P = 0.027, χ2 test) in patients who had acute liver failure from unintentional acetaminophen overdose compared with patients with acute liver failure from other causes or a race- or ethnicity-matched population. Together, these findings suggest that rs8330 is an important determinant of acetaminophen glucuronidation and could affect an individual’s risk for acetaminophen-induced liver injury. PMID:23408116

Expression of UDP-glucuronosyltransferase 1A4 in human placenta at term

PubMed Central

Østby, Lene; Stuen, Ina; Sundby, Eirik

2010-01-01

The placenta contains a large variety of metabolizing enzymes, among them UDP-glucuronosyltransferase (UGT). Several UGT2B isozymes have so far been detected in human placenta, but little is known on placental expression of UGT1A isozymes. The antiepileptic drug lamotrigine (LTG) is a UGT1A4-substrate, and its serum concentration falls by over 50% during pregnancy, leading to impaired seizure control. The placenta may be involved in this. Microsomes from term placentas of 4 LTG-users and 10 healthy control subjects were prepared. Western blot analysis detected UGT1A proteins in all placentas. The presence of UGT1A4 in placenta from LTG users was confirmed with UGT1A4 commercial standard and a specific UGT1A4 primary antibody. Since LTG is primarily metabolized by UGT1A4 and this isozyme is shown to be present in placenta at term, it may be hypothesized that the placenta is involved in the fall of LTG serum concentrations during pregnancy. PMID:21302032

UGT1A1 gene polymorphism: Impact on toxicity and efficacy of irinotecan-based regimens in metastatic colorectal cancer

PubMed Central

Schulz, Christoph; Heinemann, Volker; Schalhorn, Andreas; Moosmann, Nikolas; Zwingers, Thomas; Boeck, Stefan; Giessen, Clemens; Stemmler, Hans-Joachim

2009-01-01

AIM: To investigate the correlation between uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) gene polymorphisms and irinotecan-associated side effects and parameters of drug efficacy in patients with metastatic colorectal cancer (mCRC) receiving a low-dose weekly irinotecan chemotherapeutic regimen. METHODS: Genotypes were retrospectively evaluated by gene scan analysis on the ABI 310 sequencer of the TATAA box in the promoter region of the UGT1A1 gene in blood samples from 105 patients who had received 1st line irinotecan-based chemotherapy for mCRC. RESULTS: The distribution of the genotypes was as follows: wild type genotype (WT) (6/6) 39.0%, heterozygous genotype (6/7) 49.5%, and homozygous genotype (7/7) 9.5%. The overall response rate (OR) was similar between patients carrying the (6/7, 7/7) or the WT genotype (6/6) (44.3% vs 43.2%, P = 0.75). Neither time to progression [(TTP) 8.1 vs 8.2 mo, P = 0.97] nor overall survival [(OS) 21.2 vs 18.9 mo, P = 0.73] differed significantly in patients who carried the (6/6) when compared to the (6/7, 7/7) genotype. No significant differences in toxicity were observed: Grade 3 and 4 delayed diarrhoea [(6/7, 7/7) vs (6/6); 13.0% vs 6.2%, P = 0.08], treatment delays [(6/7, 7/7) vs (6/6); 25.1% vs 19.3%, P =0.24] or dose reductions [(6/7, 7/7) vs (6/6); 21.5% vs 27.2%, P = 0.07]. CONCLUSION: This analysis demonstrates the non-significant influence of the UGT1A1 gene polymorphism on efficacy and rate of irinotecan-associated toxicity in mCRC patients receiving low-dose irinotecan based chemotherapy. PMID:19859999

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

PubMed

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

1996-07-15

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

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

EPA Science Inventory

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

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

PubMed Central

Robotham, Scott A.; Brodbelt, Jennifer S.

2011-01-01

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

Effects of UDP-glucuronosyltransferase (UGT) polymorphisms on the pharmacokinetics of febuxostat in healthy Chinese volunteers.

PubMed

Lin, Meihua; Liu, Jian; Zhou, Huili; Wu, Minglan; Lv, Duo; Huang, Yujie; Zheng, Yunliang; Shentu, Jianzhong; Wu, Lihua

2017-02-01

The pharmacokinetics (PKs) of febuxostat varies among individuals, while the main causes are still unknown. We investigated whether the polymorphisms of UGT1A1 and UGT1A3 played an important role in the disposition of the drug after oral administration of febuxostat tablet in Chinese subjects. A total of 42 healthy subjects were from two previous independent clinical bioequivalence (BE) trials of febuxostat, in which the same reference formulation (ULORIC ® tablet, 80 mg) was taken, and thus the PK data were combined for the evaluation of pharmacogenomic effect on febuxostat PKs. Our study clearly indicated that the area under the plasma concentration-time curve (AUC) in the heterozygote and homozygote of UGT1A1*6 (c.211G > A, rs4148323) was significantly higher than that in the wild-type. Meanwhile, the clearance (CL/F) exhibited a significant reduction by 22.2%. Interestingly, UGT1A1*28, in perfect linkage disequilibrium (LD) with UGT1A3*2a, significantly increased its clearance. These results indicate that UGT1A1*6 was an important factor influencing the drug disposition, thus providing a probable explanation for interindividual variation of febuxostat PKs in Chinese subjects. In addition, by considering of the different allele distribution of UGT1A1*6 and *28 in Eastern and Western populations, these findings might further interpret the ethnic difference of febuxostat PKs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

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

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

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

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

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

PubMed Central

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

2014-01-01

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

Understanding Substrate Selectivity of Human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes

PubMed Central

Dong, Dong; Ako, Roland; Hu, Ming; Wu, Baojian

2015-01-01

The UDP-glucuronosyltransferase (UGT) enzyme catalyzes the glucuronidation reaction which is a major metabolic and detoxification pathway in humans. Understanding the mechanisms for substrate recognition by UGT assumes great importance in an attempt to predict its contribution to xenobiotic/drug disposition in vivo. Spurred on by this interest, 2D/3D-quantitative structure activity relationships (QSAR) and pharmacophore models have been established in the absence of a complete mammalian UGT crystal structure. This review discusses the recent progress in modeling human UGT substrates including those with multiple sites of glucuronidation. A better understanding of UGT active site contributing to substrate selectivity (and regioselectivity) from the homologous enzymes (i.e., plant and bacterial UGTs, all belong to family 1 of glycosyltransferase (GT1)) is also highlighted, as these enzymes share a common catalytic mechanism and/or overlapping substrate selectivity. PMID:22385482

S-Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the elimination of naproxen

PubMed Central

Bowalgaha, Kushari; Elliot, David J; Mackenzie, Peter I; Knights, Kathleen M; Swedmark, Stellan; Miners, John O

2005-01-01

Aims To characterize the kinetics of S-naproxen (‘naproxen’) acyl glucuronidation and desmethylnaproxen acyl and phenolic glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions. Methods Naproxen and desmethylnaproxen glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-performance liquid chromatography methods. Results Naproxen acyl glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent Km values (±SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 ± 13 µm (16, 43) and 473 ± 108 µm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent Km (72 µm) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective ‘probe’ fluconazole. Although data for desmethylnaproxen phenolic glucuronidation by human liver microsomes were generally adequately fitted to either the single- or two-enzyme Michaelis–Menten equation, model fitting was inconclusive for desmethylnaproxen acyl glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen glucuronidation by the recombinant UGTs. Conclusion UGT2B7 is responsible for human hepatic naproxen acyl glucuronidation, which is the primary elimination pathway for this drug. PMID:16187975

[Detection of UGT1A1*28 Polymorphism Using Fragment Analysis].

PubMed

Huang, Ying; Su, Jian; Huang, Xiaosui; Lu, Danxia; Xie, Zhi; Yang, Suqing; Guo, Weibang; Lv, Zhiyi; Wu, Hongsui; Zhang, Xuchao

2017-12-20

Uridine-diphosphoglucuronosyl transferase 1A1 (UGT1A1), UGT1A1*28 polymorphism can reduce UGT1A1 enzymatic activity, which may lead to severe toxicities in patients who receive irinotecan. This study tries to build a fragment analysis method to detect UGT1A1*28 polymorphism. A total of 286 blood specimens from the lung cancer patients who were hospitalized in Guangdong General Hospital between April 2014 to May 2015 were detected UGT1A1*28 polymorphism by fragment analysis method. Comparing with Sanger sequencing, precision and accuracy of the fragment analysis method were 100%. Of the 286 patients, 236 (82.5% harbored TA6/6 genotype, 48 (16.8%) TA 6/7 genotype and 2 (0.7%) TA7/7 genotype. Our data suggest hat the fragment analysis method is robust for detecting UGT1A1*28 polymorphism in clinical practice. It's simple, time-saving, and easy-to-carry.

Pharmacokinetic drug interactions of morphine, codeine, and their derivatives: theory and clinical reality, part I.

PubMed

Armstrong, Scott C; Cozza, Kelly L

2003-01-01

Pharmacokinetic drug-drug interactions with morphine, hydromorphone, and oxymorphone are reviewed in this column. Morphine is a naturally occurring opiate that is metabolized chiefly through glucuronidation by uridine diphosphate glucuronosyl transferase (UGT) enzymes in the liver. These enzymes produce an active analgesic metabolite and a potentially toxic metabolite. In vivo drug-drug interaction studies with morphine are few, but they do suggest that inhibition or induction of UGT enzymes could alter morphine and its metabolite levels. These interactions could change analgesic efficacy. Hydromorphone and oxymorphone, close synthetic derivatives of morphine, are also metabolized primarily by UGT enzymes. Hydromorphone may have a toxic metabolite similar to morphine. In vivo drug-drug interaction studies with hydromorphone and oxymorphone have not been done, so it is difficult to make conclusions with these drugs.

High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human.

PubMed

Akazawa, Takanori; Uchida, Yasuo; Miyauchi, Eisuke; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

2018-01-02

Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.

Identification of human UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin.

PubMed

Sadeque, Abu J M; Usmani, Khawja A; Palamar, Safet; Cerny, Matthew A; Chen, Weichao G

2012-04-01

Lorcaserin, a selective serotonin 5-HT(2C) receptor agonist, is a weight management agent in clinical development. Lorcaserin N-carbamoyl glucuronidation governs the predominant excretory pathway of lorcaserin in humans. Human UDP-glucuronosyltransferases (UGTs) responsible for lorcaserin N-carbamoyl glucuronidation are identified herein. Lorcaserin N-carbamoyl glucuronide formation was characterized by the following approaches: metabolic screening using human tissues (liver, kidney, intestine, and lung) and recombinant enzymes, kinetic analyses, and inhibition studies. Whereas microsomes from all human tissues studied herein were found to be catalytically active for lorcaserin N-carbamoyl glucuronidation, liver microsomes were the most efficient. With recombinant UGT enzymes, lorcaserin N-carbamoyl glucuronidation was predominantly catalyzed by three UGT2Bs (UGT2B7, UGT2B15, and UGT2B17), whereas two UGT1As (UGT1A6 and UGT1A9) played a minor role. UGT2B15 was most efficient, with an apparent K(m) value of 51.6 ± 1.9 μM and V(max) value of 237.4 ± 2.8 pmol/mg protein/min. The rank order of catalytic efficiency of human UGT enzymes for lorcaserin N-carbamoyl glucuronidation was UGT2B15 > UGT2B7 > UGT2B17 > UGT1A9 > UGT1A6. Inhibition of lorcaserin N-carbamoyl glucuronidation activities of UGT2B7, UGT2B15, and UGT2B17 in human liver microsomes by mefenamic acid, bisphenol A, and eugenol further substantiated the involvement of these UGT2B isoforms. In conclusion, multiple human UGT enzymes catalyze N-carbamoyl glucuronidation of lorcaserin; therefore, it is unlikely that inhibition of any one of these UGT activities will lead to significant inhibition of the lorcaserin N-carbamoyl glucuronidation pathway. Thus, the potential for drug-drug interaction by concomitant administration of a drug(s) that is metabolized by any of these UGTs is remote.

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

USDA-ARS?s Scientific Manuscript database

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

Suppression of abnormal morphology and extracytoplasmic function sigma activity in Bacillus subtilis ugtP mutant cells by expression of heterologous glucolipid synthases from Acholeplasma laidlawii.

PubMed

Matsuoka, Satoshi; Seki, Takahiro; Matsumoto, Kouji; Hara, Hiroshi

2016-12-01

Glucolipids in Bacillus subtilis are synthesized by UgtP processively transferring glucose from UDP-glucose to diacylglycerol. Here we conclude that the abnormal morphology of a ugtP mutant is caused by lack of glucolipids, since the same morphology arises after abolition of glucolipid production by disruption of pgcA and gtaB, which are involved in UDP-glucose synthesis. Conversely, expression of a monoglucosyldiacylglycerol (MGlcDG) produced by 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii (alMGS) almost completely suppressed the ugtP disruptant phenotype. Activation of extracytoplasmic function (ECF) sigmas (SigM, SigV, and SigX) in the ugtP mutant was decreased by alMGS expression, and was suppressed to low levels by MgSO 4 addition. When alMGS and alDGS (A. laidlawii 1,2-diacylglycerol-3-glucose (1-2)-glucosyltransferase producing diglucosyldiacylglycerol (DGlcDG)) were simultaneously expressed, SigX activation was repressed to wild type level. These observations suggest that MGlcDG molecules are required for maintenance of B. subtilis cell shape and regulation of ECF sigmas, and DGlcDG regulates SigX activity.

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

PubMed

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

2016-03-01

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

Involvement of three putative glucosyltransferases from the UGT72 family in flavonol glucoside/rhamnoside biosynthesis in Lotus japonicus seeds

PubMed Central

Yin, Qinggang; Shen, Guoan; Chang, Zhenzhan; Tang, Yuhong; Gao, Hongwen

2017-01-01

Abstract Flavonols are one of the largest groups of flavonoids that confer benefits for the health of plants and animals. Flavonol glycosides are the predominant flavonoids present in the model legume Lotus japonicus. The molecular mechanisms underlying the biosynthesis of flavonol glycosides as yet remain unknown in L. japonicus. In the present study, we identified a total of 188 UDP-glycosyltransferases (UGTs) in L. japonicus by genome-wide searching. Notably, 12 UGTs from the UGT72 family were distributed widely among L. japonicus chromosomes, expressed in all tissues, and showed different docking scores in an in silico bioinformatics docking analysis. Further enzymatic assays showed that five recombinant UGTs (UGT72AD1, UGT72AF1, UGT72AH1, UGT72V3, and UGT72Z2) exhibit activity toward flavonol, flavone, and isoflavone aglycones. In particular, UGT72AD1, UGT72AH1, and UGT72Z2 are flavonol-specific UGTs with different kinetic properties. In addition, the overexpression of UGT72AD1 and UGT72Z2 led to increased accumulation of flavonol rhamnosides in L. japonicus and Arabidopsis thaliana. Moreover, the increase of kaempferol 3-O-rhamnoside-7-O-rhamnoside in transgenic A. thaliana inhibited root growth as compared with the wild-type control. These results highlight the significance of the UGT72 family in flavonol glycosylation and the role of flavonol rhamnosides in plant growth. PMID:28204516

Two UGT84 Family Glycosyltransferases Catalyze a Critical Reaction of Hydrolyzable Tannin Biosynthesis in Pomegranate (Punica granatum)

PubMed Central

Ono, Nadia N.; Qin, Xiaoqiong; Wilson, Alexander E.; Li, Gang

2016-01-01

Hydrolyzable tannins (HTs) play important roles in plant herbivore deterrence and promotion of human health. A critical step in HT production is the formation of 1-O-galloyl-β-D-glucopyranoside (β-glucogallin, ester-linked gallic acid and glucose) by a UDP-glucosyltransferase (UGT) activity. We cloned and biochemically characterized four candidate UGTs from pomegranate (Punica granatum), of which only UGT84A23 and UGT84A24 exhibited β-glucogallin forming activities in enzyme assays. Although overexpression and single RNAi knockdown pomegranate hairy root lines of UGT84A23 or UGT84A24 did not lead to obvious alterations in punicalagin (the prevalent HT in pomegranate) accumulation, double knockdown lines of the two UGTs resulted in largely reduced levels of punicalagins and bis-hexahydroxydiphenyl glucose isomers. An unexpected accumulation of galloyl glucosides (ether-linked gallic acid and glucose) was also detected in the double knockdown lines, suggesting that gallic acid was utilized by an unidentified UGT activity for glucoside formation. Transient expression in Nicotiana benthamiana leaves and immunogold labeling in roots of pomegranate seedlings collectively indicated cytosolic localization of UGT84A23 and UGT84A24. Overall, functional characterization and localization of UGT84A23 and UGT84A24 open up opportunities for further understanding the regulatory control of HT metabolism in plants and its coordination with other biochemical pathways in the metabolic network. PMID:27227328

Two UGT84 Family Glycosyltransferases Catalyze a Critical Reaction of Hydrolyzable Tannin Biosynthesis in Pomegranate (Punica granatum).

PubMed

Ono, Nadia N; Qin, Xiaoqiong; Wilson, Alexander E; Li, Gang; Tian, Li

2016-01-01

Hydrolyzable tannins (HTs) play important roles in plant herbivore deterrence and promotion of human health. A critical step in HT production is the formation of 1-O-galloyl-β-D-glucopyranoside (β-glucogallin, ester-linked gallic acid and glucose) by a UDP-glucosyltransferase (UGT) activity. We cloned and biochemically characterized four candidate UGTs from pomegranate (Punica granatum), of which only UGT84A23 and UGT84A24 exhibited β-glucogallin forming activities in enzyme assays. Although overexpression and single RNAi knockdown pomegranate hairy root lines of UGT84A23 or UGT84A24 did not lead to obvious alterations in punicalagin (the prevalent HT in pomegranate) accumulation, double knockdown lines of the two UGTs resulted in largely reduced levels of punicalagins and bis-hexahydroxydiphenyl glucose isomers. An unexpected accumulation of galloyl glucosides (ether-linked gallic acid and glucose) was also detected in the double knockdown lines, suggesting that gallic acid was utilized by an unidentified UGT activity for glucoside formation. Transient expression in Nicotiana benthamiana leaves and immunogold labeling in roots of pomegranate seedlings collectively indicated cytosolic localization of UGT84A23 and UGT84A24. Overall, functional characterization and localization of UGT84A23 and UGT84A24 open up opportunities for further understanding the regulatory control of HT metabolism in plants and its coordination with other biochemical pathways in the metabolic network.

Potent and selective inhibition of magnolol on catalytic activities of UGT1A7 and 1A9.

PubMed

Zhu, Liangliang; Ge, Guangbo; Liu, Yong; He, Guiyuan; Liang, Sicheng; Fang, Zhongze; Dong, Peipei; Cao, Yunfeng; Yang, Ling

2012-10-01

1. Human exposure to magnolol can reach a high dose in daily life. Our previous studies indicated that magnolol showed high affinities to several UDP-glucuronosyltransferases (UGTs) This study was designed to examine the in vitro inhibitory effects of magnolol on UGTs, and further to evaluate the possibility of the in vivo inhibition that might happen. 2. Assays with recombinant UGTs and human liver microsomes (HLM) indicated that magnolol (10 µM) can selectively inhibit activities of UGT1A9 and extra-hepatic UGT1A7. Inhibition of magnolol on UGT1A7 followed competitive inhibition mechanism, while the inhibition on UGT1A9 obeyed either competitive or mixed inhibition mechanism, depending on substrates. The K(i) values for UGT1A7 and 1A9 are all in nanomolar ranges, lower than possible magnolol concentrations in human gut lumen and blood, indicating the in vivo inhibition on these two enzymes would likely occur. 3. In conclusion, UGT1A7 and 1A9 can be strongly inhibited by magnolol, raising the alarm for safe application of magnolol and traditional Chinese medicines containing magnolol. Additionally, given that UGT1A7 is an extra-hepatic enzyme, magnolol can serve as a selective UGT1A9 inhibitor that will act as a new useful tool in future hepatic glucuronidation phenotyping.

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

PubMed Central

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

2015-01-01

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

Biological definition of multiple chemical sensitivity from redox state and cytokine profiling and not from polymorphisms of xenobiotic-metabolizing enzymes

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

De Luca, Chiara; Scordo, Maria G.; Cesareo, Eleonora

Background: Multiple chemical sensitivity (MCS) is a poorly clinically and biologically defined environment-associated syndrome. Although dysfunctions of phase I/phase II metabolizing enzymes and redox imbalance have been hypothesized, corresponding genetic and metabolic parameters in MCS have not been systematically examined. Objectives: We sought for genetic, immunological, and metabolic markers in MCS. Methods: We genotyped patients with diagnosis of MCS, suspected MCS and Italian healthy controls for allelic variants of cytochrome P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A5), UDP-glucuronosyl transferase (UGT1A1), and glutathione S-transferases (GSTP1, GSTM1, and GSTT1). Erythrocyte membrane fatty acids, antioxidant (catalase, superoxide dismutase (SOD)) and glutathione metabolizing (GST,more » glutathione peroxidase (Gpx)) enzymes, whole blood chemiluminescence, total antioxidant capacity, levels of nitrites/nitrates, glutathione, HNE-protein adducts, and a wide spectrum of cytokines in the plasma were determined. Results: Allele and genotype frequencies of CYPs, UGT, GSTM, GSTT, and GSTP were similar in the Italian MCS patients and in the control populations. The activities of erythrocyte catalase and GST were lower, whereas Gpx was higher than normal. Both reduced and oxidised glutathione were decreased, whereas nitrites/nitrates were increased in the MCS groups. The MCS fatty acid profile was shifted to saturated compartment and IFNgamma, IL-8, IL-10, MCP-1, PDGFbb, and VEGF were increased. Conclusions: Altered redox and cytokine patterns suggest inhibition of expression/activity of metabolizing and antioxidant enzymes in MCS. Metabolic parameters indicating accelerated lipid oxidation, increased nitric oxide production and glutathione depletion in combination with increased plasma inflammatory cytokines should be considered in biological definition and diagnosis of MCS.« less

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

PubMed

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

2012-05-08

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

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

PubMed

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

2008-01-01

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

The impact of the UGT1A1*60 allele on bilirubin serum concentrations.

PubMed

Pasternak, Amy L; Crews, Kristine R; Caudle, Kelly E; Smith, Colton; Pei, Deqing; Cheng, Cheng; Broeckel, Ulrich; Gaur, Aditya H; Hankins, Jane; Relling, Mary V; Haidar, Cyrine E

2017-01-01

Identify the functional status of the uridine-diphosphate glucuronyl transferase 1A1 (UGT1A1) -3279T>G (*60) variant. Retrospective review of clinically obtained serum bilirubin concentrations in pediatric patients to evaluate the association of the UGT1A1 -3279T>G (*60) variant with bilirubin concentrations and assessed linkage disequilibrium of the UGT1A1 -3279T>G (*60) and A(TA)7TAA (*28) variants. Total bilirubin concentration did not differ between patients who had a UGT1A1*1/*1 diplotype and patients homozygous for the UGT1A1 -3279T>G (*60/*60) variant. Total bilirubin concentration was lower in patients homozygous for the UGT1A1 -3279T>G (*60/*60) variant than in patients homozygous for the UGT1A1 A(TA)7TAA (*28/*28) variant (p < 0.01). The -3279T>G (*60) and A(TA)7TAA (*28) variants were in strong incomplete linkage disequilibrium in both black and white patients. The presence of the UGT1A1 -3279T>G (*60) variant is not associated with increased bilirubin concentrations.

Disturbance of Mammary UDP-Glucuronosyltransferase Represses Estrogen Metabolism and Exacerbates Experimental Breast Cancer.

PubMed

Zhou, Xueyan; Zheng, Ziqiang; Xu, Chang; Wang, Juan; Min, Mengjun; Zhao, Yun; Wang, Xi; Gong, Yinhan; Yin, Jiale; Guo, Meng; Guo, Dong; Zheng, Junnian; Zhang, Bei; Yin, Xiaoxing

2017-08-01

The progression of breast cancer is closely related to the levels of estrogens within the body. UDP-glucuronosyltransferase (UGT) is an important class of phase II metabolizing enzymes, playing a pivotal role in detoxifying steroid hormone. In the present study, we aim at uncovering the potential dysregulation pattern of UGT and its role in estrogen metabolism and in the pathogenesis of breast cancer. Female Sprague-Dawley rats were treated with 100 mg/kg dimethylbenz(a)anthracene (DMBA) to induce breast cancer. Our results showed that the expression and activity of UGT in mammary tissues were downregulated significantly in DMBA rats. Consistent with this, levels of estradiol, 4-hydroxylated estradiol, and 2-hydroxylated estradiol were increased in both mammary tissues and serum, supporting a notable accumulation of toxic estrogen species in the target tissue of breast cancer. In addition, we also observed the decreased cell migration, cell proliferation, and DNA damage in UGT-transfected MCF-7 cells, suggesting a protective role of UGT against estrogen-induced mammary carcinogenesis. Taken together, these results indicated that accumulation of estrogens induced by UGT deficiency is a critical factor to induce the development of breast cancer. UGT contributes to estrogen elimination, and its glucuronidation capacity influences the estrogen signaling pathway and the pathogenesis of breast cancer. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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

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

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

Soy beans contain genistein, a natural compound that has estrogenic effects because it binds the estrogen receptor with relatively high affinity. Genistein is therefore the most important environmental estrogen in the human diet. Detoxification of genistein is mediated through conjugation by UDP-glucuronyltransferase 1 and 2 (UGT1 and UGT2) isoenzymes. Gunn rats have a genetic deficiency in UGT1 activity, UGT2 activities are not affected. Because our Gunn rats stopped breeding after the animal chow was changed to a type with much higher soy content, we examined the mechanism behind this soy diet induced infertility. Gunn and control rats were fed dietsmore » with and without genistein. In these rats, plasma levels of genistein and metabolites, fertility and reproductive parameters were determined. Enzyme assays showed reduced genistein UGT activity in Gunn rats, as compared to wild type rats. Female Gunn rats were completely infertile on a genistein diet, wild type rats were fertile. Genistein diet caused a persistent estrus, lowered serum progesterone and inhibited development of corpora lutea in Gunn rats. Concentrations of total genistein in Gunn and control rat plasma were identical and within the range observed in humans after soy consumption. However, Gunn rat plasma contained 25% unconjugated genistein, compared to 3.6% in control rats. This study shows that, under conditions of reduced glucuronidation, dietary genistein exhibits a strongly increased estrogenic effect. Because polymorphisms that reduce UGT1 expression are prevalent in the human population, these results suggest a cautionary attitude towards the consumption of large amounts of soy or soy supplements. -- Highlights: ► Gunn rats are partially deficient in detoxification by UDP glucuronyltransferases. ► Female Gunn rats are infertile on a soy containing diet. ► Soy contains genistein, a potent phytoestrogen. ► Inefficient glucuronidation of genistein causes female infertility.« less

Effects of Andrographis paniculata and Orthosiphon stamineus extracts on the glucuronidation of 4-methylumbelliferone in human UGT isoforms.

PubMed

Ismail, Sabariah; Hanapi, Nur Aziah; Ab Halim, Mohd Rohaimi; Uchaipichat, Verawan; Mackenzie, Peter I

2010-05-14

The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU) by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA) as the cofactor, MgCl(2), cell lysate of respective isoform, and 4MU at the approximate apparent K(m) or S(50) value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 microg/mL and 0.01, 0.10, 1.0, 10 and 50 microg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC(50) 1.70 microg/mL for UGT1A3, 2.57 microg/mL for UGT1A8, 2.82 microg/mL for UGT2B7, 5.00 micorg/mL for UGT1A1, 5.66 microg/mL for UGT1A6, 9.88 microg/mL for UGT1A7 and 15.66 microg/mL for UGT1A10). Both extracts showed less than 70% inhibition of UGT2B15, so the IC(50) values were >50 microg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa.

PubMed

Wetterhorn, Karl M; Gabardi, Kaitlyn; Michlmayr, Herbert; Malachova, Alexandra; Busman, Mark; McCormick, Susan P; Berthiller, Franz; Adam, Gerhard; Rayment, Ivan

2017-12-19

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

Functional polymorphisms in UDP-glucuronosyltransferases and recurrence in tamoxifen-treated breast cancer survivors

PubMed Central

Ahern, Thomas P.; Christensen, Mariann; Cronin-Fenton, Deirdre P.; Lunetta, Kathryn L.; Søiland, Håvard; Gjerde, Jennifer; Garne, Jens Peter; Rosenberg, Carol L.; Silliman, Rebecca A.; Sørensen, Henrik Toft; Lash, Timothy L.; Hamilton-Dutoit, Stephen

2011-01-01

Background Tamoxifen is oxidized by cytochrome-P450 enzymes (e.g., CYP2D6) to two active metabolites, which are eliminated via glucuronidation by UDP-glucuronosyltransferases (UGTs). We measured the association between functional polymorphisms in key UGTs (UGT2B15*2, UGT2B7*2, and UGT1A8*3) and the recurrence rate among breast cancer survivors. Methods We used the Danish Breast Cancer Cooperative Group registry to identify 541 cases of recurrent breast cancer among women with estrogen receptor-positive tumors treated with tamoxifen for at least one year (ER+/TAM+), and 300 cases of recurrent breast cancer among women with estrogen receptor-negative tumors who were not treated with tamoxifen (ER−/TAM−). We matched 1 control to each case on ER status, menopausal status, stage, calendar period, and county. UGT polymorphisms were genotyped from archived primary tumors. We estimated the recurrence odds ratio for the UGT polymorphisms using logistic regression models, with and without stratification on CYP2D6*4 genotype. Results No UGT polymorphism was associated with breast cancer recurrence in either the ER+/TAM+ or ER-/TAM- groups [in the ER+TAM+ group, compared with two normal alleles: adjusted OR for two UGT2B15*2 variant alleles = 1.0 (95% CI: 0.70, 1.5); adjusted OR for two for UGT2B7*2 variant alleles = 0.91 (95% CI: 0.65, 1.3); adjusted OR for 1 or 2 UGT1A8*3 variant alleles = 0.75 (0.41, 1.4)]. Associations were similar within strata of CYP2D6*4 genotype. Conclusions Functional polymorphisms in key tamoxifen-metabolizing enzymes were not associated with breast cancer recurrence risk. Impact Our results do not support the genotyping of key metabolic enzyme polymorphisms to predict response to tamoxifen therapy. PMID:21750172

Correlation of UGT1A1(*)28 and (*)6 polymorphisms with irinotecan-induced neutropenia in Thai colorectal cancer patients.

PubMed

Atasilp, Chalirmporn; Chansriwong, Pichai; Sirachainan, Ekapob; Reungwetwattana, Thanyanan; Chamnanphon, Montri; Puangpetch, Apichaya; Wongwaisayawan, Sansanee; Sukasem, Chonlaphat

2016-02-01

UDP-glucuronosyltransferase1A1 (UGT1A1) polymorphisms have been related with irinotecan toxicity. The purpose of this study was to determine the associations between UGT1A1(*)28 and (*)6 polymorphisms and irinotecan toxicity in Thai patients with metastatic colorectal cancer. 44 metastatic colorectal cancer patients received irinotecan-based chemotherapy. Hematologic toxicities were determined in the first and second cycles of treatment. The genotypes of UGT1A1(*)28 and (*)6 were analyzed by pyrosequencing technique. The frequencies of genetic testing for UGT1A1(*)28 and (*)6 polymorphisms were 22.8% (TA6/TA7; 20.5%, TA7/TA7; 2.3%) and 15.9% (GA), respectively. No patients had the homozygous UGT1A1(*)6 (AA). Neither UGT1A1(*)28 nor UGT1A1(*)6 polymorphisms were significantly associated with severe hematologic toxicities. However, analysis of UGT1A1(*)28 and (*)6 in combination revealed an association with severe neutropenia in the first and second cycles (P = 0.044, P = 0.017, respectively). Both UGT1A1(*)28 and (*)6 polymorphisms may have an increased risk of irinotecan-induced neutropenia in Thai colorectal cancer patients. Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

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

PubMed Central

2012-01-01

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

In vitro inhibition of human UGT isoforms by ritonavir and cobicistat.

PubMed

Algeelani, Sara; Alam, Novera; Hossain, Md Amin; Mikus, Gerd; Greenblatt, David J

2018-08-01

1. Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer's data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat. 2. Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors. 3. The 50% inhibitory concentrations (IC 50 ) of cobicistat and ritonavir were at least 50 µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC 50 0.7 mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC 50 17 µM). 4. Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.

Optimization of a UDP-glucuronosyltransferase assay for trout ...

EPA Pesticide Factsheets

An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosphoglucuronic acid (UDPGA; a necessary cofactor), alamethicin (a pore-forming agent added to eliminate latency), and substrate (p-nitrophenol). Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. All studies were performed under physiological conditions (pH 7.8, 11 °C) to support ongoing development of methods for extrapolating in vitro rates of biotransformation to the intact animal. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. The purpose of the present study was to optimize an existing in vitro assay for hepatic UGT activity in rainbow trout. The original assay, adapted here for use with trout S9 fractions, was updated by incorporating a membrane disrupting agent (alamethicin) to reduce latency. Additional experiments were conducted to evaluate

Pharmacokinetic drug interactions of morphine, codeine, and their derivatives: theory and clinical reality, Part II.

PubMed

Armstrong, Scott C; Cozza, Kelly L

2003-01-01

Pharmacokinetic drug-drug interactions with codeine, dihydrocodeine, hydrocodone, oxycodone, and buprenorphine are reviewed in this column. These compounds have a very similar chemical structure to morphine. Unlike morphine, which is metabolized chiefly through conjugation reactions with uridine diphosphate glucuronosyl transferase (UGT) enzymes, these five drugs are metabolized both through oxidative reactions by the cytochrome P450 (CYP450) enzyme and conjugation by UGT enzymes. There is controversy as to whether codeine, dihydrocodeine, and hydrocodone are actually prodrugs requiring activation by the CYP450 2D6 enzyme or UGT enzymes. Oxycodone and buprenorphine, however, are clearly not prodrugs and are metabolized by the CYP450 2D6 and 3A4 enzymes, respectively. Knowledge of this metabolism assists in the understanding for the potential of drug-drug interactions with these drugs. This understanding is important so that clinicians can choose the proper dosages for analgesia and anticipate potential drug-drug interactions.

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

PubMed

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

2010-07-01

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

Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077

PubMed Central

Michlmayr, Herbert; Varga, Elisabeth; Lupi, Francesca; Malachová, Alexandra; Hametner, Christian; Berthiller, Franz; Adam, Gerhard

2017-01-01

Zearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-infected cereals. Glucosylation is an important plant defense mechanism and generally reduces the acute toxicity of mycotoxins to humans and animals. Toxicological information about ZEN-glucosides is limited due to the unavailability of larger amounts required for animal studies. HvUGT14077, a recently-validated ZEN-conjugating barley UDP-glucosyltransferase was expressed in Escherichia coli, affinity purified, and characterized. HvUGT14077 possesses high affinity (Km = 3 µM) and catalytic efficiency (kcat/Km = 190 s−1·mM−1) with ZEN. It also efficiently glucosylates the phase-I ZEN-metabolites α-zearalenol and β-zearalenol, with kcat/Km of 40 and 74 s−1·mM−1, respectively. HvUGT14077 catalyzes O-glucosylation at C-14 and C-16 with preference of 14-glucoside synthesis. Furthermore, relatively slow consecutive formation of 14,16-di-glucosides was observed; their structures were tentatively identified by mass spectrometry and for ZEN-14,16-di-glucoside confirmed by nuclear magnetic resonance spectroscopy. Recombinant HvUGT14077 allowed efficient preparative synthesis of ZEN-glucosides, yielding about 90% ZEN-14-glucoside and 10% ZEN-16-glucoside. The yield of ZEN-16-glucoside could be increased to 85% by co-incubation with a β-glucosidase highly selective for ZEN-14-glucoside. Depletion of the co-substrate UDP-glucose was counteracted by a sucrose synthase based regeneration system. This strategy could also be of interest to increase the yield of minor glucosides synthesized by other glucosyltransferases. PMID:28208765

Androgen receptor signals regulate UDP-glucuronosyltransferases in the urinary bladder: a potential mechanism of androgen-induced bladder carcinogenesis.

PubMed

Izumi, Koji; Zheng, Yichun; Hsu, Jong-Wei; Chang, Chawnshang; Miyamoto, Hiroshi

2013-02-01

UDP-glucuronosyltransferases (UGTs), major phase II drug metabolism enzymes, play an important role in urinary bladder cancer initiation by detoxifying carcinogens. We aimed to determine if androgens regulate UGT expression via the androgen receptor (AR) pathway in the bladder. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to assess UGT1A levels in the normal urothelium SVHUC cell line stably expressed with AR and in bladder tissues from AR knockout (ARKO) and castrated male mice. Immunohistochemistry was also performed in radical cystectomy specimens. Dihydrotestosterone (DHT) treatment in SVHUC-AR reduced mRNA expression of all the UGT1A subtypes (19-75% decrease), and hydroxyflutamide antagonized the DHT effects. In contrast, DHT showed only marginal effects on UGT1A expression in SVHUC-Vector. Of note were higher expression levels of UGT1As in SVHUC-Vector than in SVHUC-AR. In ARKO mice, all the Ugt1a subtypes were up-regulated, compared to wild-type littermates. In wild-type male mice, castration increased the expression of Ugt1a8, Ugt1a9, and Ugt1a10. Additionally, wild-type female mice had higher levels of Ugt1a than wild-type males. Immunohistochemical studies showed strong (3+) UGT1A staining in 11/24 (46%) cancer tissues, which was significantly lower than in corresponding benign tissues [17/18 (94%) cases (P = 0.0009)]. These results suggest that androgen-mediated AR signals promote bladder carcinogenesis by down-regulating the expression of UGTs in the bladder. Copyright © 2011 Wiley Periodicals, Inc.

Inhibition of human UDP-glucuronosyltransferase enzymes by lapatinib, pazopanib, regorafenib and sorafenib: Implications for hyperbilirubinemia.

PubMed

Miners, John O; Chau, Nuy; Rowland, Andrew; Burns, Kushari; McKinnon, Ross A; Mackenzie, Peter I; Tucker, Geoffrey T; Knights, Kathleen M; Kichenadasse, Ganessan

2017-04-01

Kinase inhibitors (KIs) are a rapidly expanding class of drugs used primarily for the treatment of cancer. Data relating to the inhibition of UDP-glucuronosyltransferase (UGT) enzymes by KIs is sparse. However, lapatinib (LAP), pazopanib (PAZ), regorafenib (REG) and sorafenib (SOR) have been implicated in the development of hyperbilirubinemia in patients. This study aimed to characterise the role of UGT1A1 inhibition in hyperbilirubinemia and assess the broader potential of these drugs to perpetrate drug-drug interactions arising from UGT enzyme inhibition. Twelve recombinant human UGTs from subfamilies 1A and 2B were screened for inhibition by LAP, PAZ, REG and SOR. IC 50 values for the inhibition of all UGT1A enzymes, except UGT1A3 and UGT1A4, by the four KIs were <10μM. LAP, PAZ, REG and SOR inhibited UGT1A1-catalysed bilirubin glucuronidation with mean IC 50 values ranging from 34nM (REG) to 3734nM (PAZ). Subsequent kinetic experiments confirmed that REG and SOR were very potent inhibitors of human liver microsomal β-estradiol glucuronidation, an established surrogate for bilirubin glucuronidation, with mean K i values of 20 and 33nM, respectively. K i values for LAP and PAZ were approximately 1- and 2-orders of magnitude higher than those for REG and SOR. REG and SOR were equipotent inhibitors of human liver microsomal UGT1A9 (mean K i 678nM). REG and SOR are the most potent inhibitors of a human UGT enzyme identified to date. In vitro-in vivo extrapolation indicates that inhibition of UGT1A1 contributes significantly to the hyperbilirubinemia observed in patients treated with REG and SOR, but not with LAP and PAZ. Inhibition of other UGT1A1 substrates in vivo is likely. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of the inhibition potentials of icotinib and erlotinib against human UDP-glucuronosyltransferase 1A1.

PubMed

Cheng, Xuewei; Lv, Xia; Qu, Hengyan; Li, Dandan; Hu, Mengmeng; Guo, Wenzhi; Ge, Guangbo; Dong, Ruihua

2017-11-01

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a key role in detoxification of many potentially harmful compounds and drugs. UGT1A1 inhibition may bring risks of drug-drug interactions (DDIs), hyperbilirubinemia and drug-induced liver injury. This study aimed to investigate and compare the inhibitory effects of icotinib and erlotinib against UGT1A1, as well as to evaluate their potential DDI risks via UGT1A1 inhibition. The results demonstrated that both icotinib and erlotinib are UGT1A1 inhibitors, but the inhibitory effect of icotinib on UGT1A1 is weaker than that of erlotinib. The IC 50 values of icotinib and erlotinib against UGT1A1-mediated NCHN- O -glucuronidation in human liver microsomes (HLMs) were 5.15 and 0.68 μmol/L, respectively. Inhibition kinetic analyses demonstrated that both icotinib and erlotinib were non-competitive inhibitors against UGT1A1-mediated glucuronidation of NCHN in HLMs, with the K i values of 8.55 and 1.23 μmol/L, respectively. Furthermore, their potential DDI risks via UGT1A1 inhibition were quantitatively predicted by the ratio of the areas under the concentration-time curve (AUC) of NCHN. These findings are helpful for the medicinal chemists to design and develop next generation tyrosine kinase inhibitors with improved safety, as well as to guide reasonable applications of icotinib and erlotinib in clinic, especially for avoiding their potential DDI risks via UGT1A1 inhibition.

Comparison of the inhibitory effects of tolcapone and entacapone against human UDP-glucuronosyltransferases

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

Lv, Xia

2016-06-15

Tolcapone and entacapone are two potent catechol-O-methyltransferase (COMT) inhibitors with a similar skeleton and displaying similar pharmacological activities. However, entacapone is a very safe drug used widely in the treatment of Parkinson's disease, while tolcapone is only in limited use for Parkinson's patients and needs careful monitoring of hepatic functions due to hepatotoxicity. This study aims to investigate and compare the inhibitory effects of entacapone and tolcapone on human UDP-glucosyltransferases (UGTs), as well as to evaluate the potential risks from the view of drug-drug interactions (DDI). The results demonstrated that both tolcapone and entacapone exhibited inhibitory effects on UGT1A1, UGT1A7,more » UGT1A9 and UGT1A10. In contrast to entacapone, tolcapone exhibited more potent inhibitory effects on UGT1A1, UGT1A7, and UGT1A10, while their inhibitory potentials against UGT1A9 were comparable. It is noteworthy that the inhibition constants (K{sub i}) of tolcapone and entacapone against bilirubin-O-glucuronidation in human liver microsomes (HLM) are determined as 0.68 μM and 30.82 μM, respectively, which means that the inhibition potency of tolcapone on UGT1A1 mediated bilirubin-O-glucuronidation in HLM is much higher than that of entacapone. Furthermore, the potential risks of tolcapone or entacapone via inhibition of human UGT1A1 were quantitatively predicted by the ratio of the areas under the plasma drug concentration-time curve (AUC). The results indicate that tolcapone may result in significant increase in AUC of bilirubin or the drugs primarily metabolized by UGT1A1, while entacapone is unlikely to cause a significant DDI through inhibition of UGT1A1. - Highlights: • Tolcapone and entacapone exhibited preferential inhibition against UGT1A enzymes. • In contrast to entacapone, tolcapone exhibited more potent inhibitory effects on human UGT1A1, 1 A7 and 1 A10. • Tolcapone may lead to significant increase in AUC of bilirubin. �

Biotransformation of anthelmintics and the activity of drug-metabolizing enzymes in the tapeworm Moniezia expansa.

PubMed

Prchal, Lukáš; Bártíková, Hana; Bečanová, Aneta; Jirásko, Robert; Vokřál, Ivan; Stuchlíková, Lucie; Skálová, Lenka; Kubíček, Vladimír; Lamka, Jiří; Trejtnar, František; Szotáková, Barbora

2015-04-01

The sheep tapeworm Moniezia expansa is very common parasite, which affects ruminants such as sheep, goats as well as other species. The benzimidazole anthelmintics albendazole (ABZ), flubendazole (FLU) and mebendazole (MBZ) are often used to treat the infection. The drug-metabolizing enzymes of helminths may alter the potency of anthelmintic treatment. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (ABZ, MBZ and FLU) in M. expansa. Activities of biotransformation enzymes were determined in subcellular fractions. Metabolites of the anthelmintics were detected and identified using high performance liquid chromatography/ultra-violet/VIS/fluorescence or ultra-high performance liquid chromatography/mass spectrometry. Reduction of MBZ, FLU and oxidation of ABZ were proved as well as activities of various metabolizing enzymes. Despite the fact that the conjugation enzymes glutathione S-transferase, UDP-glucuronosyl transferase and UDP-glucosyl transferase were active in vitro, no conjugated metabolites of anthelmintics were identified either ex vivo or in vitro. The obtained results indicate that sheep tapeworm is able to deactivate the administered anthelmintics, and thus protects itself against their action.

