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Sample records for 2d6 substrate activity

  1. QSAR Models for P-450 (2D6) Substrate Activity

    Ringsted, Tine; Nikolov, Nikolai Georgiev; Jensen, Gunde Egeskov;

    2009-01-01

    activity relationship (QSAR) modelling systems. They cross validated (leave-groups-out) with concordances of 71%, 81% and 82%, respectively. Discrete organic European Inventory of Existing Commercial Chemical Substances (EINECS) chemicals were screened to predict an approximate percentage of CYP 2D6...... substrates. These chemicals are potentially present in the environment. The biological importance of the CYP 2D6 and the use of the software mentioned above were discussed....

  2. CYP2D6*1,CYP2D6*10 co-expressed with CYPOR in Bac-to-Bac expression system and activity determination%与CYPOR共表达CYP2D6*1和CYP2D6*10及其代谢活性比较

    钱鸣蓉; 陈静; 刘瑶; 余露山; 陈枢青; 曾苏

    2011-01-01

    FastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6 *1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6 *1 and CYP2D6 *10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined witb dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67±2.71 μmol·L-1 (n=3) and 666.7±56.78 pmol·nmol-1(CYP2D6)·min-1 (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36±10.89 μmol·L-1 (n=3) and 222.2±20.12 pmol·nmol-1(CYP2D6)·min-1 (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P<0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.

  3. Identification of cytochrome P450 2D6 and 2C9 substrates and inhibitors by QSAR analysis

    Jónsdóttir, Svava Ósk; Ringsted, Tine; Nikolov, Nikolai G.;

    2012-01-01

    This paper presents four new QSAR models for CYP2C9 and CYP2D6 substrate recognition and inhibitor identification based on human clinical data. The models were used to screen a large data set of environmental chemicals for CYP activity, and to analyze the frequency of CYP activity among these...... compounds. A large fraction of these chemicals were found to be CYP active, and thus potentially capable of affecting human physiology. 20% of the compounds within applicability domain of the models were predicted to be CYP2C9 substrates, and 17% to be inhibitors. The corresponding numbers for CYP2D6 were 9......% and 21%. Where the majority of CYP2C9 active compounds were predicted to be both a substrate and an inhibitor at the same time, the CYP2D6 active compounds were primarily predicted to be only inhibitors. It was demonstrated that the models could identify compound classes with a high occurrence of...

  4. The Psychostimulant Khat (Catha edulis) Inhibits CYP2D6 Enzyme Activity in Humans.

    Bedada, Worku; de Andrés, Fernando; Engidawork, Ephrem; Pohanka, Anton; Beck, Olof; Bertilsson, Leif; Llerena, Adrián; Aklillu, Eleni

    2015-12-01

    The use of khat (Catha edulis) while on medication may alter treatment outcome. In particular, the influence of khat on the metabolic activities of drug-metabolizing enzymes is not known. We performed a comparative 1-way crossover study to evaluate the effect of khat on cytochrome P450 (CYP)2D6 and CYP3A4 enzyme activity. After 1 week of khat abstinence, baseline CYP2D6 and CYP3A4 metabolic activities were determined in 40 Ethiopian male volunteers using 30 mg dextromethorphan (DM) as a probe drug and then repeated after 1 week of daily use of 400 g fresh khat leaves. Urinary concentrations of cathinone and cathine were determined to monitor the subjects' compliance to the study protocol. Genotyping for CYP2D6*3 and CYP2D6*4 was done. Plasma DM, dextrorphan and 3-methoxymorphinan concentrations were quantified. CYP2D6 and CYP3A4 enzyme activities were assessed by comparing plasma log DM/dextrorphan and log DM/methoxymorphinan metabolic ratio (MR) respectively in the presence and absence of khat. Cytochrome 2D6 MR was significantly increased from baseline by concurrent khat use (paired t test, P = 0.003; geometric mean ratio, 1.38; 95% confidence interval [95% CI], 1.12-1.53). Moreover, the inhibition of CYP2D6 activity by khat was more pronounced in CYP2D6*1/*1 compared with CYP2D6*1/*4 genotypes (P = 0.01). A marginal inhibition of CYP3A4 activity in the presence of khat was observed (P = 0.24). The mean percentage increase of CYP2D6 and CYP3A4 MR from baseline by khat use was 46% (95% CI, 20-72) and 31% (95% CI, 8-54), respectively. This is the first report linking khat use with significant inhibition of CYP2D6 metabolic activity in humans. PMID:26444948

  5. The antitussive effect of dextromethorphan in relation to CYP2D6 activity

    Abdul Manap, R; Wright, C E; Gregory, A; Rostami-Hodjegan, A; Meller, S T; Kelm, G R; Lennard, M S; Tucker, G T; Morice, A H

    1999-01-01

    Aims To test the hypothesis that inhibition of cytochrome P450 2D6 (CYP2D6) by quinidine increases the antitussive effect of dextromethorphan (DEX) in an induced cough model. Methods Twenty-two healthy extensive metaboliser phenotypes for CYP2D6 were studied according to a double-blind, randomised cross-over design after administration of: (1) Placebo antitussive preceded at 1 h by placebo inhibitor; (2) 30 mg oral DEX preceded at 1 h by placebo inhibitor (DEX30); (3) 60 mg oral DEX preceded at 1 h by placebo inhibitor (DEX60); (4) 30 mg oral DEX preceded at 1 h by 50 mg oral quinidine sulphate (QDEX30). Cough frequency following inhalation of 10% citric acid was measured at baseline and at intervals up to 12 h. Plasma concentrations of DEX and its metabolites were measured up to 96 h by h.p.l.c. Results Inhibition of CYP2D6 by quinidine caused a significant increase in the mean ratio of DEX to dextrorphan (DEX:DOR) plasma AUC(96) (0.04 vs 1.81, P < 0.001). The mean (±s.d.) decrements in cough frequency below baseline over 12 h (AUEC) were: 8% (11), 17% (14.5), 25% (16.2) and 25% (16.9) for placebo, DEX30, DEX60 and QDEX30 treatments, respectively. Statistically significant differences in antitussive effect were detected for the contrasts between DEX60/placebo (P < 0.001; 95% CI of difference +80, +327) and QDEX30/placebo (P < 0.001, +88, +336), but not for DEX30/placebo, DEX30/DEX60 or DEX30/QDEX30 (P = 0.071, −7, +241; P = 0.254, −37, +211; P = 0.187, −29, +219, respectively). Conclusions A significant antitussive effect was demonstrated after 60 mg dextromethorphan and 30 mg dextromethorphan preceded by 50 mg quinidine using an induced cough model. However, although the study was powered to detect a 10% difference in cough response, the observed differences for other contrasts were less than 10%, such that it was possible only to imply a dose effect (30 vs 60 mg) in the antitussive activity of DEX and enhancement of this effect by CYP2D6 inhibition. PMID

  6. CYP2D6 and CYP2C19 activity in a large population of Dutch healthy volunteers : indications for oral contraceptive-related gender differences

    Tamminga, WJ; Wemer, J; Oosterhuis, B; Wieling, J; Wilffert, B; de Leij, LFMH; de Zeeuw, RA; Jonkman, JHG

    1999-01-01

    Objective: We examined a large database containing results on CYP2D6 and CYP2C19 activity of 4301 Dutch volunteers phenotyped in the context of various clinical pharmacology studies. Methods: The subjects were given 22 mg dextromethorphan, 100 mg mephenytoin and 200 mg caffeine. For CYP2D6, the dext

  7. New aQTL SNPs for the CYP2D6 Identified by a Novel Mediation Analysis of Genome-Wide SNP Arrays, Gene Expression Arrays, and CYP2D6 Activity

    Guanglong Jiang

    2013-01-01

    Full Text Available Background. The genome-wide association studies (GWAS have been successful during the last few years. A key challenge is that the interpretation of the results is not straightforward, especially for transacting SNPs. Integration of transcriptome data into GWAS may provide clues elucidating the mechanisms by which a genetic variant leads to a disease. Methods. Here, we developed a novel mediation analysis approach to identify new expression quantitative trait loci (eQTL driving CYP2D6 activity by combining genotype, gene expression, and enzyme activity data. Results. 389,573 and 1,214,416 SNP-transcript-CYP2D6 activity trios are found strongly associated (P<10-5, FDR=16.6% and 11.7% for two different genotype platforms, namely, Affymetrix and Illumina, respectively. The majority of eQTLs are trans-SNPs. A single polymorphism leads to widespread downstream changes in the expression of distant genes by affecting major regulators or transcription factors (TFs, which would be visible as an eQTL hotspot and can lead to large and consistent biological effects. Overlapped eQTL hotspots with the mediators lead to the discovery of 64 TFs. Conclusions. Our mediation analysis is a powerful approach in identifying the trans-QTL-phenotype associations. It improves our understanding of the functional genetic variations for the liver metabolism mechanisms.

  8. Molecular dynamics of CYP2D6 polymorphisms in the absence and presence of a mechanism-based inactivator reveals changes in local flexibility and dominant substrate access channels.

    Parker W de Waal

    Full Text Available Cytochrome P450 enzymes (CYPs represent an important enzyme superfamily involved in metabolism of many endogenous and exogenous small molecules. CYP2D6 is responsible for ∼ 15% of CYP-mediated drug metabolism and exhibits large phenotypic diversity within CYPs with over 100 different allelic variants. Many of these variants lead to functional changes in enzyme activity and substrate selectivity. Herein, a molecular dynamics comparative analysis of four different variants of CYP2D6 was performed. The comparative analysis included simulations with and without SCH 66712, a ligand that is also a mechanism-based inactivator, in order to investigate the possible structural basis of CYP2D6 inactivation. Analysis of protein stability highlighted significantly altered flexibility in both proximal and distal residues from the variant residues. In the absence of SCH 66712, *34, *17-2, and *17-3 displayed more flexibility than *1, and *53 displayed more rigidity. SCH 66712 binding reversed flexibility in *17-2 and *17-3, through *53 remained largely rigid. Throughout simulations with docked SCH 66712, ligand orientation within the heme-binding pocket was consistent with previously identified sites of metabolism and measured binding energies. Subsequent tunnel analysis of substrate access, egress, and solvent channels displayed varied bottle-neck radii. Taken together, our results indicate that SCH 66712 should inactivate these allelic variants, although varied flexibility and substrate binding-pocket accessibility may alter its interaction abilities.

  9. The effect of TongXuan LiFei pill on the activity of CYP2D6%通宣理肺丸对CYP2D6酶活性的影响

    徐娟; 赵钢涛; 邸晓辉; 张梅; 郑绯

    2013-01-01

    目的 研究通宣理肺丸对CYP2D6酶活性的影响,探讨通宣理肺丸的代谢途径,为临床指导合理用药提供依据.方法 筛选30名健康志愿者作为受试者.以右美沙芬作为CYP2D6的探针药,研究服用通宣理肺丸对人体内代谢酶CYP2D6活性的影响.采用高效液相色谱法-质谱(HPLC-MS/MS)法测定探针药及其代谢产物的血药浓度.连续服用14 d通宣理肺丸.以曲线下面积(AUC0~12)为指标,评价CYP2D6代谢酶的活性.结果 受试者用药前CYP2D6代谢酶活性为6.1±1.5,服用通宣理肺丸后代谢酶活性为6.5±1.7,差异无统计学意义.结论 服用通宣理肺丸对CYP2D6代谢酶活性没有明显影响.

  10. Cyp2D6 catalyzes 5-hydroxylation of 1-(2-pyrimidinyl)-piperazine, an active metabolite of several psychoactive drugs, in human liver microsomes.

    Raghavan, Nirmala; Zhang, Donglu; Zhu, Mingshe; Zeng, Jianing; Christopher, Lisa

    2005-02-01

    1-(2-Pyrimidinyl)-piperazine (1-PP) is an active metabolite of several psychoactive drugs including buspirone. 1-PP is also the major metabolite in the human circulation and in rat brains following oral administration of buspirone. This study was conducted to identify the enzyme responsible for the metabolic conversion of 1-PP to 5-hydroxy-1-(2-pyrimidinyl)-piperazine (HO-1-PP) in human liver microsomes (HLMs). The product HO-1-PP was quantified by a validated liquid chromatography-tandem mass spectrometry method. In the presence of NADPH, 1-PP (100 microM) was incubated separately with human cDNA-expressed cytochrome P450 isozymes (including CYP2D6, 3A4, 1A2, 2A6, 2C9, 2C19, 2E1, and 2B6) at 37 degrees C. CYP2D6 catalyzed the formation of HO-1-PP from 1-PP. This catalytic activity was >95% inhibited by quinidine, a CYP2D6 inhibitor. HO-1-PP formation rates correlated well with the bufuralol 1-hydroxylase (CYP2D6) activities of individual HLMs. The formation of HO-1-PP followed a Michaelis-Menten kinetics with a K(m) of 171 microM and V(max) of 313 pmol/min x mg protein in HLMs. Collectively, these results indicate that polymorphic CYP2D6 is responsible for the conversion of 1-PP to HO-1-PP. PMID:15507542

  11. 以普罗帕酮为探针测定人肝微粒体中CYP2D6的活性%Determination of CYP2D6 activity in human liver microsomes using propafenone as a probe

    张顺国; 唐跃年; 卜书红

    2004-01-01

    目的:测定普罗帕酮(PPF)和5-羟基普罗帕酮(5-OHP)的比值以表达人肝微粒体中CYP2D6的活性.方法:以1g·L-1微粒体蛋白浓度37℃孵育PPF 1 h,以HPLC测定PPF和5-OHP的含量.结果:PPF和5-OHP的线性方程分别为Y=0.452 0 C+0.003 0,r=0.999 7;Y=0.749 4 C-0.020 5,r=0.997 5.结论:人肝微粒体CYP2D6酶的活性可以通过测定5-OHP与PPF的比值进行预测.

  12. Influence of CYP2D6 activity on the pharmacokinetics and pharmacodynamics of a single 20 mg dose of ibogaine in healthy volunteers.

    Glue, Paul; Winter, Helen; Garbe, Kira; Jakobi, Hannah; Lyudin, Alexander; Lenagh-Glue, Zoe; Hung, C Tak

    2015-06-01

    Conversion of ibogaine to its active metabolite noribogaine appears to be mediated primarily by CYP2D6. We compared 168 hours pharmacokinetic profiles of both analytes after a single oral 20 mg dose of ibogaine in 21 healthy subjects who had been pretreated for 6 days with placebo or the CYP2D6 inhibitor paroxetine. In placebo-pretreated subjects, ibogaine was rapidly converted to noribogaine. Median peak noribogaine concentrations occurred at 4 hours. Compared with placebo-pretreated subjects, paroxetine-pretreated subjects had rapid (Tmax  = 1.5 hours) and substantial absorption of ibogaine, with detectable levels out to 72 hours, and an elimination half-life of 10.2 hours. In this group, ibogaine was also rapidly converted to noribogaine with a median Tmax of 3 hours. Extent of noribogaine exposure was similar in both groups. CYP2D6 phenotype was robustly correlated with ibogaine AUC0-t (r = 0.82) and Cmax (r = 0.77). Active moiety (ibogaine plus noribogaine) exposure was ∼2-fold higher in paroxetine-pretreated subjects. Single 20 mg ibogaine doses were safe and well tolerated in all subjects. The doubling of exposure to active moiety in subjects with reduced CYP2D6 activity suggests it may be prudent to genotype patients awaiting ibogaine treatment, and to at least halve the intended dose of ibogaine in CYP2D6 poor metabolizers. PMID:25651476

  13. Meta-analysis of correlation between CYP2 D6 polymorphisms and tamoxifen concentrations and its activity in Chinese breast cancer patients%中国人群CYP2D6的基因多态性与乳腺癌患者他莫昔芬及其代谢物血药浓度关系的Meta分析

    熊萱; 张思超; 朱昶宇

    2015-01-01

    目的:系统评价 CYP2 D6基因型与乳腺癌患者他莫昔芬及其活性代谢物血药浓度的关系。方法计算机检索Cochrane图书馆、PubMed、EMBase、CNKI、CBM、Weipu Data、Wanfang Data等数据库,并手工检索相关文献,查找关于CYP2D6基因型与他莫昔芬及其活性代谢物血药浓度的文献。检索时间1995年1月~2014年10月。采用RevMan5.3软件进行meta分析。结果共纳入4篇文献,包含438例研究对象。 Meta分析结果显示,携带CYP2D6*10/*10基因型患者的他莫昔芬活性代谢物( HTAM、endoxifen)血药浓度明显低于携带其他基因型患者(P<0.0001);携带CYP2D6*10/*10基因型患者的他莫昔芬血药浓度低于携带CYP2D6Wt/Wt基因型患者(P<0.05)。而CYP2D6Wt/Wt及CYP2D6Wt/*10基因型携带患者间,TAM及其活性代谢物血药浓度比较,差异无统计学意义。结论中国人群CYP2D6的基因多态型对乳腺癌患者体内他莫西芬及其代谢物的浓度有影响。%Objective To systematically review the correlation between polymorphisms of CYP2D6 genotypes and concentrations of tamoxifen and its activity in Chinese breast cancer patients.Methods Such databases as Cochrane Library, PubMed, EMBase, CNKI, CBM, WeipuData and WanfangDate,from January 1995 to October 2014 were searched on line for the studies about the correlation between polymorphisms of CYP2D6 genotypes concentrations of tamoxifen and its activity in Chinese breast cancer patients.And references about it were checked.The meta-analysis was performed with RevMan 5.3 software.Results A total of 4 articles involving 438 patients were included.The results of meta-analysis showed that,the concentrations of HTAM and endoxifen in patients who had CYP2D6*10/*10 genotypes were lower than the other genotypes ( P<0.0001 ) .The concentration of tamoxifen in patients who had CYP2D6*10/*10 genotypes was lower than CYP2D6Wt/Wt ( P<0.05 ) .There was no significant

  14. Modeling Chemical Interaction Profiles: I. Spectral Data-Activity Relationship and Structure-Activity Relationship Models for Inhibitors and Non-inhibitors of Cytochrome P450 CYP3A4 and CYP2D6 Isozymes

    Richard D. Beger

    2012-03-01

    techniques, providing an independent estimator that can increase confidence in a structure-activity assessment. When modeling was applied to hazardous environmental chemicals, it was found that up to 20% of them may be substrates and up to 10% of them may be inhibitors of the CYP3A4 and CYP2D6 isoforms. The developed models provide a rare opportunity for the environmental health branch of the public health service to extrapolate to hazardous chemicals directly from human clinical data. Therefore, the pharmacological and environmental health branches are both expected to benefit from these reported models.

  15. Modeling chemical interaction profiles: I. Spectral data-activity relationship and structure-activity relationship models for inhibitors and non-inhibitors of cytochrome P450 CYP3A4 and CYP2D6 isozymes.

    McPhail, Brooks; Tie, Yunfeng; Hong, Huixiao; Pearce, Bruce A; Schnackenberg, Laura K; Ge, Weigong; Valerio, Luis G; Fuscoe, James C; Tong, Weida; Buzatu, Dan A; Wilkes, Jon G; Fowler, Bruce A; Demchuk, Eugene; Beger, Richard D

    2012-01-01

    techniques, providing an independent estimator that can increase confidence in a structure-activity assessment. When modeling was applied to hazardous environmental chemicals, it was found that up to 20% of them may be substrates and up to 10% of them may be inhibitors of the CYP3A4 and CYP2D6 isoforms. The developed models provide a rare opportunity for the environmental health branch of the public health service to extrapolate to hazardous chemicals directly from human clinical data. Therefore, the pharmacological and environmental health branches are both expected to benefit from these reported models. PMID:22421792

  16. Role of cytochrome P450 2D6 genetic polymorphism in carvedilol hydroxylation in vitro

    Wang Z

    2016-06-01

    Full Text Available Zhe Wang,1,* Li Wang,2,3,* Ren-ai Xu,4 Yun-yun Zhan,2 Cheng-ke Huang,1 Da-peng Dai,5 Jian-ping Cai,5 Guo-xin Hu2 1Department of Pharmacy, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, 2Department of Pharmacology, School of Pharmacy, Wenzhou Medical University, Wenzhou, 3Department of Pharmacy, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, 4Department of Pharmacy, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 5The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: Cytochrome P450 2D6 (CYP2D6 is a highly polymorphic enzyme that catalyzes the metabolism of a great number of therapeutic drugs. Up to now, >100 allelic variants of CYP2D6 have been reported. Recently, we identified 22 novel variants in the Chinese population in these variants. The purpose of this study was to examine the enzymatic activity of the variants toward the CYP2D6 substrate carvedilol in vitro. The CYP2D6 proteins, including CYP2D6.1 (wild type, CYP2D6.2, CYP2D6.10, and 22 other novel CYP2D6 variants, were expressed from insect microsomes and incubated with carvedilol ranging from 1.0 µM to 50 µM at 37°C for 30 minutes. After termination, the carvedilol metabolites were extracted and detected using ultra-performance liquid chromatography tandem mass-spectrometry. Among the 24 CYP2D6 variants, CYP2D6.92 and CYP2D6.96 were catalytically inactive and the remaining 22 variants exhibited significantly decreased intrinsic clearance values (ranging from ~25% to 95% compared with CYP2D6.1. The present data in vitro suggest that the newly found variants significantly reduced catalytic activities compared with CYP2D6.1. Given that CYP2D6 protein activities could affect carvedilol plasma levels, these findings are greatly relevant to

  17. High Frequency of CYP2D6 Ultrarapid Metabolizer Genotype in the Finnish Population.

    Pietarinen, Paavo; Tornio, Aleksi; Niemi, Mikko

    2016-09-01

    CYP2D6 participates in the biotransformation of many commonly used drugs. Large genetic variability in CYP2D6 results in a wide interindividual variability in the response to CYP2D6 substrate drugs. Previous studies have assessed the phenotype and genotype distributions of CYP2D6 in relatively small Finnish population samples. The aim of our study was to investigate the frequencies of CYP2D6 genotypes in a larger Finnish population cohort of 857 healthy volunteers. The volunteers were genotyped for 10 CYP2D6 genetic variants (*2, *3, *4, *5, *6, *9, *10, *17, *39, *41) and copy number variation performed with TaqMan genotyping assays and copy number assay targeting exon 9. CYP2D6 phenotypes were inferred from the genotype data with the classical and activity score methods. According to the classical method, a large majority of the study cases were extensive metabolizers (EM; 87.3%; 95% confidence interval 84.9-89.3) and the second largest group was ultrarapid metabolizers (UM; 7.2%; 5.7-9.2%). Intermediate (IM) and poor metabolizers (PM) were in clear minority (3.0%; 2.1-4.4% and 2.3%; 1.5-3.6%, respectively). The activity score method yielded similar phenotype predictions. These results show that the frequency of UM genotype is higher and that of PM and IM genotype is lower in the Finnish population than in other North European populations. Accordingly, CYP2D6 genetic profile of the Finnish population differs from its geographically close neighbours, which has implications for the effective and safe use of drugs metabolized by CYP2D6. PMID:27038154

  18. Effects of Flos carthami on CYP2D6 and on the Pharmacokinetics of Metoprolol in Rats

    Gaofeng Liu

    2011-01-01

    Full Text Available Flos carthami is a traditional Chinese herbal medicine. Clinically, the Flos carthami Injection has been used concomitantly with other Western drugs and may be used concomitantly with β-blockers, such as metoprolol, to treat cerebrovascular and coronary heart diseases, in China. Metoprolol is a CYP2D6 substrate and is predominantly metabolized by this isozyme. However, we do not know whether there is an effect of Flos carthami on CYP2D6 and the consequences of such an effect. Concern is raised regarding the possible herb-drug interaction. In this report, the effects of Flos carthami on the activity of CYP2D6 in vivo and in vitro and on the pharmacokinetics of metoprolol, in rats, are investigated. To assess the inhibitory potency of Flos carthami, the concentration associated with 50% inhibition (IC50 of dextromethorphan metabolism was determined based on the concentration-inhibition curves. The inhibitory effect of Flos carthami on CYP2D6 was also compared with cimetidine in vitro. Flos carthami could significantly inhibit CYP2D6 in rats both in vitro and in vivo (P<.05 and could slow down the metabolic rate of metoprolol as suggested by prolonged t1/2 (67.45%, by increased Cmax (74.51% and AUC0−∞ (76.89%. These results suggest that CYP2D6 is a risk factor when Flos carthami is administered concomitantly with metoprolol or other CYP2D6 substrates.

  19. The Impact of CYP2D6 Genotyping on Tamoxifen Treatment

    Ferraldeschi, Roberta; William G Newman

    2010-01-01

    Tamoxifen remains a cornerstone of treatment for patients with oestrogen-receptor-positive breast cancer. Tamoxifen efficacy depends on the biotransformation, predominantly via the cytochrome P450 2D6 (CYP2D6) isoform, to the active metabolite endoxifen. Both genetic and environmental (drug-induced) factors may alter CYP2D6 enzyme activity directly affecting the concentrations of active tamoxifen metabolites. Several studies suggest that germline genetic variants in CYP2D6 influence the clini...

  20. CYP2D6 genotype and phenotype relationship in South Indians

    Naveen A

    2006-01-01

    Full Text Available Background : Genotypes of the drug-metabolizing enzyme CYP2D6 influence plasma levels of 25% of commonlyprescribed drugs. This is the first study in India to investigate the genotype-phenotype relationship of CYP2D6. Aim : To study the influence of some CYP2D6 genotypes on the metabolism of its substrate dextromethorphanin healthy South Indian volunteers and to assess the contribution of the CYP2D6FNx0110 and CYP2D6FNx014 alleles. Materials and Methods : Twenty-six subjects from a previous CYP2D6 genotyping study of healthy volunteerswere included for phenotyping in this study. Selected volunteers belonged to any one of three genotype groups:Group I - two normal activity alleles, Group II - one reduced activity allele and one normal activity allele andGroup III - one loss of function allele along with either a wild type or reduced activity allele. Volunteers werephenotyped for the CYP2D6 enzyme using dextromethorphan as probe drug. Concentrations of the parent drugand metabolite dextrorphan were estimated using high performance liquid chromatography. Metabolic ratioswere calculated as the ratio of parent drug to metabolite in 0-8h urine samples. Statistical Analysis : Metabolic ratios from each genotype group were compared using the Mann-Whitney testat 5% significance, to observe their difference between genotype groups. Results : The mean metabolic ratios±SD in Groups I, II and III were 0.0039±0.0031, 0.0032±0.0017 and0.0391±0.0331 respectively. The mean metabolic ratio of Group III was significantly higher when comparedwith Groups I or II. In heterozygous individuals, the FNx011 or FNx012 alleles compensated for the reduced enzymeactivity due to the FNx0110 allele. However, if a heterozygous individual had a FNx014 allele, the reduced enzyme activitycould not be compensated by the FNx011 or FNx012 alleles. Conclusions : The CYP2D6 enzyme activity was found to be decreased in individuals carrying FNx014 or FNx015 alleles.The FNx011 or FNx

  1. CYP2D6 genotype determination in the Danish population

    Brøsen, K; Nielsen, P N; Brusgaard, K;

    1994-01-01

    CYP2D6 genotyping was carried out by XbaI restriction fragment length polymorphism analysis and polymerase chain reaction in 168 healthy Danish volunteers, 77 extensive metabolizers (EM) and 91 poor metabolizers (PM) of sparteine. All EM were genotyped correctly as heterozygous or homozygous for.......11-9.10). The median difference was 0.09 (95% confidence interval: 0.02-0.16). CYP2D6 phenotyping is a promising tool in tailoring the individual dose of tricyclic antidepressants, some neuroleplics and some antiarrhythmics. However if the genotype test could be improved with regard to both sensitivity in PM...... and the ability to predict CYP2D6 activity in EM then it would be of even greater clinical value in therapeutic drug monitoring. Udgivelsesdato: 1994-null...

  2. The Impact of CYP2D6 Genotyping on Tamoxifen Treatment

    Roberta Ferraldeschi

    2010-04-01

    Full Text Available Tamoxifen remains a cornerstone of treatment for patients with oestrogen-receptor-positive breast cancer. Tamoxifen efficacy depends on the biotransformation, predominantly via the cytochrome P450 2D6 (CYP2D6 isoform, to the active metabolite endoxifen. Both genetic and environmental (drug-induced factors may alter CYP2D6 enzyme activity directly affecting the concentrations of active tamoxifen metabolites. Several studies suggest that germline genetic variants in CYP2D6 influence the clinical outcomes of patients treated with adjuvant tamoxifen. Here, we review the existing data relating CYP2D6 genotypes to tamoxifen efficacy.

  3. Variation in the CYP2D6 gene is associated with a lower serum sodium concentration in patients on antidepressants

    S. Kwadijk-De Gijsel (Sonja); M.J. Bijl (Monique); L.E. Visser (Loes); R.H.N. van Schaik (Ron); A. Hofman (Albert); A.G. Vulto (Arnold); T. van Gelder (Teun); B.H.Ch. Stricker (Bruno)

    2009-01-01

    textabstractWHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Several antidepressants are metabolized by the polymorphic enzyme cytochrome P450 2D6 (CYP2D6). • The variant allele CYP2D6*4 is the main polymorphism resulting in decreased enzyme activity in Caucasians. • Decreased CYP2D6 enzyme activity poten

  4. CYP2D6 genotype and tamoxifen response in postmenopausal women with endocrine-responsive breast cancer

    Regan, Meredith M; Leyland-Jones, Brian; Bouzyk, Mark;

    2012-01-01

    Adjuvant tamoxifen therapy is effective for postmenopausal women with endocrine-responsive breast cancer. Cytochrome P450 2D6 (CYP2D6) enzyme metabolizes tamoxifen to clinically active metabolites, and CYP2D6 polymorphisms may adversely affect tamoxifen efficacy. In this study, we investigated the...... clinical relevance of CYP2D6 polymorphisms....

  5. The impact of CYP2D6-predicted phenotype on tamoxifen treatment outcome in patients with metastatic breast cancer

    L.A. Lammers (Laureen); R.H.J. Matthijsen (Ron); T. van Gelder (Teun); M.J. Bijl (Monique); A.J.M. de Graan (Anne-Joy); C.M. Seynaeve (Caroline); M.A. van Fessem (Marianne); P.M.J.J. Berns (Els); A.G. Vulto (Arnold); R.H.N. van Schaik (Ron)

    2010-01-01

    textabstractAbstract BACKGROUND: Cytochrome P450 2D6 (CYP2D6) has a crucial role in the metabolic conversion of tamoxifen into the active metabolite endoxifen. In this cohort study, the effect of CYP2D6-predicted phenotype, defined as the combined effect of CYP2D6 genetic variation and concomitant

  6. Duplication of CYP2D6 predicts high clearance of desipramine but high clearance does not predict duplication of CYP2D6

    Bergmann, T K; Bathum, L; Brøsen, Kim

    2001-01-01

    OBJECTIVE: Duplication of CYP2D6 causes very rapid metabolism of CYP2D6 substrates such as desipramine. However, we have previously shown that in the Danish population, only about 15% of very rapid metabolisers, defined as subjects with a metabolic ratio of sparteine of 0.15 or less, carried a...... duplicated allele. The question is whether gene duplication is a relatively rare cause (perhaps predictor) of very rapid metabolism or whether a low metabolic ratio is a poor predictor of this. METHODS: After measuring metabolic ratios anew, we selected six volunteers with duplication of CYP2D6 and metabolic...... duplication of CYP2D6 is poor; there must be other causes (or predictors) of very rapid metabolism and with much higher frequency than duplication of CYP2D6....

  7. Clinical Utility and Economic Impact of CYP2D6 Genotyping.

    Reynolds, Kristen K; McNally, Beth A; Linder, Mark W

    2016-09-01

    Pharmacogenetics examines an individual's genetic makeup to help predict the safety and efficacy of medications. Practical application optimizes treatment selection to decrease the failure rate of medications and improve clinical outcomes. Lack of efficacy is costly due to adverse drug reactions and increased hospital stays. Cytochrome P450 2D6 (CYP2D6) metabolizes roughly 25% of all drugs. Detecting variants that cause altered CYP2D6 enzymatic activity identifies patients at risk of adverse drug reactions or therapeutic failure with standard dosages of medications metabolized by CYP2D6. This article discusses the clinical application of pharmacogenetics to improve care and decrease costs. PMID:27514466

  8. The Effects of H2S on the Activities of CYP2B6, CYP2D6, CYP3A4, CYP2C19 and CYP2C9 in Vivo in Rat

    Xianqin Wang

    2013-12-01

    Full Text Available Hydrogen sulfide (H2S is a colorless, flammable, extremely hazardous gas with a “rotten egg” smell. The human body produces small amounts of H2S and uses it as a signaling molecule. The cocktail method was used to evaluate the influence of H2S on the activities of CYP450 in rats, which were reflected by the changes of pharmacokinetic parameters of five specific probe drugs: bupropion, metroprolol, midazolam, omeprazole and tolbutamide, respectively. The rats were randomly divided into two groups, control group and H2S group. The H2S group rats were given 5 mg/kg NaHS by oral administration once a day for seven days. The mixture of five probes was given to rats through oral administration and the blood samples were obtained at a series of time-points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by LC-MS. In comparing the H2S group with the control group, there was a statistically pharmacokinetics difference for midazolam and tolbutamide; the area under the plasma concentration-time curve (AUC was decreased for midazolam (p < 0.05 and increased for tolbutamide (p < 0.05; while there was no statistical pharmacokinetics difference for bupropion, metroprolol and omeprazole. H2S could not influence the activities of CYP2B6, CYP2D6 and CYP2C19 in rats, while H2S could induce the activity of CYP3A4 and inhibit the activity of CYP2C9 in rats.

  9. Multiplex Phenotyping for Systems Medicine: A One-Point Optimized Practical Sampling Strategy for Simultaneous Estimation of CYP1A2, CYP2C9, CYP2C19, and CYP2D6 Activities Using a Cocktail Approach.

    de Andrés, Fernando; Terán, Santiago; Bovera, Marcela; Fariñas, Humberto; Terán, Enrique; LLerena, Adrián

    2016-02-01

    Phenotyping of the CYP450 enzyme activities contributes to personalized medicine, but the past phenotyping approaches have followed a piecemeal strategy measuring single enzyme activities in vivo. A barrier to phenotyping of populations in rural and remote areas is the limited time and resources for sample collection. The CEIBA cocktail approach allows metabolic capacity estimation of multiple CYP450 enzymes in a single sample analysis, but the attendant sample collection schemes for applications in diverse global settings are yet to be optimized. The present study aimed to select an optimal matrix to simultaneously analyze CYP450 enzyme activities so as to simplify the sampling schemes in the phenotyping protocol to enhance its throughput and feasibility in native populations or in remote and underserviced geographies and social contexts. We evaluated 13 Ecuadorian healthy volunteers for CYP1A2, CYP2C9, CYP2C19, and CYP2D6 genotypes and their metabolic phenotypes, including CYP3A4, in plasma and urine after administering one reduced dose of caffeine, losartan, omeprazole, and dextromethorphan. Pharmacokinetic analyses were performed, and the correlation between AUC parent/AUC metabolite and the ratio between concentrations of probe drugs and their corresponding metabolites at timepoints ranging from 0 to 12 hours post-dose were analyzed. A single sampling timepoint, 4 hours post-dose in plasma, was identified as optimal to reflect the metabolic activity of the attendant CYP450 enzymes. This study optimizes the CEIBA multiplexed phenotyping approach and offers new ways forward for integrated drug metabolism analyses, in the pursuit of global personalized medicine applications in resource-limited regions, be they in developed or developing countries. PMID:26600202

  10. The hypoalgesic effect of oxycodone in human experimental pain models in relation to the CYP2D6 oxidation polymorphism

    Zwisler, Stine T; Enggaard, Thomas P; Noehr-Jensen, Lene;

    2009-01-01

    Oxycodone is O-demethylated by CYP2D6 to oxymorphone which is a potent micro-receptor agonist. The CYP2D6 oxidation polymorphism divides the Caucasian population in two phenotypes: approximately 8% with no enzyme activity, poor metabolizers (PM) and the remainder with preserved CYP2D6 activity...

  11. MDMA, methamphetamine, and CYP2D6 pharmacogenetics: what is clinically relevant?

    Rafael eDe La Torre

    2012-11-01

    Full Text Available In vitro human studies show that the metabolism of most amphetamine-like psychostimulants is regulated by the polymorphic cytochrome P450 isozyme CYP2D6. Two compounds, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA, were selected as archetypes to discuss the translation and clinical significance of in vitro to in vivo findings. Both compounds were chosen based on their differential interaction with CYP2D6 and their high abuse prevalence in society. Methamphetamine behaves as both a weak substrate and competitive inhibitor of CYP2D6, while MDMA acts as a high affinity substrate and potent mechanism-based inhibitor (MBI of the enzyme. The MBI behavior of MDMA on CYP2D6 implies that subjects, irrespective of their genotype/phenotype, are phenocopied to the poor metabolizer phenotype. The fraction of metabolic clearance regulated by CYP2D6 for both drugs is substantially lower than expected from in vitro studies. Other isoenzymes of cytochrome P450 and a relevant contribution of renal excretion play a part in their clearance. These facts tune down the potential contribution of CYP2D6 polymorphism in the clinical outcomes of both substances. Globally, the clinical relevance of CYP2D6 polymorphism is lower than that predicted by in vitro studies.

  12. Farnesoid X Receptor Agonist Represses Cytochrome P450 2D6 Expression by Upregulating Small Heterodimer Partner.

    Pan, Xian; Lee, Yoon-Kwang; Jeong, Hyunyoung

    2015-07-01

    Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme responsible for eliminating approximately 20% of marketed drugs. Studies have shown that differential transcriptional regulation of CYP2D6 may contribute to large interindividual variability in CYP2D6-mediated drug metabolism. However, the factors governing CYP2D6 transcription are largely unknown. We previously demonstrated small heterodimer partner (SHP) as a novel transcriptional repressor of CYP2D6 expression. SHP is a representative target gene of the farnesoid X receptor (FXR). The objective of this study is to investigate whether an agonist of FXR, 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), alters CYP2D6 expression and activity. In CYP2D6-humanized transgenic mice, GW4064 decreased hepatic CYP2D6 expression and activity (by 2-fold) while increasing SHP expression (by 2-fold) and SHP recruitment to the CYP2D6 promoter. CYP2D6 repression by GW4064 was abrogated in Shp(-/-);CYP2D6 mice, indicating a critical role of SHP in CYP2D6 regulation by GW4064. Also, GW4064 decreased CYP2D6 expression (by 2-fold) in primary human hepatocytes, suggesting that the results obtained in CYP2D6-humanized transgenic mice can be translated to humans. This proof of concept study provides evidence for CYP2D6 regulation by an inducer of SHP expression, namely, the FXR agonist GW4064. PMID:25926433

  13. Effects of 22 novel CYP2D6 variants found in Chinese population on the metabolism of dapoxetine

    Xu RA

    2016-02-01

    Full Text Available Ren-ai Xu,1,* Er-min Gu,2,* Quan Zhou,2 Lingjing Yuan,2 Xiaoxia Hu,2 Jianping Cai,3 Guoxin Hu1,2 1Department of Pharmacy, The First Affiliated Hospital of Wenzhou Medical University, 2Department of Pharmacology, School of Pharmacy, Wenzhou Medical University, Wenzhou, 3The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing, People’s Republic of China *These authors contributed equally to this work Background: CYP2D6 is one of the most important members of the cytochrome P450 superfamily. Its genetic polymorphism significantly influences the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. Methods and results: The aim of this research was mainly to explore the catalytic activities of 22 newly reported CYP2D6 isoforms (2D6*87, *88, *89, *90, *91, *92, *93, *94, *95, *96,*97, *98, *R25Q, F164L, E215K, F219S, V327M, D336N, V342M, R344Q, R440C, R497C on dapoxetine in vitro. The research was designed with an appropriate incubation system in test tubes and carried out in the constant temperature water. Through detecting its two metabolites desmethyldapoxetine and dapoxetine-N-oxide, the available data were obtained to explain the influence of CYP2D6 polymorphism on the substrate drug dapoxetine. As a result, the intrinsic clearance (Vmax/Km values of most variants were significantly altered when compared with the counterpart of CYP2D6*1, with most of these variants exhibiting either reduced Vmax and/or increased Km values. For dapoxetine demethylation pathway (which produces desmethyldapoxetine, 2D6*89 and E215K exhibited no markedly decreased relative clearance of 92.81% and 97.70%, respectively. The relative clearance of rest 20 variants exhibited decrease in different levels, ranging from 20.44% to 90.90%. For the dapoxetine oxidation pathway (which produces dapoxetine-N-oxide, the relative clearance values of three variants, 2D6*90, *94, and

  14. CYP2D6基因多态性及对药物代谢的影响%Polymorphism of CYP2D6 and the influence to the drug metabolism

    韩璐; 刘洁

    2011-01-01

    CYP2D6代谢酶是细胞色素P450家族中的成员之一,是参与I相代谢和众多内源性物质和不同药物消除的酶.虽然它在肝脏中的含量大约只占肝脏总量的200,但在临床上却参与了25%以上的常用药物的代谢活动.在所有参与药物代谢的细胞色素P450基因家族中,CYP2D6 是唯一不能被诱导的酶,这种酶具有广泛的多态性,这种多态性对酶的药物代谢功能具有重要影响,CYP2D6的这种多态性和药物代谢功能所表现的对个体活性的差异,在遗传药理学上具有重要意义.本文从CYP2 D6基因多态性和它对药物代谢的影响这两方面进行了阐述.%CYP2D6 is a member of cytochrome P450 gene family - a group of enzymes that is responsible for phase Ⅰ metabolism and elimination of numerous endogenous substrates and a diverse array of drugs.It is now thought to be involved in the metabolism of up to 25 % of the drugs that are in common use in the clinic,despite its low hepatic content (about 2 %).Among the drug-metabolizing cytochrome P450s,CYP2D6 is the only non-inducible enzyme and is highly polymorphic, which results in a large contribution of genetic variation to its inter-individual variation in enzyme activity and is an enzyme of great historical importance for pharmacogenetics.Here we summarize the polymorphism of CYP2D6 gene and their influence to drug metabolism.

  15. Cytochrome P450 2D6 variants in a Caucasian population: Allele frequencies and phenotypic consequences

    Sachse, C.; Brockmoeller, J.; Bauer, S.; Roots, I. [Humboldt Univ., Berlin (Germany)

    1997-02-01

    Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of.005 (*1 x 2), .013 (* 2 x 2), and .001 (*4 x 2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T{sub 1957}C), *2B (additional C{sub 2558}T), and *4E (additional C{sub 2938}T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EN/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment. 35 refs., 4 figs., 5 tabs.

  16. Impact of the CYP2D6 genotype on post-operative intravenous oxycodone analgesia

    Zwisler, S T; Enggaard, T P; Mikkelsen, S;

    2009-01-01

    Background: Oxycodone is a semi-synthetic opioid with a mu-receptor agonist-mediated effect in several pain conditions, including post-operative pain. Oxycodone is metabolized to its active metabolite oxymorphone by O-demethylation via the polymorphic CYP2D6. The aim of this study was to...... investigate whether CYP2D6 poor metabolizers (PMs) yield the same analgesia post-operatively from intravenous oxycodone as extensive metabolizers (EMs). Methods: Two hundred and seventy patients undergoing primarily thyroid surgery or hysterectomy were included and followed for 24 h post-operatively. The CYP2...... for the first time in patients that the oxymorphone formation depends on CYP2D6, but we found no difference in the post-operative analgesic effect of intravenous oxycodone between the two CYP2D6 genotypes....

  17. Nomenclature for human CYP2D6 alleles.

    Daly, A K; Brockmöller, J; Broly, F; Eichelbaum, M; Evans, W E; Gonzalez, F J; Huang, J D; Idle, J R; Ingelman-Sundberg, M; Ishizaki, T; Jacqz-Aigrain, E; Meyer, U A; Nebert, D W; Steen, V M; Wolf, C R; Zanger, U M

    1996-06-01

    To standardize CYP2D6 allele nomenclature, and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that alleles be designated by CYP2D6 followed by an asterisk and a combination of roman letters and arabic numerals distinct for each allele with the number specifying the key mutation and, where appropriate, a letter specifying additional mutations. Criteria for classification as a separate allele and protein nomenclature are also presented. PMID:8807658

  18. CYP2D6 genotype dependent oxycodone metabolism in postoperative patients.

    Ulrike M Stamer

    Full Text Available BACKGROUND: The impact of polymorphic cytochrome P450 CYP2D6 enzyme on oxycodone's metabolism and clinical efficacy is currently being discussed. However, there are only spare data from postoperative settings. The hypothesis of this study is that genotype dependent CYP2D6 activity influences plasma concentrations of oxycodone and its metabolites and impacts analgesic consumption. METHODS: Patients received oxycodone 0.05 mg/kg before emerging from anesthesia and patient-controlled analgesia (PCA for the subsequent 48 postoperative hours. Blood samples were drawn at 30, 90 and 180 minutes after the initial oxycodone dose. Plasma concentrations of oxycodone and its metabolites oxymorphone, noroxycodone and noroxymorphone were analyzed by liquid chromatography-mass spectrometry with electrospray ionization. CYP2D6 genotyping was performed and 121 patients were allocated to the following genotype groups: PM (poor metabolizer: no functionally active CYP2D6 allele, HZ/IM (heterozygous subjects, intermediate metabolizers with decreased CYP2D6 activity, EM (extensive metabolizers, normal CYP2D6 activity and UM (ultrarapid metabolizers, increased CYP2D6 activity. Primary endpoint was the genotype dependent metabolite ratio of plasma concentrations oxymorphone/oxycodone. Secondary endpoint was the genotype dependent analgesic consumption with calculation of equianalgesic doses compared to the standard non-CYP dependent opioid piritramide. RESULTS: Metabolism differed between CYP2D6 genotypes. Mean (95%-CI oxymophone/oxycodone ratios were 0.10 (0.02/0.19, 0.13 (0.11/0.16, 0.18 (0.16/0.20 and 0.28 (0.07/0.49 in PM, HZ/IM, EM and UM, respectively (p = 0.005. Oxycodone consumption up to the 12(th hour was highest in PM (p = 0.005, resulting in lowest equianalgesic doses of piritramide versus oxycodone for PM (1.6 (1.4/1.8; EM and UM 2.2 (2.1/2.3; p<0.001. Pain scores did not differ between genotypes. CONCLUSIONS: In this postoperative setting, the number of

  19. CYP2D6 polymorphism and mental and personality disorders in suicide attempters.

    Blasco-Fontecilla, Hilario; Peñas-Lledó, Eva; Vaquero-Lorenzo, Concepción; Dorado, Pedro; Saiz-Ruiz, Jerónimo; Llerena, Adrián; Baca-García, Enrique

    2014-12-01

    Prior studies on the association between the CYP2D6 polymorphism and suicide did not explore whether mental and personality disorders mediate this association. The main objective of the present study was to test an association between CYP2D6 polymorphism and mental and personality disorders among suicide attempters. The MINI and the DSM-IV version of the International Personality Disorder Examination Screening Questionnaire were used to diagnose mental and personality disorders, respectively, in 342 suicide attempters. Suicide attempters were divided into four groups according to their number of CYP2D6 active genes (zero, one, and two or more). Differences in mental and personality disorders across the four groups were measured using linear-by-linear association, chi square-test, and 95% confidence intervals. Suicide attempters carrying two or more active CYP2D6 genes were more likely to be diagnosed with at least one personality disorder than those with one or zero CYP2D6 active genes. PMID:25437930

  20. CYP2D6基因与药物代谢%CYP2D6 gene and drug metabolism

    施安国

    2003-01-01

    细胞色素P-450(CYP)中的CYP2D6酶在抗抑郁药、安定药及某些抗心律失常药的代谢中起重要作用,CYP2D6基因位于22号常染色体上为隐性遗传,CYP2D6基因呈多态性约有70余种等位基因变异型,也存在特异人群差别,因而导致所编码的酶活性不同,这些数据有助于理解药物代谢的个体差异、有助于预测药物之间的相互作用.

  1. Identification of novel CYP2D7-2D6 hybrids: non-functional and functional variants

    Andrea Gaedigk

    2010-10-01

    Full Text Available Polymorphic expression of CYP2D6 contributes to the wide range of activity observed for this clinically important drug metabolizing enzyme. In this report we describe novel CYP2D7/2D6 hybrid genes encoding non-functional and functional CYP2D6 protein and a CYP2D7 variant that mimics a CYP2D7/2D6 hybrid gene. Five kb long PCR products encompassing the novel genes were entirely sequenced. A quantitative assay probing in different gene regions was employed to determine CYP2D6 and 2D7 copy number variations and the relative position of the hybrid genes within the locus was assessed by long-range PCR. In addition to the previously known CYP2D6*13 and *66 hybrids, we describe three novel non-functional CYP2D7-2D6 hybrids with gene switching in exon 2 (CYP2D6*79, intron 2 (CYP2D6*80 and intron 5 (CYP2D6*67. A CYP2D7-specific T-ins in exon 1 causes a detrimental frame shift. One subject revealed a CYP2D7 conversion in the 5’-flanking region of a CYP2D6*35 allele, was otherwise unaffected (designated CYP2D6*35B. Finally, three DNAs revealed a CYP2D7 gene with a CYP2D6-like region downstream of exon 9 (designated CYP2D7[REP6]. Quantitative copy number determination, sequence analyses and long-range PCR mapping were in agreement and excluded the presence of additional gene units. Undetected hybrid genes may cause over-estimation of CYP2D6 activity (CYP2D6*1/*1 vs *1/hybrid, etc, but may also cause results that may interfere with the genotype determination. Detection of hybrid events, ‘single’ and tandem, will contribute to more accurate phenotype prediction from genotype data.

  2. National Prociency Testing Result of CYP2D6*10 Genotyping for Adjuvant Tamoxifen Therapy in China.

    Lin, Guigao; Zhang, Kuo; Yi, Lang; Han, Yanxi; Xie, Jiehong; Li, Jinming

    2016-01-01

    Tamoxifen has been successfully used for treating breast cancer and preventing cancer recurrence. Cytochrome P450 2D6 (CYP2D6) plays a key role in the process of metabolizing tamoxifen to its active moiety, endoxifen. Patients with variants of the CYP2D6 gene may not receive the full benefit of tamoxifen treatment. The CYP2D6*10 variant (the most common variant in Asians) was analyzed to optimize the prescription of tamoxifen in China. To ensure referring clinicians have accurate information for genotype-guided tamoxifen treatment, the Chinese National Center for Clinical Laboratories (NCCL) organized a national proficiency testing (PT) to evaluate the performance of laboratories providing CYP2D6*10 genotyping. Ten genomic DNA samples with CYP2D6 wild-type or CYP2D6*10 variants were validated by PCR-sequencing and sent to 28 participant laboratories. The genotyping results and pharmacogenomic test reports were submitted and evaluated by NCCL experts. Additional information regarding the number of samples tested, the accreditation/certification status, and detecting technology was also requested. Thirty-one data sets were received, with a corresponding analytical sensitivity of 98.2% (548/558 challenges; 95% confidence interval: 96.7-99.1%) and an analytic specificity of 96.5% (675/682; 95% confidence interval: 97.9-99.5%). Overall, 25/28 participants correctly identified CYP2D6*10 status in 10 samples; however, two laboratories made serious genotyping errors. Most of the essential information was included in the 20 submitted CYP2D6*10 test reports. The majority of Chinese laboratories are reliable for detecting the CYP2D6*10 variant; however, several issues revealed in this study underline the importance of PT schemes in continued external assessment and provision of guidelines. PMID:27603206

  3. Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)

    2015-12-09

    Depression; Depressive Disorder; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Intermediate Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  4. Progress on research for genetic polymorphism of drug metabolic enzyme cytochrome P450 2D6%药物代谢酶细胞色素P450 2D6的遗传多态性研究进展

    徐田雪; 杨信怡; 赵昆; 张喜川; 游雪甫

    2009-01-01

    CYP2D6是肝脏中重要的药物代谢酶,其代谢的药物占临床应用药物的20%~25%.其遗传多态性对依赖CYP2D6代谢的药物具有重要的影响.本文综述了CYP2D6在遗传多态性方面的研究进展及其临床意义.%Cytochrome P450 2D6 (CYP2D6) is an important microsome enzyme in liver which metabolizes about 20%~25% of drugs used in clinic. And these substrates of CYP2D6 are affected intensively by its genetic polymorphism. In the present article, genetic polymorphism of CYP2D6 and its clinical implications were reviewed.

  5. CYP2D6 variation, behaviour and psychopathology: implications for pharmacogenomics-guided clinical trials.

    Peñas-Lledó, Eva M; Llerena, Adrián

    2014-04-01

    Individual and population differences in polymorphic cytochrome P450 enzyme function have been known for decades. The biological significance of these differences has now been deciphered with regard to drug metabolism, action and toxicity as well as disposition of endogenous substrates, including neuroactive compounds. While the cytochrome P450 enzymes occur abundantly in the liver, they are expressed in most tissues of the body, albeit in varying amounts, including the brain. The latter location of cytochrome P450s is highly pertinent for susceptibility to neuropsychiatric diseases, not to mention local drug metabolism at the site of psychotropic drug action in the brain. In the current era of personality medicine with companion theranostics (i.e. the fusion of therapeutics with diagnostics), this article underscores that such versatile biological roles of cytochrome P450s offer multiple points of entry for personalized medicine and rational therapeutics. We focus our discussion on CYP2D6, one of the most intensively researched drug and endogenous compound metabolism pathways, with a view to relevance for, and optimization of, pharmacogenomic-guided clinical trials. Working on the premise that CYP2D6 is related to human behaviour and certain personality traits such as serotonin and dopamine system function, we further suggest that the motivation of healthy volunteers to participate in clinical trials may in part be influenced by an under- or over-representation of certain CYP2D6 metabolic groups. PMID:24033670

  6. Establishment and Optimization of Incubation System of Bactrian Camel Studying CYP2D6 Enzyme in Vitro%双峰驼 CYP2D6酶体外孵育体系的建立与优化

    王艳; 高飞; 哈斯苏荣

    2016-01-01

    为了研究双峰驼 CYP2D6酶体外活性,建立双峰驼肝微粒体孵育体系并对孵育体系中探针底物浓度、肝微粒体蛋白浓度和孵育时间等进行优化研究。首先采用改良差速离心法制备双峰驼肝微粒体、BCA 法测定双峰驼肝微粒体蛋白浓度、CO 还原差示光谱法检测 CYP 总酶含量,然后采用 HPLC 法跟踪检测孵育体系中 CYP2D6酶特异性底物的主要代谢产物去甲右美沙芬含量进而优化孵育条件。结果表明,双峰驼肝微粒体蛋白浓度为5.5650 mg/mL±0.5197 mg/mL,CYP 总酶含量为0.1777 nmol/mg±0.0503 nmol/mg;肝微粒体孵育体系的最适底物浓度为250μg/mL,肝微粒体蛋白浓度为5.5650 mg/mL,最适孵育时间为40 min 。所制备的双峰驼肝微粒体各项指标和优化后的肝微粒体孵育条件均能满足后续对双峰驼 CYP2D6酶体外活性研究的基本要求。%In order to study the in vitro activities of Bactrian Camel CYP2D6 enzyme,the liver microsome in-cubation system was established and optimized by studying the concentration of probe substrate,protein content of liver microsome and incubation time,etc.Firstly,liver microsomes of bactrian camel was pre-pared by modified differential centrifugation method,the protein content of bactrian camel liver microsomes was detected by using BCA method and the total content of CYP enzyme was determined by using CO re-duction method.Secondly,the incubation system was optimized by detecting and tracking the concentration of dextrophan,an active essential metabolite of dextromethorphan,in incubation system by using HPLC method.The results showed that the liver microsomal protein content of bactrian camel was 5.565 0 mg/mL±0.519 7 mg/mL,total CYP enzyme content was 0.177 7 nmol/mg±0.050 3 nmol/mg and the optimum substrate concentration in the liver microsome incubation system was 250 μg/mL,the op-timum concentration of protein in liver microsomes was 5

  7. Tamoxifen Pharmacogenomics: The Role of CYP2D6 as a Predictor of Drug Response

    Goetz, MP; Kamal, A; Ames, MM

    2007-01-01

    Tamoxifen continues to be a standard endocrine therapy for the prevention and treatment of estrogen receptor (ER)-positive breast cancer. Tamoxifen can be considered a classic “pro-drug,” requiring metabolic activation to elicit pharmacological activity. CYP2D6 is the rate-limiting enzyme catalyzing the conversion of tamoxifen into metabolites with significantly greater affinity for the ER and greater ability to inhibit cell proliferation. Both genetic and environmental (drug-induced) factors...

  8. Design and synthesis of novel tamoxifen analogues that avoid CYP2D6 metabolism.

    Ahmed, Nermin S; Elghazawy, Nehal H; ElHady, Ahmed K; Engel, Matthias; Hartmann, Rolf W; Abadi, Ashraf H

    2016-04-13

    Tamoxifen (TAM) is a widely used drug in the prophylaxis and treatment of breast cancer. TAM is metabolized to the more active 4-hydroxytamoxifen (4-OH-TAM) and endoxifen by cytochrome P450 (CYP) mainly CYP2D6 and CYP3A4 enzymes. Due to the genetic polymorphisms in CYP2D6 genes, high variation in the clinical outcomes of TAM treatment is observed among women of different populations. To address this issue, novel TAM analogues with possible altered activation pathways were synthesized. These analogues were tested for their antiproliferative action on MCF-7 breast cancer cell lines as well as their binding affinity for estrogen receptor (ER) ER-α and ER-β receptors. These entire novel compounds showed better antiproliferative activity than did TAM on the MCF-7 cells. Moreover, compound 10 exhibited a half maximal growth inhibition (GI50) that was 1000 times more potent than that of TAM (GI50 < 0.005 μM vs 1.58 μM, respectively). Along with a broad spectrum activity on various cancer cell lines, all the TAM analogues showed considerable activity on the ER-negative breast cancer cell line. For further study, compound 10 was incubated in human liver microsomes (HLM), human hepatocytes (hHEP) and CYP2D6 supersomes. The active hydroxyl metabolite was detected after incubation in HLM and hHEP, implicating the involvement of other enzymes in its metabolism. These results prove that this novel series of TAM analogues might provide improved clinical outcomes for poor 2D6 metabolizers. PMID:26896706

  9. Primaquine pharmacology in the context of CYP 2D6 pharmacogenomics: Current state of the art.

    Marcsisin, Sean R; Reichard, Gregory; Pybus, Brandon S

    2016-05-01

    Primaquine is the only antimalarial drug available to clinicians for the treatment of relapsing forms of malaria. Primaquine development and usage dates back to the 1940s and has been administered to millions of individuals to treat and eliminate malaria infections. Primaquine therapy is not without disadvantages, however, as it can cause life threatening hemolysis in humans with glucose-6-phosphate dehydrogenase (G6PD) deficiency. In addition, the efficacy of primaquine against relapsing malaria was recently linked to CYP 2D6 mediated activation to an active metabolite, the structure of which has escaped definitive identification for over 75years. CYP 2D6 is highly polymorphic among various human populations adding further complexity to a comprehensive understanding of primaquine pharmacology. This review aims to discuss primaquine pharmacology in the context of state of the art understanding of CYP 2D6 mediated 8-aminoquinoline metabolic activation, and shed light on the current knowledge gaps of 8-aminoquinoline mechanistic understanding against relapsing malaria. PMID:27016470

  10. In vitro metabolic interactions between black cohosh (Cimicifuga racemosa) and tamoxifen via inhibition of cytochromes P450 2D6 and 3A4.

