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Sample records for 2d6 substrate activity

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

    DEFF Research Database (Denmark)

    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. Composite functional genetic and comedication CYP2D6 activity score in predicting tamoxifen drug exposure among breast cancer patients.

    Science.gov (United States)

    Borges, Silvana; Desta, Zeruesenay; Jin, Yan; Faouzi, Azzouz; Robarge, Jason D; Philips, Sanosh; Philip, Santosh; Nguyen, Anne; Stearns, Vered; Hayes, Daniel; Rae, James M; Skaar, Todd C; Flockhart, David A; Li, Lang

    2010-04-01

    Accurate assessment of CYP2D6 phenotypes from genotype is inadequate in patients taking CYP2D6 substrate together with CYP2D6 inhibitors. A novel CYP2D6 scoring system is proposed that incorporates the impact of concomitant medications with the genotype in calculating the CYP2D6 activity score. Training (n = 159) and validation (n = 81) data sets were obtained from a prospective cohort tamoxifen pharmacogenetics registry. Two inhibitor factors were defined: 1 genotype independent and 1 genotype based. Three CYP2D6 gene scoring systems, and their combination with the inhibitor factors, were compared. These 3 scores were based on Zineh, Zanger, and Gaedigk's approaches. Endoxifen/NDM-Tam plasma ratio was used as the phenotype. The overall performance of the 3 gene scoring systems without consideration of CYP2D6-inhibiting medications in predicting CYP2D6 phenotype was poor in both the training set (R(2) = 0.24, 0.22, and 0.18) and the validation set (R(2) = 0.30, 0.24, and 0.15). Once the CYP2D6 genotype-independent inhibitor factor was integrated into the score calculation, the R(2) values in the training and validation data sets were nearly twice as high as the genotype-only scoring model: (0.44, 0.43, 0.38) and (0.53, 0.50, 0.41), respectively. The integration of the inhibitory effect of concomitant medications with the CYP2D6 genotype into the composite CYP2D6 activity score doubled the ability to predict the CYP2D6 phenotype. However, endoxifen phenotypes still varied substantially, even with incorporation of CYD2D6 genotype and inhibiting factors, suggesting that other, as yet unidentified factors must be involved in tamoxifen activation.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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...... of specific CYP activity. An overrepresentation was seen for poly-aromatic hydrocarbons (group of procarcinogens) among CYP2C9 active and mutagenic compounds compared to CYP2C9 inactive and mutagenic compounds. The mutagenicity was predicted with a QSAR model based on Ames in vitro test data.......% 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...

  4. Roles of phenylalanine at position 120 and glutamic acid at position 222 in the oxidation of chiral substrates by cytochrome P450 2D6.

    Science.gov (United States)

    Masuda, Kazufumi; Tamagake, Keietsu; Katsu, Takashi; Torigoe, Fumihiro; Saito, Keita; Hanioka, Nobumitsu; Yamano, Shigeru; Yamamoto, Shigeo; Narimatsu, Shizuo

    2006-02-01

    The roles of Phe-120 and Glu-222 in the oxidation of chiral substrates bunitrolol (BTL) and bufuralol (BF) by CYP2D6 are discussed. Wild-type CYP2D6 (CYP2D6-WT) oxidized BTL to 4-hydroxybunitrolol (4-OH-BTL) with substrate enantioselectivity of (R)-(+)-BTL > (S)-(-)-BTL. The same enzyme converted BF into 1''-hydroxybufuralol with substrate enantioselectivity of (R)-BF > (S)-BF and metabolite diastereoselectivity of (1''R)-OH kinetic parameters (K(m) and V(max) values) for (R)-BF 1''-hydroxylation remained unchanged. Furthermore, the substitution of Glu-222 as well as Glu-216 by alanine remarkably decreased both the apparent K(m) and V(max) values without changing substrate enantioselectivity or metabolite diastereoselectivity. A computer-assisted simulation study using energy minimization and molecular dynamics techniques indicated that the hydrophobic interaction of an aromatic moiety of the substrate with Phe-120 and the ionic interaction of a basic nitrogen atom of the substrate with Glu-222 in combination with Glu-216 play important roles in the binding of BF and BTL by CYP2D6 and the orientation of these substrates in the active-site cavity. This modeling yielded a convincing explanation for the reversal of substrate enantioselectivity in BTL 4-hydroxylation between CYP2D6-WT and CYP2D6-V374M having methionine in place of Val-374, which supports the validity of this modeling.

  5. CYP2D6-Genotyping in clinical practice: Impact of genotype on serum concentrations of psychiatric CYP2D6-substrates

    OpenAIRE

    Plodeck, Verena

    2010-01-01

    Cytochrom P450 2D6 spielt beim Abbau vieler Psychopharmaka eine wichtige Rolle. Es ist seit längerem bekannt, dass für CYP2D6 ein genetischer Polymorphismus existiert. Dieser führt dazu, dass die metabolischen Kapazitäten interindividuell stark variieren. Man spricht in diesem Zusammenhang von poor, intermediate, extensive und ultra-fast metabolizern. Das Ziel dieser Arbeit war es, die Auswirkungen des Genotyps auf die Serumkonzentrationen und somit auch die Verträglichkeit bzw. den Therapiee...

  6. Differential Consequences of Tramadol in Overdosing: Dilemma of a Polymorphic Cytochrome P450 2D6-Mediated Substrate.

    Science.gov (United States)

    Srinivas, Nuggehally R

    2015-09-01

    Tramadol is a centrally acting opioid analgesic that is prone to polymorphic metabolism via cytochrome P450 (CYP) 2D6. The generation of the active metabolite, O-desmethyltramadol, which occurs through the CYP 2D6 pathway, significantly contributes to the drug's activity. However, dosage adjustments of tramadol are typically not practiced in the clinic when treating patients who are homozygous extensive metabolizers, heterozygous extensive metabolizers, or poor metabolizers. In the event of a tramadol overdose, the consequences may be influenced importantly by the genotype or phenotype status of the subject. Depending on the individual subject's CYP 2D6 status, one may see excessive miotic-related toxicity driven by the excessive availability of O-desmethyltramadol or one may manifest mydriatic-related toxicity driven by the excessive availability of tramadol. This report provides pharmacokinetic perspectives in situations of tramadol overdosing.

  7. Liver kidney microsomal type 1 antibodies reduce the CYP2D6 activity in patients with chronic hepatitis C virus infection.

    Science.gov (United States)

    Girardin, F; Daali, Y; Gex-Fabry, M; Rebsamen, M; Roux-Lombard, P; Cerny, A; Bihl, F; Binek, J; Moradpour, D; Negro, F; Desmeules, J

    2012-08-01

    Liver kidney microsomal type 1 (LKM-1) antibodies have been shown to decrease the CYP2D6 activity in vitro and are present in a minority of patients with chronic hepatitis C infection. We investigated whether LKM-1 antibodies might reduce the CYP2D6 activity in vivo. All patients enrolled in the Swiss Hepatitis C Cohort Study and tested for LKM-1 antibodies were assessed (n = 1723): 10 eligible patients were matched with patients without LKM-1 antibodies. Patients were genotyped for CYP2D6 variants to exclude individuals with a poor metabolizer genotype. CYP2D6 activity was measured by a specific substrate using the dextromethorphan/dextrorphan metabolic ratio to classify patients into four activity phenotypes. All patients had a CYP2D6 extensive metabolizer genotype. The observed phenotype was concordant with the CYP2D6 genotype in most LKM-negative patients, whereas only three LKM-1 positive patients had a concordant phenotype (six presented an intermediate and one a poor metabolizer phenotype). The median DEM/DOR ratio was sixfold higher in LKM-1 positive than in LKM-1 negative patients (0.096 vs. 0.016, P = 0.004), indicating that CYP2D6 metabolic function was significantly reduced in the presence of LKM-1 antibodies. In chronic hepatitis C patients with LKM-1 antibodies, the CYP2D6 metabolic activity was on average reduced by 80%. The impact of LKM-1 antibodies on CYP2D6-mediated drug metabolism pathways warrants further translational studies. © 2012 Blackwell Publishing Ltd.

  8. Evaluation of CYP2D6 enzyme activity using a Dextromethorphan Breath Test in Women Receiving Adjuvant Tamoxifen

    Science.gov (United States)

    Safgren, Stephanie L.; Suman, Vera J.; Kosel, Matthew L.; Gilbert, Judith A; Buhrow, Sarah A.; Black, John L.; Northfelt, Donald W.; Modak, Anil S.; Rosen, David; Ingle, James N.; Ames, Matthew M.; Reid, Joel M.; Goetz, Matthew P.

    2015-01-01

    Background In tamoxifen-treated patients, breast cancer recurrence differs according to CYP2D6 genotype and endoxifen steady state concentrations (Endx Css). The 13Cdextromethorphan breath test (DM-BT), labeled with 13C at the O-CH3 moiety, measures CYP2D6 enzyme activity. We sought to examine the ability of the DM-BT to identify known CYP2D6 genotypic poor metabolizers and examine the correlation between DMBT and Endx Css. Methods DM-BT and tamoxifen pharmacokinetics were obtained at baseline (b), 3 month (3m) and 6 months (6m) following tamoxifen initiation. Potent CYP2D6 inhibitors were prohibited. The correlation between bDM-BT with CYP2D6 genotype and Endx Css was determined. The association between bDM-BT (where values ≤0.9 is an indicator of poor in vivo CYP2D6 metabolism) and Endx Css (using values ≤ 11.2 known to be associated with poorer recurrence free survival) was explored. Results 91 patients were enrolled and 77 were eligible. CYP2D6 genotype was positively correlated with b, 3m and 6m DMBT (r ranging from 0.457-0. 60 p 11.2 nM. Conclusions In patients not taking potent CYP2D6 inhibitors, DM-BT was associated with CYP2D6 genotype and 3m and 6 m Endx Css but did not provide better discrimination of Endx Css compared to CYP2D6 genotype alone. Further studies are needed to identify additional factors which alter Endx Css. PMID:25714002

  9. Linkage disequilibrium mapping identifies a 390 kb region associated with CYP2D6 poor drug metabolising activity.

    Science.gov (United States)

    Hosking, L K; Boyd, P R; Xu, C F; Nissum, M; Cantone, K; Purvis, I J; Khakhar, R; Barnes, M R; Liberwirth, U; Hagen-Mann, K; Ehm, M G; Riley, J H

    2002-01-01

    The cytochrome p450 enzyme, CYP2D6, metabolises approximately 20% of marketed drugs. CYP2D6 multiple variants are associated with altered enzyme activities. Genotyping 1018 Caucasians for CYP2D6 polymorphisms (G1846A, delT1707, delA2549 and A2935C), known to result in the recessive CYP2D6 poor drug metaboliser (PM) phenotype, identified 41 individuals with predicted PM phenotype. These 41 individuals were classified as 'cases'. Single nucleotide polymorphisms (SNPs) mapping within an 880 kb region flanking CYP2D6, were identified to evaluate potential association between genetic variation and the CYP2D6 PM phenotype. The 41 PM cases and 977 controls were genotyped and analysed for 27 SNPs. Associations were observed across a 390 kb region between 14 SNPs and the PM phenotype (P values from 6.20 x 10(-4) to 4.54 x 10(-35)). Haplotype analysis revealed more significant levels of association (P = 3.54 x 10(-56)). Strong (D' > 0.7) linkage disequilibrium (LD) between SNPs was observed across the same 390 kb region associated with the CYP2D6 phenotype. The observed phenotype:genotype association reached genome-wide levels of significance, and supports the strategy for potential application of LD mapping and whole genome association scans to pharmacogenetic studies.

  10. CYP2D6 pharmacogenomics

    Directory of Open Access Journals (Sweden)

    Mohanan Geetha Gopisankar

    2017-10-01

    Full Text Available Cytochromes are proteins that catalyze electron transfer reactions of many metabolic pathways. They are involved in drug metabolism and thus determines the therapeutic safety and efficacy of drugs in patients. Cytochrome P450 in mitochondria accounts for 90% of the oxidative metabolism of clinically used drugs during phase 1 reaction. CYP2D6 is a major gene member of this superfamily as it carries out metabolism of 25% of drugs currently available in the market. Contrary to the concept of specificity of enzyme action these can metabolize substrates of different chemistry. Since its discovery, many have intensively studied this unique hemoprotein and contributed to the elucidation of its molecular properties and physiological functions and also the structure-activity relationships of its substrates and inhibitors. Its activity ranges considerably within a population due to genetic polymorphisms which lead to varied responses to drug intake. Studying such polymorphisms which cause a significant impact in the management of patients and helps to achieve the final target of personalizing medicine. This review briefs about history, structure, and function, molecular genetics, substrates, regulators and inhibitors of CYP2D6 and its clinical pharmacogenomics.

  11. Estimation of tamoxifen metabolite concentrations in the blood of breast cancer patients through CYP2D6 genotype activity score.

    Science.gov (United States)

    Wu, Alan H B; Lorizio, Wendy; Tchu, Simone; Lynch, Kara; Gerona, Roy; Ji, Wuyang; Ruan, Weiming; Ruddy, Kathryn J; Desantis, Stephen D; Burstein, Harold J; Ziv, Elad

    2012-06-01

    Tamoxifen, a prodrug used for adjuvant breast cancer therapy, requires conversion to the active metabolite endoxifen through CYP 2D6. We aimed to construct an algorithm to predict endoxifen concentrations based on a patient’s CYP 2D6 genotype, demographic factors, and co-medication use. Eighty-eight women enrolled in the UCSF TamGen II study and 81 women enrolled in a prospective study at Dana-Farber Cancer Institute were included in this analysis. All the women had been on tamoxifen for at least 3 months before blood collection. Demographic information included the patient’s age, race/ethnicity, body mass index (where available), and self-reported and measured medications and herbals that affect 2D6 activity. DNA was extracted and genotyped for 2D6 (Amplichip, Roche Diagnostics). An activity score was calculated based on genotypes and adjusted for use of medications known to inhibit 2D6. Serum was tested for tamoxifen and metabolite concentrations and for the presence of drugs by liquid chromatography/mass spectrometry. Univariate and multivariate regression analysis were computed for age, body mass index, ethnicity, and adjusted activity score to predict tamoxifen metabolite concentrations in the training data-set of UCSF patients, and the resulting algorithm was validated in the Dana-Farber patients. For the training set, the correlation coefficient (r2) for log endoxifen and N-desmethyltamoxifen:endoxifen ratio to activity score, age, and race, were 0.520 and 0.659, respectively; 0.324 and 0.567 for the validation; and 0.396 and 0.615 for both the datasets combined. An algorithm that incorporates genotype and demographic variables can be used to predict endoxifen concentrations for women on tamoxifen therapy. If endoxifen levels are confirmed to be predictive of tamoxifen benefit, then this algorithm may be helpful to determine which women warrant endoxifen testing.

  12. The effects of desvenlafaxine and paroxetine on the pharmacokinetics of the cytochrome P450 2D6 substrate desipramine in healthy adults.

    Science.gov (United States)

    Nichols, Alice I; Fatato, Penny; Shenouda, Magdy; Paul, Jeffrey; Isler, Jennifer A; Pedersen, Ronald D; Jiang, Qin; Ahmed, Saeeduddin; Patroneva, Albena

    2009-02-01

    The potential for cytochrome P450 (CYP) 2D6 substrates to interact with desvenlafaxine (administered as desvenlafaxine succinate) and paroxetine was evaluated. In an open-label, crossover study, 20 healthy volunteers (aged 21-50) were randomized to 2 series of 9 days each of desvenlafaxine (100 mg/d) or paroxetine (20 mg/d), separated by a 5-day washout. The CYP2D6 substrate desipramine (50 mg) was administered alone on day 1 and coadministered on day 6 of dosing with either desvenlafaxine or paroxetine. CYP2D6 genotype was determined at baseline. Based on least squares geometric mean ratios between reference (desipramine alone) and test treatments, desvenlafaxine produced minor increases in desipramine area under the plasma concentration versus time curve (AUC; 36%) and peak plasma concentration (C(max); 30%) (vs paroxetine: 419%, 90%, respectively; both P Desvenlafaxine produced little change in 2-hydroxydesipramine AUC (16% increase) and C(max) (0%) versus paroxetine (18% and 82% decreases, respectively; P = .008, P desvenlafaxine, especially at the recommended therapeutic dose of 50 mg/d for major depressive disorder in the United States, has little potential to interact with CYP2D6 substrates.

  13. The effect of mirabegron, a potent and selective β3-adrenoceptor agonist, on the pharmacokinetics of CYP2D6 substrates desipramine and metoprolol.

    Science.gov (United States)

    Krauwinkel, Walter; Dickinson, James; Schaddelee, Marloes; Meijer, John; Tretter, Reiner; van de Wetering, Jeroen; Strabach, Gregory; van Gelderen, Marcel

    2014-03-01

    Mirabegron is a potent and selective β3-adrenoceptor agonist developed for the treatment of overactive bladder. In vitro studies demonstrated that mirabegron partly acts as a (quasi-) irreversible, metabolism-dependent inhibitor of CYP2D6. The effect of steady-state mirabegron on single doses of the sensitive CYP2D6 substrates metoprolol (100 mg) and desipramine (50 mg) was assessed in two open-label, one-sequence crossover studies in healthy subjects (CYP2D6 extensive metabolizers). Mirabegron 160 mg/day increased metoprolol maximum plasma concentration (C max) 1.90-fold (90 % confidence interval [CI] 1.54; 2.33) and total exposure (AUC0-∞) 3.29-fold (90 % CI 2.70; 4.00) in 12 males (study 1). Mean metoprolol half-life increased from 2.96 to 4.11 h. α-Hydroxymetoprolol C max and AUC to last measurable concentration decreased 2.6-fold and 2.2-fold, respectively. In study 2, mirabegron 100 mg/day increased desipramine C max 1.79-fold (90 % CI 1.69; 1.90) and AUC0-∞ 3.41-fold (90 % CI 3.07; 3.80) in 14 males and 14 females. Mean desipramine half-life increased from 19.5 to 35.8 h. C max of 2-hydroxydesipramine decreased ~twofold, while AUC increased ~1.3-fold. Desipramine was administered again 2 weeks after the last mirabegron dose. Desipramine C max and AUC0-∞ were still ~1.13-fold increased; the 90 % CIs fell within the 0.80-1.25 interval. All treatments were well tolerated. In conclusion, mirabegron is a moderate CYP2D6 inhibitor (ratio and 90 % CI <5.0).

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

    NARCIS (Netherlands)

    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

  15. Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6. Lack of an allosteric role of NADPH-cytochrome P450 reductase in catalytic regioselectivity.

    Science.gov (United States)

    Hanna, I H; Krauser, J A; Cai, H; Kim, M S; Guengerich, F P

    2001-10-26

    Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regioselectivity of oxidation products formed in systems containing NADPH-P450 reductase/NADPH and the model oxidant cumene hydroperoxide have been proposed by others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We examined the differences in the formation of oxidation products of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reductase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalytic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influences. The presence of the reductase had little effect on the affinity of P450 2D6 for any of these three substrates. The addition of the reaction remnants of the model oxidants (cumyl alcohol and iodobenzene) to the reductase-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from H(2)18O was quantitatively incorporated into 1'-hydroxybufuralol in the iodosylbenzene- but not in the reductase- or cumene hydroperoxide-supported reactions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distributions, not an allosteric influence of the reductase.

  16. Evidence for the activation of organophosphate pesticides by cytochromes P450 3A4 and 2D6 in human liver microsomes.

    Science.gov (United States)

    Sams, C; Mason, H J; Rawbone, R

    2000-08-16

    The role of specific cytochrome P450 isoforms in catalysing the oxidative biotransformation of the organophosphorothioate pesticides parathion, chlorpyrifos and diazinon into structures that inhibit cholinesterase has been investigated in human liver microsomes using chemical inhibitors. Pesticides were incubated with human liver microsomes and production of the anticholinergic oxon metabolite was investigated by the inhibition of human serum cholinesterase. Quinidine and ketoconazole at 10 micromol/l inhibited oxidative biotransformation. Compared to control incubations (no inhibitor) where cholinesterase activity was inhibited to between 1 and 4% of control levels, incorporation of the CYP2D6 inhibitor quinidine into the microsomal incubation resulted in cholinesterase activity of 50% for parathion, 38% for diazinon and 30% for chlorpyrifos. Addition of the CYP3A4 inhibitor ketoconazole to microsomal incubations resulted in 66% cholinesterase activity with diazinon, 20% with parathion and 5% with chlorpyrifos. The unexpected finding that CYP2D6, as well as CYP3A4, catalysed oxidative biotransformation was confirmed for chlorpyrifos and parathion using microsomes prepared from a human lymphoblastoid cell line expressing CYP2D6. While parathion has been investigated only as a model compound, chlorpyrifos and diazinon are both very important, widely used pesticides and CYP2D6 appears to be an important enzyme in their bioactivation pathway. CYP2D6 is polymorphic and hence may influence individual susceptibility to exposure to chlorpyrifos and diazinon as well as other structurally similar pesticides.