Preparation of reference material for UGT1A1 (TA)n polymorphism genotyping.

PubMed

Mlakar, Vid; Mlakar, Simona Jurković; Marc, Janja; Ostanek, Barbara

2014-08-05

Gilbert's syndrome is one of the most common metabolic syndromes in the human population characterised by mild unconjugated hyperbilirubinemia resulting from reduced activity of the bilirubin conjugating enzyme UDP-glucuronosyltransferase (UGT1A1). Although Gilbert's syndrome is usually quite benign UGT1A1(TA)n genotyping is important in exclusion of more serious causes of hyperbilirubinemia and since it has significant implications for personalised medicine. The aim of our study was to develop plasmid based reference materials which could be used for UGT1A1(TA)n genotyping. Plasmids were generated using recombinant DNA technology and their number of repeats as well as the entire sequence verified by Sanger sequencing. Their suitability as reference materials was tested using sizing by capillary electrophoresis and denaturing high performance liquid chromatography. Plasmids containing all four different alleles (TA)5, (TA)6, (TA)7 and (TA)8 that are present in the human population as well as a plasmid with (TA)4 repeats were successfully generated. Prepared plasmid reference materials allow the creation of all possible UGT1A1(TA)n polymorphism genotypes and can serve as an efficient substitute for the human genomic DNA reference material in routine genotyping and in the development of new genotyping tests. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of Isoform-Specific UGT Metabolism to Determine and Describe Rates and Profiles of Glucuronidation of Wogonin and Oroxylin A by Human Liver and Intestinal Microsomes

PubMed Central

Zhou, Qiong; Zheng, Zhijie; Xia, Bijun; Tang, Lan; Lv, Chang; Liu, Wei; Liu, Zhongqiu; Hu, Ming

2010-01-01

Purposes Glucuronidation via UDP-glucuronosyltransferases (or UGTs) is a major metabolic pathway. The purposes of this study are to determine the UGT-isoform specific metabolic fingerprint (or GSMF) of wogonin and oroxylin A, and to use isoform-specific metabolism rates and kinetics to determine and describe their glucuronidation behaviors in tissue microsomes. Methods In vitro glucuronidation rates and profiles were measured using expressed UGTs and human intestinal and liver microsomes. Results GSMF experiments indicated that both flavonoids were metabolized mainly by UGT1As, with major contributions from UGT1A3 and UGT1A7-1A10. Isoform-specific metabolism showed that kinetic profiles obtained using expressed UGT1A3 and UGT1A7-1A10 could fit to known kinetic models. Glucuronidation of both flavonoids in human intestinal and liver microsomes followed simple Michaelis-Menten kinetics. A comparison of the kinetic parameters and profiles suggests that UGT1A9 is likely the main isoform responsible for liver metabolism. In contrast, a combination of UGT1As with a major contribution from UGT1A10 contributed to their intestinal metabolism. Correlation studies clearly showed that UGT isoform-specific metabolism could describe their metabolism rates and profiles in human liver and intestinal microsomes. Conclusion GSMF and isoform-specific metabolism profiles can determine and describe glucuronidation rates and profiles in human tissue microsomes. PMID:20411407

Differences in UGT1A1, UGT1A7, and UGT1A9 polymorphisms between Uzbek and Japanese populations.

PubMed

Maeda, Hiromichi; Hazama, Shoichi; Shavkat, Abdiev; Okamoto, Ken; Oba, Koji; Sakamoto, Junichi; Takahashi, Kenichi; Oka, Masaki; Nakamura, Daisuke; Tsunedomi, Ryouichi; Okayama, Naoko; Mishima, Hideyuki; Kobayashi, Michiya

2014-06-01

Uridine-diphosphate glucuronosyltransferase 1A (UGT1A) is a key enzyme involved in irinotecan metabolism, and polymorphisms in the UGT1A gene are associated with irinotecan-induced toxicity. The aim of this study was to elucidate the allele frequencies of UGT1A polymorphisms in healthy Uzbek volunteers, and to compare them with those of the Japanese population. A total of 97 healthy volunteers from Uzbekistan were enrolled and blood samples were collected from each participant. Genotyping analysis was performed by fragment size analysis for UGT1A1*28, direct sequencing for UGT1A7*3 and UGT1A9*22, and TaqMan assays for UGT1A1*93, UGT1A1*6, UGT1A1*27, UGT1A1*60, and UGT1A7*12. The frequencies of polymorphisms were compared with the Japanese population by using the data previously reported from our study group. When the Uzbek and Japanese populations were compared, heterozygotes or homozygotes for UGT1A1*28, UGT1A1*60, and UGT1A1*93 were significantly more frequent in the Uzbek population (P < 0.01). The rate of UGT1A7*12 was not significantly different between the two populations, whereas UGT1A1*6 and UGT1A9*22 were significantly less frequent in the Uzbek population (P < 0.05). UGT1A7*1 were less prevalent in the Uzbek population than in the Japanese population (P < 0.01). The Uzbek population has different frequencies of polymorphisms in UGT1A genes compared with the Japanese population. A comprehensive study of the influence of UGT1A1 polymorphisms on the risk of irinotecan-induced toxicity is necessary for optimal use of irinotecan treatment.

UDP-Glycosyltransferases from the UGT73C Subfamily in Barbarea vulgaris Catalyze Sapogenin 3-O-Glucosylation in Saponin-Mediated Insect Resistance1[W][OA

PubMed Central

Augustin, Jörg M.; Drok, Sylvia; Shinoda, Tetsuro; Sanmiya, Kazutsuka; Nielsen, Jens Kvist; Khakimov, Bekzod; Olsen, Carl Erik; Hansen, Esben Halkjær; Kuzina, Vera; Ekstrøm, Claus Thorn; Hauser, Thure; Bak, Søren

2012-01-01

Triterpenoid saponins are bioactive metabolites that have evolved recurrently in plants, presumably for defense. Their biosynthesis is poorly understood, as is the relationship between bioactivity and structure. Barbarea vulgaris is the only crucifer known to produce saponins. Hederagenin and oleanolic acid cellobioside make some B. vulgaris plants resistant to important insect pests, while other, susceptible plants produce different saponins. Resistance could be caused by glucosylation of the sapogenins. We identified four family 1 glycosyltransferases (UGTs) that catalyze 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. Among these, UGT73C10 and UGT73C11 show highest activity, substrate specificity and regiospecificity, and are under positive selection, while UGT73C12 and UGT73C13 show lower substrate specificity and regiospecificity and are under purifying selection. The expression of UGT73C10 and UGT73C11 in different B. vulgaris organs correlates with saponin abundance. Monoglucosylated hederagenin and oleanolic acid were produced in vitro and tested for effects on P. nemorum. 3-O-β-d-Glc hederagenin strongly deterred feeding, while 3-O-β-d-Glc oleanolic acid only had a minor effect, showing that hydroxylation of C23 is important for resistance to this herbivore. The closest homolog in Arabidopsis thaliana, UGT73C5, only showed weak activity toward sapogenins. This indicates that UGT73C10 and UGT73C11 have neofunctionalized to specifically glucosylate sapogenins at the C3 position and demonstrates that C3 monoglucosylation activates resistance. As the UGTs from both the resistant and susceptible types of B. vulgaris glucosylate sapogenins and are not located in the known quantitative trait loci for resistance, the difference between the susceptible and resistant plant types is determined at an earlier stage in saponin biosynthesis. PMID:23027665

Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein

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

Higgins, Larry G.; Cavin, Christophe; Itoh, Ken

2008-02-01

Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase 1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor 2) than those lacking the transcription factor. Basal levels of mRNAs for NQO1, GSTA1,more » UGT1A6 and GCLC were lower in tissues from nrf2{sup -/-} mice than from nrf2{sup +/+} mice, but modest induction occurred in the mutant animals. Treatment of mouse embryonic fibroblasts (MEFs) from nrf2{sup +/+} mice with either coffee or the coffee-specific diterpenes cafestol and kahweol (C + K) increased NQO1 mRNA up to 9-fold. MEFs from nrf2{sup -/-} mice expressed less NQO1 mRNA than did wild-type MEFs, but NQO1 was induced modestly by coffee or C + K in the mutant fibroblasts. Transfection of MEFs with nqo1-luciferase reporter constructs showed that induction by C + K was mediated primarily by Nrf2 and required the presence of an antioxidant response element in the 5'-upstream region of the gene. Luciferase reporter activity did not increase following treatment of MEFs with 100 {mu}mol/l furan, suggesting that this ring structure within C + K is insufficient for gene induction. Priming of nrf2{sup +/+} MEFs, but not nrf2{sup -/-} MEFs, with C + K conferred 2-fold resistance towards acrolein.« less

Human hepatocytes loaded in 3D bioprinting generate mini-liver.

PubMed

Zhong, Cheng; Xie, Hai-Yang; Zhou, Lin; Xu, Xiao; Zheng, Shu-Sen

2016-10-01

Because of an increasing discrepancy between the number of potential liver graft recipients and the number of organs available, scientists are trying to create artificial liver to mimic normal liver function and therefore, to support the patient's liver when in dysfunction. 3D printing technique meets this purpose. The present study was to test the feasibility of 3D hydrogel scaffolds for liver engineering. We fabricated 3D hydrogel scaffolds with a bioprinter. The biocompatibility of 3D hydrogel scaffolds was tested. Sixty nude mice were randomly divided into four groups, with 15 mice in each group: control, hydrogel, hydrogel with L02 (cell line HL-7702), and hydrogel with hepatocyte growth factor (HGF). Cells were cultured and deposited in scaffolds which were subsequently engrafted into livers after partial hepatectomy and radiation-induced liver damage (RILD). The engrafted tissues were examined after two weeks. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total bilirubin, CYP1A2, CYP2C9, glutathione S-transferase (a-GST), and UDP-glucuronosyl transferase (UGT-2) were compared among the groups. Hematoxylin-eosin (HE) staining and immunohistochemistry of cKit and cytokeratin 18 (CK18) of engrafted tissues were evaluated. The survival time of the mice was also compared among the four groups. 3D hydrogel scaffolds did not impact the viability of cells. The levels of ALT, AST, albumin, total bilirubin, CYP1A2, CYP2C9, a-GST and UGT-2 were significantly improved in mice engrafted with 3D scaffold loaded with L02 compared with those in control and scaffold only (P<0.05). HE staining showed clear liver tissue and immunohistochemistry of cKit and CK18 were positive in the engrafted tissue. Mice treated with 3D scaffold+L02 cells had longer survival time compared with those in control and scaffold only (P<0.05). 3D scaffold has the potential of recreating liver tissue and partial liver functions and can be used in the

UGT1A1*6 polymorphism is most predictive of severe neutropenia induced by irinotecan in Japanese cancer patients.

PubMed

Onoue, Masahide; Terada, Tomohiro; Kobayashi, Masahiko; Katsura, Toshiya; Matsumoto, Shigemi; Yanagihara, Kazuhiro; Nishimura, Takafumi; Kanai, Masashi; Teramukai, Satoshi; Shimizu, Akira; Fukushima, Masanori; Inui, Ken-ichi

2009-04-01

Gene polymorphisms of the UDP-glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) contribute to individual variations in adverse events among patients administered irinotecan, and the distribution of the polymorphisms shows large interethnic differences. Variation in the solute carrier organic anion-transporter family, member 1B1 (SLCO1B1) gene also has a significant effect on the disposition of irinotecan in Asian cancer patients. In the present study, we evaluated the association of genetic polymorphisms of UGT1A1 and SLCO1B1 with irinotecanrelated neutropenia in Japanese cancer patients. One hundred and thirty-five consecutive patients treated with irinotecan were enrolled. Genotypes of UGT1A1 (*60, *28, *6, and *27) and SLCO1B1 (*1b, *5, and haplotype *15) were determined by direct sequencing. Severe neutropenia refers to events observed during the first cycle of irinotecan treatment. Severe neutropenia was observed in 29 patients (22%). Six patients were homozygous and 48 heterozygous for UGT1A1*6. Only 1 patient was homozygous for UGT1A1*28. Homozygosity for UGT1A1*6 was associated with a high risk of severe neutropenia (odds ratio [OR], 7.78; 95% confidence interval [CI], 1.36 to 44.51). No significant association was found between severe neutropenia and other UGT1A1 polymorphisms or SLCO1B1 polymorphisms. These findings suggest that the UGT1A1*6 polymorphism is a potential predictor of severe neutropenia caused by irinotecan in Japanese cancer patients.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-02-01

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

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

PubMed

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

2010-04-09

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. Copyright 2009 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-01-01

Curcuma xanthorrhiza is a native Indonesian plant and traditionally utilized for a range of illness including liver damage, hypertension, diabetes, and cancer. The study determined the effects of C. xanthorrhiza extracts (ethanol and aqueous) and their constituents (curcumene and xanthorrhizol) on UDP-glucuronosyltransferase (UGT) and glutathione transferase (GST) activities. The inhibition studies were evaluated both in rat liver microsomes and in human recombinant UGT1A1 and UGT2B7 enzymes. p-nitrophenol and beetle luciferin were used as the probe substrates for UGT assay while 1-chloro-2,4-dinitrobenzene as the probe for GST assay. The concentrations of extracts studied ranged from 0.1 to 1000 μg/mL while for constituents ranged from 0.01 to 500 μM. In rat liver microsomes, UGT activity was inhibited by the ethanol extract (IC 50 =279.74 ± 16.33 μg/mL). Both UGT1A1 and UGT2B7 were inhibited by the ethanol and aqueous extracts with IC 50 values ranging between 9.59-22.76 μg/mL and 110.71-526.65 μg/Ml, respectively. Rat liver GST and human GST Pi-1 were inhibited by ethanol and aqueous extracts, respectively (IC 50 =255.00 ± 13.06 μg/mL and 580.80 ± 18.56 μg/mL). Xanthorrhizol was the better inhibitor of UGT1A1 (IC 50 11.30 ± 0.27 μM) as compared to UGT2B7 while curcumene did not show any inhibition. For GST, both constituents did not show any inhibition. These findings suggest that C. xanthorrhiza have the potential to cause herb-drug interaction with drugs that are primarily metabolized by UGT and GST enzymes. Findings from this study would suggest which of Curcuma xanthorrhiza extracts and constituents that would have potential interactions with drugs which are highly metabolized by UGT and GST enzymes. Further clinical studies can then be designed if needed to evaluate the in vivo pharmacokinetic relevance of these interactions Abbreviations Used : BSA: Bovine serum albumin, CAM: Complementary and alternative medicine, cDNA: Complementary

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

PubMed Central

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

2018-01-01

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

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

PubMed

Fujiwara, Ryoichi; Maruo, Yoshihiro; Chen, Shujuan; Tukey, Robert H

2015-11-15

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepatic tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed Central

Fujiwara, Ryoichi; Maruo, Yoshihiro; Chen, Shujuan; Tukey, Robert H.

2015-01-01

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepatic tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression. PMID:26342858

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

PubMed Central

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

2016-01-01

20(S)-protopanaxadiol (PPD), one of the representative aglycones of ginsenosides, has a broad spectrum of pharmacological activities. Although phase I metabolism has been investigated extensively, information regarding phase II metabolism of this compound remains to be elucidated. Here, a glucuronidated metabolite of PPD in human liver microsomes (HLMs) and rat liver microsomes (RLMs) was unambiguously identified as PPD-3-O-β-d-glucuronide by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. The chemical inhibition and recombinant human UDP-Glucuronosyltransferase (UGT) isoforms assay showed that the PPD glucuronidation was mainly catalyzed by UGT1A4 in HLM, whereas UGT1A3 showed weak catalytic activity. In conclusion, PPD-3-O-β-d-glucuronide was first identified as the principal glucuronidation metabolite of PPD in HLMs, which was catalyzed by UGT1A4. PMID:27005621

Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP-glucuronosyltransferases.

PubMed

Jančová, Petra; Siller, Michal; Anzenbacherová, Eva; Křen, Vladimír; Anzenbacher, Pavel; Simánek, Vilím

2011-09-01

The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined. The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification. Hepatocytes and microsomes formed silybin A-7-O-β-D-glucuronides, B-7-O-β-D-glucuronides, A-20-O-β-D-glucuronides and B-20-O-β-D-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation. The predominant formation of 7-O-β-D-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

Impacts of the Glucuronidase Genotypes UGT1A4, UGT2B7, UGT2B15 and UGT2B17 on Tamoxifen Metabolism in Breast Cancer Patients

PubMed Central

Romero-Lorca, Alicia; Novillo, Apolonia; Gaibar, María; Bandrés, Fernando; Fernández-Santander, Ana

2015-01-01

Tamoxifen is used to prevent and treat estrogen-dependent breast cancer. It is described as a prodrug since most of its antiestrogen effects are exerted through its hydroxylated metabolites 4-OH-tamoxifen and endoxifen. In prior work, we correlated optimal plasma levels of these metabolites with certain genotypes of CYP2D6 and SULT1A2. This descriptive study examines correlations between concentrations of tamoxifen's glucuronide metabolites and genotypes UGT1A4 Pro24Thr, UGT1A4 Leu48Val, UGT2B7 His268Tyr, UGT2B15 Asp85YTyr UGT2B15 Lys523Thr and UGT2B17del in 132 patients with estrogen receptor-positive breast cancer under treatment with tamoxifen. Patients were genotyped by real-time and conventional PCR-RFLP. The glucuronides 4-OH-tamoxifen-N-glucuronide, 4-OH-tamoxifen-O-glucuronide and endoxifen-O-glucuronide were isolated from blood plasma and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Individuals who were homozygous for UGT1A448VAL showed significantly lower mean concentrations of both glucuronide metabolites compared to subjects genotyped as wt/wt plus wt/48Val (p=0.037 and p=0.031, respectively). Women homozygous for UGT2B7268Tyr also showed mean substrate/product ratios of 4-OH-tamoxifen/4-OH-tamoxifen-O-glucuronide and 4-OH-tamoxifen/4-OH-tamoxifen-N-glucuronide indicative of reduced glucuronidase activity compared to wt homozygotes or to heterozygotes for the polymorphism (p=0.005 and p=0.003, respectively). In contrast, UGT2B15 Lys523Thr and UGT2B17del were associated with possibly increased enzyme activity. Patients with at least one variant allele UGT2B15523Thr showed significantly higher 4-OH-tamoxifen-O-glucuronide and endoxifen-glucuronide levels (p=0.023 and p=0.025, respectively) indicating a variant gene-dose effect. Higher 4-OH-tamoxifen-N-glucuronide levels observed in UGT2B17del genotypes (p=0.042) could be attributed to a mechanism that compensates for the greater expression of other genes in UGT2B

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

PubMed

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

2018-02-01

More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Inhibitory Effects of Commonly Used Herbal Extracts on UDP-Glucuronosyltransferase 1A4, 1A6, and 1A9 Enzyme Activities

PubMed Central

Mohamed, Mohamed-Eslam F.

2011-01-01

The aim of this study was to investigate the effect of commonly used botanicals on UDP-glucuronosyltransferase (UGT) 1A4, UGT1A6, and UGT1A9 activities in human liver microsomes. The extracts screened were black cohosh, cranberry, echinacea, garlic, ginkgo, ginseng, milk thistle, saw palmetto, and valerian in addition to the green tea catechin epigallocatechin gallate (EGCG). Formation of trifluoperazine glucuronide, serotonin glucuronide, and mycophenolic acid phenolic glucuronide was used as an index reaction for UGT1A4, UGT1A6, and UGT1A9 activities, respectively, in human liver microsomes. Inhibition potency was expressed as the concentration of the inhibitor at 50% activity (IC50) and the volume in which the dose could be diluted to generate an IC50-equivalent concentration [volume/dose index (VDI)]. Potential inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. EGCG inhibited UGT1A4 with an IC50 value of (mean ± S.E.) 33.8 ± 3.1 μg/ml. Milk thistle inhibited both UGT1A6 and UGT1A9 with IC50 values of 59.5 ± 3.6 and 33.6 ± 3.1 μg/ml, respectively. Saw palmetto and cranberry weakly inhibited UGT1A6 and UGT1A9, respectively, with IC50 values >100 μg/ml. For each inhibition, VDI was calculated to determine the potential of achieving IC50-equivalent concentrations in vivo. VDI values for inhibitors indicate a potential for inhibition of first-pass glucuronidation of UGT1A4, UGT1A6, and UGT1A9 substrates. These results highlight the possibility of herb-drug interactions through modulation of UGT enzyme activities. Further clinical studies are warranted to investigate the in vivo extent of the observed interactions. PMID:21632963

UDP-Glucosyltransferases from Rice, Brachypodium, and Barley: Substrate Specificities and Synthesis of Type A and B Trichothecene-3-O-β-d-glucosides

PubMed Central

Malachová, Alexandra; Piątkowska, Marta; Hametner, Christian; Šofrová, Jana; Jaunecker, Günther; Häubl, Georg; Lemmens, Marc

2018-01-01

Trichothecene toxins are confirmed or suspected virulence factors of various plant-pathogenic Fusarium species. Plants can detoxify these to a variable extent by glucosylation, a reaction catalyzed by UDP-glucosyltransferases (UGTs). Due to the unavailability of analytical standards for many trichothecene-glucoconjugates, information on such compounds is limited. Here, the previously identified deoxynivalenol-conjugating UGTs HvUGT13248 (barley), OsUGT79 (rice) and Bradi5g03300 (Brachypodium), were expressed in E. coli, affinity purified, and characterized towards their abilities to glucosylate the most relevant type A and B trichothecenes. HvUGT13248, which prefers nivalenol over deoxynivalenol, is also able to conjugate C-4 acetylated trichothecenes (e.g., T-2 toxin) to some degree while OsUGT79 and Bradi5g03300 are completely inactive with C-4 acetylated derivatives. The type A trichothecenes HT-2 toxin and T-2 triol are the kinetically preferred substrates in the case of HvUGT13248 and Bradi5g03300. We glucosylated several trichothecenes with OsUGT79 (HT-2 toxin, T-2 triol) and HvUGT13248 (T-2 toxin, neosolaniol, 4,15-diacetoxyscirpenol, fusarenon X) in the preparative scale. NMR analysis of the purified glucosides showed that exclusively β-d-glucosides were formed regio-selectively at position C-3-OH of the trichothecenes. These synthesized standards can be used to investigate the occurrence and toxicological properties of these modified mycotoxins. PMID:29509722

UDP-Glucosyltransferases from Rice, Brachypodium, and Barley: Substrate Specificities and Synthesis of Type A and B Trichothecene-3-O-β-d-glucosides.

PubMed

Michlmayr, Herbert; Varga, Elisabeth; Malachová, Alexandra; Fruhmann, Philipp; Piątkowska, Marta; Hametner, Christian; Šofrová, Jana; Jaunecker, Günther; Häubl, Georg; Lemmens, Marc; Berthiller, Franz; Adam, Gerhard

2018-03-06

Trichothecene toxins are confirmed or suspected virulence factors of various plant-pathogenic Fusarium species. Plants can detoxify these to a variable extent by glucosylation, a reaction catalyzed by UDP-glucosyltransferases (UGTs). Due to the unavailability of analytical standards for many trichothecene-glucoconjugates, information on such compounds is limited. Here, the previously identified deoxynivalenol-conjugating UGTs HvUGT13248 (barley), OsUGT79 (rice) and Bradi5g03300 ( Brachypodium ), were expressed in E. coli , affinity purified, and characterized towards their abilities to glucosylate the most relevant type A and B trichothecenes. HvUGT13248, which prefers nivalenol over deoxynivalenol, is also able to conjugate C-4 acetylated trichothecenes (e.g., T-2 toxin) to some degree while OsUGT79 and Bradi5g03300 are completely inactive with C-4 acetylated derivatives. The type A trichothecenes HT-2 toxin and T-2 triol are the kinetically preferred substrates in the case of HvUGT13248 and Bradi5g03300. We glucosylated several trichothecenes with OsUGT79 (HT-2 toxin, T-2 triol) and HvUGT13248 (T-2 toxin, neosolaniol, 4,15-diacetoxyscirpenol, fusarenon X) in the preparative scale. NMR analysis of the purified glucosides showed that exclusively β-D-glucosides were formed regio-selectively at position C-3-OH of the trichothecenes. These synthesized standards can be used to investigate the occurrence and toxicological properties of these modified mycotoxins.

Influence of substrates on the in vitro kinetics of steviol glucuronidation and interaction between steviol glycosides metabolites and UGT2B7.

PubMed

Chen, Jun-Ming; Xia, Yong-Mei; Zhang, Yan-Dong; Zhang, Tong-Tong; Peng, Qing-Rui; Fang, Yun

2018-06-01

Steviol glycosides, a natural sweetener, may perform bioactivities via steviol, their main metabolite in human digestion. The metabolising kinetics, i.e. glucuronidation kinetics and interaction between steviol glycosides or their metabolites and metabolising enzyme, are important for understanding the bioactivity and cytotoxicity. The present study investigated kinetics of steviol glucuronidation in human liver microsome and a recombinant human UDP-glucuronosyltransferases isomer, UGT2B7, along with molecular docking to analyse interaction between UGT2B7 and steviol or glucose. The active pocket of UGT2B7 is consisted of Arg352, Leu347, Lys343, Phe339, Tyr354, Lys355 and Leu353. The influence of stevioside, rebaudioside A, glucose and some chemotherapy reagents on the glucuronidation was also studied. The predicted hepatic clearence suggested that steviol could be classified as high-clearence drug. The steviol glycosides did not affect the glucuronidation of steviol notably.

Effect of UDP-Glucuronosyltransferase (UGT) 1A Polymorphism (rs8330 and rs10929303) on Glucuronidation Status of Acetaminophen

PubMed Central

Tahir, Imtiaz Mahmood; Iqbal, Tahira; Saleem, Sadaf; Perveen, Sofia; Farooqi, Aboubakker

2017-01-01

Interindividual variability in polymorphic uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) ascribed to genetic diversity is associated with relative glucuronidation level among individuals. The present research was aimed to study the effect of 2 important single nucleotide polymorphisms (SNPs; rs8330 and rs10929303) of UGT1A1 gene on glucuronidation status of acetaminophen in healthy volunteers (n = 109). Among enrolled volunteers, 54.13% were male (n = 59) and 45.87% were female (n = 50). The in vivo activity of UGT1A1 was investigated by high-performance liquid chromatography-based analysis of glucuronidation status (ie, acetaminophen and acetaminophen glucuronide) in human volunteers after oral intake of a single dose (1000 mg) of acetaminophen. The TaqMan SNP genotyping assay was used for UGT1A1 genotyping. The wild-type genotype (C/C) was observed the most frequent one for both SNPs (rs8330 and rs10929303) and associated with fast glucuronidator phenotypes. The distribution of variant genotype (G/G) for SNP rs8330 was observed in 5% of male and 8% of the female population; however, for SNP rs10929303, the G/G genotype was found in 8% of both genders. A trimodal distribution (fast, intermediate, and slow) based on phenotypes was observed. Among the male participants, the glucuronidation phenotypes were observed as 7% slow, 37% intermediate, and 56% fast glucuronidators; however, these findings for the females were slightly different as 8%, 32%, and 60% respectively. The k-statistics revealed a compelling evidence for good concordance between phenotype and genotype with a k value of 1.00 for SNP rs8330 and 0.966 for SNP rs10929303 in our population. PMID:28932176

UDP-Glucuronosyltransferase 1A Compromises Intracellular Accumulation and Anti-Cancer Effect of Tanshinone IIA in Human Colon Cancer Cells

PubMed Central

Liu, Miao; Wang, Qiong; Liu, Fang; Cheng, Xuefang; Wu, Xiaolan; Wang, Hong; Wu, Mengqiu; Ma, Ying; Wang, Guangji; Hao, Haiping

2013-01-01

Background and Purpose NAD(P)H: quinone oxidoreductase 1 (NQO1) mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation is the dominant metabolic pathway of tanshinone IIA (TSA), a promising anti-cancer agent. UGTs are positively expressed in various tumor tissues and play an important role in the metabolic elimination of TSA. This study aims to explore the role of UGT1A in determining the intracellular accumulation and the resultant apoptotic effect of TSA. Experimental Approach We examined TSA intracellular accumulation and glucuronidation in HT29 (UGT1A positive) and HCT116 (UGT1A negative) human colon cancer cell lines. We also examined TSA-mediated reactive oxygen species (ROS) production, cytotoxicity and apoptotic effect in HT29 and HCT116 cells to investigate whether UGT1A levels are directly associated with TSA anti-cancer effect. UGT1A siRNA or propofol, a UGT1A9 competitive inhibitor, was used to inhibit UGT1A expression or UGT1A9 activity. Key Results Multiple UGT1A isoforms are positively expressed in HT29 but not in HCT116 cells. Cellular S9 fractions prepared from HT29 cells exhibit strong glucuronidation activity towards TSA, which can be inhibited by propofol or UGT1A siRNA interference. TSA intracellular accumulation in HT29 cells is much lower than that in HCT116 cells, which correlates with high expression levels of UGT1A in HT29 cells. Consistently, TSA induces less intracellular ROS, cytotoxicity, and apoptotic effect in HT29 cells than those in HCT116 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol can decrease TSA glucuronidation and simultaneously improve its intracellular accumulation, as well as enhance TSA anti-cancer effect. Conclusions and Implications UGT1A can compromise TSA cytotoxicity via reducing its intracellular exposure and switching the NQO1-triggered redox cycle to metabolic elimination. Our study may shed a light in understanding the cellular pharmacokinetic and

Identification of UDP-glucuronosyltransferases 1A1, 1A3 and 2B15 as the main contributors to glucuronidation of bakuchiol, a natural biologically active compound.

PubMed

Li, Feng; Wang, Shuai; Lu, Danyi; Wang, Yifei; Dong, Dong; Wu, Baojian

2017-05-01

1. Bakuchiol, one of the main active compounds of Psoralea corylifolia, possesses a variety of pharmacological activities such as anti-tumor and anti-aging effects. Here, we aimed to characterize the glucuronidation of bakuchiol using human liver microsomes (HLM) and expressed UDP-glucuronosyltransferase (UGT) enzymes. 2. The glucuronide of bakuchiol was confirmed by liquid chromatography-mass spectrometry (LC-MS) and β-glucuronidase hydrolysis assay. Glucuronidation rates and kinetic parameters were derived by enzymatic incubation and model fitting. Activity correlation analyses were performed to identify the main UGT isoforms contributing to hepatic metabolism of bakuchiol. 3. Among the three UGT enzymes (i.e., UGT1A1, UGT1A3 and UGT2B15) capable of catalyzing bakuchiol glucuronidation, UGT2B15 showed the highest activity with a CL int value of 100 μl/min/nmol. Bakuchiol glucuronidation was strongly correlated with glucuronidation of 5-hydroxyrofecoxib (r = 0.933; p < 0.001), 3-O-glucuronidation of β-estradiol (r = 0.719; p < 0.01) and significantly correlated with 24-O-glucuronidation of CDCA (r = 0.594; p < 0.05). In addition, a marked species difference existed in hepatic glucuronidation of bakuchiol. 4. In conclusion, UGT1A1, UGT1A3 and UGT2B15 were identified as the main contributors to glucuronidation of bakuchiol.

Green cocoons in silkworm Bombyx mori resulting from the quercetin 5-O-glucosyltransferase of UGT86, is an evolved response to dietary toxins.

PubMed

Xu, Xu; Wang, Meng; Wang, Ying; Sima, Yanghu; Zhang, Dayan; Li, Juan; Yin, Weiming; Xu, Shiqing

2013-05-01

The glycosylation of UDP-glucosyltransferases (UGTs) is of great importance in the control and elimination of both endogenous and exogenous toxins. Bm-UGT10286 (UGT86) is the sole provider of UGT activity against the 5-O position of quercetin and directly influences the formation of green pigment in the Bombyx cocoon. To evaluate whether cocoon coloration evolved for mimetic purposes, we concentrated on the expression pattern of Ugt86 and the activities of the enzyme substrates. The expression of Ugt86 was not only detected in the cocoon absorbing and accumulating tissues such as the digestive tube and silk glands, but also in quantity in the detoxification tissues of the malpighian tubes and fat body, as well as in the gonads. As in the green cocoon strains, Ugt86 was clearly expressed in the yellow and white cocoon strains. In vitro, the fusion protein of UGT86 showed quercetin metabolic activity. Nevertheless, Ugt86 expression of 5th instar larvae was not up-regulated in the silk gland by exogenous quercetin. However, it was significantly up-regulated in the digestive tube and gonads (P < 0.05). A similar result was observed in experiments where larvae were exposed to rutin, an insect resistance inducer and growth inhibitor typically found in plants, and to 20-hydroxylecdysone (20E), an insect endocrine and plant source hormone. On the contrary, up-regulated Ugt86 expression was almost nil in larvae exposed to juvenile hormone III (P > 0.05). The results of HPLC revealed that a new substance was formed by mixing 20E with the recombinant UGT86 protein in vitro, indicating that the effect of Ugt86 on 20E was similar to that on exogenous quercetin derived from plant food, and that the effect probably initiated the detoxification reaction against rutin. The conclusion is that the reaction of Ugt86 on the silkworm cocoon pigment quercetin is not the result of active mimetic ecogenesis, but derives from the detoxification of UGTs.

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

PubMed

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

2009-10-01

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

Diabetes mellitus reduces activity of human UDP-glucuronosyltransferase 2B7 in liver and kidney leading to decreased formation of mycophenolic acid acyl-glucuronide metabolite.

PubMed

Dostalek, Miroslav; Court, Michael H; Hazarika, Suwagmani; Akhlaghi, Fatemeh

2011-03-01

Mycophenolic acid (MPA) is an immunosuppressive agent commonly used after organ transplantation. Altered concentrations of MPA metabolites have been reported in diabetic kidney transplant recipients, although the reason for this difference is unknown. We aimed to compare MPA biotransformation and UDP-glucuronosyltransferase (UGT) expression and activity between liver (n = 16) and kidney (n = 8) from diabetic and nondiabetic donors. Glucuronidation of MPA, as well as the expression and probe substrate activity of UGTs primarily responsible for MPA phenol glucuronide (MPAG) formation (UGT1A1 and UGT1A9), and MPA acyl glucuronide (AcMPAG) formation (UGT2B7), was characterized. We have found that both diabetic and nondiabetic human liver microsomes and kidney microsomes formed MPAG with similar efficiency; however, AcMPAG formation was significantly lower in diabetic samples. This finding is supported by markedly lower glucuronidation of the UGT2B7 probe zidovudine, UGT2B7 protein, and UGT2B7 mRNA in diabetic tissues. UGT genetic polymorphism did not explain this difference because UGT2B7*2 or *1c genotype were not associated with altered microsomal UGT2B7 protein levels or AcMPAG formation. Furthermore, mRNA expression and probe activities for UGT1A1 or UGT1A9, both forming MPAG but not AcMPAG, were comparable between diabetic and nondiabetic tissues, suggesting the effect may be specific to UGT2B7-mediated AcMPAG formation. These findings suggest that diabetes mellitus is associated with significantly reduced UGT2B7 mRNA expression, protein level, and enzymatic activity of human liver and kidney, explaining in part the relatively low circulating concentrations of AcMPAG in diabetic patients.

Regiospecificity of Human UDP-glucuronosyltransferase Isoforms in Chalcone and Flavanone Glucuronidation Determined by Metal Complexation and Tandem Mass Spectrometry

PubMed Central

Niemeyer, Emily D.; Brodbelt, Jennifer S.

2013-01-01

The glucuronidation of a series of chalcones (2'-hydroxychalcone, 2',4'-dihydroxychalcone, 3,2'-dihydroxychalcone, 4,2'-dihydroxychalcone, and cardamonin) and their corresponding cyclized flavanones (7-hydroxyflavanone, 3'-hydroxyflavanone, 4'-hydroxyflavanone, and alpinetin) by nine human UDP-glucuronosyltransferase (UGT) 1A enzymes was evaluated. A post-column metal complexation LC-MS/MS strategy was used successfully to produce characteristic mass spectrometric product ions that were utilized in combination with elution order trends to identify chalcone and flavanone monoglucuronides unambiguously, thus allowing determination of the regioselectivities of the UGT1A isoforms. The presence of hydroxy groups on the A or B-ring had a significant effect on the glucuronide product yield and the site where glucuronidation occurred. For example, for reaction with UGT1A9, formation of the 2'-O-glucuronide was increased for dihydroxychalcones with A-ring hydroxy substituents. In contrast, although UGT1A8 reacted with 3,2'-dihydroxychalcone and 4,2'-dihydroxychalcone to yield 2'-O-glucuronide products, the presence of a B-ring hydroxy group at the 4' position on cardamonin and 2',4'-dihydroxychalcone quenched the reaction at the OH-2' position. Moreover, the A-ring OH-4 group promoted glucuronidation at the 2' position for the reaction of 4,2'-dihydroxychalcone with UGT1A1 and 1A3. For UGT1A7, hydroxy group substituents on the chalcone A-ring also promoted cyclization and formation of the corresponding flavanone glucuronide. PMID:23713759

Regiospecificity of human UDP-glucuronosyltransferase isoforms in chalcone and flavanone glucuronidation determined by metal complexation and tandem mass spectrometry.

PubMed

Niemeyer, Emily D; Brodbelt, Jennifer S

2013-06-28

The glucuronidation of a series of chalcones (2'-hydroxychalcone, 2',4'-dihydroxychalcone, 3,2'-dihydroxychalcone, 4,2'-dihydroxychalcone, and cardamonin) and their corresponding cyclized flavanones (7-hydroxyflavanone, 3'-hydroxyflavanone, 4'-hydroxyflavanone, and alpinetin) by eight human UDP-glucuronosyltransferase (UGT) 1A enzymes was evaluated. A postcolumn metal complexation LC-MS/MS strategy was used successfully to produce characteristic mass spectrometric product ions that were utilized in combination with elution order trends to identify chalcone and flavanone monoglucuronides unambiguously, thus allowing determination of the regioselectivities of the UGT1A isoforms. The presence of hydroxy groups on the A- or B-ring had a significant effect on the glucuronide product yield and the site where glucuronidation occurred. For example, for reaction with UGT1A9, formation of the 2'-O-glucuronide was increased for dihydroxychalcones with A-ring hydroxy substituents. In contrast, although UGT1A8 reacted with 3,2'-dihydroxychalcone and 4,2'-dihydroxychalcone to yield 2'-O-glucuronide products, the presence of a B-ring hydroxy group at the 4' position on cardamonin and 2',4'-dihydroxychalcone quenched the reaction at the OH-2' position. Moreover, the A-ring OH-4 group promoted glucuronidation at the 2' position for the reaction of 4,2'-dihydroxychalcone with UGT1A1 and 1A3. For UGT1A7, hydroxy group substituents on the chalcone A-ring also promoted cyclization and formation of the corresponding flavanone glucuronide.

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

PubMed

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. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Involvement of UDP-Glucuronosyltransferases and Sulfotransferases in the Excretion and Tissue Distribution of Resveratrol in Mice

PubMed Central

Böhmdorfer, Michaela; Szakmary, Akos; Schiestl, Robert H.; Vaquero, Javier; Riha, Juliane; Brenner, Stefan; Thalhammer, Theresia; Szekeres, Thomas; Jäger, Walter

2017-01-01

Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. It is unknown whether the expression of metabolizing enzymes correlates with resveratrol levels in organs and tissues. Therefore, we investigated the metabolism and tissue distribution of resveratrol in mice and assessed its association with the expression of UDP-glucuronosyltransferase (Ugt) and sulfotransferase (Sult) genes. Plasma, urine, feces, and various organs were analyzed using high-performance liquid chromatography at up to 8 h after intragastric resveratrol administration. The metabolism of resveratrol was pronounced, leading to the formation of resveratrol glucuronides and sulfates. Concentrations of resveratrol and its metabolites were high in the gastrointestinal organs, urine, and feces, but low in the liver and kidneys. In lung, heart, thymus, and brain tissues, parent resveratrol levels exceeded the sulfate and glucuronide concentrations. The formation of resveratrol conjugates correlated with the expression of certain Ugt and Sult genes. Reverse transcription quantitative PCR (RT-qPCR) analysis revealed high mRNA expression of Ugt1a1 and Ugt1a6a in the liver, duodenum, jejunum, ileum, and colon, leading to high concentrations of resveratrol-3-O-glucuronide in these organs. Strong correlations of resveratrol-3-O-sulfate and resveratrol-3-O-4′-O-disulfate formation with Sult1a1 mRNA expression were also observed, particularly in the liver and colon. In summary, our data revealed organ-specific expression of Sults and Ugts in mice that strongly affects resveratrol concentrations; this may also be predictive in humans following oral uptake of dietary resveratrol. PMID:29231856

Activity of xenobiotic-metabolizing enzymes in the liver of rats with multi-vitamin deficiency.

PubMed

Tutelyan, Victor A; Kravchenko, Lidia V; Aksenov, Ilya V; Trusov, Nikita V; Guseva, Galina V; Kodentsova, Vera M; Vrzhesinskaya, Oksana A; Beketova, Nina A

2013-01-01

The purpose of the study was to determine how multi-vitamin deficiency affects xenobiotic-metabolizing enzyme (XME) activities in the rat liver. Vitamin levels and XME activities were studied in the livers of male Wistar rats who were fed for 4 weeks with semi-synthetic diets containing either adequate (100 % of recommended vitamin intake) levels of vitamins (control), or decreased vitamin levels (50 % or 20 % of recommended vitamin intake). The study results have shown that moderate vitamin deficiency (50 %) leads to a decrease of vitamin A levels only, and to a slight increase, as compared with the control, in the following enzyme activities: methoxyresorufin O-dealkylase (MROD) activity of CYP1 A2 - by 34 % (p < 0.05), UDP-glucuronosyl transferase - by 26 % (p < 0.05), and quinone reductase - by 55 % (p < 0.05). Profound vitamin deficiency (20 %) led to a decrease of vitamins A, E, B1, B2, and C, and enzyme activities in the liver: MROD - to 78 % of the control level (p < 0.05), 4-nitrophenol hydroxylase - to 74 % (p < 0.05), heme oxygenase-1 - to 83 % (p < 0.05), and quinone reductase - to 60 % (p < 0.05). At the same time, the UDP-glucuronosyl transferase activity and ethoxyresorufin O-dealkylase activity of CYP1A1, pentoxyresorufin O-dealkylase activity of CYP2B1/2 and 6β-testosterone hydroxylase, as well as the total activity of glutathione transferase did not differ from the control levels. The study has demonstrated that profound multi-vitamin deficiency is associated with a decrease in the expression of CYP1A2 and CYP3A1 mRNAs to 62 % and 79 %, respectively. These data indicated that a short-term but profound multi-vitamin deficiency in rats leads to a decrease in the activities and expression of the some XME that play an important role in detoxification of xenobiotics and metabolism of drugs and antioxidant protection.

Genetic variations in UGT2B28, UGT2B17, UGT2B15 genes and the risk of prostate cancer: A case-control study.