    Li, Jinghu; Gödecke, Tanja; Chen, Shao-Nong; Imai, Ayano; Lankin, David C; Farnsworth, Norman R; Pauli, Guido F; van Breemen, Richard B; Nikolić, Dejan

    2011-08-01

    Women who experience hot flashes as a side effect of tamoxifen (TAM) therapy often try botanical remedies such as black cohosh to alleviate these symptoms. Since pharmacological activity of TAM is dependent on the metabolic conversion into active metabolites by the action of cytochromes P450 2D6 (CYP2D6) and 3A4, the objective of this study was to evaluate whether black cohosh extracts can inhibit formation of active TAM metabolites and possibly reduce its clinical efficacy. At 50 μg/mL, a 75% ethanolic extract of black cohosh inhibited formation of 4-hydroxy- TAM by 66.3%, N-desmethyl TAM by 74.6% and α-hydroxy TAM by 80.3%. In addition, using midazolam and dextromethorphan as probe substrates, this extract inhibited CYP3A4 and CYP2D6 with IC(50) values of 16.5 and 50.1 μg/mL, respectively. Eight triterpene glycosides were identified as competitive CYP3A4 inhibitors with IC(50) values ranging from 2.3-5.1 µM, while the alkaloids protopine and allocryptopine were identified as competitive CYP2D6 inhibitors with K(i) values of 78 and 122 nM, respectively. The results of this study suggests that co-administration of black cohosh with TAM might interfere with the clinical efficacy of this drug. However, additional clinical studies are needed to determine the clinical significance of these in vitro results. PMID:21827327

  11. Impact of CYP2D6 Genetic Variation on the Response of the Cardiovascular Patient to Carvedilol and Metoprolol.

    Lymperopoulos, Anastasios; McCrink, Katie A; Brill, Ava

    2015-01-01

    Carvedilol and metoprolol are two of the most commonly prescribed β-blockers in cardiovascular medicine and primarily used in the treatment of hypertension and heart failure. Cytochrome P450 2D6 (CYP2D6) is the predominant metabolizing enzyme of these two drugs. Since the first description of a CYP2D6 sparteinedebrisoquine polymorphism in the mid-seventies, substantial genetic heterogeneity has been reported in the human CYP2D6 gene, with ~100 different polymorphisms identified to date. Some of these polymorphisms render the enzyme completely inactive while others do not modify its activity. Based on all the identified variants, four metabolizer phenotypes are nowadays used to characterize drug metabolism via CYP2D6 in humans: ultra-rapid metabolizer (UM); extensive metabolizer (EM); intermediate metabolizer (IM); and poor metabolizer (PM) phenotypes. As a consequence of these CYP2D6 metabolizer phenotypes, pharmacokinetics and bioavailability of carvedilol and metoprolol can range from therapeutically ineffective levels (in the UM patients) to excessive (overdose) and potentially toxic concentrations (in PM patients). This, in turn, can result in elevated risks for either treatment failure (in terms of blood pressure reduction of hypertensive patients and of improving survival and cardiovascular function of heart failure patients) or for adverse effects (e.g. hypotension and bradycardia). The present review will discuss the impact of these CYP2D6 genetic polymorphisms on the therapeutic responses of cardiovascular patients treated with either of these two β-blockers. In addition, the potential advantages and disadvantages of implementing CYP2D6 genetic testing in the clinic to guide/personalize therapy with these two drugs will be discussed. PMID:26537419

  12. Comparison of Paeoniflorin and Albiflorin on Human CYP3A4 and CYP2D6

    Li-Na Gao

    2015-01-01

    Full Text Available Peony (Paeonia lactiflora Pall- is a plant medicine and a functional food ingredient with wide application for more than 2000 years. It can be coadministrated with many other drugs, composed of traditional Chinese medicine compound such as shaoyao-gancao decoction. In order to explore the efficacy and safety of peony, effects of paeoniflorin and albiflorin (the principal components of peony on cytochrome P450 (CYP 3A4 and CYP2D6 were analyzed in human hepatoma HepG2 cells and evaluated from the level of recombinant CYP enzymes in vitro. The findings indicated that albiflorin possessed stronger regulation on the mRNA expression of CYP3A4 and CYP2D6 than paeoniflorin. For the protein level of CYP3A4, albiflorin showed significant induction or inhibition with the concentration increasing from 10−7 M to 10−5 M, but no remarkable variation was observed in paeoniflorin-treated group. Enzyme activity assay implied that both paeoniflorin and albiflorin could regulate CYP3A4 and CYP2D6 with varying degrees. The results showed that albiflorin should be given more attention because it may play a vital role on the overall efficacy of peony. The whole behavior of both paeoniflorin and albiflorin should be focused on ensuring the rationality and effectiveness of clinical application.

  13. Stable expression of human cytochrome P450 2D6*10 in HepG2 cells

    Jian Zhuge; Ying-Nian Yu; Xiao-Dan Wu

    2004-01-01

    AIM: Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established.METHODS: Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR)from total RNA extracted from human liver tissue and cloned into pGEM-T vector, cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells. Expression of mRNA was validated by RT-PCR.Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC).RESULTS: The cloned cDNA had 4 base differences, e.g.100 C→T, 336 T→C, 408 C→G and 1 457 G→C, which resulted in P34S, and S486T amino acid substitutions, and two samesense mutations were 112 F and 136 V compared with that reported by Kimura et al(GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele. The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31±0.19 nmol.min-1.mg-1 S9 protein (n=3), but was undetectable in parental HepG2 cells.CONCLUSION: cDNA of human CYP2D6*10can be successfully doned. A cell line, HepG2-CYP2D6*10, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.

  14. Effects of cytochrome P450 2D6 and 3A5 genotypes and possible coadministered medicines on the metabolic clearance of antidepressant mirtazapine in Japanese patients.

    Okubo, Maho; Murayama, Norie; Miura, Jun; Chiba, Yasuji; Yamazaki, Hiroshi

    2015-01-01

    A personalized treatment approach should be considered with the second-generation psychiatric drug mirtazapine because of high frequencies of side effects, including characteristic drowsiness. Plasma concentrations of mirtazapine in patients are influenced by many factors, including polymorphic cytochrome P450 enzymes contributing to its transformation to 8-hydroxymirtazapine and N-demethylmirtazapine. The aim of this study was to investigate the determinant factors for individual variations of metabolic clearance of mirtazapine using in vitro and in vivo methods. In vitro analyses using liver microsomes from individual humans in correlation assays and recombinantly expressed P450 enzymes revealed that CYP2D6 was the major contributor to mirtazapine 8-hydroxylation with high affinity, and that CYP3A5 catalyzed N-demethylation in a similar high-capacity manner to that of CYP3A4. CYP1A2 was a minor contributor to mirtazapine 8-hydroxylation. Metabolic clearance of mirtazapine determined in substrate depletion assays and mirtazapine 8-hydroxylation activities in individual liver microsomes were significantly lower in CYP2D6 intermediate metabolizers (IM) and poor metabolizers (PM) than in extensive metabolizers (EM) (pCYP3A5 poor-expressors group than in the expressors group (pinhibitor), ketoconazole (a CYP3A inhibitor), and in combination with risperidone and duloxetine, possible coadministered medicines. These results suggested that mirtazapine metabolic clearance could be variously influenced by the CYP2D6 and CYP3A5 genotypes and coadministered drugs in clinical patients. PMID:25475885

  15. Untersuchung des Konzeptes der semiquantitativen CYP2D6-Gendosis bei Ultraschnellmetabolisierern im Vergleich zu Schnellmetabolisierern anhand der Pharmakokinetik von S- und R-Metoprolol

    Seeringer, Angela

    2010-01-01

    The background of this thesis was a clinical study on metoprolol as a CYP2D6 probe drug studying the influence of the CYP2D6 gene-duplication on pharmacokinetics of metoprolol in healthy volunteers. The aim of our study was to generate a more precise classification of the activitiy of CYP2D6 taking the contribution of alleles with decreased or increased activity into account. For this reason we further genotyped the study participants for CYP2D6, and established a concept of semiquantitative ...

  16. Detection of Cytochrome P450 2D6 Allele CYP2D6*6 by Using Allele-specific Amplification%ASA法测定细胞色素P450 2D6等位基因CYP2D6*6

    陈枢青; Wedl.,PJ

    1999-01-01

    细胞色素P450 2D6(CYP2D6)基因2064G→A突变,造成表达产物212位上谷氨酸变成甘氨酸,从而引起酶分子失活,被称为CYP2D6*6.利用等位基因特异扩增法,建立了ASA-PCR测定CYP2D6*6的方法.经396例测定,说明本法快捷、准确.测定结果显示CYP2D6*6与CYP2D6T共存,如果只为准确预测CYP2D6酶活性,对CYP2D6*6进行测定并无意义.

  17. CYP2D6*2 Polymorphism as a Predictor of Failed Outpatient Tramadol Therapy in Postherpetic Neuralgia Patients.

    Nasare, Namita Vilas; Banerjee, Basu Dev; Suryakantrao Deshmukh, Pravin; Mediratta, Pramod Kumari; Saxena, Ashok Kumar; Ahmed, Rafat Sultana; Bhattacharya, Sambit Nath

    2016-01-01

    Human cytochrome P4502D6 (CYP2D6) gene is highly polymorphic, leading to wide interindividual ethnic differences in CYP2D6-mediated drug metabolism. Its activity ranges from complete deficiency to excessive activity, potentially causing toxicity of the medication or therapeutic failure with recommended drug dosages. The aim of the study was to find the association of CYP2D6*2 polymorphisms with demographic characters (age, sex, and weight), pain intensity scales [numerical rating scale (NRS) sleep, global perceived effect (GPE)], and adverse drug effects in postherpetic neuralgia (PHN) patients receiving tramadol. The study comprised 246 patients [including 123 nonresponders (NRs) and 123 responders (Rs)] with PHN undergoing analgesic treatment at the pain clinic, Out Patient Department, University College of Medical Sciences, Guru Teg Bahadur Hospital, Delhi, India. Patients with any history of diabetes mellitus, human immunodeficiency virus, malignancy, hematological or liver disease, psychiatric illness, alcohol abuse, and tramadol sensitivity were excluded from the study. The NRSs of (resting and movement), NRS-sleep, and GPE were evaluated by the treating physician. Adverse drug effects during the time of the study were recorded. All samples were analyzed for CYP2D6*2 polymorphism using the polymerase chain reaction-restriction fragment length polymorphism method. The genotype distribution did not vary significantly among genders [NR (P = 0.723); R (P = 0.947)] and different age groups in NRs (P = 0.763) and Rs (P = 0.268). Clinically, statistically significant (P 0.05). In addition, CYP2D6*2 genotype was not related to the adverse effects of analgesic therapy. The overall results suggested that CYP2D6*2 polymorphism plays no role in the PHN patients receiving tramadol treatment. The CYP2D6*2 polymorphism may not be a predictor of treatment outcome of patients with respect to PHN-receiving tramadol. PMID:23567787

  18. A novel simple method for determining CYP2D6 gene copy number and identifying allele(s with duplication/multiplication.

    Taimour Langaee

    Full Text Available Cytochrome P450 2D6 (CYP2D6 gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR, restriction fragment length polymorphism (RFLP and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.

  19. Possible impact of the CYP2D6*10 polymorphism on the nonlinear pharmacokinetic parameter estimates of paroxetine in Japanese patients with major depressive disorders

    Saruwatari J

    2014-04-01

    Full Text Available Junji Saruwatari,1 Hiroo Nakashima,1 Shoko Tsuchimine,2 Miki Nishimura,1 Naoki Ogusu,1 Norio Yasui-Furukori21Division of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan; 2Department of Neuropsychiatry, Graduate School of Medicine, Hirosaki University, Hirosaki, JapanAbstract: It has been suggested that the reduced function allele with reduced cytochrome P450 (CYP 2D6 activity, CYP2D6*10, is associated with the interindividual differences in the plasma paroxetine concentrations, but there is no data presently available regarding the influence of the CYP2D6*10 polymorphism on the pharmacokinetic parameters, eg, Michaelis–Menten constant (Km and maximum velocity (Vmax, in Asian populations. The present study investigated the effects of the CYP2D6 polymorphisms, including CYP2D6*10, on the pharmacokinetic parameters of paroxetine in Japanese patients with major depressive disorders. This retrospective study included 15 Japanese patients with major depressive disorders (four males and eleven females who were treated with paroxetine. The CYP2D6*2, CYP2D6*4, CYP2D6*5, CYP2D6*10, CYP2D6*18, CYP2D6*39, and CYP2D6*41 polymorphisms were evaluated. A total of 56 blood samples were collected from the patients. The Km and Vmax values of paroxetine were estimated for each patient. The allele frequencies of CYP2D6*2, CYP2D6*4, CYP2D6*5, CYP2D6*10, CYP2D6*18, CYP2D6*39, and CYP2D6*41 were 6.7%, 0%, 10.0%, 56.7%, 0%, 26.7%, and 0%, respectively. The mean values of Km and Vmax were 50.5±68.4 ng/mL and 50.6±18.8 mg/day, respectively. Both the Km and Vmax values were significantly smaller in CYP2D6*10 allele carriers than in the noncarriers (24.2±18.3 ng/mL versus 122.5±106.3 ng/mL, P=0.008; 44.2±16.1 mg/day versus 68.3±15.0 mg/day, P=0.022, respectively. This is the first study to demonstrate that the CYP2D6*10 polymorphism could affect the nonlinear pharmacokinetic parameter estimates of

  20. Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy-N,N-dimethyltryptamine metabolism and pharmacokinetics.

    Shen, Hong-Wu; Wu, Chao; Jiang, Xi-Ling; Yu, Ai-Ming

    2010-07-01

    5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency (V(max)/K(m)), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1'-hydroxylase activities (R(2)=0.98; P<0.0001) and CYP2D6 contents (R(2)=0.77; P=0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg-CYP2D6) mouse models showed that Tg-CYP2D6 mice receiving the same dose of 5-MeO-DMT (20mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg-CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6. PMID:20206139

  1. Relationship between genotypes Sult1a2 and Cyp2d6 and tamoxifen metabolism in breast cancer patients.

    Ana Fernández-Santander

    Full Text Available Tamoxifen is a pro-drug widely used in breast cancer patients to prevent tumor recurrence. Prior work has revealed a role of cytochrome and sulfotransferase enzymes in tamoxifen metabolism. In this descriptive study, correlations were examined between concentrations of tamoxifen metabolites and genotypes for CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen receptor-positive breast cancer. Patients were genotyped using the Roche-AmpliChip® CYP450 Test, and Real-Time and conventional PCR-RFLP. Plasma tamoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen, endoxifen and tamoxifen-N-oxide were isolated and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Significantly higher endoxifen levels were detected in patients with the wt/wt CYP2D6 compared to the v/v CYP2D6 genotype (p<0.001. No differences were detected in the remaining tamoxifen metabolites among CYP2D6 genotypes. Patients featuring the SULT1A2*2 and SULT1A2*3 alleles showed significantly higher plasma levels of 4-hydroxy-tamoxifen and endoxifen (p = 0.025 and p = 0.006, respectively, as likely substrates of the SULT1A2 enzyme. Our observations indicate that besides the CYP2D6 genotype leading to tamoxifen conversion to potent hydroxylated metabolites in a manner consistent with a gene-dose effect, SULT1A2 also seems to play a role in maintaining optimal levels of both 4-hydroxy-tamoxifen and endoxifen.

  2. META-ANALYSIS OF CYP2D6 METABOLIZER PHENOTYPE AND METOPROLOL PHARMACOKINETICS

    Blake, CM; Kharasch, ED; Schwab, M.; Nagele, P.

    2013-01-01

    Metoprolol, a commonly prescribed beta-blocker, is primarily metabolized by cytochrome P450 2D6 (CYP2D6), an enzyme with substantial genetic heterogeneity. Several smaller studies have shown that metoprolol pharmacokinetics is influenced by CYP2D6 genotype and metabolizer phenotype. To increase robustness of metoprolol pharmacokinetic estimates, a systematic review and meta-analysis of pharmacokinetic studies that administered a single oral dose of immediate release metoprolol was performed. ...

  3. Active Matter on Asymmetric Substrates

    Reichhardt, C. J. Olson; Drocco, J.; Mai, T.; Wan, M. B.; Reichhardt, C.

    2011-01-01

    For collections of particles in a thermal bath interacting with an asymmetric substrate, it is possible for a ratchet effect to occur where the particles undergo a net dc motion in response to an ac forcing. Ratchet effects have been demonstrated in a variety of systems including colloids as well as magnetic vortices in type-II superconductors. Here we examine the case of active matter or self-driven particles interacting with asymmetric substrates. Active matter systems include self-motile c...

  4. Prolactin release in children treated with risperidone: impact and role of CYP2D6 metabolism.

    Troost, P.W.; Lahuis, B.E.; Hermans, M.H.; Buitelaar, J.K.; Engeland, H. van; Scahill, L.; Minderaa, R.B.; Hoekstra, P.J.

    2007-01-01

    OBJECTIVE: Little is known about the role of CYP2D6 polymorphism in risperidone-induced prolactin release in children. METHOD: Twenty-five children (aged 5-15 years) with pervasive developmental disorders were genotyped for CYP2D6 polymorphisms. Serum prolactin, risperidone, and 9-hydroxyrisperidone

  5. ASA法测定细胞色素P450 2D6酶缺陷等位基因CYP2D6E%Analysis of cytochrome P450 2D6 enzyme defect allele CYP2D6E by allele-specific amplification

    陈枢青; Wedl.,PJ

    1999-01-01

    目的:建立细胞色素P450 2D6(CYP2D6)第3023位A→C突变造成CYP2D6酶活性缺陷的等位基因CYP2D6E的测定方法.方法:利用等位基因特异扩增法(ASA)为基本原理,设计两对引物分别扩增野生型等位基因和突变型等位基因.结果:经396例测定,发现2例CYP2D6E与CYP2D6B的异突变型纯合子,其表现型均为慢代谢者.阳性对照说明本法重复性好,阴性对照显示本法无污染问题.结论:本法比PCR-RFLP法更为快捷、更少污染.对CYP2D6E的测定有助于准确预测CYP2D6表现型.

  6. Novel +90G>A Intronic Polymorphism of CYP2D6

    Monir Modaresi-nejad

    2015-04-01

    Full Text Available Objective: CYP2D6, an enzyme, metabolizes a large number of commonly prescribed drugs. Variations in CYP2D6 gene encoding this enzyme have been associated with individual differences in drug metabolism rates. The purpose of our study was to identify some allelic variants of CYP2D6 gene and to detect defective CYP2D6 alleles, as part of a pharmacogenetic screening program. Materials and Methods: A prospective study was done on 120 participants referred to Royan Institute in 2013. Allele and genotype frequencies for polymorphism of CYP2D6 gene in exons 1 and 4 were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis and sequencing on PCR products, respectively. Results: We identified a novel variant of the gene encoding cytochrome P450 2D6 (CYP2D6 at position +90 of intron 4 by sequencing method. This novel polymorphism of CYP2D6 has been deposited in GeneBank® under the accession number KF225465 in Jun 2013. Conclusion: In the current study, we identified novel polymorphism in intron 4. This single nucleotide polymorphism (SNP is known as +90G>A in the fourth intron.

  7. Mechanism-based inhibition of CYP3A4 and CYP2D6 by Indonesian medicinal plants.

    Subehan; Usia, Tepy; Iwata, Hiroshi; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2006-05-24

    Thirty samples of Indonesian medicinal plants were tested for their mechanism-based inhibition on cytochrome P450 3A4 (CYP3A4) and CYP2D6 via erythromycin N-demethylation and dextromethorphan O-demethylation activities in human liver microsomes. From screening with 0 and 20min preincubation at 0.5mg/ml of methanol extracts, five plants (Cinnamomum burmani bark, Foeniculum vulgare seed, Strychnos ligustrina wood, Tinospora crispa stem, and Zingiber cassumunar rhizome) showed more than 30% increase of CYP3A4 inhibition, while three (Alpinia galanga rhizome, Melaleuca leucadendron leaf, and Piper nigrum fruit) showed more than 30% increase of CYP2D6 inhibition. In these eight plants, Foeniculum vulgare seed, Cinnamomum burmani bark, and Strychnos ligustrina wood showed time-dependent inhibition on CYP3A4 and Piper nigrum fruit and Melaleuca leucadendron leaf on CYP2D6. Among these, four plants other than Melaleuca leucadendron revealed NADPH-dependent inhibition. Thus, Foeniculum vulgare, Cinnamomum burmani, and Strychnos ligustrina should contain mechanism-based inhibitors on CYP3A4 and Piper nigrum contain that on CYP2D6. PMID:16414224

  8. Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-Methoxy-N,N-dimethyltryptamine Metabolism and Pharmacokinetics

    Shen, Hong-Wu; Wu, Chao; Jiang, Xi-Ling; Yu, Ai-Ming

    2010-01-01

    5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibi...

  9. A poor metabolizer of both CYP2C19 and CYP2D6 identified by mechanistic pharmacokinetic simulation in a fatal drug poisoning case involving venlafaxine

    Jornil, J; Nielsen, T S; Rosendal, I;

    2013-01-01

    pharmacokinetic simulations suggested that the low metabolite ratio was the result of combined poor metabolizer (PM) status of cytochrome P450 (CYP) 2C19 and CYP2D6. This hypothesis was confirmed by genetic analysis. Simulations revealed that it was likely that the combined missing CYP2D6 and CYP2C19 activity...... combined with genotyping were considered very useful in this fatal drug poisoning case. Keywords CYP2D6; CYP2C19; Venlafaxine; Poor metabolizer; Drug poisoning; Mechanistic pharmacokinetic simulation --------------------------------------------------------------------------------...

  10. Distribution of CYP2D6 alleles and phenotypes in the Brazilian population.

    Deise C Friedrich

    Full Text Available The CYP2D6 enzyme is one of the most important members of the cytochrome P450 superfamily. This enzyme metabolizes approximately 25% of currently prescribed medications. The CYP2D6 gene presents a high allele heterogeneity that determines great inter-individual variation. The aim of this study was to evaluate the variability of CYP2D6 alleles, genotypes and predicted phenotypes in Brazilians. Eleven single nucleotide polymorphisms and CYP2D6 duplications/multiplications were genotyped by TaqMan assays in 1020 individuals from North, Northeast, South, and Southeast Brazil. Eighteen CYP2D6 alleles were identified in the Brazilian population. The CYP2D6*1 and CYP2D6*2 alleles were the most frequent and widely distributed in different geographical regions of Brazil. The highest number of CYPD6 alleles observed was six and the frequency of individuals with more than two copies ranged from 6.3% (in Southern Brazil to 10.2% (Northern Brazil. The analysis of molecular variance showed that CYP2D6 is homogeneously distributed across different Brazilian regions and most of the differences can be attributed to inter-individual differences. The most frequent predicted metabolic status was EM (83.5%. Overall 2.5% and 3.7% of Brazilians were PMs and UMs respectively. Genomic ancestry proportions differ only in the prevalence of intermediate metabolizers. The IM predicted phenotype is associated with a higher proportion of African ancestry and a lower proportion of European ancestry in Brazilians. PM and UM classes did not vary among regions and/or ancestry proportions therefore unique CYP2D6 testing guidelines for Brazilians are possible and could potentially avoid ineffective or adverse events outcomes due to drug prescriptions.

  11. Meta-Analysis of Cytochrome P2D6 Gene Polymorphisms and Susceptibility to Acute Leukemia

    Ma Limin; Ruan Linhai

    2013-01-01

    Objective:To provide a more robust assessment to the effect of cytochrome P2D6 (CYP2D6) polymorphisms on the risk of acute leukemia (AL), and to evaluate the association between the two most commonly studied CYP2D6 polymorphisms (CYP2D6*3 and CYP2D6*4) and AL risk by meta-analysis. Methods:All case-control studies investigating an association between the CYP2D6*3 or CYP2D6*4 polymorphisms and AL risk were included. Either fixed-effects or random-effects models were applied to combine odds ratios (ORs) and 95%conifdence intervals (CIs) by RevMan 5.1. Q-statistic was used to evaluate the heterogeneity, and both Egger’s test and funnel plots were used to assess publication bias. Results:Six studies were included in the meta-analysis. The results we acquired were that the OR value and 95%CI of CYP2D6*4 wild type, heterozygous mutant and homozygous mutant were 0.94 (0.66-1.35), 1.04(0.74-1.45) and 1.63 (0.95-2.81), respectively with Z=0.33, 0.23 and 1.76 (P>0.05), indicating that there was no signiifcant association between CYP2D6*4 polymorphism and the risk of AL. We also performed subgroup analysis by the AL immunophenotype for those groups with heterogeneity. The results of the combined analysis of CYP2D6*4 wild type, heterozygous mutant and acute lymphoblastic leukemia (ALL) were Z=0.08, 0.08 (P>0.05), for acute myeloblastic leukemia (AML) were Z=0.17, 0.26 (P>0.05), indicating that there was no signiifcant association between CYP2D6*4 polymorphism and the development of both ALL and AML. Conclusion:CYP2D6 polymorphisms are not associated with AL risk.

  12. Lack of association between schizophrenia and the CYP2D6 gene polymorphisms

    Pirmohamed, M.; Wild, M.J.; Kitteringham, N.R. [Univ. of Liverpool (United Kingdom)] [and others

    1996-04-09

    Approximately 5-10% of the Caucasian population lack the P450 isoform, CYP2D6. This polymorphism may be of importance in determining individual susceptibility to Parkinson`s disease. In this journal, Daniels et al. recently reported a negative association between the CYP2D6 gene locus and schizophrenia, a disease characterized by dopamine overactivity. It is important to exclude such an association because CYP2D6 is expressed in the brain and it is involved in dopamine catabolism. Between 1992 and 1993, we also performed a study similar to that, and reached the same conclusion. 7 refs., 1 tab.

  13. Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

    Jian Zhuge; Ying-Nian Yu

    2004-01-01

    AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing.METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced.Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA.Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced.RESULTS: One of the CYP2D6cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4),and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples,only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6gene were detected. The third variant was the skipped exon 3, and 153 bp was lost.CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

  14. CYP2D6 polymorphisms influence the efficacy of adjuvant tamoxifen in Thai breast cancer patients

    Sirachainan E

    2012-10-01

    Full Text Available Ekaphop Sirachainan,1 Sureerat Jaruhathai,1 Narumol Trachu,2 Ravat Panvichian,1 Thitiya Sirisinha,1 Touch Ativitavas,1 Vorachai Ratanatharathorn,1 Montri Chamnanphon,3 Chonlaphat Sukasem31Division of Medical Oncology, Department of Medicine, 2Research Center, Division of Pharmacogenomics and Personalized Medicine, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 3Division of Pharmacogenomics and Personalized Medicine, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, ThailandAim: We evaluated single nucleotide polymorphisms (SNPs of CYP2D6 to identify those that influence the efficiency of tamoxifen in adjuvant treatment of breast cancer through a matched case–control study.Methods: Peripheral blood DNA was collected from 20 patients with disease recurrence during adjuvant tamoxifen treatment and from 19 patients who had completed 5 years of tamoxifen therapy without recurrence of breast cancer. CYP2D6*4 (1846G > A; rs3892097, CYP2D6*10 (100C > T, rs1065852, and CYP2D6*5 (deletion were genotyped. The correlation between disease-free survival (DFS and genotype and clinical outcome were assessed using Kaplan–Meier analysis and a log-rank test.Results: We found the allelic frequency of CYP2D6*10 during this study. Patients with the CYP2D6*10 homozygous variant T/T genotype had a significantly shorter median of DFS than those with C/T (P = 0.036, but DFS was not significantly different from that of patients with the C/C genotype (P = 0.316. One patient who was a carrier both of CYP2D6 G/A (1846G > A and T/T (100C > T had DFS of 22.7 months.Conclusions: This study demonstrated that CYP2D6*10/*10 was significantly associated with shorter DFS in Thai breast cancer patients receiving tamoxifen. This was a pilot study investigating the correlation of CYP2D6 polymorphisms and their influence on clinical outcomes in Thai estrogen receptor

  15. Physiologically-based pharmacokinetic modeling of tamoxifen and its metabolites in women of different CYP2D6 phenotypes provides new insight into the tamoxifen mass balance

    Kristin eDickschen

    2012-05-01

    Full Text Available Tamoxifen is a first-line endocrine agent in the mechanism-based treatment of estrogen receptor positive (ER+ mammary carcinoma and applied to breast cancer patients all over the world. Endoxifen is a secondary and highly active metabolite of tamoxifen that is formed among others by the polymorphic cytochrome P450 2D6 (CYP2D6. It is widely accepted that CYP2D6 poor metabolizers (PM exert a pronounced decrease in endoxifen steady-state plasma concentrations compared to CYP2D6 extensive metabolizers (EM. Nevertheless, an in-depth understanding of the chain of cause and effect between CYP2D6 genotype, endoxifen steady-state plasma concentration, and subsequent tamoxifen treatment benefit still remains to be evolved.In this context, physiologically-based pharmacokinetic (PBPK-modeling provides a useful tool to mechanistically investigate the impact of CYP2D6 phenotype on endoxifen formation in female breast cancer patients undergoing tamoxifen therapy.It has long been thought that only a minor percentage of endoxifen is formed via 4-hydroxytamoxifen. However, the current investigation supports very recently published data that postulates a contribution of 4-hydroxytamoxifen above 20 % to total endoxifen formation. The developed PBPK-model describes tamoxifen PK in rats and humans. Moreover, tamoxifen metabolism in dependence of CYP2D6 phenotype in populations of European female individuals is well described, thus providing a good basis to further investigate the linkage of PK, mode of action, and treatment outcome in dependence of factors such as phenotype, ethnicity or co-treatment with CYP2D6 inhibitors.

  16. Dose-dependent inhibition of CYP1A2, CYP2C19 and CYP2D6 by citalopram, fluoxetine, fluvoxamine and paroxetine

    Jeppesen, U; Gram, L F; Vistisen, K;

    1996-01-01

    OBJECTIVE: The purpose of this pharmacokinetic study was to investigate the dose-dependent inhibition of model substrates for CYP2D6, CYP2C19 and CYP1A2 by four marketed selective serotonin reuptake inhibitors (SSRIs): citalopram, fluoxetine, fluvoxamine and paroxetine. METHODS: The study was...... by sparteine (CYP2D6), mephenytoin (CYP2C19) and caffeine (CYP1A2) tests. Fluoxetine was given at 3-week intervals because of the long half-life of fluoxetine and its metabolite norfluoxetine. Citalopram, fluoxetine and paroxetine were given in doses of 10, 20, 40 and 80 mg and fluvoxamine was given...... fluoxetine are potent inhibitors of CYP2D6, that fluvoxamine and fluoxetine are moderate inhibitors of CYP2C19 and that fluvoxamine is a potent inhibitor of CYP1A2 in humans in vivo. The clinical prediction of interaction from single-dose experiments may have to take the degree of accumulation during steady...

  17. Impact of Tetrahydropalmatine on the Pharmacokinetics of Probe Drugs for CYP1A2, 2D6 and 3A Isoenzymes in Beagle Dogs.

    Zhao, Yong; Liang, Aihua; Zhang, Yushi; Li, Chunying; Yi, Yan; Nilsen, Odd Georg

    2016-06-01

    Tetrahydropalmatine (Tet) exhibit multiple pharmacological activities and is used frequently by clinical practitioners. In this study, we evaluate the in vivo effects of single and repeated oral Tet administrations on CYP1A2, 2D6 and 3A activities in six beagle dogs in a randomized, controlled, open-label, crossover study. A cocktail approach, with dosages of the probe drugs caffeine (3.0 mg/kg), metoprolol (2.33 mg/kg) and midazolam (0.45 mg/kg), was used to measure cytochrome P450 (CYP) metabolic activities. The cocktail was administered orally as a single dose (12 mg/kg) 1 day prior to and 4 days after repeated oral Tet administrations (12 mg/kg three times daily). The probe drugs and their metabolites in plasma were quantified simultaneously by a validated HPLC technique, and non-compartmental parameters were used to evaluate metabolic variables for assessment of CYP inhibition or induction. Tet had no or minor impact on the pharmacokinetics and metabolism of the probe drugs caffeine and metoprolol, CYP1A2 and CYP2D6 substrates, respectively. However, Tet increased AUC0-24 h and decreased AUCratio(0-24 h) (1-hydroxymidazolam/midazolam ratio) for midazolam statistically significant, both in single or multiple dosing of Tet, with up to 39 or 57% increase for AUC0-24 h and 29% or 22 decrease for AUCratio(0-24 h), respectively, in line with previous in vitro findings for its CYP3A4 inhibition. The extensive use of Tet and herbal medicines containing Tet makes Tet a candidate for further evaluation of CYP3A-mediated herb-drug interactions. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26990021

  18. CARACTERIZACIÓN DE VARIANTES ALÉLICAS DE CITOCROMO CYP2D6 EN LA POBLACIÓN DE LA REGIÓN CENTROCCIDENTAL DE VENEZUELA

    GRIMÁN PEDRO

    2009-04-01

    Full Text Available

    RESUMEN

    El gen CYP2D6 codifica para una monooxigenasa perteneciente al citocromo P450, la cual está involucrada en la biotransformación de un gran número de drogas comúnmente prescritas, como antidepresivos, antineoplásicos y antihipertensivos. Algunos efectos adversos, así como falla terapéutica pueden ser relacionados con la actividad anormal de CYP2D6 producto de polimorfismos en el gen de dicha enzima. Con el fin de predecir la frecuencia de algunos fenotipos metabolizadores pobres de CYP2D6 en la población de la región centroccidental de Venezuela se determinaron las frecuencias alélicas y genotípicas de las variantes alélicas CYP2D6*3, *4 y *6. Se extrajo ADN genómico a partir de sangre periférica de 100 individuos voluntarios aparentemente sanos, y se procedió a la genotipificación por PCR tetra-primer alelo-específica y análisis por electroforesis en geles de agarosa. Se compararon las frecuencias obtenidas con poblaciones de otros países. El alelo más frecuente fue CYP2D6*4 con 16,5%, mostrando una diferencia significativa con la reportada con poblaciones asiáticas. Este trabajo constituye un estudio preliminar en la caracterización de un grupo más amplio de alelos de CYP2D6 con el fin de asistir al desarrollo de una farmacoterapia individualizada en nuestro país.

    Palabras clave: Citocromo P450, CYP2D6, Farmacogenética.


    ABSTRACT

    The CYP2D6 gene encodes for a monooxygenase belonging to the cytochrome P450, which is involved in the biotransformation of a large number of commonly prescribed drugs such as antidepressants, antihypertensive and antineoplastic. Some side effects, as well as therapeutic failure may be related to abnormal activity of CYP2D6 product of polymorphisms in the CYP2D6 gene. In order to predict the frequency of some poor metabolisers phenotypes of CYP2D6 in the population of the

  19. Active matter on asymmetric substrates

    Olson Reichhardt, C. J.; Drocco, J.; Mai, T.; Wan, M. B.; Reichhardt, C.

    2011-10-01

    For collections of particles in a thermal bath interacting with an asymmetric substrate, it is possible for a ratchet effect to occur where the particles undergo a net dc motion in response to an ac forcing. Ratchet effects have been demonstrated in a variety of systems including colloids as well as magnetic vortices in type-II superconductors. Here we examine the case of active matter or self-driven particles interacting with asymmetric substrates. Active matter systems include self-motile colloidal particles undergoing catalysis, swimming bacteria, artificial swimmers, crawling cells, and motor proteins. We show that a ratchet effect can arise in this type of system even in the absence of ac forcing. The directed motion occurs for certain particle-substrate interaction rules and its magnitude depends on the amount of time the particles spend swimming in one direction before turning and swimming in a new direction. For strictly Brownian particles there is no ratchet effect. If the particles reflect off the barriers or scatter from the barriers according to Snell's law there is no ratchet effect; however, if the particles can align with the barriers or move along the barriers, directed motion arises. We also find that under certain motion rules, particles accumulate along the walls of the container in agreement with experiment. We also examine pattern formation for synchronized particle motion. We discuss possible applications of this system for self-assembly, extracting work, and sorting as well as future directions such as considering collective interactions and flocking models.

  20. CYP2D6基因多态性及其临床意义

    徐艳娇; 龚森; 纪洪艳; 刘东

    2012-01-01

    CYP2D6是CYP酶系中重要的一种氧化代谢酶,参与多种药物的代谢.CYP2D6具有基因多态性,使药物代谢在不同种族之间,甚至在同种族不同人群中产生较大的差异,从而影响药物的疗效.因此,深入了解CYP2D6基因的多态性以及对药物代谢的影响,对指导临床合理用药和调整用药方案具有重大意义.

  1. Allelic distributions of CYP2D6 gene copy number variation in the Eastern Han Chinese population

    Hai-hui SHENG; Yun-lan DU; Jian SUN; Hua-sheng XIAO; Ai-ping ZENG; Wen-xiang ZHU; Ren-fang ZHU; Hong-mei LI; Zhi-dong ZHU; Ying QIN; Wei JIN; Yan LIU

    2007-01-01

    Aim: The human cytochrome P450 2D6 (CYP2D6) gene copy number variation, involving CYP2D6 gene deletion (CYP2D6*5) and duplication or multiduplication (CYP2D6*×N), can result in reduced or increased metabolism of many clinically used drugs. The identification of CYP2D6*5 and CYP2D6*×N and the investigation of their allelic distributions in ethnic populations can be important in deter-mining the right drug and dosage for each patient. Methods: The CYP2D6*5 andCYP2D6 genes, and CYP2D6 gene duplication were identified by 2 modified long PCR, respectively. To determine duplicated alleles, a novel long PCR was developed to amplify the entire duplicated CYP2D6 gene which was used as template for subsequent PCR amplification. A total of 363 unrelated Eastern Han Chinese individuals were analyzed for CYP2D6 gene copy number variation. Results: The frequency of CYP2D6*5 and CYP2D6*×N were 4.82% (n=35) and 0.69% (n=5) in the Eastern Han Chinese population, respectively. Of the 5 duplicated alleles, 3were CYP2D6*1×N and 2 were CYP2D6*10×N. One individual was a carrier of both CYP2D6*5 and CYP2D6*1×N. Taken together, the CYP2D6 gene rear-rangements were present in 10.74% of subjects. Conclusion: Allelic distributions of the CYP2D6 gene copy number variation differ among Chinese from different regions, indicating ethnic variety in Chinese. Long PCR are convenient, cost effective, specific and semiquantitative for the detection of the CYP2D6 gene copy number variation, and amplification of the entire duplicated CYP2D6 gene is necessary for the accurate identification of duplicated alleles.

  2. Ethnic background and CYP2D6 genetic polymorphisms in Costa Ricans.

    Céspedes-Garro, Carolina; Jiménez-Arce, Gerardo; Naranjo, María-Eugenia G; Barrantes, Ramiro; Llerena, Adrián

    2014-12-01

    CYP2D6 differences have already been demonstrated within Latin American populations by the CEIBA.FP Consortium of the Ibero-American Network of Pharmacogenetics (RIBEF, as per the acronym in Spanish). However, within the population of Costa Rica, no research has been conducted until now, even though this population has a trihybrid component ancestry that represents an interesting condition. Thus, the present study was aimed to determine the frequency of Ultra-rapid Metabolizers (UMs) and Poor Metabolizers (PMs) in a Costa Rican population, as well as to determine whether there are differences in the CYP2D6-predicted phenotype frequencies among three Costa Rican groups with different ethnic backgrounds. Additionally, these frequencies of PMs and UMs obtained were compared with Ibero-American populations published data. Finally, we also aimed to describe allele frequencies among different Costa Rican ethnic groups. This research has been undertaken within the framework of the RIBEF CEIBA Consortium studies on Latin American populations. A total of 385 individuals were included in the study: 139 mestizos, 197 Amerindians, and 49 Afro-Caribbeans. CYP2D6 genotypes were determined by XL-PCR and Real-Time PCR. The CYP2D6 variant alleles *2, *3, *4, *5, *6, *10, "17, *29, *35 and *41 were also determined. For the entire Costa Rican population, the frequency of PMs and UMs was 6% and 6.5%, respectively. The percentage of UMs in the mestizo population was higher than in the Amerindian population. CYP2D6 UMs vary from 3.6% to 10.1% and PMs from 1.4% to 10.2% among three Costa Rican groups. The highest frequencies of UMs (10.1%) and PMs (10.2%) were found in the mestizo and Amerindian populations, respectively. In conclusion, the frequencies of UMs and PMs for CYP2D6 varied widely across the mestizo, Amerindian and Afro-Caribbean Costa Rican populations. Future research in this population should be oriented to identify new CYP2D6 variants through sequencing methods, as well as

  3. Determination of Cytochrome P450 2D6 (CYP2D6 Gene Copy Number by Real-Time Quantitative PCR

    Laurent Bodin

    2005-01-01

    Full Text Available Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number. Using the 2−ΔΔCt calculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies, and 12 samples with duplicated genes. The average ratio ranged from 1.02 to 1.28, 1.85 to 2.21, and 2.55 to 3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

  4. Does the Medication Pattern Reflect the CYP2D6 Genotype in Patients With Diagnoses Within the Schizophrenic Spectrum?

    Jürgens, Gesche; Rasmussen, Henrik B; Werge, Thomas;

    2012-01-01

    Cytochrome P450 2D6 enzyme (CYP2D6) is an important metabolic pathway for many antipsychotics. Its genetic polymorphism causes pharmacokinetic variability that might lead to adverse drug reactions or treatment failure unless countered by appropriate dose adjustments or shift to CYP2D6-independent...

  5. The influence of the CYP2D6*4 polymorphism on drug response and disease susceptibility

    M.J. Bijl (Monique)

    2009-01-01

    textabstractThis thesis is about the role of CYP2D6, a drug-metabolizing enzyme, in today’s pharmacotherapy. Cytochrome P450 2D6 (CYP2D6) is an important member of a large family of enzymes with the name cytochrome P450 which is abundantly present in most non-monocellular living organisms. Its histo

  6. An optimized methodology for combined phenotyping and genotyping on CYP2D6 and CYP2C19

    Tamminga, C.A; Wemer, J; Oosterhuis, B; Brakenhoff, J.P G; Gerrits, M.G F; de Zeeuw, R.A; de Leij, Lou; Jonkman, J.H.G.

    2001-01-01

    A method for simultaneous phenotyping and genotyping for CYP2D6 and CYP2C19 was tested. Six healthy volunteers were selected (three extensive and three poor metabolisers for CYP2D6). CYP2D6 was probed with dextromethorphan and metoprolol and CYP2C19 was probed with omeprazole. Blood samples were col

  7. Role of Pharmacogenetics in Improving the Safety of Psychiatric Care by Predicting the Potential Risks of Mania in CYP2D6 Poor Metabolizers Diagnosed With Bipolar Disorder.

    Sánchez-Iglesias, Santiago; García-Solaesa, Virginia; García-Berrocal, Belén; Sanchez-Martín, Almudena; Lorenzo-Romo, Carolina; Martín-Pinto, Tomás; Gaedigk, Andrea; González-Buitrago, José Manuel; Isidoro-García, María

    2016-02-01

    One of the main concerns in psychiatric care is safety related to drug management. Pharmacogenetics provides an important tool to assess causes that may have contributed the adverse events during psychiatric therapy. This study illustrates the potential of pharmacogenetics to identify those patients for which pharmacogenetic-guided therapy could be appropriate. It aimed to investigate CYP2D6 genotype in our psychiatric population to assess the value of introducing pharmacogenetics as a primary improvement for predicting side effects.A broad series of 224 psychiatric patients comprising psychotic disorders, depressive disturbances, bipolar disorders, and anxiety disorders was included. The patients were genotyped with the AmpliChip CYP450 Test to analyzing 33 allelic variants of the CYP2D6 gene.All bipolar patients with poor metabolizer status showed maniac switching when CYP2D6 substrates such as selective serotonin reuptake inhibitors were prescribed. No specific patterns were identified for adverse events for other disorders.We propose to utilize pharmacogenetic testing as an intervention to aid in the identification of patients who are at risk of developing affective switching in bipolar disorder treated with selective serotonin reuptake inhibitors, CYP2D6 substrates, and inhibitors. PMID:26871771

  8. Correlation between polymorphism of CYP2D6 gene with effect of tamoxifen in patients with breast cancer%CYP2D6多态性与乳腺癌患者他莫昔芬疗效的相关性

    田超; 杨义; 李卉

    2014-01-01

    目的 探讨CYP2D6基因多态性与乳腺癌患者他莫昔芬(TAM)及其活性代谢产物4-羟基他莫昔芬(4-OH-TAM)血清浓度的相关性,并探讨CYP2D6基因多态性与乳腺癌患者预后的相关性.方法 收集2008年1月至2010年10月期间200例服用TAM的乳腺癌患者的口腔黏膜及血清标本,采用Real-time RT-PCR法检测CYP2D6* 10基因多态性,并分析其与临床病理特征和预后的关系.采用液相色谱-质谱方法(LC-MS)测定患者体内TAM及其活性代谢物4-OH-TAM的血清浓度.结果 200例乳腺癌中检测到CYP2D6* 10/* 10纯合子94例(47%),CYP2D6 wt/wt野生型48例(24%),CYP2D6 wt/* 10杂合型58例(29%).CYP2D6 wt/wt野生型和wt/* 10杂合型两组4-OH-TAM的血清浓度都明显高于*10/*10纯合型(F=4.31,P=0.01).CYP2D6* 10基因多态性与患者各项临床病理因素均无关(均P >0.05).Log-rank检验分析显示CYP2D6突变组患者平均无病生存时间(47.2个月)明显短于CYP2D6正常组患者(51.2个月),P=0.018.Cox回归分析显示CYP2D6基因型与患者无病生存时间明显相关(HR=2.755,95%CI:1.230 ~6.173,P=0.014).结论 CYP2D6*10/*10基因型与乳腺癌他莫昔芬的疗效相关,检测CYP2D6基因型可能有助于临床上有效选择他莫昔芬.%Objective To investigate the correlation of CYP2D6 gene polymorphism and serum tamoxifen (TAM) and its active metabolites (4-hydroxy tamoxifen,4-OH-TAM) in terms of prognosis of breast cancer patients.Methods The oral mucosas and serum were obtained from 200 breast cancer patients receiving oral TAM from Jan 2008 to Oct 2010.Real-time RT-PCR was used to determine CYP2D6* 10 gene polymorphism.The relationship between CYP2D6* 10 gene polymorphism and clinicopathological features and prognosis was assessed by Chi-square test and Cox proportional model.Serum TAM and 4-OH-TAM concentration were determined by liquid chromatography-trap mass spectrometry (LC-MS).Results Of 200 breast cancer patients,CYP2D6 * 10/* 10

  9. Genomics of Dementia: APOE- and CYP2D6-Related Pharmacogenetics

    Ramón Cacabelos

    2012-01-01

    Full Text Available Dementia is a major problem of health in developed societies. Alzheimer’s disease (AD, vascular dementia, and mixed dementia account for over 90% of the most prevalent forms of dementia. Both genetic and environmental factors are determinant for the phenotypic expression of dementia. AD is a complex disorder in which many different gene clusters may be involved. Most genes screened to date belong to different proteomic and metabolomic pathways potentially affecting AD pathogenesis. The ε4 variant of the APOE gene seems to be a major risk factor for both degenerative and vascular dementia. Metabolic factors, cerebrovascular disorders, and epigenetic phenomena also contribute to neurodegeneration. Five categories of genes are mainly involved in pharmacogenomics: genes associated with disease pathogenesis, genes associated with the mechanism of action of a particular drug, genes associated with phase I and phase II metabolic reactions, genes associated with transporters, and pleiotropic genes and/or genes associated with concomitant pathologies. The APOE and CYP2D6 genes have been extensively studied in AD. The therapeutic response to conventional drugs in patients with AD is genotype specific, with CYP2D6-PMs, CYP2D6-UMs, and APOE-4/4 carriers acting as the worst responders. APOE and CYP2D6 may cooperate, as pleiotropic genes, in the metabolism of drugs and hepatic function. The introduction of pharmacogenetic procedures into AD pharmacological treatment may help to optimize therapeutics.

  10. CYP2D6 function moderates the pharmacokinetics and pharmacodynamics of 3,4-methylene-dioxymethamphetamine in a controlled study in healthy individuals.

    Schmid, Yasmin; Vizeli, Patrick; Hysek, Cédric M; Prestin, Katharina; Meyer Zu Schwabedissen, Henriette E; Liechti, Matthias E

    2016-08-01

    The role of genetic polymorphisms in cytochrome (CYP) 2D6 involved in the metabolism of 3,4-methylene-dioxymethamphetamine (MDMA, ecstasy) is unclear. Effects of genetic variants in CYP2D6 on the pharmacokinetics and pharmacodynamic effects of MDMA were characterized in 139 healthy individuals (70 men, 69 women) in a pooled analysis of eight double-blind, placebo-controlled crossover studies. In CYP2D6 poor metabolizers, the maximum concentrations (Cmax) of MDMA and its active metabolite 3,4-methylene-dioxyamphetamine were +15 and +50% higher, respectively, compared with extensive metabolizers and the Cmax of the inactive metabolite 4-hydroxy-3-methoxymethamphetamine was 50-70% lower. Blood pressure and subjective drug effects increased more rapidly after MDMA administration in poor metabolizers than in extensive metabolizers. In conclusion, the disposition of MDMA and its effects in humans are altered by polymorphic CYP2D6 activity, but the effects are small because of the autoinhibition of CYP2D6. PMID:27253829

  11. Novel variant of CYP2D6*6 is undetected by a commonly used genotyping procedure

    Rasmussen, Henrik Berg; Werge, Thomas

    2011-01-01

    We report the identification of a novel and defective variant of the gene encoding cytochrome P450 2D6 (CYP2D6). This novel variant is a subtype of CYP2D6*6 that was undetected by a commercially available 5' exonuclease-based assay. Because the novel variant was found in only one of 609 individuals......, it represents a rare subtype of CYP2D6*6 that may be restricted to a single family or a subpopulation. A procedure for the identification of the novel CYP2D6*6 variant using restriction enzyme treatment of amplified fragments was developed....

  12. Influence of CYP2D6-dependent metabolism on the steady-state pharmacokinetics and pharmacodynamics of metoprolol and nicardipine, alone and in combination.

    Laurent-Kenesi, M A; Funck-Brentano, C; Poirier, J M; Decolin, D; Jaillon, P

    1993-01-01

    1 The metabolism of metoprolol depends in part on the genetically determined activity of the CYP2D6 isoenzyme. In vitro studies have shown that nicardipine is a potent inhibitor of CYP2D6 activity. Since the combination of metoprolol and nicardipine is likely to be used for the treatment of hypertension, we examined the interaction between these two drugs at steady-state. 2 Fourteen healthy volunteers, seven extensive and seven poor metabolisers of dextromethorphan were studied in a double-bl...

  13. Pharmacokinetic interactions between monoamine oxidase A inhibitor harmaline and 5-methoxy-N,N-dimethyltryptamine, and the impact of CYP2D6 status.

    Jiang, Xi-Ling; Shen, Hong-Wu; Mager, Donald E; Yu, Ai-Ming

    2013-05-01

    5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT or street name "5-MEO") is a newer designer drug belonging to a group of naturally occurring indolealkylamines. Our recent study has demonstrated that coadministration of monoamine oxidase A (MAO-A) inhibitor harmaline (5 mg/kg) increases systemic exposure to 5-MeO-DMT (2 mg/kg) and active metabolite bufotenine. This study is aimed at delineating harmaline and 5-MeO-DMT pharmacokinetic (PK) interactions at multiple dose levels, as well as the impact of CYP2D6 that affects harmaline PK and determines 5-MeO-DMT O-demethylation to produce bufotenine. Our data revealed that inhibition of MAO-A-mediated metabolic elimination by harmaline (2, 5, and 15 mg/kg) led to a sharp increase in systemic and cerebral exposure to 5-MeO-DMT (2 and 10 mg/kg) at all dose combinations. A more pronounced effect on 5-MeO-DMT PK was associated with greater exposure to harmaline in wild-type mice than CYP2D6-humanized (Tg-CYP2D6) mice. Harmaline (5 mg/kg) also increased blood and brain bufotenine concentrations that were generally higher in Tg-CYP2D6 mice. Surprisingly, greater harmaline dose (15 mg/kg) reduced bufotenine levels. The in vivo inhibitory effect of harmaline on CYP2D6-catalyzed bufotenine formation was confirmed by in vitro study using purified CYP2D6. Given these findings, a unified PK model including the inhibition of MAO-A- and CYP2D6-catalyzed 5-MeO-DMT metabolism by harmaline was developed to describe blood harmaline, 5-MeO-DMT, and bufotenine PK profiles in both wild-type and Tg-CYP2D6 mouse models. This PK model may be further employed to predict harmaline and 5-MeO-DMT PK interactions at various doses, define the impact of CYP2D6 status, and drive harmaline-5-MeO-DMT pharmacodynamics. PMID:23393220

  14. Effects of CYP2D6 and UGT2B7 polymorphisms on pharmacokinetics of tamoxifen in Thai breast cancer patients

    Areepium N

    2013-08-01

    Full Text Available N Areepium,1 D Panomvana,1 P Rungwanonchai,1 S Sathaporn,2 N Voravud3 1Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 2Department of Surgery, Phramongkutklao Hospital, Bangkok, 3Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Purpose: The objective of this study was to evaluate the impact of CYP2D6 and UGT2B7 polymorphisms on tamoxifen (TAM pharmacokinetics in Thai breast cancer patients. Methods: Thai female breast cancer patients treated with TAM were included in the study. Patients were genotyped for CYP2D6 and UGT2B7 polymorphism, and plasma levels of TAM and its potent active metabolite endoxifen (END, at steady state, were identified. Results: Fifty-nine female breast cancer patients were included in the study. The average age was 50 ± 9.3 years old; 76% were premenopausal and 85% had estrogen receptor-positive breast cancer. The allele frequencies of CYP2D6*10 and UGT2B7*2 were 53% and 28%, respectively. Patients with CYP2D6*10/*10 had lower END concentrations compared with CYP2D26*1/*10 and CYP2D6*1/*1 (9.62 ng/mL versus 15.67 ng/mL and 21.55 ng/mL, respectively, P = 0.045. Polymorphisms of UGT2B7 alone did not have any impact on TAM metabolism. However, among 20 patients with CYP2D6*10/*10, one with UGT2B7*2/*2 had higher END concentrations compared against patients with UGT2B7*1/*1 and UGT2B7*1/*2 (31.36 ng/mL versus 7.86 ng/mL, respectively, P = 0.023. Conclusion: Results from this study confirmed the impacts of CYP2D6 polymorphisms on the pharmacokinetics of TAM, while UGT2B7 polymorphisms tended to have impact on TAM metabolism in patients with homozygous CYP2D6*10. Keywords: tamoxifen, pharmacogenomics, CYP2D6, UGT2B7, breast cancer

  15. Population pharmacokinetic modelling to assess the impact of CYP2D6 and CYP3A metabolic phenotypes on the pharmacokinetics of tamoxifen and endoxifen

    ter Heine, Rob; Binkhorst, Lisette; de Graan, Anne Joy M; de Bruijn, Peter; Beijnen, Jos H; Mathijssen, Ron H J; Huitema, Alwin D R

    2014-01-01

    AIMS: Tamoxifen is considered a pro-drug of its active metabolite endoxifen. The major metabolic enzymes involved in endoxifen formation are CYP2D6 and CYP3A. There is considerable evidence that variability in activity of these enzymes influences endoxifen exposure and thereby may influence the clin

  16. An investigation of the interaction between halofantrine, CYP2D6 and CYP3A4: studies with human liver microsomes and heterologous enzyme expression systems.

    Halliday, R C; Jones, B. C.; Smith, D. A.; N. R. Kitteringham; Park, B.K.

    1995-01-01

    1. We have assessed the interaction of the antimalarial halofantrine with cytochrome P450 (CYP) enzymes in vitro, with the use of microsomes from human liver and recombinant cell lines. 2. Rac-halofantrine was a potent inhibitor (IC50 = 1.06 microM, Ki = 4.3 microM) of the 1-hydroxylation of bufuralol, a marker for CYP2D6 activity. Of a group of structurally related antimalarials tested, only quinidine (IC50 = 0.04 microM) was more potent. 3. Microsomes prepared from recombinant CYP2D6 and CY...

  17. CYP2D6 poor metabolizer status might be associated with better response to risperidone treatment.

    Almoguera, Berta; Riveiro-Alvarez, Rosa; Lopez-Castroman, Jorge; Dorado, Pedro; Vaquero-Lorenzo, Concepción; Fernandez-Piqueras, José; Llerena, Adrián; Abad-Santos, Francisco; Baca-García, Enrique; Dal-Ré, Rafael; Ayuso, Carmen

    2013-11-01

    The variability in the antipsychotic response is, to some extent, genetically determined. Several studies have attempted to establish a role for genetic variation in genes coding pharmacokinetic and pharmacodynamic targets, but to date, no definite genetic predictive marker has been identified. We aimed to explore the putative role of 19 genetic variants and risperidone clinical improvement in 76 White schizophrenic inpatients, measured as change in Positive and Negative Syndrome Scale (PANSS). CYP2D6 poor metabolism was significantly associated with greater clinical improvement in total PANSS and a trend was also found for MDR1 3435C>T to higher total PANSS scores in 3435T carriers. This study suggests the importance that genetic variability on pharmacokinetic factors may have in risperidone response and gives evidence for the need for further investigation in order to establish the actual predictive value and clinical utility that CYP2D6 genotyping might have in risperidone therapy management. PMID:24026091

  18. Use of pharmacogenetics in bioequivalence studies to reduce sample size: an example with mirtazapine and CYP2D6.

    González-Vacarezza, N; Abad-Santos, F; Carcas-Sansuan, A; Dorado, P; Peñas-Lledó, E; Estévez-Carrizo, F; Llerena, A

    2013-10-01

    In bioequivalence studies, intra-individual variability (CV(w)) is critical in determining sample size. In particular, highly variable drugs may require enrollment of a greater number of subjects. We hypothesize that a strategy to reduce pharmacokinetic CV(w), and hence sample size and costs, would be to include subjects with decreased metabolic enzyme capacity for the drug under study. Therefore, two mirtazapine studies, two-way, two-period crossover design (n=68) were re-analysed to calculate the total CV(w) and the CV(w)s in three different CYP2D6 genotype groups (0, 1 and ≥ 2 active genes). The results showed that a 29.2 or 15.3% sample size reduction would have been possible if the recruitment had been of individuals carrying just 0 or 0 plus 1 CYP2D6 active genes, due to the lower CV(w). This suggests that there may be a role for pharmacogenetics in the design of bioequivalence studies to reduce sample size and costs, thus introducing a new paradigm for the biopharmaceutical evaluation of drug products. PMID:22733239

  19. Molecular Dynamics Simulations to Investigate the Influences of Amino Acid Mutations on Protein Three-Dimensional Structures of Cytochrome P450 2D6.1, 2, 10, 14A, 51, and 62.

    Shuichi Fukuyoshi

    Full Text Available Many natural mutants of the drug metabolizing enzyme cytochrome P450 (CYP 2D6 have been reported. Because the enzymatic activities of many mutants are different from that of the wild type, the genetic polymorphism of CYP2D6 plays an important role in drug metabolism. In this study, the molecular dynamics simulations of the wild type and mutants of CYP2D6, CYP2D6.1, 2, 10, 14A, 51, and 62 were performed, and the predictions of static and dynamic structures within them were conducted. In the mutant CYP2D6.10, 14A, and 61, dynamic properties of the F-G loop, which is one of the components of the active site access channel of CYP2D6, were different from that of the wild type. The F-G loop acted as the "hatch" of the channel, which was closed in those mutants. The structure of CYP2D6.51 was not converged by the simulation, which indicated that the three-dimensional structure of CYP2D6.51 was largely different from that of the wild type. In addition, the intramolecular interaction network of CYP2D6.10, 14A, and 61 was different from that of the wild type, and it is considered that these structural changes are the reason for the decrease or loss of enzymatic activities. On the other hand, the static and dynamic properties of CYP2D6.2, whose activity was normal, were not considerably different from those of the wild type.

  20. Genetic polymorphism of CYP2D6 and its influence on the personalized usage of antipsychotics%CYP2D6基因多态性与抗精神病药物的个体化应用

    陈冰; 蔡卫民; 杨婉花

    2012-01-01

    CYP2D6是一种重要的细胞色素P450酶,存在着显著的基因多态性.CYP2D6在抗精神病类药物的代谢中发挥着重要作用,与许多抗精神病药物药动学及药效学的个体间变异存在着密切联系,检测CYP2D6基因型有助于患者抗精神病药物治疗方案的选择和调整,提高用药的安全性和有效性.本文综述CYP2D6基因多态性对抗精神病药物的药动学、不良反应及药物相互作用的影响,探讨了CYP2D6基因型检测在抗精神病个体化治疗中的应用前景.%CYP2D6 is one of the most important cytochrome P450 enzymes. There is remarkable genetic polymorphism of CYP2D6. CYP2D6 play an important role in the metabolism of antipsychotics, and has significant correlation with the inter-individual difference ol pharmacokinetics and pharmacodynamics of antipsychotics. Determination of CYP2D6 genotypes is helpful in the selection and regulation of antip-sychotic therapy regimen for the elevation of ef-ficiency and safety. The influence of CYP2D6 genetic polymorphism on the pharmacokinetics, adverse effect and drug-drug interaction was reviewed and the usefulness of CYP2D6 genoty-ping in the personalized antipsychotic therapy was discussed

  1. Genetic polymorphisms of CYP2D6 in Chinese mainland%中国大陆人群CYP2D6基因多态性的检测

    纪玲; 潘世秀; 等

    2002-01-01

    目的检测中国人群CYP2D6序列的差异性.方法在223个中国大陆人中,使用四引物聚合酶链反应、等位基因特异性扩增聚合酶链反应和多重长聚合酶链反应方法检测CYP2D6等位基因*2、*3、*4、*5、*6、*8、*10和*14.结果在中国人群中,频率最高的弱代谢(PM)等位基因是CYP2D6*5(7.2%),其次是仅存在于东方人中的CYP2D6*14(2.0%),再次为CYP2D6*4(0.2%),未发现CYP2D6*3、*6和*8等位基因.与白种人相比,中国大陆人群中分布频率最高的等位基因是CYP2D6*10(51.6%).结论在中国大陆人群中,PM等位基因CYP2D6* 5和CYP2D6* 14的频率比其它东方人高,但CYP2D6*10的频率比其它东方人低.%Objective To observe the significant differences in the frequencies of the cytochrome P450 2D6 (CYP2D6) alleles in Chinese popoulations. Methods Tetra-primer polymerase chain reaction (PCR), allele specific amplification (ASA) PCR and multiplex long PCR were developed to detect the CYP2D6 alleles * 2, * 3, * 4, * 5, * 6, * 8, * 10 and * 14 in 223 subjects from Chinese mainland.Results The CYP2D6*5 allele was the most frequent poor metabolizer (PM) allele in Chinese (7.2%), followed by CYP2D6*14 (2.0%) which was only detected in orientals. There was only 0.2% CYP2D6*4, and no CYP2D6*3, * 6 and * 8 were detected. In contrast to the Caucasians, the most frequent allele in Chinese was the * 10 allele with a frequency of 51.6%. Conclusion The frequencies of PM alleles, CYP2D6*5 and CYP2D6* 14, were higher; but the frequency of CYP2D6*10 was lower in mainland Chinese population than that in other orientals.