  17. Selective inhibition of cytochrome P450 2D6 by Sarpogrelate and its active metabolite, M-1, in human liver microsomes.

    Science.gov (United States)

    Cho, Doo-Yeoun; Bae, Soo Hyeon; Lee, Joeng Kee; Kim, Yang Weon; Kim, Bom-Taeck; Bae, Soo Kyung

    2014-01-01

    The present study was performed to evaluate the in vitro inhibitory potential of sarpogrelate and its active metabolite, M-1, on the activities of nine human cytochrome (CYP) isoforms. Using a cocktail assay, the effects of sarpogrelate on nine CYP isoforms and M-1 were measured by specific marker reactions in human liver microsomes. Sarpogrelate potently and selectively inhibited CYP2D6-mediated dextromethorphan O-demethylation with an IC50 (Ki) value of 3.05 μM (1.24 μM), in a competitive manner. M-1 also markedly inhibited CYP2D6 activity; its inhibitory effect with an IC50 (Ki) value of 0.201 μM (0.120 μM) was more potent than that of sarpogrelate, and was similarly potent as quinidine (Ki, 0.129 μM), a well-known typical CYP2D6 inhibitor. In addition, sarpogrelate and M-1 strongly inhibited both CYP2D6-catalyzed bufuralol 1'-hydroxylation and metoprolol α-hydroxylation activities. However, sarpogrelate and M-1 showed no apparent inhibition of the other following eight CYPs: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5. Upon 30-minute preincubation of human liver microsomes with sarpogrelate or M-1 in the presence of NADPH, no obvious shift in IC50 was observed in terms of inhibition of the nine CYP activities, suggesting that sarpogrelate and M-1 are not time-dependent inactivators. Sarpogrelate strongly inhibited the activity of CYP2D6 at clinically relevant concentrations in human liver microsomes. These observations suggest that sarpogrelate could have an effect on the metabolic clearance of drugs possessing CYP2D6-catalyzed metabolism as a major clearance pathway, thereby eliciting pharmacokinetic drug-drug interactions.

  18. The correlation between CYP2D6 isoenzyme activity and haloperidol efficacy and safety profile in patients with alcohol addiction during the exacerbation of the addiction

    Science.gov (United States)

    Sychev, Dmitry Alekseevich; Zastrozhin, Mikhail Sergeevich; Smirnov, Valery Valerieevich; Grishina, Elena Anatolievna; Savchenko, Ludmila Mikhailovna; Bryun, Evgeny Alekseevich

    2016-01-01

    Background Today, it is proved that isoenzymes CYP2D6 and CYP3A4 are involved in metabolism of haloperidol. In our previous investigation, we found a medium correlation between the efficacy and safety of haloperidol and the activity of CYP3A4 in patients with alcohol abuse. Objective The aim of this study was to evaluate the correlation between the activity of CYP2D6 and the efficacy and safety of haloperidol in patients with diagnosed alcohol abuse. Methods The study involved 70 men (average age: 40.83±9.92 years) with alcohol addiction. A series of psychometric scales were used in the research. The activity of CYP2D6 was evaluated by high-performance liquid chromatography with mass spectrometry using the ratio of 6-hydroxy-1,2,3,4-tetrahydro-beta-carboline to pinoline. Genotyping of CYP2D6 (1846G>A) was performed using real-time polymerase chain reaction. Results According to results of correlation analysis, statistically significant values of Spearman correlation coefficient (rs) between the activity of CYP2D6 and the difference of points in psychometric scale were obtained in patients receiving haloperidol in injection form (Sheehan Clinical Anxiety Rating Scale =−0.721 [P<0.001] and Udvald for Kliniske Undersogelser Side Effect Rating Scale =0.692 [P<0.001]) and in those receiving haloperidol in tablet form (Covi Anxiety Scale =−0.851 [P<0.001] and Udvald for Kliniske Undersogelser Side Effect Rating Scale =0.797 [P<0.001]). Conclusion This study demonstrated the correlations between the activity of CYP2D6 isozyme and the efficacy and safety of haloperidol in patients with alcohol addiction. PMID:27695358

  19. The correlation between CYP2D6 isoenzyme activity and haloperidol efficacy and safety profile in patients with alcohol addiction during the exacerbation of the addiction

    Directory of Open Access Journals (Sweden)

    Sychev DA

    2016-09-01

    Full Text Available Dmitry Alekseevich Sychev,1 Mikhail Sergeevich Zastrozhin,1–3 Valery Valerieevich Smirnov,4 Elena Anatolievna Grishina,1 Ludmila Mikhailovna Savchenko,1 Evgeny Alekseevich Bryun1,2 1Russian Medical Academy of Postgraduate Education, Ministry of Health of the Russian Federation, 2Department of Public Health, Moscow Research and Practical Centre for Narcology, 3Peoples’ Friendship University of Russia, 4National Research Center, Institute of Immunology Federal Medical-Biological Agency of Russia, Moscow, Russia Background: Today, it is proved that isoenzymes CYP2D6 and CYP3A4 are involved in metabolism of haloperidol. In our previous investigation, we found a medium correlation between the efficacy and safety of haloperidol and the activity of CYP3A4 in patients with alcohol abuse.Objective: The aim of this study was to evaluate the correlation between the activity of CYP2D6 and the efficacy and safety of haloperidol in patients with diagnosed alcohol abuse.Methods: The study involved 70 men (average age: 40.83±9.92 years with alcohol addiction. A series of psychometric scales were used in the research. The activity of CYP2D6 was evaluated by high-performance liquid chromatography with mass spectrometry using the ratio of 6-hydroxy-1,2,3,4-tetrahydro-beta-carboline to pinoline. Genotyping of CYP2D6 (1846G>A was performed using real-time polymerase chain reaction.Results: According to results of correlation analysis, statistically significant values of Spearman correlation coefficient (rs between the activity of CYP2D6 and the difference of points in psychometric scale were obtained in patients receiving haloperidol in injection form (Sheehan Clinical Anxiety Rating Scale =-0.721 [P<0.001] and Udvald for Kliniske Undersogelser Side Effect Rating Scale =0.692 [P<0.001] and in those receiving haloperidol in tablet form (Covi Anxiety Scale =-0.851 [P<0.001] and Udvald for Kliniske Undersogelser Side Effect Rating Scale =0.797 [P<0.001].Conclusion

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

    Science.gov (United States)

    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.

  1. Marmoset cytochrome P450 2D8 in livers and small intestines metabolizes typical human P450 2D6 substrates, metoprolol, bufuralol and dextromethorphan.

    Science.gov (United States)

    Uehara, Shotaro; Uno, Yasuhiro; Hagihira, Yuya; Murayama, Norie; Shimizu, Makiko; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2015-01-01

    1. Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient. 2. A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83-86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat. 3. Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting. 4. By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1'-hydroxylation. 5. These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6.

  2. 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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

    Science.gov (United States)

    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

    2012-03-15

    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.

  4. Effects of green tea catechins on cytochrome P450 2B6, 2C8, 2C19, 2D6 and 3A activities in human liver and intestinal microsomes.

    Science.gov (United States)

    Misaka, Shingen; Kawabe, Keisuke; Onoue, Satomi; Werba, José Pablo; Giroli, Monica; Tamaki, Sekihiro; Kan, Toshiyuki; Kimura, Junko; Watanabe, Hiroshi; Yamada, Shizuo

    2013-01-01

    The effects of green tea catechins on the main drug-metabolizing enzymatic system, cytochrome P450 (CYP), have not been fully elucidated. The objective of the present study was to evaluate the effects of green tea extract (GTE, total catechins 86.5%, w/w) and (-)-epigallocatechin-3-gallate (EGCG) on the activities of CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A in vitro, using pooled human liver and intestinal microsomes. Bupropion hydroxylation, amodiaquine N-deethylation, (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation were assessed in the presence or absence of various concentrations of GTE and EGCG to test their effects on CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A activities, respectively. Each metabolite was quantified using UPLC/ESI-MS, and the inhibition kinetics of GTE and EGCG on CYP enzymes was analyzed. In human liver microsomes, IC50 values of GTE were 5.9, 4.5, 48.7, 25.1 and 13.8 µg/mL, for CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A, respectively. ECGC also inhibited these CYP isoforms with properties similar to those of GTE, and produced competitive inhibitions against CYP2B6 and CYP2C8, and noncompetitive inhibition against CYP3A. In human intestinal microsomes, IC50 values of GTE and EGCG for CYP3A were 18.4 µg/mL and 31.1 µM, respectively. EGCG moderately inhibited CYP3A activity in a noncompetitive manner. These results suggest that green tea catechins cause clinically relevant interactions with substrates for CYP2B6 and CYP2C8 in addition to CYP3A.

  5. Availability of CYP2D6 genotyping results in general practitioner and community pharmacy medical records

    NARCIS (Netherlands)

    Simoons, Mirjam; Mulder, Hans; Schoevers, Robert A; Ruhé, Henricus G; van Roon, Eric N

    AIM: To investigate the availability of CYP450-2D6 (CYP2D6) genotyping results in general practitioner (GP) and/or community pharmacy records, and the influence thereof on psychotropic CYP2D6 substrate dosing. MATERIALS & METHODS: Primary outcome was the percentage of patients genotyped for CYP2D6

  6. Impact of fraction unbound, CYP3A, and CYP2D6 in vivo activities, and other potential covariates to the clearance of tramadol enantiomers in patients with neuropathic pain.

    Science.gov (United States)

    de Moraes, Natália V; Lauretti, Gabriela R; Coelho, Eduardo B; Godoy, Ana Leonor P C; Neves, Daniel V; Lanchote, Vera L

    2016-04-01

    The pharmacokinetics of tramadol is characterized by a large interindividual variability, which is partially attributed to polymorphic CYP2D6 metabolism. The contribution of CYP3A, CYP2B6, fraction unbound, and other potential covariates remains unknown. This study aimed to investigate the contribution of in vivo activities of cytochrome P450 (CYP) 2D6 and 3A as well as other potential covariates (CYP2B6 genotype to the SNP g.15631G>T, fraction unbound, age, body weight, creatinine clearance) to the enantioselective pharmacokinetics of tramadol. Thirty patients with neuropathic pain and phenotyped as CYP2D6 extensive metabolizers were treated with a single oral dose of 100 mg tramadol. Multiple linear regressions were performed to determine the contribution of CYP activities and other potential covariates to the clearance of tramadol enantiomers. The apparent total clearances were 44.9 (19.1-102-2) L/h and 55.2 (14.8-126.0) L/h for (+)- and (-)-tramadol, respectively [data presented as median (minimum-maximum)]. Between 79 and 83% of the overall variation in apparent clearance of tramadol enantiomers was explained by fraction unbound, CYP2D6, and CYP3A in vivo activities and body weight. Fraction unbound explained 47 and 41% of the variation in clearance of (+)-tramadol and (-)-tramadol, respectively. Individually, CYP2D6 and CYP3A activities were shown to have moderate contribution on clearance of tramadol enantiomers (11-16% and 11-18%, respectively). In conclusion, factors affecting fraction unbound of drugs (such as hyperglycemia or co-administration of drugs highly bound to plasma proteins) should be monitored, because this parameter dominates the elimination of tramadol enantiomers.

  7. CYP2D6 polymorphism in relation to tramadol metabolism

    DEFF Research Database (Denmark)

    Halling, Jónrit; Weihe, Pál; Brosen, Kim

    2008-01-01

    Several studies have demonstrated the impact of CYP2D6 polymorphism on the pharmacokinetics of tramadol. However, the relationship between the O-demethylation of tramadol and O-desmethyltramadol (M1) and CYP2D6 activity has not previously been investigated with tramadol in multimedicated...... outpatients under steady-state conditions. Hence, the aim of this study was to determine if the well documented pharmacokinetics of tramadol regarding CYP2D6 could be verified in a study including 88 multimedicated Faroese patients, treated with tramadol at steady-state conditions. Further, the study aimed...... collection over 12 hours. Sparteine and its metabolites were assayed by gas chromatography. Genotype analyses for the CYP2D6 3, 4, 6, and 9 alleles were performed by polymerase chain reaction and Taqman technology. Plasma and urinary concentrations of (+/-)-tramadol and (+/-)-M1 were determined by high...

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

    Directory of Open Access Journals (Sweden)

    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.

  9. CYP2D6 variability in populations from Venezuela.

    Science.gov (United States)

    Moreno, Nancy; Flores-Angulo, Carlos; Villegas, Cecilia; Mora, Yuselin

    2016-12-01

    CYP2D6 is an important cytochrome P450 enzyme that plays an important role in the metabolism of about 25% of currently prescribed drugs. The presence of polymorphisms in the CYP2D6 gene may modulate enzyme level and activity, thereby affecting individual responses to pharmacological treatments. The most prevalent diseases in the admixed population from Venezuela are cardiovascular and cancer, whereas viral, bacterial and parasitic diseases, particularly malaria, are prevalent in Amerindian populations; in the treatment of these diseases, several drugs that are metabolized by CYP2D6 are used. In this work, we reviewed the data on CYP2D6 variability and predicted metabolizer phenotypes, in healthy volunteers of two admixed and five Amerindian populations from Venezuela. The Venezuelan population is very heterogeneous as a result of the genetic admixture of three major ethnical components: Europeans, Africans and Amerindians. There are noticeable inter-regional and inter-population differences in the process of mixing of this population. Hitherto, there are few published studies in Venezuela on CYP2D6; therefore, it is necessary to increase research in this regard, in particular to develop studies with a larger sample size. There is a considerable amount of work remaining before CYP2D6 is integrated into clinical practice in Venezuela.

  10. In vitro modulatory effects of Terminalia arjuna, arjunic acid, arjunetin and arjungenin on CYP3A4, CYP2D6 and CYP2C9 enzyme activity in human liver microsomes

    Directory of Open Access Journals (Sweden)

    Alice Varghese

    2015-01-01

    Full Text Available Terminalia arjuna is a tree having an extensive medicinal potential in cardiovascular disorders. Triterpenoids are mainly responsible for cardiovascular properties. Alcoholic and aqueous bark extracts of T. arjuna, arjunic acid, arjunetin and arjungenin were evaluated for their potential to inhibit CYP3A4, CYP2D6 and CYP2C9 enzymes in human liver microsomes. We have demonstrated that alcoholic and aqueous bark extract of T. arjuna showed potent inhibition of all three enzymes in human liver microsomes with IC50 values less than 50 μg/mL. Arjunic acid, arjunetin and arjungenin did not show significant inhibition of CYP enzymes in human liver microsomes. Enzyme kinetics studies suggested that the extracts of arjuna showed reversible non-competitive inhibition of all the three enzymes in human liver microsomes. Our findings suggest strongly that arjuna extracts significantly inhibit the activity of CYP3A4, CYP2D6 and CYP2C9 enzymes, which is likely to cause clinically significant drug–drug interactions mediated via inhibition of the major CYP isozymes.

  11. Application of Mice Humanized for CYP2D6 to the Study of Tamoxifen Metabolism and Drug–Drug Interaction with Antidepressants

    Science.gov (United States)

    MacLeod, A. Kenneth; McLaughlin, Lesley A.; Henderson, Colin J.

    2017-01-01

    Tamoxifen is an estrogen receptor antagonist used in the treatment of breast cancer. It is a prodrug that is converted by several cytochrome P450 enzymes to a primary metabolite, N-desmethyltamoxifen (NDT), which is then further modified by CYP2D6 to a pharmacologically potent secondary metabolite, 4-hydroxy-N-desmethyltamoxifen (endoxifen). Antidepressants (ADs), which are often coprescribed to patients receiving tamoxifen, are also metabolized by CYP2D6 and evidence suggests that a drug–drug interaction between these agents adversely affects the outcome of tamoxifen therapy by inhibiting endoxifen formation. We evaluated this potentially important drug–drug interaction in vivo in mice humanized for CYP2D6 (hCYP2D6). The rate of conversion of NDT to endoxifen by hCYP2D6 mouse liver microsomes (MLMs) in vitro was similar to that of the most active members of a panel of 13 individual human liver microsomes. Coincubation with quinidine, a CYP2D6 inhibitor, ablated endoxifen generation by hCYP2D6 MLMs. The NDT-hydroxylation activity of wild-type MLMs was 7.4 times higher than that of hCYP2D6, whereas MLMs from Cyp2d knockout animals were inactive. Hydroxylation of NDT correlated with that of bufuralol, a CYP2D6 probe substrate, in the human liver microsome panel. In vitro, ADs of the selective serotonin reuptake inhibitor class were, by an order of magnitude, more potent inhibitors of NDT hydroxylation by hCYP2D6 MLMs than were compounds of the tricyclic class. At a clinically relevant dose, paroxetine pretreatment inhibited the generation of endoxifen from NDT in hCYP2D6 mice in vivo. These data demonstrate the potential of ADs to affect endoxifen generation and, thereby, the outcome of tamoxifen therapy. PMID:27756789

  12. Determination of CYP2D6 activity in rat liver microsomes and study on its kinetics with dextromethorphan probe in vitro%以右美沙芬为探针测定大鼠肝微粒体中CYP2D6酶活性及动力学分析

    Institute of Scientific and Technical Information of China (English)

    惠俊敏; 郭延垒; 杨竹; 李文娟; 王应雄; 于超

    2013-01-01

    目的 建立大鼠肝微粒体中细胞色素P450 (CYP) 2D6酶活性的检测方法,以右美沙芬为探针底物进行体外酶动力学分析,为相关药物代谢研究提供参考依据.方法 采用高效液相色谱-荧光检测(HPLC-FLD)方法提高检测灵敏度.通过优化大鼠肝微粒体孵育体系的反应条件,建立稳定的动力学评价方法.以Graphpad prism 5.01软件绘制米曼动力学曲线并计算Vmax与Km值.结果 右美沙芬与其代谢产物右啡烷分离良好且无其他内源性物质干扰.右啡烷检测限为5 nmol·L-1 (S/N>3),定量下限为0.015 μmol·L-1,线性范围为0.015 ~ 7.5 μmol·L-1.测定方法重现性好且稳定.经优化条件后测定,不同浓度的右美沙芬在0.2 mg· mL-1蛋白浓度下,孵育10 min,测得动力学参数V一为(1.075±0.060) nmol·min-1·mg-1 pro,Km为(7.470±0.983) μmol·L-1.结论 高效液相色谱结合荧光检测法可以有效提高灵敏度,此大鼠微粒体孵育体系的建立可应用于体外CYP2D6酶活性的测定及酶动力学研究.

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

    NARCIS (Netherlands)

    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

  14. Pharm GKB: CYP2D6 [PharmGKB

    Lifescience Database Archive (English)

    Full Text Available el for vortioxetine and CYP2D6 FDA Label for acetaminophen,tramadol and CYP2D6 FDA Label for dextromethorphan... Label for vortioxetine and CYP2D6 European Medicines Agency (EMA) Label for dextromethorphan,quinidine and ...ore of this label. Read more. last updated 10/25/2013 FDA Label for dextromethorphan, quinidine and CYP2D6 O...of NUEDEXTA is a CYP2D6 inhibitor used to increase the plasma availability of dextromethorphan, which is met... 05/02/2014 European Medicines Agency (EMA) Label for dextromethorphan, quinidine

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

    Directory of Open Access Journals (Sweden)

    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.

  16. *124030 CYTOCHROME P450, SUBFAMILY IID, POLYPEPTIDE 6; CYP2D6 [OMIM

    Lifescience Database Archive (English)

    Full Text Available nd correlated enzyme activity measure by phenotyping with dextromethorphan or debrisoquine. The frequency of...CYP2D6 gene (124030.0002). The dextromethorphan urinary metabolite ratio in this patient was 9.7, which is o... S.: Analysis of the CYP2D6 gene in relation to dextromethorphan O-demyelination

  17. Effect of Curcuma longa on CYP2D6- and CYP3A4-mediated metabolism of dextromethorphan in human liver microsomes and healthy human subjects.

    Science.gov (United States)

    Al-Jenoobi, Fahad Ibrahim; Al-Thukair, Areej A; Alam, Mohd Aftab; Abbas, Fawkeya A; Al-Mohizea, Abdullah M; Alkharfy, Khalid M; Al-Suwayeh, Saleh A

    2015-03-01

    Effect of Curcuma longa rhizome powder and its ethanolic extract on CYP2D6 and CYP3A4 metabolic activity was investigated in vitro using human liver microsomes and clinically in healthy human subjects. Dextromethorphan (DEX) was used as common probe for CYP2D6 and CYP3A4 enzymes. Metabolic activity of CYP2D6 and CYP3A4 was evaluated through in vitro study; where microsomes were incubated with NADPH in presence and absence of Curcuma extract. In clinical study phase-I, six healthy human subjects received a single dose (30 mg) of DEX syrup, and in phase-II DEX syrup was administered with Curcuma powder. The enzyme CYP2D6 and CYP3A4 mediated O- and N-demethylation of dextromethorphan into dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. Curcuma extract significantly inhibited the formation of DOR and 3-MM, in a dose-dependent and linear fashion. The 100 μg/ml dose of curcuma extract produced highest inhibition, which was about 70 % for DOR and 80 % for 3-MM. Curcuma significantly increases the urine metabolic ratio of DEX/DOR but the change in DEX/3-MM ratio was statistically insignificant. Present findings suggested that curcuma significantly inhibits the activity of CYP2D6 in in vitro as well as in vivo; which indicates that curcuma has potential to interact with CYP2D6 substrates.