PubMed

Habibi, Mohsen; Mirfakhraie, Reza; Khani, Maryam; Rakhshan, Azadeh; Azargashb, Eznollah; Pouresmaeili, Farkhondeh

2017-11-15

Glucuronidation is a major pathway for elimination of exogenous and endogenous compounds such as environmental carcinogens and androgens from the body. This biochemical pathway is mediated by enzymes called uridine diphosphoglucuronosyltransferases (UGTs). Null (del/del) genes polymorphisms in UGT2B17, and UGT2B28 and D85Y single-nucleotide polymorphism (SNP) of UGT2B15 have been reported to increase the risk of prostate cancer. The goal of this study was to determine the association of mentioned genetic variants with the risk of prostate cancer. We investigated the copy number variations (CNVs) of UGT2B17 and UGT2B28 loci and the association between rs1902023 polymorphism of UGT2B15 gene in 360 subjects consisted of 120 healthy controls, 120 prostate cancer (PC) patients and 120 benign prostatic hyperplasia (BPH) patients. No association was detected for the mentioned polymorphisms and the risk of PC. However, a significant association was detected between UGT2B17 copy number variation and BPH risk (OR=2.189; 95% CI, 1.303-3.675; p=0.003). Furthermore, we observed that the D85Y polymorphism increases the risk of BPH when analyzed in combination with the copy number variation of UGT2B17 gene (OR=0.135; 95% CI, 0.036-0.512; p=0.003). Our findings suggest that the D85Y polymorphism of UGT2B15 and CNVs in UGT2B28 and UGT2B17 genes is not associated with prostate cancer risk in Iranian patients. To our knowledge, this is the first report that implicates the role of CNV of UGT2B17 gene in BPH. Copyright © 2017 Elsevier B.V. All rights reserved.

The Contribution of Common UGT2B10 and CYP2A6 Alleles to Variation in Nicotine Glucuronidation among European Americans

PubMed Central

Bloom, A. Joseph; von Weymarn, Linda B.; Martinez, Maribel; Bierut, Laura J.; Goate, Alison; Murphy, Sharon E.

2014-01-01

UDP-glucuronosytransferase-2B10 (UGT2B10) is the primary catalyst of nicotine glucuronidation. To develop a predictive genetic model of nicotine metabolism, the conversion of deuterated (D2)-nicotine to D2-nicotine-glucuronide, D2-cotinine, D2-cotinine-glucuronide, and D2-trans-3'-hydroxycotinine were quantified in 188 European Americans, and the contribution of UGT2B10 genotype to variability in first-pass nicotine glucuronidation assessed, following a procedure previously applied to nicotine C-oxidation. The proportion of total nicotine converted to nicotine-glucuronide (D2-nicotine-glucuronide/ (D2-nicotine +D2-nicotine-glucuronide +D2-cotinine +D2-cotinine-glucuronide +D2-trans-3'-hydroxycotinine)) was the primary phenotype. The variant, rs61750900T (D67Y) (minor allele frequency (MAF) = 10%), is confirmed to abolish nicotine glucuronidation activity. Another variant, rs112561475G (N397D) (MAF = 2%), is significantly associated with enhanced glucuronidation. rs112561475G is the ancestral allele of a well-conserved amino acid, indicating that the majority of human UGT2B10 alleles are derived hypomorphic alleles. CYP2A6 and UGT2B10 genotype explain 53% of the variance in oral nicotine glucuronidation in this sample. CYP2A6 and UGT2B10 genetic variants are also significantly associated with un-deuterated (D0) nicotine glucuronidation in subjects smoking ad libitum. We find no evidence for further common variation markedly influencing hepatic UGT2B10 expression in European Americans. PMID:24192532

Novel Resveratrol-Based Substrates for Human Hepatic, Renal, and Intestinal UDP-Glucuronosyltransferases

PubMed Central

2015-01-01

Trans-Resveratrol (tRes) has been shown to have powerful antioxidant, anti-inflammatory, anticarcinogenic, and antiaging properties; however, its use as a therapeutic agent is limited by its rapid metabolism into its conjugated forms by UDP-glucuronosyltransferases (UGTs). The aim of the current study was to test the hypothesis that the limited bioavailability of tRes can be improved by modifying its structure to create analogs which would be glucuronidated at a lower rate than tRes itself. In this work, three synthetic stilbenoids, (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (NI-12a), (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzaldehyde oxime (NI-ST-05), and (E)-4-(3,5-dimethoxystyryl)-2,6-dinitrophenol (DNR-1), have been designed based on the structure of tRes and synthesized in our laboratory. UGTs recognize and glucuronidate tRes at each of the 3 hydroxyl groups attached to its aromatic rings. Therefore, each of the above compounds was designed with the majority of the hydroxyl groups blocked by methylation and the addition of other novel functional groups as part of a drug optimization program. The activities of recombinant human UGTs from the 1A and 2B families were examined for their capacity to metabolize these compounds. Glucuronide formation was identified using HPLC and verified by β-glucuronidase hydrolysis and LC–MS/MS analysis. NI-12a was glucuronidated at both the −COOH and −OH functions, NI-ST-05 formed a novel N–O-glucuronide, and no product was observed for DNR-1. NI-12a is primarily metabolized by the hepatic and renal enzyme UGT1A9, whereas NI-ST-05 is primarily metabolized by an extrahepatic enzyme, UGT1A10, with apparent Km values of 240 and 6.2 μM, respectively. The involvement of hepatic and intestinal UGTs in the metabolism of both compounds was further confirmed using a panel of human liver and intestinal microsomes, and high individual variation in activity was demonstrated between donors. In summary

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity.

PubMed

Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Fallon, John K; Barber, Jill; Smith, Philip C; Rostami-Hodjegan, Amin; Goosen, Theunis C

2017-10-01

Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes ( n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P < 0.0001; R 2 = 0.30; n = 24]. SIL-based abundance measurements correlated well with enzyme activities, with correlations ranging from moderate for UGTs 1A6, 1A9, and 2B15 (Rs = 0.52-0.59, P < 0.0001; R 2 = 0.34-0.58; n = 59) to strong correlations for UGTs 1A1, 1A3, 1A4, and 2B7 (Rs = 0.79-0.90, P < 0.0001; R 2 = 0.69-0.79). QconCAT-based data revealed generally poor correlation with activity, whereas moderate correlations were shown for UGTs 1A1, 1A3, and 2B7. Spurious abundance-activity correlations were identified in the cases of UGT1A4/2B4 and UGT2B7/2B15, which could be explained by correlations of protein expression between these enzymes. Consistent correlation of UGT abundance with catalytic activity, demonstrated by the SIL-based dataset, suggests that quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations

Establishment and Use of New MDCK II Cells Overexpressing Both UGT1A1 and MRP2 to Characterize Flavonoid Metabolism via the Glucuronidation Pathway

PubMed Central

Wang, Meifang; Yang, Guangyi; He, Yu; Xu, Beibei; Zeng, Min; Yin, Taijun; Gao, Song; Hu, Ming

2017-01-01

Scope The purpose of this study is to characterize how overexpression of an efflux transporter and an UDP-glucuronosyltransferase (UGT) affects the cellular kinetics of glucuronidation processes. Methods and Results A new MDCK II cell line overexpressing both MRP2 and UGT1A1 (MDCKII-UGT1A1/MRP2 cells) was developed and used to determine how overexpression of an efflux transporter affects the kinetics of cellular flavonoid glucuronide production. The results showed that most model flavonoids (from a total of 13) were mainly metabolized into glucuronides in the MDCKII-UGT1A1/MRP2 cells and the glucuronides were rapidly excreted. Flavonoids with three or fewer hydroxyl group at 7, 3′ or 6 hydroxyl group were also metabolized into sulfates. Mechanistic studies using 7-hydroxylflavone showed that its glucuronide was mainly (90%) effluxed by BCRP with a small (10%) but significant contribution from MRP2. Maximal velocity of glucuronide production MDCK-MRP2/UGT1A1 cells showed a fairly good correlation (R2 >0.8) with those derived using UGT1A1 microsomes, but other kinetic parameters (e.g., Km) did not correlate. Conclusion Overexpression of a second efficient efflux transporter did not significantly change the fact that BCRP is the dominant transporter for flavonoid glucuronide nor did it diminish the influence of the efflux transporter as the “gate keeper” of glucuronidation process. PMID:26833852

The Implication of the Polymorphisms of COX-1, UGT1A6, and CYP2C9 among Cardiovascular Disease (CVD) Patients Treated with Aspirin.

PubMed

Jalil, Nur Jalinna Abdul; Bannur, Zakaria; Derahman, A; Maskon, O; Darinah, Noor; Hamidi, Hamat; Gunasekaran, Osama Ali; Rafizi, Mohd; Azreen, Nur Izatul; Kek, Teh Lay; Salleh, Mohd Zaki

2015-01-01

  Enzymes potentially responsible for the pharmacokinetic variations of aspirin include cyclooxygenase-1 (COX-1), UDP-glucuronosyltransferase (UGT1A6) and P450 (CYP) (CYP2C9). We therefore aimed to determine the types and frequencies of variants of COX-1 (A-842G), UGT1A6 (UGT1A6*2; A541G and UGT1A6*3; A522C) and CYP2C9 (CYP2C9*3; A1075C) in the three major ethnic groups in Malaysia. In addition, the role of these polymorphisms on aspirin-induced gastritis among the patients was investigated. A total of 165 patients with cardiovascular disease who were treated with 75-150 mg daily dose of aspirin and 300 healthy volunteers were recruited. DNA was extracted from the blood samples and genotyped for COX-1 (A-842G), UGT1A6 (UGT1A6*2 and UGT1A6*3) and CYP2C9 (CYP2C9*3; A1075C) using allele specific polymerase chain reaction (AS-PCR). Variants UGT1A6*2,*3 and CYP2C9*3 were detected in relatively high percentage of 22.83%, 30.0% and 6.50%, respectively; while COX-1 (A-842G) was absent. The genotype frequencies for UGT1A6*2 and *3 were significantly different between Indians and Malays or Chinese. The level of bilirubin among patients with different genotypes of UGT1A6 was significantly different (p-value < 0.05). In addition, CYP2C9*3 was found to be associated with gastritis with an odd ratio of 6.8 (95 % Cl OR: 1.39 - 33.19; P = 0.033). Screening of patients with defective genetic variants of UGT1A6 and CYP2C9*3 helps in identifying patients at risk of aspirin induced gastritis. However, a randomised clinical study of bigger sample size would be needed before it is translated to clinical use.

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

PubMed

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. Copyright © 2012 John Wiley & Sons, Ltd.

Traditional Herbal Formulas to as Treatments for Musculoskeletal Disorders: Their Inhibitory Effects on the Activities of Human Microsomal Cytochrome P450s and UDP-glucuronosyltransferases

PubMed Central

Jin, Seong Eun; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Ha, Hyekyung

2016-01-01

Objective: The aim of this study was to assess the influence of traditional herbal formulas, including Bangpungtongseong-san (BPTSS; Fangfengtongsheng-san, Bofu-tsusho-san), Ojeok-san (OJS; Wuji-san, Goshaku-san), and Oyaksungi-san (OYSGS; Wuyaoshungi-san, Uyakujyunki-san), on the activities of the human cytochrome P450s (CYP450s) and UDP-glucuronosyltransferases (UGTs), which are drug-metabolizing enzymes. Materials and Methods: The activities of the major human CYP450 isozymes (CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) and UGTs (UGT1A1, UGT1A4, and UGT2B7) were investigated using in vitro fluorescence-based and luminescence-based enzyme assays, respectively. The inhibitory effects of the herbal formulas were characterized, and their IC50 values were determined. Results: BPTSS inhibited the activities of CYP1A2, CYP2C19, CYP2E1, and UGT1A1 while it exerted relatively weak inhibition on CYP2B6, CYP2C9, CYP2D6, and CYP3A4. BPTSS also negligibly inhibited the activities of UGT1A4 and UGT2B7, with IC50 values in the excess of 1000 μg/mL. OJS and OYSGS inhibited the activity of CYP2D6, whereas they exhibited no inhibition of the UGT1A4 activity at doses <1000 μg/mL. In addition, OJS inhibited the CYP1A2 activity but exerted a relatively weak inhibition on the activities of CYP2C9, CYP2C19, CYP2E1, and CYP3A4. Conversely, OJS negligibly inhibited the activities of CYP2B6, UGT1A1, and UGT2B7 with IC50 values in excess of 1000 μg/mL. OYSGS weakly inhibited the activities of CYP1A2, CYP2C19, CYP2E1, CYP3A4, and UGT1A1, with a negligible inhibition on the activities of CYP2B6, CYP2C9, and UGT2B7, with IC50 values in excess of 1000 μg/mL. Conclusions: These results provide information regarding the safety and effectiveness of BPTSS, OJS, and OYSGS when combined with conventional drugs. SUMMARY Bangpungtongseong-san inhibited the activities of human microsomal CYP1A2, CYP2C19, CYP2E1, and UGT1A1, with a negligibly inhibition on the activities of CYP2B6

Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics.

PubMed

Gröer, C; Busch, D; Patrzyk, M; Beyer, K; Busemann, A; Heidecke, C D; Drozdzik, M; Siegmund, W; Oswald, S

2014-11-01

Cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGT) are major determinants in the pharmacokinetics of most drugs on the market. To investigate their impact on intestinal and hepatic drug metabolism, we developed and validated quantification methods for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using in each case stable isotope labelled peptides as internal standard. The chromatography of the respective peptides was performed with gradient elution using a reversed phase (C18) column (Ascentis(®) Express Peptide ES-C18, 100mm×2.1mm, 2.7μm) and 0.1% formic acid (FA) as well as acetonitrile with 0.1% FA as mobile phases at a flow rate of 300μl/min. The MS/MS detection of all peptides was done simultaneously with a scheduled multiple reaction monitoring (MRM) method in the positive mode by monitoring in each case three mass transitions per proteospecific peptide and the internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (0.25-50nM), within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was successfully applied to determine the CYP and UGT protein amount in human liver and intestinal microsomes. The method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to quantify clinically relevant human CYP and UGT enzymes. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification and characterization of naturally occurring inhibitors against UDP-glucuronosyltransferase 1A1 in Fructus Psoraleae (Bu-gu-zhi)

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

Wang, Xin-Xin; Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023; Lv, Xia

As an edible traditional Chinese herb, Fructus psoraleae (FP) has been widely used in Asia for the treatment of vitiligo, bone fracture and osteoporosis. Several cases on markedly elevated bilirubin and acute liver injury following administration of FP and its related proprietary medicine have been reported, but the mechanism in FP-associated toxicity has not been well investigated yet. This study aimed to investigate the inhibitory effects of FP extract and its major constituents against human UDP-glucuronosyltransferase 1A1 (UGT1A1), the key enzyme responsible for metabolic elimination of bilirubin. To this end, N-(3-carboxy propyl)-4-hydroxy-1,8-naphthalimide (NCHN), a newly developed specific fluorescent probe formore » UGT1A1, was used to evaluate the inhibitory effects of FP extract or its fractions in human liver microsomes (HLM), while LC-UV fingerprint and UGT1A1 inhibition profile were combined to identity and characterize the naturally occurring inhibitors of UGT1A1 in FP. Our results demonstrated that both the extract of FP and five major components of FP displayed evident inhibitory effects on UGT1A1 in HLM. Among these five identified naturally occurring inhibitors, bavachin and corylifol A were found to be strong inhibitors of UGT1A1 with the inhibition kinetic parameters (K{sub i}) values lower than 1 μM, while neobavaisoflavone, isobavachalcone, and bavachinin displayed moderate inhibitory effects against UGT1A1 in HLM, with the K{sub i} values ranging from 1.61 to 9.86 μM. These findings suggested that FP contains natural compounds with potent inhibitory effects against human UGT1A1, which may be one of the important reasons for triggering FP-associated toxicity, including elevated bilirubin levels and liver injury. - Graphical abstract: LC-UV fingerprint and UGT1A1 inhibition profiles were combined to identity and characterize the natural inhibitors of UGT1A1 in F. psoraleae for the first time. Five major components in F. psoraleae were

UGT1A and UGT2B genetic variation alters nicotine and nitrosamine glucuronidation in european and african american smokers.

PubMed

Wassenaar, Catherine A; Conti, David V; Das, Soma; Chen, Peixian; Cook, Edwin H; Ratain, Mark J; Benowitz, Neal L; Tyndale, Rachel F

2015-01-01

Identifying sources of variation in the nicotine and nitrosamine metabolic inactivation pathways is important to understanding the relationship between smoking and cancer risk. Numerous UGT1A and UGT2B enzymes are implicated in nicotine and nitrosamine metabolism in vitro; however, little is known about their roles in vivo. Within UGT1A1, UGT1A4, UGT1A9, UGT2B7, UGT2B10, and UGT2B17, 47 variants were genotyped, including UGT2B10*2 and UGT2B17*2. The association between variation in these UGTs and glucuronidation activity within European and African American current smokers (n = 128), quantified as urinary ratios of the glucuronide over unconjugated compound for nicotine, cotinine, trans-3'-hydroxycotinine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was investigated in regression models assuming a dominant effect of variant alleles. Correcting for multiple testing, three UGT2B10 variants were associated with cotinine glucuronidation, rs2331559 and rs11726322 in European Americans and rs835309 in African Americans (P ≤ 0.0002). Additional variants predominantly in UGT2B10 were nominally associated with nicotine (P = 0.008-0.04) and cotinine (P = <0.001-0.02) glucuronidation in both ethnicities in addition to UGT2B10*2 in European Americans (P = 0.01, P < 0.001). UGT2B17*2 (P = 0.03) in European Americans and UGT2B7 variants (P = 0.02-0.04) in African Americans were nominally associated with 3HC glucuronidation. UGT1A (P = 0.007-0.01), UGT2B10 (P = 0.02), and UGT2B7 (P = 0.02-0.03) variants in African Americans were nominally associated with NNAL glucuronidation. Findings from this initial in vivo study support a role for multiple UGTs in the glucuronidation of tobacco-related compounds in vivo, in particular UGT2B10 and cotinine glucuronidation. Findings also provide insight into ethnic differences in glucuronidation activity, which could be contributing to ethnic disparities in the risk for smoking-related cancers. Cancer Epidemiol Biomarkers Prev

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

PubMed

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. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

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

PubMed Central

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

2016-01-01

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. PMID:27413119

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

PubMed Central

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

2014-01-01

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

TAPBPR bridges UDP-glucose:glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway

PubMed Central

Neerincx, Andreas; Hermann, Clemens; Antrobus, Robin; van Hateren, Andy; Cao, Huan; Trautwein, Nico; Stevanović, Stefan; Elliott, Tim; Deane, Janet E; Boyle, Louise H

2017-01-01

Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex. DOI: http://dx.doi.org/10.7554/eLife.23049.001 PMID:28425917

Transgenic Wheat Expressing a Barley UDP-Glucosyltransferase Detoxifies Deoxynivalenol and Provides High Levels of Resistance to Fusarium graminearum.

PubMed

Li, Xin; Shin, Sanghyun; Heinen, Shane; Dill-Macky, Ruth; Berthiller, Franz; Nersesian, Natalya; Clemente, Thomas; McCormick, Susan; Muehlbauer, Gary J

2015-11-01

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a devastating disease of wheat that results in economic losses worldwide. During infection, F. graminearum produces trichothecene mycotoxins, including deoxynivalenol (DON), that increase fungal virulence and reduce grain quality. Transgenic wheat expressing a barley UDP-glucosyltransferase (HvUGT13248) were developed and evaluated for FHB resistance, DON accumulation, and the ability to metabolize DON to the less toxic DON-3-O-glucoside (D3G). Point-inoculation tests in the greenhouse showed that transgenic wheat carrying HvUGT13248 exhibited significantly higher resistance to disease spread in the spike (type II resistance) compared with nontransformed controls. Two transgenic events displayed complete suppression of disease spread in the spikes. Expression of HvUGT13248 in transgenic wheat rapidly and efficiently conjugated DON to D3G, suggesting that the enzymatic rate of DON detoxification translates to type II resistance. Under field conditions, FHB severity was variable; nonetheless, transgenic events showed significantly less-severe disease phenotypes compared with the nontransformed controls. In addition, a seedling assay demonstrated that the transformed plants had a higher tolerance to DON-inhibited root growth than nontransformed plants. These results demonstrate the utility of detoxifying DON as a FHB control strategy in wheat.

Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius.

PubMed

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

Upregulation of UDP-Glucuronosyltransferases 1a1 and 1a7 Are Involved in Altered Puerarin Pharmacokinetics in Type II Diabetic Rats.

PubMed

Dong, Songtao; Zhang, Maofan; Niu, Huimin; Jiang, Kunyu; Jiang, Jialei; Ma, Yinglin; Wang, Xin; Meng, Shengnan

2018-06-20

Puerarin is an isoflavonoid extracted from Pueraria lobata roots, and displays a broad range of pharmacological activities, including antidiabetic activity. However, information about the pharmacokinetics of puerarin in diabetics is scarce. This study was conducted to investigate the difference in pharmacokinetic effects of puerarin in normal rats and rats with diabetes mellitus (DM), and the mechanism involved. DM was induced by a combined high-fat diet (HFD) and streptozotocin (STZ) injection. Plasma concentrations of puerarin in DM, HFD, and control rats were determined after intravenous (20 mg/kg) and oral administration (500 mg/kg) of puerarin, and pharmacokinetic parameters were estimated. The messenger RNA (mRNA) and protein expression levels of Ugt1a1 and Ugt1a7 in rat livers and intestines were measured using qRT-PCR and western blot, respectively. The area under the concentration⁻time curve and the clearance of puerarin in the DM rats statistically differed from those in the control rats ( p <0.05) with both administration routes. The hepatic and intestinal gene and protein expressions of Ugt1a1 and Ugt1a7 were significantly increased in the DM rats ( p <0.05). Therefore, the metabolic changes in diabetes could alter the pharmacokinetics of puerarin. This change could be caused by upregulated uridine diphosphate (UDP)-glucuronosyltransferase activity, which may enhance puerarin clearance, and alter its therapeutic effects.

PBPK Model of Morphine Incorporating Developmental Changes in Hepatic OCT1 and UGT2B7 Proteins to Explain the Variability in Clearances in Neonates and Small Infants.

PubMed

Emoto, Chie; Johnson, Trevor N; Neuhoff, Sibylle; Hahn, David; Vinks, Alexander A; Fukuda, Tsuyoshi

2018-06-19

Morphine has large pharmacokinetic variability, which is further complicated by developmental changes in neonates and small infants. The impacts of organic cation transporter 1 (OCT1) genotype and changes in blood-flow on morphine clearance (CL) were previously demonstrated in children, whereas changes in UDP-glucuronosyltransferase 2B7 (UGT2B7) activity showed a small effect. This study, targeting neonates and small infants, was designed to assess the influence of developmental changes in OCT1 and UGT2B7 protein expression and modified blood-flow on morphine CL using physiologically based pharmacokinetic (PBPK) modeling. The implementation of these three age-dependent factors into the pediatric system platform resulted in reasonable prediction for an age-dependent increase in morphine CL in these populations. Sensitivity of morphine CL to changes in cardiac output increased with age up to 3 years, whereas sensitivity to changes in UGT2B7 activity decreased. This study suggests that morphine exhibits age-dependent extraction, likely due to the developmental increase in OCT1 and UGT2B7 protein expression/activity and hepatic blood-flow. © 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

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

PubMed

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

2015-07-22

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

Rapid prediction of chemical metabolism by human UDP-glucuronosyltransferase isoforms using quantum chemical descriptors derived with the electronegativity equalization method.

PubMed

Sorich, Michael J; McKinnon, Ross A; Miners, John O; Winkler, David A; Smith, Paul A

2004-10-07

This study aimed to evaluate in silico models based on quantum chemical (QC) descriptors derived using the electronegativity equalization method (EEM) and to assess the use of QC properties to predict chemical metabolism by human UDP-glucuronosyltransferase (UGT) isoforms. Various EEM-derived QC molecular descriptors were calculated for known UGT substrates and nonsubstrates. Classification models were developed using support vector machine and partial least squares discriminant analysis. In general, the most predictive models were generated with the support vector machine. Combining QC and 2D descriptors (from previous work) using a consensus approach resulted in a statistically significant improvement in predictivity (to 84%) over both the QC and 2D models and the other methods of combining the descriptors. EEM-derived QC descriptors were shown to be both highly predictive and computationally efficient. It is likely that EEM-derived QC properties will be generally useful for predicting ADMET and physicochemical properties during drug discovery.

UGT2B17 and SULT1A1 gene copy number variation (CNV) detection by LabChip microfluidic technology.

PubMed

Gaedigk, Andrea; Gaedigk, Roger; Leeder, J Steven

2010-05-01

Gene copy number variations (CNVs) are increasingly recognized to play important roles in the expression of genes and hence on their respective enzymatic activities. This has been demonstrated for a number of drug metabolizing genes, such as UDP-glucuronosyltransferases 2B17 (UGT2B17) and sulfotransferase 1A1 (SULT1A1), which are subject to genetic heterogeneity, including CNV. Quantitative assays to assess gene copy number are therefore becoming an integral part of accurate genotype assessment and phenotype prediction. In this study, we evaluated a microfluidics-based system, the Bio-Rad Experion system, to determine the power and utility of this platform to detect UGT2B17 and SULT1A1 CNV in DNA samples derived from blood and tissue. UGT2B17 is known to present with 0, 1 or 2 and SULT1A1 with up to 5 gene copies. Distinct clustering (p<0.001) into copy number groups was achieved for both genes. DNA samples derived from blood exhibited less inter-run variability compared to DNA samples obtained from liver tissue. This variability may be caused by tissue-specific PCR inhibitors as it could be overcome by using DNA from another tissue, or after the DNA had undergone whole genome amplification. This method produced results comparable to those reported for other quantitative test platforms.

Correlation between plasma concentration ratios of SN-38 glucuronide and SN-38 and neutropenia induction in patients with colorectal cancer and wild-type UGT1A1 gene

PubMed Central

HIROSE, KOICHI; KOZU, CHIHIRO; YAMASHITA, KOSHIRO; MARUO, EIJI; KITAMURA, MIZUHO; HASEGAWA, JUNICHI; OMODA, KEI; MURAKAMI, TERUO; MAEDA, YORINOBU

2011-01-01

In irinotecan (CPT-11)-based chemotherapy, neutropenia and diarrhea are often induced. In the present study, the clinical significance of the concentration ratios of 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronide (SN-38G) and SN-38 in the plasma in predicting CPT-11-induced neutropenia was examined. A total of 17 patients with colorectal cancer and wild-type UDP-glucuronosyltransferase (UGT)1A1 gene were enrolled and treated with CPT-11 as part of the FOLFIRI regimen [CPT-11 and fluorouracil (5-FU)]. Blood was taken exactly 15 min following a 2-h continuous infusion of CPT-11. Plasma concentrations of SN-38, SN-38G and CPT-11 were determined by a modified high-performance liquid chromatography (HPLC) method. The median, maximum and minimum values of plasma SN-38G/SN-38 ratios were 4.25, 7.09 and 1.03, respectively, indicating that UGT activities are variable among patients with the wild-type UGT1A1 gene. The plasma SN-38G/SN-38 ratios decreased with an increase in the trial numbers of chemotherapy (r=0.741, p=0.000669), suggesting that CPT-11 treatment suppresses UGT activity, and the low plasma SN-38G/SN-38 ratios resulted in the induction of greater neutropenia. However, in this analysis, 2 clearly separated regression lines were observed between plasma SN-38G/SN-38 ratios and neutropenia induction. In conclusion, UGT activity involved in SN-38 metabolism is variable among patients with the wild-type UGT1A1 gene, and each CPT-11 treatment suppresses UGT activity. One-point determination of the plasma SN-38G/SN-38 ratio may provide indications for the prediction of CPT-11-induced neutropenia and adjustment of the optimal dose, although further studies are required. PMID:22740978

UGT1A1*6 polymorphisms are correlated with irinotecan-induced neutropenia: a systematic review and meta-analysis.

PubMed

Zhang, Xue; Yin, Jia-Fu; Zhang, Jiao; Kong, Shu-Jia; Zhang, Hong-Yin; Chen, Xue-Mei

2017-07-01

Irinotecan (IRI) chemotherapy toxicities can be severe, and may result in treatment delay, morbidity and in some rare cases death. Neutropenia is a life-threatening side effect of irinotecan, and UDP glucuronosyltransferases (UGTs) gene polymorphisms could predict the side effects in cancer patients and then reduce IRI-induced toxicity by preventative treatment or a decrease in dose. Both UGT1A1*6 and *28 were reliably demonstrated to be risk factors for IRI-induced neutropenia, with tests for both polymorphisms potentially being particularly useful in Asian cancer patients. However, some researchers reported that UGT1A1*6 could predict IRI-induced toxicities in Asian populations, controversial conclusions still remained. Thus, the association between UGT1A1*6 polymorphisms and IRI-induced severe toxicity in cancer patients is still needed to be explored. Therefore, this study aims to investigate the association between UGT1A1*6 polymorphisms and IRI-related severe neutropenia in cancer patients on a large scale. A total of 12 studies that included 746 wild genotype (G/G) cases and 394 variant genotype (G/A and A/A) cases were included on the basis of inclusion criteria. Then we assessed the methodologies quality; odds ratio (OR), risk difference (RD) and 95% confidence intervals (95% CI) were used to assess the strength of association. Overall, an increased risk of severe neutropenia in cancer patients with UGT1A1*6 polymorphisms was found. Patients with recessive models (GA + AA vs. GG) of UGT1A1*6 showed an increased risk (OR 2.03, 95% CI 1.54-2.68; RD = 0.11, P < 0.001). Specifically, the heterozygous variant of UGT1A1*6 showed an increased risk (OR 1.83, 95% CI 1.36-2.46; RD = 0.09, P < 0.001), and homozygous mutation showed also high risk (OR 2.95, 95% CI 1.83-4.75; RD = 0.18, P < 0.001) for severe neutropenia. Subgroup meta-analysis revealed that for patients harboring both heterozygous and homozygous variants, cancer types, low dose of IRI and

Effects of UGT1A9 genetic polymorphisms on monohydroxylated derivative of oxcarbazepine concentrations and oxcarbazepine monotherapeutic efficacy in Chinese patients with epilepsy.

PubMed

Lu, Yao; Fang, Youxin; Wu, Xunyi; Ma, Chunlai; Wang, Yue; Xu, Lan

2017-03-01

The human UDP-glucuronosyltransferase which is genetically polymorphic catalyzes glucuronidations of various drugs. The interactions among UGT1A4, UGT1A6, UGT1A9, and UGT2B15 genetic polymorphisms, monohydroxylated derivative (MHD) of oxcarbazepine (OXC) plasma concentrations, and OXC monotherapeutic efficacy were explored in 124 Chinese patients with epilepsy receiving OXC monotherapy. MHD is the major active metabolite of OXC, and its plasma concentration was measured using high-performance liquid chromatography when patients reached their maintenance dose of OXC. Genomic DNA was extracted from whole blood and SNP genotyping performed using PCR followed by dideoxy chain termination sequencing. We followed the patients for at least 1 year to evaluate the OXC monotherapy efficacy. Patients were divided into two groups according to their therapeutic outcome: group 1, seizure free; group 2, not seizure free. The data were analyzed using T test, one-way analysis of variance (ANOVA), Kruskal-Wallis test, chi-square test, Fisher's exact test, correlation analysis, and multivariate regression analysis. T test analysis showed that MHD plasma concentrations were significantly different between the two groups (p = 0.002). One-way ANOVA followed by Bonferroni post hoc testing of four candidate SNPs revealed that carriers of the UGT1A9 variant allele I399 C > T (TT 13.28 ± 7.44 mg/L, TC 16.41 ± 6.53 mg/L) had significantly lower MHD plasma concentrations and poorer seizure control than noncarriers (CC 22.24 ± 8.49 mg/L, p < 0.05). In our study, we have demonstrated the effects of UGT1A9 genetic polymorphisms on MHD plasma concentrations and OXC therapeutic efficacy. Through MHD monitoring, we can predict OXC therapeutic efficacy, which may be useful for the personalization of OXC therapy in epileptic patients.

UGT1A1*6 and UGT1A1*28 polymorphisms are correlated with irinotecan-induced toxicity: A meta-analysis.

PubMed

Yang, Yuwei; Zhou, MengMeng; Hu, Mingjun; Cui, Yanjie; Zhong, Qi; Liang, Ling; Huang, Fen

2018-06-22

Previous articles explored the role of UGT1A1 polymorphism on predicting irinotecan-induced toxicity, but the conclusions were still inconsistent and not comprehensive. We performed this meta-analysis to investigate the association between UGT1A1 polymorphism and irinotecan-induced toxicity. PubMed and Web of Science were searched for articles before July 2017. Inclusion and exclusion criteria were set to select eligible articles, and corresponding data were extracted from those articles. Subgroup analyses based on different cancer categories, doses and races were carried out to achieve comprehensive results. Statistical analyses were conducted using STATA 11.0. A total of 38 studies with 6742 cases were included after reading full text. Both UGT1A1*6 and UGT1A1*28 polymorphism are significantly associated with severe irinotecan-induced toxicity. Both Asian and Caucasian cancer patients with UGT1A1*28 variant had an increased risk. Compared with heterozygous variant, patients with homozygous variant suffered from a higher risk of toxicity. The effect of UGT1A1*28 polymorphism on diarrhea was less than on neutropenia. Subgroup analysis exhibited that for UGT1A1*6 polymorphism, patients treated with low-dose irinotecan were at a notable risk of toxicity. Moreover, the association between UGT1A1*6 polymorphism and irinotecan-induced toxicity was found in patients suffering from respiratory system cancers. Both UGT1A1*6 and UGT1A1*28 polymorphisms can be considered as predictors of irinotecan-induced toxicity, with effect varying by race, cancer type and irinotecan dose. © 2018 John Wiley & Sons Australia, Ltd.

Albumin Stimulates the Activity of the Human UDP-Glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the Effects Are Enzyme and Substrate Dependent

PubMed Central

Svaluto-Moreolo, Paolo; Dziedzic, Klaudyna; Yli-Kauhaluoma, Jari; Finel, Moshe

2013-01-01

Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro–in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2–4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction’s K m, increasing its V max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions’ K m are concerned. In the cases of V max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V max increases. Additionally, the BSA effects may be UGT subfamily dependent since K m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs. PMID:23372764

Predicting Flavonoid UGT Regioselectivity

PubMed Central

Jackson, Rhydon; Knisley, Debra; McIntosh, Cecilia; Pfeiffer, Phillip

2011-01-01

Machine learning was applied to a challenging and biologically significant protein classification problem: the prediction of avonoid UGT acceptor regioselectivity from primary sequence. Novel indices characterizing graphical models of residues were proposed and found to be widely distributed among existing amino acid indices and to cluster residues appropriately. UGT subsequences biochemically linked to regioselectivity were modeled as sets of index sequences. Several learning techniques incorporating these UGT models were compared with classifications based on standard sequence alignment scores. These techniques included an application of time series distance functions to protein classification. Time series distances defined on the index sequences were used in nearest neighbor and support vector machine classifiers. Additionally, Bayesian neural network classifiers were applied to the index sequences. The experiments identified improvements over the nearest neighbor and support vector machine classifications relying on standard alignment similarity scores, as well as strong correlations between specific subsequences and regioselectivities. PMID:21747849

Induced Mutagenesis in UGT74S1 Gene Leads to Stable New Flax Lines with Altered Secoisolariciresinol Diglucoside (SDG) Profiles.

PubMed

Fofana, Bourlaye; Ghose, Kaushik; Somalraju, Ashok; McCallum, Jason; Main, David; Deyholos, Michael K; Rowland, Gordon G; Cloutier, Sylvie

2017-01-01

Flax secoisolariciresinol (SECO) diglucoside (SDG) lignan is an emerging natural product purported to prevent chronic diseases in humans. SECO, the aglycone form of SDG, has shown higher intestinal cell absorption but it is not accumulated naturally in planta . Recently, we have identified and characterized a UDP-glucosyltransferase gene, UGT74S1 , that glucosylates SECO into its monoglucoside (SMG) and SDG forms when expressed in yeast. However, whether this gene is unique in controlling SECO glucosylation into SDG in planta is unclear. Here, we report on the use of UGT74S1 in reverse and forward genetics to characterize an ethyl methane sulfonate (EMS) mutagenized flax population from cultivar CDC Bethune and consisting of 1996 M2 families. EMS mutagenesis generated 73 SNP variants causing 79 mutational events in the UGT74S1 exonic regions of 93 M2 families. The mutation frequency in the exonic regions was determined to be one per 28 Kb. Of these mutations, 13 homozygous missense mutations and two homozygous nonsense mutations were observed and all were transmitted into the M3 and M4 generations. Forward genetics screening of the population showed homozygous nonsense mutants completely lacking SDG biosynthesis while the production of SMG was observed only in a subset of the M4 lines. Heterozygous or homozygous M4 missense mutants displayed a wide range of SDG levels, some being greater than those of CDC Bethune. No additional deleterious mutations were detected in these mutant lines using a panel of 10 other genes potentially involved in the lignan biosynthesis. This study provides further evidence that UGT74S1 is unique in controlling SDG formation from SECO and this is the first report of non-transgenic flax germplasm with simultaneous knockout of SDG and presence of SMG in planta .

Induced Mutagenesis in UGT74S1 Gene Leads to Stable New Flax Lines with Altered Secoisolariciresinol Diglucoside (SDG) Profiles

PubMed Central

Fofana, Bourlaye; Ghose, Kaushik; Somalraju, Ashok; McCallum, Jason; Main, David; Deyholos, Michael K.; Rowland, Gordon G.; Cloutier, Sylvie

2017-01-01

Flax secoisolariciresinol (SECO) diglucoside (SDG) lignan is an emerging natural product purported to prevent chronic diseases in humans. SECO, the aglycone form of SDG, has shown higher intestinal cell absorption but it is not accumulated naturally in planta. Recently, we have identified and characterized a UDP-glucosyltransferase gene, UGT74S1, that glucosylates SECO into its monoglucoside (SMG) and SDG forms when expressed in yeast. However, whether this gene is unique in controlling SECO glucosylation into SDG in planta is unclear. Here, we report on the use of UGT74S1 in reverse and forward genetics to characterize an ethyl methane sulfonate (EMS) mutagenized flax population from cultivar CDC Bethune and consisting of 1996 M2 families. EMS mutagenesis generated 73 SNP variants causing 79 mutational events in the UGT74S1 exonic regions of 93 M2 families. The mutation frequency in the exonic regions was determined to be one per 28 Kb. Of these mutations, 13 homozygous missense mutations and two homozygous nonsense mutations were observed and all were transmitted into the M3 and M4 generations. Forward genetics screening of the population showed homozygous nonsense mutants completely lacking SDG biosynthesis while the production of SMG was observed only in a subset of the M4 lines. Heterozygous or homozygous M4 missense mutants displayed a wide range of SDG levels, some being greater than those of CDC Bethune. No additional deleterious mutations were detected in these mutant lines using a panel of 10 other genes potentially involved in the lignan biosynthesis. This study provides further evidence that UGT74S1 is unique in controlling SDG formation from SECO and this is the first report of non-transgenic flax germplasm with simultaneous knockout of SDG and presence of SMG in planta. PMID:28983308

Magnetic Flyer Facility Correlation and UGT Simulation

DTIC Science & Technology

1978-05-01

AND UGT SIMULATION (U) Kaman Sciences Corporation L ~ P.O. Box 7463 I Colorado Springs, Colcerado 80933 ý4 May 1978DC Final Report CONTRACT No. DNA O01...selected underground test ( UGT ) environment on 3DQP; and, (2) To correlate the magnetically driven flyer plate facilities of VKSC with those of the...tailored to matcb the pressure vs. time anid total impulse measurements obtained on UGT events. This matching of experi- mental data required considerable

Toxicological responses on cytochrome P450 and metabolic transferases in liver of goldfish (Carassius auratus) exposed to lead and paraquat.

PubMed

Xu, Xiaoming; Cui, Zhaojie; Wang, Xinlei; Wang, Xixin; Zhang, Su

2018-04-30

As the producer of reactive oxygen species (ROS), both lead (Pb) and paraquat (PQ) can generate serious oxidative stress in target organs which result in irreversible toxic effects on organisms. They can disturb the normal catalytic activities of many enzymes by means of different toxicity mechanism. The changed responses of enzymes are frequently used as the biomarkers for indicating the relationship between toxicological effects and exposure levels. In this work, goldfish was exposed to a series of test groups containing lead and paraquat in the range of 0.05-10mg/L, respectively. Four hepatic enzyme activities, including 7-ethoxyresorufinO-deethylase (EROD), 7-benzyloxy-4-trifluoromethyl-coumarin-O-debenzyloxylase (BFCOD), glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UGT) were determined after 1, 7, 14, 28 days exposure. The results showed that the activities of EROD and BFCOD in fish were significantly inhibited in response to paraquat at all exposure levels during the whole experiment. Similarly, the inhibitory effects of lead exposure on BFCOD activity were found in our study, while different responses of lead on EROD were observed. There were no significant differences on EROD activity under lower concentrations of lead (less than 0.1mg/L) before 14 days until an obvious increase was occurred for the 0.5mg/L lead treatment group at day 14. Furthermore, lead showed stronger inhibition on GST activity than paraquat when the concentrations of the two toxicants were more than 0.5mg/L. However, the similar dose and time-dependent manners of UGT activity were found under lead and paraquat exposure. Our results indicated that higher exposure levels and longer accumulations caused inhibitory effects on the four enzymes regardless of lead or paraquat stress. In addition, the responses of phase I enzymes were more sensitive than that of phase II enzymes and they may be served as the acceptable biomarkers for evaluating the toxicity effects of both

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

PubMed

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

2001-02-09

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

Thio-Linked UDP–Peptide Conjugates as O-GlcNAc Transferase Inhibitors

PubMed Central

2018-01-01

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay. PMID:29723473

Association of UGT2B7 and UGT1A4 Polymorphisms with Serum Concentration of Antiepileptic Drugs in Children.

PubMed

Du, Zhongliang; Jiao, Yukun; Shi, Lianting

2016-10-31

BACKGROUND This study aimed to analyze the relationship of UGT2B7 and UGT1A4 polymorphisms with metabolism of valproic acid (VPA) and lamotrigine (LTG) in epileptic children. MATERIAL AND METHODS We administered VPA (102) and LTG (102) to 204 children with epilepsy. Blood samples were collected before the morning dose. Serum concentration of LTG was measured by high-performance liquid chromatography (HPLC). Serum VPA concentration was tested by fluorescence polarization immunoassay. UGT2B7 A268G, C802T, and G211T polymorphisms, as well as UGT1A4 L48V polymorphism, were assayed by direct automated DNA sequencing after PCR. Evaluation of efficacy was conducted using the Engel method. RESULTS The adjusted serum concentration of VPA was 4.26 μg/mL per mg/kg and LTG was 1.56 μg/mL per mg/kg. Multiple linear regression analysis revealed that VPA or LTG adjusted concentration showed a good linear relation with sex and age. UGT2B7 A268G and C802T polymorphisms were demonstrated to affect the serum concentration of VPA (F=3.147, P=0.047; F=22.754, P=0.000). UGT1A4 L48V polymorphism was not related with the serum concentration of LTG (F=5.328, P=0.006). In the efficacy analysis, we found that C802T polymorphism exerted strong effects on efficacy of VPA (χ²=9.265, P=0.010). L48V polymorphism also showed effects on efficacy of LTG (χ²=17.397, P=0.001). CONCLUSIONS UGT2B7, UGT1A4 polymorphisms play crucial roles in metabolism of VPA and LTG.

Identification of Intestinal UDP-Glucuronosyltransferase Inhibitors in Green Tea (Camellia sinensis) Using a Biochemometric Approach: Application to Raloxifene as a Test Drug via In Vitro to In Vivo Extrapolation.