  2. Polymorphism of CYP2D6 gene and the influence on the metabolism of metoprolol%CYP2D6基因多态性及对美托洛尔代谢的影响

    熊丹; 熊玉卿

    2011-01-01

    CYP2D6是一种重要的P450系氧化代谢酶,主要参与多种重要药物的代谢.CYP2D6基因多态性会引起药物代谢有显著的个体和种族差异.美托洛尔为选择性β1受体阻滞剂,临床应用上存在巨大个体差异,主要在肝脏经多条途径代谢,大约70%的代谢由CYP2D6介导,CPY2D6基因多态性对美托洛尔代谢有较大影响.本文从CYP2D6的基因多态性及它对美托洛尔代谢的影响这两方面作一综述.%As one of major cytochrome P450s, CYP2D6 is responsible for the metabolism of many important drugs. Genetic polymorphism of CYP2D6 is the basis of lager interindividual and racial variability in the metabolism of drug. Metoprolol is a selective β1-adrenoceptor antagonist, there is an obvious individual difference in clinic. It is metabolized in the liver, in vivo data indicate that approximately 70% of themetabolism of metoptolol depends upon CYP2D6. Genetic polymorphism of CYP2D6 have a great influence on the metabolism of metoprolol. Here we summarize the polymorphism of CYP2D6 gene and the influence on the metabolism of metoprolol.

  3. The prevalence of CYP2D6 and CYP2C19 genotypes in a population of healthy Dutch volunteers

    Tamminga, W.J; Wemer, J; Oosterhuis, B; de Zeeuw, R.A; de Leij, Lou; Jonkman, J.H.G.

    2001-01-01

    Aim: This study was performed in a sample of the Dutch population to estimate the prevalence of noncoding mutations of CYP2D6 and CYP2C19 as obtained by genotyping. In addition, the predictability of the genotyping strategy was assessed. Methods: The CYP2D6 and CYP2C19 status of 765 unrelated health

  4. Association of polymorphism in the cytochrome CYP2D6 and the efficacy and tolerability of simvastatin

    Mulder, A B; van Lijf, H J; Bon, M A; van den Bergh, F A; Touw, D J; Neef, C; Vermes, I

    2001-01-01

    OBJECTIVE: Because clinical data about the therapeutic consequences of polymorphic oxidation of simvastatin by CYP2D6 have not been well reported, we sought to investigate the possible link between polymorphism of CYP2D6 and the efficacy and tolerability of simvastatin treatment in a group of 88 pat

  5. Antipsychotic-induced extrapyramidal syndromes and cytochrome P-450 2D6 genotype : a case-control study

    Schillevoort, [No Value; de Boer, A; van der Weide, J; Steijns, LSW; Roos, RAC; Jansen, PAF; Leufkens, HGM

    2002-01-01

    To study the association between polymorphism of the cytochrome P-450 2D6 gene (CYP2D6) and the risk of antipsychotic-induced extrapyramidal syndromes, as measured by the use of anti parkinsonian medication. Data for this case-control study were obtained from a psychiatric hospital where newly admit

  6. CYP2D6基因多态性与他奠昔芬治疗

    杨汐

    2011-01-01

    CYP2D6是细胞色素P450酶系中的一种重要的药物代谢酶,CYP2D6基因的遗传变异可导致CYP2D6酶活性的不同,从而影响他莫昔芬的代谢,使携带不同基因型的乳腺癌病人对他莫昔芬的治疗反应不同.本文从CYP2D6基因多态性与他莫昔芬代谢、疗效及预后、副反应的关系,CYP2D6抑制剂对他莫昔芬代谢的影响等方面进行了简要综述.

  7. CARACTERIZACIÓN DE VARIANTES ALÉLICAS DE CITOCROMO CYP2D6 EN LA POBLACIÓN DE LA REGIÓN CENTROCCIDENTAL DE VENEZUELA Characterization Of Cytochrome Cyp2d6 Allele Variants In The Population Of The Central-Western Region Of Venezuela

    PEDRO GRIMÁN

    Full Text Available El gen CYP2D6 codifica para una monooxigenasa perteneciente al citocromo P450, la cual está involucrada en la biotransformación de un gran número de drogas comúnmente prescritas, como antidepresivos, antineoplásicos y antihipertensivos. Algunos efectos adversos, así como falla terapéutica pueden ser relacionados con la actividad anormal de CYP2D6 producto de polimorfismos en el gen de dicha enzima. Con el fin de predecir la frecuencia de algunos fenotipos metabolizadores pobres de CYP2D6 en la población de la región centroccidental de Venezuela se determinaron las frecuencias alélicas y genotípicas de las variantes alélicas CYP2D6*3, *4 y *6. Se extrajo ADN genómico a partir de sangre periférica de 100 individuos voluntarios aparentemente sanos, y se procedió a la genotipificación por PCR tetra-primer alelo-específica y análisis por electroforesis en geles de agarosa. Se compararon las frecuencias obtenidas con poblaciones de otros países. El alelo más frecuente fue CYP2D6*4 con 16,5%, mostrando una diferencia significativa con la reportada con poblaciones asiáticas. Este trabajo constituye un estudio preliminar en la caracterización de un grupo más amplio de alelos de CYP2D6 con el fin de asistir al desarrollo de una farmacoterapia individualizada en nuestro país.The CYP2D6 gene encodes for a monooxygenase belonging to the cytochrome P450, which is involved in the biotransformation of a large number of commonly prescribed drugs such as antidepressants, antihypertensive and antineoplastic. Some side effects, as well as therapeutic failure may be related to abnormal activity of CYP2D6 product of polymorphisms in the CYP2D6 gene. In order to predict the frequency of some poor metabolisers phenotypes of CYP2D6 in the population of the Central-Western region of Venezuela it was determined the allelic and genotypic frequencies of CYP2D6 *3, *4, *6 allelic variants. DNA was extracted from peripheral blood of 100 apparently

  8. A physiologically based pharmacokinetic model to predict disposition of CYP2D6 and CYP1A2 metabolized drugs in pregnant women.

    Ke, Alice Ban; Nallani, Srikanth C; Zhao, Ping; Rostami-Hodjegan, Amin; Isoherranen, Nina; Unadkat, Jashvant D

    2013-04-01

    Conducting pharmacokinetic (PK) studies in pregnant women is challenging. Therefore, we asked if a physiologically based pharmacokinetic (PBPK) model could be used to evaluate different dosing regimens for pregnant women. We refined and verified our previously published pregnancy PBPK model by incorporating cytochrome P450 CYP1A2 suppression (based on caffeine PK) and CYP2D6 induction (based on metoprolol PK) into the model. This model accounts for gestational age-dependent changes in maternal physiology and hepatic CYP3A activity. For verification, the disposition of CYP1A2-metabolized drug theophylline (THEO) and CYP2D6-metabolized drugs paroxetine (PAR), dextromethorphan (DEX), and clonidine (CLO) during pregnancy was predicted. Our PBPK model successfully predicted THEO disposition during the third trimester (T3). Predicted mean postpartum to third trimester (PP:T3) ratios of THEO area under the curve (AUC), maximum plasma concentration, and minimum plasma concentration were 0.76, 0.95, and 0.66 versus observed values 0.75, 0.89, and 0.72, respectively. The predicted mean PAR steady-state plasma concentration (Css) ratio (PP:T3) was 7.1 versus the observed value 3.7. Predicted mean DEX urinary ratio (UR) (PP:T3) was 2.9 versus the observed value 1.9. Predicted mean CLO AUC ratio (PP:T3) was 2.2 versus the observed value 1.7. Sensitivity analysis suggested that a 100% induction of CYP2D6 during T3 was required to recover the observed PP:T3 ratios of PAR Css, DEX UR, and CLO AUC. Based on these data, it is prudent to conclude that the magnitude of hepatic CYP2D6 induction during T3 ranges from 100 to 200%. Our PBPK model can predict the disposition of CYP1A2, 2D6, and 3A drugs during pregnancy. PMID:23355638

  9. CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping

    Amanda K Riffel

    2016-01-01

    Full Text Available TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false positive CYP2D6*15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6*15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6*35 which is also located in exon 1. Although alternative CYP2D6*15 and *35 assays resolved the issue, we discovered a novel CYP2D6*15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6*15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696 SNP of CYP2D6*43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe

  10. Cytochrome 450-2D6 Genotype Definition May Improve Therapy for Paroxysmal Atrial Fibrillation. A Case of Syncope Following

    Harry W. Daniell, MD

    2014-02-01

    Full Text Available An 81-year-old marathon runner-internist developed syncope followed by myocardial stunning two hours after conversion to normal sinus rhythm and 4 hours after adding a single 150mg dose of propafenone to 650mg of quinidine gluconate which he had ingested 3 hours earlier. Cardiac isoenzyme, electrolyte, CBC, TSH and EKG were normal. Echocardiogram one week later revealed left ventricular hypertrophy with an enlarged left atrium typical of men with habitual endurance-associated atrial fibrillation. Over the previous 24 years he had successfully converted more than 100 episodes of PAF with PIP oral quinidine sulfate (200mg-600mg or quinidine gluconate (650-975mg without utilizing other cardiac, anti-arrhythmic, or anti-coagulation therapy. Several months after we published his adverse event (37 CYP2D6 analysis demonstrated alleles 4 and 41, identifying him as an IM/PM 2D6-deficient subject. Within the next year, he successfully converted episodes of PAF on 2 occasions with 100mg of oral flecainide, but both were accompanied by mildly symptomatic EKG-documented atrial flutter with 2/1 AV block and a ventricular rate of 120-140 beats per minute, resulting in a return to PIP quinidine and successful conversion of over 60 additional mildly symptomatic but increasingly frequent episodes of PAF over the next 3 years by utilizing 200-600mg of oral quinidine sulfate each resolving within 4-8 hours, during which he was able to continue his daily exercise and other daily activities without interruption.

  11. Analysis of CYP2D6 gene duplication of Chinese population in Hubei%湖北地区人群的CYP2D6二倍体多态性基因分析

    纪玲; 马江涛; 何林; Martin Hersberger

    2004-01-01

    使用长模板PCR方法检测223例湖北地区人群中CYP2D6二倍体.在被检人群中,CYP2D6二倍体的基因频率为2.7%.检测到6例CYP2D6二倍体,基因型分别为*1×2(3/6),*2×2(2/6);*10×2(1/6).表明湖北地区人群中CYP2D6二倍体的基因频率比其他地区中国人群稍高.

  12. Stimulus control by 5methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice

    Winter, J. C.; Amorosi, D.J.; Rice, Kenner C.; Cheng, Kejun; Yu, Ai-Ming

    2011-01-01

    In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The t...

  13. CYP2D6基因多态性与药物基因组学研究的进展%Research advance in CYP2D6 genetic polymorphism and pharmacogenomics

    董天崴; 王爽; 杨军; 张志国

    2014-01-01

    CYP2D6 genetic polymorphism results in individual difference of therapeutic effect and adverse reaction of related drugs this article made an overview for that.%CYP2D6基因多态性导致有关药物疗效及不良反应的个体差异,本文就此进行综述。

  14. Lack of influence of CYP2D6 genotype on the clearance of (R)-, (S)- and racemic-methadone

    Coller, J K; Joergensen, C; Foster, D J R;

    2007-01-01

    OBJECTIVE: To investigate the influence of CYP2D6 genotype on the oral clearance of (R)-, (S)- and rac-methadone. METHODS: In this retrospective study, CYP2D6 genotypes were identified in 56 methadone maintained subjects. Plasma concentrations of (R)-, (S)- and rac-methadone were determined by...... stereoselective HPLC and sufficient data were available to estimate the apparent oral clearances of (R)-, (S)- and rac-methadone using a population kinetic model in 37 of the genotyped subjects. RESULTS: The CYP2D6 allele frequencies were similar to those previously reported in Caucasians, the most common being......: CYP2D6*1 (35.2%), CYP2D6*2 (12.0%) and CYP2D6*4 (22.2%). Three unknown SNPs were found in four subjects: 1811G > A (n = 1), 1834C > T (n = 1) and 2720G > C (n = 2). The oral clearances of (R)-, (S)- and rac-methadone varied 5.4-, 6.8- and 6.1-fold, respectively. No significant differences in methadone...

  15. Length of psychiatric hospitalization is correlated with CYP2D6 functional status in inpatients with major depressive disorder

    Ruaño, Gualberto; Szarek, Bonnie L; Villagra, David; Gorowski, Krystyna; Kocherla, Mohan; Seip, Richard L; Goethe, John W; Schwartz, Harold I

    2016-01-01

    Aim This study aimed to determine the effect of the CYP2D6 genotype on the length of hospitalization stay for patients treated for major depressive disorder. Methods A total of 149 inpatients with a diagnosis of major depressive disorder at the Institute of Living, Hartford Hospital (CT, USA), were genotyped to detect altered alleles in the CYP2D6 gene. Prospectively defined drug metabolism indices (metabolic reserve, metabolic alteration and allele alteration) were determined quantitatively and assessed for their relationship to length of hospitalization stay. Results Hospital stay was significantly longer in deficient CYP2D6 metabolizers (metabolic reserve <2) compared with functional or suprafunctional metabolizers (metabolic reserve ≥2; 7.8 vs 5.7 days, respectively; p = 0.002). Conclusion CYP2D6 enzymatic functional status significantly affected length of hospital stay, perhaps due to reduced efficacy or increased side effects of the medications metabolized by the CYP2D6 isoenzyme. Functional scoring of CYP2D6 alleles may have a substantial impact on the quality of care, patient satisfaction and the economics of psychiatric treatment. PMID:23734807

  16. Machine Learning Energies of 2 M Elpasolite (ABC$_2$D$_6$) Crystals

    Faber, Felix; von Lilienfeld, O Anatole; Armiento, Rickard

    2015-01-01

    Elpasolite is the predominant quaternary crystal structure (AlNaK$_2$F$_6$ prototype) reported in the Inorganic Crystal Structure Database. We have developed a machine learning model to calculate density functional theory quality formation energies of all the 2 M pristine ABC$_2$D$_6$ elpasolite crystals which can be made up from main-group elements (up to bismuth). Our model's accuracy can be improved systematically, reaching 0.1 eV/atom for a training set consisting of 10 k crystals. Important bonding trends are revealed, fluoride is best suited to fit the coordination of the D site which lowers the formation energy whereas the opposite is found for carbon. The bonding contribution of elements A and B is very small on average. Low formation energies result from A and B being late elements from group (II), C being a late (I) element, and D being fluoride. Out of 2 M crystals, the three degenerate pairs CaSrCs$_2$F$_6$/SrCaCs$_2$F$_6$, CaSrRb$_2$F$_6$/SrCaRb$_2$F$_6$ and CaBaCs$_2$F$_6$/BaCaCs$_2$F$_6$ yield ...

  17. CYP2D6*10B基因型对中国人普罗帕酮对映体药动学的影响%Influence of CYP2D6*10B genotype on pharmacokinetics of propafenone enantiomers in Chinese subjects

    陈冰; 蔡卫民

    2003-01-01

    AIM: To study the relationship between genotype of CYP2D6*10B and pharmacokinetics of propafenone enantiomers. METHODS: Genotype of 17 healthy Chinese HAN subjects was determined by an allele specific amplification method. The blood samples (0-15 h) of the subjects were taken after oral administration of a single dose (400 mg) of propafenone hydrochloride. Concentrations of propafenone enantiomers in plasma were mea sured by a reverse-phase HPLC with precolumn derivatization. RESULTS: Seventeen subjects characterized for CYP2D6* 1 0B genotype included (* 1/* 1) (n = 4), (* 1/* 10) (n = 5) and (* 10/* 10) (n = 8). The metabolic ratios (lg MR) of the three genotypes were -2.68+0.23, -2.2+0.7, and -1.1 +0.5, respectively. The AUC of the three groups that of *1/*10 group or *1/*1 group, and the CL of both enantiomers in *10/*10 is only half of that of *1/*10 group or * 1/* 1 group (P<0.05). CONCLUSION: CYP2D6* 10B alleles induce the declined activity of CYP2D6 and impair the metabolism of propafenone.

  18. 人CYP2D6基因体外表达体系和功能研究方法的建立%Expressing in Vitro and Functional Study of Human CYP2D6 Gene

    吴振强; 秦胜营

    2013-01-01

    根据G eneBank数据库中人CYP2D6基因构建表达载体pMA91-CYP2D6,转化酵母细胞AH22在体外进行表达,抽提所表达的CYP2D6微粒体蛋白进行Western Blot验证表明表达成功.该微粒体蛋白与探针药物异喹胍反应,利用HPLC检测生成产物4-羟基异喹胍,通过分析其得到酶动力学参数Km=10.14±0.94 μmol/L,最终建立起完整的人CYP2D6表达体系和功能研究方法,为未来测定CYP2D6各等位基因对应酶的活性,实现临床个性化用药奠定基础.

  19. CYP2D6基因克隆及在自身免疫性肝炎中的应用%CLONING OF CYP2D6 AND ITS APPLICATION IN AUTOIMMUNE HEPATITIS

    王达; 仲人前; 于嘉屏; 孔宪涛

    2000-01-01

    采用基因克隆与表达技术制备重组人CYP2D6抗原,建立免疫印迹法,检测几组肝炎患者中的抗CYP2D6抗体.605例ALT升高的成人肝炎患者组内CYP2D6自身抗体阳性检出率为3.97%(24/605),50例ALT升高的小儿肝炎患者组内阳性检出率为12%(6/50). 两组比较, 经统计学处理差异有显著性意义(P<0.01).50名正常人及40例HCV RNA阳性的肝炎患者中无一例发现该自身抗体阳性.检出的30例CYP2D6自身抗体阳性患者,30%合并有HBV感染.结果提示:CYP2D6自身抗体检测可作为自身免疫性肝炎诊断的一项重要指标.

  20. Systematic functional study of cytochrome P450 2D6 promoter polymorphisms in the Chinese Han population.

    Xueli Gong

    Full Text Available The promoter polymorphisms of drug-metabolizing genes can lead to interindividual differences in gene expression, which may result in adverse drug effects and therapeutic failure. Based on the database of CYP2D6 gene polymorphisms in the Chinese Han population established by our group, we functionally characterized the single nucleotide polymorphisms (SNPs of the promoter region and corresponding haplotypes in this population. Using site-directed mutagenesis, all the five SNPs identified and ten haplotypes with a frequency equal to or greater than 0.01 in the population were constructed on a luciferase reporter system. Dual luciferase reporter systems were used to analyze regulatory activity. The activity produced by Haplo3(-2183G>A, -1775A>G, -1589G>C, -1431C>T, -1000G>A, -678A>G, Haplo8(-2065G>A, -2058T>G, -1775A>G, -1589G>C, -1235G>A, -678A>G and MU3(-498C>A was 0.7-, 0.7-, 1.2- times respectively compared with the wild type in human hepatoma cell lines(p<0.05. These findings might be useful for optimizing pharmacotherapy and the design of personalized medicine.

  1. Variantes alélicas de CYP2D6: *4, *6 y *10 en una muestra de residentes del estado Aragua, Venezuela

    Carlos Flores-Angulo

    2015-12-01

    Full Text Available El objetivo del estudio fue determinar la frecuencia de las variantes del gen CYP2D6: *4, *6 y *10 y predecir el fenotipo metabolizador en una muestra de 145 individuos no consanguíneos, aparentemente sanos, residentes del estado Aragua, Venezuela. Los genotipos fueron determinados mediante ensayos de reacción en cadena de la polimerasa seguidos de digestión con endonucleasas de restricción. La predicción del fenotipo metabolizador se realizó con base al sistema Activity score. Las frecuencias de CYP2D6 *4, *6 y *10 fueron de 14,5%, 0,3% y 1%, respectivamente; un porcentaje significativo de individuos fueron categorizados como metabolizador rápido heterocigoto/metabolizador intermedio (23,5% y metabolizador lento (4,1%. Esta información tiene impacto clínico potencial, porque CYP2D6 interviene en el metabolismo de fármacos de prescripción frecuente como: carvedilol, captopril, cloroquina, codeína, fluoxetina, fluvastatina, haloperidol, idarrubicina, indinavir, imatinib, loperamida, nifedipina, ondansetrón y tamoxifeno

  2. Variantes alélicas de CYP2D6: *4, *6 y *10 en una muestra de residentes del estado Aragua, Venezuela

    Carlos Flores-Angulo

    2015-10-01

    Full Text Available El objetivo del estudio fue determinar la frecuencia de las variantes del gen CYP2D6: *4, *6 y *10 y predecir el fenotipo metabolizador en una muestra de 145 individuos no consanguíneos, aparentemente sanos, residentes del estado Aragua, Venezuela. Los genotipos fueron determinados mediante ensayos de reacción en cadena de la polimerasa seguidos de digestión con endonucleasas de restricción. La predicción del fenotipo metabolizador se realizó con base al sistema Activity score. Las frecuencias de CYP2D6 *4, *6 y *10 fueron de 14,5%, 0,3% y 1%, respectivamente; un porcentaje significativo de individuos fueron categorizados como metabolizador rápido heterocigoto/metabolizador intermedio (23,5% y metabolizador lento (4,1%. Esta información tiene impacto clínico potencial, porque CYP2D6 interviene en el metabolismo de fármacos de prescripción frecuente como: carvedilol, captopril, cloroquina, codeína, fluoxetina, fluvastatina, haloperidol, idarrubicina, indinavir, imatinib, loperamida, nifedipina, ondansetrón y tamoxifeno

  3. Advances in correlation between CYP2D6 and tamoxifen therapy%CYP2D6与他莫昔芬疗效相关性的研究进展

    雷蕾; 王晓稼

    2009-01-01

    CYP2D6是一种重要的P450系氧化代谢酶,是他莫昔芬在体内代谢成更强抗雌激素作用代谢产物endoxifen的重要代谢酶,因此,强代谢乳腺癌患者服用他莫昔芬后引起明显的潮热症状,而潮热症状的发生与他莫昔芬疗效正相关.除了CYP2D6基因多态性可能影响他莫昔芬体内的代谢外,帕罗西汀(paroxetine)可竞争性抑制CYP2D6对他莫昔芬的代谢,使乳腺癌患者对他莫昔芬疗效明显降低.

  4. CYP2D6 genotype predicts antipsychotic side effects in schizophrenia inpatients: a retrospective matched case-control study

    Kobylecki, Camilla J; Jakobsen, Klaus D; Hansen, Thomas;

    2009-01-01

    OBJECTIVE: The aim of the present retrospective pilot study was to examine the clinical impact of the cytochrome P450 (CYP) enzyme CYP2D6 poor metabolizer (PM) genotype in patients taking antipsychotic medication. The impaired metabolic capacity of the PM genotype results in higher steady......-state plasma concentrations at a given dose, thus increasing the risk of toxic effects from medication. METHODS: We identified 18 PM patients with a schizophrenia spectrum diagnosis from a clinical database covering all patients who have been analyzed in an ongoing standardized CYP2D6 screening program. Each...... significantly higher prevalence of noncompliance among the same PM patients. Importantly, this association was not due to differences in the use of CYP2D6-dependent or EPS/TD-causing medication across the 3 matched patient groups. CONCLUSIONS: This leads us to conclude that genetically encoded differences in...

  5. CYP2 D6的基因多态性与药物临床应用

    刘周杰

    2016-01-01

    CYP2D6是CYP酶系中重要的一种氧化代谢酶,参与多种药物的代谢。 CYP2D6具有基因多态性,这是构成药物代谢个体差异和种族差异的基础,它主要参与心血管类、抗精神病类、镇痛药、以及一些抗癌药物等的代谢。研究 CYP2D6基因多态性与药物代谢个体差异的相关性,有助于减轻药物不良反应,提高治疗效果,实施个体化给药。

  6. Stimulus control by 5methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice

    Winter, J. C.; Amorosi, D. J.; Rice, Kenner C.; Cheng, Kejun; Yu, Ai-Ming

    2011-01-01

    In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT. PMID:21624387

  7. Stimulus control by 5-methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice.

    Winter, J C; Amorosi, D J; Rice, Kenner C; Cheng, Kejun; Yu, Ai-Ming

    2011-09-01

    In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT. PMID:21624387

  8. 厚朴提取物对大鼠CYP2D6亚型酶的影响%Influence of magnolia bark extract on CYP2D6 subtype enzyme in rats

    于卫江; 张斌; 张文周

    2013-01-01

    目的 观察厚朴提取物对大鼠CYP2D6亚型酶的影响.方法 将16只成年雄性Wistar大鼠随机分为对照组和实验组,2组分别经口给予生理盐水和厚朴提取物1周,采用高效液相色谱法(HPLC)测定大鼠尿样及肝微粒体中CYP2D6的探针药物右美沙芬(DM)的代谢率,观察厚朴提取物对CYP2D6亚型酶活性的影响,并通过特异性抑制剂确定肝微粒体重组系统中厚朴提取物对CYP2D6亚型酶活性的影响.结果 实验组大鼠尿样及体外肝微粒体中DM代谢率均明显低于对照组,差异有统计学意义;厚朴提取物组肝微粒体中DM代谢率显著低于西咪替丁组和对照组,差异有统计学意义,而西咪替丁组和对照组比较,差异无统计学意义.结论 厚朴提取物可有效抑制CYP2D6亚型酶的活性,且抑制能力优于西咪替丁.

  9. CYP2D6和GST在白种人晚期肾病中的遗传变异%Genetic Variation of CYP2D6 and GST in Caucasian ESRD Patients

    严奉祥; WedlundPJ; 等

    2002-01-01

    目的:生物转化酶细胞色素P4502D6(CYP2D6)和谷胱苷肽转移酶-M1(GST-M1)、谷胱苷肽转移酶-T1(GST-T1)共同代谢内源性和外源性毒素,一部分人群由于相应基因变异导致这些酶缺乏表达,我们检测白种人群中晚期肾病(ESRD)病人这些酶的基因多态性是否比健康者有较高的频率.方法:从列克星顿及周围地区征募330名晚期肾病病人和303名健康者,均为白种人,给予他们进行CYP2D6和GST-M1和T1基因分型.结果:在ESRD病人中CYP2D6和GST-M1和GST-T1以及CYP2D6和GST-M1或GST-T1都缺乏分别为2.1%和4.2%;而在健康者中均为0.3%(P<0.01).结论:CYP2D6和GST-M1和/或GST-T1酶缺乏在ESRD病人中有较高的频率,提示这些酶缺乏可以预计慢性肾病进展的可能性.

  10. Risperidone-associated adverse drug reactions and CYP2D6 polymorphisms in a South African cohort

    Tyren M. Dodgen

    2015-06-01

    Conclusion: CYP2D6 variation appeared not to be a good pharmacogenetic marker for predicting risperidone-related ADRs in this naturalistic South African cohort. Evaluation of a larger cohort would be needed to confirm these observations, including an examination of the role of potential intermediaries between the hypothesised genetic and clinical phenotypes.

  11. 宁夏回族人群CYP2D6*10基因多态性及功能分析%Analysis on polymorphism function of CYP2D6 * 10 in Ningxia Hui

    李居怡; 王健; 高鹏; 杜娟; 李宗吉

    2010-01-01

    目的 探讨基因突变对人CYP2D6蛋白结构与功能的影响.方法 采用等位基因特异扩增(ASA-PCR)及DNA测序技术分析宁夏网族人群CYP2D6*10(C188T)基因多态性,以生物信息学方法对突变造成的肝药酶活性的下降做出合理的解释.结果 蛋白质基本性质分析工具(ProtParam)分析显示,在溶液中CYP2D6*10突变型蛋白的不稳定指数高于野生型,都高于阈值40;二级结构预测软件(DNAStar/Protean)分析显示,突变型蛋白的二级结构在第33位多了一个转角(Gamier-Robson Turn);功能佗点预测程序(Motif Scan)对蛋白功能位点进行预测,结果显示CYP2D6*10野生型蛋白有2个P450酶激活位点.而突变型没有;信号肽预测程序(Signal P)分析显示.神经网络模型(NN)C-score计算结果为突变型蛋白没有信号肽,而野牛型有.结论 基因突变可引起CYP2D6蛋白结构与功能的改变;应用生物信息学方法对CYP2D6基因突变致使的酶活性的下降做出一些可能的解释是可行的.

  12. CYP2D6 variation, behaviour and psychopathology: implications for pharmacogenomics-guided clinical trials

    Peñas-LLedó, Eva M; LLerena, Adrián

    2014-01-01

    Individual and population differences in polymorphic cytochrome P450 enzyme function have been known for decades. The biological significance of these differences has now been deciphered with regard to drug metabolism, action and toxicity as well as disposition of endogenous substrates, including neuroactive compounds. While the cytochrome P450 enzymes occur abundantly in the liver, they are expressed in most tissues of the body, albeit in varying amounts, including the brain. The latter loca...

  13. Effect of CYP2D6*10 genotype on propafenone pharmacodynamics in Chinese patients with ventricular arrhythmia%CYP2D6*10基因型对中国室性心律失常病人普罗帕酮药效学的影响

    蔡卫民; 徐军; 陈冰; 张馥敏; 黄元铸; 张银娣

    2002-01-01

    important role in plasma levelsand effects of propafenone. In 450 mg/d group, patients with homozygous mutant of CYP2D6* 10 not only had aCmax of propafenone two times as high as those of wild-type genotype, but also showed a two fold higher inhibitoryrate of VPC compared with those with homozygous CYP2D6* 1 (P<0.05). CONCLUSION: CYP2D6* 10 geno-type is relevant to decreased activity of CYP2D6 enzyme in Chinese patients. Elevated plasma concentration isconsistent with better efficacy of propafenone in patients with ventricular arrhythmia.

  14. 氧化苦参碱对大鼠CYP2D6酶的影响

    张斌; 于卫江

    2008-01-01

    目的 通过氧化苦参碱的大鼠体内、外实验,观察氧化苦参碱对大鼠CYP2D6 亚型酶的影响.方法 HPLC法测定大鼠尿液及肝微粒体中探针药物右美沙芬(DM)、代谢物去甲右美沙芬(DT)的含量.对照组和氧化苦参碱组大鼠分别经口给予生理盐水和氧化苦参碱两周,HPLC法测定大鼠尿样及肝微粒体中CYP2D6的探针药物右美沙芬的代谢率,观察氧化苦参碱对CYP2D6活性的影响.并且通过特异性抑制剂确定在肝微粒体重组系统中氧化苦参碱对CYP2D6亚型的影响.结果 实验组大鼠给予氧化苦参碱(100mg/kg),其尿样中右美沙芬的代谢率与对照组相比没有明显差别(P>0.05);实验组大鼠肝微粒体中加入右美沙芬(0.324mmol/L),其右美沙芬的代谢率与对照组相比没有明显差别(P>0.05);氧化苦参碱没有明显降低右美沙芬的代谢率(P>0.05),而CYP2D6特异性抑制剂西米替丁却明显降低右美沙芬的代谢率(P<0.01).结论 氧化苦参碱对CYP2D6酶无明显影响.

  15. 细胞色素氧化酶CYP2D6的研究进展

    张泓波; 李宝群; 王瑞婷; 梅爱敏

    2005-01-01

    细胞色素氧化酶CYP450是药物代谢中的一个重要酶系。近年来,对CYP450与药物代谢多态性的关系进行了诸多方面的研究。现已明确CYP2C9、CYP2C19、CYP2D6在表型和基因型水平上均存在遗传多态性,且对其分子机制有了较为深入的了解;而CYPlA2等其他酶可能存在多态性,但各人群研究结果不甚一致。本文着重综述CYP2D6的研究进展情况。

  16. Impact of Cytochrome P450 2D6 Function on the Chiral Blood Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine (MDMA) and Its Phase I and II Metabolites in Humans.

    Steuer, Andrea E; Schmidhauser, Corina; Tingelhoff, Eva H; Schmid, Yasmin; Rickli, Anna; Kraemer, Thomas; Liechti, Matthias E

    2016-01-01

    3,4-methylenedioxymethamphetamine (MDMA; ecstasy) metabolism is known to be stereoselective, with preference for S-stereoisomers. Its major metabolic step involves CYP2D6-catalyzed demethylenation to 3,4-dihydroxymethamphetamine (DHMA), followed by methylation and conjugation. Alterations in CYP2D6 genotype and/or phenotype have been associated with higher toxicity. Therefore, the impact of CYP2D6 function on the plasma pharmacokinetics of MDMA and its phase I and II metabolites was tested by comparing extensive metabolizers (EMs), intermediate metabolizers (IMs), and EMs that were pretreated with bupropion as a metabolic inhibitor in a controlled MDMA administration study. Blood plasma samples were collected from 16 healthy participants (13 EMs and three IMs) up to 24 h after MDMA administration in a double-blind, placebo-controlled, four-period, cross-over design, with subjects receiving 1 week placebo or bupropion pretreatment followed by a single placebo or MDMA (125 mg) dose. Bupropion pretreatment increased the maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from 0 to 24 h (AUC24) of R-MDMA (9% and 25%, respectively) and S-MDMA (16% and 38%, respectively). Bupropion reduced the Cmax and AUC24 of the CYP2D6-dependently formed metabolite stereoisomers of DHMA 3-sulfate, DHMA 4-sulfate, and 4-hydroxy-3-methoxymethamphetamine (HMMA sulfate and HMMA glucuronide) by approximately 40%. The changes that were observed in IMs were generally comparable to bupropion-pretreated EMs. Although changes in stereoselectivity based on CYP2D6 activity were observed, these likely have low clinical relevance. Bupropion and hydroxybupropion stereoisomer pharmacokinetics were unaltered by MDMA co-administration. The present data might aid further interpretations of toxicity based on CYP2D6-dependent MDMA metabolism. PMID:26967321

  17. Impact of Cytochrome P450 2D6 Function on the Chiral Blood Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine (MDMA and Its Phase I and II Metabolites in Humans.

    Andrea E Steuer

    Full Text Available 3,4-methylenedioxymethamphetamine (MDMA; ecstasy metabolism is known to be stereoselective, with preference for S-stereoisomers. Its major metabolic step involves CYP2D6-catalyzed demethylenation to 3,4-dihydroxymethamphetamine (DHMA, followed by methylation and conjugation. Alterations in CYP2D6 genotype and/or phenotype have been associated with higher toxicity. Therefore, the impact of CYP2D6 function on the plasma pharmacokinetics of MDMA and its phase I and II metabolites was tested by comparing extensive metabolizers (EMs, intermediate metabolizers (IMs, and EMs that were pretreated with bupropion as a metabolic inhibitor in a controlled MDMA administration study. Blood plasma samples were collected from 16 healthy participants (13 EMs and three IMs up to 24 h after MDMA administration in a double-blind, placebo-controlled, four-period, cross-over design, with subjects receiving 1 week placebo or bupropion pretreatment followed by a single placebo or MDMA (125 mg dose. Bupropion pretreatment increased the maximum plasma concentration (Cmax and area under the plasma concentration-time curve from 0 to 24 h (AUC24 of R-MDMA (9% and 25%, respectively and S-MDMA (16% and 38%, respectively. Bupropion reduced the Cmax and AUC24 of the CYP2D6-dependently formed metabolite stereoisomers of DHMA 3-sulfate, DHMA 4-sulfate, and 4-hydroxy-3-methoxymethamphetamine (HMMA sulfate and HMMA glucuronide by approximately 40%. The changes that were observed in IMs were generally comparable to bupropion-pretreated EMs. Although changes in stereoselectivity based on CYP2D6 activity were observed, these likely have low clinical relevance. Bupropion and hydroxybupropion stereoisomer pharmacokinetics were unaltered by MDMA co-administration. The present data might aid further interpretations of toxicity based on CYP2D6-dependent MDMA metabolism.

  18. Association of CYP2D6 and CYP1A2 gene polymorphism with tardive dyskinesia in Chinese schizophrenic patients

    Yan FU; Chang-he FAN; He-huang DENG; San-hong HU; De-peng LV; Li-hua LI; Jun-jie WANG; Xin-qiao LU

    2006-01-01

    Aim:To investigate the possible association of the CYP2D6 gene C100T polymorphism and the CYP1A2 gene C163A polymorphism with tardive dyskinesia (TD) in Chinese patients with schizophrenia.Methods:The recruited schizophrenic patients were assessed with the Abnormal Involuntary Movement Scale (AIMS),and divided into groups with TD(n=91)and without TD(n=91)according to the AIMS score.Polymorphisms of the CYP2D6 and CYP1A2 genes were determined by polymerase chain reaction(PER)-restriction fragment length polymorphism(RFLP).Results:No allele frequencies deviated from Hardy-Weinberg equilibrium.No significant differences in genotypes frequencies of the CYP2D C100T polymorphism were observed between patients with TD and without TD (x2=4.078,P>0.05),but patients with TD had a significant excess of the T allele compared with those without TD(x2=4.28,P<0.05).Moreover,the frequency of the CYP1A2 C allele in patients with TD was significantly higher than that in those without TD(x2=6.38,P<0.05).An association between TD and the CyP2D6 100T and CYP1A2 163C alleles was observed.Additionally,there were no differences in the mean AIMS scores among different genotypes in TD patients as a group or in smokers.The results of logistic regression anatysls demonstrated that mean age and duration of illness were risk factors for TD,but not sex,cumulative exposure to neuroleptic drugs in years,CYP2D6 or CYP1A2 genotype.Conclusion:The C100T polymorphism of the CYP2D6 gene and the C163A polymorphism of the CYP1A2 gene may be associated with neuroleptic drug-induced tardive dyskinesia in Chinese patients with schizophrenia.However,genetic factors have a weaker association with susceptibility to TD compared with mean age and duration of illness.

  19. Polymorphisms in the genes of citohrom oxidase P450 2D6 (CYP2D6, paraoxonase 1 (PON1 and apolipoproteine E (APOE as risk factors for Parkinson's disease

    Đurić Gordana

    2007-01-01

    Full Text Available Background/Aim. The presence of Parkinson's disease (PD among the members of a family is a clear indication of the significance of genetics in its development. In spite of that, the majority of patients with PD shows a sporadic form of the disease induced as a result of interaction of both environmental and genetic factors. The aim of this study was to examine the effects of polymorphisms in the genes of cytohrome P450 2D6(CYP2D6, paraoxonase 1 (PON 1 and apolipoprotein E (APOE, as risk factors for PD. Methods. We examined 106 patients with PD (65 men and 41 women and 75 ethnically matched control subjects. The mean age at onset of PD in the patients was 46.9±9.4 years (ranging from 30 to 70 years. Genotyping was performed using standard PCR amplification and restriction endonuclease digestion protocols described for known polymorphism in the candidate genes under study. Results. The genotype A/A polymorphisms 2D6* gene of CYP2D6 and genotype M/M polymorphisms L54M gene of PON1 were significantly more frequent in the patients with PD than in the control group. The patients with genotypes A/A and M/M had 3.4 and 3.2 higher risk of PD, respectively than the control group (p = 0.01. The relation between genotypes A/A gene of CYP2D6 and M/M gene of PON1 was modified by the age at onset. The genotypes were associated with early onset of PD (p = 0.001, p = 0.004. The carriers of the A and M alleles in homozygote had 2.4 and 4.2 years respectively earlier onset of PD than carriers of other genotypes with these polymorphisms. The frequency allele ε4 gene of APOE was higher in the PD patients with early onset (20% than in PD with later onset (7.4%, while the genotype ε3/ε3 was associated with PD late onset (p = 0.024. Combined genotype I (carriers of the two risk allels in homozygote and one alleles risk in heterozygote and combined genotype II (carriers of the three alleles risk in homozygote caused early PD. Combined genotype II was detected in 12

  20. Genotypes for the cytochrome P450 enzymes CYP2D6 and CYP2C19 in human longevitY

    Bathum, L; Andersen-Ranberg, K; Boldsen, J;

    1998-01-01

    OBJECTIVE: To test whether some genotypes for CYP2D6 or CYP2C19 could contribute to longevity, we genotyped 241 Danish nonagenarians and centenarians for CYP2D6 and CYP2C19. METHODS: For CYP2D6 we identified the alleles CYP2D6*1, CYP2D6*3 and CYP2D6*4 with allele-specific polymerase chain reaction...... (PCR). The CYP2D6*5 alleles were identified with a long PCR method. For CYP2C19 we identified the alleles CYP2C19*1, CYP2C19*2 and CYP2C19*3 with an oligonucleotide ligation assay. RESULTS: The four alleles for CYP2D6 did not occur in Hardy-Weinberg proportions. The frequency of poor metabolism was...... slightly higher (10.2%) than expected [7.7%; odds ratio (OR) = 1.36 (0.75-2.40)]. The genotypes for CYP2C19 occur in Hardy-Weinberg proportions. The frequency of poor metabolism (3.8%) was not significantly different from a young control group [3.1%; OR = 1.21 (0.26-5.75)]. CONCLUSION: CYP2D6 could play a...

  1. Effects of β1-adrenergic receptor and CYP2D6 genetic polymorphism on metoprolol pharmacokinetics and pharmacodynamics in antihypertension therapy%β1肾上腺素受体与CYP2D6基因多态性对美托洛尔抗高血压治疗的药代动力学和药效学影响

    刘洁; 刘昭前; 刘英姿; 谭志荣; 胡冬莉; 李智; 王丹; 张伟; 周宏灏

    2007-01-01

    BACKGROUND: Metoprolol is a selective β1-Blocker commonly used in essential hypertension. It is metabolized by CYP2D6. CYP2D6*10, which was identified to decrease activity of CYP2D6, is the main variance in Chinese population. β1-adrenergic receptor, with Ser49Gly and Gly389Arg polymorphisms, is the target of metoprolol. It was still unknown that whether the CYP2D6 and β1-adrenergic receptor had a synergic effect on metoprolol antihypertension therapy. AIM: To clarify the genetic polymorphism associated with metoprolol pharmacokinetics and pharmacodynamics in antihypertension therapy. METHODS: 125 mild-to-med essential hypertension patients were enrolled in this study. Patients were mono-therapied with metoprolol for 12 weeks. Blood pressure was monitored every 4 weeks. PCR-RFLP method was use to identify CYP2D6*10 and β1-adrenergic receptor Ser49Gly and Gly389Arg polymorphisms. Plasma metoprolol concentration was measured by HPLC- fluorescence detection. RESULTS: Trough blood level (C0) of metoprolol was associated with CYP2D6*10 variance in a gene-dose-effect manner, whereas the extent of blood pressure decrease was not significant different in CYP2D6*1*1, *1*10 and CYP2D6*10*10 patients. After 12 weeks metoprolol therapy, Gly49 carriers had stronger decrease in systolic and diastolic blood pressure than that of Ser49 homozygotes. Similarly, subjects homozygous for Arg389 had stronger decrease in blood pressure than that of Gly389 carriers. CONCLUSION: CYP2D6*10 variance significantly change the pharmacokinetics of metoprolol, and the genetic polymorphisms of β1-adrenergic receptor were associated with the pharmacodynamics of metopolol in antihypertension therapy.%背景: 美托洛尔是临床常用的抗高血压药物,它经由CYP2D6代谢.CYP2D6*10降低CYP2D6活性,是中国人群中最为常见的多态性.β1肾上腺素受体为美托洛尔的作用靶标,Ser49Gly与Gly389Arg多态性显著改变受体功能.CYP2D6与β1肾上腺素受体遗传多态

  2. Utility and adoption of CYP2D6 and CYP2C19 genotyping and its translation into psychiatric clinical practice

    Jürgens, G; Jacobsen, C B; Rasmussen, H B;

    2012-01-01

    To describe clinical utility and adoption of routinely offered CYP2D6 and CYP2C19 genotyping (CYP test) in daily clinical practice of a psychiatric centre.......To describe clinical utility and adoption of routinely offered CYP2D6 and CYP2C19 genotyping (CYP test) in daily clinical practice of a psychiatric centre....

  3. Single dose, CYP2D6 genotype-stratified pharmacokinetic study of atomoxetine in children with ADHD.

    Brown, J T; Abdel-Rahman, S M; van Haandel, L; Gaedigk, A; Lin, Y S; Leeder, J S

    2016-06-01

    The effect of CYP2D6 genotype on the dose-exposure relationship for atomoxetine has not been well characterized in children. Children 6-17 years of age diagnosed with attention-deficit hyperactivity disorder (ADHD) were stratified by CYP2D6 genotype into groups with 0 (poor metabolizers [PMs], n = 4), 0.5 (intermediate metabolizers [IMs], n = 3), one (extensive metabolizer [EM]1, n = 8) or two (EM2, n = 8) functional alleles and administered a single 0.5 mg/kg oral dose of atomoxetine (ATX). Plasma and urine samples were collected for 24 (IM, EM1, and EM2) or 72 hours (PMs). Dose-corrected ATX systemic exposure (area under the curve [AUC]0-∞ ) varied 29.6-fold across the study cohort, ranging from 4.4 ± 2.7 μM*h in EM2s to 5.8 ± 1.7 μM*h, 16.3 ± 2.9 μM*h, and 50.2 ± 7.3 μM*h in EM1s, IMs, and PMs, respectively (P < 0.0001). Simulated steady state profiles at the maximum US Food and Drug Administration (FDA)-recommended dose suggest that most patients are unlikely to attain adequate ATX exposures. These data support the need for individualized dosing strategies for more effective use of the medication. PMID:26660002

  4. Association of MDR1, CYP2D6, and CYP2C19 gene polymorphisms with prophylactic migraine treatment response.

    Atasayar, Gulfer; Eryilmaz, Isil Ezgi; Karli, Necdet; Egeli, Unal; Zarifoglu, Mehmet; Cecener, Gulsah; Taskapilioglu, Ozlem; Tunca, Berrin; Yildirim, Oznur; Ak, Secil; Tezcan, Gulcin; Can, Fatma Ezgi

    2016-07-15

    Prophylactic therapy response varies in migraine patients. The present study investigated the relationship between the resistance to the drugs commonly used in prophylactic therapy and the possible polymorphic variants of proteins involved in the metabolism of these drugs. Migraine patients with the MDR1 3435TT genotype exhibited a better treatment response to topiramate than migraine patients with the CC and CT genotypes (p=0.020). The MDR1 C3435T polymorphism was also found to be a higher risk factor for topiramate treatment failure in a comparison of the number of days with migraine (β2=1.152, p=0.015). However, there was no significant relationship between the treatment response to topiramate and either the CYP2D6 or CYP2C19 polymorphism, and there were no significant correlations between the treatment responses to amitriptyline, propranolol, and valproic acid and the MDR1, CYP2D6 and CYP2C19 gene polymorphisms. This is the first study to investigate the effect of the polymorphic variants on prophylactic therapy response in migraine patients. PMID:27288795

  5. Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides

    Singh, Satyender [Division of Biochemistry and Biotechnology, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India); Kumar, Vivek [Environmental Biochemistry and Molecular Biology laboratory, Department of Biochemistry, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, Delhi-110095 (India); Vashisht, Kapil; Singh, Priyanka [Division of Biochemistry and Biotechnology, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India); Banerjee, Basu Dev, E-mail: banerjeebd@hotmail.com [Environmental Biochemistry and Molecular Biology laboratory, Department of Biochemistry, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, Delhi-110095 (India); Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen [Division of Biochemistry and Biotechnology, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India); Jain, Sudhir Kumar [Centre for Epidemiology and Parasitic Diseases, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India); Rai, Arvind [Division of Biochemistry and Biotechnology, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India)

    2011-11-15

    Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p < 0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37 {+-} 2.15 vs. 6.24 {+-} 1.37 tail% DNA, p < 0.001). Further, the workers with CYP2D6*3 PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p < 0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. -- Highlights: Black-Right-Pointing-Pointer Role of CYP1A1, CYP3A5, CYP2C, CYP2D6 and PON1 genotypes on DNA damage. Black-Right-Pointing-Pointer Workers exposed to some OPs demonstrated increased DNA damage. Black-Right-Pointing-Pointer CYP2D6 *3 PM and PON1 (Q192R and L55M) genotypes are associated with DNA damage. Black-Right-Pointing-Pointer Concomitant presence of certain CYP2D6 and PON1 genotypes can increase DNA damage.

  6. Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides

    Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR–RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p < 0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37 ± 2.15 vs. 6.24 ± 1.37 tail% DNA, p < 0.001). Further, the workers with CYP2D6*3 PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p < 0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. -- Highlights: ► Role of CYP1A1, CYP3A5, CYP2C, CYP2D6 and PON1 genotypes on DNA damage. ► Workers exposed to some OPs demonstrated increased DNA damage. ► CYP2D6 *3 PM and PON1 (Q192R and L55M) genotypes are associated with DNA damage. ► Concomitant presence of certain CYP2D6 and PON1 genotypes can increase DNA damage.

  7. Mutation frequencies of the cytochrome CYP2D6 gene in Parkinson disease patients and in families

    Lucotte, G.; Turpin, J.C. [CHR, Reims (France); Gerard, N. [INSERM, Paris (France)] [and others

    1996-07-26

    The frequencies of five mutations of the debrisoquine 4-hydroxylase (CYP2D6) gene (mutations D6-A, B, C, D, and T), corresponding to poor metabolizer (PM) phenotypes, were determined by restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) in 47 patients with Parkinson disease, and compared with the findings in 47 healthy controls. These mutant alleles were about twice as frequent among patients as in controls, with an approximate relative risk ratio of 2.12 (95% confidence interval, 1.41-2.62). There seem to be no significant differences in frequencies of mutant genotypes in patients among gender and modalities of response with levodopa therapy; but frequency of the mutations was slightly enhanced after age-at-onset of 60 years. Mutations D6-B, D, and T were detected in 7 patients belonging to 10 Parkinson pedigrees. 25 refs., 1 fig., 2 tabs.

  8. No influence of the polymorphisms CYP2C19 and CYP2D6 on the efficacy of cyclophosphamide, thalidomide, and bortezomib in patients with Multiple Myeloma

    Vangsted, A. J.; Soeby, K.; Klausen, T.W.;

    2010-01-01

    patients were genotyped for the non-functional alleles CYP2C19*2 and CYP2D6*3, *4, *5 (gene deletion), *6, and CYP2D6 gene duplication. Results: In patients who were treated with bortezomib and were carriers of one or two defective CYP2D6 alleles there was a trend towards a better time-to-next treatment....... We found no association between the number of functional CYP2C19 and CYP2D6 alleles and outcome of treatment with cyclophosphamide or thalidomide. Neither was the number of functional CYP2C19 and CYP2D6 alleles associated with neurological adverse reactions to thalidomide and bortezomib. Conclusion......: There was no association between functional CYP2C19 and CYP2D6 alleles and treatment outcome in multiple myeloma patients treated with cyclophosphamide, thalidomide or bortezomib. A larger number of patients treated with bortezomib are needed to determine the role of CYP2D6 alleles in treatment outcome....

  9. No influence of the polymorphisms CYP2C19 and CYP2D6 on the efficacy of cyclophosphamide, thalidomide, and bortezomib in patients with Multiple Myeloma

    The response to treatment varies among patients with multiple myeloma and markers for prediction of treatment outcome are highly needed. Bioactivation of cyclophosphamide and thalidomide, and biodegradation of bortezomib, is dependent on cytochrome P450 metabolism. We explored the potential influence of different polymorphisms in the CYP enzymes on the outcome of treatment. Data was analyzed from 348 patients undergoing high-dose treatment and stem cell support in Denmark in 1994 to 2004. Clinical information on relapse treatment in 243 individual patients was collected. The patients were genotyped for the non-functional alleles CYP2C19*2 and CYP2D6*3, *4, *5 (gene deletion), *6, and CYP2D6 gene duplication. In patients who were treated with bortezomib and were carriers of one or two defective CYP2D6 alleles there was a trend towards a better time-to-next treatment. We found no association between the number of functional CYP2C19 and CYP2D6 alleles and outcome of treatment with cyclophosphamide or thalidomide. Neither was the number of functional CYP2C19 and CYP2D6 alleles associated with neurological adverse reactions to thalidomide and bortezomib. There was no association between functional CYP2C19 and CYP2D6 alleles and treatment outcome in multiple myeloma patients treated with cyclophosphamide, thalidomide or bortezomib. A larger number of patients treated with bortezomib are needed to determine the role of CYP2D6 alleles in treatment outcome

  10. 基于CYP2D6代谢性相互作用的儿童用药安全提示

    李娟; 李毅; 郭兴蕾

    2015-01-01

    CYP2D6是一种重要的药物代谢酶,研究基于CYP2D6的代谢性相互作用具有重要的临床意义.本文综述了关于儿科常用的各类有关于CYP2D6的药物可能产生的代谢性相互作用,旨在为临床医生合理用药提供参考.

  11. Correlation between CYP2D6 * 10 gene polymorphism and prognosis of breast cancer patients treated with tamoxifen%CYP2D6*10基因多态性与服用他莫昔芬乳腺癌患者预后的相关性

    田超; 杨义; 李卉

    2013-01-01

    目的 探讨CYP2D6 * 10基因多态性与服用他莫昔芬(TAM)乳腺癌患者预后的相关性.方法 收集四川省肿瘤医院2008年1月至2010年10月期间200例服用TAM的乳腺癌患者的口腔黏膜,采用实时RT-PCR法检测CYP2D6 * 10基因多态性,并分为CYP2D6突变组(CYP2D6 * 10/ * 10)和CYP2D6正常组(CYP2D6 wt/ * 10和CYP2D6 wt/wt).采用卡方检验及秩和检验分析CYP2D6 * 10基因多态性与临床病理特征的关系,采用Cox比例风险回归模型分析其与预后的关系.结果 在200例乳腺癌中,CYP2D6 * 10/ * 10纯合子94例(47%),CYP2D6 wt/wt野生型48例(24%),CYP2D6 wt/ * 10杂合型58例(29%).CYP2D6 * 10基因多态性与患者的组织学分级、TNM分期、HER-2表达、肿瘤类型无关(Z=-0.444,P=0.674;Z=-0.716,P=0.500;χ2=0.066,P=0.797;χ2=0.694,P=0.405).Log-rank检验分析显示CYP2D6 * 10突变组患者平均无瘤生存时间明显短于CYP2D6 * 10正常组患者(47.2个月比51.2个月,χ2=5.554,P=0.018).Cox比例风险回归模型显示,CYP2D6 * 10基因型与患者无瘤生存时间明显相关(HR=2.755,95%CI:1.230~6.173,P=0.014).结论 CYP2D6 * 10/ * 10基因型乳腺癌患者服用他莫昔芬预后较差.

  12. CYP2D6基因多态性对帕罗西汀在中国健康人药动学影响%Influence of CYP2D6 genetic polymorphism on pharmacokinetics of paroxetine in Chinese healthy people

    王蒙; 周文佳; 肖莉; 张全英

    2012-01-01

    AIM To investigate the influence of CYP2D6 genetic polymorphism on pharmacokinetics of paroxetine in Chinese healthy people. METHODS Twenty-three Chinese volunteers genotyped for CYP2D6 were separated into three groups by PCR-RFLP: CYP2D6*1/*1 group (rc = 5), CYP2D6*l/*10 group (n = 7), and CYP2D6*10/*10 group ( n - 11). After administration of a single oral dose of paroxetine hydrochloride tablet 20 mg, blood samples were collected at various time points until 96 h. The plasma concentrations of paroxetine were measured by LC-MS/MS method and the pharmacokinetics disposition of them was analyzed. RESULTS Compared with CYP2D6*1/*1 group, the tmax and t1/2l in CYP2D6*l/*10 group and tmax in CYP2D6*10/*10 group had no significant differences (P>0.05); ρmax, AUC0-96h, AUC0_∞ CL (F) and Vd of paroxetine in CYP2D6*l/*10 group and CYP2D6*10/*10 group, and t1/2 in CYP2D6*10/*10 group all had significant differences (P < 0.05 and P < 0.01). CONCLUSION The allelic gene mutations of CYP2D6*10 can cause the changes of metabolic phenotype and have effects on the metabolism of paroxetine in healthy human body.%目的 研究中国健康人CYP2D6基因多态性对帕罗西汀药动学的影响.方法 使用PCR-RFLP方法将23位志愿者分为3组:CYP2D6*1/*1组(n=5),CYP2D6* 1/* 10组(n=7),CY P2D6*10/*10组(n=11).给予帕罗西汀20 mg单剂量口服,收集给药后96 h内的一系列血样,用LC-MS/MS法测定帕罗西汀的血药浓度并做药动学分析.结果 与CYP2D6*1/*1组药动学参数相比,CYP2D6*1/*10组tmax、t1/2和CYP2D6* 10/* 10组tmax无显著差异(P>0.05);CYP2D6*1/*10、CY P2D6*1 0/*10组的ρmax、AUC0-96h、AUC0-x、CL (F)、Vd和CYP2D6*10/*10组t1/2均有显著差异(P<0.05或P<0.01).结论 CY P2D6*1D等位基因突变能引起代谢表型的改变,影响帕罗西汀在健康人的体内代谢.

  13. 中国人群细胞色素P4502D6基因多态性对曲马多药代动力学的影响%Influence of CYP2D6 genetic polymorphism on pharmacokinetics of tramadol in Chinese population

    李芹; 王睿; 郭雅; 裴斐

    2009-01-01

    目的 研究中国人群CYP2D6基因多态性对曲马多(镇痛药)药代动力学的影响.方法 不同基因型中国健康志愿者随机分为4组:第1组CYP2D6*2W*10W,第2组:CYP2D6*2M*10W,第3组:CYP2D6*2M*10H,第4组:CYP2D6*2M*10M.各组单次口服曲马多100 mg后,用高效液相色荧光检测法测定血和尿中曲马多及其M1代谢产物O-去甲基曲马多(M1)的浓度,研究不同基因型对曲马多药代动力学的影响.结果 第2组曲马多及其M1的主要药代动力学参数与第1组相比没有显著性差异;第3组与第1组、第4组与第1组、第4组与第3组比较,主要药代动力学参数均有显著性差异(P<0.05),且呈基因剂量效应.结论 CYP2D6*2对于曲马多的药代动力学过程没有影响;但CYP2D6*10可降低酶活性,且CYP2D6*10纯合子变异较杂合子变异对曲马多药代动力学的影响更大,呈基因剂量效应.%Objective To investigate on influence of CYP2D6 genetic polymorphism on pharmacokinetics of tramadol in Chinese volunteers.Methods Adult healthy Chinese volunteers with different CYP2D6 genotypes were categorized into the following four groups: group 1:CYP2D6 * 2W * 10W, group 2:CYP2D6 * 2M * 10W, group 3:CYP2D6 * 2M * 10H, group 4 : CYP2D6 * 2M * 10M. After oral ad-ministration of 100 mg tramadol, plasma and urine samples were col-lected from each subject at different time within 32 h. The plasma and urine concentrations of tramadol and its metabolite O - desmethyltramad-ol (M1) were determined by HPLC with fluorescence detection. Re-suits The main pharmacokinetic parameters of tramadol and M, in group 2 were not significantly different from those in group 1. There are significant difference for the main pharmacokinetic parameters of tram-adol and M1 between group 3 and group 1, group 4 and group 1, group 4 and group 3, respectively (P<0.05 ). Conclusion The present re-sults shown that CYP2D6 * 2 has no influence on the pharmacokinetics of tramadol, but CYP2D6 * 10 reduces CYP2

  14. No influence of the polymorphisms CYP2C19 and CYP2D6 on the efficacy of cyclophosphamide, thalidomide, and bortezomib in patients with Multiple Myeloma

    Vangsted, A. J.; Soeby, K.; Klausen, T.W.;

    2010-01-01

    Background: The response to treatment varies among patients with multiple myeloma and markers for prediction of treatment outcome are highly needed. Bioactivation of cyclophosphamide and thalidomide, and biodegradation of bortezomib, is dependent on cytochrome P450 metabolism. We explored the....... We found no association between the number of functional CYP2C19 and CYP2D6 alleles and outcome of treatment with cyclophosphamide or thalidomide. Neither was the number of functional CYP2C19 and CYP2D6 alleles associated with neurological adverse reactions to thalidomide and bortezomib. Conclusion......: There was no association between functional CYP2C19 and CYP2D6 alleles and treatment outcome in multiple myeloma patients treated with cyclophosphamide, thalidomide or bortezomib. A larger number of patients treated with bortezomib are needed to determine the role of CYP2D6 alleles in treatment outcome....

  15. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for CYP2D6 and CYP2C19 Genotypes and Dosing of Selective Serotonin Reuptake Inhibitors.