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

    DEFF Research Database (Denmark)

    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...

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

    DEFF Research Database (Denmark)

    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 investig......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...... 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....

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. CYP2D6 polymorphisms and their influence on risperidone treatment

    Directory of Open Access Journals (Sweden)

    Puangpetch A

    2016-12-01

    Full Text Available Apichaya Puangpetch,1 Natchaya Vanwong,1 Nopphadol Nuntamool,2 Yaowaluck Hongkaew,1 Monpat Chamnanphon,1 Chonlaphat Sukasem1 1Division of Pharmacogenomics and Personalized Medicine, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, 2Molecular Medicine, Faculty of Science, Mahidol University, Bangkok, Thailand Abstract: Cytochrome P450 enzyme especially CYP2D6 plays a major role in biotransformation. The interindividual variations of treatment response and toxicity are influenced by the polymorphisms of this enzyme. This review emphasizes the effect of CYP2D6 polymorphisms in risperidone treatment in terms of basic knowledge, pharmacogenetics, effectiveness, adverse events, and clinical practice. Although the previous studies showed different results, the effective responses in risperidone treatment depend on the CYP2D6 polymorphisms. Several studies suggested that CYP2D6 polymorphisms were associated with plasma concentration of risperidone, 9-hydroxyrisperidone, and active moiety but did not impact on clinical outcomes. In addition, CYP2D6 poor metabolizer showed more serious adverse events such as weight gain and prolactin than other predicted phenotype groups. The knowledge of pharmacogenomics of CYP2D6 in risperidone treatment is increasing, and it can be used for the development of personalized medication in term of genetic-based dose recommendation. Moreover, the effects of many factors in risperidone treatment are still being investigated. Both the CYP2D6 genotyping and therapeutic drug monitoring are the important steps to complement the genetic-based risperidone treatment. Keywords: CYP2D6, risperidone, polymorphisms, adverse drug reaction, pharmacogenetics, pharmacokinetics, pharmacodynamics

  2. CYP2D6 allele distribution in Macedonians, Albanians and Romanies in the Republic of Macedonia.

    Science.gov (United States)

    Kuzmanovska, M; Dimishkovska, M; Maleva Kostovska, I; Noveski, P; Sukarova Stefanovska, E; Plaseska-Karanfilska, D

    2015-12-01

    Cytochrome P450 2D6 (CYP2D6) is an enzyme of great importance for the metabolism of clinically used drugs. More than 100 variants of the CYP2D6 gene have been identified so far. The aim of this study was to investigate the allele distribution of CYP2D6 gene variants in 100 individuals of each of the Macedonian, Albanian and Romany population, by genotyping using long range polymerase chain reaction (PCR) and a multiplex single base extension method. The most frequent variants and almost equally distributed in the three groups were the fully functional alleles *1 and *2. The most common non functional allele in all groups was *4 that was found in 22.5% of the Albanians. The most common allele with decreased activity was *41 which was found in 23.0% of the Romany ethnic group, in 11.0% of the Macedonians and in 10.5% of the Albanians. Seven percent of the Albanians, 6.0% of the Romani and 4.0% of the Macedonians were poor metabolizers, while 5.0% of the Macedonians, 1.0% of Albanians and 1.0% of the Romanies were ultrarapid metabolizers. We concluded that the CYP2D6 gene locus is highly heterogeneous in these groups and that the prevalence of the CYP2D6 allele variants and genotypes in the Republic of Macedonia is in accordance with that of other European populations.

  3. The impact of experimental design on assessing mechanism-based inactivation of CYP2D6 by MDMA (Ecstasy).

    Science.gov (United States)

    Van, Linh M; Heydari, Amir; Yang, Jiansong; Hargreaves, Judith; Rowland-Yeo, Karen; Lennard, Martin S; Tucker, Geoffrey T; Rostami-Hodjegan, Amin

    2006-11-01

    MDMA (3-4-methylenedioxymethamphetamine, commonly known as Ecstasy) is a potent mechanism-based inhibitor (MBI) of cytochrome P450 2D6 (CYP2D6), causing quasi-irreversible inhibition of the enzyme in vitro. An evaluation of the in vivo implications of this phenomenon depends on the accuracy of the estimates of the parameters that define the inhibition in vitro, namely k(inact) (the maximal inhibition rate) and KI (the inactivation constant). These values are determined in two steps, pre-incubation of the enzyme with the inhibitor (enzyme inactivation), followed by dilution and further incubation to measure residual enzyme activity with a probe substrate. The aim of this study was to assess the impact of different dilutions and probe substrate concentrations on the estimates of k(inact) and KI using recombinantly expressed CYP2D6. Enzyme activity was measured by the conversion of dextromethorphan (DEX) to dextrorphan (DOR). Dilution factors of 1.25, 2, 5, 10, 25 and 50 (DEX at 30 microM) gave mean (+/-SE) values of k(inact) (min-1) of 0.20+/-0.06, 0.21+/-0.05, 0.31+/-0.06, 0.37+/-0.11, 0.51+/-0.10 and 0.58+/-0.08, respectively, and KI (microM) values (after correction for non-specific microsomal binding) of 2.22+/-1.90, 2.80+/-1.34, 5.78+/-2.07, 6.36+/-2.93, 3.99+/-1.57 and 4.86+/-1.37, respectively. Accordingly, high (e.g. 50 fold) and low (e.g. 1.25 fold) dilutions were associated with statistically significant differences in kinetic values (p <0.05). Varying DEX concentration (10-100 microM) was not associated with significant changes in k(inact) and KI values when a five-fold dilution was used (with the exception of a lower KI at 10 microM DEX). High dilution was also shown to reduce non-specific microsomal binding of MDMA. The changes in the two kinetic parameters were dependent on the experimental procedure and shown to be unlikely to have a material influence on the maximum inhibition of CYP2D6 expected in vivo after typical recreational doses of MDMA (50

  4. Cytochrome P-450 2D6 (CYP2D6) Genotype and Breast Cancer Recurrence in Tamoxifen-Treated Patients

    DEFF Research Database (Denmark)

    Ahern, Thomas P; Hertz, Daniel L; Damkier, Per;

    2017-01-01

    -infiltrated tissues, all of which showed excellent CYP2D6 genotype agreement. We applied these concordance data to a quantitative bias analysis of the subset of the 31 studies that were based on genotypes from tumor-infiltrated tissue to examine whether genotyping errors substantially biased estimates of association....... The bias analysis showed negligible bias by discordant genotypes. Summary estimates of association, with or without bias adjustment, indicated no clinically important association between CYP2D6 genotype and breast cancer survival in tamoxifen-treated women....

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

    Science.gov (United States)

    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.

  6. Genetic polymorphism of CYP2D6 in patients with systemic lupus erythematosus and systemic sclerosis.

    Science.gov (United States)

    Barańska, Małgorzata; Rychlik-Sych, Mariola; Kaszuba, Andrzej; Dziankowska-Bartkowiak, Bożena; Skrętkowicz, Jadwiga; Waszczykowska, Elżbieta

    2016-01-20

    Human organism is constantly exposed to harmful exogenous factors (xenobiotics) including drugs and carcinogenic compounds that can induce development of a large number of diseases. The processes of biotransformation in the organism are multidirectional and xenobiotics can be transformed into active or inactive metabolites via the oxidative route. The knowledge of oxidation polymorphism in the course of systemic lupus erythematosus and systemic sclerosis may be helpful in choosing more efficient and safer therapy, particularly in the case of a disease involving various organs and treated with drugs belonging to diverse therapeutic groups. The aim of the study was to evaluate the CYP2D6 polymorphism in the SLE (systemic lupus erythematosus) and SSc (systemic sclerosis) patients and to investigate a possible correlation with disease susceptibility. The study was carried out in 296 patients: 65 patients with SLE, 81 patients with SSc, and 150 healthy volunteers. The CYP2D6 genotypes were analyzed by polymerase chain reaction fragment length polymorphism (PCR-RFLP) method. The relative risk of developing SSc, expressed by the odds ratio, was three-fold higher for persons with the CYP2D6*1/CYP2D6*4 genotype (OR = 2.9; statistically significant difference, p = 0.0002). A statistically significant correlation between the CYP2D6*4 allele prevalence and the risk for developing SSc was found (OR = 1.53; p = 0.047). No effect of the CYP2D6 gene mutations on the incidence of SLE was noted. The obtained results may suggest the influence of CYP2D6*4 gene variants alleles on increased incidence of systemic sclerosis.

  7. Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

    Science.gov (United States)

    Sun, Jianguo; Peng, Ying; Wu, Hui; Zhang, Xueyuan; Zhong, Yunxi; Xiao, Yanan; Zhang, Fengyi; Qi, Huanhuan; Shang, Lili; Zhu, Jianping; Sun, Yue; Liu, Ke; Liu, Jinghan; A, Jiye; Ho, Rodney J Y; Wang, Guangji

    2015-05-01

    Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

  8. N=2, D=6 supergravity with $E_7$ gauge matter

    CERN Document Server

    Zyablyuk, K N

    1997-01-01

    The lagrangian of N=2, D=6 supergravity coupled to E_7 X SU(2) vector- and hyper-multiplets is derived. For this purpose the coset manifold E_8/E_7 X SU(2), parametrized by the scalars of the hypermultiplet, is constructed. A difference from the case of Sp(n)-matter is pointed out. This model can be considered as an intermediate step in the compactification of D=10 supergravity coupled to E_8 X E_8 matter to four-dimensional model of E_6 unification.

  9. Investigation of CYP3A4 and CYP2D6 Interactions of Withania somnifera and Centella asiatica in Human Liver Microsomes.

    Science.gov (United States)

    Savai, Jay; Varghese, Alice; Pandita, Nancy; Chintamaneni, Meena

    2015-05-01

    Withania somnifera is commonly used as a rejuvenator, whereas Centella asiatica is well known for its anxiolytic and nootropic effects. The present study aims at investigating the effect of crude extracts and principal phytoconstituents of both the medicinal plants with CYP3A4 and CYP2D6 enzyme activity in human liver microsomes (HLM). Phytoconstituents were quantified in the crude extracts of both the medicinal plants using reverse phase HPLC. Crude extracts and phytoconstituents of W. somnifera showed no significant interaction with both CYP3A4 and CYP2D6 enzymes in HLM. Of the crude extracts of C. asiatica screened in vitro, methanolic extract showed potent noncompetitive inhibition of only CYP3A4 enzyme (Ki-64.36 ± 1.82 µg/mL), whereas ethanol solution extract showed potent noncompetitive inhibition of only CYP2D6 enzyme (Ki-36.3 ± 0.44 µg/mL). The flavonoids, quercetin, and kaempferol showed potent (IC50 values less than 100 μM) inhibition of CYP3A4 activity, whereas quercetin alone showed potent inhibition of CYP2D6 activity in HLM. Because methanolic extract of C. asiatica showed a relatively high percentage content of quercetin and kaempferol than ethanol solution extract, the inhibitory effect of methanolic extract on CYP3A4 enzyme activity could be attributed to the flavonoids. Thus, co-administration of the alcoholic extracts of C. asiatica with drugs that are substrates of CYP3A4 and CYP2D6 enzymes may lead to undesirable herb-drug interactions in humans.

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

    Science.gov (United States)

    2017-08-15

    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

  11. Implications of mechanism-based inhibition of CYP2D6 for the pharmacokinetics and toxicity of MDMA.

    Science.gov (United States)

    Yang, Jiansong; Jamei, Masoud; Heydari, Amir; Yeo, Karen R; de la Torre, Rafael; Farré, Magí; Tucker, Geoffrey T; Rostami-Hodjegan, Amin

    2006-11-01

    The aim of this study was to model the in vivo kinetic consequences of mechanism-based inhibition (MBI) of CYP2D6 by 3,4 methylenedioxymethamphetamine (MDMA, ecstasy). A model with physiologically-based components of drug metabolism was developed, taking account of change in the hepatic content of active CYP2D6 due to MBI by MDMA. Based on the in vitro information, plasma concentration time profiles of MDMA after various doses were computed and compared with reported observations. The analysis suggested that a typical recreational MDMA dose could inactivate most hepatic CYP2D6 within an hour, and the return to a basal level of CYP2D6 could take at least 10 days. Thus, the genetic polymorphism of CYP2D6 and coadministration of CYP2D6 inhibitors may have less impact on MDMA pharmacokinetics and the risk of acute toxicity than previously thought. This is consistent with clinical observations that indicate no obvious link between inherited CYP2D6 deficiency and acute MDMA intoxication.

  12. 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

    Institute of Scientific and Technical Information of China (English)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  15. Improved Prediction of Endoxifen Metabolism by CYP2D6 Genotype in Breast Cancer Patients Treated with Tamoxifen

    Directory of Open Access Journals (Sweden)

    Werner Schroth

    2017-08-01

    Full Text Available Purpose: Prediction of impaired tamoxifen (TAM to endoxifen metabolism may be relevant to improve breast cancer treatment, e.g., via TAM dose increase. The polymorphic cytochrome P450 2D6 (CYP2D6 strongly determines an individual’s capacity for endoxifen formation, however, CYP2D6 phenotype assignments inferred from genotype widely differ between studies. Thus, we modeled plasma endoxifen predictability depending on variable CYP2D6 genotype groupings.Methods: CYP2D6 diplotype and metabolite plasma concentrations were assessed in 908 pre- and post-menopausal estrogen receptor (ER-positive, TAM treated early breast cancer patients of Caucasian (N = 678, Middle-Eastern Arab (N = 77, and Asian (N = 153 origin. Robust coefficients of determination (R2 were estimated for endoxifen (E or metabolic ratio endoxifen/desmethyl-TAM (E/DMT as dependent and different CYP2D6 phenotype assignments as independent variables. Allele activity scores (ASs were modified with respect to a reduced ∗10 allele activity. Predictability of endoxifen plasma concentrations above the clinical threshold of 5.9 ng/mL was investigated by receiver operating characteristic (ROC analysis.Results: CYP2D6 diplotypes (N = 898 were strongly associated with E and E/DMT independent of age (P < 10-15. Across all ethnicities, 68–82% inter-patient variability of E/DMT was explained by CYP2D6 diplotype, while plasma endoxifen was predictable by 39–58%. The previously used codeine specific phenotype classification showed worse prediction for both endpoints particularly in Asians (median R2< 20%; P < 10-9. Downgrading of ∗10 activity slightly improved the explanatory value of metabolizer phenotype (P < 0.002. Endoxifen plasma concentrations above the clinical threshold of 5.9 ng/mL were achieved in 82.3% of patients and were predictable (96% sensitivity, 57% specificity by CYP2D6 diplotypes with AS > 0.5, i.e., omitting PM/PM and PM/IM patients.Conclusion: The CYP2D6 explanatory

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

    Science.gov (United States)

    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.

  17. Effects of 22 Novel CYP2D6 Variants Found in the Chinese Population on the Bufuralol and Dextromethorphan Metabolisms In Vitro.

    Science.gov (United States)

    Cai, Jie; Dai, Da-Peng; Geng, Pei-Wu; Wang, Shuang-Hu; Wang, Hao; Zhan, Yun-Yun; Huang, Xiang-Xin; Hu, Guo-Xin; Cai, Jian-Ping

    2016-03-01

    Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that metabolizes a large number of therapeutic drugs. To date, more than 100 CYP2D6 allelic variants have been reported. Among these variants, we recently identified 22 novel variants in the Chinese population. The aim of this study was to functionally characterize the enzymatic activity of these variants in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) at high levels. Then, the insect microsomes containing expressed CYP2D6 proteins were incubated with bufuralol or dextromethorphan at 37°C for 20 or 25 min., respectively. After termination, the metabolites were extracted and used for the detection with high-performance liquid chromatography. Among the 24 CYP2D6 variants tested, two variants (CYP2D6.92 and CYP2D6.96) were found to be catalytically inactive. The remaining 22 variants exhibited significantly decreased intrinsic clearance values for bufuralol 1'-hydroxylation and 20 variants showed significantly lower intrinsic clearance values for dextromethorphan O-demethylation than those of the wild-type CYP2D6.1. Our in vitro results suggest that most of the variants exhibit significantly reduced catalytic activities compared with the wild-type, and these data provide valuable information for personalized medicine in Chinese and other Asian populations.

  18. Variable cytochrome P450 2D6 expression and metabolism of codeine and other opioid prodrugs: implications for the Australian anaesthetist.

    Science.gov (United States)

    Wilcox, R A; Owen, H

    2000-12-01

    Codeine is a popular opioid prodrug dependent on the activity of the specific cytochrome P450 enzyme 2D6 (CYP2D6). This enzyme catalyses the production of the potent analgesic metabolite morphine, but genetic studies have demonstrated that individuals from different ethnic groups exhibit considerable variability in the functional capacities of their expressed CYP2D6 enzymes, and pharmacological studies have shown many commonly prescribed drugs can reduce the action of CYP2D6 enzymes. These findings have significant clinical implications for the rational prescription of effective analgesia, especially in a multicultural country like Australia.

  19. Impact of CYP2D6 polymorphisms on clinical efficacy and tolerability of metoprolol tartrate.

    Science.gov (United States)

    Hamadeh, I S; Langaee, T Y; Dwivedi, R; Garcia, S; Burkley, B M; Skaar, T C; Chapman, A B; Gums, J G; Turner, S T; Gong, Y; Cooper-DeHoff, R M; Johnson, J A

    2014-08-01

    Metoprolol is a selective β-1 adrenergic receptor blocker that undergoes extensive metabolism by the polymorphic enzyme cytochrome P450 2D6 (CYP2D6). Our objective was to investigate the influence of CYP2D6 polymorphisms on the efficacy and tolerability of metoprolol tartrate. Two hundred and eighty-one participants with uncomplicated hypertension received 50 mg of metoprolol twice daily followed by response-guided titration to 100 mg twice daily. Phenotypes were assigned based on results of CYP2D6 genotyping and copy number variation assays. Clinical response to metoprolol and adverse effect rates were analyzed in relation to CYP2D6 phenotypes using appropriate statistical tests. Heart rate response differed significantly by CYP2D6 phenotype (P metoprolol response or tolerability.

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

    Directory of Open Access Journals (Sweden)

    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

  1. Relationship between Genotypes Sult1a2 and Cyp2d6 and Tamoxifen Metabolism in Breast Cancer Patients

    Science.gov (United States)

    Fernández-Santander, Ana; Gaibar, María; Novillo, Apolonia; Romero-Lorca, Alicia; Rubio, Margarita; Chicharro, Luis Miguel; Tejerina, Armando; Bandrés, Fernando

    2013-01-01

    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. PMID:23922954

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

    Science.gov (United States)

    Fernández-Santander, Ana; Gaibar, María; Novillo, Apolonia; Romero-Lorca, Alicia; Rubio, Margarita; Chicharro, Luis Miguel; Tejerina, Armando; Bandrés, Fernando

    2013-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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. (c) 2010 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Non-alcoholic fatty liver disease (NAFLD) potentiates autoimmune hepatitis in the CYP2D6 mouse model.

    Science.gov (United States)

    Müller, Peter; Messmer, Marie; Bayer, Monika; Pfeilschifter, Josef M; Hintermann, Edith; Christen, Urs

    2016-05-01

    Non-alcoholic fatty liver disease (NAFLD) and its more severe development non-alcoholic steatohepatitis (NASH) are increasing worldwide. In particular NASH, which is characterized by an active hepatic inflammation, has often severe consequences including progressive fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). Here we investigated how metabolic liver injury is influencing the pathogenesis of autoimmune hepatitis (AIH). We used the CYP2D6 mouse model in which wild type C57BL/6 mice are infected with an Adenovirus expressing the major liver autoantigen cytochrome P450 2D6 (CYP2D6). Such mice display several features of human AIH, including interface hepatitis, formation of LKM-1 antibodies and CYP2D6-specific T cells, as well as hepatic fibrosis. NAFLD was induced with a high-fat diet (HFD). We found that pre-existing NAFLD potentiates the severity of AIH. Mice fed for 12 weeks with a HFD displayed increased cellular infiltration of the liver, enhanced hepatic fibrosis and elevated numbers of liver autoantigen-specific T cells. Our data suggest that a pre-existing metabolic liver injury constitutes an additional risk for the severity of an autoimmune condition of the liver, such as AIH.