PubMed

Tian, Dan-Dan; Kellogg, Joshua J; Okut, Neşe; Oberlies, Nicholas H; Cech, Nadja B; Shen, Danny D; McCune, Jeannine S; Paine, Mary F

2018-05-01

Green tea ( Camellia sinensis ) is a popular beverage worldwide, raising concern for adverse interactions when co-consumed with conventional drugs. Like many botanical natural products, green tea contains numerous polyphenolic constituents that undergo extensive glucuronidation. As such, the UDP-glucuronosyltransferases (UGTs), particularly intestinal UGTs, represent potential first-pass targets for green tea-drug interactions. Candidate intestinal UGT inhibitors were identified using a biochemometrics approach, which combines bioassay and chemometric data. Extracts and fractions prepared from four widely consumed teas were screened (20-180 μ g/ml) as inhibitors of UGT activity (4-methylumbelliferone glucuronidation) in human intestinal microsomes; all demonstrated concentration-dependent inhibition. A biochemometrics-identified fraction rich in UGT inhibitors from a representative tea was purified further and subjected to second-stage biochemometric analysis. Five catechins were identified as major constituents in the bioactive subfractions and prioritized for further evaluation. Of these catechins, (-)-epicatechin gallate and (-)-epigallocatechin gallate showed concentration-dependent inhibition, with IC 50 values (105 and 59 μ M, respectively) near or below concentrations measured in a cup (240 ml) of tea (66 and 240 μ M, respectively). Using the clinical intestinal UGT substrate raloxifene, the K i values were ∼1.0 and 2.0 μ M, respectively. Using estimated intestinal lumen and enterocyte inhibitor concentrations, a mechanistic static model predicted green tea to increase the raloxifene plasma area under the curve up to 6.1- and 1.3-fold, respectively. Application of this novel approach, which combines biochemometrics with in vitro-in vivo extrapolation, to other natural product-drug combinations will refine these procedures, informing the need for further evaluation via dynamic modeling and clinical testing. Copyright © 2018 by The American Society for

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

PubMed Central

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

2014-01-01

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

The effects of clobazam treatment in rats on the expression of genes and proteins encoding glucronosyltransferase 1A/2B (UGT1A/2B) and multidrug resistance‐associated protein-2 (MRP2), and development of thyroid follicular cell hypertrophy

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

Miyawaki, Izuru, E-mail: izuru-miyawaki@ds-pharma.co.jp; Tamura, Akitoshi; Matsumoto, Izumi

Clobazam (CLB) is known to increase hepatobiliary thyroxine (T4) clearance in Sprague–Dawley (SD) rats, which results in hypothyroidism followed by thyroid follicular cell hypertrophy. However, the mechanism of the acceleration of T4-clearance has not been fully investigated. In the present study, we tried to clarify the roles of hepatic UDP-glucronosyltransferase (UGT) isoenzymes (UGT1A and UGT2B) and efflux transporter (multidrug resistance–associated protein-2; MRP2) in the CLB-induced acceleration of T4-clearance using two mutant rat strains, UGT1A-deficient mutant (Gunn) and MRP2-deficient mutant (EHBR) rats, especially focusing on thyroid morphology, levels of circulating hormones (T4 and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH), and mRNAmore » or protein expressions of UGTs (Ugt1a1, Ugt1a6, and Ugt2b1/2) and MRP2 (Mrp). CLB induced thyroid morphological changes with increases in TSH in SD and Gunn rats, but not in EHBR rats. T4 was slightly decreased in SD and Gunn rats, and T3 was decreased in Gunn rats, whereas these hormones were maintained in EHBR rats. Hepatic Ugt1a1, Ugt1a6, Ugt2b1/2, and Mrp2 mRNAs were upregulated in SD rats. In Gunn rats, UGT1A mRNAs (Ugt1a1/6) and protein levels were quite low, but UGT2B mRNAs (Ugt2b1/2) and protein were prominently upregulated. In SD and Gunn rats, MRP2 mRNA and protein were upregulated to the same degree. These results suggest that MRP2 is an important contributor in development of the thyroid cellular hypertrophy in CLB-treated rats, and that UGT1A and UGT2B work in concert with MRP2 in the presence of MRP2 function to enable the effective elimination of thyroid hormones. -- Highlights: ► Role of UGT and MRP2 in thyroid pathology was investigated in clobazam-treated rats. ► Clobazam induced thyroid cellular hypertrophy in SD and Gunn rats, but not EHBR rats. ► Hepatic Mrp2 gene and protein were upregulated in SD and Gunn rats, but not EHBR rats. ► Neither serum thyroid hormones

Activity-Based Profiling of a Physiologic Aglycone Library Reveals Sugar Acceptor Promiscuity of Family 1 UDP-Glucosyltransferases from Grape1[W

PubMed Central

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

2014-01-01

Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl β-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol β-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity. PMID:25073706

Statin Lactonization by Uridine 5'-Diphospho-glucuronosyltransferases (UGTs).

PubMed

Schirris, Tom J J; Ritschel, Tina; Bilos, Albert; Smeitink, Jan A M; Russel, Frans G M

2015-11-02

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 inducers of toxicity. They can be formed by glucuronidation mediated by uridine 5'-diphospho-glucuronosyltransferases (UGTs), but a systematic characterization of subtype specificity and kinetics of lactonization is lacking. Here, we demonstrate for six clinically relevant statins that only UGT1A1, 1A3, and 2B7 contribute significantly to their lactonization. UGT1A3 appeared to have the highest lactonization capacity with marked differences in statin conversion rates: pitavastatin ≫ atorvastatin > cerivastatin > lovastatin > rosuvastatin (simvastatin not converted). Using in silico modeling we could identify a probable statin interaction region in the UGT binding pocket. Polymorphisms in these regions of UGT1A1, 1A3, and 2B7 may be a contributing factor in statin-induced myopathies, which could be used in personalization of statin therapy with improved safety.

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

PubMed Central

Ma, Liang; Salas, Omar; Bowler, Kyle

2017-01-01

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

Studies on induction of lamotrigine metabolism in transgenic UGT1 mice

PubMed Central

Argikar, U. A.; Senekeo-Effenberger, K.; Larson, E. E.; Tukey, R. H.; Remmel, R. P.

2010-01-01

A transgenic ‘knock-in’ mouse model expressing a human UGT1 locus (Tg-UGT1) was recently developed and validated. Although these animals express mouse UGT1A proteins, UGT1A4 is a pseudo-gene in mice. Therefore, Tg-UGT1 mice serve as a ‘humanized’ UGT1A4 animal model.Lamotrigine (LTG) is primarily metabolized to its N-glucuronide (LTGG) by hUGT1A4. This investigation aimed at examining the impact of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPAR) activators on LTG glucuronidation in vivo and in vitro. Tg-UGT1 mice were administered the inducers phenobarbital (CAR), pregnenolone-16α-carbonitrile (PXR), WY-14643 (PPAR-α), ciglitazone (PPAR-γ), or L-165041 (PPAR-β), once daily for 3 or 4 days. Thereafter, LTG was administered orally and blood samples were collected over 24 h. LTG was measured in blood and formation of LTGG was measured in pooled microsomes made from the livers of treated animals.A three-fold increase in in vivo LTG clearance was seen after phenobarbital administration. In microsomes prepared from phenobarbital-treated Tg-UGT1 animals, 13-fold higher CLint (Vmax/Km) value was observed as compared with the untreated transgenic mice. A trend toward induction of catalytic activity in vitro and in vivo was also observed following pregnenolone-16α-carbonitrile and WY-14643 treatment. This study demonstrates the successful application of Tg-UGT1 mice as a novel tool to study the impact of induction and regulation on metabolism of UGT1A4 substrates. PMID:19845433

Total knee arthroplasty and Crigler-Najjar syndrome: a case report.

PubMed

Walmsley, David; Alzaharani, Khalid; Coke, William J; Gandhi, Rajiv

2010-06-01

Crigler-Najjar (CN) syndrome is a rare genetic disease characterized by hyperbilirubinemia due to a deficiency in the hepatic enzyme UDP-glucuronosyl-transferase. We describe the first case of total knee arthroplasty in a patient with CN syndrome (type II). This procedure was complicated by kernicterus 1 week after hospital discharge. He also developed Klebsiella bacteremia and sepsis, requiring a brief ICU stay. He was discharged in good condition 2 months later. It is evident that physicians involved in the care of patients with CN syndrome in the peri-operative period need to have a high index of suspicion for the development of severe hyperbilirubinemia and kernicterus in order to appropriately manage and, possibly, prevent this complication. A literature review and intra-operative observations provide insight into the possible relationship between hyperbilirubinemia and osteoarthritis as well as the peri-operative considerations to be made for this group of patients.

Establishment of the Tree Shrew as an Alcohol-Induced Fatty Liver Model for the Study of Alcoholic Liver Diseases

PubMed Central

Xing, Huijie; Jia, Kun; He, Jun; Shi, Changzheng; Fang, Meixia; Song, Linliang; Zhang, Pu; Zhao, Yue; Fu, Jiangnan; Li, Shoujun

2015-01-01

Currently, the pathogenesis of alcoholic liver diseases (ALDs) is not clear. As a result, there is no effective treatment for ALDs. One limitation is the lack of a suitable animal model for use in studying ALDs. The tree shrew is a lower primate animal, characterized by a high-alcohol diet. This work aimed to establish a fatty liver model using tree shrews and to assess the animals’ suitability for the study of ALDs. Tree shrews were treated with alcohol solutions (10% and 20%) for two weeks. Hemophysiology, blood alcohol concentrations (BACs), oxidative stress factors, alcohol metabolic enzymes and hepatic pathology were checked and assayed with an automatic biochemical analyzer, enzyme-linked immunosorbent assay (ELISA), western blot, hematoxylin-eosin (HE) staining and oil red O staining, and magnetic resonance imaging (MRI). Compared with the normal group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), total cholesterol (TC), triglyceride (TG), reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly enhanced in alcohol-treated tree shrews. However, the activity of reduced glutathione hormone (GSH) and superoxide dismutase (SOD) declined. Notable changes in alcohol dehydrogenase(ADH1), aldehyde dehydrogenase(ALDH2), CYP2E1, UDP-glucuronosyl transferase 1A1 (UGT1A1) and nuclear factor erythroid-related factor 2 (Nrf2) were observed. HE and oil red O staining showed that hepatocyte swelling, hydropic degeneration, and adipohepatic syndrome occurred in the tree shrews. Alcohol can induce fatty liver-like pathological changes and result in alterations in liver function, oxidative stress factors, alcohol metabolism enzymes and Nrf2. Therefore, the established fatty liver model of tree shrews induced by alcohol should be a promising tool for the study of ALDs. PMID:26030870

Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells

PubMed Central

Fahrmayr, C; König, J; Auge, D; Mieth, M; Fromm, MF

2012-01-01

BACKGROUND AND PURPOSE The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. EXPERIMENTAL APPROACH Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. KEY RESULTS Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. CONCLUSIONS AND IMPLICATIONS Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition. PMID:21923755

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

PubMed

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

2017-01-01

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

Role of the UGT2B17 deletion in exemestane pharmacogenetics

PubMed Central

Luo, Shaman; Chen, Gang; Truica, Cristina; Baird, Cynthia C.; Leitzel, Kim; Lazarus, Philip

2017-01-01

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of breast cancer. The major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc) by UGT2B17. The aim of the present study was to determine the effects of UGT2B17 copy number variation on the levels of urinary and plasma 17β-DHE-Gluc and 17β-DHE in patients taking EXE. Ninety-six post-menopausal Caucasian breast cancer patients with ER+ breast tumors taking 25 mg EXE daily were recruited into this study. UGT2B17 copy number was determined by a real-time PCR copy number variant assay and the levels of EXE, 17β-DHE and 17β-DHE-Gluc were quantified by UPLC/MS in patients’ urine and plasma. A 39-fold decrease (P<0.0001) in the levels of creatinine-adjusted urinary 17β-DHE-Gluc was observed among UGT2B17 (*2/*2) subjects vs. subjects with the UGT2B17 (*1/*1) genotype. The plasma levels of 17β-DHE-Gluc was decreased 29-fold (P<0.0001) in subjects with the UGT2B17 (*2/*2) genotype vs. subjects with UGT2B17 (*1/*1) genotype. The levels of plasma EXE-adjusted 17β-DHE was 28% higher (P=0.04) in subjects with the UGT2B17 (*2/*2) genotype vs. subjects with the UGT2B17 (*1/*1) genotype. These data indicate that UGT2B17 is the major enzyme responsible for 17β-DHE-Gluc formation in vivo and that the UGT2B17 copy number variant may play a role in inter-individual variability in 17β-DHE levels in vivo. PMID:28534527

Role of the UGT2B17 deletion in exemestane pharmacogenetics.

PubMed

Luo, S; Chen, G; Truica, C; Baird, C C; Leitzel, K; Lazarus, P

2018-04-01

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of breast cancer. The major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc) by UGT2B17. The aim of the present study was to determine the effects of UGT2B17 copy number variation on the levels of urinary and plasma 17β-DHE-Gluc and 17β-DHE in patients taking EXE. Ninety-six post-menopausal Caucasian breast cancer patients with ER+ breast tumors taking 25 mg EXE daily were recruited into this study. UGT2B17 copy number was determined by a real-time PCR copy number variant assay and the levels of EXE, 17β-DHE and 17β-DHE-Gluc were quantified by UPLC/MS in patients' urine and plasma. A 39-fold decrease (P<0.0001) in the levels of creatinine-adjusted urinary 17β-DHE-Gluc was observed among UGT2B17 (*2/*2) subjects vs subjects with the UGT2B17 (*1/*1) genotype. The plasma levels of 17β-DHE-Gluc was decreased 29-fold (P<0.0001) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with UGT2B17 (*1/*1) genotype. The levels of plasma EXE-adjusted 17β-DHE was 28% higher (P=0.04) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with the UGT2B17 (*1/*1) genotype. These data indicate that UGT2B17 is the major enzyme responsible for 17β-DHE-Gluc formation in vivo and that the UGT2B17 copy number variant may play a role in inter-individual variability in 17β-DHE levels in vivo.

Three-dimensional quantitative structure-activity relationship studies on UGT1A9-mediated 3-O-glucuronidation of natural flavonols using a pharmacophore-based comparative molecular field analysis model.

PubMed

Wu, Baojian; Morrow, John Kenneth; Singh, Rashim; Zhang, Shuxing; Hu, Ming

2011-02-01

Glucuronidation is often recognized as one of the rate-determining factors that limit the bioavailability of flavonols. Hence, design and synthesis of more bioavailable flavonols would benefit from the establishment of predictive models of glucuronidation using kinetic parameters [e.g., K(m), V(max), intrinsic clearance (CL(int)) = V(max)/K(m)] derived for flavonols. This article aims to construct position (3-OH)-specific comparative molecular field analysis (CoMFA) models to describe UDP-glucuronosyltransferase (UGT) 1A9-mediated glucuronidation of flavonols, which can be used to design poor UGT1A9 substrates. The kinetics of recombinant UGT1A9-mediated 3-O-glucuronidation of 30 flavonols was characterized, and kinetic parameters (K(m), V(max), CL(int)) were obtained. The observed K(m), V(max), and CL(int) values of 3-O-glucuronidation ranged from 0.04 to 0.68 μM, 0.04 to 12.95 nmol/mg/min, and 0.06 to 109.60 ml/mg/min, respectively. To model UGT1A9-mediated glucuronidation, 30 flavonols were split into the training (23 compounds) and test (7 compounds) sets. These flavonols were then aligned by mapping the flavonols to specific common feature pharmacophores, which were used to construct CoMFA models of V(max) and CL(int), respectively. The derived CoMFA models possessed good internal and external consistency and showed statistical significance and substantive predictive abilities (V(max) model: q(2) = 0.738, r(2) = 0.976, r(pred)(2) = 0.735; CL(int) model: q(2) = 0.561, r(2) = 0.938, r(pred)(2) = 0.630). The contour maps derived from CoMFA modeling clearly indicate structural characteristics associated with rapid or slow 3-O-glucuronidation. In conclusion, the approach of coupling CoMFA analysis with a pharmacophore-based structural alignment is viable for constructing a predictive model for regiospecific glucuronidation rates of flavonols by UGT1A9.

UGT74S1 is the key player in controlling secoisolariciresinol diglucoside (SDG) formation in flax.

PubMed

Fofana, Bourlaye; Ghose, Kaushik; McCallum, Jason; You, Frank M; Cloutier, Sylvie

2017-02-02

Flax lignan, commonly known as secoisolariciresinol (SECO) diglucoside (SDG), has recently been reported with health-promoting activities, including its positive impact in metabolic diseases. However, not much was reported on the biosynthesis of SDG and its monoglucoside (SMG) until lately. Flax UGT74S1 was recently reported to sequentially glucosylate SECO into SMG and SDG in vitro. However, whether this gene is the only UGT achieving SECO glucosylation in flax was not known. Flax genome-wide mining for UGTs was performed. Phylogenetic and gene duplication analyses, heterologous gene expression and enzyme assays were conducted to identify family members closely related to UGT74S1 and to establish their roles in SECO glucosylation. A total of 299 different UGTs were identified, of which 241 (81%) were duplicated. Flax UGTs diverged 2.4-153.6 MYA and 71% were found to be under purifying selection pressure. UGT74S1, a single copy gene located on chromosome 7, displayed no evidence of duplication and was deemed to be under positive selection pressure. The phylogenetic analysis identified four main clusters where cluster 4, which included UGT74S1, was the most diverse. The duplicated UGT74S4 and UGT74S3, located on chromosomes 8 and 14, respectively, were the most closely related to UGT74S1 and were differentially expressed in different tissues. Heterologous expression levels of UGT74S1, UGT74S4 and UGT74S3 proteins were similar but UGT74S4 and UGT74S3 glucosylation activity towards SECO was seven fold less than UGT74S1. In addition, they both failed to produce SDG, suggesting neofunctionalization following their divergence from UGT74S1. We showed that UGT74S1 is closely related to two duplicated genes, UGT74S4 and UGT74S3 which, unlike UGT74S1, failed to glucosylate SMG into SDG. The study suggests that UGT74S1 may be the key player in controlling SECO glucosylation into SDG in flax although its closely related genes may also contribute to a minor extent in supplying

Modulation of porcine biotransformation enzymes by anthelmintic therapy with fenbendazole and flubendazole.

PubMed

Savlík, M; Fimanová, K; Szotáková, B; Lamka, J; Skálová, L

2006-06-01

Fenbendazole (FEN) and flubendazole (FLU) are benzimidazole anthelmintics often used in pig management for the control of nematodoses. The in vivo study presented here was designed to test the influence of FLU and FEN on cytochrome P4501A and other cytochrome P450 (CYP) isoforms, UDP-glucuronosyl transferase and several carbonyl reducing enzymes. The results indicated that FEN (in a single therapeutic dose as well as in repeated therapeutic doses) caused significant induction of pig CYP1A, while FLU did not show an inductive effect towards this isoform. Some of the other hepatic and intestinal biotransformation enzymes that were assayed were moderately influenced by FEN or FLU. Strong CYP1A induction following FEN therapy in pigs may negatively affect the efficacy and pharmacokinetics of FEN itself or other simultaneously or consecutively administered drugs. From the perspective of biotransformation enzyme modulation, FLU would appear to be a more convenient anthelmintic therapy of pigs than FEN.

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

PubMed Central

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

2010-01-01

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

Effect of UGT2B10, UGT2B17, FMO3, and OCT2 Genetic Variation on Nicotine and Cotinine Pharmacokinetics and Smoking in African Americans

PubMed Central

Taghavi, Taraneh; St. Helen, Gideon; Benowitz, Neal L.; Tyndale, Rachel F.

2017-01-01

OBJECTIVES Nicotine metabolism rates differ greatly among individuals, even after controlling for variation in the major nicotine metabolizing enzyme, CYP2A6. In this study, the impact of genetic variation in alternative metabolic enzymes and transporters on nicotine and cotinine pharmacokinetics and smoking was investigated. METHODS We examined the impact of UGT2B10, UGT2B17, FMO3, NAT1, and OCT2 variation on pharmacokinetics and smoking (total nicotine equivalents and topography), before and after stratifying by CYP2A6 genotype in 60 African American smokers who received a simultaneous intravenous infusion of deuterium-labeled nicotine and cotinine. RESULTS Variants in UGT2B10 and UGT2B17 were associated with urinary glucuronidation ratios (glucuronide/free substrate). UGT2B10 rs116294140 was associated with significant alterations in cotinine and modest alterations in nicotine pharmacokinetics. These alterations, however, were not sufficient to change nicotine intake or topography. Neither UGT2B10 rs61750900, UGT2B17*2, FMO3 rs2266782, nor NAT1 rs13253389 altered nicotine or cotinine pharmacokinetics among all subjects (n=60); or among individuals with reduced CYP2A6 activity (n=23). The organic cation transporter OCT2 rs316019 significantly increased nicotine and cotinine Cmax (p=0.005, p=0.02, respectively) and decreased nicotine clearance (p=0.05). UGT2B10 rs116294140 had no significant impact on the plasma or urinary trans-3’-hydroxycotinine/cotinine ratio, commonly used as a biomarker of CYP2A6 activity. CONCLUSIONS We demonstrated that polymorphisms in genes other than CYP2A6 represent minor sources of variation in nicotine pharmacokinetics, insufficient to alter smoking in African Americans. The change in cotinine pharmacokinetics with UGT2B10 rs116294140 highlights the UGT2B10 gene as a source of variability in cotinine as a biomarker of tobacco exposure among African American smokers. PMID:28178031

Effect of UGT1A1, UGT1A3, DIO1 and DIO2 polymorphisms on L-thyroxine doses required for TSH suppression in patients with differentiated thyroid cancer.

PubMed

Santoro, Ana B; Vargens, Daniela D; Barros Filho, Mateus de Camargo; Bulzico, Daniel A; Kowalski, Luiz Paulo; Meirelles, Ricardo M R; Paula, Daniela P; Neves, Ronaldo R S; Pessoa, Cencita N; Struchine, Claudio J; Suarez-Kurtz, Guilherme

2014-11-01

To evaluate the impact of genetic polymorphisms in uridine 5'-glucuronosylytansferases UGT1A1 and UGT1A3 and iodothyronine-deiodinases types 1 and 2 on levothyroxine (T4 ; 3,5,3',5'-triiodo-L-thyronine) dose requirement for suppression of thyrotropin (TSH) secretion in patients with differentiated thyroid cancer (DTC). Patients (n = 268) submitted to total thyroidectomy and ablation by (131) I, under T4 therapy for at least 6 months were recruited in three public institutions in Brazil. Multivariate regression modelling was applied to assess the association of T4 dosing with polymorphisms in UGT1A1 (rs8175347), UGT1A3 (rs3806596 and rs1983023), DIO1 (rs11206244 and rs2235544) and DIO2 (rs225014 and rs12885300), demographic and clinical variables. A regression model including UGT1A haplotypes, age, gender, body weight and serum TSH concentration accounted for 39% of the inter-individual variation in the T4 dosage. The association of T4 dose with UGT1A haplotype is attributed to reduced UGT1A1 expression and T4 glucuronidation in liver of carriers of low expression UGT1A1 rs8175347 alleles. The DIO1 and DIO2 genotypes had no influence of T4 dosage. UGT1A haplotypes associate with T4 dosage in DTC patients, but the effect accounts for only 2% of the total variability and recommendation of pre-emptive UGT1A genotyping is not warranted. © 2014 The British Pharmacological Society.

Effect of UGT1A1, UGT1A3, DIO1 and DIO2 polymorphisms on L-thyroxine doses required for TSH suppression in patients with differentiated thyroid cancer

PubMed Central

Santoro, Ana B; Vargens, Daniela D; Barros Filho, Mateus de Camargo; Bulzico, Daniel A; Kowalski, Luiz Paulo; Meirelles, Ricardo M R; Paula, Daniela P; Neves, Ronaldo R S; Pessoa, Cencita N; Struchine, Claudio J; Suarez-Kurtz, Guilherme

2014-01-01

Aim To evaluate the impact of genetic polymorphisms in uridine 5′-glucuronosylytansferases UGT1A1 and UGT1A3 and iodothyronine-deiodinases types 1 and 2 on levothyroxine (T4; 3,5,3′,5′-triiodo-L-thyronine) dose requirement for suppression of thyrotropin (TSH) secretion in patients with differentiated thyroid cancer (DTC). Methods Patients (n = 268) submitted to total thyroidectomy and ablation by 131I, under T4 therapy for at least 6 months were recruited in three public institutions in Brazil. Multivariate regression modelling was applied to assess the association of T4 dosing with polymorphisms in UGT1A1 (rs8175347), UGT1A3 (rs3806596 and rs1983023), DIO1 (rs11206244 and rs2235544) and DIO2 (rs225014 and rs12885300), demographic and clinical variables. Results A regression model including UGT1A haplotypes, age, gender, body weight and serum TSH concentration accounted for 39% of the inter-individual variation in the T4 dosage. The association of T4 dose with UGT1A haplotype is attributed to reduced UGT1A1 expression and T4 glucuronidation in liver of carriers of low expression UGT1A1 rs8175347 alleles. The DIO1 and DIO2 genotypes had no influence of T4 dosage. Conclusion UGT1A haplotypes associate with T4 dosage in DTC patients, but the effect accounts for only 2% of the total variability and recommendation of pre-emptive UGT1A genotyping is not warranted. PMID:24910925

Exome-Wide Association Study Identifies New Low-Frequency and Rare UGT1A1 Coding Variants and UGT1A6 Coding Variants Influencing Serum Bilirubin in Elderly Subjects

PubMed Central

Oussalah, Abderrahim; Bosco, Paolo; Anello, Guido; Spada, Rosario; Guéant-Rodriguez, Rosa-Maria; Chery, Céline; Rouyer, Pierre; Josse, Thomas; Romano, Antonino; Elia, Maurizzio; Bronowicki, Jean-Pierre; Guéant, Jean-Louis

2015-01-01

Abstract Genome-wide association studies (GWASs) have identified loci contributing to total serum bilirubin level. However, no exome-wide approaches have been performed to address this question. Using exome-wide approach, we assessed the influence of protein-coding variants on unconjugated, conjugated, and total serum bilirubin levels in a well-characterized cohort of 773 ambulatory elderly subjects from Italy. Coding variants were replicated in 227 elderly subjects from the same area. We identified 4 missense rare (minor allele frequency, MAF < 0.5%) and low-frequency (MAF, 0.5%–5%) coding variants located in the first exon of the UGT1A1 gene, which encodes for the substrate-binding domain (rs4148323 [MAF = 0.06%; p.Gly71Arg], rs144398951 [MAF = 0.06%; p.Ile215Val], rs35003977 [MAF = 0.78%; p.Val225Gly], and rs57307513 [MAF = 0.06%; p.Ser250Pro]). These variants were in strong linkage disequilibrium with 3 intronic UGT1A1 variants (rs887829, rs4148325, rs6742078), which were significantly associated with total bilirubin level (P = 2.34 × 10−34, P = 7.02 × 10−34, and P = 8.27 × 10−34), as well as unconjugated, and conjugated bilirubin levels. We also identified UGT1A6 variants in association with total (rs6759892, p.Ser7Ala, P = 1.98 × 10−26; rs2070959, p.Thr181Ala, P = 2.87 × 10−27; and rs1105879, p.Arg184Ser, P = 3.27 × 10−29), unconjugated, and conjugated bilirubin levels. All UGT1A1 intronic variants (rs887829, rs6742078, and rs4148325) and UGT1A6 coding variants (rs6759892, rs2070959, and rs1105879) were significantly associated with gallstone-related cholecystectomy risk. The UGT1A6 variant rs2070959 (p.Thr181Ala) was associated with the highest risk of gallstone–related cholecystectomy (OR, 4.58; 95% CI, 1.58–13.28; P = 3.21 × 10−3). Using an exome-wide approach we identified coding variants on UGT1A1 and UGT1A6 genes in association with serum bilirubin

The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes

USDA-ARS?s Scientific Manuscript database

The dietary bioavailability of the isoflavone genistein is decreased in older rats compared to young adults. Since flavonoids are metabolized extensively by the UDP-glucuronosyltransferases (UGTs), we hypothesized that UGT flavonoid conjugating activity changes with age. The effect of age on flavono...

Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells. [Pisum sativum

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

Camirand, A.; Brummell, D.; MacLachlan, G.

1987-07-01

Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-(/sup 14/C) fucose and UDP-(/sup 14/C)xylose to xyloglucan occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with (/sup 3/H)fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.

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

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

Liu, Wei; Feng, Qian; Li, Ye

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 tomore » 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.« less

Binding pattern of intermediate UDP-4-keto-xylose to human UDP-xylose synthase: Synthesis and STD NMR of model keto-saccharides.

PubMed

Puchner, Claudia; Eixelsberger, Thomas; Nidetzky, Bernd; Brecker, Lothar

2017-01-02

Human UDP-xylose synthase (hUXS1) exclusively converts UDP-glucuronic acid to UDP-xylose via intermediate UDP-4-keto-xylose (UDP-Xyl-4O). Synthesis of model compounds like methyl-4-keto-xylose (Me-Xyl-4O) is reported to investigate the binding pattern thereof to hUXS1. Hence, selective oxidation of the desired hydroxyl function required employment of protecting group chemistry. Solution behavior of synthesized keto-saccharides was studied without enzyme via 1 H and 13 C NMR spectroscopy with respect to existent forms in deuterated potassium phosphate buffer. Keto-enol tautomerism was observed for all investigated keto-saccharides, while gem-diol hydrate forms were only observed for 4-keto-xylose derivatives. Saturation transfer difference (STD) NMR was used to study binding of synthesized keto-gylcosides to wild type hUXS1. Resulting epitope maps were correlated to earlier published molecular modeling studies of UDP-Xyl-4O. STD NMR results of Me-Xyl-4O are in good agreement with simulations of the intermediate UDP-Xyl-4O indicating a strong interaction of proton H3 with the enzyme, potentially caused by active site residue Ala 79 . In contrast, pyranoside binding pattern studies of methyl uronic acids showed some differences compared to previously published STD NMR results of UDP-glycosides. In general, obtained results can contribute to a better understanding in binding of UDP-glycosides to other UXS enzyme family members, which have high structural similarities in the active site. Copyright © 2016. Published by Elsevier Ltd.

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

PubMed

Lim, Michelle Yi-Xiu; LaMonte, Gregory; Lee, Marcus C S; Reimer, Christin; Tan, Bee Huat; Corey, Victoria; Tjahjadi, Bianca F; Chua, Adeline; Nachon, Marie; Wintjens, René; Gedeck, Peter; Malleret, Benoit; Renia, Laurent; Bonamy, Ghislain M C; Ho, Paul Chi-Lui; Yeung, Bryan K S; Chow, Eric D; Lim, Liting; Fidock, David A; Diagana, Thierry T; Winzeler, Elizabeth A; Bifani, Pablo

2016-09-19

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

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

PubMed

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

2016-07-05

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

Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

USDA-ARS?s Scientific Manuscript database

UDP-glucuronosyltransferase (UGT) activity toward the flavonoid quercetin and UGT protein were characterized in 3 equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats, n=8/age using villin to control for enterocyte content. SI microsomal intrinsic clearance of quercetin...

Morphine clearance in children: does race or genetics matter?

PubMed

Sadhasivam, Senthilkumar; Krekels, Elke H J; Chidambaran, Vidya; Esslinger, Hope R; Ngamprasertwong, Pornswan; Zhang, Kejian; Fukuda, Tsuyoshi; Vinks, Alexander A

2012-01-01

Interindividual variability in analgesic response and adverse effects of opioids because of narrow therapeutic indices are major clinical problems. Morphine is an opioid commonly used in children to manage perioperative pain. Al-though size and age often are considered primary covariates for morphine pharmacokinetic models, the impact of other factors important in personalizing care such as race and genetic variations on morphine disposition is not well documented. Genotype blinded clinical observational pharmacokinetic study. One hundred forty-six African American and Caucasian children scheduled for elective outpatient adenotonsillectomy were enrolled in our prospective genotype blinded observational study with standard perioperative clinical care. Tertiary care pediatric institution. Morphine bolus for intraoperative analgesia in children and pharmacokinetic analyses in different races. Pharmacokinetics and pharmacogenetics of intravenous morphine in a homogeneous pediatric outpatient surgical pain population were evaluated. The authors observed that African American children have higher morphine clearance than Caucasian children. The increased clearance is directed toward the formation of morphine-3-glucuronide formation, rather than the formation of morphine-6-glucuronide. Common uridine diphosphate glucuronosyl transferase (UGT) 2B7 genetic variations (2161C>T and 802C>T) were not associated with observed racial differences in morphine's clearance although the wild type of the UGT2B7 isozyme is more prevalent in the African Americans. Race of the child is an important factor in perioperative intravenous morphine's clearance and its potential role in personalizing analgesia with morphine needs further investigation.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2017-08-09

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

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

PubMed

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

2017-02-01

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

BDE-99, but not BDE-47, is a transient aryl hydrocarbon receptor agonist in zebrafish liver cells

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

Yang, Jie; Zhu, Jinyong; Chan, King Ming, E-mail:

Polybrominated diphenyl ethers (PBDEs) are endocrine-disrupting chemicals that affect the environment and the health of humans and wildlife. In this study, the zebrafish liver (ZFL) cell line was used in vitro to investigate two major PBDE contaminants: 2, 2′, 4, 4′, 5-pentabromodiphenyl ether (BDE-99) and 2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47). BDE-99 was found to significantly induce cytochrome P450 (CYP1A), uridine diphosphate glucuronosyl transferase 1 family a, b (ugt1ab), 7-ethoxyresorufin-O-deethylase activity and an aryl hydrocarbon receptor (Ahr) dependent xenobiotic response element luciferase reporter system, confirming the Ahr-mediated activation of CYP1A by BDE-99. The time-course effect indicated that the role ofmore » BDE-99 in Ahr-mediated signaling is likely to be transient and highly dependent on the ability of BDE-99 to induce CYP1A and ugt1ab, and presumably its metabolism. BDE-99 also exhibited a significant dose-response effect on a developed zebrafish pregnane X receptor luciferase reporter gene system. However, the other abundant contaminant under study, BDE-47, did not exhibit the above effects. Together, these results indicated that the molecular mechanism of PBDEs induced in ZFL cells is a chemically specific process that differs between members of the PBDE family. CYP1A induction derived by BDE-99 warrants further risk assessment as the humans, wildlife and environment are exposed to a complex mixture including dioxin-like compounds and carcinogenic compounds. - Highlights: • BDE-99 is an aryl hydrocarbon receptor (Ahr) agonist in zebrafish liver cell-line ZFL. • BDE-99 induced EROD activity, CYP1A and ugt1ab gene expression, in ZFL. • BDE-99 induced the pregnane X receptor (Pxr) luciferase reporter gene system in ZFL. • BDE-47 did not show any effects in ZFL to induce CYP1A, ugt1ab, and EROD. • BDE-47 and -99 showed no induction of Rxr and Pxr pathways in ZFL cells.« less

Gene replacement therapy for genetic hepatocellular jaundice.

PubMed

van Dijk, Remco; Beuers, Ulrich; Bosma, Piter J

2015-06-01

Jaundice results from the systemic accumulation of bilirubin, the final product of the catabolism of haem. Inherited liver disorders of bilirubin metabolism and transport can result in reduced hepatic uptake, conjugation or biliary secretion of bilirubin. In patients with Rotor syndrome, bilirubin (re)uptake is impaired due to the deficiency of two basolateral/sinusoidal hepatocellular membrane proteins, organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. Dubin-Johnson syndrome is caused by a defect in the ATP-dependent canalicular transporter, multidrug resistance-associated protein 2 (MRP2), which mediates the export of conjugated bilirubin into bile. Both disorders are benign and not progressive and are characterised by elevated serum levels of mainly conjugated bilirubin. Uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) is responsible for the glucuronidation of bilirubin; deficiency of this enzyme results in unconjugated hyperbilirubinaemia. Gilbert syndrome is the mild and benign form of inherited unconjugated hyperbilirubinaemia and is mostly caused by reduced promoter activity of the UGT1A1 gene. Crigler-Najjar syndrome is the severe inherited form of unconjugated hyperbilirubinaemia due to mutations in the UGT1A1 gene, which can cause kernicterus early in life and can be even lethal when left untreated. Due to major disadvantages of the current standard treatments for Crigler-Najjar syndrome, phototherapy and liver transplantation, new effective therapeutic strategies are under development. Here, we review the clinical features, pathophysiology and genetic background of these inherited disorders of bilirubin metabolism and transport. We also discuss the upcoming treatment option of viral gene therapy for genetic disorders such as Crigler-Najjar syndrome and the possible immunological consequences of this therapy.

Role of UGT1A1*6, UGT1A1*28 and ABCG2 c.421C>A polymorphisms in irinotecan-induced neutropenia in Asian cancer patients.

PubMed

Jada, Srinivasa Rao; Lim, Robert; Wong, Chiung Ing; Shu, Xiaochen; Lee, Soo Chin; Zhou, Qingyu; Goh, Boon Cher; Chowbay, Balram

2007-09-01

The objectives of the present study were (i) to study the pharmacogenetics of UGT1A1*6, UGT1A1*28 and ABCG2 c.421C>A in three distinct healthy Asian populations (Chinese, Malays and Indians), and (ii) to investigate the polygenic influence of these polymorphic variants in irinotecan-induced neutropenia in Asian cancer patients. Pharmacokinetic and pharmacogenetic analyses were done after administration of irinotecan as a 90-min intravenous infusion of 375 mg/m(2) once every 3 weeks (n = 45). Genotypic-phenotypic correlates showed a non-significant influence of UGT1A1*28 and ABCG2 c.421C>A polymorphisms on the pharmacokinetics of SN-38 (P > 0.05), as well as severity of neutropenia (P > 0.05). Significantly higher exposure levels to SN-38 (P = 0.018), lower relative extent of glucuronidation (REG; P = 0.006) and higher biliary index (BI; P = 0.003) were found in cancer patients homozygous for the UGT1A1*6 allele compared with patients harboring the reference genotype. The mean absolute neutrophil count (ANC) was 85% lower and the prevalence of grade 4 neutropenia (ANC < or = 500/microL) was 27% in patients homozygous for UGT1A1*6 compared with the reference group. Furthermore, the presence of the UGT1A1*6 allele was associated with an approximately 3-fold increased risk of developing severe grade 4 neutropenia compared with patients harboring the reference genotype. These exploratory findings suggest that homozygosity for UGT1A1*6 allele may be associated with altered SN-38 disposition and may increase the risk of severe neutropenia in Asian cancer patients, particularly in the Chinese cancer patients who comprised 80% (n = 36) of the patient population in the present study.

Characterization of raloxifene glucuronidation. Potential role of UGT1A8 genotype on raloxifene metabolism in vivo

PubMed Central

Sun, Dongxiao; Jones, Nathan R; Manni, Andrea; Lazarus, Philip

2014-01-01

Raloxifene is a 2nd-generation selective estrogen receptor modulator used for the prevention and treatment of osteoporosis and the prevention of breast cancer in postmenopausal women. Raloxifene is extensively metabolized by glucuronidation to form raloxifene-6-glucuronide (ral-6-Gluc) and raloxifene-4′-glucuronide (ral-4′-Gluc). The goal of the present study was to determine whether functional polymorphisms in active UGTs could play a role in altered raloxifene glucuronidation in vivo. Using homogenates from HEK293 UGT-overexpressing cell lines, raloxifene was shown to be glucuronidated primarily by the hepatic UGTs 1A1 and 1A9 and the extra-hepatic UGTs 1A8 and 1A10; no detectable raloxifene glucuronidation activity was found for UGT2B enzymes. Functional UGT1A1 transcriptional promoter genotypes were significantly (ptrend=0.005) associated with ral-6-Gluc formation in human liver microsomes, and, consistent with the decreased raloxifene glucuronidation activities observed in vitro with cell line over-expressing UGT1A8 variants, the UGT1A8*2 variant was significantly (p=0.023) correlated with total raloxifene glucuronide formation in human jejunum homogenates. While ral-4′-Gluc exhibited 1/100th the anti-estrogenic activity of raloxifene itself as measured by binding to the estrogen receptor, raloxifene glucuronides comprised ∼99% of the circulating raloxifene dose in raloxifene-treated subjects, with ral-4′-Gluc comprising ∼70% of raloxifene glucuronides. Plasma ral-6-Gluc (ptrend=0.0025), ral-4′-Gluc (ptrend=0.001), and total raloxifene glucuronides (ptrend=0.001) were increased in raloxifene-treated subjects who were predicted slow metabolizers [UGT1A8 (*1/*3)] vs intermediate metabolizers [UGT1A8 (*1/*1) or UGT1A8 (*1/*2)] vs fast metabolizers [UGT1A8 (*2/*2). These data suggest that raloxifene metabolism may be dependent on UGT1A8 genotype and that UGT1A8 genotype may play an important role in overall response to raloxifene. PMID:23682072

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

PubMed

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.

Relationship between plasma bilirubin level and oxidative stress markers in HIV-infected patients on atazanavir- vs. efavirenz-based antiretroviral therapy.

PubMed

Estrada, V; Monge, S; Gómez-Garre, M D; Sobrino, P; Masiá, M; Berenguer, J; Portilla, J; Viladés, C; Martínez, E; Blanco, J R

2016-10-01

Chronic oxidative stress (OS) may play a role in cardiovascular disease in HIV-infected patients, and increased bilirubin levels may have a beneficial role in counteracting OS. Atazanavir (ATV) inhibits UDP-glucuronosyl-transferase 1A1 (UGT1A1), thus increasing unconjugated bilirubin levels. We aimed to compare changes in OS markers in patients on ATV/ritonavir (ATV/r)- vs. efavirenz (EFV)-based first-line antiretroviral therapy (ART). A multicentre, prospective cohort study of HIV-infected patients who started first-line ART with either ATV/r or EFV was conducted. Lipoprotein-associated phospholipase A2 (Lp-PLA2), myeloperoxidase (MPO) and oxidized low-density lipoprotein (oxLDL) were measured for 145 patients in samples obtained at baseline and after at least 9 months of ART during which the initial regimen was maintained and the patient was virologically suppressed. The change in OS markers was modelled using multiple linear regressions adjusting for baseline values and confounders. After adjustment for baseline variables, patients on ATV/r had a significantly greater decrease in Lp-PLA2 [estimated difference -16.3; 95% confidence interval (CI) -31.4, -1.25; P = 0.03] and a significantly smaller increase in OxLDL (estimated difference -21.8; 95% CI -38.0, -5.6; P < 0.01) relative to those on EFV, whereas changes in MPO were not significantly different (estimated difference 1.2; 95% CI -14.3, 16.7; P = 0.88). Adjusted changes in bilirubin were significantly greater for the ATV/r group than for the EFV group (estimated difference 1.33 mg/dL; 95% CI 1.03, 1.52 mg/dL; P < 0.01). Changes in bilirubin and changes in OS markers were significantly correlated. When compared with EFV, ATV/r-based therapy was associated with lower levels of oxidative stress biomarkers, which was in part attributable to increased bilirubin levels. © 2016 British HIV Association.

Enhanced UGT1A1 Gene and Protein Expression in Endometriotic Lesions.

PubMed

Piccinato, Carla A; Neme, Rosa M; Torres, Natália; da Silva Victor, Elivane; Brudniewski, Heloísa F; Rosa E Silva, Júlio C; Ferriani, Rui A

2018-01-01

The cellular function in endometriosis lesions depends on a highly estrogenic milieu. Lately, it is becoming evident that, besides the circulating levels of estrogens, the balance of synthesis versus inactivation (metabolism) of estrogens by intralesion steroid-metabolizing enzymes also determines the local net estrogen availability. In order to extend the knowledge of the role of estrogen-metabolizing enzymes in endometriosis, we investigated the gene and protein expression of a key uridine diphospho-glucuronosyltransferase (UGT) for estrogen glucuronidation, UGT1A1, in eutopic endometrial samples obtained from nonaffected and endometriosis-affected women and also from endometriotic lesions. Although UGT1A1 messenger RNA (mRNA) expression was detected at similar frequencies in endometriotic lesions and in eutopic endometrial samples, the levels of mRNA expression were greater in deep-infiltrating endometriotic lesions and in non-deep-infiltrating lesions when compared with either control endometrium or eutopic endometrium from women with endometriosis. Overall, we observed that protein expression of UGT1A1 was significantly more frequent in samples from endometriotic lesions in comparison with endometria. In addition, expression of UGT1A1 protein was greater in deep-infiltrating than in non-deep-infiltrating endometriotic lesions. We suggest that the finding of increased expression of UGT1A1 in lesions versus endometria might be related to impairment of regulatory mechanisms, in response to a highly estrogenic milieu, and that this enzyme may be a new target for therapy.

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

PubMed

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

2010-01-08

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

Hepatic expression of transcription factors affecting developmental regulation of UGT1A1 in the Han Chinese population.