    Hicks, J K; Bishop, J R; Sangkuhl, K; Müller, D J; Ji, Y; Leckband, S G; Leeder, J S; Graham, R L; Chiulli, D L; LLerena, A; Skaar, T C; Scott, S A; Stingl, J C; Klein, T E; Caudle, K E; Gaedigk, A

    2015-08-01

    Selective serotonin reuptake inhibitors (SSRIs) are primary treatment options for major depressive and anxiety disorders. CYP2D6 and CYP2C19 polymorphisms can influence the metabolism of SSRIs, thereby affecting drug efficacy and safety. We summarize evidence from the published literature supporting these associations and provide dosing recommendations for fluvoxamine, paroxetine, citalopram, escitalopram, and sertraline based on CYP2D6 and/or CYP2C19 genotype (updates at www.pharmgkb.org). PMID:25974703

  16. Comparison of CYP2D6 genotyping by allele-specific PCR with DXT phe-notype and gene chip testing%CYP2D6 PCR基因型与DXT表型和基因芯片检测的比较

    2002-01-01

    目的:为了评价CYP2D6的基因型和表型的联系以及基因芯片在CYP2D6多基因分析中的应用.方法:242健康志愿者,口服dextromethorphan后收集尿液测定其代谢率,收集20ml血提取DNA,并通过基因特异性PCR和/(或)基因芯片分析CYP2D6*2--*11,*17和多拷贝CYP2D6基因,其中5个基因(*3、*4、*6、*7和*9)用PCR和CYD450基因芯片同时分析.结果:CYP2D6基因型比表型更富有信息和更能反映CYP2D6酶的表达.CYP2D6*3、*4、*6、*7和*9的基因检测在CYP450基因芯片和基因特异性PCR中显示高度的一致性.结论:基因芯片在检测基因多位点的多基因中是一个有发展前途和可靠的方法.%To evaluate association of genotype and phenotype of CYP2D6 and the application of oligonucleotide microarray hybridization genetic testing in CYP2D6 multiple alleles analyses. METHODS: Two hundred forty-two healthy volunteers were recruited, and a 60 mg oral dose of dextromethorphan (DXT) was administered to each for assessment of the DXT metabolic ratio [ MR]. A 20 ml blood sample was also collected for DNA isolation and testing. CYP2D6 alleles * 2-*11; * 17 and multiple CYP2D6 gene copies were tested by allele-specific PCR and/or the affymetrix CYP450 gene chip assay. Five of the CYP2D6 alleles ( * 3, * 4, *6, * 7, and * 9) were evaluated by both PCR and the CYP450 gene chip assay. RESULTS: The CYP2D6genotype was more informative and reflective in CYP2D6 enzyme expression than a phenotype. Genetic tests for the CYP2D6 * 3, * 4, * 6, * 7 and * 9 alleles showed a high degree of concordance between the CYP450 gene chip and AS-PCR methods. CONCLUSION: Oligonucleotide microarray hybridization is a promising and reliable approach for detecting multiple alleles at gene loci.

  17. Influence of the CYP2D6 isoenzyme in patients treated with venlafaxine for major depressive disorder: clinical and economic consequences.

    Antoni Sicras-Mainar

    Full Text Available Antidepressant drugs are the mainstay of drug therapy for sustained remission of symptoms. However, the clinical results are not encouraging. This lack of response could be due, among other causes, to factors that alter the metabolism of the antidepressant drug.to evaluate the impact of concomitant administration of CYP2D6 inhibitors or substrates on the efficacy, tolerability and costs of patients treated with venlafaxine for major depressive disorder in clinical practice.We designed an observational study using the medical records of outpatients. Subjects aged ≥ 18 years who started taking venlafaxine during 2008-2010 were included. Three study groups were considered: no combinations (reference, venlafaxine-substrate, and venlafaxine-inhibitor. The follow-up period was 12 months. The main variables were: demographic data, comorbidity, remission (Hamilton <7, response to treatment, adverse events and costs. The statistical analysis included logistic regression models and ANCOVA, with p values <0.05 considered significant.A total of 1,115 subjects were recruited. The mean age was 61.7 years and 75.1% were female. Approximately 33.3% (95% CI: 30.5 to 36.1 were receiving some kind of drug combination (venlafaxine-substrate: 23.0%, and venlafaxine-inhibitor: 10.3%. Compared with the venlafaxine-substrate and venlafaxine-inhibitor groups, patients not taking concomitant drugs had a better response to therapy (49.1% vs. 39.9% and 34.3%, p<0.01, greater remission of symptoms (59.9% vs. 50.2% and 43.8%, p<0.001, fewer adverse events (1.9% vs. 7.0% and 6.1%, p<0.05 and a lower mean adjusted cost (€2,881.7 vs. €4,963.3 and €7,389.1, p<0.001, respectively. All cost components showed these differences.The patients treated with venlafaxine alone showed a better response to anti-depressant treatment, greater remission of symptoms, a lower incidence of adverse events and lower healthcare costs.

  18. CYP2D6*10基因型与接受他莫昔芬治疗乳腺癌患者的生存率的相关性研究%Association between CYP2 D6*10 genotype and survival of breast cancer patients receiving tamoxifen treatment

    魏影; 徐喆

    2014-01-01

    Objective To explore the association between between CYP 2D6*10 genotype and survival of breast cancer patients receiv-ing tamoxifen(TAM) treatment.Methods Basic clinical feature,survival state and blood samples,paraffin sections were collected from 257 breast cancer patients receiving TAM treatment .CYP2D6*10 allele of breast cancer patients were checked by means of Polymerase Chain Reaction ( PCR) .We observed the association between CYP 2D6*10 genotype and survival of breast cancer patients receiving tamoxifen(TAM) treatment.Results Fifteen percent (38/257) of the patients carried the CYP2D6*10/*10 genotype,41%(105/257) the CYP2D6 wild-type (Wt)/*10 genotype and 44%(114/257) the CYP2D6 wt/wt genotype.There were no discernible cor-relations between clinicopathologic parameters and the CYP 2D6*10 genotype.We determined whether there was a correlation between the CYP2D6*10 genotype and survival and found out that the clinical outcome for patients carrying the CYP 2D6*10/*10 genotype was similar to those with other genotypes .Conclusions Our results suggest that the CYP 2D6*10 genotype is unlikely to have any clinical significance for prognosis of breast cancer patients receiving adjuvant TAM treatment .%目的:研究CYP2D6*10基因型对接受他莫昔芬(tamoxifen,TAM)治疗乳腺癌患者的生存率相关性的影响。方法选择该院乳腺外科2008-2003年收治的257名接受TAM治疗乳腺癌患者。调查TAM使用情况和生存状态等相关资料;采集患者外周静脉血5 mL或石蜡切片用于DNA提取;用PCR技术检测CYP2D6*10基因多态性;查明CYP2D6*10基因多态性与患者的生存率相关性的关系。结果该次研究中,携带CYP2D6*10/*10患者占15%(38/257),CYP2D6野生型(Wt)/*10患者占41%(105/257)和CYP2D6Wt/Wt占44%(114/257)。各CYP2D6*10基因型的临床病理参数之间没有相关性差异。在中国CYP2D6*10/*10基因型对接受TAM治疗乳

  19. Physical Properties and Microbial Activity in Forest Residual Substrate

    Many growers in the horticulture industry have expressed concern that switching from a pine bark-based substrate to one with a significant wood content will increase microbial activity, resulting in nitrogen (N) immobilization. This study evaluated four growth substrates (pine bark, peat moss and tw...

  20. No association between cytochrome P450 2D6 gene polymorphism and risk of acute leukemia: evidence based on a meta-analysis

    RUAN Xiao-lan; LI Sheng; ZENG Xian-tao; XIA Ling-hui; HU Yu

    2013-01-01

    Background Many studies indicated the human cytochrome P450 2D6 (CYP2D6) gene polymorphism was associated with acute leukemia (AL) susceptibility,however,the results were inconsistent.So we performed this meta-analysis to evaluate the relationship between CYP2D6*3 or CYP2D6*4 polymorphism and AL susceptibility.Methods We searched PubMed database up to February 20,2013,and finally yielded 9 case-control studies including 1343 cases and 1843 controls which tested the association between CYP2D6*3 or *4 polymorphism and AL.After data extraction,we conducted a meta-analysis using the Comprehensive Meta Analysis software.Results Overall,no significant association between CYP2D6*3 or *4 polymorphism and AL risk was found in this metaanalysis (+ vs.-:OR=1.13,95% CI=0.79-1.63; +/+ vs.-/-:OR=1.73,95% C/=0.99-3.02;-/+ vs.-/-:OR=1.03,95% C/=0.68-1.56; (-/+ and +/+) vs.-/-:OR=1.08,95% C/=0.72-1.63; +/+ vs.(-/+ and-/-):OR=1.76,95% C/=0.98-3.17).Similar results were also been found in stratified subgroup analysis.There was no publication bias.Conclusion CYP2D6*3 or *4 polymorphism might not be associated with AL susceptibility.However,the results need to be further confirmed by well-designed and high quality randomized controlled trials with larger sample sizes.

  1. No influence of the polymorphisms CYP2C19 and CYP2D6 on the efficacy of cyclophosphamide, thalidomide, and bortezomib in patients with Multiple Myeloma

    Vogel Ulla

    2010-08-01

    Full Text Available Abstract Background The response to treatment varies among patients with multiple myeloma and markers for prediction of treatment outcome are highly needed. Bioactivation of cyclophosphamide and thalidomide, and biodegradation of bortezomib, is dependent on cytochrome P450 metabolism. We explored the potential influence of different polymorphisms in the CYP enzymes on the outcome of treatment. Methods Data was analyzed from 348 patients undergoing high-dose treatment and stem cell support in Denmark in 1994 to 2004. Clinical information on relapse treatment in 243 individual patients was collected. The patients were genotyped for the non-functional alleles CYP2C19*2 and CYP2D6*3, *4, *5 (gene deletion, *6, and CYP2D6 gene duplication. Results In patients who were treated with bortezomib and were carriers of one or two defective CYP2D6 alleles there was a trend towards a better time-to-next treatment. We found no association between the number of functional CYP2C19 and CYP2D6 alleles and outcome of treatment with cyclophosphamide or thalidomide. Neither was the number of functional CYP2C19 and CYP2D6 alleles associated with neurological adverse reactions to thalidomide and bortezomib. Conclusion There was no association between functional CYP2C19 and CYP2D6 alleles and treatment outcome in multiple myeloma patients treated with cyclophosphamide, thalidomide or bortezomib. A larger number of patients treated with bortezomib are needed to determine the role of CYP2D6 alleles in treatment outcome.

  2. Probing small-molecule binding to cytochrome P450 2D6 and 2C9: An in silico protocol for generating toxicity alerts.

    Rossato, Gianluca; Ernst, Beat; Smiesko, Martin; Spreafico, Morena; Vedani, Angelo

    2010-12-01

    Drug metabolism, toxicity, and their interaction profiles are major issues in the drug-discovery and lead-optimization processes. The cytochromes P450 (CYPs) 2D6 and 2C9 are enzymes involved in the oxidative metabolism of a majority of marketed drugs. Therefore, the prediction of the binding affinity towards CYP2D6 and CYP2C9 would be beneficial for identifying cytochrome-mediated adverse effects triggered by drugs or chemicals (e.g., toxic reactions, drug-drug, and food-drug interactions). By identifying the binding mode by using pharmacophore prealignment, automated flexible docking, and by quantifying the binding affinity by multidimensional QSAR (mQSAR), we validated a model family of 56 compounds (46 training, 10 test) and 85 compounds (68 training, 17 test) for CYP2D6 and CYP2C9, respectively. The correlation with the experimental data (cross-validated r²=0.811 for CYP2D6 and 0.687 for CYP2C9) suggests that our approach is suited for predicting the binding affinity of compounds towards CYP2D6 and CYP2C9. The models were challenged by Y-scrambling and by testing an external dataset of binding compounds (15 compounds for CYP2D6 and 40 for CYP2C9). To assess the probability of false-positive predictions, datasets of nonbinders (64 compounds for CYP2D6 and 56 for CYP2C9) were tested by using the same protocol. The two validated mQSAR models were subsequently added to the VirtualToxLab (VTL, http://www.virtualtoxlab.org). PMID:21038340

  3. CYP2D6*10等位基因特性对临床用药安全性和有效性的影响

    谭蓉; 郑志昌; 杨继红; 孙为民

    2009-01-01

    目的:促进CYP2D6底物药物临床合理用药.方法:从CYP2D6*10等位基因对底物药物的代谢特性角度,阐述应重视该等位基因携带者用药安全有效问题.结果:CYP2D6*10等位基因表达酶蛋白的活性及稳定性的特性,可影响其对底物药物的代谢,进而影响临床用药的安全性和有效性.结论:鉴于中国人CYP2D6*10等位基因携带频率高,为了该基因携带者用药安全和有效,应重新审视CYP2D6底物药物的使用模式.

  4. Substrate utilization and VSS relations in activated sludge processes

    Droste, R.L.; Fernandes, L.; Sun, X. [Ottawa Univ., ON (Canada). Dept. of Civil Engineering

    1993-12-31

    A new empirical substrate removal model for activated sludge in continuous flow stirred tank reactor (CFSTR) and sequencing batch reactor (SBR) was developed in this study. This model includes an exponential function of volatile suspended solids to express the active biomass which is actually involved in substrate utilization. Results indicate that the proposed exponential models predict more accurately effluent COD in CFSTR and SBR systems than the first or zero order models. (author). 7 refs., 1 fig., 4 tabs.

  5. The Effect of CYP2B6, CYP2D6, and CYP3A4 Alleles on Methadone Binding: A Molecular Docking Study

    Nik Nur Syazana Bt Nik Mohamed Kamal

    2013-01-01

    Full Text Available Current methadone maintenance therapy (MMT is yet to ensure 100% successful treatment as the optimum dosage has yet to be determined. Overdose leads to death while lower dose causes the opioid withdrawal effect. Single-nucleotide polymorphisms (SNP in cytochrome P450s (CYPs, the methadone metabolizers, have been showen to be the main factor for the interindividual variability of methadone clinical effects. In this study, we investigated the effect of SNPs in three major methadone metabolizers (CYP2B6, CYP2D6, and CYP3A4 on methadone binding affinity. Results showed that CYP2B6*11, CYP2B6*12, CYP2B6*18, and CYP3A4*12 have significantly higher binding affinity to R-methadone compared to wild type. S-methadone has higher binding affinity in CYP3A4*3, CYP3A4*11, and CYP3A4*12 compared to wild type. R-methadone was shown to be the active form of methadone; thus individuals with CYP alleles that binds better to R-methadone will have higher methadone metabolism rate. Therefore, a higher dosage of methadone is necessary to obtain the opiate effect compared to a normal individual and vice versa. These results provide an initial prediction on methadone metabolism rate for individuals with mutant type CYP which enables prescription of optimum methadone dosage for individuals with CYP alleles.

  6. Distribution of CYP2D6 gene copy number variation in chinese littoral han population of zhejiang%浙江沿海地区汉族人群CYP2D6基因拷贝数多态性研究

    周仁芳; 曾爱平

    2008-01-01

    目的 了解浙江沿海地区汉族人群中CYP2D6基因拷贝数多态性分布.方法 使用改良的长模板PCR技术对浙江沿海地区汉族人群中的285例健康人群,进行CYP2D6基因拷贝数多态性进行检测.结果 CYP2D6*5缺失型和CYP2D6*×N重复基因在总样本中的发生频率分别为5.09%(n=29)和0.70%(n=4),在4个重复等位的基因中,其中3个为CYP2D6*1×2,另外1个为CYP2D6*10×2.结论 实验表明本地区CYP2D6基因拷贝数多态性分布不同于其他地区,不同地区呈现出不同的特点,表现出中华民族的遗传多样性.

  7. The impact of CYP2D6 and CYP2C19 polymorphisms on suicidal behavior and substance abuse disorder among patients with schizophrenia: a retrospective study

    Kobylecki, Camilla J; Hansen, Thomas Folkmann; Timm, Sally;

    2008-01-01

    Suicidal behavior and substance abuse are frequent phenomena among patients with schizophrenia and may be attributable in part to antipsychotic treatment failure. Individuals who carry functional variants of the CYP2D6 and CYP2C19 genes, shown to cause altered drug metabolism of psychoactive drugs......, are at risk of toxic accumulation or rapid elimination of these drugs, leading to treatment failure. We tested whether substance abuse disorder and suicidal behavior were associated with the CYP2D6 and CYP2C19 genotypes among patients with schizophrenia. Three hundred sixty-two patients with schizophrenia...... spectrum disorders (International Classification of Diseases, 10th Revision) were genotyped for functional CYP2D6 and CYP2C19 polymorphisms. Based on available medical records and clinical interviews, their suicidal behavior and substance abuse disorder were evaluated. No significant associations between...

  8. Successful treatment of schizophrenia with melperone augmentation in a patient with phenotypic CYP2D6 ultrarapid metabolization: a case report

    Gahr Maximilian

    2012-02-01

    Full Text Available Abstract Introduction There are limited treatment options for people with schizophrenia with cytochrome P450 2D6 ultrarapid metabolizer status who do not respond to amisulpride. Furthermore, the literature does not provide evidence-based guidelines for this particular constellation. Case presentation We report the case of a 50-year-old Caucasian female patient with schizophrenia and cytochrome P450 2D6 ultrarapid metabolizer status who experienced an insufficient antipsychotic effect with amisulpride. She was successfully treated with melperone-augmented haloperidol. Conclusion This report yields melperone-augmented haloperidol as a possible pharmacological strategy in the described situation. In addition, our observations support the available evidence for the potential of melperone to act as an inhibitor of cytochrome P450 2D6.

  9. The impact of CYP2D6 and CYP2C19 polymorphisms on suicidal behavior and substance abuse disorder among patients with schizophrenia: a retrospective study

    Kobylecki, C.J.; Hansen, T.; Timm, S.;

    2008-01-01

    Suicidal behavior and substance abuse are frequent phenomena among patients with schizophrenia and may be attributable in part to antipsychotic treatment failure. Individuals who carry functional variants of the CYP2D6 and CYP2C19 genes, shown to cause altered drug metabolism of psychoactive drugs......, are at risk of toxic accumulation or rapid elimination of these drugs, leading to treatment failure. We tested whether substance abuse disorder and suicidal behavior were associated with the CYP2D6 and CYP2C19 genotypes among patients with schizophrenia. Three hundred sixty-two patients with...... schizophrenia spectrum disorders (International Classification of Diseases, 10th Revision) were genotyped for functional CYP2D6 and CYP2C19 polymorphisms. Based on available medical records and clinical interviews, their suicidal behavior and substance abuse disorder were evaluated. No significant associations...

  10. CYP1A2 and CYP2D6 Gene Polymorphisms in Schizophrenic Patients with Neuroleptic Drug-Induced Side Effects.

    Ivanova, S A; Filipenko, M L; Vyalova, N M; Voronina, E N; Pozhidaev, I V; Osmanova, D Z; Ivanov, M V; Fedorenko, O Yu; Semke, A V; Bokhan, N A

    2016-03-01

    Polymorphic variants of CYP1A2 and CYP2D6 genes of the cytochrome P450 system were studied in patients with schizophrenia with drug-induced motor disorders and hyperprolactinemia against the background of long-term neuroleptic therapy. We revealed an association of polymorphic variant C-163A CYP1A2*1F of CYP1A2 gene with tardive dyskinesia and association of polymorphic variant 1846G>A CY2D6*4 and genotype A/A of CYP2D6 gene (responsible for debrisoquin-4-hydroxylase synthesis) with limbotruncal tardive dyskinesia in patients with schizophrenia receiving neuroleptics for a long time. PMID:27021090

  11. Structure-activity relationship and substrate-dependent phenomena in effects of ginsenosides on activities of drug-metabolizing P450 enzymes.

    Miao Hao

    Full Text Available Ginseng, a traditional herbal medicine, may interact with several co-administered drugs in clinical settings, and ginsenosides, the major active components of ginseng, may be responsible for these ginseng-drug interactions (GDIs. Results from previous studies on ginsenosides' effects on human drug-metabolizing P450 enzymes are inconsistent and confusing. Herein, we first evaluated the inhibitory effects of fifteen ginsenosides and sapogenins on human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes by using commercially available fluorescent probes. The structure-activity relationship of their effects on the P450s was also explored and a pharmacophore model was established for CYP3A4. Moreover, substrate-dependent phenomena were found in ginsenosides' effects on CYP3A4 when another fluorescent probe was used, and were further confirmed in tests with conventional drug probes and human liver microsomes. These substrate-dependent effects of the ginsenosides may provide an explanation for the inconsistent results obtained in previous GDI reports.

  12. Lipid metabolizing enzyme activities modulated by phospholipid substrate lateral distribution.

    Salinas, Dino G; Reyes, Juan G; De la Fuente, Milton

    2011-09-01

    Biological membranes contain many domains enriched in phospholipid lipids and there is not yet clear explanation about how these domains can control the activity of phospholipid metabolizing enzymes. Here we used the surface dilution kinetic theory to derive general equations describing how complex substrate distributions affect the activity of enzymes following either the phospholipid binding kinetic model (which assumes that the enzyme molecules directly bind the phospholipid substrate molecules), or the surface-binding kinetic model (which assumes that the enzyme molecules bind to the membrane before binding the phospholipid substrate). Our results strongly suggest that, if the enzyme follows the phospholipid binding kinetic model, any substrate redistribution would increase the enzyme activity over than observed for a homogeneous distribution of substrate. Besides, enzymes following the surface-binding model would be independent of the substrate distribution. Given that the distribution of substrate in a population of micelles (each of them a lipid domain) should follow a Poisson law, we demonstrate that the general equations give an excellent fit to experimental data of lipases acting on micelles, providing reasonable values for kinetic parameters--without invoking special effects such as cooperative phenomena. Our theory will allow a better understanding of the cellular-metabolism control in membranes, as well as a more simple analysis of the mechanisms of membrane acting enzymes. PMID:21108012

  13. CYP2D6遗传多态性对临床个体化给药的影响

    杨勇; 王友群

    2006-01-01

    本文总结了经CYP2D6代谢的临床常用药物,并对此类药物实现临床个体化给药的方法进行了探讨.同时介绍了CYP2D6的各种表型及其在不同种族之间的分布情况,以及该分布对制定临床给药方案的影响.

  14. Paroxetine, a cytochrome P450 2D6 inhibitor, diminishes the stereoselective O-demethylation and reduces the hypoalgesic effect of tramadol

    Laugesen, S; Enggaard, T P; Pedersen, R S;

    2005-01-01

    OBJECTIVE: Tramadol hydrochloride (INN, tramadol) exerts its antinociceptive action through a monoaminergic effect mediated by the parent compound and an opioid effect mediated mainly by the O-demethylated metabolite (+)-M1. O-demethylation is catalyzed by cytochrome P450 (CYP) 2D6. Paroxetine is a...... very potent inhibitor of CYP2D6. The objective of this study was to investigate the influence of paroxetine pretreatment on the biotransformation and the hypoalgesic effect of tramadol. METHODS: With and without paroxetine pretreatment (20 mg daily for 3 consecutive days), the formation of M1 and the...

  15. Does Pharmacogenetic Testing for CYP450 2D6 and 2C19 among Patients with Diagnoses within the Schizophrenic Spectrum Reduce Treatment Costs?

    Herbild, Louise; Andersen, Stig Ejdrup; Werge, Thomas;

    2013-01-01

    The effect of pharmacogenetic testing for CYP450 2D6 and 2C19 on treatment costs have not yet been documented. This study used Danish patient registers to calculate health care costs of treating patients with diagnoses within the schizophrenic spectrum for one year with or without pharmacogenetic...... testing for polymorphisms in the genes for the CYP2D6 and CYP2C19 enzymes. In a randomized, controlled trial, stratified with respect to metabolizer genotype, 104 patients were assigned to treatment based on pharmacogenetic testing and 103 patients to treatment as usual. Random exclusion of extensive and...

  16. Effects and cost-effectiveness of pharmacogenetic screening for CYP2D6 among older adults starting therapy with nortriptyline or venlafaxine : study protocol for a pragmatic randomized controlled trial (CYSCEtrial)

    Berm, Elizabeth J. J.; Hak, Eelko; Postma, Maarten; Boshuisen, Marjolein; Breuning, Laura; Brouwers, Jacobus R. B. J.; Dhondt, Ton; Jansen, Paul A. F.; Kok, Rob M.; Maring, Jan G.; van Marum, Rob; Mulder, Hans; Oude Voshaar, Richard; Risselada, Arne J.; Venema, Harry; Vleugel, Liesbeth; Wilffert, Bob

    2015-01-01

    Background: Nortriptyline and venlafaxine are commonly used antidepressants for treatment of depression in older patients. Both drugs are metabolized by the polymorphic cytochrome P450-2D6 (CYP2D6) enzyme and guidelines for dose adaptations based on the CYP2D6 genotype have been developed. The CYP2D

  17. Development of a fluorescent substrate to measure hyaluronidase activity

    Zhang, Li-Shu; Mummert, Mark E.

    2008-01-01

    A novel fluorescent substrate (termed FRET-HA) to quantitatively assess hyaluronidase activity was developed. Hyaluronan (HA), the major substrate for hyaluronidase, was dual labeled with fluorescein amine and rhodamine B amine. The fluorescein amine fluorescence signal was significantly quenched while the rhodamine B amine signal was significantly enhanced due to fluorescence resonance energy transfer (FRET). In the presence of bovine testes hyaluronidase, cleavage of HA disrupted FRET resul...

  18. Effect of the CYP2D6*10 allele on postoperative tramadol analgesia in patients after gastric cancer surgery%CYP2D6*10等位基因多态性对胃癌术后曲马多镇痛效果的影响

    汪国香; 黄丽霞; 张相彩; 陶凡

    2011-01-01

    目的:探讨中国人群CYP2D6*10等位基因对胃癌术后曲马多镇痛效果的影响.方法:70例胃癌根治术患者,手术结束前30 min静脉注入负荷剂量曲马多100 mg,随后采用持续背景剂量-PCA量的给药模式(曲马多10 mg/mL、甲氧氯普胺0.3 mg/mL)进行术后镇痛.全麻诱导后抽取血样,采用聚合酶链反应-限制性片断长度多态性方法分析患者CYP2D6*10基因多态性,根据不同CYP2D6基因型分为3组,比较不同基因组患者之间曲马多的用量.结果:CYP2D6*10等位基因频率为52.4%,不携带CYP2D6*10(I组)17例,CYP2D6*10杂合子(II组)26例,CYP2D6*10纯合子(III组)20例.48 h曲马多用量,III组明显高于I组和II组,I组和II组之间差异无统计学意义.结论:在中国人群中,CYP2D6*10等位基因对曲马多镇痛效果有显著影响.%Objective To investigate whether the CYP2D6*10 allele has an impact on the postoperative analgesia effect of tramadol in Chinese patients suffering from gastric cancer surgery. Methods Seventy gastric cancer patients suffering from gastrectomy were enrolled. After receiving a loading dose of 100 mg tramadol intravenously, patients could self-administer doses of 10 mg/mL tramadol plus 0.3 mg/mL metoclopramide via patient-controlled analgesia (PCA). Blood samples were collected after induction of anesthesia. The CYP2D6*10 polymorphism was analyzed by means of polymerase chain reaction-based restriction fragment length polymor-phism(PCR-RFLP). Demographic data among groups with different genotypes were analyzed using analysis of variance. The total consumption of tramadol between the three genotype groups for 48 h was compared. Results The allele frequency of CYP2D6*10 is 52.4%: Patients were categorized into three groups according to the CYP2D6 genotype: patients without CYP2D6*10 (group I,n=17), patients with heterozygote for CYP2D6*10 (group Ⅱ, n=26), and patients with homozygote for CYP2D6*10 (group Ⅲ, n=20). The demographic data among

  19. 细胞色素CYP2D6基因多态性与急性髓系白血病的关系研究

    里杨; 许俊锋; 王皓; 郭权; 曹楷翔

    2013-01-01

    目的:探讨细胞色素CYP2D6基因多态性与急性髓系白血病(AML)易感性的关系。方法:采用病例对照研究方法,应用聚合酶链反应-限制性片段长度多态性技术,对114例AML患者及180例健康对照者的CYP2D6(C188T)基因的多态分布进行分析。结果:AML 患者组CYP2D6各基因型频率分布与对照组之间无显著性差异;携带CYP2D6188T突变基因型的个体患AML的风险与野生型携带者相比无显著性差异。结论:CYP2D6基因多态性可能与AML遗传易感性不相关。

  20. A combined high CYP2D6-CYP2C19 metabolic capacity is associated with the severity of suicide attempt as measured by objective circumstances.

    Peñas-Lledó, E; Guillaume, S; Naranjo, M E G; Delgado, A; Jaussent, I; Blasco-Fontecilla, H; Courtet, P; LLerena, A

    2015-04-01

    This study examined, for the first time, whether a high CYP2D6-CYP2C19 metabolic capacity combination increases the likelihood of suicidal intent severity in a large study cohort. Survivors of a suicide attempt (n=587; 86.8% women) were genotyped for CYP2C19 (*2, *17) and CYP2D6 (*3, *4, *4xN, *5, *6, *10, wtxN) genetic variation and evaluated with the Beck Suicide Intent Scale (SIS). Patients with a high CYP2D6-CYP2C19 metabolic capacity showed an increased risk for a severe suicide attempt (P<0.01) as measured by the SIS-objective circumstance subscale (odds ratio (OR)=1.37; 95% confidence interval (CI)=1.05-1.78; P=0.02) after adjusting for confounders (gender, age, level of studies, marital status, mental disorders, tobacco use, family history of suicide, personal history of attempts and violence of the attempt). Importantly, the risk was greater in those without a family history of suicide (OR=1.82; CI=1.19-2.77; P=0.002). Further research is warranted to evaluate whether the observed relationship is mediated by the role of CYP2D6 and CYP2C19 involvement in the endogenous physiology or drug metabolism or both. PMID:25113522

  1. CYP2D6基因多态性与他莫昔芬及4-羟基他莫昔芬血清浓度的相关性研究%Clinical analysis of CYP2D6 gene polymorphism with serum concentration of tamoxifen and 4-Hydroxytamoxifen

    田超; 杨义; 李卉

    2014-01-01

    目的 探讨CYP2D6基因多态性与乳腺癌患者他莫昔芬(TAM)及其活性代谢产物4-羟基他莫昔芬(4-OH-TAM)血清浓度的相关性.方法 收集2008年1月~2010年10月期间200例服用TAM的乳腺癌患者的口腔粘膜及血清,采用Real-time RT-PC法检测CYP2D6*10基因多态性,采用液相色谱-质谱方法 (LC-MS)测定患者体内TAM及其活性代谢物4-OH-TAM的的血清浓度.结果 200例乳腺癌中检测到CYP2D6*10/*10纯合子94例(47%),CYP2D6 wt/wt野生型48例(24%),CYP2D6 wt/*10杂合型58例(29%).CYP2D6 wt/wt野生型和wt/*10杂合型两组4-OH-TAM的血清浓度都明显高于*10/*10纯合型(P0.05).结论 乳腺癌患者CYP2D6*10/*10基因型影响他莫昔芬的体内代谢过程,与疗效相关,服用TAM前均应推荐检测CYP2D6*10/*10基因型.

  2. Clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy": the influence of gender and genetics (CYP2D6, COMT, 5-HTT.

    Ricardo Pardo-Lozano

    Full Text Available The synthetic psychostimulant MDMA (± 3,4-methylenedioxymethamphetamine, ecstasy acts as an indirect serotonin, dopamine, and norepinephrine agonist and as a mechanism-based inhibitor of the cytochrome P-450 2D6 (CYP2D6. It has been suggested that women are more sensitive to MDMA effects than men but no clinical experimental studies have satisfactorily evaluated the factors contributing to such observations. There are no studies evaluating the influence of genetic polymorphism on the pharmacokinetics (CYP2D6; catechol-O-methyltransferase, COMT and pharmacological effects of MDMA (serotonin transporter, 5-HTT; COMT. This clinical study was designed to evaluate the pharmacokinetics and physiological and subjective effects of MDMA considering gender and the genetic polymorphisms of CYP2D6, COMT, and 5-HTT. A total of 27 (12 women healthy, recreational users of ecstasy were included (all extensive metabolizers for CYP2D6. A single oral weight-adjusted dose of MDMA was administered (1.4 mg/kg, range 75-100 mg which was similar to recreational doses. None of the women were taking oral contraceptives and the experimental session was performed during the early follicular phase of their menstrual cycle. Principal findings show that subjects reached similar MDMA plasma concentrations, and experienced similar positive effects, irrespective of gender or CYP2D6 (not taking into consideration poor or ultra-rapid metabolizers or COMT genotypes. However, HMMA plasma concentrations were linked to CYP2D6 genotype (higher with two functional alleles. Female subjects displayed more intense physiological (heart rate, and oral temperature and negative effects (dizziness, sedation, depression, and psychotic symptoms. Genotypes of COMT val158met or 5-HTTLPR with high functionality (val/val or l/* determined greater cardiovascular effects, and with low functionality (met/* or s/s negative subjective effects (dizziness, anxiety, sedation. In conclusion, the contribution

  3. The Allele and Genotype Frequences of CYP2D6*10 in Ningxia Hui Population%宁夏回族人群CYP2D6*10等位基因及基因型分布频率

    李宗吉; 王健; 王惠成; 葛立军

    2009-01-01

    目的 了解宁夏回族人群CYP2D6*10等位基因及基因型分布频率,为临床使用CYP2D6底物药物提供依据.方法 采用等位基因特异扩增(ASA-PCR)技术,分析了180例回族体检者的CYP2D6基因型.结果 CYP2D6*10基因点突变结果中野生型纯合子频率为32.3%,杂合子频率为38.3%,突变型纯合子频率为29.4%,突变型等位基因频率为48.6%.结论 宁夏回族人群CYP2D6*10突变基因频率与国内外其他民族有一定差异,在临床应用经CYP2D6代谢的药物时应考虑个体间的代谢速率差异,确保用药安全有效.

  4. Determination of CYP2D6*10 and*14 genotypes by amplification refractory mutation methods in Chinese subjects%突变阻断扩增法检测中国人CYP2D6*10、*14等位基因

    陈冰; 马涛; 蔡卫民

    2009-01-01

    目的 根据突变阻断扩增原理,建立用于检测中国人CYP2D6*10及*14等位基因的方法.方法 采用单管四引物法检测CYP2D6*10等位基因,建立等位基因特异扩增法检测CYP2D6*14等位基因,检测295名健康中国汉族人CYP2D6*10等位基因.结果 CYP2D6*10及*14等位基因基因频率分别为55.8%和1.8%,295位受试者中包括1位*14/*14、6位*1/*14、3位*10/*14,基因型分布符合Hardy-Weinberg平衡(x2=2.15,df=5,P>0.82).结论 本室建立的CYP2D6*10、*14等位基因分析法具有方便快捷、结果准确可靠的特点.

  5. Unique Gold Nanoparticle Aggregates as a Highly Active SERS Substrate

    Schwartzberg, A M; Grant, C D; Wolcott, A; Talley, C E; Huser, T R; Bogomolni, R; Zhang, J Z

    2004-04-06

    A unique gold nanoparticle aggregate (GNA) system has been shown to be an excellent substrate for surface-enhanced Raman scattering (SERS) applications. Rhodamine 6G (R6G), a common molecule used for testing SERS activity on silver, but generally difficult to detect on gold substrates, has been found to readily bind to the GNA and exhibit strong SERS activity due to the unique surface chemistry afforded by sulfur species on the surface. This GNA system has yielded a large SERS enhancement of 10{sup 7}-10{sup 9} in bulk solution for R6G, on par with or greater than any previously reported gold SERS substrate. SERS activity has also been successfully demonstrated for several biological molecules including adenine, L-cysteine, L-lysine, and L-histidine for the first time on a gold SERS substrate, showing the potential of this GNA as a convenient and powerful SERS substrate for biomolecular detection. In addition, SERS spectrum of R6G on single aggregates has been measured. We have shown that the special surface properties of the GNA, in conjunction with strong near IR absorption, make it useful for SERS analysis of a wide variety of molecules.

  6. CYP2 D6基因多态性对帕罗西汀在河南汉族健康受试者体内药代动力学的影响%Influence of CYP2 D6 genetic polymorphism on pharmacokinetics of par-oxetine in Henan Han healthy subjects

    周霖; 薛文华; 张海朋; 刘克锋; 赵杰

    2014-01-01

    目的:研究CYP2D6基因多态性对帕罗西汀在河南汉族健康受试者体内药代动力学的影响。方法:河南汉族健康受试者48人,使用PCR-RFLP技术对其进行CYP2D6基因型检测。受试者单剂量(7.5 mg)口服甲磺酸帕罗西汀胶囊后,收集120 h内的系列血样,用建立的LC-MS/MS法测定血浆帕罗西汀浓度,绘制药-时曲线,进行药代动力学分析。结果:男性CYP2D6*1/*1型受试者9人,CYP2D6*1/*10型21人;女性CYP2D6*1/*1型受试者5人,CYP2D6*1/*10型13人;未检测到CYP2D6*10/*10型;男、女CYP2D6基因型分布差异无统计学意义(χ2=0.027,P>0.999)。与CYP2D6*1/*1型男性受试者比较,CYP2D6*1/*10型男性受试者tmax、t1/2无明显变化(Z=1.145,t=1.400,P>0.05),CL(F)、Vd 降低(Z=2.557,t=2.104,P<0.05),ρmax、AUC0-120和AUC0-∞增加(Z=2.421、2.512、2.557,P<0.05)。与CYP2D6*1/*1型女性受试者比较,CYP2D6*1/*10型女性受试者ρmax、tmax、t1/2及Vd 无明显变化(Z=1.992、1.921,t=2.117、1.905,P>0.05),但 CL(F)降低(Z=2.021,P<0.05), AUC0-120和AUC0-∞增加(Z=2.021、2.021,P<0.05)。结论:CYP2D6*10突变等位基因可影响甲磺酸帕罗西汀在河南汉族健康人群体内的代谢。%Aim:To investigate the influence of CYP2D6 genetic polymorphism on pharmacokinetics of paroxetine in Henan Han healthy people .Methods:A total of 48 Han healthy volunteers from Henan Province were subjected to detect genetic polymorphied by PCR R-FLP method .The volunteers were given a single oral dose of paroxetine mesylate capsule (7.5 mg), and blood samples were collected at different time points until 120 h.The concentrations of paroxetine in plas-ma were measured by LC-MS/MS method, and the pharmacokinetics was analyzed .Results: There were 9 males with CYP2D6*1/*1, 21 with CYP2D6

  7. Substrate independent ATPase activity may complicate high throughput screening.

    Tuntland, Micheal L; Fung, L W-M

    2016-10-01

    Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening. PMID:27430931

  8. Active substrate integrated terahertz waveguide using periodic graphene stack

    Yanfei Dong; Peiguo Liu; Dingwang Yu; Bo Yi; Gaosheng Li

    2015-01-01

    The transmission properties of a substrate integrated waveguide (SIW) based on periodic graphene stacks have been theoretically investigated in the terahertz (THz) region. The effects of the dielectric-graphene-dielectric structure of the stack on the propagation properties are shown to be significant and different from the conventional active SIW based on active components. By varying the graphene chemical potential, the cut-off frequency of the proposed waveguide can be dynamically tuned fr...

  9. Comparison of Streptokinase Activity from Streptococcus mutans using Different Substrates

    Muhammad Anjum Zia*, Rana Faisal, Rao Zahid Abbas1, Gull-e-Faran, Muhammad Kashif Saleemi2 and Junaid Ali Khan3

    2013-01-01

    Streptokinase is a novel bacterial fibrinolytic enzyme that binds and activates plasminogen and is produced by several species of Streptococci. Streptococcus mutans was selected for optimum production of streptokinase using corn steep liquor, molasses, rice polishing and sugarcane bagass in liquid state fermentation. Substrates were applied in different concentrations ranging from 0.1-0.8%. Maximum fibrinolytic activity was observed by 0.3% corn steep liquor, 0.5% molasses and rice polishing ...

  10. 等位基因特异扩增法研究中国人CYP2D6中速代谢的相关基因%ALLELE SPECIFIC AMPLIFICATON FOR CYP2D6 GENE RELATED TO INTERMEDIATE METABOLIZER IN CHINESE SUBJECTS

    陈冰; 蔡卫民; 凌树森

    2001-01-01

    AIM To establish an allele specific PCR amplification (ASA-PCR) for determination of the genotype of CYP2D6*10B polymorphism in Chinese subjects. METHODS CYP2D6*10B alleles of 65 healthy Chinese subjects were analyzed by a two-step PCR assay and the correlation of genotype and phenotype was studied. RESULTS There were 20 CYP2D6*10B heterozygous genotypes subjects (wt/m) in 35 very extensive metabolizers (VEMs), which consisted the major part of VEM subjects (57%). Meanwhile, 20 subjects consisting 69% of 29 intermediate metabolizers were CYP2D6*10B homozygous mutant genotypes (m/m). The poor metabolizer was also m/m. The metabolic ratio of CYP2D6*10B m/m subjects were larger than wt/m and wild type, the values were -1.49±0.54, -2.20±0.49 and -2.47±0.61 (P<0.01). CONCLUSION PCR-ASA was shown to be a rapid and specific method. It can be used to study the genetic polymorphism, especially CYP2D6 intermediate metabolism.%目的 建立CYP2D6*10B的等位基因特异扩增法(ASA-PCR),以探讨中国人CYP2D6中速代谢的基因分型。方法 采用两步扩增法得到CYP2D6*10B等位基因特异片段,分析健康中国汉族人CYP2D6*10B等位基因,并探讨基因分型结果与右美沙芬表型分型结果的相关性。结果 35名表型为极快代谢受试者(VEMs)中,CYP2D6*10B以杂合子(wt/m)为主占57%;29名中速代谢受试者(IMs)以突变型纯合子(m/m)为主占69%;慢代谢受试者(PM)基因型为m/m。CYP2D6*10B m/m组的MR明显大于wt/m组和野生型组(wt/wt)。结论 ASA-PCR法有快速、准确的优点,可用于CYP2D6中速代谢的检测与研究。

  11. Genetic polymorphism of cytochrome P450 2D6 in Chinese population detected by gene chips%基因芯片法测定中国人群细胞色素P450 2D6的基因多态性

    李芹; 王睿; 许力; 文思远; 王升启

    2006-01-01

    目的:根据细胞色素P450 2D6基因序列的多态性分布特点,针对在中国人群药物代谢中具有潜在作用的突变位点C188T和G4268C,设计寡核苷酸探针,制备寡核苷酸芯片,探讨基因芯片技术应用于CYP2D6基因分型的可行性,观察中国人群细胞色素P450 2D6的基因多态性.方法:实验于2005-02/08进行.以克隆并已测序的细胞色素P450 2D6基因重组质粒作为标准样品,扩增产物与芯片进行杂交,制备基因芯片,测定312名中国健康志愿者(已签署知情同意书)细胞色素P4502D6基因分型,并使用直接测序法对结果进行验证.结果:312名全部进入结果分析.①312名中国健康志愿者中CYP2D6*2和CYP2D6*10的变异频率分别为CYP2D6*2W*10W 8.3%,*2H*10W 13.5%,*2M*10W 10.6%,*2M*10H 17.0%,*2M*10M 34.6%,*2H*10H 9.6%,*2H*10M 64%.②对部分样本采用直接测序的方法进行进一步确证,结果完全吻合.结论:①CYP2D6*2和CYP2D6*10突变型等位基因在中国人群中出现的频率较高.②基因芯片法简便、快捷,适用于中国人群细胞色素P4502D6基因分型研究.

  12. No association between schizophrenia and polymorphisms within the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT)

    Daniels, J.; Williams, J.; Asherson, P.; McGuffin, P.; Owen, M. [Univ of Wales College of Medicine, Cardiff (United Kingdom)

    1995-02-27

    It has been suggested that the cytochrome P450 mono-oxygenase, debrisoquine 4-hydroxylase, is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells. It is also thought to be related to the dopamine transporter that acts to take released dopamine back up into presynaptic terminals. The present study used the association approach to test the hypothesis that mutations in the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT) confer susceptibility to schizophrenia. There were no differences in allele or genotype frequencies between patients and controls in the mutations causing the poor metaboliser phenotype in CYP2D6. In addition there was no association found between schizophrenia and a 48 bp repeat within the 3{prime} untranslated region of DAT. 18 refs., 2 tabs.

  13. Escitalopram er en svag hæmmer af CYP2D6-katalyserede O-demetylering af (+)-tramadol, men ikke mindsker hypoalgesic virkning i eksperimentelle smerter

    Noehr-Jensen, L; Zwisler, S T; Larsen, F;

    2009-01-01

    Tramadol er O-demethylerede til den aktive metabolit (+)-O-desmethyltramadol ((+)- M1) via CYP2D6, et enzym, der er svagt hæmmes af escitalopram. Vi har undersøgt muligheden for en farmakokinetisk (PK) og farmakodynamiske (PD) virkningen af escitalopram på tramadol metabolisme. Femten raske...... 1,95 mikromol / LH efter escitalopram (P = 0,0027). Den gennemsnitlige ÄúÊÇ (1-12) af CPT var 4.140 og 4.388 cm.s efter placebo og escitalopram (p = 0,71). Selvom escitalopram er en svag hæmmer af CYP2D6, er det ikke forringer den analgetiske virkning af tramadol....

  14. Study of simple super-critical fluids (CO2, C2D6) through neutron scattering, Raman spectroscopy and molecular dynamic simulations

    Super-critical fluids are largely used in industrial sectors. However the knowledge of the physical phenomena in which they are involved stays insufficient because of their particular properties. A new model of adjusting molecular structures is proposed, this model has been validated through neutron scattering experiments with high momentum transfer on C2D6. The experimental representation of the critical universal function for C2D6 and CO2 has been obtained through the neutron echo spin and by relying on structure measurements made through neutron elastic scattering at small angles. Raman spectroscopy and molecular dynamics simulation have been used to feature structure and dynamics. Scattering as well as microscopic molecular density fluctuations have been analysed

  15. CYP2D6*10等位基因多态性对文拉法辛血药浓度的影响%Effect of CYP2D6*10 gene polymorphism on blood concentration of venlafaxine

    杨丽蓉; 刘天龙; 刘小雷

    2013-01-01

    目的 旨在研究细胞色素P450酶2D6*10(CYP2D6* 10)基因多态性对文拉法辛血药浓度的影响.方法 随机选择65例抑郁症患者为研究对象,提取其外周血中的DNA并利用特异性的引物对其进行扩增,对得到的扩增产物进行酶切,通过电泳表征酶切结果并分析65例抑郁患者CYP2D6*10基因的基因类型.根据所选择患者基因类型的不同,将其分为3组,即A组:纯合突变(CYP2D6*10/*14),B组:杂合体[CYP2D6*1(*2)/*14]及C组:野生型纯合体[CYP2D6*1(*2)/*5];各组患者口服文拉法辛225mg后,使用高效液相色谱仪测定患者文拉法辛(VL)和O-去甲文拉法辛(ODVL)的血药浓度以研究文拉法辛在不同基因携带者体内的代谢情况.通过汉密尔顿抑郁量表(HAMD)和药物不良反应量表(TESS)来明确文拉法辛对不同CYP2D6*10基因携带者的治疗效果和产生不良反应情况.结果 电泳结果显示,在65名患者中,纯合突变(CYP2D6*10/*14)为29例(44.6%),杂合体([CYP2D6*1(*2)/*14]为17例(26.1%),野生型纯合体[CYP2D6*1(*2)/*5]为19例(29.3%);3组间CvL CODVL及CODVL/CVL比较均具有显著地差异(P<0.05);根据CODVI/CVL值可知,A组、B组及C组对文拉法辛的代谢类型分别为快型(EM)、中型(IM)及慢型(PM).各组间TESS评分和HAMD评分差异具有统计学意义(P< 0.05).结论 不同CYP2D6* 10基因是通过决定抑郁症患者对文拉法辛的代谢类型而影响其血药浓度.%Objective To determine the effect of CYP2D6*10 gene polymorphism on blood concentration of venlafaxine. Methods A total of 65 patients with dysthymia disorders were selected as the case group and DNA extracted from their peripheral blood was amplified by PCR. The PCR products were digested by restriction endonuclease (HphI) and their CYP2D6*10 genotypes were determined by electrophoresis. According to different genotypes, 65 patients were divided into 3 groups: group A: CYP2D6*l0 /*14, group B: [ CYP2D6*1 (*2 ) /*14 ], and

  16. Consequences of CYP2D6 polymorphism for the disposition and dynamics of tolterodine : a novel drug in the treatment of urinary bladder overactivity

    Brynne, Niclas

    1998-01-01

    The pharmacokinetics and pharmacological effects of tolterodine were studied in man following administration of increasing oral and intravenous single-doses. The influence of metabolic phenotype in extensive and poor metabolizers of debrisoquine was determined. The effect of tolterodine on the major drug metabolizing cytochrome P450 (CYP) isozymes 1A2, 2C19, 2D6 and 3A4 was studied in vivo by measurements of metabolic ratios of probe drugs. Changes in tolterodine disposition...

  17. A Physiologically Based Pharmacokinetic Model to Predict Disposition of CYP2D6 and CYP1A2 Metabolized Drugs in Pregnant Women

    Ke, Alice Ban; Nallani, Srikanth C.; Zhao, Ping; Rostami-Hodjegan, Amin; Isoherranen, Nina; Unadkat, Jashvant D.

    2013-01-01

    Conducting pharmacokinetic (PK) studies in pregnant women is challenging. Therefore, we asked if a physiologically based pharmacokinetic (PBPK) model could be used to evaluate different dosing regimens for pregnant women. We refined and verified our previously published pregnancy PBPK model by incorporating cytochrome P450 CYP1A2 suppression (based on caffeine PK) and CYP2D6 induction (based on metoprolol PK) into the model. This model accounts for gestational age–dependent changes in materna...

  18. Genetic polymorphisms of CYP2D6*10 and the effectiveness of combined tamoxifen citrate and testosterone undecanoate treatment in infertile men with idiopathic oligozoospermia*

    Tang, Kai-Fa; Zhao, Yi-li; Ding, Shang-shu; Wu, Qi-Fei; Wang, Xing-yang; Shi, Jia-qi; SUN, FA; Xing, Jun-Ping

    2015-01-01

    Tamoxifen citrate, as the first line of treatment for infertile men with idiopathic oligozoospermia, was proposed by the World Health Organization (WHO), and testosterone undecanoate has shown benefits in semen values. Our objective was to assess the effectiveness of treatment with tamoxifen citrate and testosterone undecanoate in infertile men with idiopathic oligozoospermia, and whether the results would be affected by polymorphisms of CYP2D6*10. A total of 230 infertile men and 147 control...

  19. Genetic polymorphisms of CYP2D6*10 and the effectiveness of combined tamoxifen citrate and testosterone undecanoate treatment in infertile men with idiopathic oligozoospermia

    Kai-fa TANG; Yi-li ZHAO; Shang-shu DING; Qi-fei WU; Xing-yang WANG; Jia-qi SHI; Fa SUN; Jun-ping XING

    2015-01-01

      结论:CYP2D6*10基因突变型特发性少精男性不育患者接受他莫昔芬联合十一酸睾酮疗效较基因野生型组差。%Tamoxifen citrate, as the first line of treatment for infertile men with idiopathic oligozoospermia, was proposed by the World Health Organization (WHO), and testosterone undecanoate has shown benefits in semen values. Our objective was to assess the effectiveness of treatment with tamoxifen citrate and testosterone un-decanoate in infertile men with idiopathic oligozoospermia, and whether the results would be affected by polymor-phisms of CYP2D6*10. A total of 230 infertile men and 147 controls were included in the study. Patients were treated with tamoxifen citrate and testosterone undecanoate. Sex hormone, sperm parameters, and incidence of spontaneous pregnancy were detected. There were no significant differences between the control and patient groups with respect to CYP2D6*10 genotype frequencies (P>0.05). The follicle-stimulation hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels were raised, and sperm concentration and motility were increased at 3 months and became significant at 6 months, and they were higher in the wild-type al ele (C/C) than in the heterozygous variant al ele (C/T) or homozygous variant allele (T/T) subgroups (P<0.05). In addition, the percentage of normal morphology was raised at 6 months, and represented the highest percentage in the C/C subgroup (P<0.05). The incidence of spontaneous pregnancy in the C/C subgroup was higher than that in the C/T or T/T subgroups (P<0.01). This study showed that the CYP2D6*10 variant genotype demonstrated worse clinical effects in infertile men with idiopathic oligozoospermia.

  20. Role of Pharmacogenetics in Improving the Safety of Psychiatric Care by Predicting the Potential Risks of Mania in CYP2D6 Poor Metabolizers Diagnosed With Bipolar Disorder

    Sánchez-Iglesias, Santiago; García-Solaesa, Virginia; García-Berrocal, Belén; Sanchez-Martín, Almudena; Lorenzo-Romo, Carolina; Martín-Pinto, Tomás; Gaedigk, Andrea; González-Buitrago, José Manuel; Isidoro-García, María

    2016-01-01

    Abstract One of the main concerns in psychiatric care is safety related to drug management. Pharmacogenetics provides an important tool to assess causes that may have contributed the adverse events during psychiatric therapy. This study illustrates the potential of pharmacogenetics to identify those patients for which pharmacogenetic-guided therapy could be appropriate. It aimed to investigate CYP2D6 genotype in our psychiatric population to assess the value of introducing pharmacogenetics as...

  1. 用回归分割法预测4味中药对CYP2D6代谢的抑制作用

    郑春松; 陈立武; 杜建; 叶蕻芝

    2007-01-01

    细胞色素P4502D6(CYP2D6)是人体中重要的药物代谢酶,参与临床近百种药物的代谢,包括多种抗心律失常药、β受体阻滞剂、抗高血压药、抗抑郁药以及抗肿瘤药等。某些毒性或致癌物也可被CYP2D6代谢解毒或激活。除影响外源性化学物质的代谢外,CYP2D6还与某些疾病呈相关性。黄芪、女贞子、山药、白花蛇舌草有很好的抗肿瘤作用.目前研究很多,

  2. Association of polymorphism of CYP2D6 and CYP2C9 genes encoding P-450 proteins of cytochrome with arterial hypertension

    Saratsev A.V.

    2012-12-01

    Full Text Available Gene polymorphisms of cytochrome P-450 CYP2 encoding proteins of cytochrome P-450 are essential forantihy-pertensive drugs metabolism. Purpose: We study the associations of functionally defective allele variants of CYP2D6 gene and CYP2C9 gene with the degree of arterial hypertension (AH. Materials and methods: Samples of DNA of leukocytes of blood of 150 patients with AH without the associated clinical conditions (56% of women at the age of 20-59 years have been investigated. For the study of polymorphism of genes the pharmacogenetic biochip developed in the Institute of Molecular Biology n.a. V. A. Engelgardt has been used. Comparison of frequencies of occurrence of signs has been carried out on the basis of chi-square criterion. Results: It has been revealed that homozygotes by mutant A1075C, C430T alleles of CYP2C9gene and G1934A of CYP2D6 gene have been significantly more common among patients with hypertension III (p=0.01. Conclusion: The research works on genes of system of P-450 cytochrome have important clinical value for rationalization of pharmacotherapy of hypertension. The increased frequency of occurrence of mutant allele of CYP2D6 and CYP2C9 genes in patients with hypertension III requires special attention to the problem of efficiency and safety of application of hypotensive drugs for the patients.

  3. 细胞色素P450 2D6酶基因多态性与氟西汀临床效应的相关性研究%Correlation between polymorphism of cytochrome P450 2D6 gene and clinical response to fluoxetine

    侯静; 林建荣; 郑东; 何凤贞; 谭静卿; 陆欣乔; 黄煜坤; 李洁仪; 庞振泰; 林振强

    2002-01-01

    目的研究中国人P450 2D6酶(CYP2D6)基因多态性与氟西汀临床效应之间的关系.方法用氟西汀对108例抑郁患者(抑郁症89例,精神分裂症后抑郁7例,分裂情感性精神病抑郁型4例,强迫性神经症2例,焦虑性神经症1例,抑郁性神经症4例,分裂样精神病(伴抑郁症状)1例)进行治疗,用汉密尔顿抑郁量表(HAMD)和治疗中需处理的副反应症状量表(TESS)评定疗效和副反应,用聚合酶链反应-限制性片段长度多态性分析法(PCR-RFLP)分析患者CYP2D6基因型.结果 (1)PCR-RFLP分析表明,在108例抑郁患者中,CYP2D6基因型为 wt/wt者26例,wt/Ch者55例,Ch/Ch者26例, wt/A者1例,未发现B等位基因.CYP2D6等位基因频率分别为CYP2D6wt(50%),CYP2D6Ch(49.5%), CYP2D6A(0.5%). (2)入组时,不同基因型的患者之间HAMD基础评分的差异无显著性(P>0.05).经氟西汀治疗4周和8周后,不同CYP2D6基因型患者的HAMD量表评分比治疗前降低,差异均有非常显著性(P=0.000);8周后减分率达80%左右,而TESS评分较低.在不同基因型的患者之间,治疗后的HAMD评分、HAMD减分率及TESS评分的差异均无显著性(P>0.05).患者治疗前后的HAMD评分及HAMD减分率、TESS评分等与CYP2D6基因型及CYP2D6突变等位基因数目的相关关系均无显著性(P>0.05).结论未发现中国人CYP2D6基因多态性与氟西汀临床效应之间有关联.

  4. CYP2D6 Genetic Polymorphisms in Chinese Healthy and Schizophrenia Populations%中国健康人群与精神分裂症患者CYP2D6(C100T)遗传多态性的相关研究

    周健; 李虓; 王刚; 陈雪彦; 康熙雄

    2008-01-01

    目的 探讨中国健康人群与精神分裂症患者细胞色素P450 2D6(CYP2D6)遗传多态性的相关性.方法 应用PCR与DNA测序相结合的方法对88名精神分裂症患者(男40例,女48例;平均年龄40.38±13.45岁)与93名健康志愿者(男40例,女53例,平均年龄43.60±12.05岁)进行了CYP2D6的基因多态性分析,根据CYP2D6(C100T)将精神分裂症患者和健康志愿者基因型分为三组(C/C,C/T,T/T).结果 在CYP2D6(C100T)的基因型和等位基因型检测中,精神分裂症患者和健康志愿者组间的基因型和等位基因没有显著性差异(X2=0.202,P>0.05;X2=0.066,P>0.05).结论 CYP2D6(C100T)可能不是中国精神分裂症患者的一个易感因素.

  5. 他汀类药物降脂治疗的CYP2D6基因多态性及其与高脂血症的关系%Statins therapy in CYP2D6 gene polymorphism and hyperlipidemia

    李居怡; 王健; 王奕; 甘利明; 黄文静; 邓艾平

    2016-01-01

    目的:观察他汀类药物降脂治疗相关的基因多态性位点CYP4502D6* 10(CYP2D6*10)在宁夏地区的分布和对辛伐他汀降脂疗效的影响及其与高脂血症的关系.方法:采用等位基因特异扩增技术,测定150例健康回族人和200例高脂血症患者CYP2D6基因型并分析基因型与血脂水平、辛伐他汀调脂疗效的关系.结果:CYP2D6* 10基因在宁夏地区的突变频率为47.6%,CYP2D6*10等位基因与高脂血症无显著相关性,服用辛伐他汀8周后CC基因型较CT及TT基因型个体总胆固醇(TC)水平明显降低,差异有统计学意义.结论:CYP2D6* 10基因多态性存在种族和地域的差异,CYP2D6* 10基因多态性对TC水平有显著影响,该基因多态性可能成为辛伐他汀调脂疗效的预测因子.

  6. 利培酮治疗与细胞色素P4502D6/C188T酶基因多态性的关联分析%Genetic analysis of the cytochrome P450-2D6(CYP2D6)locus:screening influence of risperidone on clinical outcomes in schizophrenia

    王飚; 杨晓敏; 江三多; 钱伊萍; 汪栋祥; 江开达

    2004-01-01

    目的研究利培酮临床效应的个体差异与其代谢酶细胞色素P450 2D6(cytochrome P450 2D6, CYP2D6)酶基因多态性的相关性.方法对88例符合CCMD-3精神分裂症诊断标准的患者和96例健康对照者作病例-对照分析.精神分裂症患者给予利培酮治疗8周,用阳性和阴性症状量表(positive and negative symptom scale, PANSS)评分评价利培酮疗效.采用聚合酶链反应扩增及限制性片段长度多态性(PCR-RFLP)技术对CYP2D6 exon Ⅰ的C188T位点突变进行检测,分析利培酮临床效应与其主要代谢酶CYP2D6/C188T酶基因多态性的相关性. 结果中国上海地区人群的CYP2D6/C188T突变率(弱代谢型)为36.3%,病例组和正常对照组间基因型频率总体分布比较无显著差异(χ2=1.15, df=2, P>0.05),两组间的等位基因频率之间比较也无显著性差异(χ2=0.78, df=1, P>0.05).进行性别及有否家族史分组后分析,亦无差异存在,且CYP2D6/C188T突变与利培酮临床效应之间并无相关性(χ2=1.12, df=2; χ2=0.03, df=1, P>0.05). 结论未发现中国人CYP2D6/C188T多态性与利培酮临床效应的个体差异有相关性.

  7. CYP2D6和UGT1A6基因多态性与慢性苯中毒的危险性%Association of Genetic Polymorphism of CYP2D6 and UGT1A6 with Risks of Chronic Benzene Poisoning

    顾寿永; 张忠彬; 曹多志; 万俊香; 高晓玲; 金锡鹏; 夏昭林

    2005-01-01

    目的探讨CYP2D6基因和UGT1A6基因多态性与慢性苯中毒易感性的关系.方法采用病例一对照研究,选择152名苯中毒工人为病例组,152名接触苯而无中毒表现的工人为对照组.采用PCR-RFLP技术检测CYP2D6第9外显子g.4268位点和UGT1A6第1外显子t.181位点的多态性.结果携带CYP2D6 g.4268 C/C基因型的个体较携带有CYP2D6 g.4268 G/G或G/C基因型的个体发生苯中毒的危险性高1.72倍(95%CI:1.8~2.78,P=0.03);在不吸烟的人群中,携带CYP2D6 g.4268 C/C基因型的个体比携带G/G或G/C基因型的个体发生苯中毒的风险性高1.75倍(95%CI:1.03~2.94,P=0.04);在不饮酒的人群中,携带CYP2D6 g.4268 C/C基因型个体比携带CYP2D6 g.4268 G/G和G/C基因型的个体发生苯中毒的危险性高1.82倍(95%CI:1.09~3.03,P=0.02).UGT1A6 t.181的各基因型在病例组和对照组的分布频率差异无显著性(P>0.05).结论携带CYP2D6 g.4268 C/C基因型的个体对苯中毒可能易感.

