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

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

[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.

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.

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

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.

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.

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

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

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.

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.

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.

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.

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

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.

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

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

Inhibition of UDP-glucuronosyltransferases (UGTs) by phthalate monoesters.

PubMed

Du, Zuo; Cao, Yun-Feng; Li, Sai-Nan; Hu, Cui-Min; Fu, Zhi-Wei; Huang, Chun-Ting; Sun, Xiao-Yu; Liu, Yong-Zhe; Yang, Kun; Fang, Zhong-Ze

2018-04-01

Phthalate monoesters are important metabolites of phthalate esters (PAEs) which have been extensively utilized in industry. This study aims to investigate the inhibition of phthalate monoesters on the activity of various isoforms of UDP-glucuronosyltransferases (UGTs), trying to elucidate the toxicity mechanism of environmental endocrine disruptors from the new perspectives. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was employed to evaluate 8 kinds of phthalate monoesters on 11 sorts of main human UGT isoforms. 100 μM phthalate monoesters exhibited negligible inhibition towards the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A8, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17. The activity of UGT1A7 was strongly inhibited by monoethylhexyl phthalate (MEHP), but slightly inhibited by all the other phthalate monoesters. UGT1A9 was broadly inhibited by monobenzyl phthalate (MBZP), monocyclohexyl phthalate (MCHP), MEHP, monohexyl phthalate (MHP) and monooctyl phthalate (MOP), respectively. MEHP exhibited competitive inhibition towards UGT1A7, and MBZP, MCHP, MEHP, MHP and MOP showed competitive inhibition towards UGT1A9. The inhibition kinetic parameters (K i ) were calculated to be 11.25 μM for MEHP-UGT1A7, and 2.13, 0.09, 1.17, 7.47, 0.16 μM for MBZP-UGT1A9, MCHP-UGT1A9, MEHP-UGT1A9, MHP-UGT1A9, MOP-UGT1A9, respectively. Molecular docking indicated that both hydrogen bonds formation and hydrophobic interactions significantly contributed to the interaction between phthalate monoesters and UGT isoforms. All these information will be beneficial for understanding the adverse effects of PAEs. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

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.

[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.

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

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.

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.

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.

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

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.

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.

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.

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

PubMed

Jia, Lin; Hu, Cuimin; Wang, Haina; Liu, Yongzhe; Liu, Xin; Zhang, Yan-Yan; Li, Wei; Wang, Li-Xuan; Cao, Yun-Feng; Fang, Zhong-Ze

2015-06-01

Zaltoprofen (ZLT) is a nonsteroidal antiinflammation drug, and has been clinically employed to treat rheumatoid arthritis, osteoarthritis, and other chronic inflammatory pain conditions. The present study aims to investigate the chirality influence of zaltoprofen towards the inhibition potential towards UDP-glucuronosyltransferases (UGTs) isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation system was employed to investigate the inhibition of (R)-zaltoprofen and (S)-zaltoprofen towards UGT isoforms. The inhibition difference capability was observed for the inhibition of (R)-zaltoprofen and (S)-zaltoprofen towards UGT1A8 and UGT2B7, but not for other tested UGT isoforms. (R)-zaltoprofen exhibited noncompetitive inhibition towards UGT1A8 and competitive inhibition towards UGT2B7. The inhibition kinetic parameters were calculated to be 35.3 μM and 19.2 μM for UGT1A8 and UGT2B7. (R)-zaltoprofen and (S)-zaltoprofen exhibited a different inhibition type towards UGT1A7. Based on the reported maximum plasma concentration of (R)-zaltoprofen in vivo, a high drug-drug interaction between (R)-zaltoprofen and the drugs mainly undergoing UGT1A7, UGT1A8, and UGT2B7-catalyzed glucuronidation was indicated. © 2015 Wiley Periodicals, Inc.

[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.

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

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

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.

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.

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

PubMed Central

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

2009-01-01

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

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

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

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

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

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.

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.

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

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

Fang, Zhong-Ze; Joint Center for Translational Medicine, Dalian Institute of Chemical Physics Chinese Academy of Sciences and The first Affiliated Hospital of Liaoning Medical University, No.457, Zhongshan Road, Dalian 116023; Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892

The wide utilization of ginseng provides the high risk of herb–drug interaction (HDI) with many clinical drugs. The inhibition of ginsenosides towards drug-metabolizing enzymes (DMEs) has been regarded as an important reason for herb–drug interaction (HDI). Compared with the deep studies on the ginsenosides' inhibition towards cytochrome P450 (CYP), the inhibition of ginsenosides towards the important phase II enzymes UDP-glucuronosyltransferases (UGTs) remains to be unclear. The present study aims to evaluate the inhibition behavior of ginsenosides towards important UGT isoforms located in the liver and intestine using in vitro methods. The recombinant UGT isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employedmore » as in vitro probe reaction. The results showed that structure-dependent inhibition existed for the inhibition of ginsenosides towards UGT isoforms. To clarify the possibility of in vivo herb–drug interaction induced by this kind of inhibition, the ginsenoside Rg{sub 3} was selected as an example, and the inhibition kinetic type and parameters (K{sub i}) were determined. Rg{sub 3} competitively inhibited UGT1A7, 2B7 and 2B15-catalyzed 4-MU glucuronidation reaction, and exerted noncompetitive inhibition towards UGT1A8-catalyzed 4-MU glucuronidation. The inhibition parameters (K{sub i} values) were calculated to be 22.6, 7.9, 1.9, and 2.0 μM for UGT1A7, 1A8, 2B7 and 2B15. Using human maximum plasma concentration of Rg{sub 3} (400 ng/ml (0.5 μM)) after intramuscular injection of 60 mg Rg{sub 3}, the area under the plasma concentration-time curve (AUC) was extrapolated to increase by 2.2%, 6.3%, 26.3%, and 25% for the co-administered drugs completely undergoing the metabolism catalyzed by UGT1A7, 1A8, 2B7 and 2B15, respectively. All these results indicated that the ginsenosides' inhibition towards UGT isoforms might be an important reason for ginseng–drug interaction. - Highlights: ► Structure-dependent inhibition

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

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

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.

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.

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.

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.

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

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.

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.

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

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.

Enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7.

PubMed

Fang, Zhong-Ze; Wang, Haina; Cao, Yun-Feng; Sun, Dong-Xue; Wang, Li-Xuan; Hong, Mo; Huang, Ting; Chen, Jian-Xing; Zeng, Jia

2015-03-01

UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)-carprofen and (S)-carprofen towards multiple UGT isoforms. The results showed that (S)-carprofen exhibited stronger inhibition potential than (R)-carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)-carprofen and (S)-carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)-carprofen and (R)-carprofen towards UGT2B7. A Lineweaver-Burk plot showed that both (S)-carprofen and (R)-carprofen exhibited competitive inhibition towards UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameter (Ki ) was calculated to be 7.0 μM and 31.1 μM for (S)-carprofen and (R)-carprofen, respectively. Based on the standard for drug-drug interaction, the threshold for (S)-carprofen and (R)-carprofen to induce a drug-drug interaction is 0.7 μM and 3.1 μM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)-carprofen and (R)-carprofen to possibly induce the drug-drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)-carprofen is more important than (R)-carprofen to avoid a possible drug-drug interaction between carprofen and the drugs mainly undergoing UGT2B7-catalyzed metabolism. © 2014 Wiley Periodicals, Inc.

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.

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

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

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.

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.

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.

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.

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.

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.

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

Investigation of miR-136-5p key target genes and pathways in lung squamous cell cancer based on TCGA database and bioinformatics analysis.

PubMed

Xie, Zu-Cheng; Li, Tian-Tian; Gan, Bin-Liang; Gao, Xiang; Gao, Li; Chen, Gang; Hu, Xiao-Hua

2018-05-01

Lung squamous cell cancer (LUSC) is a common but challenging malignancy. It is important to illuminate the molecular mechanism of LUSC. Thus, we aim to explore the molecular mechanism of miR-136-5p in relation to LUSC. We used the Cancer Genome Atlas (TCGA) database to investigate the expression of miR-136-5p in relation to LUSC. Then, we identified the possible miR-136-5p target genes through intersection of the predicted miR-136-5p target genes and LUSC upregulated genes from TCGA. Bioinformatics analysis was performed to determine the key miR-136-5p targets and pathways associated with LUSC. Finally, the expression of hub genes, correlation between miR-136-5p and hub genes, and expected significance of hub genes were evaluated via the TCGA and Genotype-Tissue Expression (GTEx) project. MiR-136-5p was significantly downregulated in LUSC patients. Glucuronidation, glucuronosyltransferase, and the retinoic acid metabolic process were the most enriched metabolic interactions in LUSC patients. Ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and retinol metabolism were identified as crucial pathways. Seven hub genes (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A10, SRD5A1, and ADH7) were found to be upregulated, and UGT1A1, UGT1A3, UGT1A6, UGT1A7, and ADH7 were negatively correlated with miR-136-5p. UGT1A7 and ADH7 were the most significantly involved miR-136-5p target genes, and high expression of these genes was correlated with better overall survival and disease-free survival of LUSC patients. Downregulated miR-136-5p may target UGT1A7 and ADH7 and participate in ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and retinol metabolism. High expression of UGT1A7 and ADH7 may indicate better prognosis of LUSC patients. Copyright © 2018. Published by Elsevier GmbH.

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 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.

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

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.

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.

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.

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

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.

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

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.

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.

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.

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.

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.

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.

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

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.

Polycomb repressive complex 1 modifies transcription of active genes

PubMed Central

Pherson, Michelle; Misulovin, Ziva; Gause, Maria; Mihindukulasuriya, Kathie; Swain, Amanda; Dorsett, Dale

2017-01-01

This study examines the role of Polycomb repressive complex 1 (PRC1) at active genes. The PRC1 and PRC2 complexes are crucial for epigenetic silencing during development of an organism. They are recruited to Polycomb response elements (PREs) and establish silenced domains over several kilobases. Recent studies show that PRC1 is also directly recruited to active genes by the cohesin complex. Cohesin participates broadly in control of gene transcription, but it is unknown whether cohesin-recruited PRC1 also plays a role in transcriptional control of active genes. We address this question using genome-wide RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq). The results show that PRC1 influences transcription of active genes, and a significant fraction of its effects are likely direct. The roles of different PRC1 subunits can also vary depending on the gene. Depletion of PRC1 subunits by RNA interference alters phosphorylation of RNA polymerase II (Pol II) and occupancy by the Spt5 pausing-elongation factor at most active genes. These effects on Pol II phosphorylation and Spt5 are likely linked to changes in elongation and RNA processing detected by nascent RNA-seq, although the mechanisms remain unresolved. The experiments also reveal that PRC1 facilitates association of Spt5 with enhancers and PREs. Reduced Spt5 levels at these regulatory sequences upon PRC1 depletion coincide with changes in Pol II occupancy and phosphorylation. Our findings indicate that, in addition to its repressive roles in epigenetic gene silencing, PRC1 broadly influences transcription of active genes and may suppress transcription of nonpromoter regulatory sequences. PMID:28782042

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

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

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.

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

Association of breast-fed neonatal hyperbilirubinemia with UGT1A1 polymorphisms: 211G>A (G71R) mutation becomes a risk factor under inadequate feeding.

PubMed

Sato, Hiroko; Uchida, Toshihiko; Toyota, Kentaro; Kanno, Miyako; Hashimoto, Taeko; Watanabe, Masashi; Nakamura, Tomohiro; Tamiya, Gen; Aoki, Kuraaki; Hayasaka, Kiyoshi

2013-01-01

Breastfeeding jaundice is a well-known phenomenon, but its pathogenesis is still unclear. Increased production of bilirubin, impaired hepatic uptake and metabolism of bilirubin, and increased enterohepatic circulation of bilirubin account for most cases of pathological neonatal hyperbilirubinemia. We previously reported that 211G>A (G71R) mutation of the UGT1A1 gene is prevalent in East Asians and is associated with the development of neonatal hyperbilirubinemia. Recently, significant association of G71R mutation with hyperbilirubinemia in breast-fed neonates was reported. We enrolled 401 full-term Japanese infants, who were exclusively breast-fed without supplementation of formula before developing hyperbilirubinemia, and classified them into two groups based on the degree of maximal body weight loss during the neonatal period. We analyzed the sex, gestational age, delivery mode, body weight at birth, maximal body weight loss and genotypes of G71R and (TA)(7) polymorphic mutations of UGT1A1. Statistical analysis revealed that maximal body weight loss during the neonatal period is the only independent risk factor for the development of neonatal hyperbilirubinemia. The effect of G71R mutation on neonatal hyperbilirubinemia is significant in neonates with 5% or greater maximal body weight loss and its influence increases in parallel with the degree of maximal body weight loss. Our study indicates that G71R mutation is a risk factor for neonatal hyperbilirubinemia only in infants with inadequate breastfeeding and suggests that adequate breastfeeding may overcome the genetic predisposing factor, G71R mutation, for the development of neonatal hyperbilirubinemia.

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice

PubMed Central

Bortolussi, G; Codarin, E; Antoniali, G; Vascotto, C; Vodret, S; Arena, S; Cesaratto, L; Scaloni, A; Tell, G; Muro, A F

2015-01-01

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration. PMID:25950469

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice.

PubMed

Bortolussi, G; Codarin, E; Antoniali, G; Vascotto, C; Vodret, S; Arena, S; Cesaratto, L; Scaloni, A; Tell, G; Muro, A F

2015-05-07

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.

Effect of sucrose concentrations on Stevia rebaudiana Bertoni tissue culture and gene expression.

PubMed

Ghorbani, T; Kahrizi, D; Saeidi, M; Arji, I

2017-08-30

Stevia rebaudiana (Bert.) Bertoni is known as sweet plant which it contains a high level of steviol glycosides in the leaves.  This plant has been used from centuries ago as a sweetener for tea. One of the most important steviol glycosides is stevioside that is attractive for diabetic persons. Tissue culture is the only rapid process for the mass propagation of stevia. One of the most important factors in the medium is sucrose that is a necessary for plant growth. In the present study, we use nodal segments of the stem as explants in mediums with different sucrose concentration (50 mM, 100mM and 150mM). Several morphological traits were measured in a 28 day period. Results analysis showed a significant variation between treatments. The highest growth rate, rooting and leaf production was obtained in medium with 100mM sucrose. The correlation between measured traits was significant at the 0.01 level. To investigation of UGT74G1, UGT76G1, UGT85C2 and KS genes expression that are involved in the synthesis of SGs, RT- PCR was done with the housekeeping gene of as internal control. There were significant differences between all media. The results showed thatsucrose 100 mM containing media was more desirable than others for expression of UGT76G1 and UGT85C2 genes. Whereas, the best medium for expression of UGT74G1 was sucrose 150 mM and sucrose 50 mM for KS gene. Totally, it seems that sucrose at a concentration of 100 mMprovides the best condition for stevia growth and steviol glycosides production.

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

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

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.

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.

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

PubMed

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

2016-05-01

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

Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

PubMed

Kahrizi, Danial; Ghaheri, Matin; Yari, Zahra; Yari, Khirollah; Bahraminejad, Sohbat

2018-02-10

Stevia rebaudiana Bertoni is one of two species that contains steviol glycosides. Among steviol glycosides that extracted from leaves, stevioside and rebaudioside A are the two major and the sweetest glycosides that are about 200-300 times sweeter than sucrose with zero calories. The best method for stevia propagation is tissue culture. So, for investigation of nutrients in medium, we studied the effect of different concentrations of MS media (MS, 0.5 MS, 0.25 MS, 0 MS) on morphological traits, UGT74G1 and UGT76G1 genes expression and accumulation of steviol glycosides in stevia leaves. The best growth rate (0.472 mm/d) has occurred in plants grown in MS media. Also, the highest gene expression of UGT74G1 gene (1.000 Total lab unit) was seen under MS treatment. However, the highest expression level of UGT76G1 gene (1.701 Total lab unit) was observed at plants grown in 0 MS. The highest amount of both Stevioside and Rebaudioside A (14.23 and 8.12, respectively) were accumulated in plants under MS treatment. Obviously, dilution of MS media associated with decreasing in both expression of the intended genes and accumulation of steviol glycosides.