  7. CYP2D6*4 polymorphism: A new marker of response to hormonotherapy in male breast cancer?

    Science.gov (United States)

    Abreu, M H; Gomes, M; Menezes, F; Afonso, N; Abreu, P H; Medeiros, R; Pereira, D; Lopes, C

    2015-08-01

    Tamoxifen remains the standard hormonotherapy for Male breast cancer patients (MBC). Previous studies, in women, tried to evaluate the impact of CYP2D6 polymorphisms in tamoxifen efficacy with conflicting results. Herein we analyze the relation between CYP2D6*4 polymorphism and survival in MBC patients. Fifty-three patients, proposed to tamoxifen in adjuvant setting, were enrolled. Clinical information was collected from records and histological revision with additional immunochemistry analysis was done to better characterize the tumors. Comprehensive CYP2D6*4 genotyping from blood or tumor tissue was performed and translated into two predicted metabolic activity groups. Patients included in the two CYP2D6*4 groups did not differ concerning to age, histological characteristics, and primary treatments performed. Median age at diagnosis was 63 years-old and patients were submitted at least to mastectomy and adjuvant hormonotherapy. Recurrence was observed in 7 patients (13.2%) and 13 patients (25.5%) died with a 5-year disease-free survival of 86.2%. The poorer metabolizer group had a high risk for recurrence (p = 0.034) and this outcome effect remains in different subgroups: in tumors larger than 2 cm (p < 0.001), nodal status, N0 vs N+ (p = 0.04) and in advanced stage, stage III (p < 0.001). Poorer metabolizer patients had also a worse overall survival when tumors were larger than 2 cm (p = 0.03). In our series, there was an association between CYP2D6*4 polymorphism and a probability of recurrence, with a consistent effect in risk groups defined by classic prognostic factors. Multicentric studies with larger samples are needed to validate these results. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Distribution of CYP2D6 Alleles and Phenotypes in the Brazilian Population

    Science.gov (United States)

    Sortica, Vinicius A.; Suarez-Kurtz, Guilherme; de Moraes, Maria Elizabete; Pena, Sergio D. J.; dos Santos, Ândrea K. Ribeiro; Romano-Silva, Marco A.; Hutz, Mara H.

    2014-01-01

    Abstract 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. PMID:25329392

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

    Institute of Scientific and Technical Information of China (English)

    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.

  10. Influence of Cytochrome P450, Family 2, Subfamily D, Polypeptide 6 (CYP2D6) polymorphisms on pain sensitivity and clinical response to weak opioid analgesics.

    Science.gov (United States)

    Zahari, Zalina; Ismail, Rusli

    2014-01-01

      CYP2D6 polymorphisms show large geographical and interethnic differences. Variations in CYP2D6 activity may impact upon a patient's pain level and may contribute to interindividual variations in the response to opioids. This paper reviews the evidence on how CYP2D6 polymorphisms might influence pain sensitivity and clinical response to codeine and tramadol. For example, it is shown that (1) CYP2D6 poor metabolizers (PMs) may be less efficient at synthesizing endogenous morphine compared with other metabolizers. In contrast, ultra-rapid metabolizers (UMs) may be more efficient than other metabolizers at synthesizing endogenous morphine, thus strengthening endogenous pain modulation. Additionally, for codeine and tramadol that are bioactivated by CYP2D6, PMs may undergo no metabolite formation, leading to inadequate analgesia. Conversely, UMs may experience quicker analgesic effects but be prone to higher mu-opioid-related toxicity. The literature suggested the potential usefulness of the determination of CYP2D6 polymorphisms in elucidating serious adverse events and in preventing subsequent inappropriate selection or doses of codeine and tramadol. Notably, even though many studies investigated a possible role of the CYP2D6 polymorphisms on pain sensitivity, pharmacokinetics and pharmacodynamics of these drugs, the results of analgesia and adverse effects are conflicting. More studies are required to demonstrate genetically determined unresponsiveness and risk of developing serious adverse events for patients with pain and these should involve larger numbers of patients in different population types.

  11. A case study: neuroleptic malignant syndrome with risperidone and CYP2D6 gene variation.

    Science.gov (United States)

    Ochi, Shinichiro; Kawasoe, Koichiro; Abe, Masao; Fukuhara, Ryuji; Sonobe, Kantaro; Kawabe, Kentaro; Ueno, Shu-ichi

    2011-01-01

    We present a schizophrenic patient who experienced neuroleptic malignant syndrome with risperidone treatment due to variants of the CYP2D6 gene with reduced function. Clinicians need to be aware of this potential complication.

  12. Drug-Metabolizing Enzyme CYP2D6 Polymorphism and Phenotypes%药物代谢酶CYP2D6基因多态性及表型的研究进展

    Institute of Scientific and Technical Information of China (English)

    朱志慧; 李树春; 徐斯凡

    2009-01-01

    CYP2D6,as a member of cytochrome P450 family,is an important drug-metabolizing enzyme which catalyzes metabolism of a wide variety of drugs.It has been shown for years that the gene coding CYP2D6 enzyme has genetic polymorphism,which shows a close relationship with drug metabolism,adverse drug reaction and activation of carcinogenic substance.With the completion of human genome project and the beginning of gene polymorphism research project,the researches about polymorphism of CYP2D6 gene,and its relationship with clinical phenotypes has become one of important topics in the area of pharmacokineties.We will review CYP2D6 gene polymorphism,its production,and its influences on drug metabolism and susceptibility to certain diseases.%CYP2D6是人体内细胞色素P450家族中一种重要的药物代谢酶,其催化的药物种类广泛.多年研究表明,编码该酶的基因存在遗传多态性且该多态性与药物代谢、药物不良反应(adverse drug reaction,ADR)的产生和致癌物的活化关系密切.随着人类基因组计划的完成和基因多态性研究计划的开展,近年来对该基因多态性及其与表型关系的研究再次成为遗传药理学研究的热点.本文从CYP2D6基因多态性及其发生机制,多态性对药物代谢、相关疾病易感性的影响等四个方面对此进行综述.

  13. SNP genotyping using TaqMan technology: the CYP2D6*17 assay conundrum.

    Science.gov (United States)

    Gaedigk, Andrea; Freeman, Natalie; Hartshorne, Toinette; Riffel, Amanda K; Irwin, David; Bishop, Jeffrey R; Stein, Mark A; Newcorn, Jeffrey H; Jaime, Lazara Karelia Montané; Cherner, Mariana; Leeder, J Steven

    2015-03-19

    CYP2D6 contributes to the metabolism of many clinically used drugs and is increasingly tested to individualize drug therapy. The CYP2D6 gene is challenging to genotype due to the highly complex nature of its gene locus. TaqMan technology is widely used in the clinical and research settings for genotype analysis due to assay reliability, low cost, and the availability of commercially available assays. The assay identifying 1023C>T (rs28371706) defining a reduced function (CYP2D6*17) and several nonfunctional alleles, produced a small number of unexpected diplotype calls in three independent sets of samples, i.e. calls suggested the presence of a CYP2D6*4 subvariant containing 1023C>T. Gene resequencing did not reveal any unknown SNPs in the primer or probe binding sites in any of the samples, but all affected samples featured a trio of SNPs on their CYP2D6*4 allele between one of the PCR primer and probe binding sites. While the phenomenon was ultimately overcome by an alternate assay utilizing a PCR primer excluding the SNP trio, the mechanism causing this phenomenon remains elusive. This rare and unexpected event underscores the importance of assay validation in samples representing a variety of genotypes, but also vigilance of assay performance in highly polymorphic genes such as CYP2D6.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  15. [Identification of metabolites of epiberberine in rat liver microsomes and its inhibiting effects on CYP2D6].

    Science.gov (United States)

    Yang, Xiao-Yan; Ye, Jing; Sun, Gui-Xia; Xue, Bao-Juan; Zhao, Yuan-Yuan; Miao, Pei-Pei; Su, Jin; Zhang, Yu-Jie

    2014-10-01

    Epiberberine, one of the most important isoquinoline alkaloid in Coptidis Rhizoma, possesses extensive pharmacological activities. In this paper, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to study phase I and phase II metabolites. A Thermo HPLC system (including Surveyor AS, Surveyor LC Pump, Surveyor PDA. USA) was used. The cocktail probe drugs method was imposed to determine the content change of metoprolol, dapsone, phenacetin, chlorzoxazone and tolbutamide simultaneously for evaluating the activity of CYP2D6, CYP3A4, CYP1A2, CYP2E1 and CYP2C9 under different concentrations of epiberberine in rat liver microsomes. The result showed that epiberberine may have phase I and phase II metabolism in the rat liver and two metabolites in phase I and three metabolites in phase II are identified in the temperature incubation system of in vitro liver microsomes. Epiberberine showed significant inhibition on CYP2D6 with IC50 value of 35.22 μmol L(-1), but had no obvious inhibiting effect on the activities of CYP3A4, CYP1A2, CYP2E1 and CYP2C9. The results indicated that epiberberine may be caused drug interactions based on CYP2D6 enzyme. This study aims to provide a reliable experimental basis for its further research and development of epiberberine.

  16. Effect of genotype and methylation of CYP2D6 on smoking behaviour.

    Science.gov (United States)

    Tiili, Emmi M; Antikainen, Miia S H; Mitiushkina, Natalia V; Sukhovskaya, Olga A; Imyanitov, Evgeny N; Hirvonen, Ari P

    2015-11-01

    Cigarette smoking is one of the most influential environmental factors affecting the DNA methylation patterns. The addiction-causing substance of tobacco smoke, nicotine, has also shown the potential to alter DNA methylation patterns. However, genetics has a strong influence on DNA methylation patterns, which in turn may affect an individual's smoking behaviour. We studied eight functional gene variants of one of the most important drug-metabolizing enzymes, CYP2D6, in relation to smoking behaviour in our well-characterized study population consisting of 1230 Whites of Russian origin. In addition, potential associations between methylation levels in a CpG island in the CYP2D6 gene and sex, age, different smoking-related phenotypes and CYP2D6 genotypes were studied. Both age and sex were found to be associated with the methylation level of the CYP2D6 gene. The CYP2D6 methylation pattern also showed high genotype dependence; compared with the extensive metabolizer genotype, the poor metabolizer genotype occurred notably more frequently with higher methylation status (odds ratio 5.05, 95% confidence interval 2.14-11.90). Moreover, higher methylation levels were found to be related inversely to heavier smoking (odds ratio 0.56, 95% confidence interval 0.35-0.91). We also found associations between the CYP2D6 genotype and smoking habits; the poor metabolizer genotype tended to decrease the risk of becoming a heavy smoker compared with the extensive metabolizers, whereas the ultrarapid metabolism-related genotypes tended to increase the risk. The CYP2D6-related metabolic capacity seems to be related to cigarette consumption both through genetic and through epigenetic mechanisms.

  17. Sequencing CYP2D6 for the detection of poor-metabolizers in post-mortem blood samples with tramadol.

    Science.gov (United States)

    Fonseca, Suzana; Amorim, António; Costa, Heloísa Afonso; Franco, João; Porto, Maria João; Santos, Jorge Costa; Dias, Mário

    2016-08-01

    Tramadol concentrations and analgesic effect are dependent on the CYP2D6 enzymatic activity. It is well known that some genetic polymorphisms are responsible for the variability in the expression of this enzyme and in the individual drug response. The detection of allelic variants described as non-functional can be useful to explain some circumstances of death in the study of post-mortem cases with tramadol. A Sanger sequencing methodology was developed for the detection of genetic variants that cause absent or reduced CYP2D6 activity, such as *3, *4, *6, *8, *10 and *12 alleles. This methodology, as well as the GC/MS method for the detection and quantification of tramadol and its main metabolites in blood samples was fully validated in accordance with international guidelines. Both methodologies were successfully applied to 100 post-mortem blood samples and the relation between toxicological and genetic results evaluated. Tramadol metabolism, expressed as its metabolites concentration ratio (N-desmethyltramadol/O-desmethyltramadol), has been shown to be correlated with the poor-metabolizer phenotype based on genetic characterization. It was also demonstrated the importance of enzyme inhibitors identification in toxicological analysis. According to our knowledge, this is the first study where a CYP2D6 sequencing methodology is validated and applied to post-mortem samples, in Portugal. The developed methodology allows the data collection of post-mortem cases, which is of primordial importance to enhance the application of these genetic tools to forensic toxicology and pathology.

  18. Exploring the possible metabolism mediated interaction of Glycyrrhiza glabra extract with CYP3A4 and CYP2D6.

    Science.gov (United States)

    Pandit, Subrata; Ponnusankar, Sivasankaran; Bandyopadhyay, Arun; Ota, Sarda; Mukherjee, Pulok K

    2011-10-01

    The rhizome of Glycyrrhiza glabra L. (licorice) is used very widely in Indian and Chinese traditional medicine, and it is a popular flavor ingredient of drinks, sweets and candies. Its medicinal uses include treating bronchitis, dry cough, respiratory infections, liver disorders and diabetes. Glycyrrhizin is normally considered to be its biologically active marker, so a rapid RP-HPLC method was developed for the quantitative estimation of glycyrrhizin in the extract. The effect of the standardized extract and its marker on drug metabolizing enzymes was evaluated through CYP3A4 and CYP2D6 inhibition assays to evaluate the safety through its drug interaction potential. The inhibition of CYP3A4 and CYP2D6 isozymes was analysed by the fluorescent product formation method. In the CYP450-CO assay, the interaction potential of the standardized extract and pooled microsomes (percentage inhibition 23.23 ± 1.84%), was found to be less than the standard inhibitor. In the fluorimetric assay, G. glabra extracts showed higher IC(50) values than their positive inhibitors, ketoconazole and quinidine for CYP3A4 and CYP2D6, respectively. Furthermore, the interaction potential of the plant extract was greater than the pure compound. The results demonstrate that G. glabra and its principle bioactive compound, glycyrrhizin, when co-administered with conventional medicines showed only a weak interaction potential with drug metabolizing enzymes.

  19. Cytochrome P450-2D6 extensive metabolizers are more vulnerable to methamphetamine-associated neurocognitive impairment: preliminary findings.

    Science.gov (United States)

    Cherner, Mariana; Bousman, Chad; Everall, Ian; Barron, Daniel; Letendre, Scott; Vaida, Florin; Atkinson, J Hampton; Heaton, Robert; Grant, Igor

    2010-09-01

    While neuropsychological deficits are evident among methamphetamine (meth) addicts, they are often unrelated to meth exposure parameters such as lifetime consumption and length of abstinence. The notion that some meth users develop neuropsychological impairments while others with similar drug exposure do not, suggests that there may be individual differences in vulnerability to the neurotoxic effects of meth. One source of differential vulnerability could come from genotypic variability in metabolic clearance of meth, dependent on the activity of cytochrome P450-2D6 (CYP2D6). We compared neuropsychological performance in 52 individuals with a history of meth dependence according with their CYP2D6 phenotype. All were free of HIV or hepatitis C infection and did not meet dependence criteria for other substances. Extensive metabolizers showed worse overall neuropsychological performance and were three times as likely to be cognitively impaired as intermediate/poor metabolizers. Groups did not differ in their demographic or meth use characteristics, nor did they evidence differences in mood disorder or other substance use. This preliminary study is the first to suggest that efficient meth metabolism is associated with worse neurocognitive outcomes in humans, and implicates the products of oxidative metabolism of meth as a possible source of brain injury.

  20. Physiologically Based Pharmacokinetic Modeling of Tamoxifen and its Metabolites in Women of Different CYP2D6 Phenotypes Provides New Insight into the Tamoxifen Mass Balance

    Science.gov (United States)

    Dickschen, Kristin; Willmann, Stefan; Thelen, Kirstin; Lippert, Jörg; Hempel, Georg; Eissing, Thomas

    2012-01-01

    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 exert a pronounced decrease in endoxifen steady-state plasma concentrations compared to CYP2D6 extensive metabolizers. 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 study, physiologically based pharmacokinetic (PBPK)-modeling was applied to mechanistically investigate the impact of CYP2D6 phenotype on endoxifen formation in female breast cancer patients undergoing tamoxifen therapy. A PBPK-model of tamoxifen and its pharmacologically important metabolites N-desmethyltamoxifen (NDM-TAM), 4-hydroxytamoxifen (4-OH-TAM), and endoxifen was developed and validated. This model is able to simulate the pharmacokinetics (PK) after single and repeated oral tamoxifen doses in female breast cancer patients in dependence of the CYP2D6 phenotype. A detailed model-based analysis of the mass balance offered support for a recent hypothesis stating a more prominent role for endoxifen formation from 4-OH-TAM. In the future this model provides 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. PMID:22661948

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Physiologically Based Pharmacokinetic Predictions of Tramadol Exposure Throughout Pediatric Life: an Analysis of the Different Clearance Contributors with Emphasis on CYP2D6 Maturation.

    Science.gov (United States)

    T'jollyn, Huybrecht; Snoeys, Jan; Vermeulen, An; Michelet, Robin; Cuyckens, Filip; Mannens, Geert; Van Peer, Achiel; Annaert, Pieter; Allegaert, Karel; Van Bocxlaer, Jan; Boussery, Koen

    2015-11-01

    This paper focuses on the retrospective evaluation of physiologically based pharmacokinetic (PBPK) techniques used to mechanistically predict clearance throughout pediatric life. An intravenous tramadol retrograde PBPK model was set up in Simcyp® using adult clearance values, qualified for CYP2D6, CYP3A4, CYP2B6, and renal contributions. Subsequently, the model was evaluated for mechanistic prediction of total, CYP2D6-related, and renal clearance predictions in very early life. In two in vitro pediatric human liver microsomal (HLM) batches (1 and 3 months), O-desmethyltramadol and N-desmethyltramadol formation rates were compared with CYP2D6 and CYP3A4 activity, respectively. O-desmethyltramadol formation was mediated only by CYP2D6, while N-desmethyltramadol was mediated in part by CYP3A4. Additionally, the clearance maturation of the PBPK model predictions was compared to two in vivo maturation models (Hill and exponential) based on plasma concentration data, and to clearance estimations from a WinNonlin® fit of plasma concentration and urinary excretion data. Maturation of renal and CYP2D6 clearance is captured well in the PBPK model predictions, but total tramadol clearance is underpredicted. The most pronounced underprediction of total and CYP2D6-mediated clearance was observed in the age range of 2-13 years. In conclusion, the PBPK technique showed to be a powerful mechanistic tool capable of predicting maturation of CYP2D6 and renal tramadol clearance in early infancy, although some underprediction occurs between 2 and 13 years for total and CYP2D6-mediated tramadol clearance.

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

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

    Science.gov (United States)

    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.

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

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    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

  6. Prolactin release in children treated with risperidone - Impact and role of CYP2D6 metabolism

    NARCIS (Netherlands)

    Troost, Pieter W.; Lahuis, Bertine E.; Hermans, Mirjam H.; Buitelaar, Jan K.; van Engeland, Herman; Scahill, Lawrence; Minderaa, Ruud B.; Hoekstra, Pieter J.

    2007-01-01

    Objective: Little is known about the role of CYP2136 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-hydroxyrisperidon

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

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    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.

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

    NARCIS (Netherlands)

    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

  9. The Discriminatory Value of CYP2D6 Genotyping in Predicting the Dextromethorphan/Dextrorphan Phenotype in Women with Breast Cancer

    Science.gov (United States)

    Trojan, Andreas; Vergopoulos, Athanasios; Breitenstein, Urs; Seifert, Burkhardt; Rageth, Christoph; Joechle, Wolfgang

    2012-01-01

    Background The growth inhibitory effect of tamoxifen is used for the treatment of breast cancer. Tamoxifen efficacy is mediated by its biotransformation, predominantly via the cytochrome P450 2D6 (CYP2D6) isoenzyme, to the active metabolite endoxifen. We investigated the relationship of CYP2D6 genotypes to the metabolism of dextromethorphan (DM), which is frequently used as a surrogate marker for the formation of endoxifen. Methods The CYP2D6 genotype was determined by polymerase chain reaction (PCR) in previously untreated patients with hormone receptor-positive invasive breast cancer considered to receive antihormonal therapy. The DM/dextrorphan (DX) urinary excretion ratios were obtained in a subset of patients by high-pressure liquid chromatography (HPLC)-mediated urine analysis after intake of 25 mg DM. The relationships of genotype and corresponding phenotype were statistically analyzed for association. Results From 151 patients predicted based on their genotype data for the ‘traditional’ CYP2D6 phenotype classes poor, intermediate, extensive and ultrarapid, 83 patients were examined for their DM/DX urinary ratios. The genotype-based poor metabolizer status correlated with the DM/DX ratios, whereas the intermediate, extensive and ultrarapid genotypes could not be distinguished based on their phenotype. Citalopram intake did not significantly influence the phenotype. Conclusions The DM metabolism can be reliably used to assess the CYP2D6 enzyme activity. The correlation with the genotype can be incomplete and the metabolic ratios do not seem to be compromised by citalopram. DM phenotyping may provide a standardized tool to better assess the CYP2D6 metabolic capacity. PMID:22553469

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

    DEFF Research Database (Denmark)

    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 individua......, 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....