  8. Extracellular Protease Activity of Enteropathogenic Escherechia coli on Mucin Substrate

    SRI BUDIARTI

    2007-03-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC causes gastrointestinal infections in human. EPEC invasion was initiated by attachment and aggressive colonization on intestinal surface. Attachment of EPEC alter the intestine mucosal cells. Despite this, the pathogenic mechanism of EPEC infectior has not been fully understood. This research hypothesizes that extracellular proteolytic enzymes is necessary for EPEC colonization. The enzyme is secreted into gastrointestinal milieu and presumably destroy mucus layer cover the gastrointestinal tract. The objective of this study was to assay EPEC extracellular protease enzyme by using mucin substrate. The activity of EPEC extracellular proteolytic enzyme on 1% mucin substrate was investigated. Non-pathogenic E. coli was used as a negative control. Positive and tentative controls were Yersinia enterocolitica and Salmonella. Ten EPEC strains were assayed, seven of them were able to degrade mucin, and the highest activity was produced by K1.1 strain. Both positive and tentative controls also showed the ability to digest 0.20% mucin.

  9. Substrate modulation of enzyme activity in the herpesvirus protease family

    Lazic, Ana; Goetz, David H.; Nomura, Anson M.; Marnett, Alan B.; Craik, Charles S.

    2007-01-01

    The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 Å resolution structure of Kaposi’s Sarcoma herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 Å2 of surface are buried in the newly formed S4 pocket when tyrosin...

  10. Implication of Xenobiotic Metabolizing Enzyme gene (CYP2E1, CYP2C19, CYP2D6, mEH and NAT2 Polymorphisms in Breast Carcinoma

    Gabbouj Sallouha

    2008-04-01

    Full Text Available Abstract Background Xenobiotic Metabolizing Enzymes (XMEs contribute to the detoxification of numerous cancer therapy-induced products. This study investigated the susceptibility and prognostic implications of the CYP2E1, CYP2C19, CYP2D6, mEH and NAT2 gene polymorphisms in breast carcinoma patients. Methods The authors used polymerase chain reaction and restriction enzyme digestion to characterize the variation of the CYP2E1, CYP2C19, CYP2D6, mEH and NAT2 gene in a total of 560 unrelated subjects (246 controls and 314 patients. Results The mEH (C/C mutant and the NAT2 slow acetylator genotypes were significantly associated with breast carcinoma risk (p = 0.02; p = 0.01, respectively. For NAT2 the association was more pronounced among postmenopausal patients (p = 0.006. A significant association was found between CYP2D6 (G/G wild type and breast carcinoma risk only in postmenopausal patients (p = 0.04. Association studies of genetic markers with the rates of breast carcinoma specific overall survival (OVS and the disease-free survival (DFS revealed among all breast carcinoma patients no association to DFS but significant differences in OVS only with the mEH gene polymorphisms (p = 0.02. In addition, the mEH wild genotype showed a significant association with decreased OVS in patients with axillary lymph node-negative patients (p = 0.03 and with decreasesd DFS in patients with axillary lymph node-positive patients (p = 0.001. However, the NAT2 intermediate acetylator genotype was associated with decreased DFS in axillary lymph node-negative patients. Conclusion The present study may prove that polymorphisms of some XME genes may predict the onset of breast carcinoma as well as survival after treatment.

  11. Genetic polymorphisms of CYP2D6*10 and the effectiveness of combined tamoxifen citrate and testosterone undecanoate treatment in infertile men with idiopathic oligozoospermia*

    Tang, Kai-fa; Zhao, Yi-li; Ding, Shang-shu; Wu, Qi-fei; Wang, Xing-yang; Shi, Jia-qi; Sun, Fa; Xing, Jun-ping

    2015-01-01

    Tamoxifen citrate, as the first line of treatment for infertile men with idiopathic oligozoospermia, was proposed by the World Health Organization (WHO), and testosterone undecanoate has shown benefits in semen values. Our objective was to assess the effectiveness of treatment with tamoxifen citrate and testosterone undecanoate in infertile men with idiopathic oligozoospermia, and whether the results would be affected by polymorphisms of CYP2D6*10. A total of 230 infertile men and 147 controls were included in the study. Patients were treated with tamoxifen citrate and testosterone undecanoate. Sex hormone, sperm parameters, and incidence of spontaneous pregnancy were detected. There were no significant differences between the control and patient groups with respect to CYP2D6*10 genotype frequencies (P>0.05). The follicle-stimulation hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels were raised, and sperm concentration and motility were increased at 3 months and became significant at 6 months, and they were higher in the wild-type allele (C/C) than in the heterozygous variant allele (C/T) or homozygous variant allele (T/T) subgroups (P<0.05). In addition, the percentage of normal morphology was raised at 6 months, and represented the highest percentage in the C/C subgroup (P<0.05). The incidence of spontaneous pregnancy in the C/C subgroup was higher than that in the C/T or T/T subgroups (P<0.01). This study showed that the CYP2D6*10 variant genotype demonstrated worse clinical effects in infertile men with idiopathic oligozoospermia. PMID:25743120

  12. Genetic polymorphisms of CYP2D6*10 and the effectiveness of combined tamoxifen citrate and testosterone undecanoate treatment in infertile men with idiopathic oligozoospermia.

    Tang, Kai-fa; Zhao, Yi-li; Ding, Shang-shu; Wu, Qi-fei; Wang, Xing-yang; Shi, Jia-qi; Sun, Fa; Xing, Jun-ping

    2015-03-01

    Tamoxifen citrate, as the first line of treatment for infertile men with idiopathic oligozoospermia, was proposed by the World Health Organization (WHO), and testosterone undecanoate has shown benefits in semen values. Our objective was to assess the effectiveness of treatment with tamoxifen citrate and testosterone undecanoate in infertile men with idiopathic oligozoospermia, and whether the results would be affected by polymorphisms of CYP2D6*10. A total of 230 infertile men and 147 controls were included in the study. Patients were treated with tamoxifen citrate and testosterone undecanoate. Sex hormone, sperm parameters, and incidence of spontaneous pregnancy were detected. There were no significant differences between the control and patient groups with respect to CYP2D6*10 genotype frequencies (P>0.05). The follicle-stimulation hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels were raised, and sperm concentration and motility were increased at 3 months and became significant at 6 months, and they were higher in the wild-type allele (C/C) than in the heterozygous variant allele (C/T) or homozygous variant allele (T/T) subgroups (P<0.05). In addition, the percentage of normal morphology was raised at 6 months, and represented the highest percentage in the C/C subgroup (P<0.05). The incidence of spontaneous pregnancy in the C/C subgroup was higher than that in the C/T or T/T subgroups (P<0.01). This study showed that the CYP2D6*10 variant genotype demonstrated worse clinical effects in infertile men with idiopathic oligozoospermia. PMID:25743120

  13. Direct determination of phosphatase activity from physiological substrates in cells.

    Zhongyuan Ren

    Full Text Available A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1 mg(-1 for PPi, to 56 ± 11 nmol min(-1 mg(-1 for AMP, to 79 ± 23 nmol min(-1 mg(-1 for beta-glycerophosphate and to 73 ± 15 nmol min(-1 mg(-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  14. Interaction of drug metabolizing cytochrome P450 2D6 poor metabolizers with cytochrome P450 2C9 and 2C19 genotypes modify the susceptibility to head and neck cancer and treatment response

    Yadav, Sunishtha S.; Ruwali, Munindra [Developmental Toxicology Division, Indian Institute of Toxicology Research (Formerly: Industrial Toxicology Research Centre), Council CSIR, P.O. Box 80, Mahatma Gandhi Marg, Lucknow 226 001 (India); Pant, Mohan C.; Shukla, Pragya [Department of Radiotherapy, C.S.M. Medical University, Shahmina Road, Lucknow 226 001 (India); Singh, Ram L. [Department of Biochemistry, Dr. R.M.L. Awadh University, Faizabad 224 001, U.P. (India); Parmar, Devendra, E-mail: parmar_devendra@hotmail.com [Developmental Toxicology Division, Indian Institute of Toxicology Research (Formerly: Industrial Toxicology Research Centre), Council CSIR, P.O. Box 80, Mahatma Gandhi Marg, Lucknow 226 001 (India)

    2010-02-03

    The present case-control study attempted to investigate the association of poor metabolizer (PM) genotypes of cytochrome P450 2D6 (CYP2D6*4 and CYP2D6*10) with squamous cell carcinoma of head and neck (HNSCC) and treatment response in patients receiving chemotherapy or combination of chemo- and radiotherapy. Cases with the PM genotypes of CYP2D6 displayed a significantly increased risk for HNSCC as compared to wild type genotypes. The risk was found to further increase in cases (up to 4.8) carrying combination of PM genotypes of CYP2D6, CYP2C9 (CYP2C9*2) or CYP2C19 (CYP2C19*2), suggesting that synergism amongst the PM genotypes of drug metabolizing CYPs leads to impairment in the detoxification of the tobacco carcinogens. A small increase in the risk in tobacco (chewers or smokers) or alcohol users in cases with CYP2D6*4 allele while no change or even a small decrease in risk in cases with CYP2D6*10 allele when compared to non-tobacco or alcohol users have suggested that CYP2D6 genotypes alone do not appear to interact significantly with environmental risk factors in modifying the susceptibility to HNSCC. Furthermore, most of the cases carrying PM genotypes of CYP2D6 did not respond to the treatment. Moreover, higher prevalence of non-responders among cases carrying combination of CYP2D6*4 or CYP2D6*4, CYP2C9*2 and CYP2C19*2 have demonstrated that interaction of PM genotypes may not only significantly modify the susceptibility to HNSCC but also the treatment response.

  15. 氨苄西林与双黄连注射液单用及配伍时对大鼠CYP2D6的影响%Effects on CYP2D6 by the Single Use of Ampicillin and Shuanghuanglian or Combination of Both

    王欢; 吴东媛; 杜智敏

    2008-01-01

    目的 通过氨苄西林和双黄连注射液的大鼠体内、外实验,观察两种药物分别给药及合用时对大鼠CYP2D6的影响.方法 对照组和实验组大鼠分别经尾静脉注射给予0.9%氯化钠溶液、双黄连注射液、氨苄西林、双黄连注射液联合氨苄西林,均给药1周.高效液相色谱法测定大鼠尿样及肝微粒体中CYP2D6探针药物右美沙芬的代谢率,观察氨苄西林、双黄连注射液分别单用及合用时对大鼠CYP2D6活性的影响.通过Western blot法测定其对大鼠肝微粒体CYP2D6蛋白表达的调控.结果 实验组大鼠分别给予双黄连注射液、氨苄西林及双黄连注射液配伍氨苄西林时,尿样中右美沙芬代谢率与对照组差异无显著性(P>0.05);实验组大鼠肝微粒体中加入右美沙芬(0.324 mmol·L-1),右美沙芬代谢率与对照组差异无显著性(P>0.05);各实验组与对照组CYP2D6蛋白表达差异无显著性.结论 双黄连注射液、氨苄西林单用和两药配伍使用均不影响CYP2D6酶的活性,与同时使用经CYP2D6代谢的其他药物不会产生相互作用.

  16. 26种中药提取物对CYP3A4和CYP2D6代谢的抑制作用

    Iwata; H; 郑晓燕(摘译); 阴赪宏(校)

    2005-01-01

    研究认为,细胞色素P450(CYP)酶与药物相互作用。本次在NADPH-生成体系存在的情况下,以单味中药甲醇提取物对人肝细胞微粒体进行预培养,检测26种中药对CYP3A4和CYP2D6的抑制作用。

  17. Untersuchung zu unerwünschten Arzneimittelwirkungen von Metoprolol in Abhängigkeit vom CYP2D6-Genotyp bei ambulanten Patienten

    Pröhmer, Anne Marie Theres

    2008-01-01

    Diese prospektive klinische Studie wurde mit dem Ziel durchgeführt, den Einfluss des polymorph exprimierten Cytochrom P450 (CYP) 2D6 auf erwünschte und unerwünschte Wirkungen von Metoprolol in einem ambulanten Setting zu untersuchen. Die mittels eines Tagebuchs abgefragten unerwünschten Arzneimittelwirkungen (UAW) umfassten zentralnervöse und kardiovaskuläre Beschwerden, die sexuelle Funktion und Lebensqualität des Patienten, sowie zusätzlich bei den Patienten mit diagnostizierter Psoriasis v...

  18. CYP2D6*10 polymorphism and response to donepezil in patients with Alzheimer's disease%阿尔茨海默病CYP2D6*10基因多态性与多奈哌齐疗效

    郑雪丽; 苗雅; 钟远; 万丽丽

    2012-01-01

    目的 研究阿尔茨海默病(AD)患者CYP2D6* 10基因多态性对多奈哌齐疗效的影响.方法 筛选AD患者110例(AD组)和健康体检者124例(对照组),应用限制性片段长度多态性-聚合酶链反应(RFLP-PCR)方法对两组患者进行CYP2D6*10基因多态性测定.AD组给予多奈哌齐治疗6个月,于服药前后,应用简易精神状态量表( MMSE)和阿尔茨海默病评定量表认知分量表(ADAS-Cog)进行认知功能测定;应用高效液相色谱-质谱法测定患者血浆中多奈哌齐的稳态血药浓度.结果 AD组与对照组CYP2D6*10的基因型和等位基因频率分布差异无统计学意义(P>0.05);AD组CYP2D6*10基因突变型(T/T型与C/T型)的血药浓度、MMSE和ADAS-Cog评分与野生型(C/C型)相比,差异均有统计学意义(P< 0.05).结论 多奈哌齐对阿尔茨海默病CYP2D6* 10基因突变型患者的疗效优于野生型患者.%Objective To analyze the relation of CYP2D6* 10 polymorphism with response to donepezil in patients with Alzheimer's disease(AD). Methods A total of 110 AD patients (AD group) and 124 healthy individuals (control group) were recruited for the study. RFLP-PCR was applied to analyze the CYP2D6*10 gene polymorphisms. Besides, the AD patients were administrated with donepezil 5-10 mg/d for 6 months and evaluated by cognition scales (MMSE and ADAS-Cog) before and after therapy. The steady plasma concentration (Cp) of donepezil was measured by HPLC-MS. Results There was no statistical difference in genotypes and allele frequency of CYP2D6* 10 between AD group and control group. However, significant differences were found in AD patients on steady concentration of donepezil and cognition scores (MMSE and ADAS-Cog) between CYP2D6*10 mutant genotypes (T/T and C/T group) and wild type (C/C group) (P < 0.05). Conclusions Donepezil is more effective for AD patients carrying CYP2D6*10 mutants than those carrying wild type.

  19. Influence of CYP2D6 genetic polymorphism on pharmacokinetics of metoprolol in Chinese population%中国人群CYP2D6基因多态性对美托洛尔药代动力学的影响

    李芹; 王睿; 郭雅; 裴斐

    2008-01-01

    目的:研究中国人群CYP2D6基因多态性对美托洛尔药代动力学的影响.方法:使用基因芯片技术测定中国健康志愿者CYP2D6的基因型,按照分型结果将志愿者分为四组,第1组:CYP2D6*2W*10W,第2组:CYP2D6*2H*10W或CYP2D6*2M*10W,第3组:CYP2D6*2M*10H,第4组:CYP2D6*2M*10M,每组筛选10人,共40人.各组志愿者单次口服 100 mg 美托洛尔后,使用HPLC方法测定血和尿中美托洛尔及其代谢产物α-羟基美托洛尔(HM)的浓度,研究其在不同基因型志愿者体内的药代过程.结果:第2组美托洛尔及其HM的主要药动学参数与第1组相比均没有统计学差异.第3组美托洛尔的t1/2、AUC、Cmax显著高于第1组(P<0.05);而HM的t1/2延长 47.3%,AUC降低 56.0%(P<0.05).第4组美托洛尔的t1/2、AUC、Cmax均显著高于第1组(P<0.05)和第3组(P<0.05);HM的t1/2、AUC、Cmax与第1组和第3组相比均有统计学差异(P<0.05),且呈现基因剂量效应.第3组和第4组的口服清除率和肾清除率均低于第1组,而0~24 h 代谢比率分别为第1组的 1.82 倍和 3.96 倍.结论:CYP2D6*2对于美托洛尔的药代动力学过程没有影响;但CYP2D6*10可降低酶活性,且CYP2D6*10纯合子变异比杂合子变异对美托洛尔药代动力学的影响更大,呈现基因剂量效应.

  20. CYP1A1和CYP2D6基因多态性对白血病发生的影响%Genetic polymorphisms of CYP1A1 and CYP2D6 and their impact on leukemia morbidity

    李文凯; 刘清霞; 陈放知; 骆亚萍; 陈慧娟; 夏嘉志; 陈汉春

    2008-01-01

    目的 分析细胞色素P450 CYP1A1和CYP2D6的多态性基因型在湖南地区白血病患者和健康人群中的分布及其对白血病发生的影响.方法 采用PCR及PCR-RFLP技术分析多态性基因型频率.结果 CYP1A1和CYP2D6基因的野生型、杂合突变型及纯合突变型的分布频率在急性淋巴细胞性白血病、急性非淋巴细胞性白血病、慢性粒细胞性白血病患者组与健康对照组之间无显著性差异;携带一个突变等位基因型的个体患白血病的风险与相应野生型携带者比较均无显著性差异;急性非淋巴细胞性白血病患者组的CYP1A1杂合突变型与CYP2D6杂合突变型的联合基因型频率高于健康对照组.结论 单独的CYP1A1或CYP2D6基因的多态性变异与白血病易感性不相关;CYP1A1杂合突变与CYP2D6杂合突变的联合基因型增加患急性非淋巴细胞性白血病的风险.

  1. The correlation between ACE and CYP2D6 polymorphism and the effect of perindopril%心衰患者 ACE 及 CYP2 D6遗传多态性与培哚普利疗效的关联性研究

    彭俊; 黄自明; 郭观华

    2015-01-01

    Objective To investigate the correlation between ACE and CYP 2D6 polymorphism and the effect of perindopril.Methods The genotype was determined by PCR in 158 patients.The differences of left ventricular end-diastolic diameters ( LVDD) , left ventricular ejection fractions ( LVEF) and AngⅡlevel before and after perindo-pril treatment were detected .Results The patients with DD/CC, DD/CT and DD/TT phenotype had a great de-crease in LVDD and AngⅡ after perindopril treatment .Conclusion ACE polymorphisms correlates to the effect of perindopril treatment .CYPD2D6 polymorphism does not correlate to the perindopril treatment .%目的:探讨心衰患者ACE及CYP2D6遗传多态性与培哚普利疗效的关联性。方法选取158例心衰患者,应用PCR方法检测其ACE及CYP2D6多态性,给予培哚普利治疗,对治疗前后的左室舒张末期内径( LVDD)、左室射血分数( LVEF)和血浆血管紧张素Ⅱ( AngⅡ)进行检测。结果 DD/CC、DD/CT和DD/TT型患者经治疗后,LVDD明显缩小,且血浆AngⅡ水平下降的幅度最大。结论 ACE基因的多态性与培哚普利的治疗效果有关联性,CYP2D6基因多态性对培哚普利的疗效无明显影响。

  2. Secretory Phospholipase A(2) Activity toward Diverse Substrates

    Madsen, Jesper Jonasson; Linderoth, Lars; Subramanian, Arun Kumar;

    2011-01-01

    an inverted ester. The latter were included to study head group-enzyme interactions. Our simulation results show that the lipids are optimally placed into the binding cleft and that water molecules can freely reach the active site through a well-defined pathway; both are indicative that these...... substrates are efficiently hydrolyzed, which is in good agreement with our experimental data. The phospholipid analogue with three alkyl side chains forms aggregates of different shapes with no well-defined sizes due to its cone-shape structure. Phosphatidylglycerol and phosphatidylcholine head groups...

  3. 细胞色素P4502D6基因多态性和药物相互作用%Genetic polymorghism of CYP2D6 and drug interactions

    李芹; 王睿

    2006-01-01

    细胞色素P4502D6(CYP2D6)是一种重要的P450系氧化代谢酶,参与多种重要药物的代谢.CYP2D6具有基因多态性,对药物的代谢呈现明显的个体差异.而且CYP2D6能被多种药物竞争性抑制和诱导,药物联用时易产生相互作用.本文从CYP2D6的基因多态性与代谢表型、底物竞争作用、代谢酶的诱导和抑制等方面,探讨CYP2D6基因多态性与药物相互作用的关系.

  4. 乳腺癌患者CYP2D6多态性与Endoxifen血药浓度的相关性分析%Association between CYP2D6 polymorphism and plasma concentration of Endoxifen in breast cancer patients

    杨汐; 罗波; 吴斌

    2013-01-01

    目的:研究乳腺癌术后服用他莫昔芬(TAM)辅助治疗绝经前、雌激素受体(ER)阳性患者CYP2D6基因多态性与TAM活性代谢产物Endoxifen血药浓度的相关性,及其与TAM不良反应发生的相关性.方法:57例乳腺癌术后经TAM辅助治疗的患者(绝经前、ER阳性),采用Tetra-primer ARMS PCR检测其CYP2D6基因型,采用LC-MS/MS测定所有患者体内TAM及其活性代谢产物Endoxifen的血药浓度;根据患者自述标记存在治疗不良反应的个体.结果:CYP2D6* 10/* 10型和*1/* 10型受试者体内Endoxifen的血药浓度值分别为(23.55±4.01)和(25.90±3.93) ng/mL,均显著低于*1/*1型受试者(34.82±5.95) ng/mL,差异有统计学意义,P<0.01;但*10/* 10型和*1/* 10型之间Endoxifen的浓度值差异无统计学意义,P>0.05.CYP2D6* 1/*1型组自述无任何不良反应的比例(22.2%)低于突变组(62.5%),且差异有统计学意义,P<0.05.结论:CYP2D6基因多态性与Endoxifen血药浓度的差异及TAM治疗不良反应的发生有关;根据CYP2D6基因型,可指导绝经前、ER阳性乳腺癌患者术后的TAM个体化用药.

  5. Active substrate integrated terahertz waveguide using periodic graphene stack

    Dong, Yanfei; Liu, Peiguo; Yu, Dingwang; Yi, Bo; Li, Gaosheng

    2015-11-01

    The transmission properties of a substrate integrated waveguide (SIW) based on periodic graphene stacks have been theoretically investigated in the terahertz (THz) region. The effects of the dielectric-graphene-dielectric structure of the stack on the propagation properties are shown to be significant and different from the conventional active SIW based on active components. By varying the graphene chemical potential, the cut-off frequency of the proposed waveguide can be dynamically tuned from 3 to 3.7 THz. Moreover, the tunable waveguide displays low leakage loss and single-mode propagation with -120 dB stop-band attenuation. These primary results are very promising for THz integration devices and SIW-based systems.

  6. Active substrate integrated terahertz waveguide using periodic graphene stack

    Yanfei Dong

    2015-11-01

    Full Text Available The transmission properties of a substrate integrated waveguide (SIW based on periodic graphene stacks have been theoretically investigated in the terahertz (THz region. The effects of the dielectric-graphene-dielectric structure of the stack on the propagation properties are shown to be significant and different from the conventional active SIW based on active components. By varying the graphene chemical potential, the cut-off frequency of the proposed waveguide can be dynamically tuned from 3 to 3.7 THz. Moreover, the tunable waveguide displays low leakage loss and single-mode propagation with −120 dB stop-band attenuation. These primary results are very promising for THz integration devices and SIW-based systems.

  7. Transient Response of Aerobic and Anoxic Activated Sludge Activities to Sudden Substrate Concentration Changes

    Sin, G.; Vanrolleghem, P.A.; Gernaey, Krist

    2004-01-01

    The state-of-the-art understanding of activated sludge processes as summarized in activated sludge models (ASMs) predicts an instantaneous increase in the biomass activity (which is measured, e.g., by the corresponding respiration rate OUR, NUR, etc.) under sudden substrate concentration changes....

  8. Optically active vibrational modes of PPV derivatives on textile substrate

    In this work, MEH-PPV and BDMO-PPV films were deposited by spin-coating on “dirty” textile substrates of canvas, nylon, canvas with resin, jeans and on glass and the temperature dependence of the optical properties of them was studied by photoluminescence and Raman (300 K) techniques. The temperature dependence of the energy, of the half line width at half height of the purely electronic peak, of the integrated PL intensity and of the Huang-Rhys factor, S=I(01)/I(00), were obtained directly from the PL spectrum. For an analysis of the vibrational modes involved, Raman measurements were performed on substrates with and without polymers deposited and the results compared with those found in the literature. The films of MEH-PPV and BDMO-PPV showed optical properties similar to those films deposited on other substrates such as glass, metals, etc. It was observed an inversion of the first vibrational band in relation to the purely electronic peak with increasing temperature in the films deposited on nylon and canvas. The vibrational modes obtained by Raman were used to compose the simulation of the PL line shape of BDMO-PPV films on canvas and nylon, using a model proposed by Lin [29]. - Highlights: ► MEH-PPV and BDMO-PPV films were deposited by spin-coating on dirty textile. ► Their properties were studied by photoluminescence and Raman techniques. ► We observed inversion of first vibrational band in relation to purely electronic peak. ► Optically active vibrational modes of PPV derivatives were studied.

  9. β1肾上腺素受体与CYP2D6基因多态性对美托洛尔抗高血压治疗的影响%Effectes of β1-adrenergic receptor and CYP2D6 gene polymorphism on metoprolol antihypertension therapy

    徐承华; 杨玉雯; 曹蘅

    2011-01-01

    AIM: To investigate effects of β1-adrenergic receptor and CYP2D6 gene polymorphism on metoprolol antihypertension therapy. METHODS: 120 cases of patients with essential hypertension were detected by β1 -adrenergic receptor and CYP2D6 gene polymorphism, 118 patients with Arg 389 allele in β1- adrenergic re-ceptor gene chosen from 120 essential hypertension patients were divided into three phenotype groups according to CYP2D6 gene mutations: the poor metabolism group(PM, 55 cases), intermediate metabolism group (IM, 36 cases)and extensive metabolism group(EM, 27 cases). The subjects of PM, IM and EM were administratedwith metoprolol for the same doseQOO mg/d), taking orally twice a day. Blood pressures, heart rates and side effects were observed during 4-week following-up. RESULTS: Blood pressure and heart rate in group PM, IM and EM were decreased, especially, obviously decreased in group PM, however, the incidence of side effect was significantly increased in group PM. CONCLUSION: The genetic polymorphism of β1- ad-renergic receptor and CYP2D6 was associated with the efficiency of metoprolol antihyperten-sion therapy.%目的:探讨β1肾上腺素受体与CYP2D6基因多态性对美托洛尔抗高血压治疗的影响.方法:将2008年10月至2009年10月在安徽省皖南地区收集的120例原发性高血压患者进行岛肾上腺素能受体(β1-AR)和代谢酶细胞色素2136(CYP2D6)基因多态性检测,将β1-AR 389位携带Arg的118例患者按照CYP2D6基因表型分为弱代谢组(PM组,55例)、中等代谢组(IM组,36例)和强代谢组(EM组,27例),给予相同剂量(100 mg/d)美托洛尔,均分2次口服降压治疗,共随访4周,观察血压、心率和不良反应等指标.结果:PM、IM、EM组血压和心率均下降,尤以PM组血压和心率降幅最大,但不良反应发生率明显增加.结论:β1肾上腺素受体和CYP2D6基因多态性与美托洛尔降压疗效有一定相关性.

  10. Genetic polymorphism analysis of cytochromeP450 CYP2D6 and CYP2C19 in Han and Uyghur breast cancer patients in Xinjiang%新疆汉族与维吾尔族乳腺癌CYP2D6和CYP2C19基因多态性差异研究

    2016-01-01

    目的 探讨新疆汉族与维吾尔族绝经前乳腺癌患者CYP2D6和CYP2C19基因频率分布及他莫昔芬代谢类型以指导临床合理用药.方法 选取2011-06-01-2013-12-01新疆医科大学附属肿瘤医院绝经前激素受体阳性乳腺癌患者中汉族125例和维吾尔族121例,对CYP450中常见突变位点利用TaqMan(R)-MGB技术进行基因检测并确定他莫昔芬代谢类型.结果 CYP2D6(* 1/* 10)、CYP2D6(* 10/* 10)及CYP2C19(* 1/*1)基因型在汉族、维吾尔族两组患者中表达差异有统计学意义,x2值分别为1.123、9.746和5.935,P值分别为0.029、0.002和0.015;而CYP2D6(* 1/*5)、CYP2D6(* 5/*5)、CYP2D6(* 5/* 10)、CYP2C19(* 3/*3)基因型在两组患者中均无表达,差异无统计学意义,P>0.05.两组中CYP2D6(* 1/*1)、CYP2C19(* 1/*2)、CYP2C19(* 2/*2)、CYP2C19(* 1/*3)和CYP2C19(* 2/*3)基因型差异无统计学意义,P>0.05.汉族患者他莫昔芬快、中、慢代谢型者比例分别为72.0%、24.0%和4.0%,维吾尔族分别为76.9%、17.4%和5.7%,P>0.05.结论 汉族、维吾尔族乳腺癌患者中CYP2C19*2、CYP2C19*3基因频率均差异无统计学意义;而CYP2D6* 10等位基因的频率差异有统计学意义;他莫昔芬的代谢类型均以快代谢类型为主,两组之间差异无统计学意义.

  11. Genetic polymorphism of CYP2D6 in Karnataka and AndhraPradesh population in India%CYP2D6在印度卡纳塔克邦和安得拉邦人群中的遗传多态性

    Benny K ABRAHAM, C ADITHAN; P USHA KIRAN; Mohammed ASAD; K KOUMARAVELOU

    2000-01-01

    AIM: To study the prevalence of cytochrome P-450 2D6 (CYP2D6) polymorphism in Karnataka ( KA ) and Andhra Pradesh (AP) population. METHODS: Two hundred and eleven healthy human volunteers participated in the study (100 from KA and 111 from AP). At bed time, after voiding their bladder, the volunteers ingested 30 mg of dextromethorphan hydrobromide (DM). Urine samples were collected for 8 h. DM and its metabolite dextrorphan (DT) were estimated in the urine using HPLC. The metabolic ratio (DM/DT) was used for phenotyping. RESULTS: The prevalence of poor metabolisers (PM) in KA is 4 % and AP is 1.8 %.CONCLUSION: The frequency of PM phenotype in South Indian population is in between the Western and Oriental population.

  12. Effects of CYP2D6*10 genetic polymorphism on the protection of metoprolol for the elder during perioperative period%CYP2D6*10遗传基因多态性对美托洛尔在围术期保护老年患者心脏功能的影响

    刘芳; 张燕; 先小纲

    2014-01-01

    目的:探讨CYP2D6*10等位基因C188T单核苷酸多态性与美托洛尔(metoprolol,METO)在老年患者围术期心脏保护疗效的关系.方法:筛选323名应用METO预防围术期心血管风险的老年患者,采用PCR-RFLP法分析CYP2D6*10基因C188>T位点的单核苷酸多态性.患者用药后进行围术期临床跟踪,观测并评价METO治疗围术期血流动力学紊乱发生事件及个体服药后3h肌钙蛋白的变化.结果:本研究人群中CYP2D6* 10外显子C188>T位点的C、T等位基因频率分别为48.76%和51.24%,CYP2D6* 10基因野生型纯合子C/C、杂合子C/T、突变型纯合子T/T基因型人群分布分别为24.76%、47.98%和27.24%,其频率分布均符合Hardy-Weinberg平衡定律.统计结果显示基因型与心血管术后均有中等强度关联.对心肌梗死风险因子的散点图按照基因多态性分布情况进行分组后分析发现,其心血管风险增高与CYP2D6* 10遗传多态性存在相关的基因剂量效应.结论:CYP2D6* 10基因多态性(C188>T)可能导致METO在老年患者围术期中的心脏保护功能增强,同时还可能带来心动过缓的副作用.CYP2D6* 10突变与METO疗效密切相关,可致患者用药后收缩压显著改变,临床应关注CYP2D6遗传多态性对METO疗效的影响.

  13. An Experimental and Theoretical Study on the Kinetic Isotope Effect of C2H6 and C2D6 Reaction with OH

    Khaled, Fethi

    2015-10-30

    We report experimental and theoretical results for the deuterated kinetic isotope effect (DKIE) of the reaction of OH with ethane (C2H6) and deuterated ethane (C2D6). The reactions were investigated behind reflected shock waves over 800–1350 K by monitoring OH radicals near 306.69 nm using laser absorption. In addition, high level CCSD(T)/cc-pV(T,Q)Z//MP2/cc-pVTZ quantum chemical and statistical rate theory calculations were performed which agreed very well with the experimental findings. The results reported herein provide the first experimental evidence that DKIE for alkanes asymptotes to a value of 1.4 at high temperatures.

  14. 基因芯片技术对中国人群细胞色素P450 2D6基因多态性的研究

    李芹; 王睿

    2005-01-01

    目的:探讨基因芯片技术在细胞色素P450 2D6基因分型中应用的可行性,研究中国人群CYP2D6的基因多态性。方法建立寡核苷酸芯片测定CYP2D6基因型的方法,测定288例中国健康志愿者CYP2D6基因分型,并使用直接测序法对结果进行验证。结果:288例中国健康志愿者中CYP2D6*2和CYP2D6*10的变异频率分别高达52.8%和53.5%。对部分样本采用直接测序的方法进行进一步确证,结果完全吻合。结论:基因芯片法适用于中国人群CYP2D6基因分型研究。

  15. Substrate integrated ferrite phase shifters and active frequency selective surfaces

    Cahill, B M

    2002-01-01

    There are two distinct parts to this thesis; the first investigates the use of ferrite tiles in the construction of printed phase shifting transmission lines, culminating in the design of two compact electromagnetic controlled beam steered patch and slot antenna arrays. The second part investigates the use of active frequency selective surfaces (AFSS), which are later used to cover a uPVC constructed enclosure. Field intensity measurements are taken from within the enclosure to determine the dynamic screening effectiveness. Trans Tech G-350 Ferrite is investigated to determine its application in printed microstrip and stripline phase shifting transmission lines. 50-Ohm transmission lines are constructed using the ferrite tile and interfaced to Rogers RT Duroid 5870 substrate. Scattering parameter measurements are made under the application of variable magnetic fields to the ferrite. Later, two types of planar microwave beam steering antennas are constructed. The first uses the ferrites integrated into the Dur...

  16. Study on association of polymorphism of CYP450 2D6 with head and neck cancer and treatment response in patients receiving neoadjuvant chemotherapy paclitaxel, cisplatin, 5fu (TPF followed by chemoradiation

    Divyesh Kumar

    2014-04-01

    Results: Patients with CYP 2D6 1 showed good response to the therapy given, while CYP 2D6 4 and 10 were poor responders. Conclusion: There is a strong association of polymorphs of CYP 2D6 with occurrence of head and neck cancer. Response to treatment (TPF--CT-RT is polymorph graded. Our study thus provides an insight in to the concept of and ldquo;Right therapy to the right patient and rdquo;. [Int J Res Med Sci 2014; 2(2.000: 585-591

  17. Substrate regulation of ascorbate transport activity in astrocytes

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-[14C]ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-[14C]ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-[3H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels

  18. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    Xu, Hao; Majmudar, Jaimeen D; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H; Carlson, Heather A; Showalter, Hollis D; Martin, Brent R; Amidon, Gordon L

    2015-09-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and

  19. Substrate-competitive activity-based profiling of ester prodrug activating enzymes

    Xu, Hao; Majmudar, Jaimeen D.; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H.; Carlson, Heather A.; Showalter, Hollis D.; Martin, Brent R.; Amidon, Gordon L.

    2015-01-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating pre-clinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a 4-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse, but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design and preclinical

  20. Anthelmintics Are Substrates and Activators of Nematode P Glycoprotein▿

    Kerboeuf, Dominique; Guégnard, Fabrice

    2011-01-01

    P glycoproteins (Pgp), members of the ABC transporter superfamily, play a major role in chemoresistance. In nematodes, Pgp are responsible for resistance to anthelmintics, suggesting that they are Pgp substrates, as they are in mammalian cells. However, their binding to nematode Pgp and the functional consequences of this interaction have not been investigated. Our study showed that levamisole and most of the macrocyclic lactones (MLs) are Pgp substrates in nematodes. Ivermectin, although a v...

  1. Substrate integrated ferrite phase shifters and active frequency selective surfaces

    There are two distinct parts to this thesis; the first investigates the use of ferrite tiles in the construction of printed phase shifting transmission lines, culminating in the design of two compact electromagnetic controlled beam steered patch and slot antenna arrays. The second part investigates the use of active frequency selective surfaces (AFSS), which are later used to cover a uPVC constructed enclosure. Field intensity measurements are taken from within the enclosure to determine the dynamic screening effectiveness. Trans Tech G-350 Ferrite is investigated to determine its application in printed microstrip and stripline phase shifting transmission lines. 50-Ohm transmission lines are constructed using the ferrite tile and interfaced to Rogers RT Duroid 5870 substrate. Scattering parameter measurements are made under the application of variable magnetic fields to the ferrite. Later, two types of planar microwave beam steering antennas are constructed. The first uses the ferrites integrated into the Duroid as microstrip lines with 3 patch antennas as the radiating elements. The second uses stripline transmission lines, with slot antennas as the radiating sources etched into the ground plane of the triplate. Beam steering is achieved by the application of an external electromagnet. An AFSS is constructed by the interposition of PIN diodes into a dipole FSS array. Transmission response measurements are then made for various angles of electromagnetic wave incidence. Two states of operation exist: when a current is passed through the diodes and when the diodes are switched off. These two states form a high pass and band stop space filter respectively. An enclosure covered with the AFSS is constructed and externally illuminated in the range 2.0 - 2.8GHz. A probe antenna inside the enclosure positioned at various locations through out the volume is used to establish the effective screening action of the AFSS in 3 dimensional space. (author)

  2. Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers

    Aarnoutse, Rob E; Kleinnijenhuis, Johanneke; Koopmans, Peter P; Touw, Daan J; Wieling, Jaap; Hekster, Yechiel A; Burger, David M

    2005-01-01

    OBJECTIVE: In the treatment of human immunodeficiency virus infection, the protease inhibitor ritonavir is used in a low dose (100 mg twice daily) to inhibit cytochrome P450 (CYP) 3A4 and thereby increase plasma concentrations of coadministered protease inhibitors. When applied in a therapeutic dose

  3. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  4. Analysis of SNP genetic polymorphism in CYP3A4 and CYP2D6 in Chinese Han population%中国汉族人群CYP2D6、CYP3A4 SNP位点基因多态性分析

    杨栋; 万立华; 涂政; 石屹; 胡兰

    2014-01-01

    目的:通过对药物代谢酶CYP2D6和CYP3A4的相关单核苷酸多态性(single nucleotide polymorphism,SNP)筛选检测,获得其在中国汉族人群中遗传多态性分布的相关数据.方法:筛选11个关于CYP2D6、CYP3A4基因的SNP位点,取192份中国汉族无关个体健康自愿者的血液样本获得基因组DNA,根据单碱基延伸技术通过GenomeLabTM SNPstream(R)基因分型系统进行SNP分型.结果:本次检测的11个SNP位点在研究人群中全部具有多态性(最小等位基因频率>0.01),其中7个SNP(RS28624811、RS28670611、RS9623531、RS5758589、RS3735451、RS2246709、RS2404955、RS4646440)位点的等位基因频率在此人群与白种人相比差异具有统计学意义(P<0.01).结论:汉族人群可能有继承特定的遗传信息,导致与其他人群具有不同药物代谢率.

  5. Do CVD grown graphene films have antibacterial activity on metallic substrates?

    Dellieu, Louis; Reckinger, Nicolas; Didembourg, Christian; Letesson, Jean-Jacques; Sarrazin, Michael; Deparis, Olivier; Matroule, Jean-Yves; Colomer, Jean-François

    2014-01-01

    Accurate assessment of the antibacterial activity of graphene requires consideration of both the graphene fabrication method and, for supported films, the properties of the substrate. Large-area graphene films produced by chemical vapor deposition were grown directly on copper substrates or transferred on a gold substrate and their effect on the viability and proliferation of the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli were assessed. The viability and the proliferation of both bacterial species were not affected when they were grown on a graphene film entirely covering the gold substrate, indicating that conductivity plays no role on bacterial viability and graphene has no antibacterial activity against S. aureus and E. coli. On the other hand, antibacterial activity was observed when graphene coated the copper substrates, resulting from the release of bactericidal cupric ions in inverse proportion to the graphene surface coverage.

  6. Effect of an antiretroviral regimen containing ritonavir boosted lopinavir on intestinal and hepatic CYP3A, CYP2D6 and P-glycoprotein in HIV-infected patients.

    Wyen, C.; Fuhr, U.; Frank, D.; Aarnoutse, R.E.; Klaassen, T.; Lazar, A.; Seeringer, A.; Doroshyenko, O.; Kirchheiner, J.C.; Abdulrazik, F.; Schmeisser, N.; Lehmann, C.; Hein, W.; Schomig, E.; Burger, D.M.; Fatkenheuer, G.; Jetter, A.

    2008-01-01

    This study aimed to quantify the inhibition of cytochrome P450 (CYP3A), CYP2D6, and P-glycoprotein in human immunodeficiency virus (HIV)-infected patients receiving an antiretroviral therapy (ART) containing ritonavir boosted lopinavir, and to identify factors influencing ritonavir and lopinavir pha

  7. Epidermal growth factor stimulates substrate-selective protein-tyrosine-phosphatase activity.

    Hernández-Sotomayor, S M; Arteaga, C L; Soler, C. (Carlos); Carpenter, G

    1993-01-01

    This study investigates the regulation of protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) activity by epidermal growth factor (EGF). Cytosol from EGF-treated A-431 human epidermoid carcinoma cells was used as a source of PTPase activity, and tyrosine-phosphorylated ErbB2, EGF receptor, phospholipase C-gamma 1, and the Ras GTPase-activating protein were used as substrates to monitor PTPase activity. EGF stimulated PTPase activity that was selective toward these substrates, as it dephosphory...

  8. Identification of transglutaminase 2 kinase substrates using a novel on-chip activity assay.

    Jung, Se-Hui; Kong, Deok-Hoon; Jeon, Hye-Yoon; Ji, Su-Hyun; Han, Eun-Taek; Park, Won Sun; Hong, Seok-Ho; Kim, Min-Soo; Kim, Young-Myeong; Ha, Kwon-Soo

    2016-08-15

    Transglutaminase 2 (TG2) is an enzyme that plays a critical role in a wide variety of cellular processes through its multifunctional activities. TG2 kinase has emerged as an important regulator of apoptosis, as well as of chromatin structure and function. However, systematic investigation of TG2 kinase substrates is limited due to a lack of a suitable TG2 kinase activity assays. Thus, we developed a novel on-chip TG2 kinase activity assay for quantitative determination of TG2 kinase activity and for screening TG2 kinase substrate proteins in a high-throughput manner. Quantitative TG2 kinase activity was determined by selective detection of substrate protein phosphorylation on the surface of well-type amine arrays. The limit of detection (LOD) of this assay was 4.34μg/ml. We successfully applied this new activity assay to the kinetic analysis of 27 TG2-related proteins for TG2 kinase activity in a high-throughput manner and determined Michaelis-Menten constants (Km) of these proteins. We used the Km values and cellular locations of the TG2-related proteins to construct a substrate affinity map for TG2 kinase. Therefore, this on-chip TG2 kinase activity assay has a strong potential for the systematic investigation of substrate proteins and will be helpful for studying new physiological functions. PMID:27040940

  9. 浙南地区女性人群CYP2D6及MDR1基因多态性对曲马多镇痛效果的影响

    马光泛; 陈千煌

    2013-01-01

      目的探讨CYP2D6*10等位基因及MDR1 C3435T基因多态性对浙南地区女性人群术后曲马多镇痛效果的影响.方法收集行子宫肌瘤剥除手术患者194例,手术结束前30 min肌肉注射曲马多,采用患者自控镇痛(PCA)模式进行术后镇痛.抽取外周血,采用聚合酶链反应-限制性条带长度多态性方法(PCR-RFLP),分析患者CYP2D6*10等位基因及MDR1 C3435T基因多态性,比较不同基因型患者之间曲马多的累积量,记录镇痛起效时间、持续时间、VAS及不良反应.结果 CYP2D6*10等位基因频率为52.84%,术后2、6、12h曲马多累积量及术后2、6hVAS的比较,m/m型明显高于w/w型和m/w型,差异有统计学意义(P0.05).结论在浙南地区女性人群中,CYP2D6*10等位基因对曲马多镇痛效果有显著影响,MDR1 C3435T等位基因对曲马多镇痛效果无显著影响.%Objective To investigate the association of CYP2D6*10 and MDR1 C3435T polymorphisms with analgesic ef-ficacy of tramadol in woman patients of Southern Zhejiang province. Methods One hundred and ninety four patients with hys-termyoma undergoing uterine myomatectomy from August 2010 to March 2012. Patients were given with loading dose of tramadol intramuscularly 30 minutes before the ending of surgery and received the postoperative patient-control ed analgesia (PCA). Samples of peripheral blood were col ected for DNA isolation. Polymorphisms of CYP2D6*10 and MDR1 C3435T were detected with PCR-RFLP analysis. The differences of tramadol consumption in the genotype groups were monitored and the starting time and duration of analgesia, VAS scores and adverse reaction were observed. Results The al elic frequency of CYP2D6 was 52.84%. The tramadol consumption at 2、6h and 12h after surgery and VAS at 2h and 6h after surgery in m/m type (n=50) were higher than those in w/w (n=39) and m/w (n=105) type (P<0.05). Variant al eles C3435T in MDR1 gene was seen in frequency of 38.14%. There were no significant

  10. Association of CYP2D6 and CYP2C19 polymorphisms and disease-free survival of Thai post-menopausal breast cancer patients who received adjuvant tamoxifen

    Chamnanphon M

    2013-05-01

    Full Text Available Montri Chamnanphon,1 Khunthong Pechatanan,2 Ekapob Sirachainan,3 Narumol Trachu,4 Wasun Chantratita,5 Ekawat Pasomsub,5 Wilai Noonpakdee,6 Insee Sensorn,1,7 Chonlaphat Sukasem11Division of Pharmacogenomics and Personalized Medicine, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 2Department of Medicine, Phramongkutklao College of Medicine, 3Division of Medical Oncology, Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 4Research Center, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 5Division of Virology, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 6Department of Biochemistry, Faculty of Science, Mahidol University, 7Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, ThailandPurpose: To investigate the impact of CYP2D6 and CYP2C19 polymorphisms in predicting tamoxifen efficacy and clinical outcomes in Thai breast cancer patients.Methods: Polymorphisms of CYP2D6 and CYP2C19 were genotyped by the AmpliChip™ CYP450 Test (Roche Molecular Diagnostics, Branchburg, NJ, USA for 57 patients, who were matched as recurrent versus nonrecurrent breast cancers (n = 33 versus n = 24, respectively, with a 5-year follow-up.Results: Based on the genotype data, five CYP2D6 predicted phenotype groups were identified in this study including homozygous extensive metabolizer (13 of 57, 22.80%, extensive/intermediate metabolizer (23 of 57, 40.40%, extensive/poor metabolizer (3 of 57, 5.30%, homozygous intermediate metabolizer (14 of 57, 24.50%, and intermediate/poor metabolizer (4 of 57, 7.00%, and three CYP2C19 genotype groups including homozygous extensive metabolizer (27 of 57, 47.40%, extensive/intermediate metabolizer (27 of 57, 47.40%, and homozygous poor metabolizer (3 of 57, 5.30%. The CYP2D6 variant alleles were *10 (52 of 114, 45.60%, *5 (5 of 114, 4.40%, *41 (2 of 114, 1.80%, *4 (1 of 114, 0

  11. Electrocatalytic Oxygen Evolution on Iridium Oxide: Uncovering Catalyst-Substrate Interactions and Active Iridium Oxide Species

    Reier, T.; Teschner, D; Lunkenbein, T.; Bergmann, A; Selve, S.; Kraehnert, R.; R. Schlögl; Strasser, P.

    2014-01-01

    The morphology, crystallinity, and chemical state of well-defined Ir oxide nanoscale thin-film catalysts prepared on Ti substrates at various calcination temperatures were investigated. Special emphasis was placed on the calcination temperature-dependent interaction between Ir oxide film and Ti substrate and its impact on the electrocatalytic oxygen evolution reaction (OER) activity. The Ir oxide films were characterized by scanning electron microscopy, transmission electron microscopy, scann...

  12. Hydrolysis of particulate substrate by activated sludge under aerobic, anoxic and anaerobic conditions

    Henze, Mogens; Mladenovski, C.

    1991-01-01

    An investigation of hydrolysis of particulate organic substrate by activated sludge has been made. Raw municipal wastewater was used as substrate. It was mixed with activated sludge from a high loaded activated sludge plant with pure oxygen aeration. During 4 days batch experiments under aerobic......, anoxic and anaerobic conditions, the hydrolysis was following through the production of ammonia. The hydrolysis rate of nitrogeneous compounds is significantly affected by the electron donor available. The rate is high under aerobic conditions, medium under anaerobic conditions and low under anoxic...... conditions. The ratio between the hydrolysis rates under aerobic and under anoxic conditions are very similar to the respiration rates measured as electron equivalents....

  13. VPO catalysts synthesized on substrates with modified activated carbons

    VPO catalysts were prepared on oxidized and unoxidized activated carbons differing in initial porous structure. Carbons were oxidized under relatively soft (30% H2O2, 200 deg. C) and hard (50% H2O2, 350 deg. C) conditions. Carbon modification was carried out hydrothermally in a traditional autoclave (HTT) or a microwave reactor (MWT). The synthesis was also carried out under hydrothermal (HTS or MWS) conditions. V2O5 and NH4VO3 were used as precursors. The samples are characterized by diversified porous structure at SBET = 732-1617 m2/g and Vpor = 0.44-0.90 cm3/g, as well as various degree of VPO crystallinity. Possibility of preparation of the VPO catalysts under ecologically appropriate conditions, i.e. in aqueous solutions, was shown.

  14. Spatial variability of microbial activity and substrate utilization patterns in top- and subsoils under European beech

    Niebuhr, Jana; Heinze, Stefanie; Mikutta, Robert; Mueller, Carsten W.; Preusser, Sebastian; Marschner, Bernd

    2014-05-01

    The role of subsoils in the global carbon cycle is poorly understood and probably underestimated. This is due to an incomplete understanding of processes and mechanisms that influence carbon storage and decomposition in deeper soil horizons. Microbial communities play an important role in these processes, as their presence, structure and function are crucial for the decomposition and/or stabilization of organic compounds. In this study, carried out in a European beech (Fagus sylvatica L.) forest on a podzolic Cambisol near Hannover, the spatial variability of microbial activity and substrate utilization patterns were investigated in the subsoil. For this purpose, samples were taken from regular grids at dm distances in three soil profiles of 1.85 m depth and 3.15 m length, totaling 192 soil samples. Activities of 9 extracellular enzymes of the C-, S-, P- and N-cycle were determined with a multi-substrate enzymatic assay and for substrate utilization patterns the MicroRespTM method was applied. The results showed a strong decline of microbial activity from topsoil to subsoil. Enzyme activities varied greatly at the dm scale. The correlation of the variability of both microbial activity and substrate utilization patterns with depth and soil parameters such as pH, soil water content, total and dissolved organic carbon was tested with a principal component analysis. Existing dependencies of the variabilities on these parameters help to verify the hypotheses that microbial activity is spatially highly variable in the subsoil and this variability is due to the existence of certain hot spots of substrate availability and that outside these 'hot spots' the microbial activity and thus the decomposition of SOM are mainly limited by substrate availability.

  15. Sonochemically synthesized Ag nanoparticles as a SERS active substrate and effect of surfactant

    Highlights: • Solid state Ag SERS active substrates were sonochemically synthesized. • High intensity SERS spectra of both crystal violet and rhodamine 6G were observed. • We discovered that PVP aided synthesized substrates showed higher SERS intensity. - Abstract: Surface enhanced Raman scattering (SERS) enables the detection of substances at low concentrations using silver or gold nanostructure. The SERS technique has many applications, such as environmental detection and biosensing. Sonochemistry is an excellent and cheap deposition technique for coating substrates in a form of nanostructure at ambient temperature. It can also be utilized to prepare large SERS substrates. Here, we used the advantages of sonochemistry to deposit solid SERS substrates immobilized on GaN nanostructure. Morphology was studied by scanning electron microscopy. The elemental composition and the spatial distribution were examined by energy dispersive X-ray spectroscopy. The crystal structure and atomic presence was confirmed by X-ray diffraction. SERS substrates were examined with the analytes crystal violet (10−5 M) and rhodamine 6G (10−6 M), they showed prominent characteristic peaks. We discovered that the SERS intensity of poly-vinyl-pyrrolidinone aided sonochemical deposition of Ag nanoparticles was increased. The reason for the effect is morphological changes of the Ag nanoparticles. Smaller nanoparticles were fabricated, which increase their SERS intensity

  16. An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities.

    Moss, Marcia L; Minond, Dmitriy; Yoneyama, Toshie; Hansen, Hinrich P; Vujanovic, Nikola; Rasmussen, Fred H

    2016-08-15

    A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.3) × 10(4) M(-1) s(-1) and 2.4 (±0.3) × 10(3) M(-1) s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities. PMID:27177841

  17. Microbial decomposition in aquatic environments: combined process of extracellular enzyme activity and substrate uptake

    The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with [3H] leucine as a marker for the leucine pool and L-leucine-4-methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. Calculated on an annual average basis, turnover rates were ca. nine times higher than hydrolysis rates at the polluted station and ca., five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station

  18. Pharmacokinetic Interactions between Monoamine Oxidase A Inhibitor Harmaline and 5-Methoxy-N,N-Dimethyltryptamine, and the Impact of CYP2D6 Status

    Jiang, Xi-Ling; Shen, Hong-Wu; Mager, Donald E.; Yu, Ai-Ming

    2013-01-01

    5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT or street name “5-MEO”) is a newer designer drug belonging to a group of naturally occurring indolealkylamines. Our recent study has demonstrated that coadministration of monoamine oxidase A (MAO-A) inhibitor harmaline (5 mg/kg) increases systemic exposure to 5-MeO-DMT (2 mg/kg) and active metabolite bufotenine. This study is aimed at delineating harmaline and 5-MeO-DMT pharmacokinetic (PK) interactions at multiple dose levels, as well as the impact...

  19. Alternative nucleophilic substrates for the endonuclease activities of human immunodeficiency virus type 1 integrase

    Ealy, Julie B. [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Department of Chemistry, Penn State Lehigh Valley, 2809 E. Saucon Valley Road, Center Valley, PA 18034 (United States); Sudol, Malgorzata [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Krzeminski, Jacek; Amin, Shantu [Department of Pharmacology, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033 (United States); Katzman, Michael, E-mail: mkatzman@psu.edu [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Department of Microbiology and Immunology, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033 (United States)

    2012-11-10

    Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase.

  20. Alternative nucleophilic substrates for the endonuclease activities of human immunodeficiency virus type 1 integrase

    Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase.

  1. Activated carbon addition affects substrate pH and germination of six plant species

    Kabouw, P.; Nab, M.; Dam, van M.

    2010-01-01

    Activated carbon (AC) is widely used in ecological studies for neutralizing allelopathic compounds. However, it has been suggested that AC has direct effects on plants because it alters substrate parameters such as nutrient availability and pH. These side-effects of AC addition may interfere with al

  2. Spatiotemporal stability of neonatal rat cardiomyocyte monolayers spontaneous activity is dependent on the culture substrate.

    Jonathan Boudreau-Béland

    Full Text Available In native conditions, cardiac cells must continuously comply with diverse stimuli necessitating a perpetual adaptation. Polydimethylsiloxane (PDMS is commonly used in cell culture to study cellular response to changes in the mechanical environment. The aim of this study was to evaluate the impact of using PDMS substrates on the properties of spontaneous activity of cardiomyocyte monolayer cultures. We compared PDMS to the gold standard normally used in culture: a glass substrate. Although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 and increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol and acetylcholine. These results reveal the importance of carefully selecting the culture substrate for studies involving mechanical stimulation, especially for tissue engineering or pharmacological high-throughput screening of cardiac tissue analog.

  3. A Simple Method for the Determination of Xylanase Activity on Insoluble Substrates

    The propensity for a xylanase to convert insoluble (arabino)xylan into soluble oligosaccharides is an important parameter in the baking, pulp and paper, prebiotics, and biofuel industries. Current methods for determining xylanase activity on insoluble substrates are labor intensive, non-specific, or...

  4. Enzyme activities and substrate degradation by fungal isolates on cassava waste during solid state fermentation.

    Pothiraj, C; Eyini, M

    2007-12-01

    The growth and bioconversion potential of selected strains growing on cassava waste substrate during solid state fermentation were assessed. Rhizopus stolonifer showed the highest and the fastest utilization of starch and cellulose in the cassava waste substrate. It showed 70% starch utilization and 81% cellulose utilization within eight days. The release of reducing sugars indicating the substrate saccharification or degradation potential of the organisms reached the highest value of 406.5 mg/g by R. stolonifer on cassava waste during the eighth day of fermentation. The protein content was gradually increased (89.4 mg/g) on the eighth day of fermentation in cassava waste by R. stolonifer. The cellulase and amylase activity is higher in R. stolonifer than A. niger and P. chrysosporium. The molecular mass of purified amylase and cellulase seemed to be 75 KDal, 85 KDal respectively. PMID:24015097

  5. Preemptive Pharmacogenomic Testing for Precision Medicine: A Comprehensive Analysis of Five Actionable Pharmacogenomic Genes Using Next-Generation DNA Sequencing and a Customized CYP2D6 Genotyping Cascade.

    Ji, Yuan; Skierka, Jennifer M; Blommel, Joseph H; Moore, Brenda E; VanCuyk, Douglas L; Bruflat, Jamie K; Peterson, Lisa M; Veldhuizen, Tamra L; Fadra, Numrah; Peterson, Sandra E; Lagerstedt, Susan A; Train, Laura J; Baudhuin, Linnea M; Klee, Eric W; Ferber, Matthew J; Bielinski, Suzette J; Caraballo, Pedro J; Weinshilboum, Richard M; Black, John L

    2016-05-01

    Significant barriers, such as lack of professional guidelines, specialized training for interpretation of pharmacogenomics (PGx) data, and insufficient evidence to support clinical utility, prevent preemptive PGx testing from being widely clinically implemented. The current study, as a pilot project for the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol, was designed to evaluate the impact of preemptive PGx and to optimize the workflow in the clinic setting. We used an 84-gene next-generation sequencing panel that included SLCO1B1, CYP2C19, CYP2C9, and VKORC1 together with a custom-designed CYP2D6 testing cascade to genotype the 1013 subjects in laboratories approved by the Clinical Laboratory Improvement Act. Actionable PGx variants were placed in patient's electronic medical records where integrated clinical decision support rules alert providers when a relevant medication is ordered. The fraction of this cohort carrying actionable PGx variant(s) in individual genes ranged from 30% (SLCO1B1) to 79% (CYP2D6). When considering all five genes together, 99% of the subjects carried an actionable PGx variant(s) in at least one gene. Our study provides evidence in favor of preemptive PGx testing by identifying the risk of a variant being present in the population we studied. PMID:26947514

  6. Paraoxonase-1 activity determination via paraoxon substrate yields no significant difference in mild hyperhomocysteinemia.

    Türkeli, Hatice; Caycı, Tuncer; Akgül, Emin Özgür; Macit, Enis; Yaman, Halil; Aydın, Ibrahim; Demirin, Hilmi; Alacam, Hasan; Ozkan, Esin; Cakır, Erdinç; Deren, Ozgür; Erbil, Mehmet Kemal; Kunak, Z Ilker; Burat, Kutlay; Akman, Serif

    2010-11-01

    Elevated plasma homocystein (Hcy) level has been recognized as an important risk factor for a number of cardiovascular diseases, peripheral arterial occlusive disease and venous thrombosis. A part of Hcy in the organism is turned to homocysteine thiolactone (HcyT) via a ring closure reaction, which gains rate in hyperhomocysteinemia, and in turn undergoes a hydrolytic reaction back to Hcy by paraoxonase enzyme (PON). Since this is a protective reflex action enzyme against hyperhomocysteinemia, we investigated how a mild hyperhomocysteinemic nutritional habit affected serum PON activity in a population-based study. The difference detected via enzymatic activity using the paraoxon substrate was statistically non-significant (p=0.19), suggesting a defective performance to reflect the expected significance. Determination of serum PON activity via substrate paraoxon yielded no significant difference in an acute mild hyperhomocysteinemic diet model in humans. PMID:19419786

  7. 细胞色素CYP2D6基因多态性与海洛因海绵状白质脑病易感性的关系%Association of cytochrome P4502D6 gene polymorphism with the susceptibility of heroin spongiform leucoencephalopathy

    周亮; 陆兵勋; 尹恝

    2010-01-01

    目的 探讨细胞色素CYP2D6基因多态性与海洛因海绵状白质脑病(HSLE)易感性的关系.方法 通过PCR-RFLP技术.分析HSLE患者与吸毒及正常对照基因多态性.结果 HSLE病人CYP2D6/C188、CYP2D6/L2938、CYP2D6/G4268基因突变率均高于正常人.结论 解毒酶CYP2D6基因缺陷与HSLE发病有一定的关系.