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

Expression levels of uridine 5'-diphospho-glucuronosyltransferase genes in breast tissue from healthy women are associated with mammographic density.

PubMed

Haakensen, Vilde D; Biong, Margarethe; Lingjærde, Ole Christian; Holmen, Marit Muri; Frantzen, Jan Ole; Chen, Ying; Navjord, Dina; Romundstad, Linda; Lüders, Torben; Bukholm, Ida K; Solvang, Hiroko K; Kristensen, Vessela N; Ursin, Giske; Børresen-Dale, Anne-Lise; Helland, Aslaug

2010-01-01

Mammographic density (MD), as assessed from film screen mammograms, is determined by the relative content of adipose, connective and epithelial tissue in the female breast. In epidemiological studies, a high percentage of MD confers a four to six fold risk elevation of developing breast cancer, even after adjustment for other known breast cancer risk factors. However, the biologic correlates of density are little known. Gene expression analysis using whole genome arrays was performed on breast biopsies from 143 women; 79 women with no malignancy (healthy women) and 64 newly diagnosed breast cancer patients, both included from mammographic centres. Percent MD was determined using a previously validated, computerized method on scanned mammograms. Significance analysis of microarrays (SAM) was performed to identify genes influencing MD and a linear regression model was used to assess the independent contribution from different variables to MD. SAM-analysis identified 24 genes differentially expressed between samples from breasts with high and low MD. These genes included three uridine 5'-diphospho-glucuronosyltransferase (UGT) genes and the oestrogen receptor gene (ESR1). These genes were down-regulated in samples with high MD compared to those with low MD. The UGT gene products, which are known to inactivate oestrogen metabolites, were also down-regulated in tumour samples compared to samples from healthy individuals. Several single nucleotide polymorphisms (SNPs) in the UGT genes associated with the expression of UGT and other genes in their vicinity were identified. Three UGT enzymes were lower expressed both in breast tissue biopsies from healthy women with high MD and in biopsies from newly diagnosed breast cancers. The association was strongest amongst young women and women using hormonal therapy. UGT2B10 predicts MD independently of age, hormone therapy and parity. Our results indicate that down-regulation of UGT genes in women exposed to female sex hormones is

Expression levels of uridine 5'-diphospho-glucuronosyltransferase genes in breast tissue from healthy women are associated with mammographic density

PubMed Central

2010-01-01

Introduction Mammographic density (MD), as assessed from film screen mammograms, is determined by the relative content of adipose, connective and epithelial tissue in the female breast. In epidemiological studies, a high percentage of MD confers a four to six fold risk elevation of developing breast cancer, even after adjustment for other known breast cancer risk factors. However, the biologic correlates of density are little known. Methods Gene expression analysis using whole genome arrays was performed on breast biopsies from 143 women; 79 women with no malignancy (healthy women) and 64 newly diagnosed breast cancer patients, both included from mammographic centres. Percent MD was determined using a previously validated, computerized method on scanned mammograms. Significance analysis of microarrays (SAM) was performed to identify genes influencing MD and a linear regression model was used to assess the independent contribution from different variables to MD. Results SAM-analysis identified 24 genes differentially expressed between samples from breasts with high and low MD. These genes included three uridine 5'-diphospho-glucuronosyltransferase (UGT) genes and the oestrogen receptor gene (ESR1). These genes were down-regulated in samples with high MD compared to those with low MD. The UGT gene products, which are known to inactivate oestrogen metabolites, were also down-regulated in tumour samples compared to samples from healthy individuals. Several single nucleotide polymorphisms (SNPs) in the UGT genes associated with the expression of UGT and other genes in their vicinity were identified. Conclusions Three UGT enzymes were lower expressed both in breast tissue biopsies from healthy women with high MD and in biopsies from newly diagnosed breast cancers. The association was strongest amongst young women and women using hormonal therapy. UGT2B10 predicts MD independently of age, hormone therapy and parity. Our results indicate that down-regulation of UGT genes

Correlations between polymorphisms in the uridine diphosphate-glucuronosyltransferase 1A and C-C motif chemokine receptor 5 genes and infection with the hepatitis B virus in three ethnic groups in China.

PubMed

Zhang, Chan; He, Yan; Shan, Ke-Ren; Tan, Kui; Zhang, Ting; Wang, Chan-Juan; Guan, Zhi-Zhong

2018-02-01

Objective To determine whether genetic polymorphisms in the uridine diphosphate-glucuronosyltransferase 1A ( UGT1A) and the C-C motif chemokine receptor 5 ( CCR5) genes are associated with hepatitis B virus (HBV) infection in Yi, Yao and Han ethnic groups in the Guizhou Province of China. Methods The study enrolled subjects with and without HBV infection. Whole blood was used for DNA genotyping using standard techniques. The study determined the frequencies of several polymorphic alleles ( UGT1A6 [rs2070959], UGT1A1 [rs8175347], CCR5-59029 [rs1799987] and CCR5Δ32 [rs333]) and then characterized their relationship with HBV infection. Results A total of 404 subjects were enrolled in the study: 138 from the Yao group, 101 from the Yi group and 165 from the Han group. There was a significant difference in the frequency of UGT1A1 rs8175347 polymorphisms among the three groups. The rates of 7TA carriers of UGT1A1 rs8175347 in all three groups were significantly higher than the other genotypes. Individuals with genotype AA of UGT1A6 rs2070959 in the Yi group had a higher risk for HBV infection than in the Yao and Han groups. The frequency of genotype GG in CCR5-59029 in the Yao group was significantly higher than in the Yi group. The genotypes of CCR5Δ32 were not associated with HBV infection. Conclusion These findings provide genetic and epidemiological evidence for an association of UGT1A and CCR5-59029 polymorphisms with HBV infection in Chinese Yi and Yao populations.

Complexity and Entropy Analysis of DNMT1 Gene

USDA-ARS?s Scientific Manuscript database

Background: The application of complexity information on DNA sequence and protein in biological processes are well established in this study. Available sequences for DNMT1 gene, which is a maintenance methyltransferase is responsible for copying DNA methylation patterns to the daughter strands durin...

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.

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.

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.

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

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

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

EPA Pesticide Factsheets

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

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

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

PubMed Central

2009-01-01

Background The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). Methods Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. Results Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Conclusions Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX. PMID:19732436

Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

PubMed

Esmaeili, Fatemeh; Ghaheri, Matin; Kahrizi, Danial; Mansouri, Mohsen; Safavi, Seyed Mehdi; Ghorbani, Tayebeh; Muhammadi, Sarre; Rahmanian, Elham; Vaziri, Siavash

2018-02-10

Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

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

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.

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

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

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.

Studies on the flavonoid substrates of human UDP-glucuronosyl transferase (UGT) 2B7.

PubMed

Xie, Shenggu; You, Linya; Zeng, Su

2007-08-01

Flavonoids are found in fruits, vegetables, nuts, seeds, herbs, spices, stems and flowers, as well as in tea and red wine. They are prominent components of citrus fruits and other food sources, are consumed regularly with the human diet, and have been shown to have many biological functions, including antioxidant and chelating properties. This study suggests features of the flavonoid structure necessary for it to act as a substrate of human UGT2B7. Generally speaking, flavonol has higher glucuronidation activity than flavones and isoflavones. Differences in C3' position have an important effect on UGT2B7 glucuronidation activity, and the various substituents have different influences on glucuronidation activity. For flavonol, the bulky group at C4' can enhance glucuronidation activity. Increasing the number of hydroxyl groups of flavonoids will increase their glucuronidation activity towards UGT2B7, while conjugation of glycon will weaken the activity, and hydroxyl position can also have an important role in activity. The high glucuronidation efficiency observed with many flavonoids suggests that the contribution of UGT2B7 to the metabolism of flavonoids may be significant. The results suggest that we should not only pay attention to glucuronidation activity, but should also attach importance to the regioselectivity of glucuronidation.

Meta-analysis diagnostic accuracy of SNP-based pathogenicity detection tools: a case of UTG1A1 gene mutations.

PubMed

Galehdari, Hamid; Saki, Najmaldin; Mohammadi-Asl, Javad; Rahim, Fakher

2013-01-01

Crigler-Najjar syndrome (CNS) type I and type II are usually inherited as autosomal recessive conditions that result from mutations in the UGT1A1 gene. The main objective of the present review is to summarize results of all available evidence on the accuracy of SNP-based pathogenicity detection tools compared to published clinical result for the prediction of in nsSNPs that leads to disease using prediction performance method. A comprehensive search was performed to find all mutations related to CNS. Database searches included dbSNP, SNPdbe, HGMD, Swissvar, ensemble, and OMIM. All the mutation related to CNS was extracted. The pathogenicity prediction was done using SNP-based pathogenicity detection tools include SIFT, PHD-SNP, PolyPhen2, fathmm, Provean, and Mutpred. Overall, 59 different SNPs related to missense mutations in the UGT1A1 gene, were reviewed. Comparing the diagnostic OR, PolyPhen2 and Mutpred have the highest detection 4.983 (95% CI: 1.24 - 20.02) in both, following by SIFT (diagnostic OR: 3.25, 95% CI: 1.07 - 9.83). The highest MCC of SNP-based pathogenicity detection tools, was belong to SIFT (34.19%) followed by Provean, PolyPhen2, and Mutpred (29.99%, 29.89%, and 29.89%, respectively). Hence the highest SNP-based pathogenicity detection tools ACC, was fit to SIFT (62.71%) followed by PolyPhen2, and Mutpred (61.02%, in both). Our results suggest that some of the well-established SNP-based pathogenicity detection tools can appropriately reflect the role of a disease-associated SNP in both local and global structures.

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin.

PubMed

Brown, David A; Di Cerbo, Vincenzo; Feldmann, Angelika; Ahn, Jaewoo; Ito, Shinsuke; Blackledge, Neil P; Nakayama, Manabu; McClellan, Michael; Dimitrova, Emilia; Turberfield, Anne H; Long, Hannah K; King, Hamish W; Kriaucionis, Skirmantas; Schermelleh, Lothar; Kutateladze, Tatiana G; Koseki, Haruhiko; Klose, Robert J

2017-09-05

Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome. Copyright © 2017 The Authors. Published by Elsevier Inc. 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.

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.

Leigh syndrome associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFS1 gene.

PubMed

Martín, Miguel A; Blázquez, Alberto; Gutierrez-Solana, Luis G; Fernández-Moreira, Daniel; Briones, Paz; Andreu, Antoni L; Garesse, Rafael; Campos, Yolanda; Arenas, Joaquín

2005-04-01

Mutations in the nuclear-encoded subunits of complex I of the mitochondrial respiratory chain are a recognized cause of Leigh syndrome (LS). Recently, 6 mutations in the NDUFS1 gene were identified in 3 families. To describe a Spanish family with LS, complex I deficiency in muscle, and a novel mutation in the NDUFS1 gene. Using molecular genetic approaches, we identified the underlying molecular defect in a patient with LS with a complex I defect. The proband was a child who displayed the clinical features of LS. Muscle biochemistry results showed a complex I defect of the mitochondrial respiratory chain. Sequencing analysis of the mitochondrial DNA-encoded ND genes, the nuclear DNA-encoded NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, and NDUFAB1 genes, and the complex I assembly factor CIA30 gene revealed a novel homozygous L231V mutation (c.691C-->G) in the NDUFS1 gene. The parents were heterozygous carriers of the L231V mutation. Identifying nuclear mutations as a cause of respiratory chain disorders will enhance the possibility of prenatal diagnosis and help us understand how molecular defects can lead to complex I deficiency.

Cloning and characterization of soybean gene Fg1 encoding flavonol 3-O-glucoside/galactoside (1→6) glucosyltransferase.

PubMed

Rojas Rodas, Felipe; Di, Shaokang; Murai, Yoshinori; Iwashina, Tsukasa; Sugawara, Satoko; Mori, Tetsuya; Nakabayashi, Ryo; Yonekura-Sakakibara, Keiko; Saito, Kazuki; Takahashi, Ryoji

2016-11-01

Flavonoids are important secondary metabolites in plants. Sugar-sugar glycosyltransferases are involved in the final step of flavonoid biosynthesis and contribute to the structural diversity of flavonoids. This manuscript describes the first cloning of a sugar-sugar glucosyltransferase gene in the UGT family that attaches glucose to the 6″-position of sugar bound to a flavonol. The results provide a glimpse on the possible evolution of sugar-sugar glycosyltransferase genes and identify putative amino acids responsible for the recognition of the hydroxyl group of the sugar moiety and specification of sugar. A scheme for the genetic control of flavonol glycoside biosynthesis is proposed. Flavonol glycosides (FGs) are predominant in soybean leaves and they show substantial differences among genotypes. In previous studies, we identified two flavonoid glycoside glycosyltransferase genes that segregated in recombinant inbred lines developed from a cross between cultivars Nezumisaya and Harosoy; one was responsible for the attachment of glucose to the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol and the other was involved in the attachment of glucose to the 6″-position. This study was conducted to clone and characterize the 6″-glucosyltransferase gene. Linkage mapping indicated that the gene was located in the molecular linkage group I (chromosome 20). Based on the genome sequence, we cloned a candidate cDNA, GmF3G6"Gt from Harosoy but the corresponding cDNA could not be amplified by PCR from Nezumisaya. The coding region of GmF3G6″Gt in Harosoy is 1386 bp long encoding 462 amino acids. This gene was not expressed in leaves of Nezumisaya. The GmF3G6″Gt recombinant protein converted UDP-glucose and kaempferol 3-O-glucoside or kaempferol 3-O-galactoside to kaempferol 3-O-glucosyl-(1→6)-glucoside or kaempferol 3-O-glucosyl-(1→6)-galactoside, respectively. These results indicate that GmF3G6″Gt encodes a flavonol 3-O

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

Dose-Finding and Pharmacokinetic Study to Optimize the Dosing of Irinotecan According to the UGT1A1 Genotype of Patients With Cancer

PubMed Central

Innocenti, Federico; Schilsky, Richard L.; Ramírez, Jacqueline; Janisch, Linda; Undevia, Samir; House, Larry K.; Das, Soma; Wu, Kehua; Turcich, Michelle; Marsh, Robert; Karrison, Theodore; Maitland, Michael L.; Salgia, Ravi; Ratain, Mark J.

2014-01-01

Purpose The risk of severe neutropenia from treatment with irinotecan is related in part to UGT1A1*28, a variant that reduces the elimination of SN-38, the active metabolite of irinotecan. We aimed to identify the maximum-tolerated dose (MTD) and dose-limiting toxicity (DLT) of irinotecan in patients with advanced solid tumors stratified by the *1/*1, *1/*28, and *28/*28 genotypes. Patients and Methods Sixty-eight patients received an intravenous flat dose of irinotecan every 3 weeks. Forty-six percent of the patients had the *1/*1 genotype, 41% had the *1/*28 genotype, and 13% had the *28/*28 genotype. The starting dose of irinotecan was 700 mg in patients with the *1/*1 and *1/*28 genotypes and 500 mg in patients with the *28/*28 genotype. Pharmacokinetic evaluation was performed at cycle 1. Results In patients with the *1/*1 genotype, the MTD was 850 mg (four DLTs per 16 patients), and 1,000 mg was not tolerated (two DLTs per six patients). In patients with the *1/*28 genotype, the MTD was 700 mg (five DLTs per 22 patients), and 850 mg was not tolerated (four DLTs per six patients). In patients with the *28/*28 genotype, the MTD was 400 mg (one DLT per six patients), and 500 mg was not tolerated (three DLTs per three patients). The DLTs were mainly myelosuppression and diarrhea. Irinotecan clearance followed linear kinetics. At the MTD for each genotype, dosing by genotype resulted in similar SN-38 areas under the curve (AUCs; r2 = 0.0003; P = .97), but the irinotecan AUC was correlated with the actual dose (r2 = 0.39; P < .001). Four of 48 patients with disease known to be responsive to irinotecan achieved partial response. Conclusion The UGT1A1*28 genotype can be used to individualize dosing of irinotecan. Additional studies should evaluate the effect of genotype-guided dosing on efficacy in patients receiving irinotecan. PMID:24958824

Hepatic expression patterns of aryl hydrocarbon receptor, pregnane X receptor, two cytochrome P450s and five phase II metabolism genes responsive to 17alpha-methyltestosterone in rare minnow Gobiocypris rarus.