PubMed

Nie, Ya-Li; He, Hang; Li, Jiang-Feng; Meng, Xiang-Guang; Yan, Liang; Wang, Pei; Wang, Shu-Jie; Bi, Hong-Zheng; Zhang, Li-Rong; Kan, Quan-Cheng

2017-01-01

Complete or partial inactivity of UGT1A1, the unique enzyme responsible for bilirubin glucuronidation, is commonly associated with hyperbilirubinemia. We investigated the dynamic expression of UGT1A1, and that of the transcription factors (TFs) involved in its developmental regulation, during human hepatic growth in Han Chinese individuals. Eighty-eight prenatal, pediatric, and adult liver samples were obtained from Han Chinese individuals. Quantitative real-time polymerase chain reaction was used to evaluate mRNA expression of UGT1A1 and TFs including PXR, CAR, HNF1A, HNF4A, PPARA, etc. UGT1A1 protein levels and metabolic activity were determined by western blotting and high-performance liquid chromatography. Direct sequencing was employed to genotype UGT1A1*6 (211G˃A) and UGT1A1*28 (TA6˃TA7) polymorphisms. UGT1A1 expression was minimal in prenatal samples, but significantly elevated during pediatric and adult stages. mRNA and protein levels and metabolic activity were prominently increased (120-, 20-, and 10-fold, respectively) in pediatric and adult livers compared to prenatal samples. Furthermore, expression did not differ appreciably between pediatric and adult periods. Dynamic expression of TFs, including PXR, CAR, HNF1A, HNF4A, and PPARA, was consistent with UGT1A1 levels at each developmental stage. A pronounced correlation between expression of these TFs and that of UGT1A1 (P < 0.001) was observed. Moreover, UGT1A1*6 and UGT1A1*28 polymorphisms reduced levels of UGT1A1 by up to 40-60 %. Hepatic expression of transcription factors is associated with developmental regulation of UGT1A1 in the Han Chinese population. Moreover, UGT1A1 polymorphisms are associated with reduced expression of UGT1A1 mRNA and protein, as well as enzyme activity.

Characterization of UDP-glucuronosyltransferase genes and their possible roles in multi-insecticide resistance in Plutella xylostella (L.).

PubMed

Li, Xiuxia; Shi, Haiyan; Gao, Xiwu; Liang, Pei

2018-03-01

Uridine diphosphate-glucuronosyltransferases (UGTs), as multifunctional detoxification enzymes, play important roles in the biotransformation of various compounds. However, their roles in insecticide resistance are still unclear. This study presents a genome-wide identification of the UGTs in diamondback moth, Plutella xylostella (L.), a notorious insect pest of cruciferous crops worldwide. The possible roles of these UGTs in insecticide resistance were evaluated. A total of 21 putative UGTs in P. xylostella were identified. Quantitative real-time polymerase chain reaction (PCR)-based analyses showed that all the UGT genes were expressed in all tested developmental stages and tissues. Bioassay results indicated that a field-collected population (BL) was resistant to 9 of 10 commonly used insecticides, and 10 of 21 UGT mRNAs were upregulated in the BL population. Exposure to the LC 50 of each insecticide affected the expression of most UGT genes. Among these, the expression levels of UGT40V1, UGT45B1 and UGT33AA4 were induced by more than five insecticides, whereas indoxacarb and metaflumizone significantly repressed the expression of most UGT genes. UGTs may play important roles in the metabolism of commonly used insecticides in P. xylostella. These findings provide valuable information for further research on the physiological and toxicological functions of specific UGT genes in P. xylostella. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms.

PubMed

Brill, Shirley S; Furimsky, Anna M; Ho, Mark N; Furniss, Michael J; Li, Yi; Green, Adam G; Bradford, Wallace W; Green, Carol E; Kapetanovic, Izet M; Iyer, Lalitha V

2006-04-01

Resveratrol (trans-resveratrol, trans-3,5,4'-trihydroxystilbene) is a naturally occurring stilbene analogue found in high concentrations in red wine. There is considerable research interest to determine the therapeutic potential of resveratrol, as it has been shown to have tumour inhibitory and antioxidant properties. This study was performed to investigate the glucuronidation of resveratrol and possible drug interactions via glucuronidation. Two glucuronide conjugates, resveratrol 3-O-glucuronide and resveratrol 4'-O-glucuronide, were formed by human liver and intestinal microsomes. UGT1A1 and UGT1A9 were predominantly responsible for the formation of the 3-O-glucuronide (Km = 149 microM) and 4'-O-glucuronide (Km = 365 microM), respectively. The glucuronide conjugates were formed at higher levels (up to 10-fold) by intestinal rather than liver microsomes. Resveratrol was co-incubated with substrates of UGT1A1 (bilirubin and 7-ethyl-10-hydroxycamptothecin (SN-38)) and UGT1A9 (7-hydroxytrifluoromethyl coumarin (7-HFC)). No major changes were noted in bilirubin glucuronidation in the presence of resveratrol. Resveratrol significantly inhibited the glucuronidation of SN-38 (Ki = 6.2 +/- 2.1 microM) and 7-HFC (Ki = 0.6 +/- 0.2 microM). Hence, resveratrol has the potential to inhibit the glucuronidation of concomitantly administered therapeutic drugs or dietary components that are substrates of UGT1A1 and UGT1A9.

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

DOE PAGES

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

2017-04-03

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

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

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

Rautengarten, Carsten; Birdseye, Devon; Pattathil, Sivakumar

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

Tissue and species differences in the glucuronidation of glabridin with UDP-glucuronosyltransferases.

PubMed

Guo, Bin; Fang, Zhongze; Yang, Lu; Xiao, Ling; Xia, Yangliu; Gonzalez, Frank J; Zhu, Liangliang; Cao, Yunfeng; Ge, Guangbo; Yang, Ling; Sun, Hongzhi

2015-04-25

Glabridin (GA) has gained wide application in the cosmetics and food industry. This study was performed to investigate its metabolic inactivation and elimination by glucuronidation by use of liver and intestine microsomes from humans (HLM and HIM) and rats (RLM and RIM), and liver microsomes from cynomolgus monkeys and beagle dogs (CyLM and DLM). Both hydroxyl groups at the C2 and C4 positions of the B ring are conjugated to generate two mono-glucuronides (M1 and M2). HIM, RIM and RLM showed the most robust activity in catalyzing M2 formation with intrinsic clearance values (Clint) above 2000 μL/min/mg, with little measurable M1 formation activity. DLM displayed considerable activity both in M1 and M2 formation, with Clint values of 71 and 214 μL/min/mg, respectively, while HLM and CyLM exhibited low activities in catalyzing M1 and M2 formation, with Clint values all below 20 μL/min/mg. It is revealed that UGT1A1, 1A3, 1A9, 2B7, 2B15 and extrahepatic UGT1A8 and 1A10 are involved in GA glucuronidation. Nearly all UGTs preferred M2 formation except for UGT1A1. Notably, UGT1A8 displayed the highest activity with a Clint value more than 5-fold higher than the other isoforms. Chemical inhibition studies, using selective inhibitors of UGT1A1, 1A9, 2B7 and 1A8, further revealed that UGT1A8 contributed significantly to intestinal GA glucuronidation in humans. In summary, this in vitro study demonstrated large species differences in GA glucuronidation by liver and intestinal microsomes, and that intestinal UGTs are important for the pathway in humans. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

BDE-99, but not BDE-47, is a transient aryl hydrocarbon receptor agonist in zebrafish liver cells.

PubMed

Yang, Jie; Zhu, Jinyong; Chan, King Ming

2016-08-15

Polybrominated diphenyl ethers (PBDEs) are endocrine-disrupting chemicals that affect the environment and the health of humans and wildlife. In this study, the zebrafish liver (ZFL) cell line was used in vitro to investigate two major PBDE contaminants: 2, 2', 4, 4', 5-pentabromodiphenyl ether (BDE-99) and 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47). BDE-99 was found to significantly induce cytochrome P450 (CYP1A), uridine diphosphate glucuronosyl transferase 1 family a, b (ugt1ab), 7-ethoxyresorufin-O-deethylase activity and an aryl hydrocarbon receptor (Ahr) dependent xenobiotic response element luciferase reporter system, confirming the Ahr-mediated activation of CYP1A by BDE-99. The time-course effect indicated that the role of BDE-99 in Ahr-mediated signaling is likely to be transient and highly dependent on the ability of BDE-99 to induce CYP1A and ugt1ab, and presumably its metabolism. BDE-99 also exhibited a significant dose-response effect on a developed zebrafish pregnane X receptor luciferase reporter gene system. However, the other abundant contaminant under study, BDE-47, did not exhibit the above effects. Together, these results indicated that the molecular mechanism of PBDEs induced in ZFL cells is a chemically specific process that differs between members of the PBDE family. CYP1A induction derived by BDE-99 warrants further risk assessment as the humans, wildlife and environment are exposed to a complex mixture including dioxin-like compounds and carcinogenic compounds. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.

PubMed

Eixelsberger, Thomas; Sykora, Sabine; Egger, Sigrid; Brunsteiner, Michael; Kavanagh, Kathryn L; Oppermann, Udo; Brecker, Lothar; Nidetzky, Bernd

2012-09-07

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-D-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD(+) and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-D-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the D-glucuronyl ring accommodated by UXS features a marked (4)C(1) chair to B(O,3) boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C(4) (step 1) by aligning the enzymatic base Tyr(147) with the reactive substrate hydroxyl and it brings the carboxylate group at C(5) into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-D-glucuronic acid in the second chemical step. The protonated side chain of Tyr(147) stabilizes the enolate of decarboxylated C(4) keto species ((2)H(1) half-chair) that is then protonated from the Si face at C(5), involving water coordinated by Glu(120). Arg(277), which is positioned by a salt-link interaction with Glu(120), closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C(4) keto group by NADH, assisted by Tyr(147) as catalytic proton donor, yields UDP-xylose adopting the relaxed (4)C(1) chair conformation (step 3).

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG).

PubMed

Ghose, Kaushik; Selvaraj, Kumarakurubaran; McCallum, Jason; Kirby, Chris W; Sweeney-Nixon, Marva; Cloutier, Sylvie J; Deyholos, Michael; Datla, Raju; Fofana, Bourlaye

2014-03-28

Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax. Five UGT genes belonging to the glycosyltransferases' family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner. We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG)

PubMed Central

2014-01-01

Background Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax. Results Five UGT genes belonging to the glycosyltransferases’ family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner. Conclusion We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis. PMID:24678929

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

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

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

1990-03-25

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

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

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

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

1988-06-01

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

Characterization of three terpenoid glycosyltransferase genes in 'Valencia' sweet orange (Citrus sinensis L. Osbeck).

PubMed

Fan, Jing; Chen, Chunxian; Yu, Qibin; Li, Zheng-Guo; Gmitter, Frederick G

2010-10-01

Three putative terpenoid UDP-glycosyltransferase (UGT) genes, designated CsUGT1, CsUGT2, and CsUGT3, were isolated and characterized in 'Valencia' sweet orange (Citrus sinensis L. Osbeck). CsUGT1 consisted of 1493 nucleotides with an open reading frame encoding 492 amino acids, CsUGT2 consisted of 1727 nucleotides encoding 504 amino acids, and CsUGT3 consisted of 1705 nucleotides encoding 468 amino acids. CsUGT3 had a 145 bp intron at 730-874, whereas CsUGT1 and CsUGT2 had none. The three deduced glycosyltransferase proteins had a highly conserved plant secondary product glycosyltransferase motif in the C terminus. Phylogenetic analysis showed that CsUGT1 and CsUGT3 were classified into group L of glycosyltransferase family 1, and CsUGT2 was classified into group D. Through Southern blotting analysis, CsUGT1 was found to have two copies in the sweet orange genome, whereas CsUGT2 and CsUGT3 had at least seven and nine copies, respectively. CsUGT1, CsUGT2, and CsUGT3 were constitutively expressed in leaf, flower, and fruit tissues. The results facilitate further investigation of the function of terpenoid glycosyltransferases in citrus and the biosynthesis of terpenoid glycosides in vitro.

Characterization of ppGalNAc-T18, a member of the vertebrate-specific Y subfamily of UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases.

PubMed

Li, Xing; Wang, Jing; Li, Wei; Xu, Yingjiao; Shao, Dong; Xie, Yinyin; Xie, Wenxian; Kubota, Tomomi; Narimatsu, Hisashi; Zhang, Yan

2012-05-01

The first step of mucin-type O-glycosylation is catalyzed by members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T; EC 2.4.1.41) family. Each member of this family has unique substrate specificity and expression profiles. In this report, we describe a new subfamily of ppGalNAc-Ts, designated the Y subfamily. The Y subfamily consists of four members, ppGalNAc-T8, -T9, -T17 and -T18, in which the conserved YDX(5)WGGENXE sequence in the Gal/GalNAc-T motif of ppGalNAc-Ts is mutated to LDX(5)YGGENXE. Phylogenetic analysis revealed that the Y subfamily members only exist in vertebrates. All four Y subfamily members lack in vitro GalNAc-transferase activity toward classical substrates possibly because of the UDP-GalNAc-binding pocket mutants. However, ppGalNAc-T18, the newly identified defining member, was localized in the endoplasmic reticulum rather than the Golgi apparatus in lung carcinoma cells. The knockdown of ppGalNAc-T18 altered cell morphology, proliferation potential and changed cell O-glycosylation. ppGalNAc-T18 can also modulate the in vitro GalNAc-transferase activity of ppGalNAc-T2 and -T10, suggesting that it may be a chaperone-like protein. These findings suggest that the new Y subfamily of ppGalNAc-Ts plays an important role in protein glycosylation; characterizing their functions will provide new insight into the role of ppGalNAc-Ts.

Simultaneous determination of intracellular UDP-sugars in hyaluronic acid-producing Streptococcus zooepidemicus.

PubMed

Franke, Lukáš; Čožíková, Dagmar; Smirnou, Dzianis; Hermannová, Martina; Hanová, Tereza; Růžičková, Andrea; Velebný, Vladimír

2015-08-01

Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel associations of UDP-glucuronosyltransferase 2B gene variants with prostate cancer risk in a multiethnic study.

PubMed

Vidal, Adriana C; Tucker, Cocoa; Schildkraut, Joellen M; Richardson, Ricardo M; McPhail, Megan; Freedland, Stephen J; Hoyo, Cathrine; Grant, Delores J

2013-11-22

We have previously shown that a functional polymorphism of the UGT2B15 gene (rs1902023) was associated with increased risk of prostate cancer (PC). Novel functional polymorphisms of the UGT2B17 and UGT2B15 genes have been recently characterized by in vitro assays but have not been evaluated in epidemiologic studies. Fifteen functional SNPs of the UGT2B17 and UGT2B15 genes, including cis-acting UGT2B gene SNPs, were genotyped in African American and Caucasian men (233 PC cases and 342 controls). Regression models were used to analyze the association between SNPs and PC risk. After adjusting for race, age and BMI, we found that six UGT2B15 SNPs (rs4148269, rs3100, rs9994887, rs13112099, rs7686914 and rs7696472) were associated with an increased risk of PC in log-additive models (p < 0.05). A SNP cis-acting on UGT2B17 and UGT2B15 expression (rs17147338) was also associated with increased risk of prostate cancer (OR = 1.65, 95% CI = 1.00-2.70); while a stronger association among men with high Gleason sum was observed for SNPs rs4148269 and rs3100. Although small sample size limits inference, we report novel associations between UGT2B15 and UGT2B17 variants and PC risk. These associations with PC risk in men with high Gleason sum, more frequently found in African American men, support the relevance of genetic differences in the androgen metabolism pathway, which could explain, in part, the high incidence of PC among African American men. Larger studies are required.

[Hepatotoxicity of emodin based on UGT1A1 enzyme-mediated bilirubin in liver microsomes].

PubMed

Wang, Qi; Dai, Zhong; Zhang, Yu-Jie; Ma, Shuang-Cheng

2016-12-01

To study the hepatotoxicity of emodin based on bilirubin metabolism mediated by glucuronidation of UGT1A1 enzyme. In this study, three different incubation systems were established by using RLM, HLM, and rUGT1A1, with bilirubin as the substrate. Different concentrations of bilirubin and emodin were added in the incubation systems. The double reciprocal Michaelis equation was drawn based on the total amount of bilirubin glucuronidation. The apparent inhibition constant Ki was then calculated with the slope curve to predict the hepatotoxicity. The results indicated that emodin had a significant inhibition to the UGT1A1 enzyme in all of the three systems, with Ki=5.400±0.956(P<0.05) in HLM system, Ki =10.020±0.611(P<0.05) in RLM system, Ki=4.850±0.528(P<0.05) in rUGT1A1 system. Meanwhile, emodin had no significant difference between rat and human in terms of inhibition of UGT1A1 enzyme. Emodin had a potential risk of the hepatotoxicity by inhibiting the UGT1A1 enzyme activity. And the method established in this study provides a new thought and new method to evaluate hepatotoxicity and safety of traditional Chinese medicines. Copyright© by the Chinese Pharmaceutical Association.

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

Treesearch

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

1999-01-01

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

THE URBAN DISPERSION PROGRAM ( UDP ) NYC MSG05 EXPERIMENT

EPA Science Inventory

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

Novel associations of UDP-glucuronosyltransferase 2B gene variants with prostate cancer risk in a multiethnic study

PubMed Central

2013-01-01

Background We have previously shown that a functional polymorphism of the UGT2B15 gene (rs1902023) was associated with increased risk of prostate cancer (PC). Novel functional polymorphisms of the UGT2B17 and UGT2B15 genes have been recently characterized by in vitro assays but have not been evaluated in epidemiologic studies. Methods Fifteen functional SNPs of the UGT2B17 and UGT2B15 genes, including cis-acting UGT2B gene SNPs, were genotyped in African American and Caucasian men (233 PC cases and 342 controls). Regression models were used to analyze the association between SNPs and PC risk. Results After adjusting for race, age and BMI, we found that six UGT2B15 SNPs (rs4148269, rs3100, rs9994887, rs13112099, rs7686914 and rs7696472) were associated with an increased risk of PC in log-additive models (p < 0.05). A SNP cis-acting on UGT2B17 and UGT2B15 expression (rs17147338) was also associated with increased risk of prostate cancer (OR = 1.65, 95% CI = 1.00-2.70); while a stronger association among men with high Gleason sum was observed for SNPs rs4148269 and rs3100. Conclusions Although small sample size limits inference, we report novel associations between UGT2B15 and UGT2B17 variants and PC risk. These associations with PC risk in men with high Gleason sum, more frequently found in African American men, support the relevance of genetic differences in the androgen metabolism pathway, which could explain, in part, the high incidence of PC among African American men. Larger studies are required. PMID:24267955

IN VITRO METABOLISM OF THYROID HORMONES BY RECOMBINANT HUMAN UDP-GLUCORONOSYLTRANSFERASES AND SULFOTRANSFERASES

EPA Science Inventory

Endocrine disruptors can decrease thyroid hormone levels via the induction of hepatic uridinediphosphate-glucoronosyltransferases (UGTs) and sulfotransferases (SULTs). Due to their ability to catalyze glucuronidation and sulfation of hormones and xenobiotics, UGTs and SULTs play ...

Molecular mechanisms and cell signaling of 20-hydroxyeicosatetraenoic acid in vascular pathophysiology

PubMed Central

Fan, Fan; Ge, Ying; Lv, Wenshan; Elliott, Matthew R.; Muroya, Yoshikazu; Hirata, Takashi; Booz, George W.; Roman, Richard J.

2016-01-01

Cytochrome P450s enzymes catalyze the metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatetraenoic acid and hydroxyeicosatetraeonic acid (HETEs). 20-HETE is a vasoconstrictor that depolarizes vascular smooth muscle cells by blocking K+ channels. EETs serve as endothelial derived hyperpolarizing factors. Inhibition of the formation of 20-HETE impairs the myogenic response and autoregulation of renal and cerebral blood flow. Changes in the formation of EETs and 20-HETE have been reported in hypertension and drugs that target these pathways alter blood pressure in animal models. Sequence variants in CYP4A11 and CYP4F2 that produce 20-HETE, UDP-glucuronosyl transferase involved in the biotransformation of 20-HETE and soluble epoxide hydrolase that inactivates EETs are associated with hypertension in human studies. 20-HETE contributes to the regulation of vascular hypertrophy, restenosis, angiogenesis and inflammation. It also promotes endothelial dysfunction and contributes to cerebral vasospasm and ischemia-reperfusion injury in the brain, kidney and heart. This review will focus on the role of 20-HETE in vascular dysfunction, inflammation, ischemic and hemorrhagic stroke and cardiac and renal ischemia reperfusion injury. PMID:27100515

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

PubMed

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

1988-06-01

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

Lightweight UDP Pervasive Protocol in Smart Home Environment Based on Labview

NASA Astrophysics Data System (ADS)

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

2017-04-01

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

Clofibrate prevents and reverses the hemodynamic manifestations of hyperthyroidism in rats.

PubMed

Rodríguez-Gómez, Isabel; Cruz, Antonio; Moreno, Juan Manuel; Soler, Agatángelo; Osuna, Antonio; Vargas, Félix

2008-03-01

This study analyzed the effects of the chronic administration of clofibrate, a peroxisome proliferator-activated receptor-alpha (PPARalpha) agonist, on the development and established hemodynamic, morphologic, metabolic, and renal manifestations of hyperthyroidism in rats. The prevention study used four groups of male Wistar rats: control, clofibrate (240 mg/kg/day by gavage), T(4)(75 microg thyroxine/rat/day s.c.), and T(4)+clofibrate. All treatments were maintained for 3 weeks. Body weight (BW), tail systolic blood pressure (SBP), and heart rate (HR) were recorded weekly. Finally, temperature, SBP, pulse pressure (PP) and HR were recorded in conscious rats, and morphologic, metabolic, plasma, and renal variables were measured. The reversion study used two groups of rats, T(4)(treated for 6 weeks) and T(4)+clofibrate, measuring their hemodynamic variables and temperature for 3 weeks. T(4) increased BP, HR, PP, and temperature when compared with control rats. Clofibrate prevented and reversed the increase in SBP, HR, PP, and temperature produced by T(4) administration, reduced plasma thyroid hormone levels, and increased plasma thyroid-stimulating hormone values and phenol-uridine diphosphate-glucuronosyl-transferase (UGT) activity. However, clofibrate did not modify the cardiac or renal hypertrophy, polyphagia, polydipsia, or proteinuria of hyperthyroid rats. In normal rats, clofibrate treatment did not significantly change thyroid hormone levels, phenol-UGT activity, or any hemodynamic, morphologic, or renal variables. Chronic clofibrate treatment suppressed the hemodynamic manifestations and increased temperature of hyperthyroidism, an effect that can be produced by direct antithyroid effects. However, clofibrate administration did not modify the morphologic, metabolic, or renal alterations of hyperthyroid rats, indicating specificity in the antithyroid actions of clofibrate.

Red wine and component flavonoids inhibit UGT2B17 in vitro

PubMed Central

2012-01-01

Background The metabolism and excretion of the anabolic steroid testosterone occurs by glucuronidation to the conjugate testosterone glucuronide which is then excreted in urine. Alterations in UGT glucuronidation enzyme activity could alter the rate of testosterone excretion and thus its bioavailability. The aim of this study is to investigate if red wine, a common dietary substance, has an inhibitory effect on UGT2B17. Methods Testosterone glucuronidation was assayed using human UGT2B17 supersomes with quantification of unglucuronidated testosterone over time using HPLC with DAD detection. The selected red wine was analyzed using HPLC; and the inhibitory effects of the wine and phenolic components were tested independently in a screening assay. Further analyses were conducted for the strongest inhibitors at physiologically relevant concentrations. Control experiments were conducted to determine the effects of the ethanol on UGT2B17. Results Over the concentration range of 2 to 8%, the red wine sample inhibited the glucuronidation of testosterone by up to 70% over 2 hours. The ethanol content had no significant effect. Three red wine phenolics, identified by HPLC analyses, also inhibited the enzyme by varying amounts in the order of quercetin (72%), caffeic acid (22%) and gallic acid (9%); using a ratio of phenolic:testosterone of 1:2.5. In contrast p-coumaric acid and chlorogenic acid had no effect on the UGT2B17. The most active phenolic was selected for a detailed study at physiologically relevant concentrations, and quercetin maintained inhibitory activity of 20% at 2 μM despite a ten-fold excess of testosterone. Conclusion This study reports that in an in vitro supersome-based assay, the key steroid-metabolizing enzyme UGT2B17 is inhibited by a number of phenolic dietary substances and therefore may reduce the rate of testosterone glucuronidation in vivo. These results highlight the potential interactions of a number of common dietary compounds on

Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.

PubMed

Cao, Benjamin; Chen, Xingqiang; Yamaryo-Botte, Yoshiki; Richardson, Mark B; Martin, Kirstee L; Khairallah, George N; Rupasinghe, Thusita W T; O'Flaherty, Roisin M; O'Hair, Richard A J; Ralton, Julie E; Crellin, Paul K; Coppel, Ross L; McConville, Malcolm J; Williams, Spencer J

2013-03-15

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

UGT2B17 minor histocompatibility mismatch and clinical outcome after HLA-identical sibling donor stem cell transplantation.

PubMed

Santos, N; Rodríguez-Romanos, R; Nieto, J B; Buño, I; Vallejo, C; Jiménez-Velasco, A; Brunet, S; Buces, E; López-Jiménez, J; González, M; Ferrá, C; Sampol, A; de la Cámara, R; Martínez, C; Gallardo, D

2016-01-01

Minor histocompatibility Ags (mHags) have been implicated in the pathogenesis of GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Uridine diphospho-glucuronosyltransferase 2B17 (UGT2B17) gene deletion may act as a mHag and its association with acute GVHD (aGVHD) has been described. We retrospectively studied the clinical impact of a UGT2B17 mismatch in a cohort of 1127 patients receiving a HSCT from an HLA-identical sibling donor. UGT2B17 mismatch was present in 69 cases (6.1%). Incidence of severe aGVHD was higher in the UGT2B17 mismatched pairs (22.7% vs 14.6%), but this difference was not statistically significant (P: 0.098). We did not detect differences in chronic GVHD, overall survival, relapse-free survival, transplant-related mortality or relapse. Nevertheless, when we analyzed only those patients receiving grafts from a male donor (616 cases), aGVHD was significantly higher in the UGT2B17 mismatched group (25.1% vs 12.8%; P: 0.005) and this association was confirmed by the multivariate analysis (P: 0.043; hazard ratio: 2.16, 95% confidence interval: 1.03-4.57). Overall survival was worse for patients mismatched for UGT2B17 (P: 0.005). We conclude that UGT2B17 mismatch has a negative clinical impact in allogeneic HSCT from HLA-identical sibling donors only when a male donor is used. These results should be confirmed by other studies.

Profiling serum bile acid glucuronides in humans: gender divergences, genetic determinants and response to fenofibrate

PubMed Central

Trottier, Jocelyn; Perreault, Martin; Rudkowska, Iwona; Levy, Cynthia; Dallaire-Theroux, Amélie; Verreault, Mélanie; Caron, Patrick; Staels, Bart; Vohl, Marie-Claude; Straka, Robert J.; Barbier, Olivier

2014-01-01

Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes detoxifies cholestatic bile acids (BAs). We aimed at i) characterizing the circulating BA-glucuronide (-G) pool composition in humans, ii) evaluating how sex and UGT polymorphisms influence this composition, and iii) analyzing the effects of lipid-lowering drug fenofibrate on the circulating BA-G profile in 300 volunteers and 5 cholestatic patients. Eleven BA-Gs were determined in pre- and post-fenofibrate samples. Men exhibited higher BA-G concentrations, and various genotype/BA-G associations were discovered in relevant UGT genes. The chenodeoxycholic acid-3G concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA-3G formation. Fenofibrate exposure increased the serum levels of 5 BA-G species, including CDCA-3G, and up-regulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrates that fenofibrate stimulates BA glucuronidation in humans, and thus reduces bile acid toxicity in the liver. PMID:23756370

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

PubMed

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

2017-11-04

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

Cloning and expression studies of the Dunaliella salina UDP-glucose dehydrogenase cDNA.

PubMed

Qinghua, He; Dairong, Qiao; Qinglian, Zhang; Shunji, He; Yin, Li; Linhan, Bai; Zhirong, Yang; Yi, Cao

2005-06-01

The enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate. Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of primary cell walls. A cDNA, named DsUGDH, (GeneBank accession number: AY795899) corresponding to UGDH was cloned by RT-PCR approach from Dunaliella salina. The cDNA is 1941-bp long and has an open reading frame encoded a protein of 483 amino acids with a calculated molecular weight of 53 kDa. The derived amino acids sequence shows high homology with reported plants UGDHs, and has highly conserved amino acids motifs believed to be NAD binding site and catalytic site. Although UDP-glucose dehydrogenase is a comparatively well characterized enzyme, the cloning and characterization of the green alga Dunaliella salina UDP-glucose dehydrogenase gene is very important to understand the salt tolerance mechanism of Dunaliella salina. Northern analyses indicate that NaCl can induce the expression the DsUGDH.

Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress

PubMed Central

Mishra, Manoj K; Singh, Gaurav; Tiwari, Shalini; Singh, Ruchi; Kumari, Nishi; Misra, Pratibha

2015-01-01

Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress. PMID:26382564

Improving UDP/IP Transmission Without Increasing Congestion

NASA Technical Reports Server (NTRS)

Burleigh, Scott

2006-01-01

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

Association between liver failure and hepatic UDP-glucuronosyltransferase activity in dairy cows with follicular cysts.

PubMed

Tanemura, Kouichi; Ohtaki, Tadatoshi; Kuwahara, Yasushi; Tsumagari, Shigehisa

2017-01-20

Uridine 5'-diphospho-glucuronosyltransferase (UGT) liver activity was measured using estradiol-17β as a substrate in dairy cows with follicular cysts. The activity was significantly lower than that in dairy cows with normal estrous cycles (P<0.01). Liver disorders, such as fatty liver and hepatitis, were observed in half cows with follicular cysts, and liver UGT activity was lower than that in cows with normal estrus cycles. In addition, the liver UGT activity was significantly lower in dairy cows with follicular cysts without liver disorders than in dairy cows with normal estrous cycles. Therefore, the cows were divided into those with low, middle and high liver UGT activities, and liver disorder complication rates were investigated. The complication rate was significantly higher in the low- (78.1%) than in the middle- (22.2%) and high-level (8.3%) groups, suggesting that liver disorders are closely associated with the development of follicular cysts in dairy cows and that steroid hormone metabolism is delayed because of reduced liver UGT activity, resulting in follicular cyst formation. We conclude that reduced estradiol-17β glucuronidation in the liver and liver disorders are associated with follicular cyst occurrence in dairy cows.

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

PubMed

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

2018-03-01

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

Selective Detoxification of Phenols by Pichia pastoris and Arabidopsis thaliana Heterologously Expressing the PtUGT72B1 from Populus trichocarpa

PubMed Central

Xu, Zhi-Sheng; Lin, Ya-Qiu; Xu, Jing; Zhu, Bo; Zhao, Wei; Peng, Ri-He; Yao, Quan-Hong

2013-01-01

Phenols are present in the environment and commonly in contact with humans and animals because of their wide applications in many industries. In a previous study, we reported that uridine diphosphate-glucose-dependent glucosyltransferase PtUGT72B1 from Populus trichocarpa has high activity in detoxifying trichlorophenol by conjugating glucose. In this study, more experiments were performed to determine the substrate specificity of PtUGT72B1 towards phenolic compounds. Among seven phenols tested, three were glucosylated by PtUGT72B1 including phenol, hydroquinone, and catechol. Transgenic Arabidopsis plants expressing the enzyme PtUGT72B1 showed higher resistance to hydroquinone and catechol but more sensitivity to phenol than wild type plants. Transgenic Pichia pastoris expressing PtUGT72B1 showed enhanced resistance to all three phenols. Compared with wild type Arabidopsis plants, transgenic Arabidopsis plants showed higher removal efficiencies and exported more glucosides of phenol, phenyl β-D-glucopyranoside, to the medium after cultured with the three phenols. Protein extracts from transgenic Arabidopsis plants showed enhanced conjugating activity towards phenol, hydroquinone and catechol. PtUGT72B1 showed much higher expression level in Pichia pastoris than in Arabidopsis plants. Kinetic analysis of the PtUGT72B1 was also performed. PMID:23840543

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

PubMed

Decker, Daniel; Kleczkowski, Leszek A

2017-01-01

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

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

PubMed Central

Decker, Daniel; Kleczkowski, Leszek A.

2017-01-01

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

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

PubMed Central

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

2012-01-01

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

Association between liver failure and hepatic UDP-glucuronosyltransferase activity in dairy cows with follicular cysts

PubMed Central

TANEMURA, Kouichi; OHTAKI, Tadatoshi; KUWAHARA, Yasushi; TSUMAGARI, Shigehisa

2016-01-01

Uridine 5’-diphospho-glucuronosyltransferase (UGT) liver activity was measured using estradiol-17β as a substrate in dairy cows with follicular cysts. The activity was significantly lower than that in dairy cows with normal estrous cycles (P<0.01). Liver disorders, such as fatty liver and hepatitis, were observed in half cows with follicular cysts, and liver UGT activity was lower than that in cows with normal estrus cycles. In addition, the liver UGT activity was significantly lower in dairy cows with follicular cysts without liver disorders than in dairy cows with normal estrous cycles. Therefore, the cows were divided into those with low, middle and high liver UGT activities, and liver disorder complication rates were investigated. The complication rate was significantly higher in the low- (78.1%) than in the middle- (22.2%) and high-level (8.3%) groups, suggesting that liver disorders are closely associated with the development of follicular cysts in dairy cows and that steroid hormone metabolism is delayed because of reduced liver UGT activity, resulting in follicular cyst formation. We conclude that reduced estradiol-17β glucuronidation in the liver and liver disorders are associated with follicular cyst occurrence in dairy cows. PMID:27666462

Association between UGT2B7 gene polymorphisms and fentanyl sensitivity in patients undergoing painful orthognathic surgery

PubMed Central

Muraoka, Wataru; Nishizawa, Daisuke; Fukuda, Kenichi; Kasai, Shinya; Hasegawa, Junko; Wajima, Koichi; Nakagawa, Taneaki

2016-01-01

Background Fentanyl is often used instead of morphine for the treatment of pain because it has fewer side effects. The metabolism of morphine by glucuronidation is known to be influenced by polymorphisms of the UGT2B7 gene. Some metabolic products of fentanyl are reportedly metabolized by glucuronate conjugation. The genes that are involved in the metabolic pathway of fentanyl may also influence fentanyl sensitivity. We analyzed associations between fentanyl sensitivity and polymorphisms of the UGT2B7 gene to clarify the hereditary determinants of individual differences in fentanyl sensitivity. Results This study examined whether single-nucleotide polymorphisms (SNPs) of the UGT2B7 gene affect cold pain sensitivity and the analgesic effects of fentanyl, evaluated by a standardized pain test and fentanyl requirements in healthy Japanese subjects who underwent uniform surgical procedures. The rs7439366 SNP of UGT2B7 is reportedly associated with the metabolism and analgesic effects of morphine. We found that this SNP is also associated with the analgesic effects of fentanyl in the cold pressor-induced pain test. It suggested that the C allele of the rs7439366 SNP may enhance analgesic efficacy. Two SNPs of UGT2B7, rs4587017 and rs1002849, were also found to be novel SNPs that may influence the analgesic effects of fentanyl in the cold pressor-induced pain test. Conclusions Fentanyl sensitivity for cold pressor-induced pain was associated with the rs7439366, rs4587017, and rs1002849 SNPs of the UGT2B7 gene. Our findings may provide valuable information for achieving satisfactory pain control and open to new avenues for personalized pain treatment. PMID:28256933

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

Soybean greatly reduces valproic acid plasma concentrations: A food–drug interaction study

PubMed Central

Marahatta, Anu; Bhandary, Bidur; Jeong, Seul-Ki; Kim, Hyung-Ryong; Chae, Han-Jung

2014-01-01

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents. PMID:24618639

Soybean greatly reduces valproic acid plasma concentrations: a food-drug interaction study.

PubMed

Marahatta, Anu; Bhandary, Bidur; Jeong, Seul-Ki; Kim, Hyung-Ryong; Chae, Han-Jung

2014-03-12

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.

[Examination of UGT1A1 polymorphisms and irinotecan-induced neutropenia in patients with Colorectal cancer].

PubMed

Teruya, Tsuyoshi; Nakachi, Atsushi; Shimabukuro, Nobuhiro; Toritsuka, Daisuke; Azuma, Yasuharu; Hanashiro, Kiyotoshi; Nishiki, Takehiro; Ota, Morihito; Shimabuku, Masamori; Shiroma, Hiroshi

2015-05-01

Irinotecan is an effective drug in the treatment of colorectal cancer. However, there are reports of an association between certain UGT1A1 genetic polymorphisms and the development of adverse reactions(such as neutropenia)related to irinotecan metabolism. We retrospectively investigated UGT1A1 genetic polymorphisms and the occurrences of irinotecan-induced neutropenia in 25 patients of colorectal cancer at our hospital. Analysis of UGT1A1 genetic polymorphisms in these patients yielded the following classifications: a wild-type group( *1/*1)comprising 13 patients(52%), a heterozygous group(*1/ *28, *1/*6)of 10 patients(40%), and a homozygous group(*28/*28, *6/*6)of 2 patients(8%). The frequency of neutropenia was 15.4%(2/13)in the wild-type group, 30%(3/10)in the heterozygous group, and 100%(2/2)in the homozygous group. Grade 4 neutropenia only occurred in the homozygous group. These results suggest that a dose reduction of irinotecan should be considered for patients who fall into the homozygous group upon analysis of their UGT1A1 genetic polymorphisms, as such patients might be susceptible to grade 4 neutropenia.

Identification of the S-transferase like superfamily bacillithiol transferases encoded by Bacillus subtilis

PubMed Central

Perera, Varahenage R.; Lapek, John D.; Newton, Gerald L.; Gonzalez, David J.; Pogliano, Kit

2018-01-01

Bacillithiol is a low molecular weight thiol found in Firmicutes that is analogous to glutathione, which is absent in these bacteria. Bacillithiol transferases catalyze the transfer of bacillithiol to various substrates. The S-transferase-like (STL) superfamily contains over 30,000 putative members, including bacillithiol transferases. Proteins in this family are extremely divergent and are related by structural rather than sequence similarity, leaving it unclear if all share the same biochemical activity. Bacillus subtilis encodes eight predicted STL superfamily members, only one of which has been shown to be a bacillithiol transferase. Here we find that the seven remaining proteins show varying levels of metal dependent bacillithiol transferase activity. We have renamed the eight enzymes BstA-H. Mass spectrometry and gene expression studies revealed that all of the enzymes are produced to varying levels during growth and sporulation, with BstB and BstE being the most abundant and BstF and BstH being the least abundant. Interestingly, several bacillithiol transferases are induced in the mother cell during sporulation. A strain lacking all eight bacillithiol transferases showed normal growth in the presence of stressors that adversely affect growth of bacillithiol-deficient strains, such as paraquat and CdCl2. Thus, the STL bacillithiol transferases represent a new group of proteins that play currently unknown, but potentially significant roles in bacillithiol-dependent reactions. We conclude that these enzymes are highly divergent, perhaps to cope with an equally diverse array of endogenous or exogenous toxic metabolites and oxidants. PMID:29451913

Body Fat Percentage Is a Major Determinant of Total Bilirubin Independently of UGT1A1*28 Polymorphism in Young Obese

PubMed Central

Kohlova, Michaela; Bronze-da-Rocha, Elsa; Fernandes, João; Costa, Elísio; Catarino, Cristina; Aires, Luísa; Mansilha, Helena Ferreira; Rocha-Pereira, Petronila; Quintanilha, Alexandre; Rêgo, Carla; Santos-Silva, Alice

2014-01-01

Objectives Bilirubin has potential antioxidant and anti-inflammatory properties. The UGT1A1*28 polymorphism (TA repeats in the promoter region) is a major determinant of bilirubin levels and recent evidence suggests that raised adiposity may also be a contributing factor. We aimed to study the interaction between UGT1A1 polymorphism, hematological and anthropometric variables with total bilirubin levels in young individuals. Methods 350 obese (mean age of 11.6 years; 52% females) and 79 controls (mean age of 10.5 years; 59% females) were included. Total bilirubin and C-reactive protein (CRP) plasma levels, hemogram, anthropometric data and UGT1A1 polymorphism were determined. In a subgroup of 74 obese and 40 controls body composition was analyzed by dual-energy X-ray absorptiometry. Results The UGT1A1 genotype frequencies were 49.9%, 42.7% and 7.5% for 6/6, 6/7 and 7/7 genotypes, respectively. Patients with 7/7 genotype presented the highest total bilirubin levels, followed by 6/7 and 6/6 genotypes. Compared to controls, obese patients presented higher erythrocyte count, hematocrit, hemoglobin and CRP levels, but no differences in bilirubin or in UGT1A1 genotype distribution. Body fat percentage was inversely correlated with bilirubin in obese patients but not in controls. This inverse association was observed either in 6/7 or 6/6 genotype obese patients. UGT1A1 polymorphism and body fat percentage were the main factors affecting bilirubin levels within obese patients (linear regression analysis). Conclusion In obese children and adolescents, body fat composition and UGT1A1 polymorphism are independent determinants of total bilirubin levels. Obese individuals with 6/6 UGT1A1 genotype and higher body fat mass may benefit from a closer clinical follow-up. PMID:24901842

Epidemiological investigation of the UGT2B17 polymorphism in doping control urine samples and its correlation to T/E ratios.

PubMed

Anielski, Patricia; Simmchen, Juliane; Wassill, Lars; Ganghofner, Dirk; Thieme, Detlef

2011-10-01

The deletion polymorphism of the enzyme UGT2B17 is known to correlate with the level of the testosterone to epitestosterone (T/E) ratio in urine specimen. Due to the importance of the T/E ratio to detect testosterone abuse in doping analysis, a PCR-ELISA system (Genotype® UGT test, AmplexDiagnostics) was established to identify the UGT2B17 phenotype in urine samples. Epidemiological investigations in a set of 674 routine doping controls (in- and out-of-competition) resulted in 22.8% homozygote gene-deleted and 74.5% UGT2B17-positive athletes. The validated test system has shown to be robust and sensitive: in only 18 cases (2.7%) isolation of cell material from urine failed. Following hydrolysis of glucuronidated conjugates, steroids were analyzed as bis-TMS derivatives by gas chromatography-mass spectrometry (GC-MS), for example, testosterone (T) and epitestosterone (E). Additionally, isotope ration mass spectrometry (IRMS) analysis and luteinizing hormone (LH) measurement were applied. Mean T/E ratios significantly correlated with the UGT2B17 phenotype (del: T/E 0.9; pos: 1.7), however the values did not differ as distinctive as reported in previous studies. Additionally, the T/E ratios in the gene-deleted group did not show a normal curve of distribution (median of T/E 0.5). Obviously, beside the UGT2B17 deletion further influences have to be taken into account, for example, polymorphisms or induction of other metabolizing enzymes. Our results indicate that the UGT2B17 polymorphism might be insufficient when utilized solely as a crucial parameter for individual interpretation of T/E in urine. Nevertheless, the detection of the UGT2B17-gene deletion in urine samples would provide additional information important for gathering evidence in analysis of steroids in doping control. Copyright © 2011 John Wiley & Sons, Ltd.

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

PubMed

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

2013-11-04

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

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

PubMed

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

2016-01-01

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

UGT2B17 gene deletion associated with an increase in bone mineral density similar to the effect of hormone replacement in postmenopausal women.

PubMed

Giroux, S; Bussières, J; Bureau, A; Rousseau, F

2012-03-01

UGT2B17 is one of the most important enzymes for androgen metabolism. In addition, the UGT2B17 gene is one of the most commonly deleted regions of the human genome. The deletion was previously found associated with higher femoral bone density in men and women, and we replicated this association in a sample of postmenopausal who never used hormone therapy. Deletion of the UGT2B17 gene was previously shown to be associated with a higher hip bone mineral density (BMD). Using a PCR assay, we tried to replicate the association among a large group of 2,379 women. We examined the effect of the deletion on femoral neck BMD and lumbar spine BMD according to the menopausal status and hormone replacement therapy (HRT). We used a high-throughput PCR assay to identify the gene and the deletion in a population of well-characterized women. Two additional polymorphisms, UGT2B28 deletion and UGT2B15 rs1902023 G > T were also investigated. Only UGT2B17 deletion was associated with LS and FN BMD. Furthermore, the association was seen only among postmenopausal women who had never used hormone replacement as in the first reported association. We confirmed the association between UGT2B17 deletion and a higher LS and FN BMD. In addition, we show that the association is observed among postmenopausal women who never used HRT consistent with the enzymatic function of UGT2B17. The analysis shows that those having one or two UGT2B17 alleles benefit from HRT, which is not the case for null carriers.

Bilirubin glucuronidation revisited: proper assay conditions to estimate enzyme kinetics with recombinant UGT1A1.