  8. Possibility of formic and acetic acids as active substrates for methanogenesis in the groundwater in Horonobe, Hokkaido

    Groundwater samples in Horonobe district, Hokkaido, were analyzed to evaluate the possibility that formic and acetic acids are active substrates for methanogens in Quaternary and Neogene (Koetoi formation) formations. ΔGr corresponding to CH4-producing reactions indicates that both acids could be active substrates in almost all sampling locations. However, acetic acid was recognized to be an active substrate only in the Koetoi formation on the basis of the principle of competitive exclusion (CE) of microorganisms. The limited possibility by the CE principle suggests that dynamic equilibrium between substrate production rates and consumption rates is established only in the Koetoi formation for acetic acid. (author)

  9. Greatly Enhancing Catalytic Activity of Graphene by Doping the Underlying Metal Substrate.

    Guo, Na; Xi, Yongjie; Liu, Shuanglong; Zhang, Chun

    2015-01-01

    Graphene-based solid-state catalysis represents a new direction in applications of graphene and has attracted a lot of interests recently. However, the difficulty in fine control and large-scale production of previously proposed graphene catalysts greatly limits their industrial applications. Here we present a novel way to enhance the catalytic activity of graphene, which is highly efficient yet easy to fabricate and control. By first-principles calculations, we show that when the underlying metal substrate is doped with impurities, the catalytic activity of the supported graphene can be drastically enhanced. Graphene supported on a Fe/Ni(111) surface is chosen as a model catalyst, and the chemical reaction of CO oxidation is used to probe the catalytic activity of graphene. When the underlying Fe/Ni(111) substrate is impurity free, the graphene is catalytically inactive. When a Zn atom is doped into the substrate, the catalytic activity of the supported graphene is greatly enhanced, and the reaction barrier of the catalyzed CO oxidation is reduced to less than 0.5 eV. Intriguing reaction mechanism of catalyzed CO oxidation is revealed. These studies suggest a new class of graphene-based catalysts and pave the way for future applications of graphene in solid-state catalysis. PMID:26156332

  10. Gold Incorporated Mesoporous Silica Thin Film Model Surface as a Robust SERS and Catalytically Active Substrate

    Anandakumari Chandrasekharan Sunil Sekhar

    2016-05-01

    Full Text Available Ultra-small gold nanoparticles incorporated in mesoporous silica thin films with accessible pore channels perpendicular to the substrate are prepared by a modified sol-gel method. The simple and easy spin coating technique is applied here to make homogeneous thin films. The surface characterization using FESEM shows crack-free films with a perpendicular pore arrangement. The applicability of these thin films as catalysts as well as a robust SERS active substrate for model catalysis study is tested. Compared to bare silica film our gold incorporated silica, GSM-23F gave an enhancement factor of 103 for RhB with a laser source 633 nm. The reduction reaction of p-nitrophenol with sodium borohydride from our thin films shows a decrease in peak intensity corresponding to –NO2 group as time proceeds, confirming the catalytic activity. Such model surfaces can potentially bridge the material gap between a real catalytic system and surface science studies.

  11. Gold Incorporated Mesoporous Silica Thin Film Model Surface as a Robust SERS and Catalytically Active Substrate.

    Sunil Sekhar, Anandakumari Chandrasekharan; Vinod, Chathakudath Prabhakaran

    2016-01-01

    Ultra-small gold nanoparticles incorporated in mesoporous silica thin films with accessible pore channels perpendicular to the substrate are prepared by a modified sol-gel method. The simple and easy spin coating technique is applied here to make homogeneous thin films. The surface characterization using FESEM shows crack-free films with a perpendicular pore arrangement. The applicability of these thin films as catalysts as well as a robust SERS active substrate for model catalysis study is tested. Compared to bare silica film our gold incorporated silica, GSM-23F gave an enhancement factor of 10³ for RhB with a laser source 633 nm. The reduction reaction of p-nitrophenol with sodium borohydride from our thin films shows a decrease in peak intensity corresponding to -NO₂ group as time proceeds, confirming the catalytic activity. Such model surfaces can potentially bridge the material gap between a real catalytic system and surface science studies. PMID:27213321

  12. Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

    Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G.; Achilefu, Samuel

    2013-04-01

    Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

  13. Substrates and enzyme activities related to biotransformation of resveratrol from phenylalanine by Alternaria sp. MG1.

    Zhang, Jinhua; Shi, Junling; Liu, Yanlin

    2013-12-01

    To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO₄, and 0.2 g/L CaSO₄ and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 μg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously. PMID:24068334

  14. 2,4-Dichlorophenol hydroxylase for chlorophenol removal: Substrate specificity and catalytic activity.

    Ren, Hejun; Li, Qingchao; Zhan, Yang; Fang, Xuexun; Yu, Dahai

    2016-01-01

    Chlorophenols (CPs) are common environmental pollutants. As such, different treatments have been assessed to facilitate their removal. In this study, 2,4-dichlorophenol (2,4-DCP) hydroxylase was used to systematically investigate the activity and removal ability of 19CP congeners at 25 and 0 °C. Results demonstrated that 2,4-DCP hydroxylase exhibited a broad substrate specificity to CPs. The activities of 2,4-DCP hydroxylase against specific CP congeners, including 3-CP, 2,3,6-trichlorophenol, 2-CP, and 2,3-DCP, were higher than those against 2,4-DCP, which is the preferred substrate of previously reported 2,4-DCP hydroxylase. To verify whether cofactors are necessary to promote hydroxylase activity against CP congeners, we added FAD and found that the added FAD induced a 1.33-fold to 5.13-fold significant increase in hydroxylase activity against different CP congeners. The metabolic pathways of the CP degradation in the enzymatic hydroxylation step were preliminarily proposed on the basis of the analyses of the enzymatic activities against 19CP congeners. We found that the high activity and removal rate of 2,4-DCP hydroxylase against CPs at 0 °C enhance the low-temperature-adaptability of this enzyme to the CP congeners; as such, the proposed removal process may be applied to biochemical, bioremediation, and industrial processes, particularly in cold environments. PMID:26672451

  15. Substrate sources regulate spatial variation of metabolically active methanogens from two contrasting freshwater wetlands.

    Lin, Yongxin; Liu, Deyan; Ding, Weixin; Kang, Hojeong; Freeman, Chris; Yuan, Junji; Xiang, Jian

    2015-12-01

    There is ample evidence that methane (CH4) emissions from natural wetlands exhibit large spatial variations at a field scale. However, little is known about the metabolically active methanogens mediating these differences. We explored the spatial patterns in active methanogens of summer inundated Calamagrostis angustifolia marsh with low CH4 emissions and permanently inundated Carex lasiocarpa marsh with high CH4 emissions in Sanjiang Plain, China. In C. angustifolia marsh, the addition of (13)C-acetate significantly increased the CH4 production rate, and Methanosarcinaceae methanogens were found to participate in the consumption of acetate. In C. lasiocarpa marsh, there was no apparent increase in the CH4 production rate and no methanogen species were labeled with (13)C. When (13)CO2-H2 was added, however, CH4 production was found to be due to Fen Cluster (Methanomicrobiales) in C. angustifolia marsh and Methanobacterium Cluster B (Methanobacteriaceae) together with Fen Cluster in C. lasiocarpa marsh. These results suggested that CH4 was produced primarily by hydrogenotrophic methanogens using substrates mainly derived from plant litter in C. lasiocarpa marsh and by both hydrogenotrophic and acetoclastic methanogens using substrates mainly derived from root exudate in C. angustifolia marsh. The significantly lower CH4 emissions measured in situ in C. angustifolia marsh was primarily due to a deficiency of substrates compared to C. lasiocarpa marsh. Therefore, we speculate that the substrate source regulates both the type of active methanogens and the CH4 production pathway and consequently contributes to the spatial variations in CH4 productions observed in these freshwater marshes. PMID:26286511

  16. Mutants of Micromonospora viridifaciens sialidase have highly variable activities on natural and non-natural substrates

    Jers, Carsten; Guo, Yao; Kepp, Kasper Planeta;

    2015-01-01

    subjected to site-saturation mutagenesis and evaluated on the artificial sialidase substrate 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid and on the natural substrate casein glycomacropeptide. A considerably higher fraction of the mutants exhibited increased activity on the artificial substrate compared...... with the natural one, with the most proficient mutant showing a 13-fold improvement in kcat/Km. In contrast, no mutants displayed more than a 2-fold increase in activity on the natural substrate. To gain further insight into this important discrepancy, we analysed the stability of mutants using the Po....... Together with the minor improvement on the natural substrate this shows an important difference between naturally evolved functionality and new laboratory functionality. Our results suggest that when engineering sialidases and potentially other proteins towards non-natural substrates that are not optimized...

  17. Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate

    Wei-Lien Chuang; Joshua Pacheco; Samantha Cooper; Kingsbury, Jonathan S.; John Hinds; Pavlina Wolf; Petra Oliva; Joan Keutzer; Cox, Gerald F.; Kate Zhang

    2015-01-01

    Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann–Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann–Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the...

  18. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200.

    Kovaleva, Elena G; Rogers, Melanie S; Lipscomb, John D

    2015-09-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady-state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures determined at 1.35-1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild-type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second-sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in the steric bulk and charge of the residue at position 200 appear to be capable of altering the rate-limiting step in catalysis and, perhaps, the nature of the reactive species. PMID:26267790

  19. Enhancing antibacterial activity of surface-grafted chitosan with immobilized lysozyme on bioinspired stainless steel substrates.

    Yuan, Shaojun; Yin, Jia; Jiang, Wei; Liang, Bin; Pehkonen, S O; Choong, Cleo

    2013-06-01

    Bacterial infections have been widely recognized as a major cause of the failure of medical implants and devices. One promising strategy to reduce the incidence of infections is to impart the material surfaces with bactericidal function for inhibiting bacterial adhesion and biofilm formation. In this study, stainless steel (SS) surface was first activated by a biomimetic dopamine anchor to provide active amino groups, followed by covalently immobilizing chitosan (CS) with glutaraldehyde (GA) as a bifunctional linker. Hen egg white lysozyme, a natural defensive enzyme, was finally conjugated to the grafted chitosan to enhance biocidal functionality. The antibacterial assay results demonstrated substantial enhancement in bactericidal efficiency against Staphylococcus aureus (S. aureus) on the lysozyme-immobilized SS substrates under the neutral pH conditions as compared to the chitosan-grafted SS substrates. With the inherent advantages of robust anchoring ability of dopamine and specific functionality of lysozyme, the metallic substrates can be readily tailored with antibacterial property to combat biomaterial-centered infection for potential biomedical applications. PMID:23434686

  20. A highly active SERS sensing substrate: core–satellite assembly of gold nanorods/nanoplates

    Regiospecific core–satellite assembly of gold nanoplates (AuNPs)/gold nanorods (AuNRs) can be fabricated via ss-DNA hybridization. SERS behavior of the DNA driven assembly has been explored from inducing transition between para-ATP and DMAB through plasmon-assisted catalysis, suggesting that the core–satellite assembly can be utilized as highly active optical substrate. Moreover, a Raman label tagged thymine-rich DNA functionalized AuNRs/AuNPs assembly can be employed as in situ SERS sensing of mercury ions at the ultrasensitive ppt level, which indicates that the core–satellite assembly is appropriate as a versatile SERS substrate for the application of optical chemical or biosensing. (paper)

  1. High performance organic transistor active-matrix driver developed on paper substrate

    Peng, Boyu; Ren, Xiaochen; Wang, Zongrong; Wang, Xinyu; Roberts, Robert C.; Chan, Paddy K. L.

    2014-09-01

    The fabrication of electronic circuits on unconventional substrates largely broadens their application areas. For example, green electronics achieved through utilization of biodegradable or recyclable substrates, can mitigate the solid waste problems that arise at the end of their lifespan. Here, we combine screen-printing, high precision laser drilling and thermal evaporation, to fabricate organic field effect transistor (OFET) active-matrix (AM) arrays onto standard printer paper. The devices show a mobility and on/off ratio as high as 0.56 cm2V-1s-1 and 109 respectively. Small electrode overlap gives rise to a cut-off frequency of 39 kHz, which supports that our AM array is suitable for novel practical applications. We demonstrate an 8 × 8 AM light emitting diode (LED) driver with programmable scanning and information display functions. The AM array structure has excellent potential for scaling up.

  2. Magnetic activity of surface plasmon resonance using dielectric magnetic materials fabricated on quartz glass substrate

    Narushima, Kazuki; Ashizawa, Yoshito; Brachwitz, Kerstin; Hochmuth, Holger; Lorenz, Michael; Grundmann, Marius; Nakagawa, Katsuji

    2016-07-01

    The magnetic activity of surface plasmons in Au/MFe2O4 (M = Ni, Co, and Zn) polycrystalline bilayer films fabricated on a quartz glass substrate was studied for future magnetic sensor applications using surface plasmon resonance. The excitation of surface plasmons and their magnetic activity were observed in all investigated Au/MFe2O4 films. The magnetic activity of surface plasmons of the polycrystalline Au/NiFe2O4 film was larger than those of the other polycrystalline Au/MFe2O4 films, the epitaxial NiFe2O4 film, and metallic films. The large magnetic activity of surface plasmons of the polycrystalline film is controlled by manipulating surface plasmon excitation conditions and magnetic properties.

  3. Improvement of thermostability and activity of Trichoderma reesei endo-xylanase Xyn III on insoluble substrates.

    Matsuzawa, Tomohiko; Kaneko, Satoshi; Yaoi, Katsuro

    2016-09-01

    Trichoderma reesei Xyn III, an endo-β-1,4-xylanase belonging to glycoside hydrolase family 10 (GH10), is vital for the saccharification of xylans in plant biomass. However, its enzymatic thermostability and hydrolytic activity on insoluble substrates are low. To overcome these difficulties, the thermostability of Xyn III was improved using random mutagenesis and directed evolution, and its hydrolytic activity on insoluble substrates was improved by creating a chimeric protein. In the screening of thermostable Xyn III mutants from a random mutagenesis library, we identified two amino acid residues, Gln286 and Asn340, which are important for the thermostability of Xyn III. The Xyn III Gln286Ala/Asn340Tyr mutant showed xylanase activity even after heat treatment at 60 °C for 30 min or 50 °C for 96 h, indicating a dramatic enhancement in thermostability. In addition, we found that the addition of a xylan-binding domain (XBD) to the C-terminal of Xyn III improved its hydrolytic activity on insoluble xylan. PMID:27138202

  4. Profiling of Substrates for Zinc‐dependent Lysine Deacylase Enzymes: HDAC3 Exhibits Decrotonylase Activity In Vitro

    Madsen, Andreas Stahl; Olsen, Christian Adam

    2012-01-01

    Systematic screening of the activities of the eleven human zinc-dependent lysine deacylases against a series of fluorogenic substrates (see scheme) as well as kinetic evaluation revealed substrates for screenings of histone deacetylases HDAC10 and HDAC11 at reasonably low enzyme concentrations...

  5. Chitosan based substrates for wound infection detection based on increased lysozyme activity.

    Tegl, Gregor; Rollett, Alexandra; Dopplinger, Jasmin; Gamerith, Clemens; Guebitz, Georg M

    2016-10-20

    There is a strong need of point-of-care diagnostics for early detection of wound infection. In this study, substrates based on functionalized chitosan were developed for visual detection of elevated lysozyme activity, an infection biomarker in wound fluids. For efficient hydrolysis by lysozyme, N-acetyl chitosan with a final degree of acetylation of around 50% was synthesized. N-acetylated chitosan and a chitosan-starch composite were labeled with structurally different dyes resulting in lysozyme-responsive biomaterials. Incubation with lysozyme in buffer and artificial wound fluid lead to a release of colored hydrolysis products already after 2h incubation. Tests in human wound fluid from infected wounds indicated a clear visual color change after 2.5h compared to control samples. A higher degree of swelling of the chitosan/starch containing substrate led to faster hydrolysis by lysozyme. This study demonstrates the potential of the lysozyme-responsive materials for diagnosis of wound infection and provides different diagnostic substrates for potential incorporation in point-of-care devices. PMID:27474566

  6. Active vacuum brazing of CNT films to metal substrates for superior electron field emission performance

    The joining of macroscopic films of vertically aligned multiwalled carbon nanotubes (CNTs) to titanium substrates is demonstrated by active vacuum brazing at 820 °C with a Ag–Cu–Ti alloy and at 880 °C with a Cu–Sn–Ti–Zr alloy. The brazing methodology was elaborated in order to enable the production of highly electrically and thermally conductive CNT/metal substrate contacts. The interfacial electrical resistances of the joints were measured to be as low as 0.35 Ω. The improved interfacial transport properties in the brazed films lead to superior electron field-emission properties when compared to the as-grown films. An emission current of 150 μA was drawn from the brazed nanotubes at an applied electric field of 0.6 V μm−1. The improvement in electron field-emission is mainly attributed to the reduction of the contact resistance between the nanotubes and the substrate. The joints have high re-melting temperatures up to the solidus temperatures of the alloys; far greater than what is achievable with standard solders, thus expanding the application potential of CNT films to high-current and high-power applications where substantial frictional or resistive heating is expected. (paper)

  7. The ClpP N-Terminus Coordinates Substrate Access with Protease Active Site Reactivity

    Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpP?N exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a 'gate' controlling substrate access to the active sites, (2) binding of ClpA opens this 'gate', allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal 'gate' stimulates acyl-enzyme hydrolysis.

  8. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Domanova, Westa; Krycer, James; Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  9. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies

    Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  10. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Westa Domanova

    Full Text Available In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds, making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same

  11. Transparent conductive indium oxide film deposited on low temperature substrates by activated reactive evaporation.

    Marcovitch, O; Klein, Z; Lubezky, I

    1989-07-15

    High quality conductive coatings for the visible region were prepared on low temperature glass substrates. The conductive layer was an indium oxide film deposited by the activated reactive evaporation technique using a glow discharge hollow cathode ion gun. An antireflective layer of MgF(2) was deposited over the conductive layer. The average transmission in the visible region of the coated glass with sheet resistance of coating was durable and passed a series of environmental tests according to MIL-C-675C. PMID:20555600

  12. Transparent conductive indium oxide film deposited on low temperature substrates by activated reactive evaporation

    High quality conductive coatings for the visible region were prepared on low temperature glass substrates. The conductive layer was an indium oxide film deposited by the activated reactive evaporation technique using a glow discharge hollow cathode ion gun. An antireflective layer of MgFz was deposited over the conductive layer. The average transmission in the visible region of the coated glass with sheet resistance of 15 Ω/sq was greater than 90%. The coating was durable and passed a series of environmental tests according to MIL-C-675C

  13. Substrate structure-activity relationships guide rational engineering of modular polyketide synthase ketoreductases.

    Bailey, Constance B; Pasman, Marjolein E; Keatinge-Clay, Adrian T

    2016-01-14

    Modular polyketide synthase ketoreductases can set two chiral centers through a single reduction. To probe the basis of stereocontrol, a structure-activity relationship study was performed with three α-methyl, β-ketothioester substrates and four ketoreductases. Since interactions with the β-ketoacyl moiety were found to be most critical, residues implicated in contacting this moiety were mutated. Two mutations were sufficient to completely reverse the stereoselectivity of the model ketoreductase EryKR1, converting it from an enzyme that generates (2S,3R)-products into one that yields (2S,3S)-products. PMID:26568113

  14. Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate.

    Chuang, Wei-Lien; Pacheco, Joshua; Cooper, Samantha; Kingsbury, Jonathan S; Hinds, John; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Cox, Gerald F; Zhang, Kate

    2015-06-01

    Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann-Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann-Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls. PMID:26937397

  15. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  16. Neural substrates underlying the passive observation and active control of translational egomotion.

    Huang, Ruey-Song; Chen, Ching-Fu; Sereno, Martin I

    2015-03-11

    Moving or static obstacles often get in the way while walking in daily life. Avoiding obstacles involves both perceptual processing of motion information and controlling appropriate defensive movements. Several higher-level motion areas, including the ventral intraparietal area (VIP), medial superior temporal area, parieto-insular vestibular cortex (PIVC), areas V6 and V6A, and cingulate sulcus visual area, have been identified in humans by passive viewing of optic flow patterns that simulate egomotion and object motion. However, the roles of these areas in the active control of egomotion in the real world remain unclear. Here, we used functional magnetic resonance imaging (fMRI) to map the neural substrates underlying the passive observation and active control of translational egomotion in humans. A wide-field virtual reality environment simulated a daily scenario where doors randomly swing outward while walking in a hallway. The stimuli of door-dodging events were essentially the same in two event-related fMRI experiments, which compared passive and active dodges in response to swinging doors. Passive dodges were controlled by a computer program, while active dodges were controlled by the subject. Passive dodges activated several higher-level areas distributed across three dorsal motion streams in the temporal, parietal, and cingulate cortex. Active dodges most strongly activated the temporal-vestibular stream, with peak activation located in the right PIVC. Other higher-level motion areas including VIP showed weaker to no activation in active dodges. These results suggest that PIVC plays an active role in sensing and guiding translational egomotion that moves an observer aside from impending obstacles. PMID:25762672

  17. Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta

    The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Bronsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is in flight during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism

  18. Evaluation of Proposed In Vivo Probe Substrates and Inhibitors for Phenotyping Transporter Activity in Humans.

    Momper, Jeremiah D; Tsunoda, Shirley M; Ma, Joseph D

    2016-07-01

    Drug transporters are present in various tissues and have a significant role in drug absorption, distribution, and elimination. The International Transporter Consortium has identified 7 transporters of increasing importance from evidence of clinically significant transporter-mediated drug-drug interactions. The transporters are P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2, organic anion transporters (OAT) 1, and OAT3. Decision trees were created based on in vitro experiments to determine whether an in vivo transporter-mediated drug-drug interaction study is needed. Phenotyping is a methodology that evaluates real-time in vivo transporter activity, whereby changes in a probe substrate or probe inhibitor reflect alternations in the activity of the specified transporter. In vivo probe substrates and/or probe inhibitors have been proposed for each aforementioned transporter. In vitro findings and animal models provide the strongest evidence regarding probe specificity. However, such findings have not conclusively correlated with human phenotyping studies. Furthermore, the extent of contribution from multiple transporters in probe disposition complicates the ability to discern if study findings are the result of a specific transporter and thus provide a recommendation for a preferred probe for a drug transporter. PMID:27385182

  19. Effect of cytochrome P450 2D6*10 polymorphism on the pharmacokinetics of oral nebivolol after single and multiple doses%细胞色素 P4502 D6*10基因多态性对单次及多次口服奈必洛尔药代动力学的影响

    蔡凝芳; 程泽能; 李碧峰; 黄小红; 许可珍; 蔡梦云; 冯惠平; 何丽华; 余敏; 郭歆

    2015-01-01

    目的:评价细胞色素P4502D6*10( CYP2D6*10)基因多态性对单次及多次口服奈必洛尔药代动力学的影响。方法根据CYP2D6*10基因型选择入选15名中国健康受试者,其中CYP2D6*1携带者8名,CYP2D6*10/*10基因型7名。所有受试者单次口服奈必洛尔5 mg和多次口服奈必洛尔5 mg・ d-1,qd,连续7 d。用LC-MS/MS法测定奈必洛尔血药浓度,用WinNonlin软件计算主要的药代动力学参数。结果单次口服奈必洛尔后 CYP2D6*1携带者和CYP2D6*10/*10基因型个体中奈必洛尔的主要药代动力学参数:t1/2分别为(9.88±5.47),(12.29±6.19) h; AUCinf分别为(7.26±5.88),(8.56±5.20)μg・ L-1・ h;Cmax分别为(1.11±0.53),(1.42±0.75)μg・ L-1。多次口服奈必洛尔后CYP2D6*1携带者和CYP2D6*10/*10基因型个体中奈必洛尔的主要药代动力学参数:t1/2分别为(8.56±2.38),(7.67±4.75) h; AUCinf分别为(10.62±5.62),(12.74±7.40)μg・ L-1・ h;Cmax分别为(2.05±0.83),(2.02±0.75)μg・ L-1。奈必洛尔主要药代动力学参数在不同基因型组间比较差异无统计学意义( P>0.05)。多次给药的清除率在不同基因型中均显著低于单次给药(P<0.05)。结论 CYP2D6*10基因多态性对单次及多次口服奈必洛尔药代动力学无显著影响。多次给药后奈必洛尔体内消除减慢,且不受CYP2D6*10基因多态性影响。%Objective To evaluate the effect of cytochrome P450 2 D6*10 ( CYP2 D6*10 ) polymorphism on the pharmacokinetics of oral nebivolol after single and multiple doses. Methods Fifteen healthy volunteers which were selected according to their CYP2D6*10 genotype, consisted of 8 of CYP2D6*1 carriers and 7 of CYP2D6*10/*10 geno-types.All subjects received a single dose of 5 mg and multiple doses (5 mg・ d-1 , qd, for 7 days) .Nebivolol in plasma

  20. Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes [v2; ref status: indexed, http://f1000r.es/5ev

    Ron Ammar

    2015-05-01

    Full Text Available Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information. Phasing short read data typically requires supplemental statistical phasing based on known haplotype structure in the population or parental genotypic data. Here we demonstrate that the MinION nanopore sequencer is capable of producing very long reads to resolve both variants and haplotypes of HLA-A, HLA-B and CYP2D6 genes important in determining patient drug response in sample NA12878 of CEPH/UTAH pedigree 1463, without the need for statistical phasing. Long read data from a single 24-hour nanopore sequencing run was used to reconstruct haplotypes, which were confirmed by HapMap data and statistically phased Complete Genomics and Sequenom genotypes. Our results demonstrate that nanopore sequencing is an emerging standalone technology with potential utility in a clinical environment to aid in medical decision-making.

  1. Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition.

    Gangopadhyay, Soumyashree A; Losito, Erica L; Hougland, James L

    2014-01-21

    Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins. PMID:24344934

  2. Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate-probe distance.

    Oyamatsu, Daisuke; Hirano, Yu; Kanaya, Norihiro; Mase, Yoshiaki; Nishizawa, Matsuhiko; Matsue, Tomokazu

    2003-08-01

    The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate-probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates. PMID:12893317

  3. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro

    Pestivirus Npro is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, Npro blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. Npro's intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed Npro-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of Npro proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that Npro's catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, Npro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • Npro's autoproteolysis is studied using Npro-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • Npro prefers small amino acids with non-branched beta carbons at the P1 position

  4. Improving Polymerase Activity with Unnatural Substrates by Sampling Mutations in Homologous Protein Architectures.

    Dunn, Matthew R; Otto, Carine; Fenton, Kathryn E; Chaput, John C

    2016-05-20

    The ability to synthesize and propagate genetic information encoded in the framework of xeno-nucleic acid (XNA) polymers would inform a wide range of topics from the origins of life to synthetic biology. While directed evolution has produced examples of engineered polymerases that can accept XNA substrates, these enzymes function with reduced activity relative to their natural counterparts. Here, we describe a biochemical strategy that enables the discovery of engineered polymerases with improved activity for a given unnatural polymerase function. Our approach involves identifying specificity determining residues (SDRs) that control polymerase activity, screening mutations at SDR positions in a model polymerase scaffold, and assaying key gain-of-function mutations in orthologous protein architectures. By transferring beneficial mutations between homologous protein structures, we show that new polymerases can be identified that function with superior activity relative to their starting donor scaffold. This concept, which we call scaffold sampling, was used to generate engineered DNA polymerases that can faithfully synthesize RNA and TNA (threose nucleic acid), respectively, on a DNA template with high primer-extension efficiency and low template sequence bias. We suggest that the ability to combine phenotypes from different donor and recipient scaffolds provides a new paradigm in polymerase engineering where natural structural diversity can be used to refine the catalytic activity of synthetic enzymes. PMID:26860781

  5. Enzyme activation and catalysis: characterisation of the vibrational modes of substrate and product in protochlorophyllide oxidoreductase.

    Sytina, Olga A; Alexandre, Maxime T; Heyes, Derren J; Hunter, C Neil; Robert, Bruno; van Grondelle, Rienk; Groot, Marie Louise

    2011-02-14

    The light-dependent reduction of protochlorophyllide, a key step in the synthesis of chlorophyll, is catalyzed by the enzyme protochlorophyllide oxidoreductase (POR) and requires two photons (O. A. Sytina et al., Nature, 2008, 456, 1001-1008). The first photon activates the enzyme-substrate complex, a subsequent second photon initiates the photochemistry by triggering the formation of a catalytic intermediate. These two events are characterized by different spectral changes in the infra-red spectral region. Here, we investigate the vibrational frequencies of the POR-bound and unbound substrate, and product, and thus provide a detailed assignment of the spectral changes in the 1800-1250 cm(-1) region associated with the catalytic conversion of PChlide:NADPH:TyrOH into Chlide:NADP(+):TyrO(-). Fluorescence line narrowed spectra of the POR-bound Pchlide reveal a C=O keto group downshifted by more than 20 cm(-1) to a relatively low vibrational frequency of 1653 cm(-1), as compared to the unbound Pchlide, indicating that binding of the chromophore to the protein occurs via strong hydrogen bond(s). The frequencies of the C=C vibrational modes are consistent with a six-coordinated state of the POR-bound Pchlide, suggesting that there are two coordination interactions between the central Mg atom of the chromophore and protein residues, and/or a water molecule. The frequencies of the C=C vibrational modes of Chlide are consistent with a five-coordinated state, indicating a single interaction between the central Mg atom of the chromophore and a water molecule. Rapid-scan FTIR measurements on the Pchlide:POR:NADPH complex at 4 cm(-1) spectral resolution reveal a new band in the 1670 cm(-1) region. The FTIR spectra of the enzyme activation phase indicate involvement of a nucleotide-binding structural motif, and an increased exposure of the protein to solvent after activation. PMID:21103538

  6. Electrodeposition of metallic tungsten coating from binary oxide molten salt on low activation steel substrate

    Tungsten is considered a promising plasma facing armor material for future fusion devices. An electrodeposited metallic tungsten coating from Na2WO4–WO3 binary oxide molten salt on low activation steel (LAS) substrate was investigated in this paper. Tungsten coatings were deposited under various pulsed currents conditions at 1173 K in atmosphere. Cathodic current density and pulsed duty cycle were investigated for pulsed current electrolysis. The crystal structure and microstructure of tungsten coatings were characterized by X-ray diffractometry, scanning electron microscopy, and energy X-ray dispersive analysis techniques. The results indicated that pulsed current density and duty cycle significantly influence tungsten nucleation and electro-crystallization phenomena. The average grain size of the coating becomes much larger with increasing cathodic current density, which demonstrates that appropriate high cathodic current density can accelerate the growth of grains on the surface of the substrate. The micro-hardness of tungsten coatings increases with the increasing thickness of coatings; the maximum micro-hardness is 482 HV. The prepared tungsten coatings have a smooth surface, a porosity of less than 1%, and an oxygen content of 0.024 wt%

  7. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

  8. A selection assay for haloalkane dehalogenase activity based on toxic substrates.

    Fibinger, Michael P C; Davids, Timo; Böttcher, Dominique; Bornscheuer, Uwe T

    2015-11-01

    Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 10(6) cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases. PMID:25998660

  9. The construction of hierarchical structure on Ti substrate with superior osteogenic activity and intrinsic antibacterial capability

    Huang, Ying; Zha, Guangyu; Luo, Qiaojie; Zhang, Jianxiang; Zhang, Feng; Li, Xiaohui; Zhao, Shifang; Zhu, Weipu; Li, Xiaodong

    2014-08-01

    The deficient osseointegration and implant-associated infections are pivotal issues for the long-term clinical success of endosteal Ti implants, while development of functional surfaces that can simultaneously overcome these problems remains highly challenging. This study aimed to fabricate sophisticated Ti implant surface with both osteogenic inducing activity and inherent antibacterial ability simply via tailoring surface topographical features. Micro/submciro/nano-scale structure was constructed on Ti by three cumulative subtractive methods, including sequentially conducted sandblasting as well as primary and secondary acid etching treatment. Topographical features of this hierarchical structure can be well tuned by the time of the secondary acid treatment. Ti substrate with mere micro/submicro-scale structure (MS0-Ti) served as a control to examine the influence of hierarchical structures on surface properties and biological activities. Surface analysis indicated that all hierarchically structured surfaces possessed exactly the same surface chemistry as that of MS0-Ti, and all of them showed super-amphiphilicity, high surface free energy, and high protein adsorption capability. Biological evaluations revealed surprisingly antibacterial ability and excellent osteogenic activity for samples with optimized hierarchical structure (MS30-Ti) when compared with MS0-Ti. Consequently, for the first time, a hierarchically structured Ti surface with topography-induced inherent antibacterial capability and excellent osteogenic activity was constructed.

  10. Amorphous silicon thin film transistor active-matrix organic light-emitting diode displays fabricated on flexible substrates

    Nichols, Jonathan A.

    Organic light-emitting diode (OLED) displays are of immense interest because they have several advantages over liquid crystal displays, the current dominant flat panel display technology. OLED displays are emissive and therefore are brighter, have a larger viewing angle, and do not require backlights and filters, allowing thinner, lighter, and more power efficient displays. The goal of this work was to advance the state-of-the-art in active-matrix OLED display technology. First, hydrogenated amorphous silicon (a-Si:H) thin film transistor (TFT) active-matrix OLED pixels and arrays were designed and fabricated on glass substrates. The devices operated at low voltages and demonstrated that lower performance TFTs could be utilized in active-matrix OLED displays, possibly allowing lower cost processing and the use of polymeric substrates. Attempts at designing more control into the display at the pixel level were also made. Bistable (one bit gray scale) active-matrix OLED pixels and arrays were designed and fabricated. Such pixels could be used in novel applications and eventually help reduce the bandwidth requirements in high-resolution and large-area displays. Finally, a-Si:H TFT active-matrix OLED pixels and arrays were fabricated on a polymeric substrate. Displays fabricated on a polymeric substrates would be lightweight; flexible, more rugged, and potentially less expensive to fabricate. Many of the difficulties associated with fabricating active-matrix backplanes on flexible substrates were studied and addressed.

  11. Probing substrate interactions in the active tunnel of a catalytically deficient cellobiohydrolase (Cel7)

    Westh, Peter; Colussi, Francieli; Sørensen, Trine Holst;

    2015-01-01

    Cellobiohydrolases (CBHs) break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be...... sites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with DP of 2-3 the binding constants were around 105 M-1, and for longer ligands (DP 5-8) this value was about 107 M-1. Within each of......) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel...

  12. SUBSTRATE UTILIZATION IS INFLUENCED BY ACUTE DIETARY CARBOHYDRATE INTAKE IN ACTIVE, HEALTHY FEMALES

    Sara Gregory

    2011-03-01

    Full Text Available The present study compared the metabolic responses between a single low-carbohydrate (LC and low-fat (LF meal followed by an aerobic exercise bout in females. Subjects included 8 active, premenopausal females. Subjects completed a LC and LF testing session. Respiratory gas exchange (RER measurements were taken for 20 min fasted, for 55 min postprandial (PP, and during 30 min of exercise. Blood was collected for assessment of glucose (G, insulin (IN, triglycerides (TG, and free fatty acids (FFA during the final 10 min of each time period. The LF meal provided 396 kcal (78% carbohydrate, 7% fat, and 15% protein. The LC meal provided 392 kcal (15% carbohydrate, 68% fat, and 18% protein. No significant differences existed between test meals for fasting blood measurements. PP IN (µU·mL-1 levels were significantly lower following LC compared to LF [10.7 (6.1 vs. 26.0 (21.0]. Postexercise (PE FFA (mEq·L-1 levels were significantly greater following LC [1.1 (0.3 vs. 0.5 (0.3]. PE TG (mg·dL-1 levels were significantly greater following LC [152.0 (53.1 vs. 114.4 (40.9]. RER was significantly lower at all time points following LC compared to LF. In moderately active adult females, ingestion of a single LC meal resulted in greater lipid oxidation at rest and during exercise as compared to a single LF meal. Although macronutrient distribution appears to have dictated substrate utilization in the present study, more research is needed regarding the long-term effects of macronutrient redistribution with and without exercise on substrate utilization.

  13. Isolation of novel ribozymes that ligate AMP-activated RNA substrates

    Hager, A. J.; Szostak, J. W.

    1997-01-01

    BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

  14. Bacillus subtilis BY-kinase PtkA controls enzyme activity and localization of its protein substrates

    Jers, Carsten; Pedersen, Malene Mejer; Paspaliari, Dafni Katerina;

    2010-01-01

    P>Bacillus subtilis BY-kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. Our recent phosphoproteome study identified nine new tyrosine-phosphorylated prote......P>Bacillus subtilis BY-kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. Our recent phosphoproteome study identified nine new tyrosine......-phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single-stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA-dependent phosphorylation. Because enzyme activity was not...

  15. Calcite precipitation on glass substrates and active stalagmites in Katerloch Cave (Austria): Constraints from environmental monitoring

    Sakoparnig, Marlene; Boch, Ronny; Wang, Xianfeng; Lin, Ke; Spötl, Christoph; Leis, Albrecht; Gollowitsch, Anna; Dietzel, Martin

    2016-04-01

    Located near Graz at the SE-rim of the Alps Katerloch is well-known for its impressive dripstone decoration, e.g. several metres tall and relatively fast growing (0.2-0.7 mm/yr on average) candle-stick-type stalagmites. In the course of an ongoing multi-annual and partially high-resolution cave monitoring program we study modern (active) sites of carbonate deposition focusing on the site-specific growth dynamics and connection of modern regional and cave environmental conditions with petrographic, chemical and stable isotopic information captured in the speleothems. Fresh calcite precipitates on artificial (glass) substrates underneath active drip sites were collected continuously from 2006 to 2014 (eight years!). The samples (up to 7 mm thick) represent cave sections of different temperature and drip sites of partially different characteristics (e.g. drip rate). We also recovered short drill cores (up to 3 cm length, 1 cm diameter) from the top of active stalagmites probably representing the last decades to centuries of calcite crystallization. Moreover, an actively growing stalagmite (K10) comprising both modern and past calcite deposition was collected. 238U-234U-230Th dating using MC-ICP-MS of K10 (71 cm long) revealed several distinct growth intervals (separated by growth interruptions) starting at 129.1 ±1.2 kyr BP (Last Interglacial) up to now, mostly reflecting warm and humid climate intervals. High-resolution (100 μm) isotope profiles micromilled from the multi-annual modern calcite precipitates on artificial substrates revealed low δ13C values of -12.8 to -8.3 ‰ (VPDB) and relatively high δ18O of -6.9 to -4.9 ‰Ṫhe δ18O curves from all collection sites (different growth rate) record a pronounced decrease during their most recent growth period most likely corresponding to a significant decrease towards lower oxygen isotope values observed in drip waters collected in the year 2014 compared with samples from 2005 to 2007. Drip water δ2H /δ18O

  16. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  17. Temperature and substrate chemistry as major drivers of interregional variability of leaf microbial decomposition and cellulolytic activity in headwater streams.

    Fenoy, Encarnación; Casas, J Jesús; Díaz-López, Manuel; Rubio, Juan; Guil-Guerrero, J Luís; Moyano-López, Francisco J

    2016-11-01

    Abiotic factors, substrate chemistry and decomposers community composition are primary drivers of leaf litter decomposition. In soil, much of the variation in litter decomposition is explained by climate and substrate chemistry, but with a significant contribution of the specialisation of decomposer communities to degrade specific substrates (home-field advantage, HFA). In streams, however, HFA effects on litter decomposition have not been explicitly tested. We evaluated responses of microbial decomposition and β-glucosidase activity to abiotic factors, substrate and decomposer assemblages, using a reciprocal litter transplant experiment: 'ecosystem type' (mountain vs lowland streams) × 'litter chemistry' (alder vs reed). Temperature, pH and ionic concentration were higher in lowland streams. Decomposition for both species was faster in lowland streams. Decomposition of reed was more accelerated in lowland compared with mountain streams than that of alder, suggesting higher temperature sensitivity of decomposition in reed. Q10 (5°C-15°C) values of β-glucosidase activity were over 2. The alkaline pH and high ionic concentration of lowland streams depleted enzyme activity. We found similar relationships of decomposition or enzyme activity with abiotic factors for both species, suggesting limited support to the HFA hypothesis. Overall, our results suggest a prime role of temperature interacting with substrate chemistry on litter decomposition. PMID:27515735

  18. Active-site models for complexes of quinolinate synthase with substrates and intermediates

    Soriano, Erika V.; Zhang, Yang; Colabroy, Keri L.; Sanders, Jennie M.; Settembre, Ethan C.; Dorrestein, Pieter C.; Begley, Tadhg P.; Ealick, Steven E., E-mail: see3@cornell.edu [Cornell University, Ithaca, NY 14853-1301 (United States)

    2013-09-01

    Structural studies of quinolinate synthase suggest a model for the enzyme–substrate complex and an enzyme–intermediate complex with a [4Fe–4S] cluster. Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel β-sheet flanked by four α-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe–4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005 ▶), J. Biol. Chem.280, 26645–26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe–4S] cluster, prior to cluster assembly.

  19. Programmable SERS active substrates for chemical and biosensing applications using amorphous/crystalline hybrid silicon nanomaterial

    Powell, Jeffery Alexander; Venkatakrishnan, Krishnan; Tan, Bo

    2016-01-01

    We present the creation of a unique nanostructured amorphous/crystalline hybrid silicon material that exhibits surface enhanced Raman scattering (SERS) activity. This nanomaterial is an interconnected network of amorphous/crystalline nanospheroids which form a nanoweb structure; to our knowledge this material has not been previously observed nor has it been applied for use as a SERS sensing material. This material is formed using a femtosecond synthesis technique which facilitates a laser plume ion condensation formation mechanism. By fine-tuning the laser plume temperature and ion interaction mechanisms within the plume, we are able to precisely program the relative proportion of crystalline Si to amorphous Si content in the nanospheroids as well as the size distribution of individual nanospheroids and the size of Raman hotspot nanogaps. With the use of Rhodamine 6G (R6G) and Crystal Violet (CV) chemical dyes, we have been able to observe a maximum enhancement factor of 5.38 × 106 and 3.72 × 106 respectively, for the hybrid nanomaterial compared to a bulk Si wafer substrate. With the creation of a silicon-based nanomaterial capable of SERS detection of analytes, this work demonstrates a redefinition of the role of nanostructured Si from an inactive to SERS active role in nano-Raman sensing applications.

  20. Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates

    Lavrik O. I.

    2012-06-01

    Full Text Available Aim. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ using 5-formyluridine (5-foU containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence of replication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.

  1. Enzyme Activities and Substrate Degradation by Fungal Isolates on Cassava Waste During Solid State Fermentation

    Pothiraj, C.; Eyini, M.

    2007-01-01

    The growth and bioconversion potential of selected strains growing on cassava waste substrate during solid state fermentation were assessed. Rhizopus stolonifer showed the highest and the fastest utilization of starch and cellulose in the cassava waste substrate. It showed 70% starch utilization and 81% cellulose utilization within eight days. The release of reducing sugars indicating the substrate saccharification or degradation potential of the organisms reached the highest value of 406.5 m...

  2. Glucuronoyl esterases are active on the polymeric substrate methyl esterified glucuronoxylan.

    Biely, Peter; Malovíková, Anna; Uhliariková, Iveta; Li, Xin-Liang; Wong, Dominic W S

    2015-08-19

    Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic deesterification was monitored by (1)H NMR spectroscopy and evaluated on the basis of the decrease of the signal of the ester methyl group and increase of the signal of methanol. The results show for the first time the action of enzymes on polymeric substrate, which imitates more closely the natural substrate in plant cell walls than the low molecular mass artificial substrates used up to present. PMID:26216754

  3. Rapid Functional Definition of Extended Spectrum β-Lactamase Activity in Bacterial Cultures via Competitive Inhibition of Fluorescent Substrate Cleavage

    Sallum, Ulysses W; Zheng, Xiang; Verma, Sarika; Hasan, Tayyaba

    2010-01-01

    The functional definition of extended-spectrum β-lactamase (ESBL) activity is a clinical challenge. Here we report a rapid and convenient assay of β-lactamase activity through the competitive inhibition of fluorescent substrate hydrolysis that provides a read-out nearly 40x more rapidly than conventional techniques for functional definition. A panel of β-lactam antibiotics was used for competition against β-lactamase enzyme activated photosensitizer (β-LEAP) yielding a competitive index (Ci) ...

  4. Neuronal Activity Stimulated by Liquid Substrates Injection at Zusanli (ST36 Acupoint: The Possible Mechanism of Aquapuncture

    Chun-Yen Chen

    2014-01-01

    Full Text Available Aquapuncture is a modified acupuncture technique and it is generally accepted that it has a greater therapeutic effect than acupuncture because of the combination of the acupoint stimulation and the pharmacological effect of the drugs. However, to date, the mechanisms underlying the effects of aquapuncture remain unclear. We hypothesized that both the change in the local spatial configuration and the substrate stimulation of aquapuncture would activate neuronal signaling. Thus, bee venom, normal saline, and vitamins B1 and B12 were injected into a Zusanli (ST36 acupoint as substrate of aquapuncture, whereas a dry needle was inserted into ST36 as a control. After aquapuncture, activated neurons expressing Fos protein were mainly observed in the dorsal horn of the spinal cord in lumbar segments L3–5, with the distribution nearly identical among all groups. However, the bee venom injection induced significantly more Fos-expressing neurons than the other substrates. Based on these data, we suggest that changes in the spatial configuration of the acupoint activate neuronal signaling and that bee venom may further strengthen this neuronal activity. In conclusion, the mechanisms for the effects of aquapuncture appear to be the spatial configuration changes occurring within the acupoint and the ability of injected substrates to stimulate neuronal activity.

  5. Synthesis of carbon nanofibers on impregnated powdered activated carbon as cheap substrate

    A.A. Mamun

    2016-07-01

    Full Text Available The catalysis and characterization of carbon nanofibers (CNFs composite are reported in this work. Carbon nanofibers were produced on oil palm shell powdered activated carbon (PAC, which was impregnated with nickel. Chemical Vapor Deposition (CVD of C2H2 was used in the presence of hydrogen at ∼650 °C. The flow rates of carbon source and hydrogen were fixed. The CNFs formed directly on the surface of the impregnated PAC. Variable weight percentages (1%, 3%, 5%, 7% and 9% of the catalyst salt (Ni+2 were used for the impregnation. However, the best catalysis was observed on the substrate with 3% Ni+2. The product displayed a relatively high surface area, essentially constituted by the external surface. New functional groups also appeared compared to those in the PAC. Field Emission Scanning Microscopy (FESEM, Transmission Electron Microscopy (TEM, Fourier Transform Infrared (FTIR, BET surface area analysis and energy dispersive X-ray (EDX were used for the characterization of the new carbon nano product, which was produced through a clean novel process.

  6. Effect of substrate (ZnO morphology on enzyme immobilization and its catalytic activity

    Huang Xuelei

    2011-01-01

    Full Text Available Abstract In this study, zinc oxide (ZnO nanocrystals with different morphologies were synthesized and used as substrates for enzyme immobilization. The effects of morphology of ZnO nanocrystals on enzyme immobilization and their catalytic activities were investigated. The ZnO nanocrystals were prepared through a hydrothermal procedure using tetramethylammonium hydroxide as a mineralizing agent. The control on the morphology of ZnO nanocrystals was achieved by varying the ratio of CH3OH to H2O, which were used as solvents in the hydrothermal reaction system. The surface of as-prepared ZnO nanoparticles was functionalized with amino groups using 3-aminopropyltriethoxysilane and tetraethyl orthosilicate, and the amino groups on the surface were identified and calculated by FT-IR and the Kaiser assay. Horseradish peroxidase was immobilized on as-modified ZnO nanostructures with glutaraldehyde as a crosslinker. The results showed that three-dimensional nanomultipod is more appropriate for the immobilization of enzyme used further in catalytic reaction.

  7. Effect of substrate (ZnO) morphology on enzyme immobilization and its catalytic activity

    Zhang, Yan; Wu, Haixia; Huang, Xuelei; Zhang, Jingyan; Guo, Shouwu

    2011-07-01

    In this study, zinc oxide (ZnO) nanocrystals with different morphologies were synthesized and used as substrates for enzyme immobilization. The effects of morphology of ZnO nanocrystals on enzyme immobilization and their catalytic activities were investigated. The ZnO nanocrystals were prepared through a hydrothermal procedure using tetramethylammonium hydroxide as a mineralizing agent. The control on the morphology of ZnO nanocrystals was achieved by varying the ratio of CH3OH to H2O, which were used as solvents in the hydrothermal reaction system. The surface of as-prepared ZnO nanoparticles was functionalized with amino groups using 3-aminopropyltriethoxysilane and tetraethyl orthosilicate, and the amino groups on the surface were identified and calculated by FT-IR and the Kaiser assay. Horseradish peroxidase was immobilized on as-modified ZnO nanostructures with glutaraldehyde as a crosslinker. The results showed that three-dimensional nanomultipod is more appropriate for the immobilization of enzyme used further in catalytic reaction.

  8. Memory characteristics of poly-Si using MIC as an active layer on glass substrates

    In this paper the electrical properties of nonvolatile memory (NVM) using multi-stack gate insulators of oxide-nitride-oxynitride (ONOn) and an active layer of low temperature polycrystalline silicon (LTPS) were investigated. From hydrogenated amorphous silicon (a-Si : H), the LTPS thin film with a high crystalline fraction of 96% and a low surface roughness of 1.28 nm was fabricated by metal induced crystallization (MIC) with annealing conditions of 650 0C for 5 h on glass substrates. The LTPS thin film transistor or the NVM had a field effect mobility of (μFE) 10 cm2 V-1 s-1 and a threshold voltage (VTH) of -3.5 V. The results demonstrated that the NVM had a memory window of 1.6 V with a programming and erasing (P/E) voltage of -14 V and 14 V in 1 ms. Moreover, retention properties of the memory were shown to exceed 80% after 10 years. Therefore, the LTPS fabricated by MIC has become a potential material for NVM application which is employed for the system integration of panel displays.

  9. Memory characteristics of poly-Si using MIC as an active layer on glass substrates

    Nguyen, Thanh Nga; Jung, Sungwook; Nguyen, Van Duy; Yi, Junsin, E-mail: yi@yurim.skku.ac.k [School of Information and Communication Engineering, Sungkyunkwan University, Suwon, Kyouggi 440-746 (Korea, Republic of)

    2010-03-17

    In this paper the electrical properties of nonvolatile memory (NVM) using multi-stack gate insulators of oxide-nitride-oxynitride (ONOn) and an active layer of low temperature polycrystalline silicon (LTPS) were investigated. From hydrogenated amorphous silicon (a-Si : H), the LTPS thin film with a high crystalline fraction of 96% and a low surface roughness of 1.28 nm was fabricated by metal induced crystallization (MIC) with annealing conditions of 650 {sup 0}C for 5 h on glass substrates. The LTPS thin film transistor or the NVM had a field effect mobility of ({mu}{sub FE}) 10 cm{sup 2} V{sup -1} s{sup -1} and a threshold voltage (V{sub TH}) of -3.5 V. The results demonstrated that the NVM had a memory window of 1.6 V with a programming and erasing (P/E) voltage of -14 V and 14 V in 1 ms. Moreover, retention properties of the memory were shown to exceed 80% after 10 years. Therefore, the LTPS fabricated by MIC has become a potential material for NVM application which is employed for the system integration of panel displays.

  10. Effects of a TiC substrate on the catalytic activity of Pt for NO reduction.

    Chu, Xingli; Fu, Zhaoming; Li, Shasha; Zhang, Xilin; Yang, Zongxian

    2016-05-11

    Density functional theory calculations are used to elucidate the catalytic properties of a Pt monolayer supported on a TiC(001) substrate (Pt/TiC) toward NO reduction. It is found that the compound system of Pt/TiC has a good stability due to the strong Pt-TiC interaction. The diverse dissociation paths (namely the direct dissociation mechanism and the dimeric mechanism) are investigated. The transition state searching calculations suggest that NO has strong diffusion ability and small activation energy for dissociation on the Pt/TiC. For NO reduction on the Pt/TiC surface, we have found that the direct dissociation mechanisms (NO + N + O → NO2 + N and NO + N + O → N2 + O + O) are easier with a smaller dissociation barrier than those on the Pt(111) surface; and the dimeric process (NO + NO → (NO)2 → N2O + O → N2 + O + O) is considered to be dominant or significant with even a lower energy barrier than that of the direct dissociation. The results show that Pt/TiC can serve as an efficient catalyst for NO reduction. PMID:27117987

  11. Posttranslational ruling of xanthine oxidase activity in bovine milk by its substrates

    The aims of this study were to test the hypothesis that the substrates of xanthine oxidase (XO), xanthine and hypoxanthine, are consumed while the milk is stored in the gland between milkings, and to explore how XO activity responds to bacteria commonly associated with subclinical infections in the mammary gland. Freshly secreted milk was obtained following complete evacuation of the gland and induction of milk ejection with oxytocin. In bacteria-free fresh milk xanthine and hypoxanthine were converted to uric acid within 30 min (T1/2 ∼ 10 min), which in turn provides electrons for formation of hydrogen peroxide and endows the alveolar lumen with passive protection against invading bacteria. On the other hand, the longer residence time of milk in the cistern compartment was not associated with oxidative stress as a result of XO idleness caused by exhaustion of its physiological fuels. The specific response of XO to bacteria species and the resulting bacteria-dependent nitrosative stress further demonstrates that it is part of the gland immune system

  12. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu;

    2016-01-01

    -ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its CDR-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to...

  13. Opioids and Efflux Transporters. 1. P-Glycoprotein Substrate Activity of N-Substituted Analogs of Meperidine.

    Mercer, Susan L.; Cunningham, Christopher W.; Hassan, Hazem; Eddington, Natalie D.; Coop, Andrew

    2006-01-01

    P-Glycoprotein (P-gp) is an efflux transporter which is up-regulated at the blood brain barrier in both morphine and oxycodone tolerant rats. Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-gp, suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of compounds, and show that a meperidine anal...

  14. Pt monolayer coating on complex network substrate with high catalytic activity for the hydrogen evolution reaction

    Li, Man; Ma, Qiang; Zi, Wei; Liu, Xiaojing; Zhu, Xuejie; Liu, Shengzhong (Frank)

    2015-01-01

    A deposition process has been developed to fabricate a complete-monolayer Pt coating on a large-surface-area three-dimensional (3D) Ni foam substrate using a buffer layer (Ag or Au) strategy. The quartz crystal microbalance, current density analysis, cyclic voltammetry integration, and X-ray photoelectron spectroscopy results show that the monolayer deposition process accomplishes full coverage on the substrate and the deposition can be controlled to a single atomic layer thickness. To our kn...

  15. Molecular Modeling of Peroxidase and Polyphenol Oxidase: Substrate Specificity and Active Site Comparison

    Lalida Shank; Vannajan Sanghiran Lee; Prontipa Nokthai

    2010-01-01

    Peroxidases (POD) and polyphenol oxidase (PPO) are enzymes that are well known to be involved in the enzymatic browning reaction of fruits and vegetables with different catalytic mechanisms. Both enzymes have some common substrates, but each also has its specific substrates. In our computational study, the amino acid sequence of grape peroxidase (ABX) was used for the construction of models employing homology modeling method based on the X-ray structure of cytosolic ascorbate peroxidase from ...

  16. Reactions of hypochlorous acid with biological substrates are activated catalytically by tertiary amines.

    Prütz, W A

    1998-09-15

    The activation of reactions of HOCl with a variety of model substrates by tertiary amines was investigated spectroscopically by tandem-mix and stopped-flow techniques. HOCl-induced chlorination of salicylate can be sped up by several orders of magnitude by catalytic amounts of trimethylamine (TMN). The effect is obviously due to the fast generation of reactive quarternary chloramonium ions, TMN+ Cl, which act as chain carrier in a catalytic reaction cycle. Of various catalysts tested, quinine shows the highest activity; this is attributable to the quinuclidine (QN) substituent, a bicyclic tertiary amine, forming a particularly reactive chloro derivative, QN+ Cl, which does not decompose autocatalytically. The rate of catalytic salicylate chlorination as a function of pH (around pH 7) depends not at least on the basicity of the tertiary amine; the rate increases with pH in the cases of TMN and quinuclidine (high basicity), but decreases with pH in the case of MES (low basicity). Tertiary amines also catalyze the interaction between HOCl and alkenes, as shown using sorbate as model. Reaction of HOCl with the nucleotides GMP and CMP is sped up remarkably by catalytic amounts of tertiary amines. In the case of GMP the same product spectrum is produced by HOCl in absence and presence of catalyst, but a change in the product spectra is obtained when AMP and CMP are reacted with HOCl in presence of catalyst. Using poly(dA-dT).poly(dA-dT) as DNA model, it is shown that HOCl primarily induces an absorbance increase at 263 nm, which indicates unfolding of the double strand due to fast chlorination of thymidine; a subsequent secondary absorbance decrease can be explained by slow chlorination of adenosine. Both the primary and secondary processes are activated by catalytic amounts of quinine. No evidence was found for a radical pathway in TMN-mediated oxidation of formate by HOCl. The present results suggest that low concentrations of certain tertiary amines have the potential

  17. 褪黑素对大鼠肝微粒体细胞色素P450酶亚型CYP2C9、CYP2D6、CYP3A4活性的影响

    刘明远; 杨光远; 王跃新; 张春斌; 白雪; 张建华

    2011-01-01

    目的 观察褪黑素对大鼠肝微粒体内细胞色素P450 (CYP450) 酶亚型CYP2C9、CYP2D6、CYP3A4活性的影响.方法 以生理盐水为对照,大鼠灌胃0.54 mg·kg-1·d-1的褪黑素,连续7 d,然后测定其肝微体中CYP2C9、CYP2D6、CYP3A4活性.结果 与对照组比较,褪黑素组CYP2C9、CYP2D6、CYP3A4的活性无变化(P>0.05).结论 褪黑素对CYP450酶 CYP2C9、CYP2D6、CYP3A4活性无影响.

  18. Ex Vivo Antioxidant Activity of Selected Medicinal Plants against Fenton Reaction-Mediated Oxidation of Biological Lipid Substrates

    Namratha Pai Kotebagilu

    2015-01-01

    Full Text Available Free radical-mediated oxidation is often linked to various degenerative diseases. Biological substrates with lipids as major components are susceptible to oxygen-derived lipid peroxidation due to their composition. Lipid peroxide products act as biomarkers in evaluating the antioxidant potential of various plants and functional foods. The study focused on evaluation of the antioxidant potential of two extracts (methanol and 80% methanol of four medicinal plants, Andrographis paniculata, Costus speciosus, Canthium parviflorum, and Abrus precatorius, against Fenton reaction-mediated oxidation of three biological lipid substrates; cholesterol, low-density lipoprotein, and brain homogenate. The antioxidant activity of the extracts was measured by thiobarbituric acid reactive substances method. Also, the correlation between the polyphenol, flavonoid content, and the antioxidant activity in biological substrates was analyzed. Results indicated highest antioxidant potential by 80% methanol extract of Canthium parviflorum (97.55%, methanol extract of Andrographis paniculata (72.15%, and methanol extract of Canthium parviflorum (49.55% in cholesterol, low-density lipoprotein, and brain, respectively. The polyphenol and flavonoid contents of methanol extract of Andrographis paniculata in cholesterol (r=0.816 and low-density lipoprotein (r=0.948 and Costus speciosus in brain (r=0.977, polyphenols, and r=0.949, flavonoids correlated well with the antioxidant activity. The findings prove the antioxidant potential of the selected medicinal plants against Fenton reaction in biological lipid substrates.

  19. Ex Vivo Antioxidant Activity of Selected Medicinal Plants against Fenton Reaction-Mediated Oxidation of Biological Lipid Substrates.

    Pai Kotebagilu, Namratha; Reddy Palvai, Vanitha; Urooj, Asna

    2015-01-01

    Free radical-mediated oxidation is often linked to various degenerative diseases. Biological substrates with lipids as major components are susceptible to oxygen-derived lipid peroxidation due to their composition. Lipid peroxide products act as biomarkers in evaluating the antioxidant potential of various plants and functional foods. The study focused on evaluation of the antioxidant potential of two extracts (methanol and 80% methanol) of four medicinal plants, Andrographis paniculata, Costus speciosus, Canthium parviflorum, and Abrus precatorius, against Fenton reaction-mediated oxidation of three biological lipid substrates; cholesterol, low-density lipoprotein, and brain homogenate. The antioxidant activity of the extracts was measured by thiobarbituric acid reactive substances method. Also, the correlation between the polyphenol, flavonoid content, and the antioxidant activity in biological substrates was analyzed. Results indicated highest antioxidant potential by 80% methanol extract of Canthium parviflorum (97.55%), methanol extract of Andrographis paniculata (72.15%), and methanol extract of Canthium parviflorum (49.55%) in cholesterol, low-density lipoprotein, and brain, respectively. The polyphenol and flavonoid contents of methanol extract of Andrographis paniculata in cholesterol (r = 0.816) and low-density lipoprotein (r = 0.948) and Costus speciosus in brain (r = 0.977, polyphenols, and r = 0.949, flavonoids) correlated well with the antioxidant activity. The findings prove the antioxidant potential of the selected medicinal plants against Fenton reaction in biological lipid substrates. PMID:26933511

  20. Formation of gold nanostructures on copier paper surface for cost effective SERS active substrate - Effect of halide additives

    Desmonda, Christa; Kar, Sudeshna; Tai, Yian

    2016-03-01

    In this study, we report the simple fabrication of an active substrate assisted by gold nanostructures (AuNS) for application in surface-enhanced Raman scattering (SERS) using copier paper, which is a biodegradable and cost-effective material. As cellulose is the main component of paper, it can behave as a reducing agent and as a capping molecule for the synthesis of AuNS on the paper substrate. AuNS can be directly generated on the surface of the copier paper by addition of halides. The AuNS thus synthesized were characterized by ultraviolet-visible spectroscopy, SEM, XRD, and XPS. In addition, the SERS effect of the AuNS-paper substrates synthesized by using various halides was investigated by using rhodamine 6G and melamine as probe molecules.