PubMed

Gao, Jiancao; Liu, Shaozhen; Zhang, Yingying; Yuan, Cong; Yang, Yanping; Wang, Zaizhao

2014-05-01

17Alpha-methyltestosterone (MT), a synthetic androgen, is widely used in aquaculture. Aquatic organisms can receive continuous exposure to residual MT throughout their lives. Aiming to evaluate the effects of MT on genes involved in biotransformation pathway, meanwhile attempting to unravel the MT metabolic pathway at the transcriptional level in fish, here we isolated the cDNAs of previously unreported AHR2, Sult1 st1, Ugt2a1 and Ugt2b6 in rare minnow, and predominantly investigated the hepatic transcriptional patterns of AHR2, PXR and five biotransformation genes after MT exposure in both genders adult rare minnow Gobiocypris rarus. The present findings suggest that AHR2 and PXR should play important roles in regulating biotransformation enzymes related to MT catabolism, moreover, CYP1A, CYP3A, SULT1 ST4, SULT1 ST6 and UGT2A1 may play certain roles in catabolism of MT in adult G. rarus. Additionally, UGT2A1 may make greater contribution than SULT1 ST4 and SULT1 ST6 in MT catabolism in males. Copyright © 2014 Elsevier B.V. All rights reserved.

GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes

PubMed Central

Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

2014-01-01

Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12–ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex. PMID:25046113

An Association Between Functional Polymorphisms of the Interleukin 1 Gene Complex and Schizophrenia Using Transmission Disequilibrium Test.

PubMed

Kapelski, Pawel; Skibinska, Maria; Maciukiewicz, Malgorzata; Pawlak, Joanna; Dmitrzak-Weglarz, Monika; Szczepankiewicz, Aleksandra; Zaremba, Dorota; Twarowska-Hauser, Joanna

2016-12-01

IL1 gene complex has been implicated in the etiology of schizophrenia. To assess whether IL1 gene complex is associated with susceptibility to schizophrenia in Polish population we conducted family-based study. Functional polymorphisms from IL1A (rs1800587, rs17561, rs11677416), IL1B (rs1143634, rs1143643, rs16944, rs4848306, rs1143623, rs1143633, rs1143627) and IL1RN (rs419598, rs315952, rs9005, rs4251961) genes were genotyped in 143 trio with schizophrenia. Statistical analysis was performed using transmission disequilibrium test. We have found a trend toward an association of rs1143627, rs16944, rs1143623 in IL1B gene with the risk of schizophrenia. Our results show a protective effect of allele T of rs4251961 in IL1RN against schizophrenia. We also performed haplotype analysis of IL1 gene complex and found a trend toward an association with schizophrenia of GAGG haplotype (rs1143627, rs16944, rs1143623, rs4848306) in IL1B gene, haplotypes: TG (rs315952, rs9005) and TT (rs4251961, rs419598) in IL1RN. Haplotype CT (rs4251961, rs419598) in IL1RN was found to be associated with schizophrenia. After correction for multiple testing associations did not reach significance level. Our results might support theory that polymorphisms of interleukin 1 complex genes (rs1143627, rs16944, rs1143623, rs4848306 in IL1B gene and rs4251961, rs419598, rs315952, rs9005 in IL1RN gene) are involved in the pathogenesis of schizophrenia, however, none of the results reach significance level after correction for multiple testing.

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.

Gene-Gene and Gene-Environment Interactions in the Etiology of Breast Cancer

DTIC Science & Technology

2007-06-01

When you eat fried or baked pork or beef , you normally prefer that: Entire surface is brown with a slight burnt flavor 1...Uridine diphospho-glucuronosyltransferase 1A1 (UGT1A1) is involved in catalyzing estrogen, the hormone that plays a central role in the etiology of...relationship of UGT1A1 genotypes with plasma levels of estrone, estrone sulfate, estradiol, testosterone, and sex hormone binding globulins (SHBG

Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes

PubMed Central

Pratt, Victoria M.; Everts, Robin E.; Aggarwal, Praful; Beyer, Brittany N.; Broeckel, Ulrich; Epstein-Baak, Ruth; Hujsak, Paul; Kornreich, Ruth; Liao, Jun; Lorier, Rachel; Scott, Stuart A.; Smith, Chingying Huang; Toji, Lorraine H.; Turner, Amy; Kalman, Lisa V.

2017-01-01

Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention–based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing. PMID:26621101

Cosegregation of intragenic markers with a novel mutation that causes Crigler-Najjar syndrome type I: implication in carrier detection and prenatal diagnosis.

PubMed Central

Moghrabi, N; Clarke, D J; Burchell, B; Boxer, M

1993-01-01

Crigler-Najjar syndrome type 1 (CN-1) is a familial disorder characterized by severe unconjugated hyperbilirubinemia and jaundice and leads to kernicterus, neurological damage, and eventual death unless treated with liver transplantation. Previous reports identified mutations in the UGT1 gene complex to be the cause of the disease. The total absence of all phenol/bilirubin UGT proteins and their activities in liver homogenate of a CN-1 patient was determined by enzymological and immunochemical analysis. A novel homozygous nonsense mutation (CGA-->TGA) was identified in the patient by the combined techniques of PCR and direct sequencing. This mutation was located in exon 3 of the constant region in the gene complex which is common to all phenol and bilirubin UGTs. The segregation of the mutation in the patient's family was analyzed and confirmed the recessive nature of the disease. Newly developed intragenic polymorphic probes (UGT1* 4 and UGT-Const) were used on Southern blots of MspI-digested genomic DNA of the patient and his family. The segregation of individual alleles within the family was observed from haplotypes generated. Comparison of the segregation of haplotypes with the mutation for the patient and his family revealed the allele identified by the A1-B1-C2 haplotype to be carrying the mutation. The risk of recombination occurring is negligible, because of the intragenic nature of the probes. This study demonstrates the potential usefulness of these probes in carrier detection and prenatal/presymptomatic diagnosis. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8102509

Unraveling the Mechanism Underlying the Glycosylation and Methylation of Anthocyanins in Peach1[C][W

PubMed Central

Cheng, Jun; Wei, Guochao; Zhou, Hui; Gu, Chao; Vimolmangkang, Sornkanok; Liao, Liao; Han, Yuepeng

2014-01-01

Modification of anthocyanin plays an important role in increasing its stability in plants. Here, six anthocyanins were identified in peach (Prunus persica), and their structural diversity is attributed to glycosylation and methylation. Interestingly, peach is quite similar to the wild species Prunus ferganensis but differs from both Prunus davidiana and Prunus kansueasis in terms of anthocyanin composition in flowers. This indicates that peach is probably domesticated from P. ferganensis. Subsequently, genes responsible for both methylation and glycosylation of anthocyanins were identified, and their spatiotemporal expression results in different patterns of anthocyanin accumulation in flowers, leaves, and fruits. Two tandem-duplicated genes encoding flavonoid 3-O-glycosyltransferase (F3GT) in peach, PpUGT78A1 and PpUGT78A2, showed different activity toward anthocyanin, providing an example of divergent evolution of F3GT genes in plants. Two genes encoding anthocyanin O-methyltransferase (AOMT), PpAOMT1 and PpAOMT2, are expressed in leaves and flowers, but only PpAOMT2 is responsible for the O-methylation of anthocyanins at the 3′ position in peach. In addition, our study reveals a novel branch of UGT78 genes in plants that lack the highly conserved intron 2 of the UGT gene family, with a great variation of the amino acid residue at position 22 of the plant secondary product glycosyltransferase box. Our results not only provide insights into the mechanisms underlying anthocyanin glycosylation and methylation in peach but will also aid in future attempts to manipulate flavonoid biosynthesis in peach as well as in other plants. PMID:25106821

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

PubMed

Bock, Karl Walter

2016-01-01

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

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.

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.

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

PubMed Central

Bortolussi, Giulia; Zentilin, Lorena; Baj, Gabriele; Giraudi, Pablo; Bellarosa, Cristina; Giacca, Mauro; Tiribelli, Claudio; Muro, Andrés F.

2012-01-01

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer. PMID:22094718

The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

PubMed

Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

2016-07-01

The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

The NSL Complex Regulates Housekeeping Genes in Drosophila

PubMed Central

Raja, Sunil Jayaramaiah; Holz, Herbert; Luscombe, Nicholas M.; Manke, Thomas; Akhtar, Asifa

2012-01-01

MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP–seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP–seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication–related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription. PMID:22723752

Recruitment by the Repressor Freud-1 of Histone Deacetylase-Brg1 Chromatin Remodeling Complexes to Strengthen HTR1A Gene Repression.

PubMed

Souslova, Tatiana; Mirédin, Kim; Millar, Anne M; Albert, Paul R

2017-12-01

Five-prime repressor element under dual repression binding protein-1 (Freud-1)/CC2D1A is genetically linked to intellectual disability and implicated in neuronal development. Freud-1 represses the serotonin-1A (5-HT1A) receptor gene HTR1A by histone deacetylase (HDAC)-dependent or HDAC-independent mechanisms in 5-HT1A-negative (e.g., HEK-293) or 5-HT1A-expressing cells (SK-N-SH), respectively. To identify the underlying mechanisms, Freud-1-associated proteins were affinity-purified from HEK-293 nuclear extracts and members of the Brg1/SMARCCA chromatin remodeling and Sin3A-HDAC corepressor complexes were identified. Pull-down assays using recombinant proteins showed that Freud-1 interacts directly with the Brg1 carboxyl-terminal domain; interaction with Brg1 required the carboxyl-terminal of Freud-1. Freud-1 complexes in HEK-293 and SK-N-SH cells differed, with low levels of BAF170/SMARCC2 and BAF57/SMARCE1 in HEK-293 cells and low-undetectable BAF155/SMARCC1, Sin3A, and HDAC1/2 in SK-N-SH cells. Similarly, by quantitative chromatin immunoprecipitation, Brg1-BAF170/57 and Sin3A-HDAC complexes were observed at the HTR1A promoter in HEK-293 cells, whereas in SK-N-SH cells, Sin3A-HDAC proteins were not detected. Quantifying 5-HT1A receptor mRNA levels in cells treated with siRNA to Freud-1, Brg1, or both RNAs addressed the functional role of the Freud-1-Brg1 complex. In HEK-293 cells, 5-HT1A receptor mRNA levels were increased only when both Freud-1 and Brg1 were depleted, but in SK-N-SH cells, depletion of either protein upregulated 5-HT1A receptor RNA. Thus, recruitment by Freud-1 of Brg1, BAF155, and Sin3A-HDAC complexes appears to strengthen repression of the HTR1A gene to prevent its expression inappropriate cell types, while recruitment of the Brg1-BAF170/57 complex is permissive to 5-HT1A receptor expression. Alterations in Freud-1-Brg1 interactions in mutants associated with intellectual disability could impair gene repression leading to altered neuronal

Recruitment by the Repressor Freud-1 of Histone Deacetylase-Brg1 Chromatin Remodeling Complexes to Strengthen HTR1A Gene Repression

PubMed Central

Souslova, Tatiana; Mirédin, Kim; Millar, Anne M.

2017-01-01

Five-prime repressor element under dual repression binding protein-1 (Freud-1)/CC2D1A is genetically linked to intellectual disability and implicated in neuronal development. Freud-1 represses the serotonin-1A (5-HT1A) receptor gene HTR1A by histone deacetylase (HDAC)-dependent or HDAC-independent mechanisms in 5-HT1A-negative (e.g., HEK-293) or 5-HT1A-expressing cells (SK-N-SH), respectively. To identify the underlying mechanisms, Freud-1-associated proteins were affinity-purified from HEK-293 nuclear extracts and members of the Brg1/SMARCCA chromatin remodeling and Sin3A-HDAC corepressor complexes were identified. Pull-down assays using recombinant proteins showed that Freud-1 interacts directly with the Brg1 carboxyl-terminal domain; interaction with Brg1 required the carboxyl-terminal of Freud-1. Freud-1 complexes in HEK-293 and SK-N-SH cells differed, with low levels of BAF170/SMARCC2 and BAF57/SMARCE1 in HEK-293 cells and low-undetectable BAF155/SMARCC1, Sin3A, and HDAC1/2 in SK-N-SH cells. Similarly, by quantitative chromatin immuno-precipitation, Brg1-BAF170/57 and Sin3A-HDAC complexes were observed at the HTR1A promoter in HEK-293 cells, whereas in SK-N-SH cells, Sin3A-HDAC proteins were not detected. Quantifying 5-HT1A receptor mRNA levels in cells treated with siRNA to Freud-1, Brg1, or both RNAs addressed the functional role of the Freud-1-Brg1 complex. In HEK-293 cells, 5-HT1A receptor mRNA levels were increased only when both Freud-1 and Brg1 were depleted, but in SK-N-SH cells, depletion of either protein upregulated 5-HT1A receptor RNA. Thus, recruitment by Freud-1 of Brg1, BAF155, and Sin3A-HDAC complexes appears to strengthen repression of the HTR1A gene to prevent its expression inappropriate cell types, while recruitment of the Brg1-BAF170/57 complex is permissive to 5-HT1A receptor expression. Alterations in Freud-1-Brg1 interactions in mutants associated with intellectual disability could impair gene repression leading to altered neuronal

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.

Genetic variation of genes involved in dihydrotestosterone metabolism and the risk of prostate cancer.

PubMed

Setlur, Sunita R; Chen, Chen X; Hossain, Ruhella R; Ha, Jung Sook; Van Doren, Vanessa E; Stenzel, Birgit; Steiner, Eberhard; Oldridge, Derek; Kitabayashi, Naoki; Banerjee, Samprit; Chen, Jin Yun; Schäfer, Georg; Horninger, Wolfgang; Lee, Charles; Rubin, Mark A; Klocker, Helmut; Demichelis, Francesca

2010-01-01

Dihydrotestosterone (DHT) is an important factor in prostate cancer (PCA) genesis and disease progression. Given PCA's strong genetic component, we evaluated the possibility that variation in genes involved in DHT metabolism influence PCA risk. We investigated copy number variants (CNV) and single nucleotide polymorphisms (SNP). We explored associations between CNV of uridine diphospho-glucuronosyltransferase (UGT) genes from the 2B subclass, given their prostate specificity and/or involvement in steroid metabolism and PCA risk. We also investigated associations between SNPs in genes (HSD3B1, SRD5A1/2, and AKR1C2) involved in the conversion of testosterone to DHT, and in DHT metabolism and PCA risk. The population consisted of 426 men (205 controls and 221 cases) who underwent prostate-specific antigen screening as part of a PCA early detection program in Tyrol, Austria. No association between CNV in UGT2B17 and UGT2B28 and PCA risk was identified. Men carrying the AA genotype at SNP rs6428830 (HSD3B1) had an odds ratio (OR) of 2.0 [95% confidence intervals (95% CI), 1.1-4.1] compared with men with GG, and men with AG or GG versus AA in rs1691053 (SRD5A1) had an OR of 1.8 (95% CI, 1.04-3.13). Individuals carrying both risk alleles had an OR of 3.1 (95% CI, 1.4-6.7) when compared with men carrying neither (P = 0.005). Controls with the AA genotype on rs7594951 (SRD5A2) tended toward higher serum DHT levels (P = 0.03). This is the first study to implicate the 5alpha-reductase isoform 1 (SRD5A1) and PCA risk, supporting the rationale of blocking enzymatic activity of both isoforms of 5alpha-reductase for PCA chemoprevention.

RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

PubMed

Merrick, B Alex; Phadke, Dhiral P; Auerbach, Scott S; Mav, Deepak; Stiegelmeyer, Suzy M; Shah, Ruchir R; Tice, Raymond R

2013-01-01

Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the

CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene.