  11. CYP2D6 polymorphisms may predict occurrence of adverse effects to tamoxifen: a preliminary retrospective study

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    Wickramage I

    2017-03-01

    Full Text Available Ishani Wickramage,1 Kamani Hemamala Tennekoon,1 Merenchi Arachchige Yasantha Ariyaratne,2 Asanka Sudeshini Hewage,1 Tharmini Sundralingam,1 1Institute of Biochemistry, Molecular Biology and Biotechnology (IBMBB, University of Colombo, Colombo, Sri Lanka; 2National Cancer Institute, Maharagama, Sri Lanka Introduction and aims: Tamoxifen is an adjuvant drug effective in treating hormone ­receptor – positive breast cancer. However, 30%–50% of patients relapse and many develop adverse effects, such as hot flashes and fatty liver. Allelic variations altering the activity of cytochrome P450-2D6 enzyme affect response to tamoxifen by modulating metabolism of tamoxifen into its pharmacologically active metabolite endoxifen. Although association between CYP2D6 polymorphisms and recurrence of breast cancer in patients on tamoxifen had been reported, little evidence exists on association between these polymorphisms and adverse effects to tamoxifen. This study explored the association between CYP2D6 polymorphisms and tamoxifen effects, hitherto not studied in Sri Lanka. Methods: A retrospective preliminary study was carried out on 24 breast cancer patients on tamoxifen for minimally 3 months attending National Cancer Institute, Maharagama, Sri Lanka. They were not on CYP2D6-inhibiting drugs, chemotherapy or other endocrine therapy, and had no conditions that could occur as adverse effects to tamoxifen before starting the therapy. Their blood samples were collected, DNA was extracted and genotyped using SNaPshot Multiplex sequencing based single-nucleotide polymorphism (SNP assay. Results: SNP/allele frequencies detected: 1846G>A (confirmatory of *4 null allele=8.3%; 2549delA (confirmatory of *3 null allele=50%; 100C>T (suggestive of *10 reduced functional allele, in addition to other alleles=0%; combination of 2988G>A, -1584C and 2850C>T (strongly suggestive of *41 or other reduced functional allele=4.8%. Occurrence of heterozygous 2988G>A SNP with

  12. Practical interpretation of CYP2D6 haplotypes: Comparison and integration of automated and expert calling.

    Science.gov (United States)

    Ruaño, Gualberto; Kocherla, Mohan; Graydon, James S; Holford, Theodore R; Makowski, Gregory S; Goethe, John W

    2016-05-01

    We describe a population genetic approach to compare samples interpreted with expert calling (EC) versus automated calling (AC) for CYP2D6 haplotyping. The analysis represents 4812 haplotype calls based on signal data generated by the Luminex xMap analyzers from 2406 patients referred to a high-complexity molecular diagnostics laboratory for CYP450 testing. DNA was extracted from buccal swabs. We compared the results of expert calls (EC) and automated calls (AC) with regard to haplotype number and frequency. The ratio of EC to AC was 1:3. Haplotype frequencies from EC and AC samples were convergent across haplotypes, and their distribution was not statistically different between the groups. Most duplications required EC, as only expansions with homozygous or hemizygous haplotypes could be automatedly called. High-complexity laboratories can offer equivalent interpretation to automated calling for non-expanded CYP2D6 loci, and superior interpretation for duplications. We have validated scientific expert calling specified by scoring rules as standard operating procedure integrated with an automated calling algorithm. The integration of EC with AC is a practical strategy for CYP2D6 clinical haplotyping.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    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...... ratios ranging from 0.07 to 0.17 and six volunteers without duplication with metabolic ratios ranging from 0.08 to 0.21. Each subject took 100 mg of desipramine. Blood and urine were collected for 48 h. RESULTS: The median total oral clearance of desipramine was 372 l/h and 196 l/h [median difference 108...... l/h (95.9% c.i., -304-598 l/h)] and the median partial clearance of desipramine by 2-hydroxylation was 155 l/h and 87 l/h [median difference 47 l/h (95.9% c.i., -124-141 l/h)] for the group with duplication and the group without duplication, respectively. CONCLUSION: The predictive value...

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

    NARCIS (Netherlands)

    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. CYP2D6 phenotypes are associated with adverse outcomes related to opioid medications

    Science.gov (United States)

    St Sauver, Jennifer L; Olson, Janet E; Roger, Veronique L; Nicholson, Wayne T; Black, John L; Takahashi, Paul Y; Caraballo, Pedro J; Bell, Elizabeth J; Jacobson, Debra J; Larson, Nicholas B; Bielinski, Suzette J

    2017-01-01

    Background Variation in the CYP2D6 gene may affect response to opioids in both poor and ultrarapid metabolizers, but data demonstrating such associations have been mixed, and the impact of variants on toxicity-related symptoms (e.g., nausea) is unclear. Therefore, we examined the association between CYP2D6 phenotype and poor pain control or other adverse symptoms related to the use of opioids in a sample of primary care patients. Materials and methods We identified all patients in the Mayo Clinic RIGHT Protocol who were prescribed an opioid medication between July 01, 2013 and June 30, 2015, and categorized patients into three phenotypes: poor, intermediate to extensive, or ultrarapid CYP2D6 metabolizers. We reviewed the electronic health record of these patients for indications of poor pain control or adverse symptoms related to medication use. Associations between phenotype and outcomes were assessed using Chi-square tests and logistic regression. Results Overall, 257 (25% of RIGHT Protocol participants) patients received at least one opioid prescription; of these, 40 (15%) were poor metabolizers, 146 (57%) were intermediate to extensive metabolizers, and 71 (28%) were ultrarapid metabolizers. We removed patients that were prescribed a CYP2D6 inhibitor medication (n=38). After adjusting for age and sex, patients with a poor or ultrarapid phenotype were 2.7 times more likely to experience either poor pain control or an adverse symptom related to the prescription compared to patients with an intermediate to extensive phenotype (odds ratio: 2.68; 95% CI: 1.39, 5.17; p=0.003). Conclusion Our results suggest that >30% of patients with a poor or ultrarapid CYP2D6 phenotype may experience an adverse outcome after being prescribed codeine, tramadol, oxycodone, or hydrocodone. These medications are frequently prescribed for pain relief, and ~39% of the US population is expected to carry one of these phenotypes, suggesting that the population-level impact of these gene

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

    Science.gov (United States)

    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.

  18. 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

    Institute of Scientific and Technical Information of China (English)

    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

  19. CYP2D6 genotype and phenotype in Amerindians of Tepehuano origin and Mestizos of Durango, Mexico.

    Science.gov (United States)

    Sosa-Macías, Martha; Elizondo, Guillermo; Flores-Pérez, Carmen; Flores-Pérez, Janet; Bradley-Alvarez, Francisco; Alanis-Bañuelos, Ruth E; Lares-Asseff, Ismael

    2006-05-01

    Although the drug-metabolizing enzyme CYP2D6 has been studied extensively in subjects of differing ethnicities, limited CYP2D6 pharmacogenetic data are available for the Amerindian population and Mestizos of Mexico. Dextromethorphan hydroxylation phenotype was studied in Tepehuano Amerindian (n = 58) and Mestizo (n = 88) subjects, and 195 individuals (85 Tepehuano Amerindians and 110 Mestizos) were genotyped by polymerase chain reaction-restriction fragment length polymorphism methods to identify the frequencies of the CYP2D6*3, *4, *6, and *10 alleles. Tepehuano Amerindian subjects lacked the poor metabolizer (PM) phenotype, whereas in Mestizos the PM phenotype frequency was 6.8%. The CYP2D6*3, *6, and *10 alleles were not found in Tepehuano Amerindians. The CYP2D6*4 allele had a low frequency (0.006) in this Amerindian group. In the Mestizo group, the CYP2D6*3, *4, and *10 alleles had frequencies of 0.009, 0.131, and 0.023, respectively. The CYP2D6*6 allele was not found in Mestizos. The genotype-phenotype association was strongly statistically significant (r(2) = .45; P = .005) in Mestizos. The Tepehuano population was found to have a low phenotypic and genotypic CYP2D6 diversity and differed from other Amerindian groups. On the other hand, the frequencies of the CYP2D6 variant alleles in Mestizos were similar to those reported for whites.

  20. CYP2D6 and CYP2A6 biotransform dietary tyrosol into hydroxytyrosol.

    Science.gov (United States)

    Rodríguez-Morató, Jose; Robledo, Patricia; Tanner, Julie-Anne; Boronat, Anna; Pérez-Mañá, Clara; Oliver Chen, C-Y; Tyndale, Rachel F; de la Torre, Rafael

    2017-02-15

    The dietary phenol tyrosol has been reported to be endogenously transformed into hydroxytyrosol, a potent antioxidant with multiple health benefits. In this work, we evaluated whether tyrosine hydroxylase (TH) and cytochrome P450s (CYPs) catalyzed this process. To assess TH involvement, Wistar rats were treated with α-methyl-L-tyrosine and tyrosol. Tyrosol was converted into hydroxytyrosol whilst α-methyl-L-tyrosine did not inhibit the biotransformation. The role of CYP was assessed in human liver microsomes (HLM) and tyrosol-to-hydroxytyrosol conversion was observed. Screening with selective enzymatic CYP inhibitors identified CYP2A6 as the major isoform involved in this process. Studies with baculosomes further demonstrated that CYP2D6 and CYP3A4 could transform tyrosol into hydroxytyrosol. Experiments using human genotyped livers showed an interindividual variability in hydroxytyrosol formation and supported findings that CYP2D6 and CYP2A6 mediated this reaction. The dietary health benefits of tyrosol-containing foods remain to be evaluated in light of CYP pharmacogenetics.

  1. Tafenoquine treatment of Plasmodium vivax malaria: suggestive evidence that CYP2D6 reduced metabolism is not associated with relapse in the Phase 2b DETECTIVE trial.

    Science.gov (United States)

    St Jean, Pamela L; Xue, Zhengyu; Carter, Nick; Koh, Gavin C K W; Duparc, Stephan; Taylor, Maxine; Beaumont, Claire; Llanos-Cuentas, Alejandro; Rueangweerayut, Ronnatrai; Krudsood, Srivicha; Green, Justin A; Rubio, Justin P

    2016-02-18

    Tafenoquine (TQ) and primaquine (PQ) are 8-aminoquinolines (8-AQ) with anti-hypnozoite activity against vivax malaria. PQ is the only FDA-approved medicine for preventing relapsing Plasmodium vivax infection and TQ is currently in phase 3 clinical trials for the same indication. Recent studies have provided evidence that cytochrome P450 (CYP) metabolism via CYP2D6 plays a role in PQ efficacy against P. vivax and have suggested that this effect may extend to other 8-AQs, including TQ. Here, a retrospective pharmacogenetic (PGx) investigation was performed to assess the impact of CYP2D6 metabolism on TQ and PQ efficacy in the treatment of P. vivax in the DETECTIVE study (TAF112582), a recently completed, randomized, phase 2b dose-ranging clinical trial. The impact of CYP2D6 on TQ pharmacokinetics (PK) was also investigated in TAF112582 TQ-treated subjects and in vitro CYP metabolism of TQ was explored. A limitation of the current study is that TAF112582 was not designed to be well powered for PGx, thus our findings are based on TQ or PQ efficacy in CYP2D6 intermediate metabolizers (IM), as there were insufficient poor metabolizers (PM) to draw any conclusion on the impact of the PM phenotype on efficacy. The impact of genetically-predicted CYP2D6 reduced metabolism on relapse-free efficacy six months post-dosing of TQ or PQ, both administered in conjunction with chloroquine (CQ), was assessed using exact statistical methods in 198 P. vivax-infected study participants comparing IM to extensive metabolizers (EM). The influence of CYP2D6 metabolizer phenotypes on TQ PK was assessed comparing median TQ area under the curve (AUC). In vitro metabolism of TQ was investigated using recombinant, over-expressed human CYP enzymes and human hepatocytes. Metabolite identification experiments were performed using liquid chromatography-mass spectrometry. Reduction of CYP2D6 activity was not associated with an increase in relapse-rate in TQ-treated subjects (p = 0.57). In contrast

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

    DEFF Research Database (Denmark)

    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...... 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 in doses...... after fluoxetine intake, although no volunteers changed phenotype from extensive metabolisers to poor metabolisers. Three of the six volunteers changed phenotype from extensive metabolisers to poor metabolisers after intake of 40 or 80 mg paroxetine. There was a statistically significant increase...

  3. Studies of novel deuterides RMn2D6 (R — rare earth) compressed in DAC up to 30 GPa

    Science.gov (United States)

    Filipek, S. M.; Sugiura, H.; Paul-Boncour, V.; Wierzbicki, R.; Liu, R. S.; Bagkar, N.

    2008-07-01

    The exposure of RMn2 (C15 or C14 cubic Laves phase, where R = Y; Dy; Ho or Er) to high deuterium pressure leads to formation of novel, unique YMn2D6, DyMn2D6, HoMn2D6 and ErMn2D6 deuterides with cubic Fm-3m symmetry. In spite of different structures and molecular volumes of parent RMn2 compounds, the molar volumes of RMn2D6 deuterides are almost identical. In this paper, we present results of studies on RMn2Dx (where R = Y, Dy, Ho and Er) submitted to compression up to 30 GPa in diamond anvil cell (DAC) combined with energy dispersive X-ray diffraction. The EOS (equation of state) parameters of the above four RMn2D6 samples and YMn2Dx, with x RMn2D6 but very different than those of interstitial deuterides YMn2Dx (x RMn2D6 up to 30 GPa.

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

    NARCIS (Netherlands)

    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

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

    NARCIS (Netherlands)

    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

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

    NARCIS (Netherlands)

    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

  7. The Influence of CYP2D6 Phenotype on the Pharmacokinetic Profile of Atomoxetine in Caucasian Healthy Subjects

    Directory of Open Access Journals (Sweden)

    Todor Ioana

    2017-06-01

    Full Text Available Objective: To analyze a potential phenotypic variation within the studied group based on the pharmacokinetic profile of atomoxetine and its active metabolite, and to further investigate the impact of CYP2D6 phenotype on atomoxetine pharmacokinetics. Methods: The study was conducted as an open-label, non-randomized clinical trial which included 43 Caucasian healthy volunteers. Each subject received a single oral dose of atomoxetine 25 mg. Subsequently, atomoxetine and 4-hydroxyatomoxetine-O-glucuronide (glucuronidated active metabolite plasma concentrations were determined and a noncompartmental method was used to calculate the pharmacokinetic parameters of both compounds. Further on, the CYP2D6 metabolic phenotype was assessed using the area under the curve (AUC metabolic ratio (atomoxetine/ 4-hydroxyatomoxetine-O-glucuronide and specific statistical tests (Lilliefors (Kolgomorov-Smirnov and Anderson-Darling test. The phenotypic differences in atomoxetine disposition were identified based on the pharmacokinetic profile of the parent drug and its metabolite. Results: The statistical analysis revealed that the AUC metabolic ratio data set did not follow a normal distribution. As a result, two different phenotypes were identified, respectively the poor metabolizer (PM group which included 3 individuals and the extensive metabolizer (EM group which comprised the remaining 40 subjects. Also, it was demonstrated that the metabolic phenotype significantly influenced atomoxetine pharmacokinetics, as PMs presented a 4.5-fold higher exposure to the parent drug and a 3.2-fold lower exposure to its metabolite in comparison to EMs. Conclusions: The pharmacokinetic and statistical analysis emphasized the existence of 2 metabolic phenotypes: EMs and PMs. Furthermore, it was proved that the interphenotype variability had a marked influence on atomoxetine pharmacokinetic profile.

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

    Directory of Open Access Journals (Sweden)

    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

  9. Stiff substrates enhance cultured neuronal network activity.

    Science.gov (United States)

    Zhang, Quan-You; Zhang, Yan-Yan; Xie, Jing; Li, Chen-Xu; Chen, Wei-Yi; Liu, Bai-Lin; Wu, Xiao-an; Li, Shu-Na; Huo, Bo; Jiang, Lin-Hua; Zhao, Hu-Cheng

    2014-08-28

    The mechanical property of extracellular matrix and cell-supporting substrates is known to modulate neuronal growth, differentiation, extension and branching. Here we show that substrate stiffness is an important microenvironmental cue, to which mouse hippocampal neurons respond and integrate into synapse formation and transmission in cultured neuronal network. Hippocampal neurons were cultured on polydimethylsiloxane substrates fabricated to have similar surface properties but a 10-fold difference in Young's modulus. Voltage-gated Ca(2+) channel currents determined by patch-clamp recording were greater in neurons on stiff substrates than on soft substrates. Ca(2+) oscillations in cultured neuronal network monitored using time-lapse single cell imaging increased in both amplitude and frequency among neurons on stiff substrates. Consistently, synaptic connectivity recorded by paired recording was enhanced between neurons on stiff substrates. Furthermore, spontaneous excitatory postsynaptic activity became greater and more frequent in neurons on stiff substrates. Evoked excitatory transmitter release and excitatory postsynaptic currents also were heightened at synapses between neurons on stiff substrates. Taken together, our results provide compelling evidence to show that substrate stiffness is an important biophysical factor modulating synapse connectivity and transmission in cultured hippocampal neuronal network. Such information is useful in designing instructive scaffolds or supporting substrates for neural tissue engineering.

  10. Comparison of pharmacokinetic variability of fesoterodine vs. tolterodine extended release in cytochrome P450 2D6 extensive and poor metabolizers.

    Science.gov (United States)

    Malhotra, Bimal; Darsey, Edress; Crownover, Penelope; Fang, Juanzhi; Glue, Paul

    2011-08-01

    Tolterodine and 5-hydroxymethyl tolterodine (5-HMT) are equipotent active moieties of tolterodine; 5-HMT is the singular active moiety of fesoterodine. The formation of 5-HMT from tolterodine occurs via CYP2D6, and some subjects are poor metabolizers CYP2D6. On the other hand, the formation of 5-HMT from fesoterodine occurs via ubiquitous esterases. Cross-study comparisons of data from phase 1 studies suggest that active moiety exposures are considerably more variable following tolterodine extended release vs. fesoterodine. This head-to-head study confirmed the findings of reduced pharmacokinetic variability of fesoterodine and further delineates that tolterodine, and not 5-HMT, was the principal source of variability after administration of tolterodine extended release. The data suggest that fesoterodine delivers 5-HMT consistently, regardless of CYP2D6 status, with up to 40% higher bioavailability compared with tolterodine. Tolterodine and 5-hydroxymethyl tolterodine (5-HMT) are equipotent active moieties of tolterodine; 5-HMT is the singular active moiety of fesoterodine. Formation of 5-HMT from fesoterodine and tolterodine occurs via esterases and CYP2D6 respectively. This randomized, crossover, open-label, multiple-dose study in CYP2D6 extensive metabolizers (EMs) and poor metabolizers (PMs) compared the pharmacokinetics of fesoterodine vs. tolterodine extended release (ER). Subjects received fesoterodine and tolterodine ER with a ≥3-day washout period. Treatment comprised 4-mg once daily doses for 5 days escalated to 8-mg once daily for 5 days. Pharmacokinetics of active moieties were compared by drug, dose and genotype. Active moiety exposures following fesoterodine and tolterodine ER increased proportional to dose in EMs and PMs. In EMs only, coefficients of variation for AUC and C(max) following fesoterodine (up to 46% and 48% respectively) were lower than those following tolterodine ER (up to 87% and 87% respectively). Following fesoterodine and

  11. 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

    Directory of Open Access Journals (Sweden)

    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

  12. 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

    Institute of Scientific and Technical Information of China (English)

    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。

  13. Machine Learning Energies of 2 Million Elpasolite (A B C2D6) Crystals

    Science.gov (United States)

    Faber, Felix A.; Lindmaa, Alexander; von Lilienfeld, O. Anatole; Armiento, Rickard

    2016-09-01

    Elpasolite is the predominant quaternary crystal structure (AlNaK2F6 prototype) reported in the Inorganic Crystal Structure Database. We develop a machine learning model to calculate density functional theory quality formation energies of all ˜2 ×106 pristine A B C2D6 elpasolite crystals that can be made up from main-group elements (up to bismuth). Our model's accuracy can be improved systematically, reaching a mean absolute error of 0.1 eV /atom for a training set consisting of 10 ×103 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 the 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 (group I) element, and D being fluoride. Out of 2 ×106 crystals, 90 unique structures are predicted to be on the convex hull—among which is NFAl2Ca6, with a peculiar stoichiometry and a negative atomic oxidation state for Al.

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

    CERN Document Server

    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 ...

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

    DEFF Research Database (Denmark)

    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...