PubMed

Zhou, Jin; Tracy, Timothy S; Remmel, Rory P

2010-11-01

Bilirubin, an end product of heme catabolism, is primarily eliminated via glucuronic acid conjugation by UGT1A1. Impaired bilirubin conjugation, caused by inhibition of UGT1A1, can result in clinical consequences, including jaundice and kernicterus. Thus, evaluation of the ability of new drug candidates to inhibit UGT1A1-catalyzed bilirubin glucuronidation in vitro has become common practice. However, the instability of bilirubin and its glucuronides presents substantial technical challenges to conduct in vitro bilirubin glucuronidation assays. Furthermore, because bilirubin can be diglucuronidated through a sequential reaction, establishment of initial rate conditions can be problematic. To address these issues, a robust high-performance liquid chromatography assay to measure both bilirubin mono- and diglucuronide conjugates was developed, and the incubation conditions for bilirubin glucuronidation by human embryonic kidney 293-expressed UGT1A1 were carefully characterized. Our results indicated that bilirubin glucuronidation should be assessed at very low protein concentrations (0.05 mg/ml protein) and over a short incubation time (5 min) to assure initial rate conditions. Under these conditions, bilirubin total glucuronide formation exhibited a hyperbolic (Michaelis-Menten) kinetic profile with a K(m) of ∼0.2 μM. In addition, under these initial rate conditions, the relative proportions between the total monoglucuronide and the diglucuronide product were constant across the range of bilirubin concentration evaluated (0.05-2 μM), with the monoglucuronide being the predominant species (∼70%). In conclusion, establishment of appropriate incubation conditions (i.e., very low protein concentrations and short incubation times) is necessary to properly characterize the kinetics of bilirubin glucuronidation in a recombinant UGT1A1 system.

Genome-wide identification of glycosyltransferases converting phloretin to phloridzin in Malus species.

PubMed

Zhou, Kun; Hu, Lingyu; Li, Pengmin; Gong, Xiaoqing; Ma, Fengwang

2017-12-01

Phloridzin (phloretin 2'-O-glucoside) is the most abundant phenolic compound in Malus species, accounting for up to 18% of the dry weight in leaves. Glycosylation of phloretin at the 2' position is the last and key step in phloridzin biosynthesis. It is catalyzed by a uridine diphosphate (UDP)-glucose:phloretin 2'-O-glucosyltransferase (P2'GT), which directly determines the concentration of phloridzin. However, this process is poorly understood. We conducted a large-scale investigation of phloridzin accumulations in leaves from 64 Malus species and cultivars. To identify the responsible P2'GT, we performed a genome-wide analysis of the expression patterns of UDP-dependent glycosyltransferase genes (UGTs). Two candidates were screened preliminarily in Malus spp. cv. Adams (North American Begonia). Results from further qRT-PCR analyses of the genotypes showed a divergence in phloridzin production. Our assays of enzyme activity also suggested that MdUGT88F4 and MdUGT88F1 regulate the conversion of phloretin to phloridzin in Malus plants. Finally, when they were silenced in 'GL-3' ('Royal Gala'), the concentrations of phloridzin and phloretin (and trilobatin) were significantly reduced and increased, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

The EGF Repeat-Specific O-GlcNAc-Transferase Eogt Interacts with Notch Signaling and Pyrimidine Metabolism Pathways in Drosophila

PubMed Central

Müller, Reto; Jenny, Andreas; Stanley, Pamela

2013-01-01

The O-GlcNAc transferase Eogt modifies EGF repeats in proteins that transit the secretory pathway, including Dumpy and Notch. In this paper, we show that the Notch ligands Delta and Serrate are also substrates of Eogt, that mutation of a putative UDP-GlcNAc binding DXD motif greatly reduces enzyme activity, and that Eogt and the cytoplasmic O-GlcNAc transferase Ogt have distinct substrates in Drosophila larvae. Loss of Eogt is larval lethal and disrupts Dumpy functions, but does not obviously perturb Notch signaling. To identify novel genetic interactions with eogt, we investigated dominant modification of wing blister formation caused by knock-down of eogt. Unexpectedly, heterozygosity for several members of the canonical Notch signaling pathway suppressed wing blister formation. And importantly, extensive genetic interactions with mutants in pyrimidine metabolism were identified. Removal of pyrimidine synthesis alleles suppressed wing blister formation, while removal of uracil catabolism alleles was synthetic lethal with eogt knock-down. Therefore, Eogt may regulate protein functions by O-GlcNAc modification of their EGF repeats, and cellular metabolism by affecting pyrimidine synthesis and catabolism. We propose that eogt knock-down in the wing leads to metabolic and signaling perturbations that increase cytosolic uracil levels, thereby causing wing blister formation. PMID:23671640

Transcriptomic and Proteomic Responses of Sweetpotato Whitefly, Bemisia tabaci, to Thiamethoxam

PubMed Central

Yang, Nina; Xie, Wen; Yang, Xin; Wang, Shaoli; Wu, Qingjun; Li, Rumei; Pan, Huipeng; Liu, Baiming; Shi, Xiaobin; Fang, Yong; Xu, Baoyun; Zhou, Xuguo; Zhang, Youjun

2013-01-01

Background The sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is one of the most widely distributed agricultural pests. Although it has developed resistance to many registered insecticides including the neonicotinoid insecticide thiamethoxam, the mechanisms that regulate the resistance are poorly understood. To understand the molecular basis of thiamethoxam resistance, “omics” analyses were carried out to examine differences between resistant and susceptible B. tabaci at both transcriptional and translational levels. Results A total of 1,338 mRNAs and 52 proteins were differentially expressed between resistant and susceptible B. tabaci. Among them, 11 transcripts had concurrent transcription and translation profiles. KEGG analysis mapped 318 and 35 differentially expressed genes and proteins, respectively, to 160 and 59 pathways (p<0.05). Thiamethoxam treatment activated metabolic pathways (e.g., drug metabolism), in which 118 transcripts were putatively linked to insecticide resistance, including up-regulated glutathione-S-transferase, UDP glucuronosyltransferase, glucosyl/glucuronosyl transferase, and cytochrome P450. Gene Ontology analysis placed these genes and proteins into protein complex, metabolic process, cellular process, signaling, and response to stimulus categories. Quantitative real-time PCR analysis validated “omics” response, and suggested a highly overexpressed P450, CYP6CX1, as a candidate molecular basis for the mechanistic study of thiamethoxam resistance in whiteflies. Finally, enzymatic activity assays showed elevated detoxification activities in the resistant B. tabaci. Conclusions This study demonstrates the applicability of high-throughput omics tools for identifying molecular candidates related to thiamethoxam resistance in an agricultural important insect pest. In addition, transcriptomic and proteomic analyses provide a solid foundation for future functional investigations into the complex molecular mechanisms

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

DOE PAGES

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry; ...

2017-01-01

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat ofmore » uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.« less

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

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

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat ofmore » uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.« less

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

PubMed

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

2006-02-15

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

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

PubMed

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

2018-05-15

Most organisms, from Bacteria to Eukarya , synthesize UDP- N -acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP- N -acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea , the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii , predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii , and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic

PROTEOMIC ANALYSIS OPTIMIZATION: SELECTIVE PROTEIN SAMPLE ON-COLUMN RETENTION IN REVERSE-PHASE LIQUID CHROMATOGRAPHY

EPA Science Inventory

Why work was done?

To be able to identify, on a proteomic level, cytochromes P450 (CYP) and UDP-glucuronosyltransferases (UGT) in mouse liver microsomes for the conazole exposure study IRP # NHEERL-ECD-SCN-CZ-2002-01-R1_Addendum 1. The new enrichment method was necessary beca...

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage[OPEN

PubMed Central

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry; Sanhueza, Dayan; Ejsmentewicz, Troy; Sandoval-Ibañez, Omar; Parra-Rojas, Juan Pablo; Ebert, Berit; Reyes, Francisca C.

2017-01-01

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix. PMID:28062750

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

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

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

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

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

PubMed

Xie, Ying; Miranda, Sonia R; Hoskins, Janelle M; Hawke, Roy L

2017-01-15

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.

Developmental control of apiogalacturonan biosynthesis and UDP-apiose production in a duckweed. [Spirodela polyrrhiza

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

Longland, J.M.; Fry, S.C.; Trewavas, A.J.

1989-07-01

Vegetative fronds of Spirodela polyrrhiza were induced to form dormant turions by the addition of 1 micromolar abscisic acid or by shading. The cell wall polymers of fronds contained a high proportion of the branched-chain pentose, D-apiose (about 20% of total noncellulosic wall sugar residues), whereas turion cell walls contained only trace amounts (about 0.2%). When the fronds were fed D-({sup 3}H)glucuronic acid for 30 minutes, the accumulated UDP-({sup 3}H)apiose pool accounted for about 27% of the total phosphorylated ({sup 3}H)pentose derivatives; in turions, the UDP({sup 3}H)apiose pool accounted for only about 4% of the total phosphorylated ({sup 3}H)pentose derivatives.more » They conclude that the developmentally regulated decrease in the biosynthesis of a wall polysaccharide during turion formation involves a reduction in the supply of the relevant sugar nucleotide. One controlling enzyme activity is suggested to be UDP-apiose/UDP-xylose synthase. However, since there was a 100-fold decrease in the rate of polysaccharide synthesis and only a 9-fold decrease in UDP-apiose accumulation, there is probably also control of the activity of the relevant polysaccharide synthase.« less

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

PubMed

Kärkönen, Anna; Fry, Stephen C

2006-03-01

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

Differences in the Glucuronidation of Resveratrol and Pterostilbene: Altered Enzyme Specificity and Potential Gender Differences

PubMed Central

Dellinger, Ryan W.; Gomez Garcia, Angela M.; Meyskens, Frank L.

2015-01-01

Summary Resveratrol, a natural polyphenol found in grapes, berries and other plants, has been proposed as an ideal chemopreventative agent due to its plethora of health promoting activities. However, despite its lofty promise as a cancer prevention agent its success in human clinical trials has been limited due to its poor bioavailability. Thus, interest in other natural polyphenols is intensifying including the naturally occurring dimethylated analog of resveratrol, pterostilbene. The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the metabolism of both resveratrol and pterostilbene. The current study sought to elucidate the UGT family members responsible for the metabolism of pterostilbene and to examine gender differences in the glucuronidation of resveratrol and pterostilbene. We demonstrate that UGT1A1 and UGT1A3 are mainly responsible for pterostilbene glucuronidation although UGT1A8, UGT1A9 and UGT1A10 also had detectable activity. Intriguingly, UGT1A1 exhibits the highest activity against both resveratrol and pterostilbene despite altered hydroxyl group specificity. Using pooled human liver microsomes, enzyme kinetics were determined for pterostilbene and resveratrol glucuronides. In all cases females were more efficient than males, indicating potential gender differences in stilbene metabolism. Importantly, the glucuronidation of pterostilbene is much less efficient than that of resveratrol, indicating that pterostilbene will have dramatically decreased metabolism in humans. PMID:23965644

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

PubMed

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

2015-02-01

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

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

PubMed

Smith, James Amor; Bar-Peled, Maor

2017-01-01

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

Hibiscus cannabinus feruloyl-coa:monolignol transferase

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

Wilkerson, Curtis; Ralph, John; Withers, Saunia

The invention relates to isolated nucleic acids encoding a feruloyl-CoA:monolignol transferase and feruloyl-CoA:monolignol transferase enzymes. The isolated nucleic acids and/or the enzymes enable incorporation of monolignol ferulates into the lignin of plants, where such monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate. The invention also includes methods and plants that include nucleic acids encoding a feruloyl-CoA:monolignol transferase enzyme and/or feruloyl-CoA:monolignol transferase enzymes.

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

PubMed

Richter, Lilian H J; Kaminski, Yeda Rumi; Noor, Fozia; Meyer, Markus R; Maurer, Hans H

2016-09-01

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

Enzymatic Glycosylation by Transferases

NASA Astrophysics Data System (ADS)

Blixt, Ola; Razi, Nahid

Glycosyltransferases are important biological catalysts in cellular systems generating complex cell surface glycans involved in adhesion and signaling processes. Recent advances in glycoscience have increased the demands to access significant amount of glycans representing the glycome. Glycosyltransferases are now playing a key role for in vitro synthesis of oligosaccharides and the bacterial genome are increasingly utilized for cloning and over expression of active transferases in glycosylation reactions. This chapter highlights the recent progress towards preparative synthesis of oligosaccharides representing terminal sequences of glycoproteins and glycolipids using recombinant transferases. Transferases are also being explored in the context of solid-phase synthesis, immobilized on resins and over expression in vivo by engineered bacteria.

Protective effects of coffee against oxidative stress induced by the tobacco carcinogen benzo[α]pyrene.

PubMed

Kalthoff, Sandra; Landerer, Steffen; Reich, Julia; Strassburg, Christian P

2017-07-01

Coffee consumption has been epidemiologically associated with a lower risk for liver cirrhosis and cancer. UDP-glucuronosyltransferases (UGT1A) catalyze the detoxification of reactive metabolites thereby acting as indirect antioxidants. Aim of the study was to examine UGT1A regulation in response to Benzo[α]pyrene (BaP) to elucidate the potentially protective effects of coffee on BaP-induced oxidative stress and toxicity. In cell culture (HepG2, KYSE70 cells) and in htgUGT1A-WT mice, UGT1A transcription was activated by BaP, while it was reduced or absent htgUGT1A-SNP (containing 10 commonly occurring UGT1A-SNPs) mice. siRNA-mediated knockdown identified aryl hydrocarbon receptor (AhR) and nuclear factor erythroid2-related factor-2 (Nrf2) as mediators of BaP-induced UGT1A upregulation. Exposure to coffee led to a reduction of BaP-induced production of reactive oxygen species in vitro and in htgUGT1A-WT and -SNP mice. After UGT1A silencing by UGT1A-specific siRNA in cell culture, the coffee-mediated reduction of ROS production was significantly impaired compared to UGT1A expressing cells. A common UGT1A haplotype, prevalent in 9% (homozygous) of the White population, significantly impairs the expression of UGT1A enzymes in response to the putative tobacco carcinogen BaP and is likely to represent a significant risk factor for reduced detoxification and increased genotoxicity. Coffee was demonstrated to inhibit BaP-induced production of oxidative stress by UGT1A activation, and is therefore an attractive candidate for chemoprotection in risk groups for HCC or other tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of in vitro glucuronidation clearance of a range of drugs in human kidney microsomes: comparison with liver and intestinal glucuronidation and impact of albumin.

PubMed

Gill, Katherine L; Houston, J Brian; Galetin, Aleksandra

2012-04-01

Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL(int, UGT)) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL(int, UGT) on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL(int, UGT) in different tissues. Although BSA increased CL(int, UGT) in all tissues, the extent was tissue- and drug-dependent. Scaled CL(int, UGT) in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min(-1) · g tissue(-1) in liver, kidney, and intestinal microsomes. Renal CL(int, UGT) (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL(int, UGT) for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CL(int, UGT) (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL(int, UGT) is particularly important for UGT1A9 substrates.

Characterization of In Vitro Glucuronidation Clearance of a Range of Drugs in Human Kidney Microsomes: Comparison with Liver and Intestinal Glucuronidation and Impact of Albumin

PubMed Central

Gill, Katherine L.; Houston, J. Brian

2012-01-01

Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CLint, UGT) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CLint, UGT on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CLint, UGT in different tissues. Although BSA increased CLint, UGT in all tissues, the extent was tissue- and drug-dependent. Scaled CLint, UGT in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min−1 · g tissue−1 in liver, kidney, and intestinal microsomes. Renal CLint, UGT (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CLint, UGT for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CLint, UGT (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CLint, UGT is particularly important for UGT1A9 substrates. PMID:22275465

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

USDA-ARS?s Scientific Manuscript database

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

Elucidation and functional characterization of CsPSY and CsUGT promoters in Crocus sativus L.

PubMed Central

Bhat, Archana; Mishra, Sonal; Kaul, Sanjana

2018-01-01

The dried stigmas of Crocus sativus constitute the saffron, which is considered to be the costliest spice of the world. Saffron is valuable for its constituents, which are mainly apocarotenoids. In order to enhance the production of apocarotenoids, it is imperative to understand the regulation of apocarotenoid biosynthetic pathway. In C. sativus, although the pathway has been elucidated, the information regarding the regulation of the pathwaygenes is scanty. During the present investigation, the characterization of promoters regulating the expression of two important genes i.e. CsPSY and CsUGT was performed. We successfully cloned the promoters of both the genes, which were functionally characterized in Crocus sativus and Nicotiana tabaccum. In silico analysis of the promoters demonstrated the presence of several important cis regulatory elements responding tolight, hormonesand interaction with transcription factors (TFs). Further analysis suggested the regulation of CsPSY promoter by Abscisic acid (ABA) and that of CsUGT by Gibberellic acid (GA). In addition, we also observed ABA and GA mediated modulation in the expression of significant TFs and CsPSY and CsUGT transcripts. Overall, the study addresses issues related to regulation of key genes of apocarotenoid pathway in C.sativus. PMID:29634744

Elucidation and functional characterization of CsPSY and CsUGT promoters in Crocus sativus L.

PubMed

Bhat, Archana; Mishra, Sonal; Kaul, Sanjana; Dhar, Manoj K

2018-01-01

The dried stigmas of Crocus sativus constitute the saffron, which is considered to be the costliest spice of the world. Saffron is valuable for its constituents, which are mainly apocarotenoids. In order to enhance the production of apocarotenoids, it is imperative to understand the regulation of apocarotenoid biosynthetic pathway. In C. sativus, although the pathway has been elucidated, the information regarding the regulation of the pathwaygenes is scanty. During the present investigation, the characterization of promoters regulating the expression of two important genes i.e. CsPSY and CsUGT was performed. We successfully cloned the promoters of both the genes, which were functionally characterized in Crocus sativus and Nicotiana tabaccum. In silico analysis of the promoters demonstrated the presence of several important cis regulatory elements responding tolight, hormonesand interaction with transcription factors (TFs). Further analysis suggested the regulation of CsPSY promoter by Abscisic acid (ABA) and that of CsUGT by Gibberellic acid (GA). In addition, we also observed ABA and GA mediated modulation in the expression of significant TFs and CsPSY and CsUGT transcripts. Overall, the study addresses issues related to regulation of key genes of apocarotenoid pathway in C.sativus.

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

PubMed

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

2005-12-05

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

Mycophenolic acid AUC in Thai kidney transplant recipients receiving low dose mycophenolate and its association with UGT2B7 polymorphisms.

PubMed

Pithukpakorn, Manop; Tiwawanwong, Tiwat; Lalerd, Yupaporn; Assawamakin, Anunchai; Premasathian, Nalinee; Tasanarong, Adis; Thongnoppakhun, Wanna; Vongwiwatana, Attapong

2014-01-01

Despite use of a lower mycophenolate dose in Thai kidney transplant patients, acceptable graft and patient outcomes can be achieved. We therefore examined the pharmacokinetics of mycophenolic acid (MPA) by area under the curve (AUC) and investigated genetic contribution in mycophenolate metabolism in this population. Kidney transplant recipients with stable graft function who were receiving mycophenolate mofetil 1,000 mg/d in combination with either cyclosporine or tacrolimus, and prednisolone were studied. The MPA concentration was measured by fluorescence polarization immunoassay (FPIA), at predose and 1, 1.5, 2, 4, 6, 8, 10, and 12 hours after dosing. Genetic polymorphisms in UGT1A8, UGT1A9, and UGT2B7 were examined by denaturing high-performance liquid chromatography (DHPLC)-based single-base extension (SBE) analysis. A total 138 patients were included in study. The mean AUC was 39.49 mg-h/L (28.39-89.58 mg-h/L), which was in the therapeutic range. The correlation between the predose MPA concentration and AUC was poor. The mean AUC in the tacrolimus group was higher than that in the cyclosporine group. Polymorphisms in UGT2B7 showed significant association with AUC. Most of our patients with reduced mycophenolate dose had the AUC within the therapeutic range. Genetic polymorphisms in UGT2B7 may play a role in MPA metabolism in Thai kidney transplant patients.

Milk thistle, a herbal supplement, decreases the activity of CYP3A4 and uridine diphosphoglucuronosyl transferase in human hepatocyte cultures.

PubMed

Venkataramanan, R; Ramachandran, V; Komoroski, B J; Zhang, S; Schiff, P L; Strom, S C

2000-11-01

Milk thistle extract is one of the most commonly used nontraditional therapies, particularly in Germany. Milk thistle is known to contain a number of flavonolignans. We evaluated the effect of silymarin, on the activity of various hepatic drug-metabolizing enzymes in human hepatocyte cultures. Treatment with silymarin (0.1 and 0.25 mM) significantly reduced the activity of CYP3A4 enzyme (by 50 and 100%, respectively) as determined by the formation of 6-beta-hydroxy testosterone and the activity of uridine diphosphoglucuronosyl transferase (UGT1A6/9) (by 65 and 100%, respectively) as measured by the formation of 4-methylumbelliferone glucuronide. Silymarin (0.5 mM) also significantly decreased mitochondrial respiration as determined by MTT reduction in human hepatocytes. These observations point to the potential of silymarin to impair hepatic metabolism of certain coadministered drugs in humans. Indiscriminate use of herbal products may lead to altered pharmacokinetics of certain drugs and may result in increased toxicity of certain drugs.

UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation.

PubMed

Wen, Chao; Huang, Wei; Zhu, Xue-Lin; Li, Xiao-San; Zhang, Fan; Jiang, Ren-Wang

2018-02-02

A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C 21 steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

SCN1A, ABCC2 and UGT2B7 gene polymorphisms in association with individualized oxcarbazepine therapy.

PubMed

Ma, Chun-Lai; Wu, Xun-Yi; Jiao, Zheng; Hong, Zhen; Wu, Zhi-Yuan; Zhong, Ming-Kang

2015-01-01

Associations between the effects of SCN1A, SCN2A, ABCC2 and UGT2B7 genetic polymorphisms and oxcarbazepine (OXC) maintenance doses in Han Chinese epileptic patients were investigated. Genetic polymorphisms were detected in 184 epileptic patients receiving OXC monotherapy by high-resolution melting curve and TaqMan method. Carriers of the SCN1A IVS5-91G>A, UGT2B7 c.802T>C and ABCC2 c.1249G>A variant alleles required significantly higher OXC maintenance doses than noncarriers (p < 0.05). Corresponding relative ln (concentration-dose ratios) values for SCN1A IVS5-91 variants differed by the genotypic order GG > GA > AA. SCN1A, UGT2B7 and ABCC2 genetic polymorphisms are associated with OXC maintenance doses and may be useful for the personalization of OXC therapy in epileptic patients. Further studies are needed. Original submitted 6 June 2014; Revision submitted 5 September 2014.

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

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

Kopacz-Jodczyk, T.; Galasinski, W.

1987-10-01

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

Feruloyl-CoA:monolignol transferase

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

Wilkerson, Curtis; Ralph, John; Withers, Saunia

The invention relates to nucleic acids encoding a feruloyl-CoA:monolignol transferase and the feruloyl-CoA:monolignol transferase enzyme that enables incorporation of monolignol ferulates, for example, including p-coumaryl ferulate, coniferyl ferulate, and sinapyl ferulate, into the lignin of plants.

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

PubMed

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

2012-08-01

During the past century, several epidemics of human African trypanosomiasis, a deadly disease caused by the protist Trypanosoma brucei, have afflicted sub-Saharan Africa. Over 10 000 new victims are reported each year, with hundreds of thousands more at risk. As current drug treatments are either highly toxic or ineffective, novel trypanocides are urgently needed. The T. brucei galactose synthesis pathway is one potential therapeutic target. Although galactose is essential for T. brucei survival, the parasite lacks the transporters required to intake galactose from the environment. UDP-galactose 4'-epimerase (TbGalE) is responsible for the epimerization of UDP-glucose to UDP-galactose and is therefore of great interest to medicinal chemists. Using molecular dynamics simulations, we investigate the atomistic motions of TbGalE in both the apo and holo states. The sampled conformations and protein dynamics depend not only on the presence of a UDP-sugar ligand, but also on the chirality of the UDP-sugar C4 atom. This dependence provides important insights into TbGalE function and may help guide future computer-aided drug discovery efforts targeting this protein. © 2012 John Wiley & Sons A/S.

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

PubMed

Kopacz-Jodczyk, T; Gałasiński, W

1987-10-01

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

Significantly decreased and more variable expression of major CYPs and UGTs in liver microsomes prepared from HBV-positive human hepatocellular carcinoma and matched pericarcinomatous tissues determined using an isotope label-free UPLC-MS/MS method.

PubMed

Yan, Tongmeng; Gao, Song; Peng, Xiaojuan; Shi, Jian; Xie, Cong; Li, Qiang; Lu, Linlin; Wang, Ying; Zhou, Fuyuan; Liu, Zhongqiu; Hu, Ming

2015-03-01

To determine the liver expression of cytochrome P450 (CYPs) and uridine 5'-diphosphate-glucuronosyltransferases (UGTs), the major phase I and II metabolism enzymes responsible for clearance and detoxification of drugs, xenobiotic and endogenous substances. A validated isotope label-free method was established for absolute and simultaneous quantification of 9 CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D, 2E1 and 3A4) and 5 UGTs (1A1, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes using LC-MS/MS. The LC-MS/MS method displayed excellent dynamic range (at least 250-fold) and high sensitivity for each of the signature peptides with acceptable recovery, accuracy and precision. The protein expression profile of CYP and UGT isoforms were then determined in match microsomes samples prepared from patients with HBV-positive human hepatocellular carcinoma (HCC). In the tumor microsomes, the average absolute amounts of 8 major CYP isoforms (except CYP2C19) and 3 UGT isoforms (UGT1A1, UGT1A4 and UGT2B7) were decreased significantly (p < 0.05), whereas UGT1A6 and UGT1A9 levels were unchanged (p > 0.05). In addition, among isoforms with altered expression, 6 of 8 CYP isoforms and all three UGT isoforms were much more variable in tumor microsomes. Lastly, the importance of CYP3A4 was greatly diminished whereas the importance of UGT1A6 was enhanced in tumor microsomes. The use of an isotope label-free absolute quantification method for the simultaneous determination of 9 CYPs and 5 UGTs in human liver microsomes reveals that expression levels of CYPs and UGTs in human liver are severely impact by HCC, which could impact drug metabolism, disposition and pharmacotherapy.

Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

PubMed Central

Guleria, Praveen; Yadav, Sudesh Kumar

2013-01-01

Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2017-04-01

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

Biotransformation of Bisphenol AF to Its Major Glucuronide Metabolite Reduces Estrogenic Activity

PubMed Central

Yin, Jie; Zhang, Jing; Feng, Yixing; Shao, Bing

2013-01-01

Bisphenol AF (BPAF), an endocrine disrupting chemical, can induce estrogenic activity through binding to estrogen receptor (ER). However, the metabolism of BPAF in vivo and the estrogenic activity of its metabolites remain unknown. In the present study, we identified four metabolites including BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrated and BPAF sulfate in the urine of Sprague-Dawley (SD) rats. BPAF-G was further characterized by nuclear magnetic resonance (NMR). After treatment with a single dose of BPAF, BPAF was metabolized rapidly to BPAF-G, as detected in the plasma of SD rats. Biotransformation of BPAF to BPAF-G was confirmed with human liver microsomes (HLM), and Vmax of glucuronidation for HLM was 11.6 nmol/min/mg. We also found that BPAF glucuronidation could be mediated through several human recombinant UDP-glucuronosyltransferases (UGTs) including UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, among which UGT2B7 showed the highest efficiency of glucuronidation. To explain the biological function of BPAF biotransformation, the estrogenic activities of BPAF and BPAF-G were evaluated in ER-positive breast cancer T47D and MCF7 cells. BPAF significantly stimulates ER-regulated gene expression and cell proliferation at the dose of 100 nM and 1 μM in breast cancer cells. However, BPAF-G did not show any induction of estrogenic activity at the same dosages, implying that formation of BPAF-G is a potential host defense mechanism against BPAF. Based on our study, biotransformation of BPAF to BPAF-G can eliminate BPAF-induced estrogenic activity, which is therefore considered as reducing the potential threat to human beings. PMID:24349450

Grouping and characterization of putative glycosyltransferase genes from Panax ginseng Meyer.

PubMed

Khorolragchaa, Altanzul; Kim, Yu-Jin; Rahimi, Shadi; Sukweenadhi, Johan; Jang, Moon-Gi; Yang, Deok-Chun

2014-02-15

Glycosyltransferases are members of the multigene family of plants that can transfer single or multiple activated sugars to a range of plant molecules, resulting in the glycosylation of plant compounds. Although the activities of many glycosyltransferases and their products have been recognized for a long time, only in recent years were some glycosyltransferase genes identified and few have been functionally characterized in detail. Korean ginseng (Panax ginseng Meyer), belonging to Araliaceae, has been well known as a popular mysterious medicinal herb in East Asia for over 2,000 years. A total of 704 glycosyltransferase unique sequences have been found from a ginseng expressed sequence tag (EST) library, and these sequences encode enzymes responsible for the secondary metabolite biosynthesis. Finally, twelve UDP glycosyltransferases (UGTs) were selected as the candidates most likely to be involved in triterpenoid synthesis. In this study, we classified the candidate P. ginseng UGTs (PgUGTs) into proper families and groups, which resulted in eight UGT families and six UGT groups. We also investigated those gene candidates encoding for glycosyltransferases by analysis of gene expression in methyl jasmonate (MeJA)-treated ginseng adventitious roots and different tissues from four-year-old ginseng using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). For organ-specific expression, most of PgUGT transcription levels were higher in leaves and roots compared with flower buds and stems. The transcription of PgUGTs in adventitious roots treated with MeJA increased as compared with the control. PgUGT1 and PgUGT2, which belong to the UGT71 family genes expressed in MeJA-treated adventitious roots, were especially sensitive, showing 33.32 and 38.88-fold expression increases upon 24h post-treatments, respectively. © 2013 Elsevier B.V. All rights reserved.

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

PubMed

Horynová, Milada; Takahashi, Kazuo; Hall, Stacy; Renfrow, Matthew B; Novak, Jan; Raška, Milan

2012-02-01

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system. Copyright © 2011 Elsevier Inc. All rights reserved.

The inverse relationship between bladder and liver in 4-aminobiphenyl-induced DNA damage

PubMed Central

Stablewski, Aimee B.; Vouros, Paul; Zhang, Yuesheng

2015-01-01

Bladder cancer risk is significantly higher in men than in women. 4-Aminobiphenyl (ABP) is a major human bladder carcinogen from tobacco smoke and other sources. In mice, male bladder is more susceptible to ABP-induced carcinogenesis than female bladder, but ABP is more carcinogenic in the livers of female mice than of male mice. Here, we show that castration causes male mice to acquire female phenotype regarding susceptibility of bladder and liver to ABP. However, spaying has little impact on organ susceptibility to ABP. Liver UDP-glucuronosyltransferases (UGTs) are believed to protect liver against but sensitize bladder to ABP, as glucuronidation of ABP and its metabolites generally reduces their toxicity and promotes their elimination via urine, but the metabolites are labile in urine, delivering carcinogenic species to the bladder. Indeed, liver expression of ABP-metabolizing human UGT1A3 transgene in mice increases bladder susceptibility to ABP. However, ABP-specific liver UGT activity is significantly higher in wild-type female mice than in their male counterparts, and castration also significantly increases ABP-specific UGT activity in the liver. Taken together, our data suggest that androgen increases bladder susceptibility to ABP via liver, likely by modulating an ABP-metabolizing liver enzyme, but exclude UGT as an important mediator. PMID:25596734

Ectopic expression of UGT84A2 delayed flowering by indole-3-butyric acid-mediated transcriptional repression of ARF6 and ARF8 genes in Arabidopsis.

PubMed

Zhang, Gui-Zhi; Jin, Shang-Hui; Li, Pan; Jiang, Xiao-Yi; Li, Yan-Jie; Hou, Bing-Kai

2017-12-01

Ectopic expression of auxin glycosyltransferase UGT84A2 in Arabidopsis can delay flowering through increased indole-3-butyric acid and suppressed transcription of ARF6, ARF8 and flowering-related genes FT, SOC1, AP1 and LFY. Auxins are critical regulators for plant growth and developmental processes. Auxin homeostasis is thus an important issue for plant biology. Here, we identified an indole-3-butyric acid (IBA)-specific glycosyltransferase, UGT84A2, and characterized its role in Arabidopsis flowering development. UGT84A2 could catalyze the glycosylation of IBA, but not indole-3-acetic acid (IAA). UGT84A2 transcription expression was clearly induced by IBA. When ectopically expressing in Arabidopsis, UGT84A2 caused obvious delay in flowering. Correspondingly, the increase of IBA level, the down-regulation of AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8, and the down-regulation of flowering-related genes such as FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO1(SOC1), APETALA1 (AP1), and LEAFY(LFY) were observed in transgenic plants. When exogenously applying IBA to wild-type plants, the late flowering phenotype, the down-regulation of ARF6, ARF8 and flowering-related genes recurred. We examined the arf6arf8 double mutants and found that the expression of flowering-related genes was also substantially decreased in these mutants. Together, our results suggest that glycosyltransferase UGT84A2 may be involved in flowering regulation through indole-3-butyric acid-mediated transcriptional repression of ARF6, ARF8 and downstream flowering pathway genes.

Maternal drug abuse and human term placental xenobiotic and steroid metabolizing enzymes in vitro.

PubMed

Paakki, P; Stockmann, H; Kantola, M; Wagner, P; Lauper, U; Huch, R; Elovaara, E; Kirkinen, P; Pasanen, M

2000-02-01

We evaluated the impact of maternal drug abuse at term on human placental cytochrome P450 (CYP)-mediated (Phase I) xenobiotic and steroid-metabolizing activities [aromatase, 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), pyrene 1-hydroxylase (P1OH), and testosterone hydroxylase], and androstenedione-forming isomerase, NADPH quinone oxidoreductase (Phase II), UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) activities in vitro. Overall, the formation of androstenedione, P1OH, and testosterone hydroxylase was statistically significant between control and drug-abusing subjects; we observed no significant differences in any other of the phase I and II activities. In placentas from drug-abusing mothers, we found significant correlations between ECOD and P1OH activities (p < 0. 001), but not between ECOD and aromatase or P1OH and EROD activities; we also found significant correlations between blood cotinine and UGT activities (p < 0.01). In contrast, in controls (mothers who did not abuse drugs but did smoke cigarettes), the P1OH activity correlated with ECOD, EROD (p < 0.001), and testosterone hydroxylase (p < 0.001) activities. Our results (wider variation in ECOD activity among tissue from drug-abusing mothers and the significant correlation between P1OH and ECOD activities, but not with aromatase or EROD activities) indicate that maternal drug abuse results in an additive effect in enhancing placental xenobiotic metabolizing enzymes when the mother also smokes cigarettes; this may be due to enhancing a "silent" CYP form, or a new placental CYP form may be activated. The change in the steroid metabolism profile in vitro suggests that maternal drug abuse may alter normal hormonal homeostasis during pregnancy.

Development of a new DHPLC assay for genotyping UGT1A (TA)n polymorphism associated with Gilbert's syndrome.

PubMed

Mlakar, Simona Jurkovic; Ostanek, Barbara

2011-01-01

Gilbert's syndrome is the most common hereditary disorder of bilirubin metabolism. The causative mutation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the UGT1A1 promoter. Affected individuals are homozygous for the variant promoter and have 7 TA repeats instead of 6. Promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians. The aim of our study was to develop denaturing high-performance liquid chromatography (DHPLC) assay for genotyping UGT1A1(TA)n polymorphism and to compare it with a previously described single-strand conformation polymorphism (SSCP) assay. Fifty DNA samples with common genotypes ((TA)6/6, (TA)6/7, (TA)7/7) as well as 7 samples with one of the following rare genotypes- (TA)5/6, (TA)5/7, (TA)6/8 or (TA)7/8 were amplified by polymerase chain reaction (PCR) and genotyped by DHPLC using sizing mode. All samples were previously genotyped by SSCP assay which was validated by sequencing analysis. All samples with either common or rare genotypes showed completely concordant results between DHPLC and SSCP assays. Our results show that sizing DHPLC assay is more efficient compared to classical SSCP assay due to shorter time of genotyping analysis, ability of genotyping increased number of samples per day, higher robustness, reproducibility and cost-effectiveness with no loss of accuracy in detection of all UGT1A1(TA)n genotypes. We developed a new DHPLC assay which is suitable for accurate, automated, highthroughput, robust genotyping of all UGT1A1(TA)n polymorphism variants, compared to a labour intensive and time-consuming SSCP assay.

UDP-glucuronosyltransferase 1A1*6 and *28 polymorphisms as indicators of initial dose level of irinotecan to reduce risk of neutropenia in patients receiving FOLFIRI for colorectal cancer.

PubMed

Miyata, Yoshinori; Touyama, Tetsuo; Kusumi, Takaya; Morita, Yoshitaka; Mizunuma, Nobuyuki; Taniguchi, Fumihiro; Manabe, Mitsuaki

2016-08-01

Irinotecan (CPT-11)-induced neutropenia is associated with UDP-glucuronosyltransferase (UGT) 1A1*6 and *28 polymorphisms. This prospective study investigated whether using these polymorphisms to adjust the initial dose of CPT-11 as part of FOLFIRI treatment in colorectal cancer patients might improve safety. All data were collected by a physician. The relationship between UGT1A1 polymorphisms and first-cycle neutropenia, reasons for treatment discontinuation, and time-to-treatment failure were evaluated. Multivariate analysis was used to assess the risk of neutropenia. A total of 795 patients were divided into wild-type (*1/*1) (50.1 %), heterozygous (*28/*1, *6/*1) (41.1 %), and homozygous (*28/*28, *6/*6, *28/*6) (8.8 %) groups, in which the median starting dose of CPT-11 was 143.0, 143.0, and 115.0 mg/m(2), respectively. First-cycle grade ≥3 neutropenia occurred in 17.3, 25.4, and 28.6 % of these patients, respectively. Multivariate analysis revealed that the incidence of grade ≥3 neutropenia was significantly greater in the heterozygous and homozygous groups than in the wild-type group [odds ratio (OR) 1.67; 95 % confidence interval (CI) 1.16-2.42; p = 0.0060, and OR 2.22; 95 % CI 1.22-4.02; p = 0.0088, respectively]. Age (OR 1.77; 95 % CI 1.24-2.53; p = 0.0017), coelomic fluid (OR 1.84; 95 % CI 1.05-3.25; p = 0.0343), and non-reduction in starting dose (OR 1.53; 95 % CI 1.08-2.18; p = 0.0176) were also identified as significant risk factors. The risk of neutropenia was higher in the heterozygous and homozygous groups at initiation of CPT-11 treatment. This suggests that when a reduction in dose is required in patients harboring two variant alleles, the decrease should be approximately 20 %.

Metabolism of the Folate Precursor p-Aminobenzoate in Plants

PubMed Central

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

2008-01-01

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

The mitochondrial O-linked N-acetylglucosamine transferase (mOGT) in the diabetic patient could be the initial trigger to develop Alzheimer disease.

PubMed

Lozano, Liliana; Lara-Lemus, Roberto; Zenteno, Edgar; Alvarado-Vásquez, Noé

2014-10-01

Diabetes mellitus (DM) is considered a risk factor for the development of Alzheimer disease (AD); however, how DM favors evolution of AD is still insufficiently understood. Hyperglycemia in DM is associated to an increase in mitochondrial reactive oxygen species (ROS) generation, as well as damage of hippocampal cells, reflected by changes in morphological and mitochondrial functionality. Similar mitochondrial damage has been observed when amyloid beta (Aβ) accumulates in the brain of AD patients. In DM, the excess of glucose in the brain induces higher activity of the hexosamine biosynthesis pathway (HBP), it synthesizes UDP-N-acetylglucosamine (UDP-GlcNAc), which is used by O-linked N-acetylglucosamine transferase (OGT) to catalyze O-GlcNAcylation of numerous proteins. Although O-GlcNAcylation plays an important role in maintaining structure and cellular functionality, chronic activity of this pathway has been associated with insulin resistance and hyperglycemia-induced glucose toxicity. Three different forms of OGT are known: nucleocytoplasmic (ncOGT), short (sOGT), and mitochondrial (mOGT). Previous reports showed that overexpression of ncOGT is not toxic to the cell; in contrast, overexpression of mOGT is associated with cellular apoptosis. In this work, we suggest that hyperglycemia in the diabetic patient could induce greater expression and activity of mOGT, modifying the structure and functionality of mitochondria in hippocampal cells, accelerating neuronal damage, and favoring the start of AD. In consequence, mOGT activity could be a key point for AD development in patients with DM. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2016-01-01

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. PMID:27551039

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

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

Wang, Tsung-Pao; Pan, Yun-Ru; Fu, Chien-Yu

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 multicellularmore » 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.« less

Evolution of a Major Drug Metabolizing Enzyme Defect in the Domestic Cat and Other Felidae: Phylogenetic Timing and the Role of Hypercarnivory

PubMed Central

Shrestha, Binu; Reed, J. Michael; Starks, Philip T.; Kaufman, Gretchen E.; Goldstone, Jared V.; Roelke, Melody E.; O'Brien, Stephen J.; Koepfli, Klaus-Peter; Frank, Laurence G.; Court, Michael H.

2011-01-01

The domestic cat (Felis catus) shows remarkable sensitivity to the adverse effects of phenolic drugs, including acetaminophen and aspirin, as well as structurally-related toxicants found in the diet and environment. This idiosyncrasy results from pseudogenization of the gene encoding UDP-glucuronosyltransferase (UGT) 1A6, the major species-conserved phenol detoxification enzyme. Here, we established the phylogenetic timing of disruptive UGT1A6 mutations and explored the hypothesis that gene inactivation in cats was enabled by minimal exposure to plant-derived toxicants. Fixation of the UGT1A6 pseudogene was estimated to have occurred between 35 and 11 million years ago with all extant Felidae having dysfunctional UGT1A6. Out of 22 additional taxa sampled, representative of most Carnivora families, only brown hyena (Parahyaena brunnea) and northern elephant seal (Mirounga angustirostris) showed inactivating UGT1A6 mutations. A comprehensive literature review of the natural diet of the sampled taxa indicated that all species with defective UGT1A6 were hypercarnivores (>70% dietary animal matter). Furthermore those species with UGT1A6 defects showed evidence for reduced amino acid constraint (increased dN/dS ratios approaching the neutral selection value of 1.0) as compared with species with intact UGT1A6. In contrast, there was no evidence for reduced amino acid constraint for these same species within UGT1A1, the gene encoding the enzyme responsible for detoxification of endogenously generated bilirubin. Our results provide the first evidence suggesting that diet may have played a permissive role in the devolution of a mammalian drug metabolizing enzyme. Further work is needed to establish whether these preliminary findings can be generalized to all Carnivora. PMID:21464924

Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver.

PubMed

Papageorgiou, Ioannis; Freytsis, Marina; Court, Michael H

2016-10-01

Acetaminophen is the leading cause of acute liver failure (ALF) in many countries including the United States. Hepatic glucuronidation by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes is the major route of acetaminophen elimination. Reduced glucuronidation may predispose some individuals to acetaminophen-induced ALF, but mechanisms underlying reduced glucuronidation are poorly understood. We hypothesized that specific microRNAs (miRNAs) may reduce UGT1A activity by direct effects on the UGT1A 3'-UTR shared by all UGT1A enzyme transcripts, or by indirect effects on transcription factors regulating UGT1A expression. We performed an unbiased miRNA whole transcriptome association analysis using a bank of human livers with known acetaminophen glucuronidation activities. Of 754 miRNAs evaluated, 9 miRNAs were identified that were significantly overexpressed (p<0.05; >2-fold) in livers with low acetaminophen glucuronidation activities compared with those with high activities. miR-375 showed the highest difference (>10-fold), and was chosen for further mechanistic validation. We demonstrated using in silico analysis and luciferase reporter assays that miR-375 has a unique functional binding site in the 3'-UTR of the aryl hydrocarbon receptor (AhR) gene. Furthermore overexpression of miR-375 in LS180 cells demonstrated significant repression of endogenous AhR protein (by 40%) and mRNA (by 10%), as well as enzyme activity and/or mRNA of AhR regulated enzymes including UGT1A1, UGT1A6, and CYP1A2, without affecting UGT2B7, which is not regulated by AhR. Thus miR-375 is identified as a novel repressor of UGT1A-mediated hepatic acetaminophen glucuronidation through reduced AhR expression, which could predispose some individuals to increased risk for acetaminophen-induced ALF. Published by Elsevier Inc.