PubMed

Uchida, Shusaku; Teubner, Brett J W; Hevi, Charles; Hara, Kumiko; Kobayashi, Ayumi; Dave, Rutu M; Shintaku, Tatsushi; Jaikhan, Pattaporn; Yamagata, Hirotaka; Suzuki, Takayoshi; Watanabe, Yoshifumi; Zakharenko, Stanislav S; Shumyatsky, Gleb P

2017-01-10

Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets. Copyright © 2017 The Author(s). Published by Elsevier Inc. 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

Effects of life cycle and leaves location on gene expression and glycoside biosynthesis pathway in Stevia rebaudiana Bertoni.

PubMed

Ghaheri, Matin; Adibrad, Elaheh; Safavi, Seyed Mehdi; Kahrizi, Danial; Soroush, Ali; Muhammadi, Saare; Ghorbani, Tayebeh; Sabzevari, Ali; Ansarypour, Zahra; Rahmanian, Elham

2018-02-10

Stevia rebaudiana Bertoni is One of the most important biologically sourced and low-calorie sweeteners that known as "Sweet Weed". It contains steviol glycosides that they are about 200-300 times sweeter than sucrose. Tissue culture is the best method with high efficiency that can overcome to problems of traditional methods, and it is the most useful tools for studying stress tolerance mechanisms under in vitro conditions to obtain drought tolerance. In the present research, we investigated the impact of life cycle, leaves location and the harvesting time on expression of UGT74G1 and UGT76G1 as well as steviol glycosides accumulation. The highest gene expression of both UGT74G1 and UGT76G1 (207.677 and 208.396 Total Lab unit, respectively) was observed in young leaves in the second vegetative year. Also, the highest amount of stevioside accumulation (13.04) was due to the old leaves in vegetative stage which had significant differences with other effects whereas the lowest accumulation (7.47) was seen at young leaves at vegetative stage. Interestingly, the highest level of rebaudioside a production (15.74) was occurred at the young leaves at vegetative stage. There was significant differences between life cycle and leaves location on steviol glycoside production in stevia.

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.

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.

The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

PubMed Central

Myster, S H; Knott, J A; O'Toole, E; Porter, M E

1997-01-01

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex. Images PMID:9247642

Candidate gene polymorphisms in patients with acetaminophen-induced acute liver failure.

PubMed

Court, Michael H; Peter, Inga; Hazarika, Suwagmani; Vasiadi, Magdalini; Greenblatt, David J; Lee, William M

2014-01-01

Acetaminophen is a leading cause of acute liver failure (ALF). Genetic differences might predispose some individuals to develop ALF. In this exploratory study, we evaluated genotype frequency differences among patients enrolled by the ALF Study Group who had developed ALF either intentionally from a single-time-point overdose of acetaminophen (n = 78), unintentionally after chronic high doses of acetaminophen (n = 79), or from causes other than acetaminophen (n = 103). The polymorphisms evaluated included those in genes encoding putative acetaminophen-metabolizing enzymes (UGT1A1, UGT1A6, UGT1A9, UGT2B15, SULT1A1, CYP2E1, and CYP3A5) as well as CD44 and BHMT1. Individuals carrying the CYP3A5 rs776746 A allele were overrepresented among ALF patients who had intentionally overdosed with acetaminophen, with an odds ratio of 2.3 (95% confidence interval, 1.1-4.9; P = 0.034) compared with all other ALF patients. This finding is consistent with the enhanced bioactivation of acetaminophen by the CYP3A5 enzyme. Persons homozygous for the CD44 rs1467558 A allele were also overrepresented among patients who had unintentionally developed ALF from chronic acetaminophen use, with an odds ratio of 4.0 (1.0-17.2, P = 0.045) compared with all other ALF subjects. This finding confirms a prior study that found elevated serum liver enzyme levels in healthy volunteers with the CD44 rs1467558 AA genotype who had consumed high doses of acetaminophen for up to 2 weeks. However, both genetic associations were considered relatively weak, and they were not statistically significant after adjustment for multiple comparisons testing. Nevertheless, both CYP3A5 rs776746 and CD44 rs1467558 warrant further investigation as potential genomic markers of enhanced risk of acetaminophen-induced ALF.

Microarray gene expression analysis of uterosacral ligaments in uterine prolapse.

PubMed

Ak, Handan; Zeybek, Burak; Atay, Sevcan; Askar, Niyazi; Akdemir, Ali; Aydin, Hikmet Hakan

2016-11-01

Pelvic organ prolapse (POP) is a major health problem that impairs the quality of life with a wide clinical spectrum. Since the uterosacral ligaments provide primary support for the uterus and the upper vagina, we hypothesize that the disruption of these ligaments may lead to a loss of support and eventually contribute to POP. In this study, we therefore investigated whether there are any differences in the transcription profile of uterosacral ligaments in patients with POP when compared to those of the control samples. Seventeen women with POP and 8 non-POP controls undergoing hysterectomy for benign conditions were included in the study. Affymetrix® Gene Chip microarrays (Human Hu 133 plus 2.0) were used for whole genome gene expression profiling analysis. There was 1 significantly down-regulated gene, NKX2-3 in patients with POP compared to the controls (p=4.28464e-013). KIF11 gene was found to be significantly down-regulated in patients with ≥3 deliveries compared to patients with <3 deliveries (p=0.0156237). UGT1A1 (p=2.43388e-005), SCARB1 (p=1.19001e-006) and NKX2-3 (p=2.17966e-013) genes were found to be significantly down-regulated in the premenopausal patients compared to the premenopausal controls. UGT1A1 gene was also found to be significantly down-regulated in the post menopausal patients compared to the postmenopausal controls (p=0.0005). This study provides evidence for a significant down-regulation of the genes that take role in cell cycle, proliferation and embryonic development along with cell adhesion process on the development of POP for the first time. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. 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

A novel gene, MdSSK1, as a component of the SCF complex rather than MdSBP1 can mediate the ubiquitination of S-RNase in apple.

PubMed

Yuan, Hui; Meng, Dong; Gu, Zhaoyu; Li, Wei; Wang, Aide; Yang, Qing; Zhu, Yuandi; Li, Tianzhong

2014-07-01

As a core factor in S-RNase-based gametophytic self-incompatibility (GSI), the SCF (SKP1-Cullin1-F-box-Rbx1) complex (including pollen determinant SLF, S-locus-F-box) functions as an E3 ubiquitin ligase on non-self S-RNase. The SCF complex is formed by SKP1 bridging between SLF, CUL1, and Rbx1; however, it is not known whether an SCF complex lacking SKP1 can mediate the ubiquitination of S-RNase. Three SKP1-like genes from pollen were cloned based on the structural features of the SLF-interacting-SKP1-like (SSK) gene and the 'Golden Delicious' apple genome. These genes have a motif of five amino acids following the standard 'WAFE' at the C terminal and, in addition, contain eight sheets and two helices. All three genes were expressed exclusively in pollen. In the yeast two-hybrid and pull-down assays only one was found to interact with MdSFBB and MdCUL1, suggesting it is the SLF-interacting SKP1-like gene in apple which was named MdSSK1. In vitro experiments using MdSSK1, S2-MdSFBB1 (S2-Malus domestica S-locus-F-box brother) and MdCUL1 proteins incubated with S 2-RNase and ubiquitin revealed that the SCF complex ubiquitinylates S-RNase in vitro, while MdSBP1 (Malus domestica S-RNase binding protein 1) could not functionally replace MdSSK1 in the SCF complex in ubiquitinylating S-RNase. According to the above experiments, MdSBP1 is probably the only factor responsible for recognition with S-RNase, while not a component of the SCF complex, and an SCF complex containing MdSSK1 is required for mediating the ubiquitination of S-RNase. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Effect of NET-1 siRNA conjugated sub-micron bubble complex combined with low-frequency ultrasound exposure in gene transfection

PubMed Central

Wu, Bolin; Liang, Xitian; Jing, Hui; Han, Xue; Sun, Yixin; Guo, Cunli; Liu, Ying; Cheng, Wen

2018-01-01

The present study evaluated the effect of NET-1 siRNA-conjugated sub-micron bubble (SMB) complexes combined with low-frequency ultrasound exposure in gene transfection. The NET-1 gene was highly expressed level in SMMC-7721 human hepatocellular carcinoma cell line. The cells were divided into seven groups and treated with different conditions. The groups with or without low-frequency ultrasound exposure, groups of adherent cells, and suspension cells were separated. The NET-1 siRNA-conjugated SMB complexes were made in the laboratory and tested by Zetasizer Nano ZS90 analyzer. Flow cytometry was used to estimate the transfection efficiency and cellular apoptosis. Western blot and quantitative real-time polymerase chain reaction (qPCR) were used for the estimation of the protein and mRNA expressions, respectively. Transwell analysis determined the migration and invasion capacities of the tumor cells. The results did not show any difference in the transfection efficiency between adherent and suspension cells. However, the NET-1 siRNA-SMB complexes combined with low-frequency ultrasound exposure could enhance the gene transfection effectively. In summary, the NET-1 siRNA-SMB complexes appeared to be promising gene vehicle. PMID:29423111

RNA-Seq Profiling Reveals Novel Hepatic Gene Expression Pattern in Aflatoxin B1 Treated Rats

PubMed Central

Merrick, B. Alex; Phadke, Dhiral P.; Auerbach, Scott S.; Mav, Deepak; Stiegelmeyer, Suzy M.; Shah, Ruchir R.; Tice, Raymond R.

2013-01-01

Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1’s carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT’s) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find

Complex phylogeny and gene expression patterns of members of the NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) in wheat

PubMed Central

Buchner, Peter; Hawkesford, Malcolm J.

2014-01-01

NPF (formerly referred to as low-affinity NRT1) and ‘high-affinity’ NRT2 nitrate transporter genes are involved in nitrate uptake by the root, and transport and distribution of nitrate within the plant. The NPF gene family consists of 53 members in Arabidopsis thaliana, however only 11 of these have been functionally characterized. Although homologous genes have been identified in genomes of different plant species including some cereals, there is little information available for wheat (Triticum aestivum). Sixteen genes were identified in wheat homologous to characterized Arabidopsis low-affinity nitrate transporter NPF genes, suggesting a complex wheat NPF gene family. The regulation of wheat NFP genes by plant N-status indicated involvement of these transporters in substrate transport in relation to N-metabolism. The complex expression pattern in relation to tissue specificity, nitrate availability and senescence may be associated with the complex growth patterns of wheat depending on sink/source demands, as well as remobilization during grain filling. PMID:24913625

Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes: A GeT-RM Collaborative Project.

PubMed

Pratt, Victoria M; Everts, Robin E; Aggarwal, Praful; Beyer, Brittany N; Broeckel, Ulrich; Epstein-Baak, Ruth; Hujsak, Paul; Kornreich, Ruth; Liao, Jun; Lorier, Rachel; Scott, Stuart A; Smith, Chingying Huang; Toji, Lorraine H; Turner, Amy; Kalman, Lisa V

2016-01-01

Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

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

Influence of Genetic Ancestry on INDEL Markers of NFKβ1, CASP8, PAR1, IL4 and CYP19A1 Genes in Leprosy Patients.

PubMed

Pinto, Pablo; Salgado, Claudio; Santos, Ney Pereira Carneiro; Santos, Sidney; Ribeiro-dos-Santos, Ândrea

2015-01-01

Leprosy is an insidious infectious disease caused by the obligate intracellular bacteria Mycobacterium leprae, and host genetic factors can modulate the immune response and generate distinct categories of leprosy susceptibility that are also influenced by genetic ancestry. We investigated the possible effects of CYP19A1 [rs11575899], NFKβ1 [rs28362491], IL1α [rs3783553], CASP8 [rs3834129], UGT1A1 [rs8175347], PAR1 [rs11267092], CYP2E1 [INDEL 96pb] and IL4 [rs79071878] genes in a group of 141 leprosy patients and 180 healthy individuals. The INDELs were typed by PCR Multiplex in ABI PRISM 3130 and analyzed with GeneMapper ID v3.2. The NFKβ1, CASP8, PAR1 and IL4 INDELs were associated with leprosy susceptibility, while NFKβ1, CASP8, PAR1 and CYP19A1 were associated with the MB (Multibacilary) clinical form of leprosy. NFKβ1 [rs28362491], CASP8 [rs3834129], PAR1 [rs11267092] and IL4 [rs79071878] genes are potential markers for susceptibility to leprosy development, while the INDELs in NFKβ1, CASP8, PAR1 and CYP19A1 (rs11575899) are potential markers for the severe clinical form MB. Moreover, all of these markers are influenced by genetic ancestry, and European contribution increases the risk to leprosy development, in other hand an increase in African contribution generates protection against leprosy.

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

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

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

Cytokinin-Specific Glycosyltransferases Possess Different Roles in Cytokinin Homeostasis Maintenance.

PubMed

Šmehilová, Mária; Dobrůšková, Jana; Novák, Ondřej; Takáč, Tomáš; Galuszka, Petr

2016-01-01

Plant hormones cytokinins (CKs) are one of the major mediators of physiological responses throughout plant life span. Therefore, a proper homeostasis is maintained by regulation of their active levels. Besides degradation, CKs are deactivated by uridine diphosphate glycosyltransferases (UGTs). Physiologically, CKs active levels decline in senescing organs, providing a signal to nutrients that a shift to reproductive tissues has begun. In this work, we show CK glucosides distribution in Arabidopsis leaves during major developmental transition phases. Besides continuous accumulation of N-glucosides we detected sharp maximum of the glucosides in senescence. This is caused prevalently by N7-glucosides followed by N9-glucosides and specifically also by trans-zeatin-O-glucoside (tZOG). Interestingly, we observed a similar trend in response to exogenously applied CK. In Arabidopsis, only three UGTs deactivate CKs in vivo: UGT76C1, UGT76C2 and UGT85A1. We thereby show that UGT85A1 is specifically expressed in senescent leaves whereas UGT76C2 is activated rapidly in response to exogenously applied CK. To shed more light on the UGTs physiological roles, we performed a comparative study on UGTs loss-of-function mutants, characterizing a true ugt85a1-1 loss-of-function mutant for the first time. Although no altered phenotype was detected under standard condition we observed reduced chlorophyll degradation with increased anthocyanin accumulation in our experiment on detached leaves accompanied by senescence and stress related genes modulated expression. Among the mutants, ugt76c2 possessed extremely diminished CK N-glucosides levels whereas ugt76c1 showed some specificity toward cis-zeatin (cZ). Besides tZOG, a broader range of CK glucosides was decreased in ugt85a1-1. Performing CK metabolism gene expression profiling, we revealed that activation of CK degradation pathway serves as a general regulatory mechanism of disturbed CK homeostasis followed by decreased CK signaling in

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.

[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

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.

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.

MUC1-C activates polycomb repressive complexes and downregulates tumor suppressor genes in human cancer cells.

PubMed

Rajabi, Hasan; Hiraki, Masayuki; Kufe, Donald

2018-04-01

The PRC2 and PRC1 complexes are aberrantly expressed in human cancers and have been linked to decreases in patient survival. MUC1-C is an oncoprotein that is also overexpressed in diverse human cancers and is associated with a poor prognosis. Recent studies have supported a previously unreported function for MUC1-C in activating PRC2 and PRC1 in cancer cells. In the regulation of PRC2, MUC1-C (i) drives transcription of the EZH2 gene, (ii) binds directly to EZH2, and (iii) enhances occupancy of EZH2 on target gene promoters with an increase in H3K27 trimethylation. Regarding PRC1, which is recruited to PRC2 sites in the hierarchical model, MUC1-C induces BMI1 transcription, forms a complex with BMI1, and promotes H2A ubiquitylation. MUC1-C thereby contributes to the integration of PRC2 and PRC1-mediated repression of tumor suppressor genes, such as CDH1, CDKN2A, PTEN and BRCA1. Like PRC2 and PRC1, MUC1-C is associated with the epithelial-mesenchymal transition (EMT) program, cancer stem cell (CSC) state, and acquisition of anticancer drug resistance. In concert with these observations, targeting MUC1-C downregulates EZH2 and BMI1, inhibits EMT and the CSC state, and reverses drug resistance. These findings emphasize the significance of MUC1-C as a therapeutic target for inhibiting aberrant PRC function and reprogramming the epigenome in human cancers.