  16. SNP genotyping using TaqMan® technology: the CYP2D6*17 assay conundrum

    Science.gov (United States)

    Gaedigk, Andrea; Freeman, Natalie; Hartshorne, Toinette; Riffel, Amanda K.; Irwin, David; Bishop, Jeffrey R.; Stein, Mark A.; Newcorn, Jeffrey H.; Jaime, Lazara Karelia Montané; Cherner, Mariana; Leeder, J. Steven

    2015-01-01

    CYP2D6 contributes to the metabolism of many clinically used drugs and is increasingly tested to individualize drug therapy. The CYP2D6 gene is challenging to genotype due to the highly complex nature of its gene locus. TaqMan® technology is widely used in the clinical and research settings for genotype analysis due to assay reliability, low cost, and the availability of commercially available assays. The assay identifying 1023C>T (rs28371706) defining a reduced function (CYP2D6*17) and several nonfunctional alleles, produced a small number of unexpected diplotype calls in three independent sets of samples, i.e. calls suggested the presence of a CYP2D6*4 subvariant containing 1023C>T. Gene resequencing did not reveal any unknown SNPs in the primer or probe binding sites in any of the samples, but all affected samples featured a trio of SNPs on their CYP2D6*4 allele between one of the PCR primer and probe binding sites. While the phenomenon was ultimately overcome by an alternate assay utilizing a PCR primer excluding the SNP trio, the mechanism causing this phenomenon remains elusive. This rare and unexpected event underscores the importance of assay validation in samples representing a variety of genotypes, but also vigilance of assay performance in highly polymorphic genes such as CYP2D6. PMID:25788121

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

    Science.gov (United States)

    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

  18. Effects of polymorphisms in CYP2D6 and ABC transporters and side effects induced by gefitinib on the pharmacokinetics of the gefitinib metabolite, O-desmethyl gefitinib.

    Science.gov (United States)

    Kobayashi, Hiroyuki; Sato, Kazuhiro; Niioka, Takenori; Takeda, Masahide; Okuda, Yuji; Asano, Mariko; Ito, Hiroshi; Miura, Masatomo

    2016-06-01

    We investigated the effects of polymorphisms in CYP2D6, ABCB1, and ABCG2 and the side effects induced by gefitinib on the pharmacokinetics of O-desmethyl gefitinib, the active metabolite of gefitinib. On day 14 after beginning therapy with gefitinib, plasma concentrations of gefitinib and O-desmethyl gefitinib were measured. Patients were grouped into three groups according to their combination of CYP2D6 alleles: homozygous extensive metabolisers (EMs; *1/*1, *1/*2, and *2/*2; n = 13), heterozygous EMs (*1/*5, *2/*5, *1/*10, and *2/*10; n = 18), and intermediate metabolisers (IMs; *5/*10 and *10/*10; n = 5). The median AUC0-24 of O-desmethyl gefitinib in CYP2D6 IMs was 1460 ng h/mL, whereas that in homozygous EMs was 12,523 ng h/mL (P = 0.021 in univariate analysis). The median AUC ratio of O-desmethyl gefitinib to gefitinib differed among homozygous EMs, heterozygous EMs, and IMs at a ratio of 1.41:0.86:0.24 (P = 0.030). On the other hand, there were no significant differences in the AUC0-24 of O-desmethyl gefitinib between ABCB1 and ABCG2 genotypes. In a multivariate analysis, CYP2D6 homozygous EMs (P = 0.012) were predictive for a higher AUC0-24 of O-desmethyl gefitinib. The side effects of diarrhoea, skin rash, and hepatotoxicity induced by gefitinib were unrelated to the AUC0-24 of O-desmethyl gefitinib. CYP2D6 polymorphisms were associated with the formation of O-desmethyl gefitinib from gefitinib. In CYP2D6 homozygous EMs, the plasma concentrations of O-desmethyl gefitinib were higher over 24 h after taking gefitinib than those of the parent compound; however, side effects induced by gefitinib were unrelated to O-desmethyl gefitinib exposure.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Substituted Imidazole of 5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates Cytochrome P450 2D6 by Protein Adduction

    Science.gov (United States)

    Nagy, Leslie D.; Mocny, Catherine S.; Diffenderfer, Laura E.; Hsi, David J.; Butler, Brendan F.; Arthur, Evan J.; Fletke, Kyle J.; Palamanda, Jairam R.; Nomeir, Amin A.

    2011-01-01

    5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) is a potent mechanism-based inactivator of human cytochrome P450 2D6 that displays type I binding spectra with a Ks of 0.39 ± 0.10 μM. The partition ratio is ∼3, indicating potent inactivation that addition of exogenous nucleophiles does not prevent. Within 15 min of incubation with SCH 66712 and NADPH, ∼90% of CYP2D6 activity is lost with only ∼20% loss in ability to bind CO and ∼25% loss of native heme over the same time. The stoichiometry of binding to the protein was 1.2:1. SDS-polyacrylamide gel electrophoresis with Western blotting and autoradiography analyses of CYP2D6 after incubations with radiolabeled SCH 66712 further support the presence of a protein adduct. Metabolites of SCH 66712 detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron. PMID:21422192

  1. An overview on the allelic variant of CYP2D6 genotype

    African Journals Online (AJOL)

    Yomi

    to metabolize as many as 20% of commonly prescribed drugs. Its substrates ... an affordable molecular diagnostic technology and is already in use in ..... involves competition with another drug for the enzyme ... dose escalation trials. This can ...

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

    Directory of Open Access Journals (Sweden)

    Carlos Flores-Angulo

    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. Melperone but not bisoprolol or metoprolol is a clinically relevant inhibitor of CYP2D6: evidence from a therapeutic drug monitoring survey.

    Science.gov (United States)

    Hefner, Gudrun; Unterecker, Stefan; Shams, Mohamed E E; Wolf, Margarete; Falter, Tanja; Haen, Ekkehard; Hiemke, Christoph

    2015-11-01

    Cytochrome P450 enzymes (CYP) can be inhibited or induced by drugs, resulting in clinically significant drug-drug interactions that can cause unanticipated adverse reactions or therapeutic failures. The objective of the study was to analyze the in vivo inhibitory potential of the beta-blockers bisoprolol and metoprolol as well as the low-potency antipsychotic melperone on CYP2D6. By utilizing a large therapeutic drug monitoring database of 2874 samples, data from patients who had been treated with venlafaxine (VEN) either without (control group) or with a concomitant medication with bisoprolol, metoprolol or melperone were evaluated retrospectively to study the CYP2D6-catalyzed O-demethylation to O-desmethylvenlafaxine (ODVEN). Dose-adjusted serum levels (C/D) of VEN and ODVEN as well as the metabolic ratios (ODVEN/VEN) were computed for the four groups and compared using Kruskal-Wallis test. In total, 381 patients could be included for analysis. No significant difference was found in the median C/D (VEN), C/D (ODVEN) or C/D of the active moiety (VEN + ODVEN) in either the metoprolol (N = 103) or bisoprolol group (N = 101), compared to the control group (N = 108). In contrast, a significantly higher median C/D (VEN) (0.79 ng/ml/mg, range 0.13-5.73 ng/ml/mg) (P metoprolol has a clinically relevant inhibitory potential on CYP2D6.

  4. Understanding outliers on the usual dose-response curve: venlafaxine as a way to phenotype patients in terms of their CYP 2D6 status and why it matters.

    Science.gov (United States)

    Preskorn, Sheldon H

    2010-01-01

    Venlafaxine is a model substrate for the drug metabolizing cytochrome P450 (CYP) enzyme 2D6. The desvenlafaxine/venlafaxine ratio, either after a single dose or at steady state, can be used to determine whether a patient is functionally (i.e., phenotypically) a CYP 2D6 extensive or poor metabolizer (EM or PM). In turn, CYP 2D6 EM and PM status is important in determining the efficacy of venlafaxine as an antidepressant. Based on a secondary analysis of four of the venlafaxine registration trials, venlafaxine was effective in patients who were CYP 2D6 EMs versus a parallel placebo-treated control group, whereas it was not effective in patients who were CYP 2D6 PMs. Thus, venlafaxine is a useful example of how drugs can be used to quantify differences in drug metabolizing capacity among patients and how such differences can in turn affect the efficacy of a drug (i.e., make a patient an outlier on the usual dose-response curve).

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Influence of CYP2D6 Polymorphisms on Serum Levels of Tamoxifen Metabolites in Spanish Women with Breast Cancer

    Science.gov (United States)

    Zafra-Ceres, Mercedes; de Haro, Tomas; Farez-Vidal, Esther; Blancas, Isabel; Bandres, Fernando; de Dueñas, Eduardo Martinez; Ochoa-Aranda, Enrique; Gomez-Capilla, Jose A.; Gomez-Llorente, Carolina

    2013-01-01

    Background Estrogen receptor-positive breast cancer tumors depend on estrogen signaling for their growth and replication and can be treated by anti-estrogen therapy with tamoxifen. Polymorphisms of the CYP2D6 and CYP2C19 genes are associated with an impaired response to tamoxifen. The study objective was to investigate the impact of genetic polymorphisms in CYP2D6 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Spanish women with estrogen receptor-positive breast cancer who were candidates for tamoxifen therapy. Methods: We studied 90 women with estrogen receptor-positive breast cancer, using the AmpliChip CYP450 test to determine CYP2D6 and CYP2C19 gene variants. Plasma levels of tamoxifen and its metabolites were quantified by high-performance liquid chromatography. Results The CYP2D6 phenotype was extensive metabolizer in 80%, intermediate metabolizer in 12.2%, ultra-rapid metabolizer in 2.2%, and poor metabolizer in 5.6% of patients, and the allele frequency was 35.0% for allele *1, 21.0% for *2, and 18.9% for *4. All poor metabolizers in this series were *4/*4, and their endoxifen and 4-hydroxy tamoxifen levels were 25% lower than those of extensive metabolizers. CYP2C19*2 allele, which has been related to breast cancer outcomes, was detected in 15.6% of the studied alleles. Conclusion CYP2D6*4/*4 genotype was inversely associated with 4-hydroxy tamoxifen and endoxifen levels. According to these results, CYP2D6 and CYP2C19 genotyping appears advisable before the prescription of tamoxifen therapy. PMID:23781139

  8. The burden and management of cytochrome P450 2D6 (CYP2D6)-mediated drug–drug interaction (DDI) : Co-medication of metoprolol and paroxetine or fluoxetine in the elderly

    NARCIS (Netherlands)

    Bahar, Muh Akbar; Hak, Eelko; Bos, Jens H. J.; Borgsteede, Sander D.; Wilffert, Bob

    Purpose: Metoprolol and paroxetine/fluoxetine are inevitably co-prescribed because cardiovascular disorders and depression often coexist in the elderly. This leads to CYP2D6-mediated drug-drug interactions (DDI). Because systematic evaluations are lacking, we assessed the burden of

  9. Effect of genetic polymorphism on the inhibition of dopamine formation from p-tyramine catalyzed by brain cytochrome P450 2D6.

    Science.gov (United States)

    Niwa, Toshiro; Shizuku, Marina; Yamano, Kaori

    2017-04-15

    The inhibitory effects of steroid hormones, including glucocorticoids such as cortisol, and related compounds on dopamine formation from p-tyramine, catalyzed by cytochrome P450 (CYP) 2D6.2 (Arg296Cys, Ser486Thr) and CYP2D6.10 (Pro34Ser, Ser486Thr) were compared with the effects of those catalyzed by CYP2D6.1 (wild type), to investigate the effect of a CYP2D6 polymorphism on neuroactive amine metabolism in the brain. Inhibition constants (Ki) or 50% inhibitory concentrations of six steroid hormones (cortisol, cortisone, corticosterone, dehydroepiandrosterone, progesterone, and pregnenolone) and quinidine and quinine-typical potent inhibitors of the human CYP2D6 and rat CYP2D subfamily, respectively-toward dopamine formation catalyzed by CYP2D6.1, CYP2D6.2, and CYP2D6.10 expressed in recombinant Escherichia coli were compared. Although most steroid hormones had no or minor inhibitory effects on the dopamine formation by all CYP2D6 variants, progesterone inhibited the metabolism and Ki value against CYP2D6.10 was approximately twice that for CYP2D6.1 and CYP2D6.2. Quinidine exhibited stronger inhibition than quinine; however, these two compounds inhibited the CYP2D6.10-mediated reaction more weakly than the CYP2D6.1 and CYP2D6.2 reactions. These results suggest that CYP2D6 polymorphism would affect drug interaction through dopamine formation in the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Cost-effectiveness of Cyp2d6 genotyping in older depressed patients, starting with nortriptyline therapy

    NARCIS (Netherlands)

    Berm, E.J.; Gout-Zwart, J.J.; Luttjeboer, J.; Maring, J.G.; Wilffert, B.; Postma, M.J.

    2015-01-01

    Objectives: Genotyping for the cytochrome P450-2D6 has the potency to predict differences in metabolism of nortriptyline. This information could optimize treatment. We explored if possible benefits could outweigh genotyping costs for Dutch depressed patients in clinical psychiatry. Methods: First, a

  11. An assessment of drug-drug interactions: the effect of desvenlafaxine and duloxetine on the pharmacokinetics of the CYP2D6 probe desipramine in healthy subjects.

    Science.gov (United States)

    Patroneva, Albena; Connolly, Sandra M; Fatato, Penny; Pedersen, Ron; Jiang, Qin; Paul, Jeffrey; Guico-Pabia, Christine; Isler, Jennifer A; Burczynski, Michael E; Nichols, Alice I

    2008-12-01

    A number of antidepressants inhibit the activity of the cytochrome P450 2D6 enzyme system, which can lead to drug-drug interactions. Based on its metabolic profile, desvenlafaxine, administered as desvenlafaxine succinate, a new serotonin-norepinephrine reuptake inhibitor, is not expected to have an impact on activity of CYP2D6. This single-center, randomized, open-label, four-period, crossover study was undertaken to evaluate the effect of multiple doses of desvenlafaxine (100 mg/day, twice the recommended therapeutic dose for major depressive disorder in the United States) and duloxetine (30 mg b.i.d.) on the pharmacokinetics (PK) of a single dose of desipramine (50 mg). A single dose of desipramine was given first to assess its PK. Desvenlafaxine or duloxetine was then administered, in a crossover design, so that steady-state levels were achieved; a single dose of desipramine was then coadministered. The geometric least-square mean ratios (coadministration versus desipramine alone) for area under the plasma concentration versus time curve (AUC) and peak plasma concentrations (C(max)) of desipramine and 2-hydroxydesipramine were compared using analysis of variance. Relative to desipramine alone, increases in AUC and C(max) of desipramine associated with duloxetine administration (122 and 63%, respectively) were significantly greater than those associated with desvenlafaxine (22 and 19%, respectively; P desvenlafaxine and desipramine (-24 versus 9%; P desvenlafaxine had a minimal impact on the PK of desipramine compared with duloxetine, suggesting a lower risk for CYP2D6-mediated drug interactions.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    PM patient was carefully matched on age, gender and diagnosis with an intermediate metabolizer (IM) and an extensive metabolizer (EM) from the same database to generate 18 triplets. Clinical data, primarily on side effects of treatment, were obtained from medical records by an experienced research......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...

  13. Generation and validation of rapid computational filters for cyp2d6 and cyp3a4.

    Science.gov (United States)

    Ekins, Sean; Berbaum, Jennifer; Harrison, Richard K

    2003-09-01

    CYP2D6 and CYP3A4 represent two particularly important members of the cytochrome p450 enzyme family due to their involvement in the metabolism of many commercially available drugs. Avoiding potent inhibitory interactions with both of these enzymes is highly desirable in early drug discovery, long before entering clinical trials. Computational prediction of this liability as early as possible is desired. Using a commercially available data set of over 1750 molecules to train computer models that were generated with commercially available software enabled predictions of inhibition for CYP2D6 and CYP3A4, which were compared with empirical data. The results suggest that using a recursive partitioning (tree) technique with augmented atom descriptors enables a statistically significant rank ordering of test-set molecules (Spearman's rho of 0.61 and 0.48 for CYP2D6 and CYP3A4, respectively), which represents an increased rate of identifying the best compounds when compared with the random rate. This approach represents a valuable computational filter in early drug discovery to identify compounds that may have p450 inhibition liabilities prior to molecule synthesis. Such computational filters offer a new approach in which lead optimization in silico can occur with virtual molecules simultaneously tested against multiple enzymes implicated in drug-drug interactions, with a resultant cost savings from a decreased level of molecule synthesis and in vitro screening.

  14. Pharmacokinetics of venlafaxine extended release 75  mg and desvenlafaxine 50  mg in healthy CYP2D6 extensive and poor metabolizers: a randomized, open-label, two-period, parallel-group, crossover study.

    Science.gov (United States)

    Nichols, Alice I; Focht, Kristen; Jiang, Qin; Preskorn, Sheldon H; Kane, Cecelia P

    2011-01-01

    Genetically driven variations in the level of cytochrome P450 (CYP) 2D6 metabolic activity have been shown to significantly affect the pharmacokinetic behaviour of medications that are substrates of this enzyme. To evaluate the impact of CYP2D6 extensive metabolizer (EM) and poor metabolizer (PM) phenotypes on the pharmacokinetics of single doses of venlafaxine extended release (ER) and desvenlafaxine (administered as desvenlafaxine succinate). This study used a randomized, open-label, two-period, parallel-group, crossover design. The enrolled healthy subjects participated in the study for approximately 8 weeks, which included ≤ 6 weeks of screening procedures and two separate 1-week partial inpatient confinement periods (separated by a 4-day washout period), during which venlafaxine ER or desvenlafaxine was administered and blood samples were collected. Subjects were admitted to partial inpatient confinement in a laboratory setting for the two separate study periods where each study drug was individually administered. Blood samples for pharmacokinetic analyses were collected during the 120 hours following administration of each study drug. Plasma concentrations of the study drugs were measured by a third-party analyst using liquid chromatography-tandem mass spectrometry. Healthy subjects were recruited through newspaper advertisements and genotyped to determine their CYP2D6 metabolic phenotype (i.e. EM or PM) using internally developed and commercially available assays. Subjects were reimbursed for their participation in this study. Single, sequentially administered oral doses of the dual-acting, serotonin and norepinephrine reuptake inhibiting antidepressants venlafaxine ER (75  mg) and desvenlafaxine (50  mg) were administered. The main outcome measures were differences in the geometric means for area under the plasma concentration-time curve from time zero to infinity (AUC(∞)) and peak plasma concentration (C(max)) between EMs and PMs. Comparisons were

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  17. Rough Set Theory as an Interpretable Method for Predicting the Inhibition of Cytochrome P450 1A2 and 2D6.

    Science.gov (United States)

    Burton, Julien; Petit, Joachim; Danloy, Emeric; Maggiora, Gerald M; Vercauteren, Daniel P

    2013-07-01

    Early prediction of ADME properties such as the cytochrome P450 (CYP) mediated drug-drug interactions is an important challenge in the drug discovery area. In this study, we propose to couple an original data mining approach based on Rough Set Theory (RST) to a structural description of molecules. The latter was achieved by using two types of structural keys: (1) the MACCS keys and (2) a set of five in-house fingerprints based on properties of the electron density distributions of chemical groups. The compounds considered are involved in the inhibition of CYP1A2 and CYP2D6. RST allowed the extraction of rules further used as classifiers to predict the inhibitory profile of an independent set of molecules. The results reached prediction accuracies of 90.6 and 88.2 % for CYP1A2 and CYP2D6, respectively. In addition, these classifiers were analyzed to determine which structural fragments were most used for building the rules, revealing relationships between the occurrence of particular molecular fragments and CYP inhibition. The results assessed RST as a suitable tool to build strongly predictive models and infer structure-activity rules associated with potency.

  18. Enantioselective analysis of citalopram and escitalopram in postmortem blood together with genotyping for CYP2D6 and CYP2C19.

    Science.gov (United States)

    Carlsson, Björn; Holmgren, Anita; Ahlner, Johan; Bengtsson, Finn

    2009-03-01

    Citalopram is marketed as a racemate (50:50) mixture of the S(+)-enantiomer and R(-)-enantiomer and the active S(+)-enantiomer (escitalopram) that possess inhibitory effects. Citalopram was introduced in Sweden in 1992 and is the most frequently used antidepressant to date in Sweden. In 2002, escitalopram was introduced onto the Swedish market for treatment of depression and anxiety disorders. The main objective of this study was to investigate S(+)-citalopram [i.e., the racemic drug (citalopram) or the enantiomer (escitalopram)] present in forensic autopsy cases positive for the presence of citalopram in routine screening using a non-enantioselective bioanalytical method. Fifty out of the 270 samples found positive by gas chromatography-nitrogen-phosphorus detection were further analyzed using enantioselective high-performance liquid chromatography. The 50 cases were genotyped for CYP2D6 and CYP2C19, as these isoenzymes are implicated in the metabolism of citalopram and escitalopram. In samples positive for racemic citalopram using the screening method for forensic autopsy cases, up to 20% would have been misinterpreted in the absence of an enantioselective method. An enantioselective method is thus necessary for correct interpretation of autopsy cases, after the enantiomer has been introduced onto the market. The percentage of poor metabolizers was 6% for CYP2D6 and 8% for CYP2C19.