  1. Fabrication of highly active and cost effective SERS plasmonic substrates by electrophoretic deposition of gold nanoparticles on a DVD template

    Highlights: • Simple and cost effective electrophoretic method to fabricate plasmonic substrates. • SERS performance at three different excitation laser lines. • Promising applicability in SERS based biosensing. - Abstract: In this work we present a simple, rapid and cost effective method to fabricate highly active SERS substrates. This method consists in an electrophoretic deposition of gold nanoparticles on a metallic nanostructured template of a commercial digital versatile disk (DVD). The negatively charged gold nanoparticles self-assemble on the positively charged DVD metallic film connected to a positive terminal of a battery, due to the influence of the electric field. When gold nanoparticles self-assembled on DVD metallic film, a 10-fold additional enhancement of Raman signal was observed when compared with the case of GNPs self-assembled on a polycarbonate DVD substrate only. Finite-difference time-domain simulations demonstrated that the additional electromagnetic field arising in the hot-spots created between gold nanoparticles and DVD metallic film induces an additional enhancement of the Raman signal. SERS efficiency of the fabricated plasmonic substrate was successfully demonstrated through detection of para-aminothiophenol molecule with three different excitation laser lines (532, 633 and 785 nm). The enhancement factor was calculated to be 106 and indicates that plasmonic substrates fabricated through this method could be a promising platform for future SERS based sensors

  2. Fabrication of highly active and cost effective SERS plasmonic substrates by electrophoretic deposition of gold nanoparticles on a DVD template

    Leordean, Cosmin; Marta, Bogdan; Gabudean, Ana-Maria; Focsan, Monica; Botiz, Ioan; Astilean, Simion, E-mail: simion.astilean@phys.ubbcluj.ro

    2015-09-15

    Highlights: • Simple and cost effective electrophoretic method to fabricate plasmonic substrates. • SERS performance at three different excitation laser lines. • Promising applicability in SERS based biosensing. - Abstract: In this work we present a simple, rapid and cost effective method to fabricate highly active SERS substrates. This method consists in an electrophoretic deposition of gold nanoparticles on a metallic nanostructured template of a commercial digital versatile disk (DVD). The negatively charged gold nanoparticles self-assemble on the positively charged DVD metallic film connected to a positive terminal of a battery, due to the influence of the electric field. When gold nanoparticles self-assembled on DVD metallic film, a 10-fold additional enhancement of Raman signal was observed when compared with the case of GNPs self-assembled on a polycarbonate DVD substrate only. Finite-difference time-domain simulations demonstrated that the additional electromagnetic field arising in the hot-spots created between gold nanoparticles and DVD metallic film induces an additional enhancement of the Raman signal. SERS efficiency of the fabricated plasmonic substrate was successfully demonstrated through detection of para-aminothiophenol molecule with three different excitation laser lines (532, 633 and 785 nm). The enhancement factor was calculated to be 10{sup 6} and indicates that plasmonic substrates fabricated through this method could be a promising platform for future SERS based sensors.

  3. Fabrication of a metal membrane on a perforated polymer substrate by palladium aerosol activation and subsequent electroless plating.

    Byeon, Jeong Hoon; Hwang, Jungho

    2009-02-01

    Fabrication of a metal membrane on a perforated flexible poly(tetrafluoroethylene) (PTFE) substrate was developed by employing spark-generated palladium (Pd) aerosol activation and the subsequent electroless plating of Pd. After aerosol activation, Pd agglomerates of spark-generated primary particles (approximately 2.6 nm in diameter) with a face-centered-cubic structure were deposited uniformly on the PTFE substrate. Homogeneous Pd particles with an average size of 188 nm were tightly packed together to form a Pd membrane after Pd plating. The average plating rate of Pd during 30 min of plating at an activation intensity of 25 microg/cm(2) was 14.2 microg/cm(2) x min. PMID:20353212

  4. Redox-coupled substrate water reorganization in the active site of Photosystem II-The role of calcium in substrate water delivery.

    Ugur, Ilke; Rutherford, A William; Kaila, Ville R I

    2016-06-01

    Photosystem II (PSII) catalyzes light-driven water splitting in nature and is the key enzyme for energy input into the biosphere. Important details of its mechanism are not well understood. In order to understand the mechanism of water splitting, we perform here large-scale density functional theory (DFT) calculations on the active site of PSII in different oxidation, spin and ligand states. Prior to formation of the O-O bond, we find that all manganese atoms are oxidized to Mn(IV) in the S3 state, consistent with earlier studies. We find here, however, that the formation of the S3 state is coupled to the movement of a calcium-bound hydroxide (W3) from the Ca to a Mn (Mn1 or Mn4) in a process that is triggered by the formation of a tyrosyl radical (Tyr-161) and its protonated base, His-190. We find that subsequent oxidation and deprotonation of this hydroxide on Mn1 result in formation of an oxyl-radical that can exergonically couple with one of the oxo-bridges (O5), forming an O-O bond. When O2 leaves the active site, a second Ca-bound water molecule reorients to bridge the gap between the manganese ions Mn1 and Mn4, forming a new oxo-bridge for the next reaction cycle. Our findings are consistent with experimental data, and suggest that the calcium ion may control substrate water access to the water oxidation sites. PMID:26826591

  5. Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

    Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

    1999-01-01

    BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

  6. Synthesis and structure-activity relationship of liposomal substrates for phospholipase A(2)

    Viart, Helene Marie-France; Clausen, Mads Hartvig

    2011-01-01

    A recent innovation in the use of liposomes as drug delivery systems consists of covalently attaching an anticancer drug at the sn-2 position of phospholipids. However, some of those lipids could not be hydrolyzed by sPLA2. Steric bulk in the vicinity of the sn-2 position appears to prevent hydro...... hydrolysis of the substrate. Structurally different lipids have been synthesized and formulated as liposomes, subjected to sPLA2 and the hydrolysis rates have been compared to Molecular Dynamics simulations of the enzyme/substrate complexes....

  7. 细胞色素P4502D6基因拷贝数多态性与血铅尿铅水平的关系研究%The relationship between copy number polymorphisms of CYP2D6 gene and lead level of blood and urine in workers exposed to lead

    蒋琴琴; 刘斌; 刘移民

    2013-01-01

    目的 探讨铅作业人群细胞色素氧化酶P450酶系中的CYP2D6基因拷贝数多态性与铅毒性引起的血铅、尿铅水平的关系.方法 选择广州市某蓄电池行业233名铅作业工人,按铅尘接触水平分为高浓度组和低浓度组2组,采用统一调查表进行职业性健康问卷调查;采用石墨炉原子吸收光谱法测定现场空气铅尘及铅作业工人血铅、尿铅水平;提取铅作业人群周围血DNA,采用实时荧光定量聚合酶链式反应(PCR)法和△Ct值法检测铅作业工人的CYP2D6基因拷贝数,分析不同铅尘作业环境下作业人员CYP2D6基因拷贝数多态性与血铅、尿铅水平的关系.结果 铅作业工人CYP2D6基因拷贝数与血铅、尿铅水平有关,2组中拷贝数小于2的作业人员血铅浓度均高于拷贝数为2的人群(P<0.001),低浓度组拷贝数小于2的研究对象尿铅浓度比拷贝数为2的人群高(P=0.03),尚未发现年龄、性别、工龄、学历与血铅水平和尿铅水平的关系差异有统计学意义.结论 CYP2D6基因拷贝数多态性与铅毒性引起的血铅尿铅水平有关,拷贝数少于2的CYP2D6基因可能是铅中毒易感基因型.%OBJECTIVE To probe into the the relationship between blood lead and urine lead level and copy number polymorphisms of CYP2D6 gene in workers exposed to lead. METHODS 233 cases of workers exposed to lead were scheduled for an interview to collect the subjects' data on occupational information. Their blood lead, urine lead and the ambient air lead were tested with graphite furnace atomic absorption spectrometry. CYP2D6 gene copy number of these workers was detected with realtime fluorescence quantitative PCR method and ACt method. Under different concentrations of environmental lead dust, analyze the relationship between CYP2D6 gene copy number polymorphisms and blood lead, urine lead level. RESULTS CYP2D6 gene copy number polymorphisms was relative with blood lead and urine lead level in

  8. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper;

    2016-01-01

    ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (OSR1, HDGF and ccdc82...

  9. Investigation of photocatalytic activity of titanium dioxide deposited on metallic substrates by DC magnetron sputtering

    Daviðsdóttir, Svava; Canulescu, Stela; Dirscherl, Kai;

    2013-01-01

    The photocatalytic properties of titanium dioxide (TiO2) coating in the anatase crystalline structure deposited on aluminium AA1050 alloy and stainless steel S316L substrates were investigated. The coating was prepared by DC magnetron sputtering. The microstructure and surface morphology of the...

  10. Preparation and characterization of spray deposited Cd1-xZnxS thin films on activated substrate

    Sankarasubramanian, Kaliappan; Sethuraman, Kunjithapatham; Babu, Ramraj Ramesh; Ramamurthi, Kandasamy

    2013-10-01

    Cd1-xZnxS thin films were deposited on the glass substrates (GS) and on the potassium permanganate activated substrates (KMAS) at 350 °C using spray pyrolysis technique. X-ray diffraction studies confirmed that the films are polycrystalline and belong to the hexagonal structure. Scanning electron microscopic studies revealed an effective change on the surface morphology of the film due to potassium permanganate activation (KMA). Transmittance spectra of Cd1-xZnxS thin films were recorded in the range of 320-700 nm and the transmittance was found to be high for the film coated on KMAS than GS. Photoluminescence spectra show a strong emission peak at 585 nm for the film coated on the GS and it is shifted to 592 nm for the film coated on KMAS. Thus the results authenticate that KMA influences the structural and optical properties of the deposited films.

  11. Functional imaging of proteases: recent advances in the design and application of substrate- and activity- based probes

    Edgington, Laura E.; Verdoes, Martijn; Bogyo, Matthew

    2011-01-01

    Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity...

  12. Shallow optically active structural defect in wurtzite GaN epilayers grown on stepped 4H-SiC substrates

    Xu, SJ; Xie, MH; Wang, HJ; Cheung, SH; Li, Q.; Dai, XQ; Tong, SY

    2003-01-01

    Shallow optically active structural defect in wurtzite GaN epilayers grown on stepped 4H-SiC substrates was investigated. The GaN epilayers grown with plasma-assisted molecular-beam epitaxy were optically characterized by photoluminescence and excitation spectra. Results showed that the localized states which were induced by the structural defect located about 100 meV above the maximum valence band of GaN.

  13. Restricted Active Site Docking by Enzyme-bound Substrate Enforces the Ordered Cleavage of Prothrombin by Prothrombinase*

    Hacisalihoglu, Ayse; Panizzi, Peter; Bock, Paul E.; Camire, Rodney M.; Krishnaswamy, Sriram

    2007-01-01

    The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg320 to produce meizothrombin, which is then cleaved at Arg271 to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in Vmax for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the indi...

  14. Effects of Hard Surface Grinding and Activation on Electroless-Nickel Plating on Cast Aluminium Alloy Substrates

    Olawale Olarewaju Ajibola

    2014-01-01

    Full Text Available This work examined effects of hard surface polishing grits and activation on electroless-nickel (EN plating on cast aluminium alloy substrates in sodium hypophosphite baths. As-received aluminium alloy sample sourced from automobile hydraulic brake master cylinder piston was melted in electric furnace and sand cast into rod. The cast samples were polished using different grits (60 μm–1200 μm before plating. The effects on adhesion, appearance, and quantity of EN deposits on substrates were studied. Observation shows that the quantity of EN deposit is partly dependent on the alloy type and roughness of the surface of the substrates, whereas the adhesion and brightness are not solely controlled by the degree of surface polishing. The best yield in terms of adhesion and appearance was obtained from the activation in zincate and palladium chloride solutions. Higher plating rates (g/mm2/min of 3.01E-05, 2.41E-05, and 2.90E-05 were obtained from chromate, zincate, and chloride than 8.49E-06, 8.86E-06, and 1.69E-05 as obtained from HCl etched, NaOH, and H2O activated surfaces, respectively.

  15. Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray.

    Jie Shi

    Full Text Available O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT and O-GlcNAcase (OGA. O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity.

  16. Modulation of Pantothenate Kinase 3 Activity by Small Molecules that Interact with the Substrate/Allosteric Regulatory Domain

    Leonardi, Roberta; Zhang, Yong-Mei; Yun, Mi-Kyung; Zhou, Ruobing; Zeng, Fu-Yue; Lin, Wenwei; Cui, Jimmy; Chen, Taosheng; Rock, Charles O.; White, Stephen W.; Jackowski, Suzanne (SJCH)

    2010-09-27

    Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3 acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.

  17. Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

    Németh, E.; Körtvélyesi, T.; Kožíšek, Milan; Thulstrup, P. W.; Christensen, H. E. M.; Asaka, M. N.; Nagata, K.; Gyurcsik, B.

    2014-01-01

    Roč. 19, č. 8 (2014), s. 1295-1303. ISSN 0949-8257 Grant ostatní: OPPC(XE) CZ.2.16/3.1.00/24016; Seventh Framework Programme of the European Union(XE) FP7-312284 Institutional support: RVO:61388963 Keywords : binding affinity * calorimetry * zinc nuclease * substrate induced folding * protein engineering Subject RIV: CE - Biochemistry Impact factor: 2.538, year: 2014

  18. Addition of Aromatic Substrates Restores Trichloroethylene Degradation Activity in Pseudomonas putida F1

    Morono, Yuki; Unno, Hajime; TANJI, Yasunori; Hori, Katsutoshi

    2004-01-01

    The rate of trichloroethylene (TCE) degradation by toluene dioxygenase (TDO) in resting cells of Pseudomonas putida F1 gradually decreased and eventually stopped within 1.5 h, as in previous reports. However, the subsequent addition of toluene, which is the principal substrate of TDO, resulted in its immediate degradation without a lag phase. After the consumption of toluene, degradation of TCE restarted at a rate similar to its initial degradation, suggesting that this degradation was mediat...

  19. Substrate dependent in vitro antifungal activity of Bacillus sp strain AR2

    Singh, Anil Kumar; Rautela, Ria; Cameotra, Swaranjit Singh

    2014-01-01

    Background Biosurfactants are a structurally diverse group of secondary metabolites with lots of potential to serve mankind. Depending upon the structure and composition they may exhibit properties that make them suitable for a particular application. Structural and compositional diversity of biosurfactant is unambiguously substrate dependent. The present study investigates the qualitative and quantitative effect of different water soluble carbon source on the biosurfactant produced by Bacill...

  20. Structure of active IspH enzyme from escherichia coli provides mechanistic insights into substrate reduction

    Gräwert, Tobias

    2009-07-20

    The terminal step of the non-mevalonate pathway of terpene biosynthesis is catalyzed by IspH (see scheme). In the crystal structure of IspH from E. coli, a bound inorganic diphosphate ligand occupies the position of the diphosphate residue of the substrate. Together with mutation studies and theoretical calculations, these data support a mechanism which is analogous to the Birch reduction of allylic alcohols. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

  1. Control of Substrate Access to the Active Site in Methane Monooxygenase

    Lee, Seung Jae; McCormick, Michael S.; Lippard, Stephen J.; Cho, Uhn-Soo

    2013-01-01

    Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrat...

  2. Gold nano-island arrays on silicon as SERS active substrate for organic molecule detection

    Gold islands forming highly controlled arrays have been fabricated by two potential step electrochemical deposition method using nanopatterned Si surface templates. In the present work, the Raman scattering studies realized using 11-mercaptoundecanoic probe molecule showed that such structures exhibit an enhanced Raman signal compared with nanostructured physical deposited thin gold film on flat silicon substrate and can be valued as surface-enhanced Raman scattering substrates. Besides the more appropriate management of nano-island arrays distribution, the high ratio of their Raman signals can be explain by the epitaxial-like growth mechanism of the metallic nano-islands, clearly showed by X-ray diffraction studies. Furthermore, the substrates enabled reproducibility and stability detection due to the chemically assembling of organothiol molecules, the X-ray photoelectron spectroscopy studies confirming formation of the thiolate species which corresponds to Au-S bonds, and also, the unwanted ‘hot-spots’ are missing, which make them suitable for high sensitivity biosensing applications. - Highlights: • Gold nano-islands are electrochemical deposited on nanopatterned silicon. • The X-ray diffraction studies revealed the epitaxial-like growth mechanism. • Enhanced Raman signal of Au nano-islands was observed compared with Au nano-film

  3. Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

    Dawn L Taylor

    Full Text Available Vibrio cholerae encodes six resistance-nodulation-division (RND efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels.

  4. Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

    Taylor, Dawn L; Ante, Vanessa M; Bina, X Renee; Howard, Mondraya F; Bina, James E

    2015-01-01

    Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels. PMID:25695834

  5. Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).

    Chufan, Eduardo E; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T; Durell, Stewart R; Ambudkar, Suresh V

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each

  6. Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1.

    Eduardo E Chufan

    Full Text Available P-glycoprotein (Pgp, ABCB1 is an ATP-Binding Cassette (ABC transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982 with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available

  7. β1肾上腺素受体与CYP2D6基因多态性对美托洛尔抗高血压治疗的药代动力学和药效学影响

    HU Jie; HU Zhao-qian; HU Ying-zi; TAN Zhi-rong; HU Dong-li; LI Zhi; WANG Dan; ZHANG Wei; ZHOU Hong-hao

    2007-01-01

    背景:美托洛尔是临床常用的抗高血压药物,它经由CYP2D6代谢。CYP2D6*10降低CYP2D6活性,是中国人群中最为常见的多态性。β1肾上腺素受体为美托洛尔的作用靶标,Ser49Gly与Gly389Arg多态性显著改变受体功能。CYP2D6与β1肾上腺素受体遗传多态性对美托洛尔降压疗效的联合影响仍属未知。目的:发现与美托洛尔药代动力学与药效动力学相关的基因多态性位点。为提高高血压病的疗效和减少不良反应提供实验依据。方法:符合WHO/ISH高血压诊断标准的轻、中度高血压患者125例,服用美托洛尔单药治疗12周,每四周检测血压。在临床观察疗效的同时,应用PCR-RFLP方法对患者进行CYP2D6*10与β1肾上腺素受体Ser49Gly和Gly389Arg基因型分析。同时抽取静脉血5mL,高效液相色谱-荧光检测法测定患者美托洛尔谷浓度。结果:美托洛尔谷浓度与CYP2D610基因型显著相关,并呈基因剂量效应。但高血压患者血压降低程度在CYP2D6*1*1、1*10与CYP2D6*10*10组间无差异。Gly49携带者服用美托洛尔后收缩压与舒张压的降低显著大于Ser49Ser纯合子;与Gly389携带者相比,Arg389Arg服用美托洛尔后收缩压与舒张压的降低更为显著,表明Gly49与Arg389型受体对美托洛尔治疗有较好的敏感性。结论:CYP2D6*10突变显著改变美托洛尔的药代动力学,但对美托洛尔的降压效果无显著性影响。β1肾上腺素受体遗传多态性与β受体阻滞药的降压敏感性有一定相关性。

  8. Fuel-Stimulated Insulin Secretion Depends upon Mitochondria Activation and the Integration of Mitochondrial and Cytosolic Substrate Cycles

    Gary W. Cline

    2011-10-01

    Full Text Available The pancreatic islet β-cell is uniquely specialized to couple its metabolism and rates of insulin secretion with the levels of circulating nutrient fuels, with the mitochondrial playing a central regulatory role in this process. In the β-cell, mitochondrial activation generates an integrated signal reflecting rates of oxidativephosphorylation, Kreb's cycle flux, and anaplerosis that ultimately determines the rate of insulin exocytosis. Mitochondrial activation can be regulated by proton leak and mediated by UCP2, and by alkalinization to utilize the pH gradient to drive substrate and ion transport. Converging lines of evidence support the hypothesis that substrate cycles driven by rates of Kreb's cycle flux and by anaplerosis play an integral role in coupling responsive changes in mitochondrial metabolism with insulin secretion. The components and mechanisms that account for the integrated signal of ATP production, substrate cycling, the regulation of cellular redox state, and the production of other secondary signaling intermediates are operative in both rodent and human islet β-cells.

  9. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  10. Chemically-modified graphene sheets as an active layer for eco-friendly metal electroplating on plastic substrates

    Oh, Joon-Suk; Hwang, Taeseon; Nam, Gi-Yong; Hong, Jung-Pyo [Department of Polymer Science and Engineering, Sungkyunkwan University, Chunchun-dong, Jangan-gu, Suwon, 440-746 (Korea, Republic of); Bae, Ah-Hyun; Son, Sang-Ik; Lee, Geun-Ho; Sung, Hak kyung [Manufacturing Tech. Center, Samsung Electronics Co., Ltd., Maetan-dong, Yeongtong-gu, Suwon, 443-742 (Korea, Republic of); Choi, Hyouk Ryeol; Koo, Ja Choon [School of Mechanical Engineering, Sungkyunkwan University, Suwon (Korea, Republic of); Nam, Jae-Do, E-mail: jdnam@skku.edu [Department of Polymer Science and Engineering, Sungkyunkwan University, Chunchun-dong, Jangan-gu, Suwon, 440-746 (Korea, Republic of); Department of Energy Science, Sungkyunkwan University, Suwon (Korea, Republic of)

    2012-10-30

    Eco-friendly nickel (Ni) electroplating was carried out on a plastic substrate using chemically modified graphene sheets as an active and conductive layer to initiate electroplating without using conventional pre-treatment or electroless metal-seeding processes. A graphene oxide (GO) solution was self-assembled on a polyethylene terephthalate (PET) film followed by evaporation to give GO layers (thickness around 6.5 {mu}m) on PET (GO/PET) film. Then, the GO/PET film was chemically and thermally reduced to convert the GO layers to reduced graphene oxide (RGO) layers on the PET substrate. The RGO-coated PET (RGO/PET) film showed the sheet resistance of 100 {Omega} per square. On RGO/PET film, Ni electroplating was conducted under the constant-current condition and the entire surface of the PET film was completely metalized with Ni without any voids.

  11. Salinosporamides A and B Inhibit Proteasome Activity and Delay the Degradation of N-end Rule Model Substrates

    Shin, Seungkyun; Bang, Daein; Choi, Wonhoon; Lee, Minjae [Kyung Hee Univ., Yongin (Korea, Republic of); Kim, Seonghwan; Oh, Dongchan [Seoul National Univ., Seoul (Korea, Republic of)

    2013-05-15

    The proteasome, which is highly evolutionarily conserved, is responsible for the degradation of most short-lived proteins in cells. Small-molecule inhibitors targeting the proteasome's degradative activity have been extensively developed as lead compounds for various human diseases. An exemplified molecule is bortezomib, which was approved by FDA in 2003 for the treatment of multiple myeloma. Here, using transiently and stably expressed N-end rule model substrates in mammalian cells, we evaluated and identified that salinosporamide A and salinosporamide B effectively inhibited the proteasomal degradation. Considering that a variety of proteasome substrates are implicated in the pathogenesis of many diseases, they have the potential to be clinically applicable as therapeutic agents.

  12. Chemically-modified graphene sheets as an active layer for eco-friendly metal electroplating on plastic substrates

    Eco-friendly nickel (Ni) electroplating was carried out on a plastic substrate using chemically modified graphene sheets as an active and conductive layer to initiate electroplating without using conventional pre-treatment or electroless metal-seeding processes. A graphene oxide (GO) solution was self-assembled on a polyethylene terephthalate (PET) film followed by evaporation to give GO layers (thickness around 6.5 μm) on PET (GO/PET) film. Then, the GO/PET film was chemically and thermally reduced to convert the GO layers to reduced graphene oxide (RGO) layers on the PET substrate. The RGO-coated PET (RGO/PET) film showed the sheet resistance of 100 Ω per square. On RGO/PET film, Ni electroplating was conducted under the constant-current condition and the entire surface of the PET film was completely metalized with Ni without any voids.

  13. Effects of the propeptide of group X secreted phospholipase A(2) on substrate specificity and interfacial activity on phospholipid monolayers.

    Point, Vanessa; Bénarouche, Anaïs; Jemel, Ikram; Parsiegla, Goetz; Lambeau, Gérard; Carrière, Frédéric; Cavalier, Jean-François

    2013-01-01

    Group X secreted phospholipase A(2) (GX sPLA(2)) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA(2) activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA(2) activity of recombinant mouse GX sPLA(2) (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA(2) activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30-35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA(2) activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA(2). This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance. PMID:22967966

  14. Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples

    Coêlho, Diego F.; Thais Peron Saturnino; Fernanda Freitas Fernandes; Priscila Gava Mazzola; Edgar Silveira; Elias Basile Tambourgi

    2016-01-01

    Given the importance of protease’s worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit o...

  15. Substrate specificity of Micrococcus luteus uv endonuclease and its overlap with DNA photolyase activity

    The action of an endonuclease from Micrococcus luteus that operates on uv damage in DNA overlaps with that of DNA photolyase from yeast: homo- and heterocyclobutane dipyrimidines in DNA are substrates for both enzymes, but pyrimidine adducts or the spore photoproduct in DNA are not. As expected from this overlap, the action of the two enzymes is mutually interfering: single-strand nicks introduced by the endonuclease effectively preclude photoreactivation; conversely, formation of a photolyase-cyclobutane dipyrimidine complex can prevent nicking by the endonuclease

  16. Investigation of photocatalytic activity of titanium dioxide coating deposited on aluminium alloy substrate by plasma technique

    Daviðsdóttir, Svava; Soyama, Juliano; Dirscherl, Kai;

    2011-01-01

    Nowadays, there is an increased need for functionalized surfaces with self-cleaning and antibacterial properties. Titanium dioxide (TiO2) in the anatase crystalline structure is one of the most powerful photocatalytic materials available today, which can provide above functionalities. The....... Literature consists of large number of publications on titanium dioxide coating for self-cleaning applications, with glass as the main substrate. Only little work is available on TiO2 coating of metallic alloys used for engineering applications. Engineering materials, such as light-weight aluminium and steel...

  17. A Fungal α-Galactosidase from Tricholoma matsutake with Broad Substrate Specificity and Good Hydrolytic Activity on Raffinose Family Oligosaccharides

    Xueran Geng

    2015-07-01

    Full Text Available An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS, indicating the importance of tryptophan residue(s at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.

  18. Physiological characterization of the ARO10-dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces cerevisiae.

    Vuralhan, Zeynep; Luttik, Marijke A H; Tai, Siew Leng; Boer, Viktor M; Morais, Marcos A; Schipper, Dick; Almering, Marinka J H; Kötter, Peter; Dickinson, J Richard; Daran, Jean-Marc; Pronk, Jack T

    2005-06-01

    Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity. PMID:15933030

  19. Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples

    Diego F. Coêlho

    2016-01-01

    Full Text Available Given the importance of protease’s worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM. We also quantified the limit of detection (LoD and limit of quantification (LoQ in the above-mentioned optimum (0.072 and 0.494 mg·mL−1 of azocasein, resp. and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%, which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise.

  20. Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples.

    Coêlho, Diego F; Saturnino, Thais Peron; Fernandes, Fernanda Freitas; Mazzola, Priscila Gava; Silveira, Edgar; Tambourgi, Elias Basile

    2016-01-01

    Given the importance of protease's worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL(-1) of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise. PMID:26925415

  1. Substrate-mediated enhanced activity of Ru nanoparticles in catalytic hydrogenation of benzene

    Liu, Xin

    2012-01-01

    The impact of carbon substrate-Ru nanoparticle interactions on benzene and hydrogen adsorption that is directly related to the performance in catalytic hydrogenation of benzene has been investigated by first-principles based calculations. The stability of Ru 13 nanoparticles is enhanced by the defective graphene substrate due to the hybridization between the dsp states of the Ru 13 particle with the sp 2 dangling bonds at the defect sites. The local curvature formed at the interface will also raise the Ru atomic diffusion barrier, and prohibit the particle sintering. The strong interfacial interaction results in the shift of averaged d-band center of the deposited Ru nanoparticle, from -1.41 eV for a freestanding Ru 13 particle, to -1.17 eV for the Ru/Graphene composites, and to -1.54 eV on mesocellular foam carbon. Accordingly, the adsorption energies of benzene are increased from -2.53 eV for the Ru/mesocellular foam carbon composites, to -2.62 eV on freestanding Ru 13 particles, to -2.74 eV on Ru/graphene composites. A similar change in hydrogen adsorption is also observed, and all these can be correlated to the shift of the d-band center of the nanoparticle. Thus, Ru nanoparticles graphene composites are expected to exhibit both high stability and superior catalytic performance in hydrogenation of arenes. © 2012 The Royal Society of Chemistry.

  2. The inhibition of major human hepatic cytochrome P450 enzymes by 18 pesticides: comparison of the N-in-one and single substrate approaches.

    Abass, Khaled; Pelkonen, Olavi

    2013-08-01

    In the present study on human hepatic microsomes, the N-in-one assay with ten probe substrates for nine cytochrome-P450 enzymes (CYPs) was compared with the single substrate assays to investigate pesticides-CYP interactions. CYP inhibition was measured by liquid chromatography-tandem mass spectrometry (LC/MS-MS). As illustrated by the initial screening at 100 μM concentration of 18 pesticides, CYPs are more sensitive to organophosphates (OPs) than to other pesticide groups. Chlorpyrifos and fenitrothion were most effective in inhibiting CYP1A1/2, and CYP2B6. Profenofos was also inhibitory towards multiple CYPs. Pyrethroids, e.g. deltamethrin, fenvalerate and lambda-cyhalothrin, potently inhibited CYP2D6. CYP3A4 activity was moderately inhibited by fenvalerate and potently by alpha-cypermethrin. The correlations between IC50 values obtained from the N-in-one and single substrate approaches were highly significant for CYP2Cs (r(2)=0.94), CYP3A4, omeprazole-sulfoxidation, (r(2)=0.89), followed by CYP1A2 and CYP2B6 (r(2)=0.82), and CYP2D6 (r(2)=0.80). In contrast no correlation was observed with CYP2E1 and CYP3A4 (midazolam-1'-hydroxylation). The N-in-one screening assay seems useful and reliable for most CYP activities when a comprehensive and quick evaluation of potential interactions with CYPs is needed. However, at the present moment, it does not enable discrimination on the basis of mechanism of inhibition. A strict comparison between single and N-in-one assays is a prerequisite for more extensive routine use. PMID:22634058

  3. Control of substrate oxidation in MOD ceramic coating on low-activation ferritic steel with reduced-pressure atmosphere

    Tanaka, Teruya, E-mail: teru@nifs.ac.jp; Muroga, Takeo

    2014-12-15

    Highlights: • A Cr{sub 2}O{sub 3} layer was produced on a ferritic steel substrate with a reduced-pressure. • The Cr{sub 2}O{sub 3} layer prevents further substrate oxidation in following coating process. • The Cr{sub 2}O{sub 3} layer has a function as a hydrogen permeation barrier. • A smooth MOD Er{sub 2}O{sub 3} coating was successfully made on the Cr{sub 2}O{sub 3} layer by dip coating. • The Cr{sub 2}O{sub 3} layer would enhance flexibility in MOD coating process and performances. - Abstract: An Er{sub 2}O{sub 3} ceramic coating fabricated using the metal–organic decomposition (MOD) method on a Cr{sub 2}O{sub 3}-covered low-activation ferritic steel JLF-1 substrate was examined to improve hydrogen permeation barrier performance of the coating. The Cr{sub 2}O{sub 3} layer was obtained before coating by heat treating the substrate at 700 °C under reduced pressures of <5 × 10{sup −3} Pa and 5 Pa. The Cr{sub 2}O{sub 3} layer was significantly stable even with heat treatment at 700 °C in air. This layer prevented further production of Fe{sub 2}O{sub 3}, which has been considered to degrade coating performance. An MOD Er{sub 2}O{sub 3} coating with a smooth surface was successfully obtained on a Cr{sub 2}O{sub 3}-covered JLF-1 substrate by dip coating followed by drying and baking. Preprocessing to obtain a Cr{sub 2}O{sub 3} layer would provide flexibility in the coating process for blanket components and ducts. Moreover, the Cr{sub 2}O{sub 3} layer suppressed hydrogen permeation through the JLF-1 substrate. While further optimization of the coating fabrication process is required, it would be possible to suppress hydrogen permeation significantly by multilayers of Cr{sub 2}O{sub 3} and MOD oxide ceramic.

  4. Microbial community dynamics linked to enhanced substrate availability and biogas production of electrokinetically pre-treated waste activated sludge.

    Westerholm, Maria; Crauwels, Sam; Houtmeyers, Sofie; Meerbergen, Ken; Van Geel, Maarten; Lievens, Bart; Appels, Lise

    2016-10-01

    The restricted hydrolytic degradation rate of complex organic matter presents a considerable challenge in anaerobic digestion of waste activated sludge (WAS). Within this context, application of pre-treatment of digester substrate has potential for improved waste management and enhanced biogas production. Anaerobic degradation of untreated or electrokinetically pre-treated WAS was performed in two pilot-scale digesters for 132days. WAS electrokinetically pre-treated with energy input 0.066kJ/kg sludge was used in a first phase of operation and WAS pre-treated with energy input 0.091kJ/kg sludge was used in a second phase (each phase lasted at least three hydraulic retention times). Substrate characteristics before and after pre-treatment and effects on biogas digester performance were comprehensively analysed. To gain insights into influences of altered substrate characteristics on microbial communities, the dynamics within the bacterial and archaeal communities in the two digesters were investigated using 16S rRNA gene sequencing (pyrosequencing) and quantitative PCR (qPCR). Specific primers targeting dominant operation taxonomic units (OTUs) and members of the candidate phylum Cloacimonetes were designed to further evaluate their abundance and dynamics in the digesters. Electrokinetic pre-treatment significantly improved chemical oxygen demand (COD) and carbohydrate solubility and increased biogas production by 10-11% compared with untreated sludge. Compositional similarity of the bacterial community during initial operation and diversification during later operation indicated gradual adaptation of the community to the higher solubility of organic material in the pre-treated substrate. Further analyses revealed positive correlations between gene abundance of dominant OTUs related to Clostridia and Cloacimonetes and increased substrate availability and biogas production. Among the methanogens, the genus Methanosaeta dominated in both digesters. Overall, the

  5. Aromatic Amino Acid Mutagenesis at the Substrate Binding Pocket of Yarrowia lipolytica Lipase Lip2 Affects Its Activity and Thermostability

    Guilong Wang

    2014-01-01

    Full Text Available The lipase2 from Yarrowia lipolytica (YLLip2 is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100 with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM to introduce aromatic amino acid mutations. Two mutants (V94W and I100F were created. The enzymatic properties of the mutant lipases were detected and compared with the wild-type. The activities of mutant enzymes dropped to some extent towards p-nitrophenyl palmitate (pNPC16 and their optimum temperature was 35°C, which was 5°C lower than that of the wild-type. However, the thermostability of I100F increased 22.44% after incubation for 1 h at 40°C and its optimum substrate shifted from p-nitrophenyl laurate (pNPC12 to p-nitrophenyl caprate (pNPC10. The above results demonstrated that the two substituted amino acid residuals have close relationship with such enzymatic properties as thermostability and substrate selectivity.

  6. A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser-His-Asp Containing C-C Bond Hydrolase

    Ruzzini, Antonio C.; Bhowmik, Shiva; Ghosh, Subhangi; Yam, Katherine C.; Bolin, Jeffrey T.; Eltis, Lindsay D. [Purdue; (UBC)

    2013-11-12

    The meta-cleavage product (MCP) hydrolases utilize a Ser–His–Asp triad to hydrolyze a carbon–carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ESred, which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ESred decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2nuc ~ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His–Asp pair does not play an essential role. The data further suggest that ESred represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

  7. An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.

    Oliveira, G S; Ulhoa, C J; Silveira, M H L; Andreaus, J; Silva-Pereira, I; Poças-Fonseca, M J; Faria, F P

    2013-01-01

    Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl β-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation. PMID:23054694

  8. Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities.

    Gasparello-Clemente, Elaine; Silveira, Paulo Flávio

    2002-11-01

    In the present study we examined the feasibility of using the fluorometry of naphthylamine derivatives for revealing peptidase activities in venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpions Tityus serrulatus and Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. Neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) activities were presented in all snake venoms, with the highest levels in B. alternatus. Although all examined peptidase activities showed relatively low levels in arthropod venoms, basic aminopeptidase (APB) activity from P. nigriventer venom was the exception. Compared to the other peptidase activities, relatively high levels of acid aminopeptidase (APA) activity were restricted to B. arietans venom. B. arietans also exhibited a prominent content of APB activity which was lower in other venoms. Relatively low prolyl endopeptidase and proline iminopeptidase activities were, respectively, detectable only in T. bahiensis and B. insularis. Pyroglutamate aminopeptidase activity was undetectable in all venoms. All examined peptidase activities were undetectable in T. serrulatus venom. In this study, the specificities of a diverse array of peptidase activities from representative venoms were demonstrated for the first time, with a description of their distribution which may contribute to guiding further investigations. The expressive difference between snake and arthropod venoms was indicated by APN and DPP IV activities while APA and APB activities distinguished the venom of B. arietans from those of Brazilian snakes. The data reflected the relatively uniform qualitative distribution of the peptidase activities investigated, together with their unequal quantitative distribution, indicating the evolutionary divergence in the processing of peptides in these different

  9. ACTIVITIES OF AMMONIA ASSIMILATION ENZYMES AS INDICATORS OF THE RELATIVE SUPPLY OF NITROGEN SUBSTRATES FOR MARINE BACTERIOPLANKTON IN SUB-TROPICAL COASTAL WATER

    The supply of nitrogen substrates available for bacterial production in seawater was determined using the activities of ammonia assimilation enzymes, glutamine synthetase (GS) and glutamate dehydrogenase (GDH). Expression of GS and GDH by bacteria in pure culture is generally ind...

  10. Simultaneous LC-MS/MS analysis of the plasma concentrations of a cocktail of 5 cytochrome P450 substrate drugs and their metabolites.

    Tanaka, Shimako; Uchida, Shinya; Inui, Naoki; Takeuchi, Kazuhiko; Watanabe, Hiroshi; Namiki, Noriyuki

    2014-01-01

    A "cocktail" approach, which involves simultaneous administration of multiple CYP-specific probes, concurrently detects the activity of multiple CYP enzymes. We developed and validated a rapid and selective LC-MS/MS method for determining the plasma concentrations of 5 CYP probe drugs and metabolites (caffeine/paraxanthine, CYP1A2 substrate; losartan/losartan carboxylic acid (E3174), CYP2C9 substrate; omeprazole/5-hydroxyomeprazole, CYP2C19 substrate; dextromethorphan/dextrorphan, CYP2D6 substrate; and midazolam/1'-hydroxymidazolam, CYP3A4 substrate) by single-step extraction, followed by a single LC-MS/MS run. An Ostro™ 96-well plate was used for extraction of CYP substrates and metabolites from human plasma and urine. Following optimization of the chromatographic conditions, all the peaks were well separated, and retention times ranged between 4.4 and 11.7 min. The total run time for a single injection was within 13 min. The accuracy and precision values suggested that the assay had high accuracy and reliability in plasma and urine samples. No significant matrix interference was observed. To demonstrate the efficacy of this method, plasma and urine concentrations of 5 CYP probe substrates and their metabolites were determined after simultaneous oral administration of 5 drugs to 4 healthy volunteers. All the substrates and metabolites were detected over an 8 h period, and the plasma concentrations of each substrate at 8 h after administration were above the lower limit of quantification. Urine concentrations of drugs and their metabolic ratio were evaluated after the administration. In conclusion, the advantage of our cocktail approach is that it enables in vivo assessment of the activity of various drug-metabolizing enzymes in a single assay. PMID:24389476

  11. Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity.

    Zandonella, G; Haalck, L; Spener, F; Faber, K; Paltauf, F; Hermetter, A

    1996-01-01

    A new type of fluorogenic alkyldiacyl glycerols was synthesized and used as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain perylene as a fluorophore and the trinitrophenylamino (TNP) residue as a quencher. Both substituents are covalently bound to the omega-ends of the sn-2 and sn-1 (3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase activity by the continuous increase in fluorescence intensity which is caused by dequenching. Using enantiomeric pairs of these compounds, we were able to analyze lipase stereoselectivity depending on the reaction medium. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labelled with pyrene or perylene as fluorophores, can be used for a dual-wavelength "stereoassay" of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be determined simultaneously, and the difference in the rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase. Hydrolysis rates measured with perylene-substituted lipids are generally lower than those obtained with the pyrene analogs. Thus, with a mixture of perylene and pyrene-substituted lipids, we observe a higher apparent stereoselectivity of lipases since we measure a combination of stereo- and substrate selectivity. In the presence of albumin, all microbial lipases tested so far exhibit stereopreference for the sn-1 glycerol position. In our assay, the apparent stereoselectivities are highest if in the presence of albumin, the sn-1 position carries pyrene and the sn-3 position is substituted with perylene. The lipase stereoselectivity assay described here requires the simultaneous measurement of the fluorescence intensities at two different wavelengths in a single cuvette and can thus be carried out using existing and cheap

  12. A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method.

    Mustafa, Ahmad; Karmali, Amin; Abdelmoez, Wael

    2016-01-01

    The present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR pH was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37°C and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4°C compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations. PMID:27581492

  13. Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

    Németh, Eszter; Körtvélyesi, Tamás; Kožíšek, Milan;

    2014-01-01

    The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn(2+)-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues res...

  14. Arrayed SU-8 polymer thermal actuators with inherent real-time feedback for actively modifying MEMS’ substrate warpage

    Wang, Xinghua; Xiao, Dingbang; Chen, Zhihua; Wu, Xuezhong

    2016-09-01

    This paper describes the design, fabrication and characterization of a batch-fabricated micro-thermal actuators array with inherent real-time self-feedback, which can be used to actively modify micro-electro-mechanical systems’ (MEMS’) substrate warpage. Arrayed polymer thermal actuators utilize SU-8 polymer (a thick negative photoresist) as a functional material with integrated Ti/Al film-heaters as a microscale heat source. The electro-thermo-mechanical response of a micro-fabricated actuator was measured. The resistance of the Al/Ti film resistor varies obviously with ambient temperature, which can be used as inherent feedback for observing real-time displacement of activated SU-8 bumps (0.43 μm Ω‑1). Due to the high thermal expansion coefficient, SU-8 bumps tend to have relatively large deflection at low driving voltage and are very easily integrated with MEMS devices. Experimental results indicated that the proposed SU-8 polymer thermal actuators (array) are able to achieve accurate rectification of MEMS’ substrate warpage, which might find potential applications for solving stress-induced problems in MEMS.

  15. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A. [Brookhaven National Lab., Upton, NY (United States). Biology Dept.; Lees-Miller, S.P.; Lintott, L.G. [Univ. of Calgary, Alberta (Canada). Dept. of Biological Sciences; Sakaguchi, Kazuyasu; Appella, E. [National Institutes of Health, Bethesda, MD (United States). Lab. of Cell Biology

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  16. Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

    Beverly Z Packard; Akira Komoriya

    2008-01-01

    Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clinical efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.

  17. AKTIVITAS HIALURONIDASE BAKTERI STREPTOKOKUS GRUP B PADA SUBSTRAT ASAM HIALURONAT THE HYALURONIDASE ACTIVITY OF GROUP B STREPTOCOCCUS IN HYALURONIC ACID SUBSTRATE

    Zinatul Hayati

    2007-06-01

    Full Text Available Streptococcus agalactiae atau yang juga dikenal dengan Streptokokus grup B (SGB telah diketahui sebagai agen penyebab pneumonia, septikemia dan meningitis neonatal pada manusia dan hewan. Hialuronidase merupakan produk ekstraseluler dari SGB yang menentukan virulensinya. Penelitian ini dilakukan untuk menguji akitvitas hialuronidase dari 10 isolat SGB yang diisolasi dari kasus komplikase obstetri, serta memurnikan dan mengkarakterisasi hialuronidase yang diisolasi dari SGB SV-14 pada substrat asam hialuronat.

  18. Nanostructure, solvation dynamics, and nanotemplating of plasmonically active SERS substrate in reverse vesicles

    Saha, Ranajay; Rakshit, Surajit [S.N. Bose National Centre for Basic Sciences, Department of Chemical, Biological and Macromolecular Sciences (India); Majumdar, Dipanwita; Singha, Achintya [Bose Institute, Department of Physics (India); Mitra, Rajib Kumar; Pal, Samir Kumar, E-mail: skpal@bose.res.in [S.N. Bose National Centre for Basic Sciences, Department of Chemical, Biological and Macromolecular Sciences (India)

    2013-04-15

    Reverse vesicles (RVs) are the organic counterparts to vesicles and are spherical containers in oils consisting of an oily core surrounded by reverse bilayers with water layers present in between. We present here a facile route for forming stable RV from nontoxic surfactants and oil components. The RV formation is characterized by dynamic light scattering and further confirmed by transmission electron microscopic (TEM) techniques. The water channels present in between the bilayers are found to be a potential template for inorganic nanoparticles' (NPs) synthesis. Both the UV-Vis absorption spectroscopy and the TEM study reveal successful formation of highly clustered silver NPs within the water layers of the RVs. X-ray powder diffraction analyzes the crystalline nature of the NPs. FTIR spectroscopy shows the signature of different kinds of water molecules in between the RV bilayers. The dynamical description of the templating water, dictating the controlled formation of the NPs in the RV, is well revealed in the picosecond-resolved solvation dynamics study of a hydrophilic fluorescence probe 2 Prime -(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) -benzimidazo-2-yl-benzimidazole] (H258). The rotational anisotropy study successfully describes geometrical restriction of the probe molecule in the RV. Notably, this study provides the first proof-of-concept data for the ability of the RV to be a template of synthesizing metal NPs. The as-prepared NP clusters are evaluated to be potential surface-enhanced Raman scattering substrate in solution using crystal violet as a model analyte. The present study offers a new RV, which is a prospective nontoxic nanotemplate and is believed to contribute potentially in the emerging NP-vesicle hybrid assembly-based plasmonic applications.Nanotemplating of metal clusters for the efficient SERS detection in liquid phase is reported in a new nontoxic reverse vesicle.

  19. Nanostructure, solvation dynamics, and nanotemplating of plasmonically active SERS substrate in reverse vesicles

    Reverse vesicles (RVs) are the organic counterparts to vesicles and are spherical containers in oils consisting of an oily core surrounded by reverse bilayers with water layers present in between. We present here a facile route for forming stable RV from nontoxic surfactants and oil components. The RV formation is characterized by dynamic light scattering and further confirmed by transmission electron microscopic (TEM) techniques. The water channels present in between the bilayers are found to be a potential template for inorganic nanoparticles’ (NPs) synthesis. Both the UV–Vis absorption spectroscopy and the TEM study reveal successful formation of highly clustered silver NPs within the water layers of the RVs. X-ray powder diffraction analyzes the crystalline nature of the NPs. FTIR spectroscopy shows the signature of different kinds of water molecules in between the RV bilayers. The dynamical description of the templating water, dictating the controlled formation of the NPs in the RV, is well revealed in the picosecond-resolved solvation dynamics study of a hydrophilic fluorescence probe 2′-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) -benzimidazo-2-yl-benzimidazole] (H258). The rotational anisotropy study successfully describes geometrical restriction of the probe molecule in the RV. Notably, this study provides the first proof-of-concept data for the ability of the RV to be a template of synthesizing metal NPs. The as-prepared NP clusters are evaluated to be potential surface-enhanced Raman scattering substrate in solution using crystal violet as a model analyte. The present study offers a new RV, which is a prospective nontoxic nanotemplate and is believed to contribute potentially in the emerging NP-vesicle hybrid assembly-based plasmonic applications.Nanotemplating of metal clusters for the efficient SERS detection in liquid phase is reported in a new nontoxic reverse vesicle.

  20. Wetland restoration and methanogenesis: the activity of microbial populations and competition for substrates at different temperatures

    V. Jerman

    2009-02-01

    Full Text Available Ljubljana marsh in Slovenia is a 16 000 ha area of partly drained fen, intended to be flooded to restore its ecological functions. The resultant water-logging may create anoxic conditions, eventually stimulating production and emission of methane, the most important greenhouse gas next to carbon dioxide. We examined the upper layer (~30 cm of Ljubljana marsh soil for microbial processes that would predominate in water-saturated conditions, focusing on the potential for iron reduction, carbon mineralization (CO2 and CH4 production, and methane emission. Methane emission from water-saturated microcosms was near minimum detectable levels even after extended periods of flooding (>5 months. Methane production in anoxic soil slurries started only after a lag period and was inversely related to iron reduction, which suggested that iron reduction out-competed methanogenesis for electron donors, such as H2 and acetate. Methane production was observed only in samples incubated at 14–38°C. At the beginning of methanogenesis, acetoclastic methanogenesis dominated. In accordance with the preferred substrate, most (91% mcrA (encoding the methyl coenzyme-M reductase, a key gene in methanogenesis clone sequences could be affiliated to the acetoclastic genus Methanosarcina. No methanogens were detected in the original soil. However, a diverse community of iron-reducing Geobacteraceae was found. Our results suggest that methane emission can remain transient and low if water-table fluctuations allow re-oxidation of ferrous iron, sustaining iron reduction as the most important process in terminal carbon mineralization.

  1. Activation of ion-implanted polycrystalline silicon thin films prepared on glass substrates

    Phosphorous-implanted polycrystalline Si thin films were subjected to thermal annealing between 300 °C and 650 °C. The thermal activation was monitored electrically and structurally using Hall measurements, Raman spectroscopy, UV–visible spectrophotometry, and transmission electron microscopy. Charge transport information was correlated to the corresponding structural evolution in thermal activation. Phosphorous-implanted activation is divided into short-range ordering at low temperatures and long-range ordering at high temperatures, with the boundary between low and high temperatures set at 425 °C. Short-range ordering allows for significant increase in electronic concentration through substitution of P for Si. Higher temperatures are attributed to long-range ordering, thereby increasing electronic mobility.

  2. Influence of soil management practices and substrate availability on microbial biomass and its activities in some haplic luvisols

    Friedel, Jurgen K. [University Hohenheeim, Stuttgart (Germany)

    1996-07-01

    Soil microbial biomass and activities are sensitive indicators of management effects. Higher contents of microbial biomass and higher activities, for example, are found with crop rotations in contrast to bare fallow and mono culture systems. The main reason for these differences is a higher input of crop and root residues in crop rotation systems, leading to more microbial available substrate. The objectives of this study were to describe indices for microbial available substrate in arable soils depending on management practices, and to relate them with soil microbial biomass and activities. At two locations (Muttergarten and hinger Hof near the University of Hohenheim, Stuttgart, SW-Germany), adenosine triphosphate (ATP) contents and microbial activities were measured in haplic Luviosls. As indices for microbial available substrate, water soluble organic carbon compounds in soils were determined and decomposable young soil organic matter was calculated from organic fertilizers and crop and root residues using empirical decomposition functions. Higher ATP contents and microbial activities were observed along with organic fertilization (liquid cattle manure) than with mineral fertilization. Shallow cultivation with a rotary cultivator led to higher values of microbial properties in the upper part of the Ap horizon than ploughing. Soil microbial parameters were higher in plots under a rape-cereals crop rotation, compared to a legumes-cereals crop rotation. Microbial biomass and its activities were related more closely to decomposable young soil organic matter than to soil humus content or to any other soil property. Water soluble organic carbon compounds did not prove as an indicator of microbial available substrate. [Spanish] La biomasa y la actividad microbianas son indicadores sensibles de los efectos del manejo del suelo. Por ejemplo, con la rotacion de cultivos se obtiene un contenido y una actividad mayores de la biomasa microbiana en contraste con el simple

  3. An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase

    True, A.E.; Orville, A.M.; Pearce, L.L.; Lipscomb, J.D.; Que, L. Jr. (Univ. of Minnesota, Minneapolis (USA))

    1990-12-01

    X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 {angstrom} and 2 O/N ligands at 2.08 {angstrom}. The 2.08-{angstrom} bonds are assigned to the two histidines, while the 1.90-{angstrom} bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 {angstrom}. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions. For the iron site to remain 5-coordinate and the HPCA to be chelated to the iron, the substrate must displace not only the hydroxide but also a ligand from the protein backbone, probably a histidine. The mechanistic implications of the displacement of hydroxide and a protein ligand in the active site are discussed.

  4. Effects of negative air ions on activity of neural substrates involved in autonomic regulation in rats

    Suzuki, Satoko; Yanagita, Shinya; Amemiya, Seiichiro; Kato, Yumi; Kubota, Natsuko; Ryushi, Tomoo; Kita, Ichiro

    2008-07-01

    The neural mechanism by which negative air ions (NAI) mediate the regulation of autonomic nervous system activity is still unknown. We examined the effects of NAI on physiological responses, such as blood pressure (BP), heart rate (HR), and heart rate variability (HRV) as well as neuronal activity, in the paraventricular nucleus of the hypothalamus (PVN), locus coeruleus (LC), nucleus ambiguus (NA), and nucleus of the solitary tract (NTS) with c-Fos immunohistochemistry in anesthetized, spontaneously breathing rats. In addition, we performed cervical vagotomy to reveal the afferent pathway involved in mediating the effects of NAI on autonomic regulation. NAI significantly decreased BP and HR, and increased HF power of the HRV spectrum. Significant decreases in c-Fos positive nuclei in the PVN and LC, and enhancement of c-Fos expression in the NA and NTS were induced by NAI. After vagotomy, these physiological and neuronal responses to NAI were not observed. These findings suggest that NAI can modulate autonomic regulation through inhibition of neuronal activity in PVN and LC as well as activation of NA neurons, and that these effects of NAI might be mediated via the vagus nerves.

  5. Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases.

    Dayalan Naidu, Sharadha; Sutherland, Calum; Zhang, Ying; Risco, Ana; de la Vega, Laureano; Caunt, Christopher J; Hastie, C James; Lamont, Douglas J; Torrente, Laura; Chowdhry, Sudhir; Benjamin, Ivor J; Keyse, Stephen M; Cuenda, Ana; Dinkova-Kostova, Albena T

    2016-09-15

    Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response. PMID:27354066

  6. Human intestinal P-glycoprotein activity estimated by the model substrate digoxin

    Larsen, U L; Hyldahl Olesen, L; Nyvold, Charlotte Guldborg;

    2007-01-01

    P-glycoprotein (Pgp) plays a part in the intestinal uptake of xenobiotics and has been associated with susceptibility to ulcerative colitis. The aim of this study was to examine Pgp activity in relation to age, gender, medical treatment (rifampicin or ketoconazole) and the multidrug resistance (M...

  7. B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

    Mohammad, Dara K.; Ali, Raja H.; Turunen, Janne J.; Nore, Beston F.; Smith, C. I. Edvard

    2016-01-01

    Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins. PMID:27487157

  8. Programmable SERS active substrates for chemical and biosensing applications using amorphous/crystalline hybrid silicon nanomaterial

    Jeffery Alexander Powell; Krishnan Venkatakrishnan; Bo Tan

    2016-01-01

    We present the creation of a unique nanostructured amorphous/crystalline hybrid silicon material that exhibits surface enhanced Raman scattering (SERS) activity. This nanomaterial is an interconnected network of amorphous/crystalline nanospheroids which form a nanoweb structure; to our knowledge this material has not been previously observed nor has it been applied for use as a SERS sensing material. This material is formed using a femtosecond synthesis technique which facilitates a laser plu...

  9. CHEMICAL, PHYSICAL AND SENSORY ANALYSIS OF ACTIVITY DIFFERENT YEAST SPECIES ON IDENTICAL SUBSTRATE IN WINE PRODUCTION

    Vladimír Vietoris; Hana Balková,Tatiana Bojňanská; Ľubomír Bennár; Peter Czako

    2013-01-01

    Rizling vlašský is the second most important variety in Slovakia. The science of wine production includes a summary of knowledge and experience in the field of grape growing and wine making, or the production of different types of wines using specific methods of production. Wine quality is the result of the interaction between yeast, bacteria and microscopic funguses. In this research, we studied the effects of active dry wine yeasts on chemical, physical and sensory parameters in wine produc...

  10. Structure of Human Acid Sphingomyelinase Reveals the Role of the Saposin Domain in Activating Substrate Hydrolysis.

    Xiong, Zi-Jian; Huang, Jingjing; Poda, Gennady; Pomès, Régis; Privé, Gilbert G

    2016-07-31

    Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction. PMID:27349982

  11. The improvement of fermentation activity of yeast and mould by gamma irradiation on irradiated sago and onggok substrates

    For the purpose of increasing the product of fermentation and enzyme activity produced by microorganism, an experiment has been carried out using irradiated yeast and mould for fermenting irradiated sago (Metroxylon sago) and tapioca waste (onggok). Sago and onggok starches were irradiated with gamma-rays with a dose of 25 kGy. Onggok starch was irradiated on dried and wet conditions. Local isolated yeast and mould (R. oryza e) were irradiated in suspension with doses of 0.4 and 4 kGy. The measurement of enzymes activities such as amylase, AMG, cellulase and protease, and the fermentation products i.e. glucose and reductase d glucose were carried out for analyzing the influence of radiation on the fermentation process. The fermentation using yeast and onggok substrate produced glucose 28 % higher than sago. On the contrary, the fermentation of sago increased the production of reductase glucose by 7.3% higher than that of onggok. At the end of experiment, on the 14th. however, the activity of cellulase enzyme produced by irradiated mould in the fermentation with dried and wet onggok increase 13 to 15 times higher than the activity enzyme produced by the control. In a similar way fermentation of irradiated dry onggok produced amylase 57.9% higher than that on irradiated wet onggok. (author), 16 refs., 2 figs

  12. Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

    Styles, Nathan A; Shonsey, Erin M; Falany, Josie L; Guidry, Amber L; Barnes, Stephen; Falany, Charles N

    2016-07-01

    Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with β-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity. PMID:27230263

  13. Synthesis and characterization of Ag@Cu nano/microstructure ordered arrays as SERS-active substrates

    Zhang, Pinhua; Cui, Guangliang; Xiao, Chuanhai; Zhang, Mingzhe; Chen, Li; Shi, Changmin

    2016-06-01

    We fabricated an Ag decorated Cu (Ag@Cu) nano/microstructure ordered array by facile template-free 2D electrodeposition combined with a galvanic reduction method for SERS applications. The Cu nano/microstructure ordered arrays were first synthesized by a 2D electrodeposition method, then Ag nanocubes were decorated on the arrays by galvanic reduction without any capping agent. The pollution-free surface and edge-to-face heterostructure of Ag nanocubes and Cu nano/microstructure arrays provide the powerful field-enhancements for SERS performance. The results verified that the Ag@Cu nano/microstructure ordered arrays have excellent activity for 4-Mercaptopyridine, and the sensitivity limit is as low as 10‑8 M. Therefore, this facile route provides a useful platform for the fabrication of a SERS substrate based on nano/microstructure ordered arrays.

  14. Characterization of the Helicase Activity and Substrate Specificity of Mycobacterium tuberculosis UvrD▿

    Curti, Elena; Smerdon, Stephen J; Davis, Elaine O.

    2006-01-01

    UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. ...

  15. Synthesis of carbon nanofibers on impregnated powdered activated carbon as cheap substrate

    Mamun, A. A.; Y.M. Ahmed; S.A. Muyibi; M.F.R. Al-Khatib; A.T. Jameel; M.A. AlSaadi

    2016-01-01

    The catalysis and characterization of carbon nanofibers (CNFs) composite are reported in this work. Carbon nanofibers were produced on oil palm shell powdered activated carbon (PAC), which was impregnated with nickel. Chemical Vapor Deposition (CVD) of C2H2 was used in the presence of hydrogen at ∼650 °C. The flow rates of carbon source and hydrogen were fixed. The CNFs formed directly on the surface of the impregnated PAC. Variable weight percentages (1%, 3%, 5%, 7% and 9%) of the catalyst s...

  16. Understanding the solar photo-catalytic activity of TiO2-ITO nanocomposite deposited on low cost substrates

    In this work, we report on the photo-catalytic properties of TiO2-ITO nanocomposite deposited on low cost conventional clay ceramic substrates. The nanocomposite was formed by spraying a solution prepared from the P25 TiO2 powder (Degussa) mixed with an organometallic paste of a dissolved combination of indium and tin. A TiO2-ITO powder-like nanocomposite was prepared for X-ray diffraction (XRD) and transmission electron microscopy (TEM) characterization. The mean particle size of the TiO2-ITO nanocomposite was found to be larger than that of pure TiO2. The optical features of TiO2-ITO-based layers (deposited on glass substrates) were investigated using UV-vis spectroscopy. The TiO2-ITO nanocomposite deposited layers were found to have higher light absorption than the P25 TiO2 powder. The photo-catalytic properties of the TiO2-ITO nanocomposite (deposited on low cost clay ceramic substrates) were tested under solar irradiation using a well-known polluting dye. It was shown that the TiO2-ITO nanocomposite exhibits higher degradation rates towards the pollutant dye than the P25 TiO2 powder. The optical band gap of the TiO2-ITO nanocomposite (2.79 eV) was found to be lower than that of pure TiO2 (3.1 eV), while ITO (indium tin oxide) has a band gap of about 4.2 eV. ITO was found to be entirely transparent to sun light, while it exhibits a slight photo-catalytic activity, signifying the possible existence of an indirect photo-catalysis phenomenon (sensitized semiconductor photocalysis) and potential degradation (oxidation) of the pollutant through electron transfer from the dye to conduction band of the semiconductor. All photo-catalytic activity results were discussed in light of the optical band gap of the various compounds.