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

PubMed

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

2018-05-17

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

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

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

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

1986-05-01

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

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

PubMed Central

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

2015-01-01

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

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

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

Maruyama, Daisuke; Nishitani, Yuichi; Nonaka, Tsuyoshi

2006-12-01

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

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

PubMed

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

1992-01-01

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

Modulation of bilirubin neurotoxicity by the Abcb1 transporter in the Ugt1-/- lethal mouse model of neonatal hyperbilirubinemia.

PubMed

Bockor, Luka; Bortolussi, Giulia; Vodret, Simone; Iaconcig, Alessandra; Jašprová, Jana; Zelenka, Jaroslav; Vitek, Libor; Tiribelli, Claudio; Muro, Andrés F

2017-01-01

Moderate neonatal jaundice is the most common clinical condition during newborn life. However, a combination of factors may result in acute hyperbilirubinemia, placing infants at risk of developing bilirubin encephalopathy and death by kernicterus. While most risk factors are known, the mechanisms acting to reduce susceptibility to bilirubin neurotoxicity remain unclear. The presence of modifier genes modulating the risk of developing bilirubin-induced brain damage is increasingly being recognised. The Abcb1 and Abcc1 members of the ABC family of transporters have been suggested to have an active role in exporting unconjugated bilirubin from the central nervous system into plasma. However, their role in reducing the risk of developing neurological damage and death during neonatal development is still unknown.To this end, we mated Abcb1a/b-/- and Abcc1-/- strains with Ugt1-/- mice, which develop severe neonatal hyperbilirubinemia. While about 60% of Ugt1-/- mice survived after temporary phototherapy, all Abcb1a/b-/-/Ugt1-/- mice died before postnatal day 21, showing higher cerebellar levels of unconjugated bilirubin. Interestingly, Abcc1 role appeared to be less important.In the cerebellum of Ugt1-/- mice, hyperbilirubinemia induced the expression of Car and Pxr nuclear receptors, known regulators of genes involved in the genotoxic response.We demonstrated a critical role of Abcb1 in protecting the cerebellum from bilirubin toxicity during neonatal development, the most clinically relevant phase for human babies, providing further understanding of the mechanisms regulating bilirubin neurotoxicity in vivo. Pharmacological treatments aimed to increase Abcb1 and Abcc1 expression, could represent a therapeutic option to reduce the risk of bilirubin neurotoxicity. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Disposition of lasofoxifene, a next-generation selective estrogen receptor modulator, in healthy male subjects.

PubMed

Prakash, Chandra; Johnson, Kim A; Gardner, Mark J

2008-07-01

Disposition of lasofoxifene, a next-generation selective estrogen receptor modulator, was investigated in male volunteers after p.o. administration of a single 20-mg dose of [(14)C]lasofoxifene. Approximately 72% of the administered dose was recovered from the urine and feces, with majority of dose excreted in the feces, probably via bile. The absorption of lasofoxifene in humans was slow with T(max) values typically exceeding 6 h. The C(max) and area under plasma concentration-time profile from time 0 to the last quantifiable time point values of lasofoxifene were lower than those determined for total radioactivity, indicating presence of circulating metabolites. The primary clearance mechanisms for lasofoxifene in humans were direct conjugation (glucuronide and sulfate conjugates) and phase I oxidation, each accounting for about half of the metabolism. Several oxidative metabolites were identified by liquid chromatography/tandem mass spectrometry. The primary phase I metabolites were the result of hydroxylations on the tetraline moiety and the phenyl rings attached to the tetraline, and oxidation on the pyrrolidine moiety. Considering the numerous metabolites seen in vivo, additional in vitro studies using human liver and intestinal microsomes, recombinant cytochromes P450 (P450s), and UDP glucuronosyltransferases (UGTs) were performed. The turnover of lasofoxifene was very slow in liver microsomes, and only two metabolites were identified as two regioisomers of the catechol metabolite. The results from in vitro experiments with recombinant isoforms and P450 isoform-selective inhibitors suggested that the oxidative metabolism of lasofoxifene is catalyzed primarily by CYP3A and CYP2D6. In addition, its glucuronidation is catalyzed by UGTs that are expressed in both the liver (UGT1A1, UGT1A3, UGT1A6, and UGT1A9) and the intestine (UGT1A8 and UGT1A10).

Cancer Chemoprevention by Traditional Chinese Herbal Medicine and Dietary Phytochemicals: Targeting Nrf2-Mediated Oxidative Stress/Anti-Inflammatory Responses, Epigenetics, and Cancer Stem Cells

PubMed Central

Hun Lee, Jong; Shu, Limin; Fuentes, Francisco; Su, Zheng-Yuan; Tony Kong, Ah-Ng

2013-01-01

Excessive oxidative stress induced by reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive metabolites of carcinogens alters cellular homeostasis, leading to genetic/epigenetic changes, genomic instability, neoplastic transformation, and cancer initiation/progression. As a protective mechanism against oxidative stress, antioxidant/detoxifying enzymes reduce these reactive species and protect normal cells from endo-/exogenous oxidative damage. The transcription factor nuclear factor-erythroid 2 p45 (NF-E2)-related factor 2 (Nrf2), a master regulator of the antioxidative stress response, plays a critical role in the expression of many cytoprotective enzymes, including NAD(P)H:quinine oxidoreductase (NQO1), heme oxygenase-1 (HO-1), UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). Recent studies demonstrated that many dietary phytochemicals derived from various vegetables, fruits, spices, and herbal medicines induce Nrf2-mediated antioxidant/detoxifying enzymes, restore aberrant epigenetic alterations, and eliminate cancer stem cells (CSCs). The Nrf2-mediated antioxidant response prevents many age-related diseases, including cancer. Owing to their fundamental contribution to carcinogenesis, epigenetic modifications and CSCs are novel targets of dietary phytochemicals and traditional Chinese herbal medicine (TCHM). In this review, we summarize cancer chemoprevention by dietary phytochemicals, including TCHM, which have great potential as a safer and more effective strategy for preventing cancer. PMID:24716158

Cancer chemoprevention by traditional chinese herbal medicine and dietary phytochemicals: targeting nrf2-mediated oxidative stress/anti-inflammatory responses, epigenetics, and cancer stem cells.

PubMed

Hun Lee, Jong; Shu, Limin; Fuentes, Francisco; Su, Zheng-Yuan; Tony Kong, Ah-Ng

2013-01-01

Excessive oxidative stress induced by reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive metabolites of carcinogens alters cellular homeostasis, leading to genetic/epigenetic changes, genomic instability, neoplastic transformation, and cancer initiation/progression. As a protective mechanism against oxidative stress, antioxidant/detoxifying enzymes reduce these reactive species and protect normal cells from endo-/exogenous oxidative damage. The transcription factor nuclear factor-erythroid 2 p45 (NF-E2)-related factor 2 (Nrf2), a master regulator of the antioxidative stress response, plays a critical role in the expression of many cytoprotective enzymes, including quinine oxidoreductase (NQO1), heme oxygenase-1 (HO-1), UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). Recent studies demonstrated that many dietary phytochemicals derived from various vegetables, fruits, spices, and herbal medicines induce Nrf2-mediated antioxidant/detoxifying enzymes, restore aberrant epigenetic alterations, and eliminate cancer stem cells (CSCs). The Nrf2-mediated antioxidant response prevents many age-related diseases, including cancer. Owing to their fundamental contribution to carcinogenesis, epigenetic modifications and CSCs are novel targets of dietary phytochemicals and traditional Chinese herbal medicine (TCHM). In this review, we summarize cancer chemoprevention by dietary phytochemicals, including TCHM, which have great potential as a safer and more effective strategy for preventing cancer.

Transcriptome sequencing for identification of diapause-associated genes in fall webworm, Hyphantria cunea Drury.

PubMed

Deng, Yu; Li, Fei; Rieske, Lynne K; Sun, Li-Li; Sun, Shou-Hui

2018-08-20

Fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae) is extremely adaptable and highly invasive in China as a defoliator of ornamental and forest trees. Both voltinism and diapause strategies of fall webworm in China are variable, and this variability contributes to it invasiveness. Little is known about molecular regulation of diapause in fall webworm. To gain insight into possible mechanisms of diapause induction, high-throughput RNA-seq data were generated from non-diapause pupae (NDP) and diapause pupae (DP). A total of 58,151 unigenes were assembled and researched against nine public databases. In total, 29,013 up-regulated and 3451 down-regulated unigenes were differentially expressed by DP when compared with those of NDP. Genes encoding proteins such as UDP-glycosyl transferase (UGT), cytochrome P450 and Hsp70 were predicted to be involved in diapause. Moreover, GO function and KEGG pathway enrichments were performed on all differentially expressed genes (DEGs) and showed that cell cycle and insulin signaling pathways may be related to the diapause of the fall webworm. This study provides valuable information about the fall webworm transcriptome for future gene function research, especially as it relates to diapause. Copyright © 2018 Elsevier B.V. All rights reserved.

Nrf2 the rescue: effects of the antioxidative/electrophilic response on the liver.

PubMed

Klaassen, Curtis D; Reisman, Scott A

2010-04-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that positively regulates the basal and inducible expression of a large battery of cytoprotective genes. These gene products include proteins that catalyze reduction reactions (NAD(P)H:quinone oxidoreductase 1, Nqo1), conjugation reactions (glutathione-S-transferases, Gsts and UDP-glucuronosyltransferases, Ugts), as well as the efflux of potentially toxic xenobiotics and xenobiotic conjugates (multidrug resistance-associated proteins, Mrps). The significance of Nrf2 in the liver has been established, as livers of Nrf2-null mice are more susceptible to various oxidative/electrophilic stress-induced pathologies than wild-type mice. In contrast, both pharmacological and genetic models of hepatic Nrf2 activation are protective against oxidative/electrophilic stress. Furthermore, because certain Nrf2-target genes in the liver could affect the distribution, metabolism, and excretion of xenobiotics, the effects of Nrf2 on the kinetics of drugs and other xenobiotics should also be considered, with a special emphasis on metabolism and excretion. Therefore, this review highlights the research that has contributed to the understanding of the importance of Nrf2 in toxicodynamics and toxicokinetics, especially that which pertains to the liver. 2010 Elsevier Inc. All rights reserved.

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

PubMed

Beerens, Koen; Soetaert, Wim; Desmet, Tom

2013-09-01

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

DPD and UGT1A1 deficiency in colorectal cancer patients receiving triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan

PubMed Central

Falvella, Felicia Stefania; Cheli, Stefania; Martinetti, Antonia; Mazzali, Cristina; Iacovelli, Roberto; Maggi, Claudia; Gariboldi, Manuela; Pierotti, Marco Alessandro; Di Bartolomeo, Maria; Sottotetti, Elisa; Mennitto, Roberta; Bossi, Ilaria; de Braud, Filippo; Clementi, Emilio; Pietrantonio, Filippo

2015-01-01

Aims Triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan is a standard therapy for metastatic colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) in DPYD and UGT1A1 influence fluoropyrimdines and irinotecan adverse events (AEs). Low frequency DPYD variants (c.1905 + 1G > A, c.1679 T > G, c.2846A > T) are validated but more frequent ones (c.496A > G, c.1129-5923C > G and c.1896 T > C) are not. rs895819 T > C polymorphism in hsa-mir-27a is associated with reduced DPD activity. In this study, we evaluated the clinical usefulness of a pharmacogenetic panel for patients receiving triplet combinations. Methods Germline DNA was available from 64 CRC patients enrolled between 2008 and 2013 in two phase II trials of capecitabine, oxaliplatin and irinotecan plus bevacizumab or cetuximab. SNPs were determined by Real-Time PCR. We evaluated the functional variants in DPYD (rare: c.1905 + 1G > A, c.1679 T > G, c.2846A > T; most common: c.496A > G, c.1129-5923C > G, c.1896 T > C), hsa-mir-27a (rs895819) and UGT1A1 (*28) genes to assess their association with grade 3–4 AEs. Results None of the patients carried rare DPYD variants. We found DPYD c.496A > G, c.1129-5923C > G, c.1896 T > C in heterozygosity in 19%, 5% and 8%, respectively, homozygous rs895819 in hsa-mir-27a in 9% and homozygous UGT1A1*28 in 8%. Grade 3–4 AEs were observed in 36% patients and were associated with DPYD c.496A > G (odds ratio (OR) 4.93, 95% CI 1.29, 18.87; P = 0.021) and homozygous rs895819 in hsa-mir-27a (OR 11.11, 95% CI 1.21, 102.09; P = 0.020). Carriers of DPYD c.1896 T > C and homozygous UGT1A1*28 showed an OR of 8.42 (95% CI 0.88, 80.56; P = 0.052). Multivariate analysis confirmed an independent value for DPYD c.496A > G and c.1896 T > C. Conclusions Concomitant assessment of DPYD variants and the UGT1A1*28 allele is a promising strategy needing further validation for dose personalization. PMID:25782327

Glucosylation of 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone, the Key Strawberry Flavor Compound in Strawberry Fruit1

PubMed Central

Hong, Xiaotong; Zhao, Shuai; Liu, Jingyi; Schulenburg, Katja; Huang, Fong-Chin; Franz-Oberdorf, Katrin

2016-01-01

Strawberries emit hundreds of different volatiles, but only a dozen, including the key compound HDMF [4-hydroxy-2,5-dimethyl-3(2H)-furanone] contribute to the flavor of the fruit. However, during ripening, a considerable amount of HDMF is metabolized to the flavorless HDMF β-d-glucoside. Here, we functionally characterize nine ripening-related UGTs (UDP-glucosyltransferases) in Fragaria that function in the glucosylation of volatile metabolites by comprehensive biochemical analyses. Some UGTs showed a rather broad substrate tolerance and glucosylated a range of aroma compounds in vitro, whereas others had a more limited substrate spectrum. The allelic UGT71K3a and b proteins and to a lesser extent UGT73B24, UGT71W2, and UGT73B23 catalyzed the glucosylation of HDMF and its structural homolog 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone. Site-directed mutagenesis to introduce single K458R, D445E, D343E, and V383A mutations and a double G433A/I434V mutation led to enhanced HDMF glucosylation activity compared to the wild-type enzymes. In contrast, a single mutation in the center of the plant secondary product glycosyltransferase box (A389V) reduced the enzymatic activity. Down-regulation of UGT71K3 transcript expression in strawberry receptacles led to a significant reduction in the level of HDMF-glucoside and a smaller decline in HDMF-glucoside-malonate compared with the level in control fruits. These results provide the foundation for improvement of strawberry flavor and the biotechnological production of HDMF-glucoside. PMID:26993618

Preclinical discovery of candidate genes to guide pharmacogenetics during phase I development: the example of the novel anticancer agent ABT-751

PubMed Central

Innocenti, Federico; Ramírez, Jacqueline; Obel, Jennifer; Xiong, Julia; Mirkov, Snezana; Chiu, Yi-Lin; Katz, David A.; Carr, Robert A.; Zhang, Wei; Das, Soma; Adjei, Araba; Moyer, Ann M.; Chen, Pei Xian; Krivoshik, Andrew; Medina, Diane; Gordon, Gary B.; Ratain, Mark J.; Sahelijo, Leonardo; Weinshilboum, Richard M.; Fleming, Gini F.; Bhathena, Anahita

2013-01-01

Objective ABT-751, a novel orally available antitubulin agent, is mainly eliminated as inactive glucuronide (ABT-751G) and sulfate (ABT-751S) conjugates. We performed a pharmacogenetic investigation of ABT-751 pharmacokinetics using in-vitro data to guide the selection of genes for genotyping in a phase I trial of ABT-751. Methods UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes were screened for ABT-751 metabolite formation in vitro. Forty-seven cancer patients treated with ABT-751 were genotyped for 21 variants in these genes. Results UGT1A1, UGT1A4, UGT1A8, UGT2B7, and SULT1A1 were found to be involved in the formation of inactive ABT-751 glucuronide (ABT-751G) and sulfate (ABT-751S). SULT1A1 copy number (> 2) was associated with an average 34% increase in ABT-751 clearance (P= 0.044), an 18% reduction in ABT-751 AUC (P = 0.045), and a 50% increase in sulfation metabolic ratios (P=0.025). UGT1A8 rs6431558 was associated with a 28% increase in glucuronidation metabolic ratios (P =0.022), and UGT1A4*2 was associated with a 65% decrease in ABT-751 Ctrough (P = 0.009). Conclusion These results might represent the first example of a clinical pharmacokinetic effect of the SULT1A1 copy number variant on the clearance of a SULT1A1 substrate. A-priori selection of candidate genes guided by in-vitro metabolic screening enhanced our ability to identify genetic determinants of interpatient pharmacokinetic variability. PMID:23670235

Glucosylation of 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone, the Key Strawberry Flavor Compound in Strawberry Fruit.

PubMed

Song, Chuankui; Hong, Xiaotong; Zhao, Shuai; Liu, Jingyi; Schulenburg, Katja; Huang, Fong-Chin; Franz-Oberdorf, Katrin; Schwab, Wilfried

2016-05-01

Strawberries emit hundreds of different volatiles, but only a dozen, including the key compound HDMF [4-hydroxy-2,5-dimethyl-3(2H)-furanone] contribute to the flavor of the fruit. However, during ripening, a considerable amount of HDMF is metabolized to the flavorless HDMF β-d-glucoside. Here, we functionally characterize nine ripening-related UGTs (UDP-glucosyltransferases) in Fragaria that function in the glucosylation of volatile metabolites by comprehensive biochemical analyses. Some UGTs showed a rather broad substrate tolerance and glucosylated a range of aroma compounds in vitro, whereas others had a more limited substrate spectrum. The allelic UGT71K3a and b proteins and to a lesser extent UGT73B24, UGT71W2, and UGT73B23 catalyzed the glucosylation of HDMF and its structural homolog 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone. Site-directed mutagenesis to introduce single K458R, D445E, D343E, and V383A mutations and a double G433A/I434V mutation led to enhanced HDMF glucosylation activity compared to the wild-type enzymes. In contrast, a single mutation in the center of the plant secondary product glycosyltransferase box (A389V) reduced the enzymatic activity. Down-regulation of UGT71K3 transcript expression in strawberry receptacles led to a significant reduction in the level of HDMF-glucoside and a smaller decline in HDMF-glucoside-malonate compared with the level in control fruits. These results provide the foundation for improvement of strawberry flavor and the biotechnological production of HDMF-glucoside. © 2016 American Society of Plant Biologists. All Rights Reserved.

Selective reduction in the expression of UGTs and SULTs, a novel mechanism by which piperine enhances the bioavailability of curcumin in rat.

PubMed

Zeng, Xiaohui; Cai, Dake; Zeng, Qiaohuang; Chen, Zhao; Zhong, Guoping; Zhuo, Juncheng; Gan, Haining; Huang, Xuejun; Zhao, Ziming; Yao, Nan; Huang, Dane; Zhang, Chengzhe; Sun, Dongmei; Chen, Yuxing

2017-01-01

Curcumin (CUR) is known to exert numerous health-promoting effects in pharmacological studies, but its low bioavailability hinders the development of curcumin as a feasible therapeutic agent. Piperine (PIP) has been reported to enhance the bioavailability of curcumin, but the underlying mechanism remains poorly understood. In an attempt to find the mechanism by which piperine enhances the bioavailability of curcumin, the dosage ratio (CUR: PIP) and pre-treatment with piperine were hypothesized as key factors for improving the bioavailability in this combination. Therefore, combining curcumin with piperine at various dose ratios (1:1 to 100:1) and pre-dosing with piperine (0.5-8 h prior to curcumin) were designed to investigate their contributions to the pharmacokinetic parameters of curcumin in rats and their effects on the expression of UGT and SULT isoforms. It was shown that the C max and AUC 0-t of curcumin were slightly increased by 1.29 and 1.67 fold at a ratio of 20:1, while curcumin exposure was enhanced significantly in all the piperine pre-treated rats (0.5-8 h), peaking at 6 h (a 6.09-fold and 5.97-fold increase in C max and AUC 0-t , p < 0.01), regardless of the unchanged t 1/2 and T max . Also observed was a time-dependent inhibition of the hepatic expression of UGT1A6, 1A8, SULT1A1, 1A3, and the colonic expression of UGT1A6 that occurred within 6 h of piperine pre-treatment but was reversed at 8 h, which correlated with the changes in curcumin exposure. Similarly, the inhibitory effect of piperine on most of the UGTs and SULTs are time-dependent in Caco-2 and HepG2 cells. It is concluded that piperine pre-treatment time-dependently improves the bioavailability of curcumin through the reversible and selective inhibition of UGTs and SULTs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

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

DOE PAGES

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

2016-10-26

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

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

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

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

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

Cremophor EL-based nanoemulsion enhances transcellular permeation of emodin through glucuronidation reduction in UGT1A1-overexpressing MDCKII cells.

PubMed

Zhang, Tianpeng; Dong, Dong; Lu, Danyi; Wang, Shuai; Wu, Baojian

2016-03-30

Oral emodin, a natural anthraquinone and active component of many herbal medicines, is poorly bioavailable because of extensive first-pass glucuronidation. Here we aimed to prepare emodin nanoemulsion (EMO-NE) containing cremophor EL, and to assess its potential for enhancing transcellular absorption of emodin using UGT1A1-overexpressing MDCKII cells (or MDCK1A1 cells). EMO-NE was prepared using a modified emulsification technique and subsequently characterized by particle size, morphology, stability, and drug release. MDCKII cells were stably transfected with UGT1A1 using the lentiviral transfection approach. Emodin transport and metabolism were evaluated in Transwell-cultured MDCK1A1 cells after apical dosing of EMO-NE or control solution. The obtained EMO-NE (116 ± 6.5 nm) was spherical and stable for at least 2 months. Emodin release in vitro was a passive diffusion-driven process. EMO-NE administration increased the apparent permeability of emodin by a 2.3-fold (p<0.001) compared to the pure emodin solution (1.2 × 10(-5) cm/s vs 5.3 × 10(-6) cm/s). Further, both apical and basolateral excretion of emodin glucuronide (EMO-G) were significantly decreased (≥56.5%, p<0.001) in EMO-NE group. This was accompanied by a marked reduction (57.4%, p<0.001) in total emodin glucuronidation. It was found that the reduced glucuronidation was due to inhibition of cellular metabolism by cremophor EL. Cremophor EL inhibited UGT1A1-mediated glucuronidation of emodin using the mixed-type inhibition mechanism. In conclusion, cremophor EL-based nanoemulsion greatly enhanced transcellular permeation of emodin through inhibition of UGT metabolism. This cremophor EL-based nanoformulation may be a promising strategy to improve the oral bioavailability of emodin. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular reproductive characteristics of the reef coral Pocillopora damicornis.

PubMed

Rougée, Luc R A; Richmond, Robert H; Collier, Abby C

2015-11-01

Coral reefs are an indispensible worldwide resource, accounting for billions of dollars in cultural, economic, and ecological services. An understanding of coral reproduction is essential to determining the effects of environmental stressors on coral reef ecosystems and their persistence into the future. Here, we describe the presence of and changes in steroidal hormones along with associated steroidogenic and steroid removal enzymes during the reproductive cycle of the brooding, pan-Pacific, hermaphroditic coral, Pocillopora damicornis. Detectable levels of 17β-estradiol, estrone, progesterone and testosterone were consistently detected over two consecutive lunar reproductive cycles in coral tissue. Intra-colony variation in steroid hormone levels ranged between 1.5- and 2.2-fold and were not statistically different. Activities of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase and cytochrome P450 (CYP) 17 dehydrogenase were detectable and did not fluctuate over the reproductive cycle. Aromatase-like activity was detected during the lunar reproductive cycle with no significant fluctuations. Activities of regeneration enzymes did not fluctuate over the lunar cycle; however, activity of the clearance enzyme UDP-glucuronosyl transferases increased significantly (ANOVA, post hoc p<0.01) during the two weeks before and after peak larval release (planulation), suggesting that the activity of this enzyme family may be linked to the reproductive state of the coral. Sulfotransferase enzymes could not be detected. Our findings provide the first data defining normal physiological and lunar/reproductive variability in steroidal enzymes in a coral species with respect to their potential role in coral reproduction. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of sprout extract from Tuscan black cabbage on xenobiotic-metabolizing and antioxidant enzymes in rat liver.

PubMed

Melega, Simone; Canistro, Donatella; Pagnotta, Eleonora; Iori, Renato; Sapone, Andrea; Paolini, Moreno

2013-02-18

In recent years, health protection by natural products has received considerable attention, and a multitude of nutraceuticals have been characterized and their use promoted. Dietary consumption of Cruciferous vegetables, rich in glucosinolates (GLs), and their myrosinase-mediated hydrolysis products isothiocyanates (ITCs), were associated with reductions in cancer risk. In this study, the chemo-preventive potential of sprout extract of Tuscan black cabbage (Brassica oleracea L. var. acephala subvar. Laciniata L.) (TBCSE), through modulation of the xenobiotic-metabolizing apparatus and antioxidant defenses, was investigated in Sprague-Dawley rat liver. TBCSE was administered either orally or intraperitoneally, at a dose of 15mg/kg b.w., daily for twenty-one consecutive days, in the absence or presence of exogenous myrosinase, β-thioglucoside glucohydrolase (MYR), to distinguish the effects of intact GLs and ITCs, in the context of the extract. A complex, mild modulation pattern of P450-related monooxygenases was observed, mainly regarding CYP content (up to 36% loss), NADPH cytochrome (P450) c-reductase (up to 26% loss), CYP1A1 (up to 23% loss), but no evident distinctions among the effects of the extracts containing GLs or ITCs, were noted. In contrast, significant inductions of phase-II enzymes (up to 107% for UDP-glucuronosyl-transferase, and up to 36% for glutathione S-transferase) were recorded only where the GLs to ITCs conversion had occurred. A boosting effect on catalase (up to 38%), NAD(P)H:quinone reductase (up to 70%), glutathione reductase and glutathione peroxidase (up to 10%) was also recorded, suggesting an indirect antioxidant capacity of the extracts. Overall, the general phase-I inhibition, together with the up-regulation of detoxifying phase-II and antioxidant enzymes, exerted by the TBCSE supplementation, seem to be in line with the classical chemopreventive theory, but whether the addition of exogenous MYR is relevant, still remains to be

Extended mathematical model for "in vivo" quantification of the interaction betweeen atazanavir and bilirubin.

PubMed

Lozano, Roberto; Domeque, Nieves; Apesteguia, Alberto-Fermín

2014-02-01

The objective of the present work was to conduct an "in vivo" analysis of the atazanavir-bilirubin interaction. We developed a new mathematical approach to PK/PDPK models for competitive interaction based on the Michaelis-Menten equation, which was applied to patients with polymorphisms in the gene for UDP-glucuronosyltransferase 1A1 (UGT1A1). Atazanavir is known to induce concentration-dependent increases in bilirubin plasma levels. Thus, we employed our mathematical model to analyse rises in steady state atazanavir and bilirubin concentrations, ultimately plotting a nomogram for detection of suboptimal atazanavir exposure. Application of our model revealed that an absolute value or a steady state increase in bilirubin falling below 3.8Φ µmol/L (where Φ is a correction factor, =1 for UGT1A1 wild type and ≠1 for UGT1A1 variants) could be used to predict suboptimal atazanavir exposure and treatment failure. Thus, we have successfully established a new mathematical approach for pharmacodynamic-pharmacokinetic modelling of the interaction between atazanavir and bilirubin, as it relates to genetic variants of UGT1A1. Taken together, our findings indicate that bilirubin plasma levels represent a valuable marker of atazanavir exposure. © 2013, The American College of Clinical Pharmacology.

Panax ginseng genome examination for ginsenoside biosynthesis.

PubMed

Xu, Jiang; Chu, Yang; Liao, Baosheng; Xiao, Shuiming; Yin, Qinggang; Bai, Rui; Su, He; Dong, Linlin; Li, Xiwen; Qian, Jun; Zhang, Jingjing; Zhang, Yujun; Zhang, Xiaoyan; Wu, Mingli; Zhang, Jie; Li, Guozheng; Zhang, Lei; Chang, Zhenzhan; Zhang, Yuebin; Jia, Zhengwei; Liu, Zhixiang; Afreh, Daniel; Nahurira, Ruth; Zhang, Lianjuan; Cheng, Ruiyang; Zhu, Yingjie; Zhu, Guangwei; Rao, Wei; Zhou, Chao; Qiao, Lirui; Huang, Zhihai; Cheng, Yung-Chi; Chen, Shilin

2017-11-01

Ginseng, which contains ginsenosides as bioactive compounds, has been regarded as an important traditional medicine for several millennia. However, the genetic background of ginseng remains poorly understood, partly because of the plant's large and complex genome composition. We report the entire genome sequence of Panax ginseng using next-generation sequencing. The 3.5-Gb nucleotide sequence contains more than 60% repeats and encodes 42 006 predicted genes. Twenty-two transcriptome datasets and mass spectrometry images of ginseng roots were adopted to precisely quantify the functional genes. Thirty-one genes were identified to be involved in the mevalonic acid pathway. Eight of these genes were annotated as 3-hydroxy-3-methylglutaryl-CoA reductases, which displayed diverse structures and expression characteristics. A total of 225 UDP-glycosyltransferases (UGTs) were identified, and these UGTs accounted for one of the largest gene families of ginseng. Tandem repeats contributed to the duplication and divergence of UGTs. Molecular modeling of UGTs in the 71st, 74th, and 94th families revealed a regiospecific conserved motif located at the N-terminus. Molecular docking predicted that this motif captures ginsenoside precursors. The ginseng genome represents a valuable resource for understanding and improving the breeding, cultivation, and synthesis biology of this key herb. © The Author 2017. Published by Oxford University Press.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2013-07-01

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

DPYD and UGT1A1 genotyping to predict adverse events during first-line FOLFIRI or FOLFOXIRI plus bevacizumab in metastatic colorectal cancer.

PubMed

Cremolini, Chiara; Del Re, Marzia; Antoniotti, Carlotta; Lonardi, Sara; Bergamo, Francesca; Loupakis, Fotios; Borelli, Beatrice; Marmorino, Federica; Citi, Valentina; Cortesi, Enrico; Moretto, Roberto; Ronzoni, Monica; Tomasello, Gianluca; Zaniboni, Alberto; Racca, Patrizia; Buonadonna, Angela; Allegrini, Giacomo; Ricci, Vincenzo; Di Donato, Samantha; Zagonel, Vittorina; Boni, Luca; Falcone, Alfredo; Danesi, Romano

2018-01-30

Our study addresses the issue of the clinical reliability of three candidate DPYD and one UGT single nucleotide polymorphisms in predicting 5-fluorouracil- and irinotecan-related adverse events. To this purpose, we took advantage of a large cohort of metastatic colorectal cancer patients treated with first-line 5-fluorouracil- and irinotecan-based chemotherapy regimens (i.e., FOLFIRI or FOLFOXIRI) plus bevacizumab in the randomized clinical trial TRIBE by GONO (clinicaltrials.gov: NCT00719797), in which adverse events were carefully and prospectively collected at each treatment cycle. Here we show that patients bearing DPYD c.1905+1G/A and c.2846A/T genotypes, together with UGT1A1*28 variant carriers, have an increased risk of experiencing clinically relevant toxicities, including hematological AEs and stomatitis. No carrier of the DPYD c.1679T>G minor allele was identified. Present results support the preemptive screening of mentioned DPYD and UGT1A1 variants to identify patients at risk of clinically relevant 5-fluoruracil- and irinotecan-related AEs, in order to improve treatments' safety through a "genotype-guided" approach.

Effects of UGTs on the ionosphere

NASA Astrophysics Data System (ADS)

Argo, P. E.; Fitzgerald, T. J.

The processes that propagate local effects of underground nuclear tests from the ground into the upper atmosphere, and produce a detectable signal in the ionosphere are described. Initially, the blast wave from a underground test (UGT) radially expands, until it reaches the surface of the earth. The wave is both reflected and transmitted at this sharp discontinuity in propagation media. Tne reflected wave combines with the incident wave to form an 'Airy surface,' at which very strong ripping forces tear the earth apart. This broken region is called the 'spat zone,' and is launched into ballistic motion. The resultant ground motion launches an acoustical wave into the atmosphere. This acoustic wave, with overpressures of a few tenths of one percent, propagates upwards at the speed of sound. Assuming purely linear propagation, the path of the acoustic energy can be tracked using raytracing models. Most of the wave energy, which is radiated nearly vertically, tends to propagate into the upper atmosphere, while wave energy radiated at angles greater than about 30 degrees to the vertical will be reflected back to earth and is probably what is seen by most infrasonde measurements.

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

PubMed Central

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

1993-01-01

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

Catechol-O-Methyltransferase and UDP-Glucuronosyltransferases in the Metabolism of Baicalein in Different Species.

PubMed

Zhang, Ruiya; Cui, Yonglei; Wang, Yan; Tian, Xiangge; Zheng, Lu; Cong, HaiJian; Wu, Bin; Huo, Xiaokui; Wang, Chao; Zhang, BaoJing; Wang, Xiaobo; Yu, Zhonghui

2017-12-01

Baicalein is the major bioactive flavonoid in some herb medicines and dietary plants; however, the detailed metabolism pathway of its major metabolite oroxylin A-7-O-β-D-glucuronide in human was not clear. It was important to illustrate the major metabolic enzymes that participate in its elimination for the clinic use of baicalein. We first revealed a two-step metabolism profile for baicalein and illustrated the combination of catechol-O-methyltransferase (COMT) and uridine diphosphate-glucuronosyltransferases (UGTs) in drug metabolism, further evaluated its bioactivity variation during drug metabolism. The metabolism profiles were systematically characterized in different human biology preparations; after then, the anti-inflammatory activities of metabolites were evaluated in LPS-induced RAW264.7 cell. The first-step metabolite of baicalein was isolated and identified as oroxylin A; soluble-bound COMT (S-COMT) was the major enzyme responsible for its biotransformation. Specially, position 108 mutation of S-COMT significantly decreases the elimination. Meantime, oroxylin A was rapidly metabolized by UGTs, UGT1A1, -1A3, -1A6, -1A7, -1A8, -1A9, and -1A10 which were involved in the glucuronidation. Considerable species differences were observed with 1060-fold K m (3.05 ± 1.86-3234 ± 475 μM) and 330-fold CL int (5.93-1973 μL/min/mg) variations for baicalein metabolism. Finally, the middle metabolite oroxylin A exhibited a potent anti-inflammatory activity with the IC 50 value of 28 μM. The detailed kinetic parameters indicated that COMT provide convenience for the next glucuronidation; monkey would be a preferred animal model for the preclinical investigation of baicalein. Importantly, oroxylin A should be reconsidered in evaluating baicalein efficacy against inflammatory diseases.

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

PubMed

Srivastava, Ankita; Chandra, Deepak

2017-06-05

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

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

NASA Astrophysics Data System (ADS)

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

2015-12-01

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

Dose-dependent testosterone sensitivity of the steroidal passport and GC-C-IRMS analysis in relation to the UGT2B17 deletion polymorphism.

PubMed

Strahm, Emmanuel; Mullen, Jenny E; Gårevik, Nina; Ericsson, Magnus; Schulze, Jenny J; Rane, Anders; Ekström, Lena

2015-01-01

The newly implemented Steroid Module of the Athlete Biological Passport has improved doping tests for steroids. A biomarker included in this passport is the urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratio, a ratio greatly affected by a deletion polymorphism in UGT2B17. Suspect urine doping tests are further analyzed with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the origin of the androgen. In this study, we investigated the sensitivity of the steroidal module and the IRMS analysis, in subjects administered with three doses of testosterone enanthate (500, 250, and 125 mg), in relation to the UGT2B17 polymorphism. All subjects carrying the UGT2B17 enzyme reached the traditionally used threshold, a T/E ratio of 4, after all three administered doses, whereas none of the subjects devoid of this enzyme reached a T/E of 4. On the other hand, using the athlete biological passport and IRMS analysis, all three doses could be detected to a high degree of sensitivity. The concentrations of all steroids included in the steroidal module were dose dependently increased, except for epitestosterone which decreased independent of dose. The decrease in epitestosterone was significantly associated with circulatory levels of testosterone post dose (rs =0.60 and p=0.007). In conclusion, these results demonstrate that administration of a single dose of 125-500 mg testosterone enanthate could be detected using the athlete biological passport, together with IRMS. Since IRMS is sensitive to testosterone doping independent of UGT2B17 genotype, also very small changes in the steroidal passport should be investigated with IRMS. Copyright © 2015 John Wiley & Sons, Ltd.

21 CFR 862.1535 - Ornithine carbamyl transferase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine carbamyl transferase test system is a device intended to measure the activity of the enzyme ornithine... and treatment of liver diseases, such as infectious hepatitis, acute cholecystitis (inflammation of...

Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.

PubMed Central

Hartweck, Lynn M; Scott, Cheryl L; Olszewski, Neil E

2002-01-01

The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting. PMID:12136030

[Induction of uridine 5'-diphosphate-glucuronosyltransferase gene expression by sulforaphane and its mechanism: experimental study in human colon cancel cells].

PubMed

Wang, Min; Li, Yan-Qing; Zhong, Ning; Chen, Jian; Xu, Xiao-Qun; Yuan, Meng-Biao

2005-03-30

To study the induction of expression of uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) 1A in colon cancer cells by sulforaphane (SFN) and its possible mechanism. Human colon cancer cells of the line Caco-2 were cultured and added with SFN of different terminal concentrations, all below the concentration of IC(50). RT-PCR was used to examine the expression of UGT1A mRNA induced by SFN. Western blotting was used to detect the expression of UGT1A protein. The glucuronidation rate of N-hydroxy-PhIP was measured by high performance liquid chromatography (HPLC). The nuclear localization of transcription factor Nrf2 was observed by confocal laser microscopy. (1) Expression of UGT1A mRNA was observed in the Cac0-2 cells induced by SFN of the concentrations of 10 micromol/L approximately 35 micromol/L in a dose-independent manner (P < 0.05). Sulforaphane of the concentration of 25 micromol/L induced the UGT1A mRNA expression time-dependently. The levels of UGT1A1, UGT1A8, and UGT1A10 mRNA expression were significantly increased in the cells treated with 25 micromol/L sulforaphane compared to that in the controls (P = 0.006, P = 0.017, and P = 0.008 respectively). (2) The UGT1A protein band intensity increased significantly in the Coco-2 cells treated with sulforaphane of the concentrations 10 micromol/L approximately 30 micromol/L for 24 h in comparison with the control cells. (3) When the microsomes from the untreated Caco-2 cells were incubated with N-hydroxy-PhIP there was a minor HPLC peak at the expected retention time for N-hydroxy-PhIP-N2-glucuronide. This peak was dramatically increased in the sulforaphane-treated cells, suggesting higher activities of glucuronidation of N-hydroxy-PhIP. (4) Cytoplasmic labeling of NF-E2-related factor 2 (Nrf2), a transcription factor, with no nuclear staining was observed in the non-stimulated cells, whereas an intense nuclear labeling was observed in the sulforaphane-treated cells, indicating the induction of nuclear

Association between the low-dose irinotecan regimen-induced occurrence of grade 4 neutropenia and genetic variants of UGT1A1 in patients with gynecological cancers

PubMed Central

MORIYA, HIROYUKI; SAITO, KATSUHIKO; HELSBY, NUALA; SUGINO, SHIGEKAZU; YAMAKAGE, MICHIAKI; SAWAGUCHI, TAKERU; TAKASAKI, MASAHIKO; KATO, HIDENORI; KUROSAWA, NAHOKO

2014-01-01

The occurrence of severe neutropenia during treatment with irinotecan (CPT-11) is associated with the *6 and *28 alleles of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). However, the correlation between these variants and the occurrence of severe neutropenia in a low-dose CPT-11 regimen for the treatment of gynecological cancers has not been extensively studied. There are also no studies regarding the association between the 421C>A mutation in ATP-binding cassette sub-family G member 2 (ABCG2) and the occurrence of severe neutropenia in CPT-11-treated patients with gynecological cancers. The present study was designed to determine the factors associated with the occurrence of grade 4 neutropenia during chemotherapy for gynecological cancers with combinations of CPT-11 and cisplatin or mitomycin C. In total, 44 patients with gynecological cancer were enrolled in the study. The association between the absolute neutrophil count (ANC) nadir values, the total dose of CPT-11 and the genotypes of UGT1A1 or ABCG2 was studied. No correlation was observed between the ANC nadir values and the total dose of CPT-11. The ANC nadir values in the UGT1A1*6/*28 and *6/*6 groups were significantly lower compared with those in the *1/*1 group (P<0.01). Univariate analysis showed no association between the occurrence of grade 4 neutropenia and the ABCG2 421C>A mutation. Subsequent to narrowing the factors by univariate analysis, multivariate logistic regression analysis only detected significant correlations between the occurrence of grade 4 neutropenia and the UGT1A1*6/*6 and *6/*28 groups (P=0.029; odds ratio, 6.90; 95% confidence interval, 1.22–38.99). No associations were detected between the occurrence of grade 4 neutropenia and the heterozygous variant (*1/*6 or *1/*28) genotype, type of regimen or age. In conclusion, the UGT1A1*6/*28 and *6/*6 genotypes were found to be associated with the occurrence of severe neutropenia in the low-dose CPT-11 regimen for

Effects of ABCB1, ABCC2, UGT2B7 and HNF4α genetic polymorphisms on oxcarbazepine concentrations and therapeutic efficacy in patients with epilepsy.

PubMed

Shen, Chunhong; Zhang, Bijun; Liu, Zhirong; Tang, Yelei; Zhang, Yinxi; Wang, Shan; Guo, Yi; Ding, Yao; Wang, Shuang; Ding, Meiping

2017-10-01

The aim of the study is to investigate the effects of ABCB1, ABCC2, UGT2B7 and HNF4α genetic polymorphisms on plasma oxcarbazepine (OXC) concentrations and therapeutic efficacy in Han Chinese patients with epilepsy. We recruited 116 Han Chinese patients with epilepsy who were receiving OXC monotherapy. Blood samples were taken and OXC levels were measured. The polymorphisms of ABCB1 rs1045642, ABCC2 rs2273697, UGT2B7 rs7439366, and HNF4α rs2071197 were determined. The therapeutic efficacy of OXC at the 1-year time-point was assessed. Data analysis was performed using IBM SPSS Statistics 22.0. The genetic polymorphism of ABCB1 rs1045642 was found to be associated with normalized OXC concentration and therapeutic efficacy in patients with epilepsy (P<0.05). As for UGT2B7 rs7439366, the allele polymorphism exhibited a correlation with treatment outcome, but not OXC concentration. The polymorphisms of ABCC2 rs2273697 and HNF4α rs2071197 was not associated with OXC concentrations and therapeutic efficacy. These results suggested that ABCB1 rs1045642 and UGT2B7 rs7439366 may affect OXC pharmacokinetics and therapeutic efficacy in Han Chinese patients with epilepsy. However, further studies in larger populations and other ethnic groups are required. Copyright © 2017 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

21 CFR 862.1535 - Ornithine carbamyl transferase test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine...

21 CFR 862.1535 - Ornithine carbamyl transferase test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine...

Reversible grade 4 hyperbilirubinemia in a patient with UGT1A1 7/7 genotype treated with irinotecan and cetuximab.

PubMed

Gupta, Bhavna; LeVea, Charles; Litwin, Alan; Fakih, Marwan G

2007-03-01

Irinotecan-induced gastrointestinal toxicities are common and typically present in the form of diarrhea or nausea and vomiting. However, severe hyperbilirubinemia (grade 3/4) has not been previously reported in association with this chemotherapeutic agent. We report a case of prolonged grade 4 hyperbilirubinemia after a single dose of irinotecan at 125 mg/m(2). This severe toxicity was attributed to a UGT1A1 7/7 genotype and resolved to grade 2 after 8 weeks of supportive care. This case outlines the possibility of severe hepatic toxicity with moderate doses of irinotecan in patients with a UGT1A1 7/7 genotype. Despite the severity and prolonged duration of the associated irinotecan-induced hepatic toxicity, the management of similar cases should focus on intensive supportive measures because the toxicity is likely to resolve eventually.