Rsp5-Bul1/2 complex is necessary for the HSE-mediated gene expression in budding yeast.

PubMed

Kaida, Daisuke; Toh-e, Akio; Kikuchi, Yoshiko

2003-07-11

Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae and is concerned with many functions such as endocytosis and transcription through ubiquitination of various substrates. Bul1 or its homologue Bul2 binds to Rsp5 through the PY-motif and the bul1 bul2 double mutant is sensitive to various stresses. We demonstrate here that heat shock element (HSE)-mediated gene expression was defective in both rsp5-101 and bul1 bul2 mutants under high temperature condition. The bul1 gene containing mutations in the PY motif region did not recover this defective gene expression of the bul1 bul2 mutant. The protein level and phosphorylation state of the HSE-binding transcription factor, Hsf1, was not affected by these mutations. Thus, the Rsp5-Bul1/2 complex has a new function for the HSE-mediated gene expression and may regulate it through other factors than Hsf1.

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

Fli1 Represses Transcription of the Human α2(I) Collagen Gene by Recruitment of the HDAC1/p300 Complex

PubMed Central

Asano, Yoshihide; Trojanowska, Maria

2013-01-01

Fli1, a member of the Ets transcription factor family, is a key repressor of the human α2(I) collagen (COL1A2) gene. Although our previous studies have delineated that TGF-β induces displacement of Fli1 from the COL1A2 promoter through sequential post-translational modifications, the detailed mechanism by which Fli1 functions as a potent transcriptional repressor of the COL1A2 gene has not been fully investigated. To address this issue, we carried out a series of experiments especially focusing on protein-protein interaction and epigenetic transcriptional regulation. The combination of tandem affinity purification and mass spectrometry identified HDAC1 as a Fli1 interacting protein. Under quiescent conditions, HDAC1 induced deacetylation of Fli1 resulting in an increase of Fli1 DNA binding ability and p300 enhanced this process by promoting the formation of a Fli1-HDAC1-p300 complex. TGF-β-induced phosphorylation of Fli1 at threonine 312 led to disassembly of this protein complex. In quiescent dermal fibroblasts Fli1, HDAC1, and p300 occupied the −404 to −237 region, including the Fli1 binding site, of the COL1A2 promoter. TGF-β induced Fli1 and HDAC1 dissociation from the COL1A2 promoter, while promoting Ets1 and p300 recruitment. Furthermore, acetylation levels of histone H3 around the Fli1 binding site in the COL1A2 promoter inversely correlated with the DNA occupancy of Fli1 and HDAC1, while positively correlating with that of Ets1 and p300. In the functional studies, HDAC1 overexpression magnified the inhibitory effect of Fli1 on the COL1A2 promoter. Moreover, pharmacological blockade of HDAC1 by entinostat enhanced collagen production in dermal fibroblasts. Collectively, these results indicate that under quiescent conditions Fli1 recruits HDAC1/p300 to the COL1A2 promoter and suppresses the expression of the COL1A2 gene by chromatin remodeling through histone deacetylation. TGF-β-dependent phosphorylation of Fli1 at threonine 312 is a critical step

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.

Mutations in the gene encoding the Sigma 2 subunit of the adaptor protein 1 complex, AP1S2, cause X-linked mental retardation.

PubMed

Tarpey, Patrick S; Stevens, Claire; Teague, Jon; Edkins, Sarah; O'Meara, Sarah; Avis, Tim; Barthorpe, Syd; Buck, Gemma; Butler, Adam; Cole, Jennifer; Dicks, Ed; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Hinton, Jonathon; Jones, David; Menzies, Andrew; Mironenko, Tatiana; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alexandra; Tofts, Calli; Varian, Jennifer; West, Sofie; Widaa, Sara; Yates, Andy; Catford, Rachael; Butler, Julia; Mallya, Uma; Moon, Jenny; Luo, Ying; Dorkins, Huw; Thompson, Deborah; Easton, Douglas F; Wooster, Richard; Bobrow, Martin; Carpenter, Nancy; Simensen, Richard J; Schwartz, Charles E; Stevenson, Roger E; Turner, Gillian; Partington, Michael; Gecz, Jozef; Stratton, Michael R; Futreal, P Andrew; Raymond, F Lucy

2006-12-01

In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles.

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.

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

Saponin Biosynthesis in Saponaria vaccaria. cDNAs Encoding β-Amyrin Synthase and a Triterpene Carboxylic Acid Glucosyltransferase1[OA

PubMed Central

Meesapyodsuk, Dauenpen; Balsevich, John; Reed, Darwin W.; Covello, Patrick S.

2007-01-01

Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a β-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria. PMID:17172290

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.

HoxBlinc RNA recruits Set1/MLL complexes to activate Hox gene expression patterns and mesoderm lineage development

PubMed Central

Deng, Changwang; Li, Ying; Zhou, Lei; Cho, Joonseok; Patel, Bhavita; Terada, Nao; Li, Yangqiu; Bungert, Jörg; Qiu, Yi; Huang, Suming

2015-01-01

Summary Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1+ mesoderm and then promotes hematopoietic differentiation through regulating hoxb gene pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated KD or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb gene expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1+ precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1+ precursors and differentiation of Flk1+ cells into hematopoietic lineages. PMID:26725110

The CRB1 Complex: Following the Trail of Crumbs to a Feasible Gene Therapy Strategy.

PubMed

Quinn, Peter M; Pellissier, Lucie P; Wijnholds, Jan

2017-01-01

Once considered science fiction, gene therapy is rapidly becoming scientific reality, targeting a growing number of the approximately 250 genes linked to hereditary retinal disorders such as retinitis pigmentosa and Leber's congenital amaurosis. Powerful new technologies have emerged, leading to the development of humanized models for testing and screening these therapies, bringing us closer to the goal of personalized medicine. These tools include the ability to differentiate human induced pluripotent stem cells (iPSCs) to create a "retina-in-a-dish" model and the self-formed ectodermal autonomous multi-zone, which can mimic whole eye development. In addition, highly specific gene-editing tools are now available, including the CRISPR/Cas9 system and the recently developed homology-independent targeted integration approach, which allows gene editing in non-dividing cells. Variants in the CRB1 gene have long been associated with retinopathies, and more recently the CRB2 gene has also been shown to have possible clinical relevance with respect to retinopathies. In this review, we discuss the role of the CRB protein complex in patients with retinopathy. In addition, we discuss new opportunities provided by stem cells and gene-editing tools, and we provide insight into how the retinal therapeutic pipeline can be improved. Finally, we discuss the current state of adeno-associated virus-mediated gene therapy and how it can be applied to treat retinopathies associated with mutations in CRB1 .

Validation of variants in SLC28A3 and UGT1A6 as genetic markers predictive of anthracycline-induced cardiotoxicity in children.

PubMed

Visscher, H; Ross, C J D; Rassekh, S R; Sandor, G S S; Caron, H N; van Dalen, E C; Kremer, L C; van der Pal, H J; Rogers, P C; Rieder, M J; Carleton, B C; Hayden, M R

2013-08-01

The use of anthracyclines as effective antineoplastic drugs is limited by the occurrence of cardiotoxicity. Multiple genetic variants predictive of anthracycline-induced cardiotoxicity (ACT) in children were recently identified. The current study was aimed to assess replication of these findings in an independent cohort of children. . Twenty-three variants were tested for association with ACT in an independent cohort of 218 patients. Predictive models including genetic and clinical risk factors were constructed in the original cohort and assessed in the current replication cohort. . We confirmed the association of rs17863783 in UGT1A6 and ACT in the replication cohort (P = 0.0062, odds ratio (OR) 7.98). Additional evidence for association of rs7853758 (P = 0.058, OR 0.46) and rs885004 (P = 0.058, OR 0.42) in SLC28A3 was found (combined P = 1.6 × 10(-5) and P = 3.0 × 10(-5), respectively). A previously constructed prediction model did not significantly improve risk prediction in the replication cohort over clinical factors alone. However, an improved prediction model constructed using replicated genetic variants as well as clinical factors discriminated significantly better between cases and controls than clinical factors alone in both original (AUC 0.77 vs. 0.68, P = 0.0031) and replication cohort (AUC 0.77 vs. 0.69, P = 0.060). . We validated genetic variants in two genes predictive of ACT in an independent cohort. A prediction model combining replicated genetic variants as well as clinical risk factors might be able to identify high- and low-risk patients who could benefit from alternative treatment options. Copyright © 2013 Wiley Periodicals, Inc.

Silencing of Agamma-globin gene expression during adult definitive erythropoiesis mediated by GATA-1-FOG-1-Mi2 complex binding at the -566 GATA site.

PubMed

Harju-Baker, Susanna; Costa, Flávia C; Fedosyuk, Halyna; Neades, Renee; Peterson, Kenneth R

2008-05-01

Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 and -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the epsilon-globin gene was transcriptionally silenced in adult beta-globin locus yeast artificial chromosome (beta-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the (A)gamma-globin -566 or (G)gamma-globin -567 GATA site when gamma-globin expression was low (day 18) but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.

Downregulation of the expression of mitochondrial electron transport complex genes in autism brains.

PubMed

Anitha, Ayyappan; Nakamura, Kazuhiko; Thanseem, Ismail; Matsuzaki, Hideo; Miyachi, Taishi; Tsujii, Masatsugu; Iwata, Yasuhide; Suzuki, Katsuaki; Sugiyama, Toshiro; Mori, Norio

2013-05-01

Mitochondrial dysfunction (MtD) and abnormal brain bioenergetics have been implicated in autism, suggesting possible candidate genes in the electron transport chain (ETC). We compared the expression of 84 ETC genes in the post-mortem brains of autism patients and controls. Brain tissues from the anterior cingulate gyrus, motor cortex, and thalamus of autism patients (n = 8) and controls (n = 10) were obtained from Autism Tissue Program, USA. Quantitative real-time PCR arrays were used to quantify gene expression. We observed reduced expression of several ETC genes in autism brains compared to controls. Eleven genes of Complex I, five genes each of Complex III and Complex IV, and seven genes of Complex V showed brain region-specific reduced expression in autism. ATP5A1 (Complex V), ATP5G3 (Complex V) and NDUFA5 (Complex I) showed consistently reduced expression in all the brain regions of autism patients. Upon silencing ATP5A1, the expression of mitogen-activated protein kinase 13 (MAPK13), a p38 MAPK responsive to stress stimuli, was upregulated in HEK 293 cells. This could have been induced by oxidative stress due to impaired ATP synthesis. We report new candidate genes involved in abnormal brain bioenergetics in autism, supporting the hypothesis that mitochondria, critical for neurodevelopment, may play a role in autism. © 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.

LHX3 interacts with inhibitor of histone acetyltransferase complex subunits LANP and TAF-1β to modulate pituitary gene regulation.

PubMed

Hunter, Chad S; Malik, Raleigh E; Witzmann, Frank A; Rhodes, Simon J

2013-01-01

LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex.

LHX3 Interacts with Inhibitor of Histone Acetyltransferase Complex Subunits LANP and TAF-1β to Modulate Pituitary Gene Regulation

PubMed Central

Witzmann, Frank A.; Rhodes, Simon J.

2013-01-01

LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex. PMID:23861948

Increased Transcript Complexity in Genes Associated with Chronic Obstructive Pulmonary Disease

PubMed Central

Lackey, Lela; McArthur, Evonne; Laederach, Alain

2015-01-01

Genome-wide association studies aim to correlate genotype with phenotype. Many common diseases including Type II diabetes, Alzheimer’s, Parkinson’s and Chronic Obstructive Pulmonary Disease (COPD) are complex genetic traits with hundreds of different loci that are associated with varied disease risk. Identifying common features in the genes associated with each disease remains a challenge. Furthermore, the role of post-transcriptional regulation, and in particular alternative splicing, is still poorly understood in most multigenic diseases. We therefore compiled comprehensive lists of genes associated with Type II diabetes, Alzheimer’s, Parkinson’s and COPD in an attempt to identify common features of their corresponding mRNA transcripts within each gene set. The SERPINA1 gene is a well-recognized genetic risk factor of COPD and it produces 11 transcript variants, which is exceptional for a human gene. This led us to hypothesize that other genes associated with COPD, and complex disorders in general, are highly transcriptionally diverse. We found that COPD-associated genes have a statistically significant enrichment in transcript complexity stemming from a disproportionately high level of alternative splicing, however, Type II Diabetes, Alzheimer’s and Parkinson’s disease genes were not significantly enriched. We also identified a subset of transcriptionally complex COPD-associated genes (~40%) that are differentially expressed between mild, moderate and severe COPD. Although the genes associated with other lung diseases are not extensively documented, we found preliminary data that idiopathic pulmonary disease genes, but not cystic fibrosis modulators, are also more transcriptionally complex. Interestingly, complex COPD transcripts are more often the product of alternative acceptor site usage. To verify the biological importance of these alternative transcripts, we used RNA-sequencing analyses to determine that COPD-associated genes are frequently

Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase.

PubMed

Di, Shaokang; Yan, Fan; Rodas, Felipe Rojas; Rodriguez, Tito O; Murai, Yoshinori; Iwashina, Tsukasa; Sugawara, Satoko; Mori, Tetsuya; Nakabayashi, Ryo; Yonekura-Sakakibara, Keiko; Saito, Kazuki; Takahashi, Ryoji

2015-05-23

-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

Common Genetic Variation In Cellular Transport Genes and Epithelial Ovarian Cancer (EOC) Risk.