  17. Non-invasive imaging of tumors by monitoring autotaxin activity using an enzyme-activated near-infrared fluorogenic substrate.

    Damian Madan

    Full Text Available Autotaxin (ATX, an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA from lysophosphatidylcholine (LPC. Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2 that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.

  18. Active lakes in Antarctica survive on a sedimentary substrate – Part 1: Theory

    S. P. Carter

    2015-03-01

    Full Text Available Over the past decade satellite observations have revealed that active subglacial lake systems are widespread under the Antarctic ice sheet, including the ice streams, yet we have insufficient understanding of the lake-drainage process to incorporate it into ice sheet models. Process models for drainage of ice-dammed lakes based on conventional "R-channels" incised into the base of the ice through melting are unable to reproduce the timing and magnitude of drainage from Antarctic subglacial lakes estimated from satellite altimetry given the low hydraulic gradients along which such lakes drain. We developed a process model in which channels are mechanically eroded into deformable subglacial sediment (till instead ("T-channel". When applied to the known lakes of the Whillans/Mercer system, the model successfully reproduced the key characteristics of estimated lake volume changes for the period 2003–2009. If our model is realistic, it implies that most active lakes are shallow and only exist in the presence of saturated sediment, explaining why they are difficult to detect with classical radar methods. It also implies that the lake-drainage process is sensitive to the composition and strength of the underlying till, suggesting that models could be improved with a realistic treatment of sediment – interfacial water exchange.

  19. Tracking Dynamics of Plant Biomass Composting by Changes in Substrate Structure, Microbial Community, and Enzyme Activity

    Wei, H.; Tucker, M. P.; Baker, J. O.; Harris, M.; Luo, Y. H.; Xu, Q.; Himmel, M. E.; Ding, S. Y.

    2012-04-01

    Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars. However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process. In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera) wood-chips and mown lawn grass clippings (85:15 in dry-weight) and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed. The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP) and solid-state fermentation for the production of cellulolytic enzymes and biofuels.

  20. Tracking dynamics of plant biomass composting by changes in substrate structure, microbial community, and enzyme activity

    Wei Hui

    2012-04-01

    Full Text Available Abstract Background Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars. However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process. Results In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera wood-chips and mown lawn grass clippings (85:15 in dry-weight and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed. Conclusion The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP and solid-state fermentation for the production of cellulolytic enzymes and biofuels.

  1. The role of active site aromatic residues in substrate degradation by the human chitotriosidase.

    Eide, Kristine Bistrup; Stockinger, Linn Wilhelmsen; Lewin, Anna Sofia; Tøndervik, Anne; Eijsink, Vincent G H; Sørlie, Morten

    2016-02-01

    Human chitotriosidase (HCHT) is a glycoside hydrolase family 18 chitinase synthesized and secreted in human macrophages thought be an innate part of the human immune system. It consists of a catalytic domain with the (β/α)8 TIM barrel fold having a large area of solvent-exposed aromatic amino acids in the active site and an additional family 14 carbohydrate-binding module. To gain further insight into enzyme functionality, especially the effect of the active site aromatic residues, we expressed two variants with mutations in subsites on either side of the catalytic acid, subsite -3 (W31A) and +2 (W218A), and compared their catalytic properties on chitin and high molecular weight chitosans. Exchange of Trp to Ala in subsite -3 resulted in a 12-fold reduction in extent of degradation and a 20-fold reduction in kcat(app) on chitin, while the values are 5-fold and 10-fold for subsite +2. Moreover, aromatic residue mutation resulted in a decrease of the rate of chitosan degradation contrasting previous observations for bacterial family 18 chitinases. Interestingly, the presence of product polymers of 40 sugar moieties and higher starts to disappear already at 8% degradation for HCHT50-W31A. Such behavior contrast that of the wild type and HCHT-W218A and resembles the action of endo-nonprocessive chitinases. PMID:26621384

  2. CHEMICAL, PHYSICAL AND SENSORY ANALYSIS OF ACTIVITY DIFFERENT YEAST SPECIES ON IDENTICAL SUBSTRATE IN WINE PRODUCTION

    Vladimír Vietoris

    2013-02-01

    Full Text Available Rizling vlašský is the second most important variety in Slovakia. The science of wine production includes a summary of knowledge and experience in the field of grape growing and wine making, or the production of different types of wines using specific methods of production. Wine quality is the result of the interaction between yeast, bacteria and microscopic funguses. In this research, we studied the effects of active dry wine yeasts on chemical, physical and sensory parameters in wine production. We have applied five kinds of yeasts (FERMIVIN, FERMIVIN PDV, FERMICRU AR2, FERMIFLOR and FERMICRU VB1. It can be concluded that the application of active dry wine yeasts is beneficial for the production of rizling vlašský. The best showing were yeasts FERMIFLOR and FERMIVIN PDM. In the last sample where they were left the original yeasts the varietal aroma was preserved. It can be noted that the wine was right technologically produced and all wines were harmonious with a pleasant fresh taste.

  3. Fabrication of tunable plasmonic substrates using a table-top gold coater and a hot plate, their optical characterization, and surface enhanced Raman activity

    Arora, A.; Krishnan, A.

    2015-10-01

    We present a simple scalable technique for repeatable fabrication of large area (cm2) electromagnetic hot spots using tunable Localized Surface Plasmon Resonance (LSPR) substrates and their k-space microscopic imaging characterization. The substrates were fabricated simply using a low vacuum air plasma scanning electron microscope gold coater and annealing using a hot plate. The measured permittivity profile and optical transmission characteristics of such substrates showed large changes before and after annealing, with clear changes in the occurrence and position of the LSPR in the visible spectrum. Furthermore, the LSPR wavelength of these substrates was tuned from 537 nm to 630 nm using cyclic deposition and annealing. It was observed that every anneal step could be used to blue shift the resonance, while a deposition step could be used to red shift the resonance, thus giving rise to a wide tunability. We also present the k-space images of the substrates using narrowband fluorescence leakage radiation microscopy and broadband polarization microscopy. The enhanced scattering in these substrates was clearly imaged in the k-space, and the color content in the broadband k-space images correlates well with the spectral characteristics of these substrates that can be used in commercial quality testing without a spectrometer. The optical characteristics of the substrates were attributed to the morphology evolution verified using scanning probe microscopy. A single particle model based simulation was used to evaluate the optical response. The substrates were then tested for surface enhanced Raman spectroscopy (SERS) activity using control experiments involving Rhodamine 6G dye in PMMA matrix of different concentrations with analyte volumes of approximately 200 pl and analytical enhancements of >3 ×104 (net enhancement >1.8 ×107 ) were obtained. The limit of detection was ≈ 10-8 M in low volume (≈200 pl) analyte, reaching the regime of few molecule detection. To

  4. Diazomethyl ketone substrate derivatives as active-site-directed inhibitors of thiol proteases. Papain

    Leary, R.; Larsen, D.; Watanabe, H.; Shaw, E.

    1977-12-27

    The diazomethyl ketones of z-Phe and z-Phe-Phe inactivate papain by a stoichiometric reaction at the active-center thiol. Since the reagents are stable in mercaptoethanol, their reaction with papain is judged to be the result of complex formation characteristic of affinity-labeling reagents. The diazomethyl ketones react by a mechanism different from that of chloromethyl ketones, since the pH dependence of their inactivation of papain is different, the rate increasing with decreasing pH. This relationship has been observed in other cases, such as the reaction of azaserine with glutamine amidotransferases (Buchanan, J. M. (1973), Adv. Enzmol. Relat. Areas Mol. Biol. 39, 91), and is interpreted as an indication of reaction with a thiol group in its protonated form.

  5. Synchronizing Substrate Activation Rates in Multicomponent Reactions with Metal-Organic Framework Catalysts.

    Aguirre-Díaz, Lina María; Iglesias, Marta; Snejko, Natalia; Gutiérrez-Puebla, Enrique; Monge, M Ángeles

    2016-05-01

    A study on the influence of the cation coordination number, number of Lewis acid centers, concurrent existence of Lewis base sites, and structure topology on the catalytic activity of six new indium MOFs, has been carried out for multicomponent reactions (MCRs). The new indium polymeric frameworks, namely [In8 (OH)6 (popha)6 (H2 O)4 ]⋅3 H2 O (InPF-16), [In(popha)(2,2'-bipy)]⋅3 H2 O (InPF-17), [In3 (OH)3 (popha)2 (4,4'-bipy)]⋅4 H2 O (InPF-18), [In2 (popha)2 (4,4'-bipy)2 ]⋅3 H2 O (InPF-19), [In(OH)(Hpopha)]⋅0.5 (1,7-phen) (InPF-20), and [In(popha)(1,10-phen)]⋅4 H2 O (InPF-21) (InPF=indium polymeric framework, H3 popha=5-(4-carboxy-2-nitrophenoxy)isophthalic acid, phen=phenanthroline, bipy=bipyridine), have been hydrothermally obtained by using both conventional heating (CH) and microwave (MW) procedures. These indium frameworks show efficient Lewis acid behavior for the solvent-free cyanosilylation of carbonyl compounds, the one pot Passerini 3-component (P-3CR) and the Ugi 4-component (U-4CR) reactions. In addition, InPF-17 was found to be a highly reactive, recyclable, and environmentally benign catalyst, which allows the efficient synthesis of α-aminoacyl amides. The relationship between the Lewis base/acid active site and the catalytic performance is explained by the 2D seven-coordinated indium framework of the catalyst InPF-17. This study is an attempt to highlight the main structural and synthetic factors that have to be taken into account when planning a new, effective MOF-based heterogeneous catalyst for multicomponent reactions. PMID:27010759

  6. Thermodynamics of the localized D2-D6 system

    Gomez-Reino, Marta [Martin Fisher School of Physics, Brandeis University, Waltham, MA 02454 (United States)]. E-mail: marta@brandeis.edu; Naculich, Stephen G. [Department of Physics, Bowdoin College, Brunswick, ME 04011 (United States)]. E-mail: naculich@bowdoin.edu; Schnitzer, Howard J. [Martin Fisher School of Physics, Brandeis University, Waltham, MA 02454 (United States)]. E-mail: schnitzer@brandeis.edu

    2005-05-02

    An exact fully-localized extremal supergravity solution for N{sub 2} D2-branes and N{sub 6} D6-branes, which is dual to 3-dimensional supersymmetric SU(N{sub 2}) gauge theory with N{sub 6} fundamentals, was found by Cherkis and Hashimoto. In order to consider the thermal properties of the gauge theory we present the non-extremal extension of this solution to first order in an expansion near the core of the D6-branes. We compute the Hawking temperature and the black-brane horizon area/entropy. The leading-order entropy, which is proportional to N{sub 2}{sup 3/2}N{sub 6}{sup 1/2}T{sub H}{sup 2}, is not corrected to first order in the expansion. This result is consistent with the analogous weak-coupling result at the correspondence point N{sub 2} similar to N{sub 6}.

  7. Thermodynamics of the localized D2-D6 system

    An exact fully-localized extremal supergravity solution for N2 D2-branes and N6 D6-branes, which is dual to 3-dimensional supersymmetric SU(N2) gauge theory with N6 fundamentals, was found by Cherkis and Hashimoto. In order to consider the thermal properties of the gauge theory we present the non-extremal extension of this solution to first order in an expansion near the core of the D6-branes. We compute the Hawking temperature and the black-brane horizon area/entropy. The leading-order entropy, which is proportional to N23/2N61/2TH2, is not corrected to first order in the expansion. This result is consistent with the analogous weak-coupling result at the correspondence point N2 similar to N6

  8. Thermodynamics of the localized D2-D6 system

    Gomez-Reino, Marta; Naculich, Stephen; Schnitzer, Howard

    2004-01-01

    An exact fully-localized extremal supergravity solution for N_2 D2 branes and N_6 D6 branes, which is dual to 3-dimensional supersymmetric SU(N_2) gauge theory with N_6 fundamentals, was found by Cherkis and Hashimoto. In order to consider the thermal properties of the gauge theory we present the non-extremal extension of this solution to first order in an expansion near the core of the D6 branes. We compute the Hawking temperature and the black brane horizon area/entropy. The leading order e...

  9. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  10. Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study.

    Flannelly, David F; Aoki, Thalia G; Aristilde, Ludmilla

    2015-09-01

    The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs. PMID:26160737

  11. Xanthine oxidoreductase activity assay in tissues using stable isotope-labeled substrate and liquid chromatography high-resolution mass spectrometry.

    Murase, Takayo; Nampei, Mai; Oka, Mitsuru; Ashizawa, Naoki; Matsumoto, Koji; Miyachi, Atsushi; Nakamura, Takashi

    2016-01-01

    Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1±0.7, 158±5, 928±25pmol/min/mg of protein, respectively (mean±SD, n=5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [(15)N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9-8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter

  12. Impurities analysis of polycrystalline silicon substrates: Neutronic Activation Analysis (NAA) and Secondary Ion Mass Spectrometry (SIMS)

    Lounis, A.; Lenouar, K.; Gritly, Y.; Abbad, B.; Azzaz, M.; Taïbi, K.

    2010-01-01

    In this study we have determined the concentration of some impurities such as carbon, iron, copper, titanium, nickel of the flat product (polycrystalline silicon). These impurities generate a yield decrease in the photovoltaic components. The material (polycrystalline silicon) used in this work is manufactured by the Unit of Silicon Technology Development (UDTS Algiers, Algeria). The 80 kg ingot has been cutted into 16 briquettes in order to have plates (flat product) of 100 mm×100 mm dimensions. Each briquette is divided into three parts top (T), middle (M) and bottom (B). For this purpose, the following instrumental analysis techniques have been employed: neutronic analysis (neutronic activation analysis) and secondary ion mass spectrometry (SIMS). Masses of 80 mg are sampled and form of discs 18 mm in diameter, then exposed to a flux of neutron of 2.1012neutron cm-2 s-1 during 15 min. The energetic profile of incidental flux is constituted of fast neutrons (ΦR = 3.1012n.cm-2 s-1; E = 2 Mev), thermal neutrons (ΦTH = 1013n.cm-2 s-1; E = 0.025 ev) and epithermal neutrons (Φepi = 7.1011 n cm-2 s-1; E>4.9 ev), irradiation time 15 mn, after 20 mn of decrement, acquisitions of 300 s are carried out. The results are expressed by disintegration per second which does not exceed the 9000 Bq, 500 Bq and 2600 Bq, respectively for copper, titanium and nickel. It is observed that the impurities concentrations in the medium are higher. The impurities in the bottom of the ingots originate from the crucible. The impurities in the top originate from impurities dissolved in the liquid silicon, which have segregated to the top layer of the ingot and after solidification diffuse. Silicon corresponds to a mixture of three isotopes 28Si, 29Si and 30Si. These elements clearly appear on the mass spectrum (SIMS). The presence of iron and the one of nickel has been noticed.

  13. Impurities analysis of polycrystalline silicon substrates : Neutronic Activation Analysis (NAA) and Secondary Ion Mass Spectrometry (SIMS)

    Full text: In this study we have determined the concentration of some impurities such as carbon, iron, copper, titanium, nickel of the flat product (polycrystalline silicon). These impurities generate a yield decrease in the photovoltaic components. The material (polycrystalline silicon) used in this work is manufactured by the Unit of Silicon Technology Development (UDTS Algiers, Algeria). The 80 kg ingot has been cutted into 16 briquettes in order to have plates (flat product) of 100 mm x 100 mm dimensions. Each briquette is divided into three parts top (T), middle (M) and bottom (B). For this purpose, the following instrumental analysis techniques have been employed: neutronic analysis ( neutronic activation analysis) and secondary ion mass spectrometry (SIMS). Masses of 80 mg are sampled and form of discs 18 mm in diameter, then exposed to a flux of neutron of 2.1012 neutron cm-2 s-1 during 15 min . The R= 3.1012 energetic profile of incidental flux is constituted of fast neutrons ( TH = 1013 ncm-2 s-1; E = 0.025 eV) ncm-2 s-1; E = 2 MeV), thermal neutrons ( epi = 7.1011 n cm-2 s-1; E and epithermal neutrons (> 4.9 eV), irradiation time 15 mn, after 20 mn of decrement, acquisitions of 300 s are carried out, the results are expressed by disintegration per second which does not exceed the 9000, 500 and 2600 Bq, respectively for copper, titanium and nickel. It is observed that the impurities concentrations in the medium are higher. The impurities in the bottom of the ingots originate from the crucible. The impurities in the top originate from impurities dissolved in the liquid silicon, which have segregated to the top layer of the ingot and after solidification diffuse. Silicon corresponds to a mixture of three isotopes 28Si, 29Si and 30Si. These elements clearly appear on the mass spectrum (SIMS).The presence of iron and the one of nickel has been noticed. (author)

  14. Facile synthesis of large-scale Ag nanosheet-assembled films with sub-10 nm gaps as highly active and homogeneous SERS substrates

    Li, Zhongbo; Meng, Guowen; Liang, Ting; Zhang, Zhuo; Zhu, Xiaoguang

    2013-01-01

    We report a facile low-cost synthetic approach to large-scale Ag nanosheet-assembled films with a high density of uniformly distributed sub-10 nm gaps between the adjacent nanosheets on Si substrates via galvanic cell reactions. The distribution density of Ag nanosheets on substrates could be tailored by tuning the duration of the HF-etching and the concentration of citric acid in the solution. Furthermore, in conjunction with a conventional photolithography, highly uniform patterned Ag nanosheet-assembled structures with different morphologies can be achieved on Si substrates via galvanic-cell-induced growth. By using rhodamine 6G as a standard test molecule, the large-scale Ag nanosheet-assembled films exhibit highly active and homogenous surface-enhanced Raman scattering (SERS) effect and also show promising potentials as reliable SERS substrates for rapid detection of trace polychlorinated biphenyls (PCBs).

  15. Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

    Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred

    2014-07-01

    Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry. PMID:24687155

  16. Evaluation of Hydrolytic Activity of Different Pectinases on Sugar Beet (Beta vulgaris Substrate Using FT-MIR Spectroscopy

    Adina CHIŞ

    2011-11-01

    Full Text Available The aim of this study was to evaluate the pectinase activity of two commercial enzymes Rohament PL and Rohapect 10L, using as substrate the sugar beet pulp, in different conditions. The method applied to check the rate of hydrolysis was Fourier-transformed infrared spectroscopy (FT-MIR, by recording the absorption spectra of different carbohydrates in the specific, MIR (middle IR spectral range (650-4000 cm-1. In order to calibrate the method, it has been recorded the FT-MIR spectrum of standard solutions of glucose, fructose, sucrose and galacturonic acid, at different concentrations, establishing also the peaks and the fingerprint regions specific to these compounds. Considering the specific peak intensities identified for glucose (at 1033 cm-1, fructose (at 1063 cm-1, sucrose (at 995 cm-1, and based on peak area for galacturonic acid (1500-700 cm-1, it has been calculated their concentrations, as a result of the Rohament PL or Rohapect 10L activity. Based on calibration curves for each sugar type, it has been calculated their concentrations in the sugar beet samples, and after their release by enzymatic treatment, establishing an optimized protocol of action for the two enzymes. Differences among the two enzymes activity were identified, specifically their optimum concentration and hydrolysis timing. FT-MIR spectroscopy proves to be an adequate method to evaluate the enzymatic activity of the different enzyme types, having certain advantages in comparison with the chromatographic methods, being a rapid, non-destructive method, at relatively low costs.

  17. Electrodeposition for preparation of efficient surface-enhanced Raman scattering-active silver nanoparticle substrates for neurotransmitter detection

    Highlights: ► We demonstrate a facile, rapid, harmless and low-cost electrochemical method for SERS substrate preparation. ► Cyclic voltammetry is an efficient method for nanoparticles deposition. ► Electrodeposited AgNPs are suitable for SERS substrates to detect neurotransmitters. ► These SERS substrates reveal an excellent signal reproducibility. ► These SERS substrates exhibit an enhancement factor of 107 for non-resonance analytes. -- Abstract: A stable and efficient surface-enhanced Raman scattering (SERS) substrate for neurotransmitter and cholinergic neurotransmission precursor detection was obtained by silver nanoparticle (AgNP) electrodeposition onto tin-doped indium oxide (ITO) using cyclic voltammetry. The size and surface coverage of the deposited AgNPs were controlled by changing the scan rate and the number of scans. The SERS performance of these substrates was analyzed by studying its reproducibility, repeatability and signal enhancement measured from p-aminothiophenol (p-ATP) covalently bonded to the substrate. We compared the SERS performance for samples with different Ag particle coverage and particle sizes. The performance was also compared with a commercial substrate. Our substrates exhibited a SERS enhancement factor of around 107 for p-ATP which is three orders of magnitude larger than for the commercial substrate. Apart from this high enhancement effect the substrate also shows extremely good reproducibility. The average spectral correlation coefficient (Γ) is 0.96. This is larger than for the commercial substrate (0.85) exhibiting a much lower SERS signal intensity. Finally, the application of our substrates as SERS bio-sensors was demonstrated with the detection of the neurotransmitters acetylcholine, dopamine, epinephrine and choline, the precursor for acetylcholine. The intensive SERS spectra observed for low concentrations of choline (2 × 10−6 M), acetylcholine (4 × 10−6 M), dopamine (1 × 10−7 M) and epinephrine (7

  18. Bactericidal Activity of Aqueous Acrylic Paint Dispersion for Wooden Substrates Based on TiO2 Nanoparticles Activated by Fluorescent Light

    Diana Di Gioia

    2013-08-01

    Full Text Available The photocatalytic effect of TiO2 has great potential for the disinfection of surfaces. Most studies reported in the literature use UV activation of TiO2, while visible light has been used only in a few applications. In these studies, high concentrations of TiO2, which can compromise surface properties, have been used. In this work, we have developed an acrylic-water paint dispersion containing low TiO2 content (2 vol % for the inactivation of microorganisms involved in hospital-acquired infections. The nanoparticles and the coating have been characterized using spectroscopic techniques and transmission electron microscopy, showing their homogenous dispersion in the acrylic urethane coating. A common fluorescent light source was used to activate the photocatalytic activity of TiO2. The paint dispersion showed antimicrobial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The coating containing the TiO2 nanoparticles maintained good UV stability, strong adhesion to the substrate and high hardness. Therefore, the approach used is feasible for paint formulation aimed at disinfection of healthcare surfaces.

  19. The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations

    Mykuliak V. V.

    2014-03-01

    Full Text Available Aim. To study the mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase (MtTyrRS. Methods. Complexes of MtTyrRS with tyrosine, ATP and tyrosyl adenylate were constructed by superposition of the MtTyrRS structure and crystallographic structures of bacterial TyrRS. All complexes of MtTyrRS with substrates were investigated by molecular dynamics (MD simulations in solution. Results. It was shown the formation of network of hydrogen bonds between substrates and the MtTyrRS active center, which were stable in the course of MD simulations. ATP binds in the active site both by hydrogen bonds and via electrostatic interactions with Lys231 and Lys234 of catalytic KFGKS motif. Conclusions. The L-tyrosine binding site in the enzyme active site is negatively charged, whereas the ATP binding site contains positive Lys231 and Lys234 residues of catalytic KFGKS motif. The occupancy of H-bonds between substrates and the enzyme evidences a significant conformational mobility of the active site.

  20. Interfacial Structure and Photocatalytic Activity of Magnetron Sputtered TiO2 on Conducting Metal Substrates

    Daviðsdóttir, Svava; Petit, Jean-Pierre; Mermoux, Michel;

    2014-01-01

    using X-ray diffraction, Raman spectroscopy, atomic force microscope, and scanning and transmission electron microscopy. Photocatalytic behavior of the coating was explored by using optical spectrophotometry and electrochemical methods via photovoltage, photocurrent, and scanning kelvin probe microscopy......The photocatalytic behavior of magnetron sputtered anatase TiO2 coatings on copper, nickel, and gold was investigated with the aim of understanding the effect of the metallic substrate and coating-substrate interface structure. Stoichiometry and nanoscale structure of the coating were investigated...... measurements. The nature of the metal substrate and coating-substrate interface had profound influence on the photocatalytic behavior. Less photon energy was required for TiO2 excitation on a nickel substrate, whereas TiO2 coating on copper showed a higher band gap attributed to quantum confinement. However...

  1. Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

    Stefano Pascarella

    2012-01-01

    Full Text Available Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  2. TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization.

    Jouannet, Stéphanie; Saint-Pol, Julien; Fernandez, Laurent; Nguyen, Viet; Charrin, Stéphanie; Boucheix, Claude; Brou, Christel; Milhiet, Pierre-Emmanuel; Rubinstein, Eric

    2016-05-01

    The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aβ, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization. PMID:26686862

  3. Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

    Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the β-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission

  4. Development of Novel Rifampicin-Derived P-Glycoprotein Activators/Inducers. Synthesis, In Silico Analysis and Application in the RBE4 Cell Model, Using Paraquat as Substrate

    Vilas-Boas, Vânia; Silva, Renata; Palmeira, Andreia; Sousa, Emília; Ferreira, Luísa Maria; Branco, Paula Sério; Carvalho, Félix; Bastos, Maria de Lourdes; Remião, Fernando

    2013-01-01

    P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modula...

  5. Silver nanocube on gold microplate as a well-defined and highly active substrate for SERS detection.

    Xia, Xiaohu; Rycenga, Matthew; Qin, Dong; Xia, Younan

    2013-10-14

    Strong enhancement and good reproducibility in Raman signals are two major requirements for a surface-enhanced Raman scattering (SERS) substrate to be used for sensitive detection of an analyte. Here we report a new type of SERS substrate that was fabricated by depositing a Ag nanocube (AgNC) on the surface of a Au microplate (AuMP). Owing to the strong and reproducible hot spots formed at corner sites of the AgNC in proximity with the AuMP surface, the new substrate showed high sensitivity and reproducibility. Using 1,4-benzenedithiol as a probe, the SERS enhancement factor of a typical "AgNC on AuMP" substrate could reach a level as high as 4.7×10(7). In addition to the high sensitivity and reproducibility, the "AgNC on AuMP" substrate also displayed very good stability. Potential use of the "AgNC on AuMP" substrate was demonstrated by detecting crystal violet with high sensitivity. PMID:24187611

  6. Surface-enhanced Raman scattering (SERS) activity of Ag, Au and Cu nanoclusters on TiO2-nanotubes/Ti substrate

    Tubular arrays of TiO2 nanotubes (ranging in diameter from 40 to 110 nm) on a Ti substrate were used as a support for Ag, Au or Cu deposits obtained by the sputter deposition technique, where the amount of metal varied from 0.01 to 0.2 mg/cm2. Those composite supports were intended for surface-enhanced Raman scattering (SERS) investigations. Composite samples were studied with the aid of scanning electron microscopy (SEM) and Auger electron spectroscopy (AES) to reveal their characteristic morphological and chemical features. Raman spectra of pyridine (as a probe molecule) were measured at different cathodic potentials ranging from -0.2 down to -1.2 V after the pyridine had been adsorbed on the metal-covered TiO2 nanotube/Ti substrates. In addition, SERS spectra on a bulk standard activated Ag, Au and Cu substrates were also measured. The SERS activity of the composite samples was strongly dependent on the amount of metal deposit, e.g. at and above 0.06 mg Ag/cm2, the intensity of SERS signal was even higher than that for the Ag reference substrate. The high activity of these composites is mainly a result of their specific morphology. The high SERS sensitivity on the surface morphology of the substrate made it possible to monitor very small temporal changes in the Ag metal clusters. This rearrangement was not detectable with microscopic (SEM) or microanalytical (AES) methods. The SERS activity of Au or Cu clusters was distinctly lower than those of Ag. The spectral differences exhibited by the three kinds of composites as compared to the reference metal samples are discussed.

  7. Determination of the photocatalytic activity of TiO2 coatings on clay roofing tile substrates methylene blue as model pollutant

    Skapin Andrea S.

    2009-01-01

    Full Text Available The photocatalytically active mesoporous coatings, based on titanium dioxide sols (Degussa, of the fired clay roofing tiles substrate were prepared by using poly(ethylene glycol (PEG M-600 and M-4000, as the structure directing agents. The coatings were deposited using spray technique followed by thermal treatment. Photocatalytic activity of the TiO2 coatings was evaluated by aqueous solution of methylene blue as model dye, deposited on the top of the coatings, after irradiation with UV light. The results were compared with the photocatalytic efficiency of some commercial self-cleaning products (clay roofing tiles, glass. The newly design coatings showed an interesting decolourisation performance (over 30 % after 24 h. It appeared that the procedure of photocatalytic activity determination, in the case of porous substrates, should be renewed by a preadsorption process.

  8. Promoting the Hydrosilylation of Benzaldehyde by Using a Dicationic Antimony-Based Lewis Acid: Evidence for the Double Electrophilic Activation of the Carbonyl Substrate.

    Hirai, Masato; Cho, Junsang; Gabbaï, François P

    2016-05-01

    The concomitant activation of carbonyl substrates by two Lewis acids has been investigated by using [1,2-(Ph2 MeSb)2 C6 H4 ](2+) ([1](2+) ), an antimony-based bidentate Lewis acid obtained by methylation of the corresponding distibine. Unlike the simple stibonium cation [Ph3 MeSb](+) , dication [1](2+) efficiently catalyzes the hydrosilylation of benzaldehyde under mild conditions. The catalytic activity of this dication is correlated to its ability to doubly activate the carbonyl functionality of the organic substrate. This view is supported by the isolation of [1-μ2 -DMF][OTf]2 , an adduct, in which the DMF oxygen atom bridges the two antimony centers. PMID:26934491

  9. Rapid shifts in Atta cephalotes fungus-garden enzyme activity after a change in fungal substrate (Attini, Formicidae)

    Kooij, P W; Schiøtt, M; Boomsma, J J;

    2011-01-01

    Fungus gardens of the basidiomycete Leucocoprinus gongylophorus sustain large colonies of leaf-cutting ants by degrading the plant material collected by the ants. Recent studies have shown that enzyme activity in these gardens is primarily targeted toward starch, proteins and the pectin matrix......, we measured the changes in enzyme activity after a controlled shift in fungal substrate offered to six laboratory colonies of Atta cephalotes. An ant diet consisting exclusively of grains of parboiled rice rapidly increased the activity of endo-proteinases and some of the pectinases attacking the...... associated with cell walls, rather than toward structural cell wall components such as cellulose and hemicelluloses. Substrate constituents are also known to be sequentially degraded in different sections of the fungus garden. To test the plasticity in the extracellular expression of fungus-garden enzymes...

  10. Enzymatic activity in turkey, duck, quail and chicken liver microsomes against four human cytochrome P450 prototype substrates and aflatoxin B1

    Hansen W. Murcia

    2011-10-01

    Full Text Available Cytochrome P450 enzymes (CYP are a group of monooxygenases able to biotransform several kinds of xenobiotics including aflatoxin B1 (AFB1, a highly toxic mycotoxin. These enzymes have been widely studied in humans and others mammals, but there is not enough information in commercial poultry species about their biochemical characteristics or substrate specificity. The aim of the present study was to identify CYPs from avian liver microsomes with the use of prototype substrates specific for human CYP enzymes and AFB1. Biochemical characterization was carried out in vitro and biotransformation products were detected by high-performance liquid chromatography (HPLC. Enzymatic constants were calculated and comparisons between turkey, duck, quail and chicken activities were done. The results demonstrate the presence of four avian ortholog enzyme activities possibly related with a CYP1A1, CYP1A2, CYP2A6 (activity not previously identified and CYP3A4 poultry orthologs, respectively. Large differences in enzyme kinetics specific for prototype substrates were found among the poultry species studied. Turkey liver microsomes had the highest affinity and catalytic rate for AFB1 whereas chicken enzymes had the lowest affinity and catalytic rate for the same substrate. Quail and duck microsomes showed intermediate values. These results correlate well with the known in vivo sensitivity for AFB1 except for the duck. A high correlation coefficient between 7-ethoxyresorufin-Odeethylase (EROD and 7-methoxyresorufin- O-deethylase (MROD activities was found in the four poultry species, suggesting that these two enzymatic activities might be carried out by the same enzyme. The results of the present study indicate that four prototype enzyme activities are present in poultry liver microsomes, possibly related with the presence of three CYP avian orthologs. More studies are needed in order to further characterize these enzymes.

  11. Comparative assessment of plasmid and oligonucleotide DNA substrates in measurement of in vitro base excision repair activity

    Hou, Esther W.; Prasad, Rajendra; Asagoshi, Kenjiro; Masaoka, Aya; Wilson, Samuel H.

    2007-01-01

    Mammalian base excision repair (BER) is mediated through at least two subpathways designated ‘single-nucleotide’ (SN) and ‘long-patch’ (LP) BER (2-nucleotides long/more repair patch). Two forms of DNA substrate are generally used for in vitro BER assays: oligonucleotide- and plasmid-based. For plasmid-based BER assays, the availability of large quantities of substrate DNA with a specific lesion remains the limiting factor. Using sequence-specific endonucleases that cleave only one strand of D...

  12. 4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase : Synthesis and evaluation of 2-fluoro substrate analogues

    Johnson, William H; Wang, Susan C; Stanley, Thanuja M; Czerwinski, Robert M; Almrud, Jeffrey J; Poelarends, Gerrit J; Murzin, Alexey G; Whitman, Christian P

    2004-01-01

    A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in B

  13. Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

    Parikh, Kaushal; Diks, Sander H.; Tuynman, Jurriaan H. B.; Verhaar, Auke; Lowenberg, Mark; Hommes, Daan W.; Joore, Jos; Pandey, Akhilesh; Peppelenbosch, Maikel P.

    2009-01-01

    Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to pred

  14. Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate Modeling and Active Site Mutations

    Momb, Jessica; Wang, Canhui; Liu, Dali; Thomas, Pei W.; Petsko, Gregory A.; Guo, Hua; Ringe, Dagmar; Fast, Walter (UNM); (Brandeis); (Texas)

    2008-12-02

    The N-acyl-l-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo-{beta}-lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme-product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily.

  15. Control over the morphology of AlN during molecular beam epitaxy with the plasma activation of nitrogen on Si (111) substrates

    The results of studies of the growth kinetics of AlN layers during molecular beam epitaxy with the plasma activation of nitrogen using Si (111) substrates are presented. The possibility of the growth of individual AlN/Si (111) nanocolumns using growth conditions with enrichment of the surface with metal near the formation mode of Al drops, at a substrate temperature close to maximal, during molecular beam epitaxy with the plasma activation of nitrogen (Ts ≈ 850°C) is shown. The possibility of growing smooth AlN layers on a nanocolumnar AlN/Si (111) buffer with the use of Ts ≈ 750°C and growth conditions providing enrichment with metal is shown

  16. Flower-like gold nanostructures electrodeposited on indium tin oxide (ITO) glass as a SERS-active substrate for sensing dopamine

    Flower-like gold nanostructures (so called gold nanoflowers) were electrodeposited on indium tine oxide (ITO) glass and served as a SERS-active substrate for sensing dopamine. A double-potential method was applied for the deposition of gold nanostructures on the glass. The density, size, and morphology of the gold nanostructures were controlled by adjusting parameters such as deposition potentials, duration, and concentrations of gold precursors. The particle density on the glass was controlled by changing the overvoltage potentials, while the size and morphology were controlled by changing the concentration of gold precursors and growth times of Gold crystal seeds. The intensity of the Raman spectrum of dopamine was distinctly enhanced on the gold nanostructures with unique flower-like topography, thereby demonstrating the utility of this SERS-active substrate that is facilely obtained by the double-potential deposition method without supporting electrolytes. (author)

  17. Development of Novel Rifampicin-Derived P-Glycoprotein Activators/Inducers. Synthesis, In Silico Analysis and Application in the RBE4 Cell Model, Using Paraquat as Substrate

    Vilas-Boas, Vânia; Silva, Renata; Palmeira, Andreia; Sousa, Emília; Ferreira, Luísa Maria; Branco, Paula Sério; Carvalho, Félix; Bastos, Maria de Lourdes; Remião, Fernando

    2013-01-01

    P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp. PMID:23991219

  18. Acid-Denatured Green Fluorescent Protein (GFP as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase

    Marco A. Ramos

    2011-07-01

    Full Text Available Green fluorescent protein (GFP has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for

  19. SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.

    Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M; Lin, Jung-Hsin

    2009-07-01

    Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design. PMID:19433508

  20. Post-Heparin LPL Activity Measurement Using VLDL As a Substrate: A New Robust Method for Routine Assessment of Plasma Triglyceride Lipolysis Defects

    Di Filippo, Mathilde; Marçais, Christophe; Charrière, Sybil; Marmontel, Oriane; Broyer, Martine; Delay, Mireille; Merlin, Micheline; Nollace, Axel; Valéro, René; Lagarde, Michel; Pruneta-Deloche, Valérie; Moulin, Philippe; Sassolas, Agnès

    2014-01-01

    Background Determination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. Consequently by using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles. Methods Post-heparin plasma was added to the VLDL substrate prepared by ultracentrifugation of heat inactivated normolipidemic human serums, diluted in buffer, pH 8.15. Following incubation at 37°c, the NEFA (non esterified fatty acids) produced were assayed hourly for 4 hours. LPL activity was expressed as µmol/l/min after subtraction of hepatic lipase (HL) activity, obtained following LPL inhibition with NaCl 1.5 mmol/l. Molecular analysis of LPL, GPIHBP1, APOA5, APOC2, APOE genes was available for 62 patients. Results Our method was reproducible (coefficient of variation (CV): intra-assay 5.6%, inter-assay 7.1%), and tightly correlated with the conventional radiolabelled triolein emulsion method (n = 26, r = 0.88). Normal values were established at 34.8±12.8 µmol/l/min (mean±SD) from 20 control subjects. LPL activities obtained from 71 patients with documented history of major hypertriglyceridemia showed a trimodal distribution. Among the 11 patients with a very low LPL activity (10 µmol/l/min. Conclusion This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects. PMID:24788417

  1. Ex Vivo Antioxidant Activity of Selected Medicinal Plants against Fenton Reaction-Mediated Oxidation of Biological Lipid Substrates

    Namratha Pai Kotebagilu; Vanitha Reddy Palvai; Asna Urooj

    2015-01-01

    Free radical-mediated oxidation is often linked to various degenerative diseases. Biological substrates with lipids as major components are susceptible to oxygen-derived lipid peroxidation due to their composition. Lipid peroxide products act as biomarkers in evaluating the antioxidant potential of various plants and functional foods. The study focused on evaluation of the antioxidant potential of two extracts (methanol and 80% methanol) of four medicinal plants, Andrographis paniculata, Cost...

  2. Probing the structural dependency of photoinduced properties of colloidal quantum dots using metal-oxide photo-active substrates

    Patty, Kira; Campbell, Quinn; Hamilton, Nathan; West, Robert G. [Department of Physics, University of Alabama in Huntsville, Huntsville, Alabama 35899 (United States); Sadeghi, Seyed M., E-mail: seyed.sadeghi@uah.edu [Department of Physics, University of Alabama in Huntsville, Huntsville, Alabama 35899 (United States); Nano and Micro Device Center, University of Alabama in Huntsville, Huntsville, Alabama 35899 (United States); Mao, Chuanbin [Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, Oklahoma 73019 (United States)

    2014-09-21

    We used photoactive substrates consisting of about 1 nm coating of a metal oxide on glass substrates to investigate the impact of the structures of colloidal quantum dots on their photophysical and photochemical properties. We showed during irradiation these substrates can interact uniquely with such quantum dots, inducing distinct forms of photo-induced processes when they have different cores, shells, or ligands. In particular, our results showed that for certain types of core-shell quantum dot structures an ultrathin layer of a metal oxide can reduce suppression of quantum efficiency of the quantum dots happening when they undergo extensive photo-oxidation. This suggests the possibility of shrinking the sizes of quantum dots without significant enhancement of their non-radiative decay rates. We show that such quantum dots are not influenced significantly by Coulomb blockade or photoionization, while those without a shell can undergo a large amount of photo-induced fluorescence enhancement via such blockade when they are in touch with the metal oxide.

  3. Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity*

    Akopian, Tatos; Kandror, Olga; Tsu, Christopher; Lai, Jack H.; Wu, Wengen; Liu, Yuxin; Zhao, Peng; Park, Annie; Wolf, Lisa; Dick, Lawrence R.; Rubin, Eric J.; Bachovchin, William; Goldberg, Alfred L.

    2015-01-01

    The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents. PMID:25759383

  4. α--AMYLASES OF Aspergillus flavus var. oryzae AND Bacillus subtilis: THE SUBSTRATE SPECIFICITY AND RESISTANCE TO A NUMBER OF CHEMICALLY ACTIVE SUBSTANCES

    K. V. Avdiyuk

    2013-06-01

    Full Text Available The ability of Aspergillus flavus var. oryzae 80428 and Bacillus subtilis 147 α-amylases to split different carbohydrate-containing substrates, such as maltose, sucrose, trehalose, dextrin, α- and β-cyclodextrin, amylose, amylopectin, glycogen, pullulan, soluble starch, insoluble starch, corn starch, wheat starch, dextran 500 has been studied. It was shown that investigated enzymes differ by substrate specificity. α-Amylase of A. flavus var. oryzae 80428 rapidly hydrolysed soluble potato and wheat starch, while the α-amylase of B. subtilis 147 — only wheat starch. Both enzymes don’t cleave maltose, α-cyclodextrin and dextran 500. A. flavus var. oryzae 80428 α-amylase display very small ability to hydrolyze pullulan, while α-amylase of B. subtilis 147 it does not act in general. The lowest values of Michaelis constant for both enzymes at splitting of glycogen have been obtained, indicating that enzymes have the greatest affinity to this substrate. The studies of influence of chemically active substances on activity of A. flavus var. oryzae 80428 and B. subtilis 147 ?-amylases show there are resistant to urea, deoxycholic acid, Tween-80, Triton X-100 and hydrogen peroxide. It’s indicate the enzymes tested may be competitive in compare with earlier described in literature enzymes. The obtained results give a possibility to propose in future usage these enzymes in different fields of industry, foremost in detergent industry.

  5. Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

    Warren, Jeremy G; Lincoln, James E; Kirkpatrick, Bruce C

    2015-01-01

    Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

  6. 5´AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4

    Fritzen, Andreas Mæchel; Lundsgaard, Anne-Marie; Jeppesen, Jacob;

    2015-01-01

    It is well known that exercise has a major impact on substrate metabolism for many hours after exercise. However, the regulatory mechanisms increasing lipid oxidation and facilitating glycogen resynthesis in the post-exercise period are unknown. To address this, substrate oxidation was measured...... in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 -deficiency not affecting the exercise-induced activation of the PDK4 transcriptional regulators, HDAC4 and SIRT1. Interestingly, PDK4 protein content...... regulates muscle metabolism post-exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently creating conditions for increased fatty acid oxidation. This article is protected by copyright. All rights reserved....

  7. Nitrification in a zeoponic substrate

    McGilloway, R. L.; Weaver, R. W.; Ming, D. W.; Gruener, J. E.

    2003-01-01

    Clinoptilolite is a zeolite mineral with high cation exchange capacity used in zeoponic substrates that have been proposed as a solid medium for growing plants or as a fertilizer material. The kinetics of nitrification has not been measured for NH4+ saturated zeoponic substrate. Experiments were conducted to evaluate the production of NO2- and NO3-, and nitrifier populations in zeoponic substrates. Small columns were filled with zeoponic substrate inoculated with a commercial inoculum or soil enrichment culture of nitrifying bacteria. In addition to column studies, a growth chamber study was conducted to evaluate the kinetics of nitrification in zeoponic substrates used to grow radishes (Raphanus sativus L.). The zeoponic substrate provided a readily available source of NH4+, and nitrifying bacteria were active in the substrate. Ammonium oxidation rates in column studies ranged from 5 to 10 micrograms N g-1 substrate h-1, and NO2- oxidation rates were 2 to 9.5 micrograms N g-1 substrate h-1. Rates determined from the growth chamber study were approximately 1.2 micrograms N g-1 substrate h-1. Quantities of NH4+ oxidized to NO2- and NO3- in inoculated zeoponic substrate were in excess of plant up-take. Acidification as a result of NH4+ oxidation resulted in a pH decline, and the zeoponic substrate showed limited buffering capacity.

  8. Micropatterning of biomolecules on a glass substrate in fused silica microchannels by using photolabile linker-based surface activation

    We report on a straightforward method for creating micropatterns of multiple biomolecules. The anti-fouling agent 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and a photolabile linker (PL) were covalently linked to an amino-terminated silane surface. Patterns were generated by selective removal of the MPC polymer via UV irradiation. Multiple micropatterns of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) and rhodamine-labeled goat fragment antigen-binding fragments (FAB) were deposited on a same glass substrate. We also employed micropatterning of multiple biomolecules in that Texas red-labeled BSA and FITC-labeled rabbit anti-mouse IgG were placed inside a microchannel. (author)

  9. Aromatic Amino Acid Mutagenesis at the Substrate Binding Pocket of Yarrowia lipolytica Lipase Lip2 Affects Its Activity and Thermostability

    Guilong Wang; Zimin Liu; Li Xu; Yunjun Yan

    2014-01-01

    The lipase2 from Yarrowia lipolytica (YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100) with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM) to introduce aromatic amino acid mutations. Two mutants (V94W and I100F...

  10. Estimation of nitrogenase activity in the presence of ethylene biosynthesis by use of deuterated acetylene as a substrate

    Nitrogenase reduces deuterated acetylene primarily to cis dideuterated ethylene. This can be distinguished from undeuterated ethylene by the use of Fourier transform infrared spectroscopy. Characteristic bands in the region from 800 to 3,500 cm-1 can be used to identify and quantitate levels of these products. This technique is applicable to field studies of nitrogen fixation where ethylene biosynthesis by plants or bacteria is occurring. We have verified the reaction stoichiometry by using Klebsiella pneumoniae and Bradyrhizobium japonicum in soybeans. The most useful bands for quantitation of substrate purity and product distribution are as follows: acetylene-d0, 3,374 cm-1; acetylene-d1, 2,584 cm-1; acetylene-d2, 2,439 cm-1; cis-ethylene-d2, 843 cm-1; trans-ethylene-d2, 988 cm-1; ethylene-d1, 943 cm-1; ethylene-d0, 949 cm-1. (The various deuterated ethylenes and acetylenes are designated by a lowercase d and subscript to indicate the number, but not the position, of deuterium atoms in the molecule.) Mass spectrometry coupled to a gas chromatograph system has been used to assist in quantitation of the substrate and product distributions. Significant amounts of trans-ethylene-d2 were produced by both wild-type and nifV mutant K. pneumoniae. Less of this product was observed with the soybean system

  11. Electrochemical biosensor based on enzyme substrate as a linker: Application for aldolase activity with pectin-thionine complex as recognization element and signal amplification probe.

    Wang, Xiaonan; Wang, Meiwen; Zhang, Yuanyuan; Miao, Xiaocao; Huang, Yuanyuan; Zhang, Juan; Sun, Lizhou

    2016-09-15

    A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate, thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe. The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p-phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability. PMID:27107145

  12. Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.

    Bjelic, Sinisa; Aqvist, Johan

    2004-11-23

    The histo-aspartic protease (HAP) from the malaria parasite P. falciparum is one of several new promising targets for drug intervention. The enzyme possesses a novel type of active site, but its 3D structure and mechanism of action are still unknown. Here we use a combination of homology modeling, automated docking searches, and molecular dynamics/reaction free energy profile simulations to predict the enzyme structure, conformation of bound substrate, catalytic mechanism, and rate of the peptide cleavage reaction. We find that the computational tools are sufficiently reliable both for identifying substrate binding modes and for distinguishing between different possible reaction mechanisms. It is found that the favored pathway only involves direct participation by the catalytic aspartate, with the neighboring histidine providing critical stabilization (by a factor of approximately 10000) along the reaction. The calculated catalytic rate constant of about 0.1 s(-1) for a hexapeptide substrate derived from the alpha chain of human hemoglobin is in excellent agreement with experimental kinetic data for a similar peptide fragment. PMID:15544322

  13. Characterization of functional microorganism groups and substrate in activated sludge and wastewater by AUR, NUR and OUR

    Kristensen, G. Holm; Jørgensen, P. Elberg; Henze, Mogens

    1992-01-01

    Activated sludge functional microorganism groups: nitrifiers, denitrifiers and heterotrophs, were characterized through determinations of maximum specific utilization rates of ammonia (AUR) nitrate (NUR) and oxygen (OUR). Characterizations of the functional groups were done on activated sludges...

  14. Evaluation of polysaccharides content in fruit bodies and their antimicrobial activity of four Ganoderma lucidum (W Curt.: Fr. P. Karst. strains cultivated on different wood type substrates

    Krystyna Skalicka-Woźniak

    2012-03-01

    Full Text Available Quantitative determination of polysaccharides in Ganoderma lucidum fruit bodies from different sawdust cultivation substrates and their antibacterial activity was done. Thirty six samples were analyzed. Four strains of Ganoderma lucidum (GL01, GL02, GL03 and GL04 were cultivated on the growth substrates of three different sawdust types: birch (Bo, maple (Kl or alder (Ol amended with wheat bran in three different concentrations: 10, 20 and 30% (w/w. Even though the richest in polysaccharides was GL01 strain, the highest yields of the polysaccharides were determined in GL04Kl3 sample and was 112.82 mg/g of dry weight. The antibacterial activity of polysaccharides was determined in vitro using micro-dilution broth method. The panel of eight reference bacterial strains was used. All the polysaccharide samples tested showed the broad spectrum and the moderate antibacterial activity. Micrococcus luteus ATCC 10240 strain was the most sensitive with MIC (minimal inhibitory concentration = 0.63 − 1.25 mg/mL.

  15. Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using C-13-labeled substrates

    Santos, Maria Margarida M. dos; Gombert, A.K.; Christensen, B.;

    2003-01-01

    role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon...... metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate...... kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO2 via the tricarboxylic acid cycle for a...

  16. Alumina Encapsulated Strain Gage Not Mechanically Attached To The Substrate, Used to Temperature Compensate an Active High Temperature Gage In A Half-Bridge Configuration

    Piazza, Anthony (Inventor)

    2001-01-01

    A temperature compensation element for a high-temperature strain gage and the method of fabricating the same. Preferably, the element is a "dummy" strain gage not mechanically attached to the substrate. The element is encapsulated in an insulative material and used to compensate an active high-temperature strain gage and wired in a half-bridge configuration. The temperature compensation element and high-temperature strain gage are fabricated using the method of the present invention. This method includes temporarily adhering the element to a heat sink, encapsulated in an insulative material and then removed from the heat sink. Next, the element is either stacked or placed near the active gage. Ideally, the element and the active gage have substantially similar heat transfer and electrical properties.

  17. Genetic variation in the 3'-UTR of CYP1A2, CYP2B6, CYP2D6, CYP3A4, NR1I2 and UGT2B7: potential effect on regulation by microRNA and pharmacogenomics relevance

    Marelize eSwart

    2014-06-01

    Full Text Available Introduction: Pharmacogenomics research has concentrated on variation in genes coding for drug metabolising enzymes, transporters and nuclear receptors. However, variation affecting microRNA could also play a role in drug response. This project set out to investigate potential microRNA target sites in 11 genes and the extent of variation in the 3'-UTR of six selected genes; CYP1A2, CYP2B6, CYP2D6, CYP3A4, NR1I2 and UGT2B7. Methods: Fifteen microRNA target prediction algorithms were used to identify microRNAs predicted to regulate 11 genes. The 3'-UTR of the 6 genes which topped the list of potential microRNA targets was sequenced in 30 black South Africans. In addition, genetic variants within these genes were investigated for interference with mRNA-microRNA interactions. Potential effects of observed variants were determined using in silico prediction tools. Results: The 11 genes coding for DMEs, transporters and nuclear receptors were predicted to be targets of microRNAs with CYP2B6, NR1I2 (PXR, CYP3A4 and CYP1A2, interacting with the most microRNAs. The majority of identified genetic variants were predicted to interfere with microRNA regulation. For example, the variant, rs1054190C in NR1I2 was predicted to result in the presence of a binding site for the microRNA miR-1250-5p, while the variant rs1054191G was predicted to result in the absence of a recognition site for miR-371b-3p, miR-4258 and miR-4707-3p. Fifteen of the seventeen, novel variants occurred within microRNA target sequences.Conclusion: The 3'-UTR harbours variation that is likely to influence regulation of specific genes by microRNA. In silico prediction followed by functional validation could aid in decoding the contribution of variation in the 3'-UTR, to some unexplained heritability that affects drug response. Understanding the specific role of each microRNA may lead to identification of markers for targeted therapy and therefore improve personalized drug treatment.

  18. Complex Hydrocarbon Chemistry in Interstellar and Solar System Ices Revealed: A Combined Infrared Spectroscopy and Reflectron Time-of-flight Mass Spectrometry Analysis of Ethane (C2H6) and D6-Ethane (C2D6) Ices Exposed to Ionizing Radiation

    Abplanalp, Matthew J.; Kaiser, Ralf I.

    2016-08-01

    The irradiation of pure ethane (C2H6/C2D6) ices at 5.5 K, under ultrahigh vacuum conditions was conducted to investigate the formation of complex hydrocarbons via interaction with energetic electrons simulating the secondary electrons produced in the track of galactic cosmic rays. The chemical modifications of the ices were monitored in situ using Fourier transform infrared spectroscopy (FTIR) and during temperature-programmed desorption via mass spectrometry exploiting a quadrupole mass spectrometer with electron impact ionization (EI-QMS) as well as a reflectron time-of-flight mass spectrometer coupled to a photoionization source (PI-ReTOF-MS). FTIR confirmed previous ethane studies by detecting six molecules: methane (CH4), acetylene (C2H2), ethylene (C2H4), the ethyl radical (C2H5), 1-butene (C4H8), and n-butane (C4H10). However, the TPD phase, along with EI-QMS, and most importantly, PI-ReTOF-MS, revealed the formation of at least 23 hydrocarbons, many for the first time in ethane ice, which can be arranged in four groups with an increasing carbon-to-hydrogen ratio: C n H2n+2 (n = 3, 4, 6, 8, 10), C n H2n (n = 3–10), {{{C}}}n{{{H}}}2n-2 (n = 3–10), and {{{C}}}n{{{H}}}2n-4 (n = 4–6). The processing of simple ethane ices is relevant to the hydrocarbon chemistry in the interstellar medium, as ethane has been shown to be a major product of methane, as well as in the outer solar system. These data reveal that the processing of ethane ices can synthesize several key hydrocarbons such as C3H4 and C4H6 isomers, which ha­ve been found to synthesize polycyclic aromatic hydrocarbons like indene (C9H8) and naphtha­lene (C10H8) in the ISM and in hydrocarbon-rich atmospheres of planets and their moons such as Titan.

  19. Antifungal activity of Ag:hydroxyapatite thin films synthesized by pulsed laser deposition on Ti and Ti modified by TiO2 nanotubes substrates

    Eraković, S.; Janković, A.; Ristoscu, C.; Duta, L.; Serban, N.; Visan, A.; Mihailescu, I. N.; Stan, G. E.; Socol, M.; Iordache, O.; Dumitrescu, I.; Luculescu, C. R.; Janaćković, Dj.; Miškovic-Stanković, V.

    2014-02-01

    Hydroxyapatite (HA) is a widely used biomaterial for implant thin films, largely recognized for its excellent capability to chemically bond to hard tissue inducing the osteogenesis without immune response from human tissues. Nowadays, intense research efforts are focused on development of antimicrobial HA doped thin films. In particular, HA doped with Ag (Ag:HA) is expected to inhibit the attachment of microbes and contamination of metallic implant surface. We herewith report on nano-sized HA and Ag:HA thin films synthesized by pulsed laser deposition on pure Ti and Ti modified with 100 nm diameter TiO2 nanotubes (fabricated by anodization of Ti plates) substrates. The HA-based thin films were characterized by SEM, AFM, EDS, FTIR, and XRD. The cytotoxic activity was tested with HEp2 cells against controls. The antifungal efficiency of the deposited layers was tested against the Candida albicans and Aspergillus niger strains. The Ti substrates modified with TiO2 nanotubes covered with Ag:HA thin films showed the highest antifungal activity.

  20. Direct growth of NiCo2O4 nanostructures on conductive substrates with enhanced electrocatalytic activity and stability for methanol oxidation.

    Qian, Lei; Gu, Li; Yang, Li; Yuan, Hongyan; Xiao, Dan

    2013-08-21

    In this report, NiCo2O4 nanostructures with different morphologies were directly grown on conductive substrates (stainless steel and ITO) by a facile electrodeposition method in addition to a post-annealing process. The morphology changes on different conductive substrates are discussed in detail. The NiCo2O4 on stainless steel (SS) had a high surface area (119 m(2) g(-1)) and was successfully used in the electrocatalytic oxidation of methanol. The electrocatalytic performance was investigated by cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS) measurements. Impressively, the NiCo2O4 showed much higher electrocatalytic activity, lower overpotential and greater stability compared to that of only NiO or Co3O4 synthesized by the same method. The higher electrocatalytic activity is due to the high electron conductivity, large surface area of NiCo2O4 and the fast ion/electron transport in the electrode and at the electrolyte-electrode interface. This is important for further development of high performance non-platinum electrocatalysts for application in direct methanol fuel cells. PMID:23828628

  1. NIR and visible investigation of some potential SERS-active substrates for studying antitumour agent all- trans retinoic acid

    Beljebbar, A.; Sockalingum, G. D.; Morjani, H.; Angiboust, J. F.; Manfait, M.

    1997-01-01

    Red and near-infrared excited Fourier transform surface-enhanced Raman spectra of an anticancer agent, all- trans retinoic acid (ATRA), adsorbed on gold island films are reported. Best results have been obtained with plates 80 Å and 40 Å thick respectively in the red and near-infrared and at concentrations of 10 -5 and 5 × 10 -6 M with a spinning system. The use of near-infrared laser excitation with low photon energy, allows us to overcome the problems of isomerisation when the sample is exposed for a long time to the laser radiation. Comparison between the Raman and SERS spectra in the visible shows that the adsorption on the surface does not perturb the structure of ATRA and confirms the long range enhancement of the island films with this type of molecule. Spectral data show that while gold island films and colloids are appropriate substrates for use with red excitation, silver and gold colloids as well as gold island films exhibit satisfactory enhancement levels in the near-infrared. This study will in the future allow us to choose the appropriate system that will serve to investigate the interaction of ATRA with its target in vitro and the effect of this differentiating agent in human leukaemia cell lines such as K562 and HL60.

  2. Activation and control of microlens liquid arrays on functionalized polar electric crystal substrates by electro-wetting effect and temperature

    In recent years a variety of liquid bases optical elements have been conceived, designed and fabricated even for commercial products like digital cameras o cellular phone cameras. The impressive development of microfluidic systems in conjunction with optics has led to the creation of a completely new Science field of investigation named optofludics. Optofludics, among others topics, deals with investigation and methods for realizing liquid micro-lenses. A variety of liquid micro-lenses have been designed and realized by using different configurations. We demonstrate that a lensing effect can be obtained in an open microfluidic system by using a thin layer of liquid on a polar electric crystal such as Lithium Niobate (LiNbO3). Electrowetting patterning on LiNbO3 surface is obtained by pyroelectric effect consisting in a simple but reliable electrodes-less and circuit-less configuration. The electrodes are intrinsically embedded into the substrate. The material is functionalised by means of a micro-engineering electric filed poling process. Lens array with variable focus has been demonstrated with a large number of lens elements (10x10) on micrometric scale (aperture of single lens 100 microns).

  3. Fuel-Stimulated Insulin Secretion Depends upon Mitochondria Activation and the Integration of Mitochondrial and Cytosolic Substrate Cycles

    Gary W Cline

    2011-01-01

    The pancreatic islet β-cell is uniquely specialized to couple its metabolism and rates of insulin secretion with the levels of circulating nutrient fuels, with the mitochondrial playing a central regulatory role in this process. In the β-cell, mitochondrial activation generates an integrated signal reflecting rates of oxidativephosphorylation, Kreb's cycle flux, and anaplerosis that ultimately determines the rate of insulin exocytosis. Mitochondrial activation can be regulated by proton leak ...

  4. Nutrition Intensity in Ternary Diagrams Interpretation for Some Ornamental Species Cultivated on Organic Substrate with Increased Biological Activity

    Roxana Maria MADJAR

    2014-12-01

    Full Text Available Nowadays, many biodegradable organic wastes no longer need to represent an environmental hazard and as a consequence, they could be recycled to obtain horticultural substrates. An experiment was conducted on two deciduous (Tamarix tetrandra, Ligustrum ovalifolium ‘Aureum’ and two coniferous species (Chamaecyparis pisifera ‘Boulevard’, Chamaecyparis lawsoniana ‘Stardust’ grown on a soil mixture of forestry compost, leaves compost, peat and grape marc compost. The aim of the research was to investigate the response to fertilization and to obtain valuable information regarding absorption rate of nutritive elements during v