  19. 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.

    Directory of Open Access Journals (Sweden)

    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.

  20. Inference of the Genetic Polymorphisms of CYP2D6 in Six Subtribes of the Malaysian Orang Asli from Whole-Genome Sequencing Data.

    Science.gov (United States)

    Yu, Choo Yee; Ang, Geik Yong; Subramaniam, Vinothini; Johari James, Richard; Ahmad, Aminuddin; Abdul Rahman, Thuhairah; Mohd Nor, Fadzilah; Shaari, Syahrul Azlin; Teh, Lay Kek; Salleh, Mohd Zaki

    2017-07-01

    CYP2D6 is one of the major enzymes in the cytochrome P450 monooxygenase system. It metabolizes ∼25% of prescribed drugs and hence, the genetic diversity of a CYP2D6 gene has continued to be of great interest to the medical and pharmaceutical industries. This study was designed to perform a systematic analysis of the CYP2D6 gene in six subtribes of the Malaysian Orang Asli. Genomic DNAs were extracted from the blood samples followed by whole-genome sequencing. The reads were aligned to the reference human genome hg19 and variants in the CYP2D6 gene were analyzed. CYP2D6*5 and duplication of CYP2D6 were analyzed using previously established methods. A total of 72 single nucleotide polymorphisms were identified. CYP2D6*1, *2, *4, *5, *10,*41, and duplication of the gene were found in the Orang Asli, whereby CYP2D6*2 and *41 alleles are reported for the first time in the Malaysian population. The findings in this study provide insights into the genetic polymorphisms of CYP2D6 in the Orang Asli of Peninsular Malaysia.

  1. Inhibition of CYP2D6-mediated tramadol O-demethylation in methadone but not buprenorphine maintenance patients

    Science.gov (United States)

    Coller, Janet K; Michalakas, Jennifer R; James, Heather M; Farquharson, Aaron L; Colvill, Joel; White, Jason M; Somogyi, Andrew A

    2012-01-01

    AIMS To compare the O- (CYP2D6 mediated) and N- (CYP3A4 mediated) demethylation metabolism of tramadol between methadone and buprenorphine maintained CYP2D6 extensive metabolizer subjects. METHODS Nine methadone and seven buprenorphine maintained subjects received a single 100 mg dose of tramadol hydrochloride. Blood was collected at 4 h and assayed for tramadol, methadone, buprenorphine and norbuprenorphine (where appropriate) and all urine over 4 h was assayed for tramadol and its M1 and M2 metabolites. RESULTS The urinary metabolic ratio [median (range)] for O-demethylation (M1) was significantly lower (P= 0.0002, probability score 1.0) in the subjects taking methadone [0.071 (0.012–0.103)] compared with those taking buprenorphine [0.192 (0.108–0.392)], but there was no significant difference (P= 0.21, probability score 0.69) in N-demethylation (M2). The percentage of dose [median (range)] recovered as M1 was significantly lower in subjects taking methadone compared with buprenorphine (0.069 (0.044–0.093) and 0.126 (0.069–0.187), respectively, P= 0.04, probability score 0.19), M2 was significantly higher in subjects taking methadone compared with buprenorphine (0.048 (0.033–0.085) and 0.033 (0.014–0.049), respectively, P= 0.04, probability score 0.81). Tramadol was similar (0.901 (0.635–1.30) and 0.685 (0.347–1.04), respectively, P= 0.35, probability score 0.65). CONCLUSIONS Methadone inhibited the CYP2D6-mediated metabolism of tramadol to M1. Hence, as the degree of opioid analgesia is largely dependent on M1 formation, methadone maintenance patients may not receive adequate analgesia from oral tramadol. PMID:22369095

  2. Substrate utilization during exercise in active people.

    Science.gov (United States)

    Coyle, E F

    1995-04-01

    When people walk at low intensity after fasting, the energy needed is provided mostly by oxidation of plasma fatty acids. As exercise intensity increases (eg, to moderate running), plasma fatty acid turnover does not increase and the additional energy is obtained by utilization of muscle glycogen, blood glucose, and intramuscular triglyceride. Further increases in exercise intensity are fueled mostly by increases in muscle glycogen utilization with some additional increase in blood glucose oxidation. Muscle glycogen and blood glucose contribute equally to carbohydrate energy production over 2-3 h of moderate-intensity exercise; fatigue develops when these substrates are depleted. Active people can deplete muscle glycogen with 30-60 min of high intensity, intermittent exercise. When the ingestion of dietary carbohydrate is optimal, it is possible to resynthesize muscle glycogen to high concentrations in approximately 24 h, which is the major factor in recovery of exercise tolerance. However, this requires that a 70-kg person eat at least 50 g carbohydrate per every 2 h, beginning soon after exercise, and ingest 500-600 g in 24 h (ie; approximately 7-9 g/kg body wt). Carbohydrate foods eliciting high glycemic and insulinemic responses promote more rapid glycogen resynthesis than do foods eliciting lower glycemic responses. Therefore, foods ingested for energy before, during, or after exercise should be classified according to their glycemic index. Although carbohydrate ingestion before and during exercise adds exogenous substrate to the body, it usually attenuates plasma fatty acid mobilization and oxidation.

  3. Intermediate metabolizer: increased side effects in psychoactive drug therapy. The key to cost-effectiveness of pretreatment CYP2D6 screening?

    Science.gov (United States)

    Laika, B; Leucht, S; Heres, S; Steimer, W

    2009-12-01

    The cytochrome P450 2D6 (CYP2D6) isoenzyme metabolizes about 25% of clinically used drugs. The impact of CYP2D6 metabolizer status on therapeutic outcome was assessed in 365 psychiatric in-patients treated with neuroleptics or antidepressants. Length of hospitalization and response onset were prolonged for patients receiving CYP2D6 drugs. Intermediate metabolizers (IMs) receiving CYP2D6 doses above the population median had more side effects after 4 weeks than extensive metabolizers with above-median doses (9/13, 69% vs 4/23, 17%, P = 0.003), than IMs with below-median doses (5/22, 23%, P = 0.012) and IMs with other medication (24/84, 29%, P = 0.009). The Clinical Global Impression scale response was lower for IMs treated with CYP2D6 drugs (3/42, 7%) than for IMs with other medication (21/84, 25%, P = 0.017) probably due to increased side effects. Identification of IM status (38% of study population) may help to reduce side effects and length/cost of hospitalization. Thus, not only poor and ultrarapid metabolizer but also IMs may benefit from CYP2D6 genotyping. This is of paramount interest since it greatly improves cost/benefit estimations for pretreatment CYP2D6 screening.

  4. Free energies of binding of R - and S -propranolol to wild-type and F483A mutant cytochrome P450 2D6 from molecular dynamics simulations

    NARCIS (Netherlands)

    Commandeur, Jan; Graaf, de Chris; Keizers, Peter; Oostenbrink, Chris; Vugt-Lussenburg, van Barbara; Vermeulen, Nico

    2007-01-01

    Detailed molecular dynamics (MD) simulations have been performed to reproduce and rationalize the experimental finding that the F483A mutant of CYP2D6 has lower affinity for R-propranolol than for S-propranolol. Wild-type (WT) CYP2D6 does not show this stereospecificity. Four different approaches t

  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

    Energy Technology Data Exchange (ETDEWEB)

    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. Amylase activity in substrate deficiency aerobic granules.

    Science.gov (United States)

    Lee, Chuen-Chi; Lee, Duu-Jong; Lai, Juin-Yih

    2009-01-01

    Immunohistochemical staining was applied together with the multicolor fluorescent scheme to demonstrate the amylase activity for polysaccharide hydrolysis in stored or starved aerobic granules that are in substrate deficiency. If sufficient nutrients were present, alpha-amylase and beta-amylase were found close to the surface layer of the original granules. Following storage or starvation during which most external nutrients were depleted, the alpha-amylase and beta-amylase were distributed over the entire granule interior, suggesting endogenous respiration at the core of the granule. In particular, the fluorescent intensities of alpha-amylase and beta-amylase were enriched 5-20 microm from the edge of the internal cavity, suggesting the strong correlation between polysaccharide hydrolysis and the formation of interior cavities. The secreted amylase was located near the living cells, suggesting that the polysaccharide hydrolysis is restricted to local environment that occurs near the functional strains. Internal hydrolysis within the core, for the case of both proteins and polysaccharides should correspond in principle to the loss of granule stability.

  7. Influence of CYP2D6 deletion, multiplication, -1584C-->G, 31G-->A and 2988G-->a gene polymorphisms on dextromethorphan metabolism among Mexican tepehuanos and mestizos.

    Science.gov (United States)

    Sosa-Macías, Martha; Dorado, Pedro; Alanis-Bañuelos, Ruth E; Llerena, Adrián; Lares-Asseff, Ismael

    2010-01-01

    The aim of this study was to explain the variability of CYP2D6 activity by the identification of CYP2D6 deletion and multiplications, and the single-nucleotide polymorphisms (SNPs) -1584C-->G, 31G-->A and 2988G-->A in Mexican Mestizo and Tepehuano subjects. One hundred twelve Mestizos and 99 Tepehuano Amerindians were studied, who were previously phenotyped with dextromethorphan. The frequencies of CYP2D6*2A [-1584C-->G] and *35 [-1584C-->G, 31G-->A] were 10.7 and 4.1%, respectively, in Mestizos, which is evidently a trend towards an extensive metabolism in carriers of the -1584G change. In Tepehuanos, *2A was identified with a frequency of 20%, and the allele *35 was not found. The frequencies of CYP2D6*5 (deletion) and *41[2988G-->A] were 1.3 and 2.2% in Mestizos and 0.5 and 1% in Tepehuanos, respectively. The SNP 2988A was found to be significantly related with the intermediate metabolizer phenotype in Mestizos (R = 0.309; n = 88; p = 0.006). The multiplications had frequencies of 4.1% in Mestizos and 1.5% in Tepehuanos. Only in the Mestizos did the presence of multiplications significantly decrease the DM/DX (dextromethorphan/dextrorphan) values (R = 0.273; n = 88; p = 0.016). The polymorphisms studied had different frequencies between Tepehuanos and Mestizos (p < 0.001); however, in the Tepehuano group these had a low influence on their phenotypic expression. It helps to understand the genotype-phenotype relationships of CYP2D6 in our studied populations.

  8. Effect of Concomitant CYP2D6 Inhibitor Use and Tamoxifen Adherence on Breast Cancer Recurrence in Early-Stage Breast Cancer

    National Research Council Canada - National Science Library

    Vincent O. Dezentjé; Nico J.C. van Blijderveen; Hans Gelderblom; Hein Putter; Myrthe P.P. van Herk-Sukel; Mariel K. Casparie; Antoine C.G. Egberts; Johan W.R. Nortier; Henk-Jan Guchelaar

    2010-01-01

    .... Concomitant use of CYP2D6 inhibitors, such as selective serotonin reuptake inhibitors, as well as low tamoxifen adherence may negatively impact tamoxifen efficacy in patients with breast cancer...

  9. Clinical Pharmacology of 3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”): The Influence of Gender and Genetics (CYP2D6, COMT, 5-HTT)

    OpenAIRE

    Ricardo Pardo-Lozano; Magí Farré; Samanta Yubero-Lahoz; Brian O'Mathúna; Marta Torrens; Cristina Mustata; Clara Pérez-Mañá; Klaus Langohr; Elisabet Cuyàs; Marcel lí Carbó; Rafael de la Torre

    2012-01-01

    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 pharmac...

  10. 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

    Institute of Scientific and Technical Information of China (English)

    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.

  11. 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

    Institute of Scientific and Technical Information of China (English)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

    Singh, Satyender; Kumar, Vivek; Vashisht, Kapil; Singh, Priyanka; Banerjee, Basu Dev; Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen; Jain, Sudhir Kumar; Rai, Arvind

    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 (pdamage compared to control subjects (14.37±2.15 vs. 6.24±1.37 tail% DNA, pdamage when compared to other genotypes (pdamage 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. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities

    National Research Council Canada - National Science Library

    Ismail H Al-abdullah; Karine Bagramyan; Shiela Bilbao; Meirigeng Qi; Markus Kalkum

    2017-01-01

    A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay...

  17. Substrate utilization and VSS relations in activated sludge processes

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    , and 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......We have studied secretory phospholipase A(2)-IIA (sPLA(2)) activity toward different phospholipid analogues by performing biophysical 1 characterizations and molecular dynamics simulations. The phospholipids were natural substrates, triple alkyl phospholipids, a prodrug anticancer etherlipid...... charged residues, but relatively large fluctuations are observed, suggesting that these interactions are not necessarily important for stabilizing substrate binding to the enzyme....

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    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 between...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    PEDRO GRIMÁN

    2009-01-01

    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 común- mente 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 genó- mico 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 pobla- ciones 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 consti- tuye 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.

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

    DEFF Research Database (Denmark)

    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...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    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....... 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...

  5. Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

    Science.gov (United States)

    Shisler, Krista A; Broderick, Joan B

    2014-03-15

    The glycyl radical enzyme activating enzymes (GRE-AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe-4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE-AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE-AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE-AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes. Copyright © 2014. Published by Elsevier Inc.

  6. Substrate stiffness regulates filopodial activities in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Yu-Ren Liou

    Full Text Available Microenvironment stiffening plays a crucial role in tumorigenesis. While filopodia are generally thought to be one of the cellular mechanosensors for probing environmental stiffness, the effects of environmental stiffness on filopodial activities of cancer cells remain unclear. In this work, we investigated the filopodial activities of human lung adenocarcinoma cells CL1-5 cultured on substrates of tunable stiffness using a novel platform. The platform consists of an optical system called structured illumination nano-profilometry, which allows time-lapsed visualization of filopodial activities without fluorescence labeling. The culturing substrates were composed of polyvinyl chloride mixed with an environmentally friendly plasticizer to yield Young's modulus ranging from 20 to 60 kPa. Cell viability studies showed that the viability of cells cultured on the substrates was similar to those cultured on commonly used elastomers such as polydimethylsiloxane. Time-lapsed live cell images were acquired and the filopodial activities in response to substrates with varying degrees of stiffness were analyzed. Statistical analyses revealed that lung cancer cells cultured on softer substrates appeared to have longer filopodia, higher filopodial densities with respect to the cellular perimeter, and slower filopodial retraction rates. Nonetheless, the temporal analysis of filopodial activities revealed that whether a filopodium decides to extend or retract is purely a stochastic process without dependency on substrate stiffness. The discrepancy of the filopodial activities between lung cancer cells cultured on substrates with different degrees of stiffness vanished when the myosin II activities were inhibited by treating the cells with blebbistatin, which suggests that the filopodial activities are closely modulated by the adhesion strength of the cells. Our data quantitatively relate filopodial activities of lung cancer cells with environmental stiffness and

  7. An investigation of CYP2D6 genotype and response to metoprolol CR/XL during dose titration in patients with heart failure: a MERIT-HF substudy.

    Science.gov (United States)

    Batty, J A; Hall, A S; White, H L; Wikstrand, J; de Boer, R A; van Veldhuisen, D J; van der Harst, P; Waagstein, F; Hjalmarson, Å; Kjekshus, J; Balmforth, A J

    2014-03-01

    To explore the pharmacogenetic effects of the cytochrome P450 (CYP)2D6 genotype in patients with systolic heart failure treated using controlled/extended-release (CR/XL) metoprolol, this study assessed the CYP2D6 locus for the nonfunctional *4 allele (1846G>A; rs3892097) in the Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF; n = 605). Participants were characterized as extensive, intermediate, or poor metabolizers (EMs, IMs, or PMs, respectively), based on the presence of the CYP2D6*4 allele (EM: *1*1, 60.4%; IM: *1*4, 35.8%; and PM: *4*4, 3.8%). Plasma metoprolol concentrations were 2.1-/4.6-fold greater in the IM/PM groups as compared with the EM group (P Metoprolol induced significantly lower heart rates and diastolic blood pressures during early titration, indicating a CYP2D6*4 allele dose-response effect (P metoprolol pharmacokinetics/pharmacodynamics during early titration; however, the MERIT-HF-defined titration schedule remains recommended for all patients, regardless of genotype.

  8. 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

    Institute of Scientific and Technical Information of China (English)

    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

  9. 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)

    NARCIS (Netherlands)

    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

  10. Increased prevalence of functional minor allele variants of drug metabolizing CYP2B6 and CYP2D6 genes in Roma population samples.

    Science.gov (United States)

    Weber, Agnes; Szalai, Renata; Sipeky, Csilla; Magyari, Lili; Melegh, Marton; Jaromi, Luca; Matyas, Petra; Duga, Balazs; Kovesdi, Erzsebet; Hadzsiev, Kinga; Melegh, Bela

    2015-06-01

    Cytochrome P450 2B6 and 2D6 are important enzymes in human drug metabolism. These phase I enzymes are known to contribute the biotransformation of clinically important pharmaceuticals, including antidepressants, anticancer and anxiolytic drugs. The aim of this work was to determine the pharmacogenetic profile of CYP2B6 and CYP2D6 in Roma and Hungarian population samples. A study population of 426 healthy Roma and 431 healthy Hungarian subjects were characterized for CYP2B6 c.516G>T, CYP2D6 c.100C>T and c.1846G>A polymorphisms using predesigned TaqMan Drug Metabolism Genotyping Real Time-PCR assays. We found significant differences in the presence of CYP2B6 c.516G>T (pT (p=0.003) and c.1846G>A (p=0.022) between Hungarian and Roma population. The 516T allele frequency was 33.6% in the Roma group, 21.4% in Hungarians, whereas the minor CYP2D6 100T allele was present in 26.6% in Romas and 20.5% in Hungarians. The 1864A allele frequency was 22.5% in Roma and 18.1% in Hungarian samples. A significant increase was found in genotype frequencies for homozygous minor allele carrier Roma participants compared to Hungarians for CYP2B6 516TT and CYP2D6 100TT. The following CYP2D6 genotypes were identified in Roma samples: *1/*1 (55.4%), *1/*4 (2.1%), *1/*10 (3.1%), *4/*10 (38.7%), *10/*10 (0.7%). Our results demonstrate an increased minor allele frequency for CYP2B6 and CYP2D6 polymorphisms in Roma samples that implies clinical significance in this ethnic group. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  12. Substrate Activity Screening (SAS) and Related Approaches in Medicinal Chemistry.

    Science.gov (United States)

    Gladysz, Rafaela; Lambeir, Anne-Marie; Joossens, Jurgen; Augustyns, Koen; Van der Veken, Pieter

    2016-03-04

    Substrate activity screening (SAS) was presented a decade ago by Ellman and co-workers as a straightforward methodology for the identification of fragment-sized building blocks for enzyme inhibitors. Ever since, SAS and variations derived from it have been successfully applied to the discovery of inhibitors of various families of enzymatically active drug targets. This review covers key achievements and challenges of SAS and related methodologies, including the modified substrate activity screening (MSAS) approach. Special attention is given to the kinetic and thermodynamic aspects of these methodologies, as a thorough understanding thereof is crucial for successfully transforming the identified fragment-sized hits into potent inhibitors.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Correlations of CYP2C9∗3/CYP2D6∗10/CYP3A5∗3 gene polymorphisms with efficacy of etanercept treatment for patients with ankylosing spondylitis

    Science.gov (United States)

    Chen, Yuan-Yuan

    2017-01-01

    Abstract Background: The tumor necrosis factor alpha (TNF-α) inhibitor etanercept has been proven to be effective in the treatment of ankylosing spondylitis (AS), while genetic polymorphism may affect drug metabolism or drug receptor, resulting in interindividual variability in drug disposition and efficacy. The purpose of this study is to investigate the correlations between CYP2C9∗3/CYP2D6∗10/CYP3A5∗3 gene polymorphisms and the efficacy of etanercept treatment for patients with AS. Methods: From March 2012 to June 2015, 312 AS patients (174 males and 138 females, mean age: 35.2 ± 5.83 years) from 18 to 56 years old were enrolled in this study. Polymerase chain reaction-restriction fragment length polymorphism was applied to detect the allele and genotype frequencies of CYP2C9∗3, CYP2D6∗10, and CYP3A5∗3 gene polymorphisms. The joint swelling score, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) level of AS patients were compared before and after 24-week etanercept treatment. Assessment in Ankylosing Spondylitis (ASAS) and bath ankylosing spondylitis disease activity index (BASDAI) scores were recorded to assess the efficacy of etanercept treatment. Results: The AS patients with wild-type ∗1/∗1 and heterozygous ∗1/∗3 genotypes of CYP2C9∗3 polymorphism accounted for 93.59% and 6.41%, respectively, without ∗3/∗3 genotype. The AS patients with wild-type CC, heterozygous CT, and mutation homozygous TT genotypes of CYP2D6∗10 polymorphism accounted for 19.23%, 39.10%, and 41.67%, respectively. The AS patients with wild-type ∗1/∗1, heterozygous ∗1/∗3, and mutation homozygous ∗3/∗3 genotypes of CYP3A5∗3 polymorphism accounted for 7.69%, 36.22%, and 56.09%, respectively. After 24-week treatment, AS patients with wild-type ∗1/∗1 genotype of CYP2C9∗3, CC genotype of CYP2D6∗10, and ∗3/∗3 genotype of CYP3A5∗3 polymorphisms had lower joint swelling score, ESR, and CRP level. The joint swelling

  15. Investigation of morphine and morphine glucuronide levels and cytochrome P450 isoenzyme 2D6 genotype in codeine-related deaths.