PubMed

Chornokur, Ganna; Lin, Hui-Yi; Tyrer, Jonathan P; Lawrenson, Kate; Dennis, Joe; Amankwah, Ernest K; Qu, Xiaotao; Tsai, Ya-Yu; Jim, Heather S L; Chen, Zhihua; Chen, Ann Y; Permuth-Wey, Jennifer; Aben, Katja K H; Anton-Culver, Hoda; Antonenkova, Natalia; Bruinsma, Fiona; Bandera, Elisa V; Bean, Yukie T; Beckmann, Matthias W; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A; Brooks-Wilson, Angela; Bunker, Clareann H; Butzow, Ralf; Campbell, Ian G; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; du Bois, Andreas; Despierre, Evelyn; Dicks, Ed; Doherty, Jennifer A; Dörk, Thilo; Dürst, Matthias; Easton, Douglas F; Eccles, Diana M; Edwards, Robert P; Ekici, Arif B; Fasching, Peter A; Fridley, Brooke L; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G; Glasspool, Rosalind; Goodman, Marc T; Gronwald, Jacek; Harrington, Patricia; Harter, Philipp; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A T; Hillemanns, Peter; Hogdall, Claus K; Hogdall, Estrid; Hosono, Satoyo; Jakubowska, Anna; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y; Kelemen, Linda E; Kellar, Mellissa; Kiemeney, Lambertus A; Krakstad, Camilla; Kjaer, Susanne K; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D; Lee, Alice W; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A; Liang, Dong; Lim, Boon Kiong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F A G; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R; McNeish, Iain; Menon, Usha; Milne, Roger L; Modugno, Francesmary; Moysich, Kirsten B; Ness, Roberta B; Nevanlinna, Heli; Eilber, Ursula; Odunsi, Kunle; Olson, Sara H; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Paul, James; Pearce, Celeste L; Pejovic, Tanja; Pelttari, Liisa M; Pike, Malcolm C; Poole, Elizabeth M; Risch, Harvey A; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H; Rudolph, Anja; Runnebaum, Ingo B; Rzepecka, Iwona K; Salvesen, Helga B; Schernhammer, Eva; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C; Spiewankiewicz, Beata; Sucheston, Lara; Teo, Soo-Hwang; Terry, Kathryn L; Thompson, Pamela J; Thomsen, Lotte; Tangen, Ingvild L; Tworoger, Shelley S; van Altena, Anne M; Vierkant, Robert A; Vergote, Ignace; Walsh, Christine S; Wang-Gohrke, Shan; Wentzensen, Nicolas; Whittemore, Alice S; Wicklund, Kristine G; Wilkens, Lynne R; Wu, Anna H; Wu, Xifeng; Woo, Yin-Ling; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Hasmad, Hanis N; Berchuck, Andrew; Iversen, Edwin S; Schildkraut, Joellen M; Ramus, Susan J; Goode, Ellen L; Monteiro, Alvaro N A; Gayther, Simon A; Narod, Steven A; Pharoah, Paul D P; Sellers, Thomas A; Phelan, Catherine M

2015-01-01

Defective cellular transport processes can lead to aberrant accumulation of trace elements, iron, small molecules and hormones in the cell, which in turn may promote the formation of reactive oxygen species, promoting DNA damage and aberrant expression of key regulatory cancer genes. As DNA damage and uncontrolled proliferation are hallmarks of cancer, including epithelial ovarian cancer (EOC), we hypothesized that inherited variation in the cellular transport genes contributes to EOC risk. In total, DNA samples were obtained from 14,525 case subjects with invasive EOC and from 23,447 controls from 43 sites in the Ovarian Cancer Association Consortium (OCAC). Two hundred seventy nine SNPs, representing 131 genes, were genotyped using an Illumina Infinium iSelect BeadChip as part of the Collaborative Oncological Gene-environment Study (COGS). SNP analyses were conducted using unconditional logistic regression under a log-additive model, and the FDR q<0.2 was applied to adjust for multiple comparisons. The most significant evidence of an association for all invasive cancers combined and for the serous subtype was observed for SNP rs17216603 in the iron transporter gene HEPH (invasive: OR = 0.85, P = 0.00026; serous: OR = 0.81, P = 0.00020); this SNP was also associated with the borderline/low malignant potential (LMP) tumors (P = 0.021). Other genes significantly associated with EOC histological subtypes (p<0.05) included the UGT1A (endometrioid), SLC25A45 (mucinous), SLC39A11 (low malignant potential), and SERPINA7 (clear cell carcinoma). In addition, 1785 SNPs in six genes (HEPH, MGST1, SERPINA, SLC25A45, SLC39A11 and UGT1A) were imputed from the 1000 Genomes Project and examined for association with INV EOC in white-European subjects. The most significant imputed SNP was rs117729793 in SLC39A11 (per allele, OR = 2.55, 95% CI = 1.5-4.35, p = 5.66x10-4). These results, generated on a large cohort of women, revealed associations between inherited cellular transport

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

Characterization of 107 Genomic DNA Reference Materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1

PubMed Central

Pratt, Victoria M.; Zehnbauer, Barbara; Wilson, Jean Amos; Baak, Ruth; Babic, Nikolina; Bettinotti, Maria; Buller, Arlene; Butz, Ken; Campbell, Matthew; Civalier, Chris; El-Badry, Abdalla; Farkas, Daniel H.; Lyon, Elaine; Mandal, Saptarshi; McKinney, Jason; Muralidharan, Kasinathan; Noll, LeAnne; Sander, Tara; Shabbeer, Junaid; Smith, Chingying; Telatar, Milhan; Toji, Lorraine; Vairavan, Anand; Vance, Carlos; Weck, Karen E.; Wu, Alan H.B.; Yeo, Kiang-Teck J.; Zeller, Markus; Kalman, Lisa

2010-01-01

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research. PMID:20889555

The complexity of gene expression dynamics revealed by permutation entropy

PubMed Central

2010-01-01

Background High complexity is considered a hallmark of living systems. Here we investigate the complexity of temporal gene expression patterns using the concept of Permutation Entropy (PE) first introduced in dynamical systems theory. The analysis of gene expression data has so far focused primarily on the identification of differentially expressed genes, or on the elucidation of pathway and regulatory relationships. We aim to study gene expression time series data from the viewpoint of complexity. Results Applying the PE complexity metric to abiotic stress response time series data in Arabidopsis thaliana, genes involved in stress response and signaling were found to be associated with the highest complexity not only under stress, but surprisingly, also under reference, non-stress conditions. Genes with house-keeping functions exhibited lower PE complexity. Compared to reference conditions, the PE of temporal gene expression patterns generally increased upon stress exposure. High-complexity genes were found to have longer upstream intergenic regions and more cis-regulatory motifs in their promoter regions indicative of a more complex regulatory apparatus needed to orchestrate their expression, and to be associated with higher correlation network connectivity degree. Arabidopsis genes also present in other plant species were observed to exhibit decreased PE complexity compared to Arabidopsis specific genes. Conclusions We show that Permutation Entropy is a simple yet robust and powerful approach to identify temporal gene expression profiles of varying complexity that is equally applicable to other types of molecular profile data. PMID:21176199

Food restriction increase the expression of mTORC1 complex genes in the skeletal muscle of juvenile pacu (Piaractus mesopotamicus)

PubMed Central

de Paula, Tassiana Gutierrez; Zanella, Bruna Tereza Thomazini; Fantinatti, Bruno Evaristo de Almeida; de Moraes, Leonardo Nazário; Duran, Bruno Oliveira da Silva; de Oliveira, Caroline Bredariol; Salomão, Rondinelle Artur Simões; da Silva, Rafaela Nunes; Padovani, Carlos Roberto; dos Santos, Vander Bruno; Mareco, Edson Assunção; Carvalho, Robson Francisco; Dal-Pai-Silva, Maeli

2017-01-01

Skeletal muscle is capable of phenotypic adaptation to environmental factors, such as nutrient availability, by altering the balance between muscle catabolism and anabolism that in turn coordinates muscle growth. Small noncoding RNAs, known as microRNAs (miRNAs), repress the expression of target mRNAs, and many studies have demonstrated that miRNAs regulate the mRNAs of catabolic and anabolic genes. We evaluated muscle morphology, gene expression of components involved in catabolism, anabolism and energetic metabolism and miRNAs expression in both the fast and slow muscle of juvenile pacu (Piaractus mesopotamicus) during food restriction and refeeding. Our analysis revealed that short periods of food restriction followed by refeeding predominantly affected fast muscle, with changes in muscle fiber diameter and miRNAs expression. There was an increase in the mRNA levels of catabolic pathways components (FBXO25, ATG12, BCL2) and energetic metabolism-related genes (PGC1α and SDHA), together with a decrease in PPARβ/δ mRNA levels. Interestingly, an increase in mRNA levels of anabolic genes (PI3K and mTORC1 complex: mTOR, mLST8 and RAPTOR) was also observed during food restriction. After refeeding, muscle morphology showed similar patterns of the control group; the majority of genes were slightly up- or down-regulated in fast and slow muscle, respectively; the levels of all miRNAs increased in fast muscle and some of them decreased in slow muscle. Our findings demonstrated that a short period of food restriction in juvenile pacu had a considerable impact on fast muscle, increasing the expression of anabolic (PI3K and mTORC1 complex: mTOR, mLST8 and RAPTOR) and energetic metabolism genes. The miRNAs (miR-1, miR-206, miR-199 and miR-23a) were more expressed during refeeding and while their target genes (IGF-1, mTOR, PGC1α and MAFbx), presented a decreased expression. The alterations in mTORC1 complex observed during fasting may have influenced the rates of protein

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.

A case of concomitant Gilbert's syndrome and hereditary spherocytosis

PubMed Central

Lee, Hee Jung; Moon, Hee Seok; Lee, Eaum Seok; Kim, Seok Hyun; Sung, Jae Kyu; Lee, Byung Seok; Jeong, Hyun Yong; Eu, Young Jae

2010-01-01

We describe moderate hyperbilirubinemia in a 28-year-old man who suffered from gallstones and splenomegaly, with combined disorders of hereditary spherocytosis (HS) and Gilbert's syndrome (GS). Since it is difficult to diagnose HS in the absence of signs of anemia, we evaluated both the genetic mutation in the UGT1A1 gene and abnormalities in the erythrocyte membrane protein; the former was heterozygous for a UGT1A1 allele with three mutations and the latter was partially deficient in ankyrin expression. This is the first report of the concomitance of HS and GS with three heterozygous mutations [T-3279G, A (TA)7TAA, and G211A] in the UGT1A1 gene. PMID:20924216

Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

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

Hong, Wei, E-mail: hongwei@tijmu.edu.cn; College of Basic Medicine, Tianjin Medical University, 300070 Tianjin; Li, Jinru

Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by whichmore » Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.« less

HoxBlinc RNA Recruits Set1/MLL Complexes to Activate Hox Gene Expression Patterns and Mesoderm Lineage Development.

PubMed

Deng, Changwang; Li, Ying; Zhou, Lei; Cho, Joonseok; Patel, Bhavita; Terada, Naohiro; Li, Yangqiu; Bungert, Jörg; Qiu, Yi; Huang, Suming

2016-01-05

Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1(+) mesoderm and then promotes hematopoietic differentiation through regulation of hoxb pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated knockdown or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1(+) precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1(+) precursors and differentiation of Flk1(+) cells into hematopoietic lineages. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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.

Plasmodium vivax Isolates from Cambodia and Thailand Show High Genetic Complexity and Distinct Patterns of P. vivax Multidrug Resistance Gene 1 (pvmdr1) Polymorphisms

PubMed Central

Lin, Jessica T.; Patel, Jaymin C.; Kharabora, Oksana; Sattabongkot, Jetsumon; Muth, Sinuon; Ubalee, Ratawan; Schuster, Anthony L.; Rogers, William O.; Wongsrichanalai, Chansuda; Juliano, Jonathan J.

2013-01-01

Plasmodium vivax accounts for an increasing fraction of malaria infections in Thailand and Cambodia. We compared P. vivax genetic complexity and antimalarial resistance patterns in the two countries. Use of a heteroduplex tracking assay targeting the merozoite surface protein 1 gene revealed that vivax infections in both countries are frequently polyclonal (84%), with parasites that are highly diverse (HE = 0.86) but closely related (GST = 0.18). Following a history of different drug policies in Thailand and Cambodia, distinct patterns of antimalarial resistance have emerged: most Cambodian isolates harbor the P. vivax multidrug resistance gene 1 (pvmdr1) 976F mutation associated with chloroquine resistance (89% versus 8%, P < 0.001), whereas Thai isolates more often display increased pvmdr1 copy number (39% versus 4%, P < 0.001). Finally, genotyping of paired isolates from individuals suspected of suffering relapse supports a complex scheme of relapse whereby recurrence of multiple identical variants is sometimes accompanied by the appearance of novel variants. PMID:23509126

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

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site▿ †

PubMed Central

Harju-Baker, Susanna; Costa, Flávia C.; Fedosyuk, Halyna; Neades, Renee; Peterson, Kenneth R.

2008-01-01

Autonomous silencing of γ-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the Aγ-globin gene was identified between −730 and −378 relative to the mRNA start site. A marked copy of the Aγ-globin gene inserted between locus control region 5′ DNase I-hypersensitive site 1 and the ɛ-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the −566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low γ-globin levels, whereas only a weak signal was detected when γ-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the Aγ-globin −566 or Gγ-globin −567 GATA site when γ-globin expression was low (day 18) but not when γ-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, γ-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the −566/−567 GATA sites of the proximal γ-globin promoters. PMID:18347053

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.

Estrogens and Progesterone Promote Persistent CCND1 Gene Activation during G1 by Inducing Transcriptional Derepression via c-Jun/c-Fos/Estrogen Receptor (Progesterone Receptor) Complex Assembly to a Distal Regulatory Element and Recruitment of Cyclin D1 to Its Own Gene Promoter

PubMed Central

Cicatiello, Luigi; Addeo, Raffaele; Sasso, Annarita; Altucci, Lucia; Petrizzi, Valeria Belsito; Borgo, Raphaelle; Cancemi, Massimo; Caporali, Simona; Caristi, Silvana; Scafoglio, Claudio; Teti, Diana; Bresciani, Francesco; Perillo, Bruno; Weisz, Alessandro

2004-01-01

Transcriptional activation of the cyclin D1 gene (CCND1) plays a pivotal role in G1-phase progression, which is thereby controlled by multiple regulatory factors, including nuclear receptors (NRs). Appropriate CCND1 gene activity is essential for normal development and physiology of the mammary gland, where it is regulated by ovarian steroids through a mechanism(s) that is not fully elucidated. We report here that CCND1 promoter activation by estrogens in human breast cancer cells is mediated by recruitment of a c-Jun/c-Fos/estrogen receptor α complex to the tetradecanoyl phorbol acetate-responsive element of the gene, together with Oct-1 to a site immediately adjacent. This process coincides with the release from the same DNA region of a transcriptional repressor complex including Yin-Yang 1 (YY1) and histone deacetylase 1 and is sufficient to induce the assembly of the basal transcription machinery on the promoter and to lead to initial cyclin D1 accumulation in the cell. Later on in estrogen stimulation, the cyclin D1/Cdk4 holoenzyme associates with the CCND1 promoter, where E2F and pRb can also be found, contributing to the long-lasting gene enhancement required to drive G1-phase completion. Interestingly, progesterone triggers similar regulatory events through its own NRs, suggesting that the gene regulation cascade described here represents a crossroad for the transcriptional control of G1-phase progression by different classes of NRs. PMID:15282324

Environmental cues induce changes of steviol glycosides contents and transcription of corresponding biosynthetic genes in Stevia rebaudiana.

PubMed

Yang, Yongheng; Huang, Suzhen; Han, Yulin; Yuan, Haiyan; Gu, Chunsun; Wang, Zhongwei

2015-01-01

Plant growth and secondary metabolism are commonly regulated by external cues such as light, temperature and water availability. In this study, the influences of low and high temperatures, dehydration, photoperiods, and different growing stages on the changes of steviol glycosides (SGs) contents and transcription levels of fifteen genes involved in SGs biosynthesis of Stevia rebaudiana Bertoni were examined using HPLC and RT-PCR. The observations showed that the transcript levels of all the fifteen genes were maximum under 25 °C treatment, and the transcription of SrDXS, SrDXR, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI, SrGGDPS, SrCPPS1, SrUGT85C2 and SrUGT76G1 were restrained both in low temperature (15 °C) and high temperature (35 °C). Most genes in SGs biosynthesis pathway exhibited down-regulation in dehydration. To elucidate the effect of photoperiods, the plants were treated by different simulated photoperiods (8 L/16 D, 1 0L/14 D, 14 L/10 D and 16 L/8 D), but no significant transcription changes were observed. In the study of growing stages, there were evident changes of SGs contents, and the transcript levels of all the fifteen genes were minimal in fast growing period, and exhibited evident increase both in flower-bud appearing stage and flowering stage. The obtained results strongly suggest that the effect of environmental cues on steviol glycosides contents and transcription of corresponding biosynthetic genes in S. rebaudiana is significant. It is worth to study deeply. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Integrating Gene Expression with Summary Association Statistics to Identify Genes Associated with 30 Complex Traits.

PubMed

Mancuso, Nicholas; Shi, Huwenbo; Goddard, Pagé; Kichaev, Gleb; Gusev, Alexander; Pasaniuc, Bogdan

2017-03-02

Although genome-wide association studies (GWASs) have identified thousands of risk loci for many complex traits and diseases, the causal variants and genes at these loci remain largely unknown. Here, we introduce a method for estimating the local genetic correlation between gene expression and a complex trait and utilize it to estimate the genetic correlation due to predicted expression between pairs of traits. We integrated gene expression measurements from 45 expression panels with summary GWAS data to perform 30 multi-tissue transcriptome-wide association studies (TWASs). We identified 1,196 genes whose expression is associated with these traits; of these, 168 reside more than 0.5 Mb away from any previously reported GWAS significant variant. We then used our approach to find 43 pairs of traits with significant genetic correlation at the level of predicted expression; of these, eight were not found through genetic correlation at the SNP level. Finally, we used bi-directional regression to find evidence that BMI causally influences triglyceride levels and that triglyceride levels causally influence low-density lipoprotein. Together, our results provide insight into the role of gene expression in the susceptibility of complex traits and diseases. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

PubMed Central

2011-01-01

Abstract Background Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway. Results One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with β-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with β-AS in various B. chinense tissues. Conclusions A collection of high

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

Effect of salinity on gene expression, morphological and biochemical characteristics of stevia rebaudiana Bertoni under in vitro conditions.