Polymorphisms of glutathione S-transferase Mu 1, glutathione S-transferase theta 1 and glutathione S-transferase Pi 1 genes in Hodgkin's lymphoma susceptibility and progression.

PubMed

Lourenço, Gustavo J; Néri, Iramaia A; Sforni, Vitor C S; Kameo, Rodolfo; Lorand-Metze, Irene; Lima, Carmen S P

2009-06-01

We tested in this study whether the polymorphisms of the glutathione S-transferase Mu1 (GSTM1), glutathione S-transferase Theta 1 (GSTT1) and glutathione S-transferase Pi 1 (GSTP1), involved in metabolism of chemical agents, cell proliferation and cell survival, alter the risk for Hodgkin lymphoma (HL). Genomic DNA from 110 consecutive patients with HL and 226 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. Similar frequencies of the GSTM1 and GSTT1 genotypes were seen in patients and controls. In contrast, the frequency of the GSTP1 wild genotype (59.1%versus 36.3%, P = 0.004) was higher in patients than in controls. Individuals with the wild genotype had a 2.68 (95%CI: 1.38-5.21)-fold increased risk for the disease than others. An excess of the GSTP1 wild genotype was also observed in patients with tumors of stages III + IV when compared with those with tumors of stages I + II (39.1%versus 20.0%, P = 0.03). These results suggest that the wild allele of the GSTP1 gene is linked to an increased risk and high aggressiveness of the HL in our cases but they should be confirmed by further studies with larger cohorts of patients and controls.

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

PubMed

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

2016-12-01

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

METABOLISM AND DISPOSITION OF ACETAMINOPHEN: RECENT ADVANCES IN RELATION TO HEPATOTOXICITY AND DIAGNOSIS

PubMed Central

McGill, Mitchell R.; Jaeschke, Hartmut

2013-01-01

Acetaminophen (APAP) is one of the most widely used drugs. Though safe at therapeutic doses, overdose causes mitochondrial dysfunction and centrilobular necrosis in the liver. The first studies of APAP metabolism and activation were published more than forty years ago. Most of the drug is eliminated by glucuronidation and sulfation. These reactions are catalyzed by UDP-glucuronosyltransferases (UGT1A1 and 1A6) and sulfotransferases (SULT1A1, 1A3/4, and 1E1), respectively. However, some is converted by CYP2E1 and other cytochrome P450 enzymes to a reactive intermediate that can bind to sulfhydryl groups. The metabolite can deplete liver glutathione (GSH) and modify cellular proteins. GSH binding occurs spontaneously, but may also involve GSH-S-transferases. Protein binding leads to oxidative stress and mitochondrial damage. The glucuronide, sulfate, and GSH conjugates are excreted by transporters in the canalicular (Mrp2 and Bcrp) and basolateral (Mrp3 and Mrp4) hepatocyte membranes. Conditions that interfere with metabolism and metabolic activation can alter the hepatotoxicity of the drug. Recent data providing novel insights into these processes, particularly in humans, are reviewed in the context of earlier work, and the effects of altered metabolism and reactive metabolite formation are discussed. Recent advances in the diagnostic use of serum adducts are covered. PMID:23462933

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

PubMed

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

2003-08-01

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

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

PubMed

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

2016-08-01

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

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

PubMed

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

2000-05-01

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

Pharmacokinetics of bisphenol A in neonatal and adult rhesus monkeys

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

Doerge, Daniel R., E-mail: daniel.doerge@fda.hhs.go; Twaddle, Nathan C.; Woodling, Kellie A.

Bisphenol A (BPA) is a high-production volume industrial chemical used in the manufacture of polycarbonate plastic products and epoxy resin-based food can liners. The presence of BPA in urine of > 90% of Americans aged 6-60 is controversial because of the potential for endocrine disruption, particularly during perinatal development, as suggested by in vitro, experimental animal, and epidemiological studies. The current study used LC/MS/MS to measure serum pharmacokinetics of aglycone (active) and conjugated (inactive) BPA in adult and neonatal rhesus monkeys by oral (PND 5, 35, 70) and intravenous injection (PND 77) routes using d6-BPA to avoid sample contamination. Themore » concentration-time profiles observed in adult monkeys following oral administration of 100 {mu}g/kg bw were remarkably similar to those previously reported in human volunteers given a similar dose; moreover, minimal pharmacokinetic differences were observed between neonatal and adult monkeys for the receptor-active aglycone form of BPA. Circulating concentrations of BPA aglycone were quite low following oral administration (< 1% of total), which reflects the redundancy of active UDP-glucuronosyl transferase isoforms in both gut and liver. No age-related changes were seen in internal exposure metrics for aglycone BPA in monkeys, a result clearly different from developing rats where significant inverse age-related changes, based on immaturity of Phase II metabolism and renal excretion, were recently reported. These observations imply that any toxicological effect observed in rats from early postnatal exposures to BPA could over-predict those possible in primates of the same age, based on significantly higher internal exposures and overall immaturity at birth.« less

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

PubMed

Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

2003-09-01

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

THE EFFECT OF BACULOVIRUS INFECTION ON ECDYSTEROID TITER IN GYPSY MOTH LARVAE (LYMANTRIA DISPAR).

EPA Science Inventory

Insect baculovirus carries a gene refered to as egt. This gene encodes an enzyme known as ecdysteroid UDP-glucosyl transferase which catalyzes the sugar conjugation of ecdysteroids. Using a gypsy moth embryonic cell line EGT activity of Lymantria dispar nuclear polyhedrosis virus...

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

PubMed Central

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

2003-01-01

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

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

PubMed

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

2003-11-01

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

Differences in response of glucuronide and glutathione conjugating enzymes to aflatoxin B/sub 1/ and N-acetylaminofluorene in underfed rats

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

Rajpurohit, R.; Krishnaswamy, K.

Changes in the hepatic drug/xenobiotic-metabolizing enzymes in underfed rats exposed to aflatoxin B/sub 1/ and N-acetylaminofluorene were investigated. Neither carcinogen, fed at the level of 10 ..mu..g and 0.667 mg per 100 g body weight, respectively, over a period of 3 wk, had any significant influence on cytochrome P-450 and aryl hydrocarbon hydroxylase in the undernourished rats. Significantly low activities of UDP-glucuronyltransferase and glutathione S-transferase were observed in food-restricted animals fed on aflatoxin B/sub 1/. N-acetylaminofluorene, on the other hand stimulated both the enzyme activities in the underfed group, to as much observed in the respective well-fed treated group. UDP-Glucuronyltransferasemore » and glutathione S-transferase in undernutrition seem to respond differently to aflatoxin B/sub 1/ and N-acetylaminofluorene. Further studies are needed to assess the possible consequences of such alterations.« less

Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

PubMed

Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

2005-01-01

The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

PubMed Central

Danielson, U H; Esterbauer, H; Mannervik, B

1987-01-01

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism. PMID:3426557

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

PubMed

Timson, David J; Lindert, Steffen

2013-09-10

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

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

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

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle

2015-10-15

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

[Interactions of food and drug metabolism].

PubMed

Delzenne, N M; Verbeeck, R K

2001-01-01

The nutritional state, and/or the ingestion of specific nutrients, is/are able to modify drug disposition, by interfering with drug absorption, distribution, storage, and metabolism. Recent data report that nutrients interfere with drug metabolism either by modifying key enzymes of phase I (cytochromeP450 dependent mixed function oxidase) and II (glucuronosyl, sulfonyl- ... transferases), or by modulating coenzymes availability (NADPH, UDPglucuronic acid...). Food components involved in drug metabolism modifications are either macro-nutrients (carbohydrates, lipids, proteins, ethanol), micronutriments (vitamins, minerals), or phytochemicals. Drug-nutrients interactions may be beneficials, and thus could constitute, i.e. a way to improve drug therapeutic index, or generate adverse effects.

Prognostic Significance of ESR1 Amplification and ESR1 PvuII, CYP2C19*2, UGT2B15*2 Polymorphisms in Breast Cancer Patients

PubMed Central

Markiewicz, Aleksandra; Wełnicka-Jaśkiewicz, Marzena; Skokowski, Jarosław; Jaśkiewicz, Janusz; Szade, Jolanta; Jassem, Jacek; Żaczek, Anna J.

2013-01-01

Introduction Amplification of the ESR1 gene, coding for estrogen receptor alpha, was shown to predict responsiveness to tamoxifen, however its prognostic impact in breast cancer patients has not been thoroughly investigated. Other factors that could contribute to responsiveness to tamoxifen treatment are polymorphisms in ESR1 gene and genes involved in tamoxifen metabolism. The aim of this study was to assess the prognostic role of ESR1 gene dosage in a consecutive group of breast cancer patients and to correlate this feature with clinico-pathological factors. Additionally, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphisms were analyzed in the tamoxifen-treated subgroup of patients. Materials and Methods Primary tumor samples from 281 stage I-III consecutive breast cancer patients were analyzed for ESR1 gene dosage using real-time PCR with locked nucleic acids hydrolysis probes. In the tamoxifen-treated subgroup of patients, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphism in leukocytes genomic DNA were analyzed. Results were correlated with clinico-pathological factors and with disease-free survival (DFS) and overall survival (OS). Results ESR1 amplification (with a cut-off level of 2.0) was found in 12% of the entire group of breast cancer patients, and in 18% of the ER-negative subgroup. This feature was associated with decreased DFS both in the entire group (P=0.007) and in the ER-negative subgroup (P=0.03), but not in the tamoxifen-treated patients. Patients with ESR1 PvuII wt/wt genotype and at least one UGT2B15 wt allele had a worse DFS (P=0.03) and showed a trend towards decreased Os (P=0.08) in comparison to patients with ESR1 PvuII wt/vt or vt/vt genotype and UGT2B15 *2/*2 genotype. Conclusions ESR1 amplification can occur in ER-negative tumors and may carry poor prognosis. In the tamoxifen-treated subgroup, poor prognosis was related to the combined presence of ESR1 PvuII wt/wt and UGT2B15wt/wt or wt/*2 genotype. PMID:23951298

Prognostic significance of ESR1 amplification and ESR1 PvuII, CYP2C19*2, UGT2B15*2 polymorphisms in breast cancer patients.

PubMed

Markiewicz, Aleksandra; Wełnicka-Jaśkiewicz, Marzena; Skokowski, Jarosław; Jaśkiewicz, Janusz; Szade, Jolanta; Jassem, Jacek; Zaczek, Anna J

2013-01-01

Amplification of the ESR1 gene, coding for estrogen receptor alpha, was shown to predict responsiveness to tamoxifen, however its prognostic impact in breast cancer patients has not been thoroughly investigated. Other factors that could contribute to responsiveness to tamoxifen treatment are polymorphisms in ESR1 gene and genes involved in tamoxifen metabolism. The aim of this study was to assess the prognostic role of ESR1 gene dosage in a consecutive group of breast cancer patients and to correlate this feature with clinico-pathological factors. Additionally, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphisms were analyzed in the tamoxifen-treated subgroup of patients. Primary tumor samples from 281 stage I-III consecutive breast cancer patients were analyzed for ESR1 gene dosage using real-time PCR with locked nucleic acids hydrolysis probes. In the tamoxifen-treated subgroup of patients, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphism in leukocytes genomic DNA were analyzed. Results were correlated with clinico-pathological factors and with disease-free survival (DFS) and overall survival (OS). ESR1 amplification (with a cut-off level of 2.0) was found in 12% of the entire group of breast cancer patients, and in 18% of the ER-negative subgroup. This feature was associated with decreased DFS both in the entire group (P=0.007) and in the ER-negative subgroup (P=0.03), but not in the tamoxifen-treated patients. Patients with ESR1 PvuII wt/wt genotype and at least one UGT2B15 wt allele had a worse DFS (P=0.03) and showed a trend towards decreased Os (P=0.08) in comparison to patients with ESR1 PvuII wt/vt or vt/vt genotype and UGT2B15 *2/*2 genotype. ESR1 amplification can occur in ER-negative tumors and may carry poor prognosis. In the tamoxifen-treated subgroup, poor prognosis was related to the combined presence of ESR1 PvuII wt/wt and UGT2B15wt/wt or wt/*2 genotype.

Glucuronidation and its impact on the bioactivity of [6]-shogaol.

PubMed

Wang, Pei; Zhao, Yantao; Zhu, Yingdong; Sang, Shengmin

2017-09-01

-shogaol (6S) from ginger has been reported to have diverse bioactivities and can be widely metabolized in animals and humans; however, the impact of glucuronidation on its bioactivity is still largely unknown. This study investigates the glucuronidation of 6S and its effect on cell cytotoxicity and Nrf2-inducing activities of 6S. The glucuronidated metabolite of 6S, 4-O-monoglucuronide 6S (6S-G), was synthesized and characterized for the first time. Glucuronidation of 6S in humans was studied using microsomes of the liver and intestine and recombinant UDP-glucuronosyltransferase (UGTs). The kinetics of 6S glucuronidation by human liver and intestinal microsomes followed the substrate inhibition kinetics model. The intrinsic glucuronidation clearance (CL int ) of 6S in human liver microsomes was higher than that in human intestine microsomes. Among the recombinant UGTs examined, UGT1A1, 1A3, 1A6, 1A8, 1A10, 2B7, 2B15, and 2B17 exhibited glucuronidation activity toward 6S, with UGT2B7 being the most potent one. Compared with 6S, the glucuronidation of 6S largely eliminated its cell cytotoxicity against human colon cancer cell lines HT-116 and HT-29, and its Nrf2-inducing activity. The findings from current study provide foundations for understanding the role of glucuronidation in biotransformation and biological activities of 6S. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

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

2006-01-01

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

Base substitution mutations in uridinediphosphate-dependent glycosyltransferase 76G1 gene of Stevia rebaudiana causes the low levels of rebaudioside A: mutations in UGT76G1, a key gene of steviol glycosides synthesis.

PubMed

Yang, Yong-Heng; Huang, Su-Zhen; Han, Yu-Lin; Yuan, Hai-Yan; Gu, Chun-Sun; Zhao, Yan-Hai

2014-07-01

Steviol glycosides, extracted from the leaves of Stevia rebaudiana (Bert) Bertoni, are calorie-free sugar substitute of natural origin with intensely sweet (Boileau et al., 2012). Stevioside and rebaudioside A are the two main kinds of the diterpenic glycosides. We analyzed the concentration of stevioside and rebaudioside A in Stevia leaves of about 500 samples (hybrid progenies) and discovered a mutation plant "Z05" with very low levels of rebaudioside A. Because UGT76G1, a uridinediphosphate-dependent glycosyltransferases, is responsible for the conversion from stevioside to rebaudioside A (Richman et al., 2005), so mutation identification was done by sequencing the candidate gene, UGT76G1. In this study molecular analysis of two strains revealed a heterozygotic nonsense mutation of c.389T > G (p.L121X) in UGT76G1. Meanwhile, we found some amino acid substitutions significant change the protein structure. And the difference of enzyme activity between two strains proved the lack of functionality of UGT76G1 of the mutation "Z05". So the nonsense mutation and amino acid substitution mutation resulted in the low levels of rebaudioside A. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Natural variations in xenobiotic-metabolizing enzymes: developing tools for coral monitoring

NASA Astrophysics Data System (ADS)

Rougée, L. R. A.; Richmond, R. H.; Collier, A. C.

2014-06-01

The continued deterioration of coral reefs worldwide demonstrates the need to develop diagnostic tools for corals that go beyond general ecological monitoring and can identify specific stressors at sublethal levels. Cellular diagnostics present an approach to defining indicators (biomarkers) that have the potential to reflect the impact of stress at the cellular level, allowing for the detection of intracellular changes in corals prior to outright mortality. Detoxification enzymes, which may be readily induced or inhibited by environmental stressors, present such a set of indicators. However, in order to apply these diagnostic tools for the detection of stress, a detailed understanding of their normal, homeostatic levels within healthy corals must first be established. Herein, we present molecular and biochemical evidence for the expression and activity of major Phase I detoxification enzymes cytochrome P450 (CYP450), CYP2E1, and CYP450 reductase, as well as the Phase II enzymes UDP, glucuronosyltransferase (UGT), β-glucuronidase, glutathione- S-transferase (GST), and arylsulfatase C (ASC) in the coral Pocillopora damicornis. Additionally, we characterized enzyme expression and activity variations over a reproductive cycle within a coral's life history to determine natural endogenous changes devoid of stress exposure. Significant changes in enzyme activity over the coral's natural lunar reproductive cycle were observed for CYP2E1 and CYP450 reductase as well as UGT and GST, while β-glucuronidase and ASC did not fluctuate significantly. The data represent a baseline description of `health' for the expression and activity of these enzymes that can be used toward understanding the impact of environmental stressors on corals. Such knowledge can be applied to address causes of coral reef ecosystem decline and to monitor effectiveness of mitigation strategies. Achieving a better understanding of cause-and-effect relationships between putative stressors and biological

Spectrofluorimetric assay method for glutathione and glutathione transferase using monobromobimane.

PubMed

Yakubu, S I; Yakasai, I A; Musa, A

2011-06-01

The primary role of glutathione transferase is to defend an organism from toxicities through catalyzing the reaction of glutathione (GSH) with potentially toxic compounds or metabolites to their chemically and biologically inert conjugates. The objective of the study was to develop a simple and sensitive spectrofluorimetric assay method for glutathione transferase using monobromobimane (MBB), a non fluorescent compound with electrophilic site. MBB slowly reacted with glutathione to form fluorescent glutathione conjugate and that the reaction was catalysed by glutathione transferase. Both non-enzymatic and enzymatic reaction products of MBB, in presence of GSH in phosphate buffer (pH 6.5), were measured by following increase of fluorescence at wavelength of 475nm. For validation of the assay method, the kinetic parameters such as the apparent Michaelis-Mente constants and maximum rates of conjugate formation as well as the specific activity of rat hepatic glutathione transferase were determined. The method was found to be sensitive, thus, applied to measure glutathione contents of crude preparation of rat hepatic cytosol fraction.

Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits.

PubMed Central

Singh, S V; Partridge, C A; Awasthi, Y C

1984-01-01

Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively. Images Fig. 1. Fig. 5. PMID:6433888

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

PubMed

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

2003-07-01

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

[Regulation of terpene metabolism]. Annual progress report, March 15, 1988--March 14, 1989

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

Croteau, R.

1989-12-31

Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

[Regulation of terpene metabolism]. [Mentha piperita, Mentha spicata

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

Croteau, R.

1989-01-01

Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

Urinary Elimination of Bile Acid Glucuronides under Severe Cholestatic Situations: Contribution of Hepatic and Renal Glucuronidation Reactions.

PubMed

Perreault, Martin; Wunsch, Ewa; Białek, Andrzej; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Poirier, Guy G; Milkiewicz, Piotr; Barbier, Olivier

2018-01-01

Biliary obstruction, a severe cholestatic complication, causes accumulation of toxic bile acids (BAs) in liver cells. Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic BAs. Using liquid chromatography coupled to tandem mass spectrometry, 11 BA glucuronide (-G) species were quantified in prebiliary and postbiliary stenting serum and urine samples from 17 patients with biliary obstruction. Stenting caused glucuronide- and fluid-specific changes in BA-G levels and BA-G/BA metabolic ratios. In vitro glucuronidation assays with human liver and kidney microsomes revealed that even if renal enzymes generally displayed lower K M values, the two tissues shared similar glucuronidation capacities for BAs. By contrast, major differences between the two tissues were observed when four human BA-conjugating UGTs 1A3, 1A4, 2B4, and 2B7 were analyzed for mRNA and protein levels. Notably, the BA-24G producing UGT1A3 enzyme, abundant in the liver, was not detected in kidney microsomes. In conclusion, the circulating and urinary BA-G profiles are hugely impacted under severe cholestasis. The similar BA-glucuronidating abilities of hepatic and renal extracts suggest that both the liver and kidney may contribute to the urine BA-G pool.

Urinary Elimination of Bile Acid Glucuronides under Severe Cholestatic Situations: Contribution of Hepatic and Renal Glucuronidation Reactions

PubMed Central

Perreault, Martin; Białek, Andrzej; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Poirier, Guy G.

2018-01-01

Biliary obstruction, a severe cholestatic complication, causes accumulation of toxic bile acids (BAs) in liver cells. Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic BAs. Using liquid chromatography coupled to tandem mass spectrometry, 11 BA glucuronide (-G) species were quantified in prebiliary and postbiliary stenting serum and urine samples from 17 patients with biliary obstruction. Stenting caused glucuronide- and fluid-specific changes in BA-G levels and BA-G/BA metabolic ratios. In vitro glucuronidation assays with human liver and kidney microsomes revealed that even if renal enzymes generally displayed lower KM values, the two tissues shared similar glucuronidation capacities for BAs. By contrast, major differences between the two tissues were observed when four human BA-conjugating UGTs 1A3, 1A4, 2B4, and 2B7 were analyzed for mRNA and protein levels. Notably, the BA-24G producing UGT1A3 enzyme, abundant in the liver, was not detected in kidney microsomes. In conclusion, the circulating and urinary BA-G profiles are hugely impacted under severe cholestasis. The similar BA-glucuronidating abilities of hepatic and renal extracts suggest that both the liver and kidney may contribute to the urine BA-G pool. PMID:29850459

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

PubMed

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

2004-11-01

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

Improving oral bioavailability of resveratrol by a UDP-glucuronosyltransferase inhibitory excipient-based self-microemulsion.

PubMed

Yang, Fei-Fei; Zhou, Jing; Hu, Xiao; Cong, Zhao-Qing; Liu, Chun-Yu; Pan, Rui-Le; Chang, Qi; Liu, Xin-Min; Liao, Yong-Hong

2018-03-01

Self-microemulsifying (SME) drug delivery system has been developed to increase oral bioavailabilities, and inhibitory excipients are capable of improving oral bioavailability by inhibiting enzyme mediated intestinal metabolism. However, the potential of enzyme inhibitory excipients containing SME in boosting resveratrol bioavailability remains largely uninvestigated. In this study, we set out to prepare SME-1 with UGT inhibitory excipients (excipients without inhibitory activities named SME-2 as control) to increase the bioavailability of RES by inhibiting intestinal metabolism. Results demonstrated that similar physicochemical properties such as size, polydistribution index and in vitro release, cellular uptake and permeability in Caco-2 cells as well as in vivo lymphatic distribution between inhibitory SME-1 and non-inhibitory SME-2 were observed. In vivo study demonstrated that the molar ratios of RES-G/RES were 7.25±0.48 and 5.06±2.42 for free drug and SME-2, respectively, and the molar ratio decreased to 0.36±0.10 in SME-1 group. Pharmacokinetic study confirmed that the inhibitory excipients containing SME demonstrated potential in increasing bioavailability of RES from 6.5% for the free RES and 12.9% for SME-2 to 76.1% in SME-1 through modulating the glucuronidation by UGT inhibitory excipients. Copyright © 2018 Elsevier B.V. All rights reserved.

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

DOEpatents

Jarvis, Eric E.; Roessler, Paul G.

1999-01-01

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

Molecular and biochemical characterization of tomato farnesyl-protein transferase.

PubMed

Schmitt, D; Callan, K; Gruissem, W

1996-10-01

The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity. In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified. We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties. Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities. Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro. LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development. Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1. Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth.

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

PubMed Central

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

2003-01-01

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

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

PubMed

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

2014-01-17

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

UGT1A1 (TA)n genotyping in sickle-cell disease: high resolution melting (HRM) curve analysis or direct sequencing, what is the best way?

PubMed

Thomas, Vincent; Mazard, Blandine; Garcia, Caroline; Lacan, Philippe; Gagnieu, Marie-Claude; Joly, Philippe

2013-09-23

Minucci et al. have proposed in 2010 a rapid, simple and cost-effective HRM method on the LightCycler 480® apparatus (Roche) for the determination of the 6/6, 6/7 and 7/7 genotypes of the (TA)n UGT1A1 promoter polymorphism. However, they have not studied the n=5 and n=8 alleles which can be quite frequent in sickle-cell disease patients. The aim of our study was to test this HRM protocol to all the 10 possible (TA)n UGT1A1 genotypes (i.e. 5/5, 5/6, 5/7, 5/8, 6/6, 6/7, 6/8, 7/7, 7/8 and 8/8) by using our SCD cohort of patients. All genotypes could be unambiguously identified except 6/7 and 6/8 which give a similar HRM profile. For those two genotypes, the differentiation necessitates either a direct Sanger sequencing or a second PCR protocol followed by a 3% agarose gel migration. For the (TA)n UGT1A1 promoter genotyping of African patients, each lab has to wonder what is the best way between (i) direct Sanger sequencing of all patients and (ii) HRM protocol for all patients followed by a complementary analysis to differentiate the 6/7 and 6/8 genotypes. © 2013. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2012-03-01

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

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

DOEpatents

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

1999-07-27

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

Xenobiotic metabolizing enzyme activities in cells used for testing skin sensitization in vitro.

PubMed

Fabian, E; Vogel, D; Blatz, V; Ramirez, T; Kolle, S; Eltze, T; van Ravenzwaay, B; Oesch, F; Landsiedel, R

2013-09-01

For ethical and regulatory reasons, in vitro tests for scoring potential toxicities of cosmetics are essential. A test strategy for investigating potential skin sensitization using two human keratinocytic and two human dendritic cell lines has been developed (Mehling et al. Arch Toxicol 86:1273–1295, 2012). Since prohaptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (XME) activities in these cell lines is of high interest. In this study, XME activity assays, monitoring metabolite or cofactor, showed the following: all three passages of keratinocytic (KeratinoSens® and LuSens) and dendritic (U937 und THP-1) cells displayed N-acetyltransferase 1 (NAT1) activities (about 6–60 nmol/min/mg S9-protein for acetylation of para-aminobenzoic acid). This is relevant since reactive species of many cosmetics are metabolically controlled by cutaneous NAT1. Esterase activities of about 1–4 nmol fluorescein diacetate/min/mg S9-protein were observed in all passages of investigated keratinocytic and about 1 nmol fluorescein diacetate/min/mg S9-protein in dendritic cell lines. This is also of practical relevance since many esters and amides are detoxified and others activated by cutaneous esterases. In both keratinocytic cell lines, activities of aldehyde dehydrogenase (ALDH) were observed (5–17 nmol product/min/mg cytosolic protein). ALDH is relevant for the detoxication of reactive aldehydes. Activities of several other XME were below detection, namely the investigated cytochrome P450-dependent alkylresorufin O-dealkylases 7-ethylresorufin O-deethylase, 7-benzylresorufin O-debenzylase and 7-pentylresorufin O-depentylase (while NADPH cytochrome c reductase activities were much above the limit of quantification), the flavin-containing monooxygenase, the alcohol dehydrogenase as well as the UDP glucuronosyl transferase activities.

Raphanus sativus cv. Sango Sprout Juice Decreases Diet-Induced Obesity in Sprague Dawley Rats and Ameliorates Related Disorders.

PubMed

Vivarelli, Fabio; Canistro, Donatella; Sapone, Andrea; De Nicola, Gina Rosalinda; Babot Marquillas, Clara; Iori, Renato; Antonazzo, Ippazio Cosimo; Gentilini, Fabio; Paolini, Moreno

2016-01-01

Obesity is recognized as a leading global health problem, correlated with an increased risk for several chronic diseases. One strategy for weight control management includes the use of vegetables rich in bioactive compounds to counteract weight gain, improve the antioxidant status and stimulate lipid catabolism. The aim of this study was to investigate the role of Raphanus sativus Sango sprout juice (SSJ), a Brassica extraordinarily rich in anthocyanins (AC) and isothiocyanates (ITCs), in a non-genetic model of obesity (high fat diet-HFD induced). Control groups were fed with HFD or regular diet (RD). After a 10-week period, animals were assigned to experimental units and treated by gavage for 28 days as follows: HFD and RD control groups (rats fed HFD or RD and treated with vehicle only) and HFD-treated groups (rats fed HFD and treated with 15, 75 or 150 mg/kg b.w. of SSJ). Body weight and food consumption were recorded and serum lipid profile was measured (total cholesterol, triglycerides, and non-esterified fatty acids). Hepatic phase-I, phase-II as well as antioxidant enzymatic activities were assessed. SSJ lowered total cholesterol level, food intake and liver weight compared with HFD rodents. SSJ at medium dose proved effective in reducing body-weight (~19 g reduction). SSJ was effective in up-regulating the antioxidant enzymes catalase, quinone reductase, oxidised glutathione reductase and superoxide dismutase, which reached or exceeded RD levels, as well as the phase II metabolic enzyme UDP-glucuronosyl transferase (up to about 43%). HFD up-regulated almost every cytochrome P450 isoform tested, and a mild down-regulation to baseline was observed after SSJ intervention. This work reveals, for the first time, the antioxidant, hypolipidemic and antiobesity potential of SSJ, suggesting its use as an efficient new functional food/nutraceutical product.

Raphanus sativus cv. Sango Sprout Juice Decreases Diet-Induced Obesity in Sprague Dawley Rats and Ameliorates Related Disorders

PubMed Central

Sapone, Andrea; De Nicola, Gina Rosalinda; Babot Marquillas, Clara; Iori, Renato; Antonazzo, Ippazio Cosimo; Gentilini, Fabio; Paolini, Moreno

2016-01-01

Background Obesity is recognized as a leading global health problem, correlated with an increased risk for several chronic diseases. One strategy for weight control management includes the use of vegetables rich in bioactive compounds to counteract weight gain, improve the antioxidant status and stimulate lipid catabolism. Aim of the Study The aim of this study was to investigate the role of Raphanus sativus Sango sprout juice (SSJ), a Brassica extraordinarily rich in anthocyanins (AC) and isothiocyanates (ITCs), in a non-genetic model of obesity (high fat diet-HFD induced). Methods Control groups were fed with HFD or regular diet (RD). After a 10-week period, animals were assigned to experimental units and treated by gavage for 28 days as follows: HFD and RD control groups (rats fed HFD or RD and treated with vehicle only) and HFD-treated groups (rats fed HFD and treated with 15, 75 or 150 mg/kg b.w. of SSJ). Body weight and food consumption were recorded and serum lipid profile was measured (total cholesterol, triglycerides, and non-esterified fatty acids). Hepatic phase-I, phase-II as well as antioxidant enzymatic activities were assessed. Results SSJ lowered total cholesterol level, food intake and liver weight compared with HFD rodents. SSJ at medium dose proved effective in reducing body-weight (~19 g reduction). SSJ was effective in up-regulating the antioxidant enzymes catalase, NAD(P)H:quinone reductase, oxidised glutathione reductase and superoxide dismutase, which reached or exceeded RD levels, as well as the phase II metabolic enzyme UDP-glucuronosyl transferase (up to about 43%). HFD up-regulated almost every cytochrome P450 isoform tested, and a mild down-regulation to baseline was observed after SSJ intervention. Conclusion This work reveals, for the first time, the antioxidant, hypolipidemic and antiobesity potential of SSJ, suggesting its use as an efficient new functional food/nutraceutical product. PMID:26987061

Removal of glucuronic acid from xylan is a strategy to improve the conversion of plant biomass to sugars for bioenergy.

PubMed

Lyczakowski, Jan J; Wicher, Krzysztof B; Terrett, Oliver M; Faria-Blanc, Nuno; Yu, Xiaolan; Brown, David; Krogh, Kristian B R M; Dupree, Paul; Busse-Wicher, Marta

2017-01-01

Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised. Here, we show using Arabidopsis gux mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of gux biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce ( Picea glauca ) gene, PgGUX , encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis gux mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation. Removal of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant

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

PubMed

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

2018-04-04

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

Albumin's Influence on Carprofen Enantiomers-Hymecromone Interaction.

PubMed

Tang, Mingjie; Guo, Yanjie; Gao, Youshui; Tang, Chao; Dang, Xiaoqian; Zhou, Zubin; Sun, Yuqiang; Wang, Kunzheng

2016-03-01

Hymecromone is an important coumarin drug, and carprofen is one of the most important nonsteroidal antiinflammatory drugs (NSAIDs). The present study aims to determine the influence of bovine serum albumin (BSA) on the carprofen-hymecromone interaction. The inhibition of carprofen enantiomers on the UDP-glucuronosyltransferase (UGT) 2B7-catalyzed glucuronidation of hymecromone was investigated in the UGTs incubation system with and without BSA. The inhibition capability of increased by 20% (P < 0.001) of (R)-carprofen after the addition of 0.5% BSA in the incubation mixture. In contrast, no significant difference was observed for the inhibition of (S)-carprofen on UGT2B7 activity in the absence or presence of 0.5% BSA in the incubation system. The Lineweaver-Burk plot showed that the intersection point was located in the vertical axis, indicating the competitive inhibition of (R)-carprofen on UGT2B7 in the incubation system with BSA, which is consistent with the inhibition kinetic type of (R)-carprofen on UGT2B7 in the incubation system without BSA. Furthermore, the second plot using the slopes from the Lineweaver-Burk versus the concentrations of (R)-carprofen showed that the fitting equation was y=39.997x+50. Using this equation, the inhibition kinetic parameter was calculated to be 1.3 μM. For (S)-carprofen, the intersection point was located in the horizontal axis in the Lineweaver-Burk plot for the incubation system with BSA, indicating the noncompetitive inhibition of (S)-carprofen on the activity of UGT2B7. The fitting plot of the second plot was y=24.6x+180, and the inhibition kinetic parameter was 7.3 μM. In conclusion, the present study gives a short summary of BSA's influence on the carprofen enantiomers-hymecromone interaction, which will guide the clinical application of carprofen and hymecromone. © 2015 Wiley Periodicals, Inc.

Phenobarbital Induction and Chemical Synergism Demonstrate the Role of UDP-Glucuronosyltransferases in Detoxification of Naphthalophos by Haemonchus contortus Larvae

PubMed Central

Ruffell, Angela P.; Ingham, Aaron B.

2014-01-01

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. PMID:25288079

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

PubMed

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.

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

PubMed

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

2014-01-01

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

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

PubMed

Miszkiel, Aleksandra; Wojciechowski, Marek

2017-11-01

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

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

PubMed

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

2014-05-01

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

The UDP-glucose dehydrogenase of Escherichia coli K-12 displays substrate inhibition by NAD that is relieved by nucleotide triphosphates.

PubMed

Mainprize, Iain L; Bean, Jordan D; Bouwman, Catrien; Kimber, Matthew S; Whitfield, Chris

2013-08-09

UDP-glucose dehydrogenase (Ugd) generates UDP-glucuronic acid, an important precursor for the production of many hexuronic acid-containing bacterial surface glycostructures. In Escherichia coli K-12, Ugd is important for biosynthesis of the environmentally regulated exopolysaccharide known as colanic acid, whereas in other E. coli isolates, the same enzyme is required for production of the constitutive group 1 capsular polysaccharides, which act as virulence determinants. Recent studies have implicated tyrosine phosphorylation in the activation of Ugd from E. coli K-12, although it is not known if this is a feature shared by bacterial Ugd proteins. The activities of Ugd from E. coli K-12 and from the group 1 capsule prototype (serotype K30) were compared. Surprisingly, for both enzymes, site-directed Tyr → Phe mutants affecting the previously proposed phosphorylation site retained similar kinetic properties to the wild-type protein. Purified Ugd from E. coli K-12 had significant levels of NAD substrate inhibition, which could be alleviated by the addition of ATP and several other nucleotide triphosphates. Mutations in a previously identified UDP-glucuronic acid allosteric binding site decreased the binding affinity of the nucleotide triphosphate. Ugd from E. coli serotype K30 was not inhibited by NAD, but its activity still increased in the presence of ATP.

[Human drug metabolizing enzymes. II. Conjugation enzymes].

PubMed

Vereczkey, L; Jemnitz, K; Gregus, Z

1998-09-01

In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

GLUTATHIONE S-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE

EPA Science Inventory

GLUTATHIONE s-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE. M K Ross1 and R A Pegram2. 1Curriculum in Toxicology, University of North Carolina at Chapel Hill; 2Experimental Toxicology Division, NHEERL/ORD, United States Environmental Protection Agency, Research Triangl...

Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors

PubMed Central

Kosa, Nicolas M.; Foley, Timothy L.; Burkart, Michael D.

2016-01-01

Phosphopantetheinyl transferase (E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human phosphopantetheinyl transferase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity. PMID:24192555

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

PubMed

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

2017-01-01

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

Molecular Cloning of Adenosinediphosphoribosyl Transferase.

DTIC Science & Technology

1987-09-08

nature of the blocking group is unknown, except its identity with pyroglutamic acid was ruled out by its insensitivity to pyroglutaminase (not shown...AdenosinediphosphoribOSyl Transferase (ADPRT) is: 1) the complete amino acid sequence of this large protein is best determined -from the DNA !equence of the gene, 2...enzyme (I), determination of its peptide structure (II) and application of synthetic DNA probes (III) derived from amino acid sequences, resulting in the

Impact of blue, red, and far-red light treatments on gene expression and steviol glycoside accumulation in Stevia rebaudiana.

PubMed

Yoneda, Yuki; Nakashima, Hiroshi; Miyasaka, Juro; Ohdoi, Katsuaki; Shimizu, Hiroshi

2017-05-01

Stevia rebaudiana (Bertoni) Bertoni is a plant that biosynthesizes a group of natural sweeteners that are up to approximately 400 times sweeter than sucrose. The sweetening components of S. rebaudiana are steviol glycosides (SGs) that partially share their biosynthesis pathway with gibberellins (GAs). However, the molecular mechanisms through which SGs levels can be improved have not been studied. Therefore, transcription levels of several SG biosynthesis-related genes were analyzed under several light treatments involved in GA biosynthesis. We detected higher transcription of UGT85C2, which is one of the UDP-glycosyltransferases (UGTs) involved in catalyzing the sugar-transfer reaction, under red/far-red (R/FR) 1.22 light-emitting diodes (LEDs) and blue LEDs treatment. In this study, it was demonstrated that transcription levels of SG-related genes and the SGs content are affected by light treatments known to affect the GA contents. It is expected that this approach could serve as a practical way to increase SG contents using specific light treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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

2004-05-06

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

Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues.

PubMed

Vieira, R P; Mulloy, B; Mourão, P A

1991-07-25

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.

Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

PubMed Central

Rabausch, U.; Juergensen, J.; Ilmberger, N.; Böhnke, S.; Fischer, S.; Schubach, B.; Schulte, M.

2013-01-01

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. PMID:23686272

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-01-01

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

Prostate cancer and polymorphism D85Y in gene for dihydrotestosterone degrading enzyme UGT2B15: Frequency of DD homozygotes increases with Gleason Score.

PubMed

Hajdinjak, Tine; Zagradisnik, Boris

2004-06-01

Although, a functional rationale for influence of polymorphism D85Y in gene UGT2B15 on prostate cancer (PCa) exists (different V(max) of enzyme), conflicting results have been reported. DNA from 178 controls and 206 PCa patients with known Gleason score were genotyped using a newly developed RFLP assay, which allowed the detection of both alleles in an individual after single PCR amplification. 16% DD, 52% DY; PCa patients: 23% DD, 49% DY. Subgroups of PCa: well differentiated: 11% DD, 37% DY; moderately differentiated: 22% DD, 50% DY; poorly differentiated: 34% DD, 50% DY. Correlation was confirmed between Gleason score and number of D alleles (P = 0.018) and persisted after age adjustment. When comparing controls to patients with a Gleason score of 7 or more, difference for the frequency of homozygosity DD was significant between the groups (P = 0.032, OR = 2.04). Polymorphism D85Y in gene UGT2B15 correlates with differentiation of PCa. Copyright 2004 Wiley-Liss, Inc.

Characterization of Panax ginseng UDP-Glycosyltransferases Catalyzing Protopanaxatriol and Biosyntheses of Bioactive Ginsenosides F1 and Rh1 in Metabolically Engineered Yeasts.

PubMed

Wei, Wei; Wang, Pingping; Wei, Yongjun; Liu, Qunfang; Yang, Chengshuai; Zhao, Guoping; Yue, Jianmin; Yan, Xing; Zhou, Zhihua

2015-09-01

Ginsenosides, the main pharmacologically active natural compounds in ginseng (Panax ginseng), are mostly the glycosylated products of protopanaxadiol (PPD) and protopanaxatriol (PPT). No uridine diphosphate glycosyltransferase (UGT), which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPg1, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside F1. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity (>84%) with UGTPg1. We demonstrate that UGTPg100 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rh1, and UGTPg101 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rg1 from F1. However, UGTPg102 and UGTPg103 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rh1 by introducing the genetically engineered PPT-producing pathway and UGTPg1 or UGTPg100. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Biosynthesis of 2-Hydroxyacid-Containing Polyhydroxyalkanoates by Employing butyryl-CoA Transferases in Metabolically Engineered Escherichia coli.

PubMed

David, Yokimiko; Joo, Jeong Chan; Yang, Jung Eun; Oh, Young Hoon; Lee, Sang Yup; Park, Si Jae

2017-11-01

The authors previously reported the production of polyhydroxyalkanoates (PHAs) containing 2-hydroxyacid monomers by expressing evolved Pseudomonas sp. 6-19 PHA synthase and Clostridium propionicum propionyl-CoA transferase in engineered microorganisms. Here, the authors examined four butyryl-CoA transferases from Roseburia sp., Eubacterium hallii, Faecalibacterium prausnitzii, and Anaerostipes caccae as potential CoA-transferases to support synthesis of polymers having 2HA monomer. In vitro activity analyses of the four butyryl-CoA transferases suggested that each butyryl-CoA transferase has different activities towards 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB), and lactate (LA). When Escherichia coli XL1-Blue expressing Pseudomonas sp. 6-19 PhaC1437 along with one butyryl-CoA transferase is cultured in chemically defined MR medium containing 20 g L -1 of glucose, 2 g L -1 of sodium 3-hydroxybutyrate, and various concentrations of sodium 2-hydroxybutyrate, PHAs consisting of 3HB, 2HB, and LA are produced. The monomer composition of PHAs agreed well with the substrate specificities of butyryl-CoA transferases from E. hallii, F. prausnitzii, and A. caccae, but not Roseburia sp. When E. coli XL1-Blue expressing PhaC1437 and E. hallii butyryl-CoA transferase is cultured in MR medium containing 20 g L -1 of glucose and 2 g L -1 of sodium 2-hydroxybutyrate, P(65.7 mol% 2HB-co-34.3 mol% LA) is produced with the highest PHA content of 30 wt%. Butyryl-CoA transferases also supported the production of P(3HB-co-2HB-co-LA) from glucose as the sole carbon source in E. coli XL1-Blue strains when one of these bct genes is expressed with phaC1437, cimA3.7, leuBCD, panE, and phaAB genes. Butyryl-CoA transferases characterized in this study can be used for engineering of microorganisms that produce PHAs containing novel 2-hydroxyacid monomers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna

PubMed Central

David, Rhiannon M.; Jones, Huw S.; Panter, Grace H.; Winter, Matthew J.; Hutchinson, Thomas H.; Kevin Chipman, J.

2012-01-01

Organic solvents, such as dimethylsulfoxide (DMSO) and methanol are widely used as vehicles to solubilise lipophilic test compounds in toxicity testing. However, the effects of such solvents upon innate detoxification processes in aquatic organisms are poorly understood. This study assessed the effect of solvent exposure upon cytochrome P450 (CYP)-mediated xenobiotic metabolism in Daphnia magna and zebrafish larvae (4 d post fertilisation). Adult D. magna were demonstrated to have a low, but detectable, metabolism of ethoxyresorufin in vivo and this activity was not modulated by pre-exposure to DMSO or methanol (24 h, up to 0.1% and 0.05% v/v, respectively). In contrast, the metabolism of ethoxyresorufin in zebrafish larvae was significantly reduced by both solvents (0.1% and 0.05% v/v, respectively) after 24 h of exposure. In zebrafish, these observed decreases in activity towards ethoxyresorufin were accompanied by decreased expression of a variety of genes coding for drug metabolising enzymes (corresponding to CYP1, CYP2, CYP3 and UDP-glucuronyl transferase [UGT] family enzymes), measured by quantitative PCR. Reduction of gene expression and CYP1 enzyme activities by methanol (0.05% v/v) in zebrafish larvae was partially reversed by co-exposure with Aroclor 1254 (100 μg L−1). Overall this study suggests that relatively low concentrations of organic solvents can impact upon the biotransformation of certain xenobiotics in zebrafish larvae, and that this warrants consideration when assessing compounds for metabolism and toxicity in this species. PMID:22472102

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2016-10-01

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

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

PubMed Central

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

2000-01-01

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

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