PubMed

Fallah, F; Nokhasi, F; Ghaheri, M; Kahrizi, D; Beheshti Ale Agha, A; Ghorbani, T; Kazemi, E; Ansarypour, Z

2017-08-15

Stevia rebaudiana Bertoni is a famous medicinal plant for its low calorific value compounds which are named steviol glycosides (SGs) and they are 150-300 times sweeter than sugar. Among various SGs, stevioside and rebaudioside A considered to be the main sweetening compounds.  Soil salinity is one of the most essential stress in the world. Salinity affects the survival and yield of crops. In current study the effects of salinity and osmotic stress caused by different concentration of NaCl (0, 20, 40, 60 and 80 mM) on morphological traits, genes expressionand amount of both stevioside and rebaudioside Aunder in vitro conditions has been investigated. The morphological traits such as bud numbers, root numbers, shoot length (after 15 and 30 days) were evaluated. With increasing salinity, the values of all studied morphological traits decreased. To investigation of UGT74G1 and UGT76G1 genes expression that are involved in the synthesis of SGs, RT-PCR was done and there were significant differences between all media. The highest expression of both genes was observed in plantlets grown on MS media (with NaCl-free). Also, the lowest amounts of gene expression of the both genes were seen in MS+ 60 mM NaCl. Based on HPLC results, the highest amount of both stevioside and rebaudioside A were observed in plantlets grown in MS media (with NaCl-free). Finally, it can be concluded that stevia can survive under salt stress, but it has the best performance in the lower salinity.

Polymorphisms in heterocyclic aromatic amines metabolism-related genes are associated with colorectal adenoma risk

PubMed Central

Eichholzer, Monika; Rohrmann, Sabine; Barbir, Aline; Hermann, Silke; Teucher, Birgit; Kaaks, Rudolf; Linseisen, Jakob

2012-01-01

Colorectal adenoma (CRA) and colorectal cancer (CRC) risks have been linked to the intake of red and processed meat. Heterocyclic aromatic amines (HCA) formed herein during high temperature cooking, are metabolized by a variety of enzymes, and allelic variation in the coding genes could influence individual CRA risk. Associations of polymorphisms in NAT1, NAT2, GSTA1, SULT1A1, CYP1A2, UGT1A7, UGT1A9, GSTP1 genes with colorectal adenoma risk were investigated in a nested case-control study of the EPIC-Heidelberg cohort including 428 cases matched by age, sex and year of recruitment with one or two controls (n=828) with negative colonoscopy per case. Genoyping was preformed with the Sequenom MassArray system and the LightCycler 480. Conditional logistic regression was used to compute odds ratios (OR) and corresponding 95% confidence intervals (CI). For rs15561 (NAT1) and rs1057126 (NAT1), the rarer allel was significantly inversely associated with adenoma risk OR=0.80 (95% CI 0.65-0.97) and (OR=0.81 (95% CI 0.65-0.99) and, respectively). For the combined NAT2 alleles encoding for enzymes with medium (versus slow) activity we also observed a significantly inverse association with adenoma risk (OR=0.75; 95% CI 0.85-0.97). In addition, homozygous carriers of the A allele of rs3957357 (GSTA1), i.e., those with a decreased enzyme activity, had a decreased risk of colorectal adenoma with an OR of 0.68 (95% CI 0.50-0.92; AA versus GG/GA). Polymorphisms in the other tested genes did not modify the risk of colorectal adenomas. In conclusion, polymorphisms in NAT1, NAT2, and GSTA1 are related to colorectal adenoma risk in this German cohort. PMID:22724046

Polymorphisms in heterocyclic aromatic amines metabolism-related genes are associated with colorectal adenoma risk.

PubMed

Eichholzer, Monika; Rohrmann, Sabine; Barbir, Aline; Hermann, Silke; Teucher, Birgit; Kaaks, Rudolf; Linseisen, Jakob

2012-01-01

Colorectal adenoma (CRA) and colorectal cancer (CRC) risks have been linked to the intake of red and processed meat. Heterocyclic aromatic amines (HCA) formed herein during high temperature cooking, are metabolized by a variety of enzymes, and allelic variation in the coding genes could influence individual CRA risk. Associations of polymorphisms in NAT1, NAT2, GSTA1, SULT1A1, CYP1A2, UGT1A7, UGT1A9, GSTP1 genes with colorectal adenoma risk were investigated in a nested case-control study of the EPIC-Heidelberg cohort including 428 cases matched by age, sex and year of recruitment with one or two controls (n=828) with negative colonoscopy per case. Genoyping was preformed with the Sequenom MassArray system and the LightCycler 480. Conditional logistic regression was used to compute odds ratios (OR) and corresponding 95% confidence intervals (CI). For rs15561 (NAT1) and rs1057126 (NAT1), the rarer allel was significantly inversely associated with adenoma risk OR=0.80 (95% CI 0.65-0.97) and (OR=0.81 (95% CI 0.65-0.99) and, respectively). For the combined NAT2 alleles encoding for enzymes with medium (versus slow) activity we also observed a significantly inverse association with adenoma risk (OR=0.75; 95% CI 0.85-0.97). In addition, homozygous carriers of the A allele of rs3957357 (GSTA1), i.e., those with a decreased enzyme activity, had a decreased risk of colorectal adenoma with an OR of 0.68 (95% CI 0.50-0.92; AA versus GG/GA). Polymorphisms in the other tested genes did not modify the risk of colorectal adenomas. In conclusion, polymorphisms in NAT1, NAT2, and GSTA1 are related to colorectal adenoma risk in this German cohort.

Biogenesis of the yeast cytochrome bc1 complex.

PubMed

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-01-01

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

Safety study and characterization of E1A-liposome complex gene-delivery protocol in an ovarian cancer model.

PubMed

Xing, X; Zhang, S; Chang, J Y; Tucker, S D; Chen, H; Huang, L; Hung, M C

1998-11-01

A phase I clinical trial of E1A-liposome complex is currently ongoing in patients with HER-2/neu-overexpressing breast or ovarian cancers. To optimize the E1A-liposome complex for a further stage of clinical trial, several aspects of the current protocol have been examined in an animal model. In the orthotopic ovarian cancer model, different doses of lipid in the the E1A-liposome complex, which is currently used in clinical trials, were tested for the in vivo gene-transfer efficacy and tumor-suppression function. A lowered lipid dose--1/13 of the previous amount--produced gene expression level and E1A tumor-suppression efficacy similar to that of the original protocol. Mini-E1A, an E1A construct without its immortalization domain and yet capable of repressing HER-2/neu, was proved to be as potent as E1A in suppressing tumor development in vivo. These changes in the E1A-liposome complex will significantly reduce any potential adverse effects caused by lipid vector and E1A DNA. To examine further whether residual E1A DNA may still exist in normal organs after the E1A-liposome treatment, PCR was used to detect E1A DNA in mice that survived for 1 1/2 years after the last treatment. E1A DNA was detected only in the lungs and kidneys, but not in livers, hearts, spleens, brains, uterus or the ovaries. Furthermore, resistance of the E1A DNA extracted from tissues to the digestion of Dpnl restriction enzyme, which can cleave the methylated E1A plasmid DNA generated by methylation-competent bacteria, suggested integration of E1A DNA into the chromosome of the lungs and kidneys. Experimental results presented here provide important information for safety concerns and for the design of future phase II and phase III trials.

The intratumoral balance between metabolic and immunologic gene expression is associated with anti-PD-1 response in patients with renal cell carcinoma

PubMed Central

Ascierto, Maria Libera; McMiller, Tracee L.; Berger, Alan E.; Danilova, Ludmila; Anders, Robert A.; Netto, George J.; Xu, Haiying; Pritchard, Theresa S.; Fan, Jinshui; Cheadle, Chris; Cope, Leslie; Drake, Charles G.; Pardoll, Drew M.; Taube, Janis M.; Topalian, Suzanne L.

2016-01-01

Pretreatment tumor PD-L1 expression correlates with response to anti-PD-1/PD-L1 therapies. Yet, most patients with PD-L1+ tumors do not respond to treatment. The current study was undertaken to investigate mechanisms underlying the failure of PD-1–targeted therapies in patients with advanced renal cell carcinoma (RCC) whose tumors express PD-L1. Formalin-fixed, paraffin-embedded (FFPE) pretreatment tumor biopsies expressing PD-L1 were derived from 13 RCC patients. RNA was isolated from PD-L1+ regions and subjected to whole genome microarray and multiplex quantitative (q)RT-PCR gene expression analysis. A balance between gene expression profiles reflecting metabolic pathways and immune functions was associated with clinical outcomes following anti-PD-1 therapy. In particular, the expression of genes involved in metabolic and solute transport functions such as UGT1A family members, also found in kidney cancer cell lines, was associated with treatment failure in patients with PD-L1+ RCC. Conversely, tumors from responding patients overexpressed immune markers such as BACH2, a regulator of CD4+ T cell differentiation, and CCL3, involved in leukocyte migration. These findings suggest that tumor cell–intrinsic metabolic factors may contribute to treatment resistance in RCC, thus serving as predictive markers for treatment outcomes and potential new targets for combination therapy regimens with anti-PD-1. PMID:27491898

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.

Acetylation of histone deacetylase 1 regulates NuRD corepressor complex activity.

PubMed

Yang, Tao; Jian, Wei; Luo, Yi; Fu, Xueqi; Noguchi, Constance; Bungert, Jörg; Huang, Suming; Qiu, Yi

2012-11-23

HDAC1-containing NuRD complex is required for GATA-1-mediated repression and activation. GATA-1 associated with acetylated HDAC1-containing NuRD complex, which has no deacetylase activity, for gene activation. Acetylated HDAC1 converts NuRD complex from a repressor to an activator during GATA-1-directed erythroid differentiation program. HDAC1 acetylation may function as a master regulator for the activity of HDAC1 containing complexes. Histone deacetylases (HDACs) play important roles in regulating cell proliferation and differentiation. The HDAC1-containing NuRD complex is generally considered as a corepressor complex and is required for GATA-1-mediated repression. However, recent studies also show that the NuRD complex is involved in GATA-1-mediated gene activation. We tested whether the GATA-1-associated NuRD complex loses its deacetylase activity and commits the GATA-1 complex to become an activator during erythropoiesis. We found that GATA-1-associated deacetylase activity gradually decreased upon induction of erythroid differentiation. GATA-1-associated HDAC1 is increasingly acetylated after differentiation. It has been demonstrated earlier that acetylated HDAC1 has no deacetylase activity. Indeed, overexpression of an HDAC1 mutant, which mimics acetylated HDAC1, promotes GATA-1-mediated transcription and erythroid differentiation. Furthermore, during erythroid differentiation, acetylated HDAC1 recruitment is increased at GATA-1-activated genes, whereas it is significantly decreased at GATA-1-repressed genes. Interestingly, deacetylase activity is not required for Mi2 remodeling activity, suggesting that remodeling activity may be required for both activation and repression. Thus, our data suggest that NuRD can function as a coactivator or repressor and that acetylated HDAC1 converts the NuRD complex from a repressor to an activator during GATA-1-directed erythroid differentiation.

Clinical validity of new genetic biomarkers of irinotecan neutropenia: an independent replication study.

PubMed

Crona, D J; Ramirez, J; Qiao, W; de Graan, A-J; Ratain, M J; van Schaik, R H N; Mathijssen, R H J; Rosner, G L; Innocenti, F

2016-02-01

The overall goal of this study was to provide evidence for the clinical validity of nine genetic variants in five genes previously associated with irinotecan neutropenia and pharmacokinetics. Variants associated with absolute neutrophil count (ANC) nadir and/or irinotecan pharmacokinetics in a discovery cohort of cancer patients were genotyped in an independent replication cohort of 108 cancer patients. Patients received single-agent irinotecan every 3 weeks. For ANC nadir, we replicated UGT1A1*28, UGT1A1*93 and SLCO1B1*1b in univariate analyses. For irinotecan area under the concentration-time curve (AUC0-24), we replicated ABCC2 -24C>T; however, ABCC2 -24C>T only predicted a small fraction of the variance. For SN-38 AUC0-24 and the glucuronidation ratio, we replicated UGT1A1*28 and UGT1A1*93. In addition to UGT1A1*28, this study independently validated UGT1A1*93 and SLCO1B1*1b as new predictors of irinotecan neutropenia. Further demonstration of their clinical utility will optimize irinotecan therapy in cancer patients.

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.

Common Genetic Variation In Cellular Transport Genes and Epithelial Ovarian Cancer (EOC) Risk

PubMed Central

Chornokur, Ganna; Lin, Hui-Yi; Tyrer, Jonathan P.; Lawrenson, Kate; Dennis, Joe; Amankwah, Ernest K.; Qu, Xiaotao; Tsai, Ya-Yu; Jim, Heather S. L.; Chen, Zhihua; Chen, Ann Y.; Permuth-Wey, Jennifer; Aben, Katja KH.; Anton-Culver, Hoda; Antonenkova, Natalia; Bruinsma, Fiona; Bandera, Elisa V.; Bean, Yukie T.; Beckmann, Matthias W.; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A.; Brooks-Wilson, Angela; Bunker, Clareann H.; Butzow, Ralf; Campbell, Ian G.; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S.; Cramer, Daniel W.; Cunningham, Julie M.; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; du Bois, Andreas; Despierre, Evelyn; Dicks, Ed; Doherty, Jennifer A.; Dörk, Thilo; Dürst, Matthias; Easton, Douglas F.; Eccles, Diana M.; Edwards, Robert P.; Ekici, Arif B.; Fasching, Peter A.; Fridley, Brooke L.; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G.; Glasspool, Rosalind; Goodman, Marc T.; Gronwald, Jacek; Harrington, Patricia; Harter, Philipp; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A. T.; Hillemanns, Peter; Hogdall, Claus K.; Hogdall, Estrid; Hosono, Satoyo; Jakubowska, Anna; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y.; Kelemen, Linda E.; Kellar, Mellissa; Kiemeney, Lambertus A.; Krakstad, Camilla; Kjaer, Susanne K.; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D.; Lee, Alice W.; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A.; Liang, Dong; Lim, Boon Kiong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F. A. G.; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R.; McNeish, Iain; Menon, Usha; Milne, Roger L.; Modugno, Francesmary; Moysich, Kirsten B.; Ness, Roberta B.; Nevanlinna, Heli; Eilber, Ursula; Odunsi, Kunle; Olson, Sara H.; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Paul, James; Pearce, Celeste L.; Pejovic, Tanja; Pelttari, Liisa M.; Pike, Malcolm C.; Poole, Elizabeth M.; Risch, Harvey A.; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H.; Rudolph, Anja; Runnebaum, Ingo B.; Rzepecka, Iwona K.; Salvesen, Helga B.; Schernhammer, Eva; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B.; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C.; Spiewankiewicz, Beata; Sucheston, Lara; Teo, Soo-Hwang; Terry, Kathryn L.; Thompson, Pamela J.; Thomsen, Lotte; Tangen, Ingvild L.; Tworoger, Shelley S.; van Altena, Anne M.; Vierkant, Robert A.; Vergote, Ignace; Walsh, Christine S.; Wang-Gohrke, Shan; Wentzensen, Nicolas; Whittemore, Alice S.; Wicklund, Kristine G.; Wilkens, Lynne R.; Wu, Anna H.; Wu, Xifeng; Woo, Yin-Ling; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Hasmad, Hanis N.; Berchuck, Andrew; Iversen, Edwin S.; Schildkraut, Joellen M.; Ramus, Susan J.; Goode, Ellen L.; Monteiro, Alvaro N. A.; Gayther, Simon A.; Narod, Steven A.; Pharoah, Paul D. P.; Sellers, Thomas A.; Phelan, Catherine M.

2015-01-01

Background Defective cellular transport processes can lead to aberrant accumulation of trace elements, iron, small molecules and hormones in the cell, which in turn may promote the formation of reactive oxygen species, promoting DNA damage and aberrant expression of key regulatory cancer genes. As DNA damage and uncontrolled proliferation are hallmarks of cancer, including epithelial ovarian cancer (EOC), we hypothesized that inherited variation in the cellular transport genes contributes to EOC risk. Methods In total, DNA samples were obtained from 14,525 case subjects with invasive EOC and from 23,447 controls from 43 sites in the Ovarian Cancer Association Consortium (OCAC). Two hundred seventy nine SNPs, representing 131 genes, were genotyped using an Illumina Infinium iSelect BeadChip as part of the Collaborative Oncological Gene-environment Study (COGS). SNP analyses were conducted using unconditional logistic regression under a log-additive model, and the FDR q<0.2 was applied to adjust for multiple comparisons. Results The most significant evidence of an association for all invasive cancers combined and for the serous subtype was observed for SNP rs17216603 in the iron transporter gene HEPH (invasive: OR = 0.85, P = 0.00026; serous: OR = 0.81, P = 0.00020); this SNP was also associated with the borderline/low malignant potential (LMP) tumors (P = 0.021). Other genes significantly associated with EOC histological subtypes (p<0.05) included the UGT1A (endometrioid), SLC25A45 (mucinous), SLC39A11 (low malignant potential), and SERPINA7 (clear cell carcinoma). In addition, 1785 SNPs in six genes (HEPH, MGST1, SERPINA, SLC25A45, SLC39A11 and UGT1A) were imputed from the 1000 Genomes Project and examined for association with INV EOC in white-European subjects. The most significant imputed SNP was rs117729793 in SLC39A11 (per allele, OR = 2.55, 95% CI = 1.5-4.35, p = 5.66x10-4). Conclusion These results, generated on a large cohort of women, revealed associations

Mi2β Is Required for γ-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in β-YAC Transgenic Mice

PubMed Central

Costa, Flávia C.; Fedosyuk, Halyna; Chazelle, Allen M.; Neades, Renee Y.; Peterson, Kenneth R.

2012-01-01

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. PMID:23284307

Mi2β is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice.

PubMed

Costa, Flávia C; Fedosyuk, Halyna; Chazelle, Allen M; Neades, Renee Y; Peterson, Kenneth R

2012-01-01

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (A)γ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of (A)γ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting -566 (A)γ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

PubMed

Kubešová, Kateřina; Dořičáková, Aneta; Trávníček, Zdeněk; Dvořák, Zdeněk

2016-07-25

The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effect of KH2PO4 on gene expression, morphological and biochemical characteristics of stevia rebaudiana Bertoni under in vitro conditions.

PubMed

Kahrizi, D; Ghari, S M; Ghaheri, M; Fallah, F; Ghorbani, T; Beheshti Ale Agha, A; Kazemi, E; Ansarypour, Z

2017-08-15

Stevia rebaudiana is one of the most important biologically sourced and low-calorie sweeteners Bertoni that has a lot of steviol glycosides. Tissue culture is the best for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. Therefore, the effect of different concentrations of KH2PO4on stevia growth factors and gene expression had been studied by tissue culture methods, RT-PCR and HPLC. According the results, bud numbers had increased significantly in MS + 0.034 mMKH2PO4 media and the highest measured length was seen in plants grown under MS + 0.034 mM KH2PO4 treatment. Also, the highest growth rate (1.396 mm/d) was observed in MS + 0.034 mMKH2PO4.The best concentration of KH2PO4 for expression of UGT74G1 was 0.00425mMand the best one for UGT76G1 expression was 0.017mM. Interestingly, the best media for both stevioside and rebaudioside A accumulation was 0.017mM KH2PO4containing media. There was positive correlation between the best media for gene expression and the best one for steviol glycosides production.

LWD-TCP complex activates the morning gene CCA1 in Arabidopsis.

PubMed

Wu, Jing-Fen; Tsai, Huang-Lung; Joanito, Ignasius; Wu, Yi-Chen; Chang, Chin-Wen; Li, Yi-Hang; Wang, Ying; Hong, Jong Chan; Chu, Jhih-Wei; Hsu, Chao-Ping; Wu, Shu-Hsing

2016-10-13

A double-negative feedback loop formed by the morning genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and the evening gene TIMING OF CAB EXPRESSION1 (TOC1) contributes to regulation of the circadian clock in Arabidopsis. A 24-h circadian cycle starts with the peak expression of CCA1 at dawn. Although CCA1 is targeted by multiple transcriptional repressors, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and CCA1 HIKING EXPEDITION (CHE), activators of CCA1 remain elusive. Here we use mathematical modelling to infer a co-activator role for LIGHT-REGULATED WD1 (LWD1) in CCA1 expression. We show that the TEOSINTE BRANCHED 1-CYCLOIDEA-PCF20 (TCP20) and TCP22 proteins act as LWD-interacting transcriptional activators. The concomitant binding of LWD1 and TCP20/TCP22 to the TCP-binding site in the CCA1 promoter activates CCA1. Our study reveals activators of the morning gene CCA1 and provides an action mechanism that ensures elevated expression of CCA1 at dawn to sustain a robust clock.

LWD–TCP complex activates the morning gene CCA1 in Arabidopsis

PubMed Central

Wu, Jing-Fen; Tsai, Huang-Lung; Joanito, Ignasius; Wu, Yi-Chen; Chang, Chin-Wen; Li, Yi-Hang; Wang, Ying; Hong, Jong Chan; Chu, Jhih-Wei; Hsu, Chao-Ping; Wu, Shu-Hsing

2016-01-01

A double-negative feedback loop formed by the morning genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and the evening gene TIMING OF CAB EXPRESSION1 (TOC1) contributes to regulation of the circadian clock in Arabidopsis. A 24-h circadian cycle starts with the peak expression of CCA1 at dawn. Although CCA1 is targeted by multiple transcriptional repressors, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and CCA1 HIKING EXPEDITION (CHE), activators of CCA1 remain elusive. Here we use mathematical modelling to infer a co-activator role for LIGHT-REGULATED WD1 (LWD1) in CCA1 expression. We show that the TEOSINTE BRANCHED 1-CYCLOIDEA-PCF20 (TCP20) and TCP22 proteins act as LWD-interacting transcriptional activators. The concomitant binding of LWD1 and TCP20/TCP22 to the TCP-binding site in the CCA1 promoter activates CCA1. Our study reveals activators of the morning gene CCA1 and provides an action mechanism that ensures elevated expression of CCA1 at dawn to sustain a robust clock. PMID:27734958

Fanconi Anemia Core Complex Gene Promoters Harbor Conserved Transcription Regulatory Elements

PubMed Central

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5′ region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3′ regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters. PMID:21826217

Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

PubMed

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

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.

Characterization of the telomere complex, TERF1 and TERF2 genes in muntjac species with fusion karyotypes

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

Hartmann, Nils; Scherthan, Harry

The telomere binding proteins TRF1 and TRF2 maintain and protect chromosome ends and confer karyotypic stability. Chromosome evolution in the genus Muntiacus is characterized by numerous tandem (end-to-end) fusions. To study TRF1 and TRF2 telomere binding proteins in Muntiacus species, we isolated and characterized the TERF1 and -2 genes from Indian muntjac (Muntiacus muntjak vaginalis; 2n = 6 female) and from Chinese muntjac (Muntiacus reveesi; 2n = 46). Expression analysis revealed that both genes are ubiquitously expressed and sequence analysis identified several transcript variants of both TERF genes. Control experiments disclosed a novel testis-specific splice variant of TERF1 in humanmore » testes. Amino acid sequence comparisons demonstrate that Muntiacus TRF1 and in particular TRF2 are highly conserved between muntjac and human. In vivo TRF2-GFP and immuno-staining studies in muntjac cell lines revealed telomeric TRF2 localization, while deletion of the DNA binding domain abrogated this localization, suggesting muntjac TRF2 represents a functional telomere protein. Finally, expression analysis of a set of telomere-related genes revealed their presence in muntjac fibroblasts and testis tissue, which suggests the presence of a conserved telomere complex in muntjacs. However, a deviation from the common theme was noted for the TERT gene, encoding the catalytic subunit of telomerase; TERT expression could not be detected in Indian or Chinese muntjac cDNA or genomic DNA using a series of conserved primers, while TRAP assay revealed functional telomerase in Chinese muntjac testis tissues. This suggests muntjacs may harbor a diverged telomerase sequence.« less

The DREAM complex: Master coordinator of cell cycle dependent gene expression

PubMed Central

Sadasivam, Subhashini; DeCaprio, James A.

2014-01-01

Preface The dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM) complex provides a previously unsuspected unifying role in the cell cycle by directly linking p130, p107, E2F, BMYB and FOXM1. DREAM mediates gene repression during G0 and coordinates periodic gene expression with peaks during G1/S and G2/M. Perturbations in DREAM regulation shift the balance from quiescence towards proliferation and contribute to increased mitotic gene expression levels frequently observed in cancers with poor prognosis. PMID:23842645

Acetylation of Histone Deacetylase 1 Regulates NuRD Corepressor Complex Activity*

PubMed Central

Yang, Tao; Jian, Wei; Luo, Yi; Fu, Xueqi; Noguchi, Constance; Bungert, Jörg; Huang, Suming; Qiu, Yi

2012-01-01

Histone deacetylases (HDACs) play important roles in regulating cell proliferation and differentiation. The HDAC1-containing NuRD complex is generally considered as a corepressor complex and is required for GATA-1-mediated repression. However, recent studies also show that the NuRD complex is involved in GATA-1-mediated gene activation. We tested whether the GATA-1-associated NuRD complex loses its deacetylase activity and commits the GATA-1 complex to become an activator during erythropoiesis. We found that GATA-1-associated deacetylase activity gradually decreased upon induction of erythroid differentiation. GATA-1-associated HDAC1 is increasingly acetylated after differentiation. It has been demonstrated earlier that acetylated HDAC1 has no deacetylase activity. Indeed, overexpression of an HDAC1 mutant, which mimics acetylated HDAC1, promotes GATA-1-mediated transcription and erythroid differentiation. Furthermore, during erythroid differentiation, acetylated HDAC1 recruitment is increased at GATA-1-activated genes, whereas it is significantly decreased at GATA-1-repressed genes. Interestingly, deacetylase activity is not required for Mi2 remodeling activity, suggesting that remodeling activity may be required for both activation and repression. Thus, our data suggest that NuRD can function as a coactivator or repressor and that acetylated HDAC1 converts the NuRD complex from a repressor to an activator during GATA-1-directed erythroid differentiation. PMID:23014989

Identifying driving gene clusters in complex diseases through critical transition theory

NASA Astrophysics Data System (ADS)

Wolanyk, Nathaniel; Wang, Xujing; Hessner, Martin; Gao, Shouguo; Chen, Ye; Jia, Shuang

A novel approach of looking at the human body using critical transition theory has yielded positive results: clusters of genes that act in tandem to drive complex disease progression. This cluster of genes can be thought of as the first part of a large genetic force that pushes the body from a curable, but sick, point to an incurable diseased point through a catastrophic bifurcation. The data analyzed is time course microarray blood assay data of 7 high risk individuals for Type 1 Diabetes who progressed into a clinical onset, with an additional larger study requested to be presented at the conference. The normalized data is 25,000 genes strong, which were narrowed down based on statistical metrics, and finally a machine learning algorithm using critical transition metrics found the driving network. This approach was created to be repeatable across multiple complex diseases with only progression time course data needed so that it would be applicable to identifying when an individual is at risk of developing a complex disease. Thusly, preventative measures can be enacted, and in the longer term, offers a possible solution to prevent all Type 1 Diabetes.

Genomic and Coexpression Analyses Predict Multiple Genes Involved in Triterpene Saponin Biosynthesis in Medicago truncatula[C][W

PubMed Central

Naoumkina, Marina A.; Modolo, Luzia V.; Huhman, David V.; Urbanczyk-Wochniak, Ewa; Tang, Yuhong; Sumner, Lloyd W.; Dixon, Richard A.

2010-01-01

Saponins, an important group of bioactive plant natural products, are glycosides of triterpenoid or steroidal aglycones (sapogenins). Saponins possess many biological activities, including conferring potential health benefits for humans. However, most of the steps specific for the biosynthesis of triterpene saponins remain uncharacterized at the molecular level. Here, we use comprehensive gene expression clustering analysis to identify candidate genes involved in the elaboration, hydroxylation, and glycosylation of the triterpene skeleton in the model legume Medicago truncatula. Four candidate uridine diphosphate glycosyltransferases were expressed in Escherichia coli, one of which (UGT73F3) showed specificity for multiple sapogenins and was confirmed to glucosylate hederagenin at the C28 position. Genetic loss-of-function studies in M. truncatula confirmed the in vivo function of UGT73F3 in saponin biosynthesis. This report provides a basis for future studies to define genetically the roles of multiple cytochromes P450 and glycosyltransferases in triterpene saponin biosynthesis in Medicago. PMID:20348429

TGF-β1 gene-engineered mesenchymal stem cells induce rat cartilage regeneration using nonviral gene vector.

PubMed

He, Cai-Xia; Zhang, Tian-Yuan; Miao, Pei-Hong; Hu, Zhong-Jie; Han, Min; Tabata, Yasuhiko; Hu, Yu-Lan; Gao, Jian-Qing

2012-01-01

This study evaluated the potential of utilizing transfected pTGFβ-1 gene-engineered rat mesenchymal stem cells (MSCs) using nonviral vector to promote cartilage regeneration. Pullulan-spermine was used as the nonviral gene vector and gelatin sponge was used as the scaffold. MSCs were engineered with TGF-β1 gene with either the three-dimensional (3D) reverse transfection system or the two-dimensional (2D) conventional transfection system. For the 3D reverse transfection system, pullulan-spermine/pTGF-β1 gene complexes were immobilized to the gelatin sponge, followed by the seeding of MSCs. Pullulan-spermine/pTGF-β1 gene complexes were delivered to MSCs cultured in the plate to perform the 2D conventional transfection system, and then MSCs were seeded to the gelatin sponge. Then, TGF-β1 gene-transfected MSC seeded gelatin sponge was implanted to the full-thickness cartilage defect. Compared with the control group, both groups of TGF-β1 gene-engineered MSCs improved cartilage regeneration through optical observation and histology staining. So, with pullulan-spermine as the nonviral vector, TGF-β1-gene engineered MSCs can induce cartilage regeneration in vivo. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Nrf2 target genes are induced under marginal selenium-deficiency

PubMed Central

Müller, Mike; Banning, Antje; Brigelius-Flohé, Regina

2010-01-01

A suboptimal selenium supply appears to prevail in Europe. The current study, therefore, was focused on the changes in gene expression under a suboptimal selenium intake. Previous microarray analyses in the colon of mice fed either a selenium-adequate or a moderately deficient diet revealed a change in genes of several pathways. Severe selenium-deficiency has been found previously to influence Nrf2-regulated genes of the adaptive response. Since the previous pathway analyses were done with a program not searching for Nrf2 target genes, respective genes were manually selected and confirmed by qPCR. qPCR revealed an induction of phase II (Nqo1, Gsts, Sult1b1 and Ugt1a6) and antioxidant enzymes (Hmox1, Mt2, Prdx1, Srxn1, Sod1 and Gclc) under the selenium-poor diet, which is considered to compensate for the loss of selenoproteins. The strongest effects were observed in the duodenum where preferentially genes for antioxidant enzymes were up-regulated. These also include the mRNA of the selenoproteins TrxR1 and GPx2 that would enable their immediate translation upon selenium refeeding. The down-regulation of Gsk3β in moderate selenium-deficiency observed in the previous paper provides a possible explanation for the activation of the Nrf2 pathway, because inhibition of GSK3β results in the nuclear accumulation of Nrf2. PMID:21189866