    Science.gov (United States)

    Frost, Joachim; Helland, Arne; Nordrum, Ivar S; Slørdal, Lars

    2012-07-10

    Compared to morphine and morphine-6-glucuronide (M6G), codeine and its other major metabolites codeine-6-glucuronide and norcodeine have weak affinity to opioid μ-receptors. Analgesic effects of codeine are thus largely dependent on metabolic conversion to morphine by the polymorphic cytochrome P450 isoenzyme 2D6 (CYP2D6). How this relates to toxicity and post-mortem whole blood levels is not known. This paper presents a case series of codeine-related deaths where concentrations of morphine, M6G and morphine-3-glucuronide (M3G), as well as CYP2D6 genotype, are taken into account. Post-mortem toxicological specimens from a total of 1444 consecutive forensic autopsy cases in Central Norway were analyzed. Among these, 111 cases with detectable amounts of codeine in femoral blood were identified, of which 34 had femoral blood concentrations exceeding the TIAFT toxicity threshold of 0.3mg/L. Autopsy records of these 34 cases were retrieved and reviewed. In the 34 reviewed cases, there was a large variability in individual morphine to codeine concentration ratios (M/C ratios), and morphine levels could not be predicted from codeine concentrations, even when CYP2D6 genotype was known. 13 cases had codeine concentrations exceeding the TIAFT threshold for possibly lethal serum concentrations (1.6 mg/L). Among these, 8 individuals had morphine concentrations below the toxic threshold according to TIAFT (0.15 mg/L). In one case, morphine as well as M6G and M3G concentrations were below the limit of detection. A comprehensive investigation of codeine-related fatalities should, in addition to a detailed case history, include quantification of morphine and morphine metabolites. CYP2D6 genotyping may be of interest in cases with unexpectedly high or low M/C ratios.

  16. Do CYP2D6 genotypes reflect oxycodone requirements for cancer patients treated for cancer pain? A cross-sectional multicentre study.

    Science.gov (United States)

    Andreassen, Trine Naalsund; Eftedal, Ingrid; Klepstad, Pål; Davies, Andrew; Bjordal, Kristin; Lundström, Staffan; Kaasa, Stein; Dale, Ola

    2012-01-01

    Opioids are recommended by the World Health Organization for moderate to severe cancer pain. Oxycodone is one of the most commonly used opioids and is metabolized in the liver by CYP3A4 and CYP2D6 enzymes. The aim of this cross-sectional study was to assess the relationship between oxycodone pharmacokinetics, pharmacodynamics and the CYP2D6 genotypes "poor metaboliser" (PM), "extensive metaboliser" (EM) and "ultra-rapid metaboliser" (URM) in a cohort of patients with cancer pain. The patients were genotyped for the most common CYP2D6 variants and serum concentrations of oxycodone and metabolites were determined. Pain was assessed using the Brief Pain Inventory (BPI). The EORTC QLQ-C30 was used to assess the symptoms of tiredness and nausea. Cognitive function was assessed by the Mini Mental State (MMS) examination. Associations were examined by analyses of variance (ANOVA) and covariance (ANCOVA), or ordinal logistic regressions with and without covariates. The sample consisted of 27 PM, 413 EM (including heterozygotes) and 10 URM. PM had lower oxymorphone and noroxymorphone serum concentrations and oxymorphone to oxycodone ratios than EM and URM. No differences between PM, EM and URM in pain intensity, nausea, tiredness or cognitive function was found. CYP2D6 genotypes caused expected differences in pharmacokinetics, but they had no pharmacodynamic consequence. CYP2D6 genotypes did not influence pain control, the adverse symptoms nausea and sedation or the risk for cognitive failure in this study of patients treated with oxycodone for cancer pain.

  17. CYP2B6, CYP2D6, and CYP3A4 catalyze the primary oxidative metabolism of perhexiline enantiomers by human liver microsomes.

    Science.gov (United States)

    Davies, Benjamin J; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

    2007-01-01

    The cytochrome P450 (P450)-mediated 4-monohydroxylations of the individual enantiomers of the racemic antianginal agent perhexiline (PHX) were investigated in human liver microsomes (HLMs) to identify stereoselective differences in metabolism and to determine the contribution of the polymorphic enzyme CYP2D6 and other P450s to the intrinsic clearance of each enantiomer. The cis-, trans1-, and trans2-4-monohydroxylation rates of (+)- and (-)-PHX by human liver microsomes from three extensive metabolizers (EMs), two intermediate metabolizers (IMs), and two poor metabolizers (PMs) of CYP2D6 were measured with a high-performance liquid chromatography assay. P450 isoform-specific inhibitors, monoclonal antibodies directed against P450 isoforms, and recombinantly expressed human P450 enzymes were used to define the P450 isoform profile of PHX 4-monohydroxylations. The total in vitro intrinsic clearance values (mean +/- S.D.) of (+)- and (-)-PHX were 1376 +/- 330 and 2475 +/- 321, 230 +/- 225 and 482 +/- 437, and 63.4 +/- 1.6 and 54.6 +/- 1.2 microl/min/mg for the EM, IM, and PM HLMs, respectively. CYP2D6 catalyzes the formation of cis-OH-(+)-PHX and trans1-OH-(+)-PHX from (+)-PHX and cis-OH-(-)-PHX from (-)-PHX with high affinity. CYP2B6 and CYP3A4 each catalyze the trans1- and trans2-4-monohydroxylation of both (+)- and (-)-PHX with low affinity. Both enantiomers of PHX are subject to significant polymorphic metabolism by CYP2D6, although this enzyme exhibits distinct stereoselectivity with respect to the conformation of metabolites and the rate at which they are formed. CYP2B6 and CYP3A4 are minor contributors to the intrinsic P450-mediated hepatic clearance of both enantiomers of PHX, except in CYP2D6 PMs.

  18. CYP3A4 and seasonal variation in vitamin D status in addition to CYP2D6 contribute to therapeutic endoxifen level during tamoxifen therapy.

    Science.gov (United States)

    Teft, Wendy A; Gong, Inna Y; Dingle, Brian; Potvin, Kylea; Younus, Jawaid; Vandenberg, Theodore A; Brackstone, Muriel; Perera, Francisco E; Choi, Yun-Hee; Zou, Guangyong; Legan, Robin M; Tirona, Rommel G; Kim, Richard B

    2013-05-01

    Tamoxifen is a widely utilized adjuvant anti-estrogen agent for hormone receptor-positive breast cancer, known to undergo CYP2D6-mediated bioactivation to endoxifen. However, little is known regarding additional genetic and non-genetic determinants of optimal endoxifen plasma concentration. Therefore, 196 breast cancer patients on tamoxifen were enrolled in this prospective study over a 24-month period. Blood samples were collected for pharmacogenetic and drug-level analysis of tamoxifen and metabolites. Regression analysis indicated that besides CYP2D6, the recently described CYP3A4*22 genotype, seasonal variation, and concomitant use of CYP2D6-inhibiting antidepressants were significant predictors of endoxifen concentration. Of note, genetic variation explained 33 % of the variability while non-genetic variables accounted for 13 %. Given the proposed notion of a sub-therapeutic endoxifen concentration for predicting breast cancer recurrence, we set the therapeutic threshold at 18 nM, the 20th percentile for endoxifen level among enrolled patients in this cohort. Nearly 70 % of CYP2D6 poor metabolizers as well as extensive metabolizers on potent CYP2D6-inhibiting antidepressants exhibited endoxifen levels below 18 nM, while carriers of CYP3A4*22 were twofold less likely to be in sub-therapeutic range. Unexpectedly, endoxifen levels were 20 % lower during winter months than mean levels across seasons, which was also associated with lower vitamin D levels. CYP3A4*22 genotype along with sunshine exposure and vitamin D status may be unappreciated contributors of tamoxifen efficacy. The identified covariates along with demographic variables were integrated to create an endoxifen concentration prediction algorithm to pre-emptively evaluate the likelihood of individual patients falling below the optimal endoxifen concentration.

  19. Fibrinogen patterns and activity on substrates with tailored hydroxy density.

    Science.gov (United States)

    Rodríguez Hernández, José Carlos; Rico, Patricia; Moratal, David; Monleón Pradas, Manuel; Salmerón-Sánchez, Manuel

    2009-08-11

    The influence of the surface fraction of OH groups on fibrinogen (FG) adsorption is investigated in copolymers of ethyl acrylate and hydroxy ethylacrylate. The amount of adsorbed FG, quantified by western-blotting combined with image analysis of the corresponding bands, decreases as the hydrophilicity of the substrate increases. The influence of substrate wettability on FG conformation and distribution is observed by atomic force microscopy (AFM). The most hydrophobic substrate promotes FG fibrillogenesis, which leads to a fibrin-like appearance in the absence of any thrombin. The degree of FG interconnection was quantified by calculating the fractal dimension of the adsorbed protein from image analysis of the AFM results. The biological activity of the adsorbed FG is correlated to cell adhesion on FG-coated substrates.

  20. Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

    Institute of Scientific and Technical Information of China (English)

    Pei-pei DONG; Chang-xiao LIU; Ling YANG; Zhong-ze FANG; Yan-yan ZHANG; Guang-bo GE; Yu-xi MAO; Liang-liang ZHU; Yan-qing QU; Wei Li; Li-ming WANG

    2011-01-01

    Aim: To ascertain the effects of erlotinib on CYP3A, to investigate the amplitude and kinetics of erlotinib-mediated inhibition of seven major CYP isoforms in human liver microsomes (HLMs) for evaluating the magnitude of erlotinib in drug-drug interaction in vivo.Methods:The activities of 7 major CYP isoforms (CYP1A2, CYP2A6, CYP3A, CYP2C9, CYP2D6, CYP2C8, and CYP2E1) were assessed in HLMs using HPLC or UFLC analysis. A two-step incubation method was used to examine the time-dependent inhibition of erlotinib on CYP3A.Results: The activity of CYP2C8 was inhibited with an IC50 value of 6.17±2.0μmol/L. Erlotinib stimulated the midazolam 1'-hydroxy reaction, but inhibited the formation of 6β-hydroxytestosterone and oxidized nifedipine. Inhibition of CYP3A by erlotinib was substratedependent: the IC50 values for inhibiting testosterone 6β-hydroxylation and nifedipine metabolism were 31.3±8.0 and 20.5±5.3μmol/L, respectively. Erlotinib also exhibited the time-dependent inhibition on CYP3A, regardless of the probe substrate used: the value of K1 and k1nack were 6.3μmol/L and 0.035 min-1 for midazolam; 9.0μmol/L and 0.045 min-1 for testosterone; and 10.1μmol/L and 0.058 min-1 for nifedipine.Conclusion: The inhibition of CYP3A by erlotinib was substrate-dependent, while its time-dependent inhibition on CYP3A was substrateindependent. The time-dependent inhibition of CYP3A may be a possible cause of drug-drug interaction, suggesting that attention should be paid to the evaluation of erlotinib's safety, especially in the context of combination therapy.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. A general method for determining secondary active transporter substrate stoichiometry.

    Science.gov (United States)

    Fitzgerald, Gabriel A; Mulligan, Christopher; Mindell, Joseph A

    2017-01-25

    The number of ions required to drive substrate transport through a secondary active transporter determines the protein's ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter's mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na(+)-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.

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

    DEFF Research Database (Denmark)

    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 analgesic effect of 150 mg of tramadol were studied in 16 healthy extensive metabolizers of sparteine in a randomized, double-blind, placebo-controlled, 4-way crossover study by use of experimental pain models. RESULTS: With paroxetine pretreatment, the area under the plasma concentration-time curve (AUC...

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

    DEFF Research Database (Denmark)

    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 analgesic effect of 150 mg of tramadol were studied in 16 healthy extensive metabolizers of sparteine in a randomized, double-blind, placebo-controlled, 4-way crossover study by use of experimental pain models. RESULTS: With paroxetine pretreatment, the area under the plasma concentration-time curve (AUC...

  5. Does pharmacogenetic testing for CYP450 2D6 and 2C19 among patients with diagnoses within the schizophrenic spectrum reduce treatment costs?

    Science.gov (United States)

    Herbild, Louise; Andersen, Stig E; Werge, Thomas; Rasmussen, Henrik B; Jürgens, Gesche

    2013-10-01

    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 healthcare costs of treating patients with diagnoses within the schizophrenic spectrum for 1 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 intermediate metabolizers was used to increase the frequency of extreme metabolizers (poor metabolizers and ultrarapid metabolizers for CYP2D6) to 20% in both groups. Cost differences were analysed at several levels including (i) overall healthcare expenditure, (ii) psychiatric hospital cost (iii) nonpsychiatric hospital cost, (iv) primary care spending and (v) pharmaceuticals. Statistically significant differences in costs of psychiatric care dependent on metabolizer status were found between intervention groups. Pharmacogenetic testing significantly reduced costs among the extreme metabolizers (poor metabolizers and ultrarapid metabolizers) to 28%. Use of primary care services and pharmaceuticals was also affected by the intervention.This study confirms earlier findings that extreme metabolizers (poor and ultrarapid metabolizers) incur higher costs than similar patients with a normal metabolizer genotype. However, this study shows that these excess costs can be reduced by pharmacogenetic testing. Pharmacogenetic testing for CYP2D6 and CYP2C19 could thus be considered as a means of curtailing high psychiatric treatment costs among extreme metabolizers.

  6. Tailoring adjuvant endocrine therapy for postmenopausal breast cancer: a CYP2D6 multiple-genotype-based modeling analysis and validation.

    Directory of Open Access Journals (Sweden)

    Ke-Da Yu

    Full Text Available PURPOSE: Previous studies have suggested that postmenopausal women with breast cancer who present with wild-type CYP2D6 may actually have similar or superior recurrence-free survival outcomes when given tamoxifen in place of aromatase inhibitors (AIs. The present study established a CYP2D6 multiple-genotype-based model to determine the optimal endocrine therapy for patients harboring wild-type CYP2D6. METHODS: We created a Markov model to determine whether tamoxifen or AIs maximized 5-year disease-free survival (DFS for extensive metabolizer (EM patients using annual hazard ratio (HR data from the BIG 1-98 trial. We then replicated the model by evaluating 9-year event-free survival (EFS using HR data from the ATAC trial. In addition, we employed two-way sensitivity analyses to explore the impact of HR of decreased-metabolizer (DM and its frequency on survival by studying a range of estimates. RESULTS: The 5-year DFS of tamoxifen-treated EM patients was 83.3%, which is similar to that of genotypically unselected patients who received an AI (83.7%. In the validation study, we further demonstrated that the 9-year EFS of tamoxifen-treated EM patients was 81.4%, which is higher than that of genotypically unselected patients receiving tamoxifen (78.4% and similar to that of patients receiving an AI (83.2%. Two-way sensitivity analyses demonstrated the robustness of the results. CONCLUSIONS: Our modeling analyses indicate that, among EM patients, the DFS/EFS outcome of patients receiving tamoxifen is similar to that of patients receiving an AI. Further prospective clinical trials are needed to evaluate the value of the CYP2D6 genotype in the selection of endocrine therapy.

  7. Comparison of the pharmacokinetics of venlafaxine extended release and desvenlafaxine in extensive and poor cytochrome P450 2D6 metabolizers.

    Science.gov (United States)

    Preskorn, Sheldon; Patroneva, Albena; Silman, Heather; Jiang, Qin; Isler, Jennifer A; Burczynski, Michael E; Ahmed, Saeeduddin; Paul, Jeffrey; Nichols, Alice I

    2009-02-01

    The goal of this study was to evaluate the impact of cytochrome P450 2D6 extensive metabolizer (EM) or poor metabolizer (PM) status on the pharmacokinetics of single doses of venlafaxine extended release (ER) and desvenlafaxine (administered as desvenlafaxine succinate) in healthy adults. In an open-label, crossover study, 14 healthy volunteers (aged 18-55 years; 7 EMs and 7 PMs) received, in randomized sequence, single doses of venlafaxine ER 75 mg/d or desvenlafaxine 100 mg/d. Cytochrome P450 2D6 genotyping was performed, and plasma drug levels were measured. The arithmetic means and standard deviation (SD) for area under the plasma concentration-versus-time curve (AUC) and peak plasma concentration (Cmax) were calculated. Comparisons of AUC and Cmax between cytochrome P450 2D6 EMs and PMs were calculated using a Wilcoxon exact test. After administration of venlafaxine ER, mean Cmax and AUC of venlafaxine were significantly greater in PMs compared with EMs, whereas mean Cmax and AUC of its metabolite, desvenlafaxine, were significantly lower for PMs than for EMs (P = 0.001, all comparisons). In contrast, mean Cmax and AUC of desvenlafaxine after administration of desvenlafaxine were comparable between EMs and PMs. Cytochrome P450 2D6 genetic polymorphisms had no discernible impact on exposure to desvenlafaxine after desvenlafaxine administration; in contrast, compared with an EM phenotype, a PM phenotype had a significant effect on venlafaxine and desvenlafaxine plasma concentrations after venlafaxine ER administration. This reduced pharmacokinetic variability of desvenlafaxine may translate into better uniformity of response for patients receiving desvenlafaxine versus venlafaxine, but additional studies are required to test this hypothesis.

  8. 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

    Institute of Scientific and Technical Information of China (English)

    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

  9. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    Directory of Open Access Journals (Sweden)

    Fiona Karen Harlan

    Full Text Available Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research

  10. The role of CYP2D6 and TaqI A polymorphisms in malignant neuroleptic syndrome: two case reports with three episodes.

    Science.gov (United States)

    Zivković, Maja; Mihaljević-Peles, Alma; Sagud, Marina; Silić, Ante; Mihanović, Mate

    2010-03-01

    Malignant neuroleptic syndrome (MNS) is a serious and potentially fatal side-effect of neuroleptic treatment. Beside antipsychotic drugs, other psychotropic drugs such as antidepressants and lithium carbonate can cause this life threatening side-effect. Underlying mechanism of this side-effect is still unknown and debated. So far some risk factors have been identified, with clinical observations and recent pharmacogenetic research suggesting (with inconsistent findings) correlation between genetic mechanisms and predisposition to MNS. Polymorphisms of CYP2D6 enzyme through which most psychotropic drugs are metabolized and TaqIA DRD2 which is target for antipsychotic drugs could be the link between pharmacogenetic factors and potential for development of MNS. In this paper we present two case reports with clinical presentation of three consecutive MNS. One patient developed MNS while he was taking combination of drugs: first time haloperidol, promazine and fluphenazine, second time fluphenazine and perazine and third time clozapine, promazine and valproic acid consecutively. The other patient developed MNS while taking following combination of drugs: first time haloperidol and lithium carbonate, second time risperidone and third time clozapine consecutively. Pharmacogenetic analysis for CYP2D6 and TaqI A DRD2 polymorphisms for both patients was done. Genotypisation of CYP2D6*1*3*4*5*6 in both patients showed no evidence of poor metabolizer phenotype. On the other hand, first patient was heterozygous for CYP2D6*4 (genotype *1/*4). CYP2D6 polymorphisms could have clinical significance because may lead to toxicity and unwanted side-effects in standard usual antipsychotic dose ranges. Analysis Taql A DRD2 polymorphism for first patient showed that he is heterozygous for A1 allele (genotype A1A2) which is commonly associated with predisposition to MNS. According to our literature three consecutive MNS are rarely described, and incidence of MNS generally is too low to

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

    Science.gov (United States)

    Real, M D; Ferré, J; Chapa, F J

    1991-05-01

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

  12. Large area flexible SERS active substrates using engineered nanostructures

    Science.gov (United States)

    Chung, Aram J.; Huh, Yun Suk; Erickson, David

    2011-07-01

    Surface enhanced Raman scattering (SERS) is an analytical sensing method that provides label-free detection, molecularly specific information, and extremely high sensitivity. The Raman enhancement that makes this method attractive is mainly attributed to the local amplification of the incident electromagnetic field that occurs when a surface plasmon mode is excited at a metallic nanostructure. Here, we present a simple, cost effective method for creating flexible, large area SERS-active substrates using a new technique we call shadow mask assisted evaporation (SMAE). The advantage of large, flexible SERS substrates such as these is they have more area for multiplexing and can be incorporated into irregular surfaces such as clothing. We demonstrate the formation of four different types of nanostructure arrays (pillar, nib, ellipsoidal cylinder, and triangular tip) by controlling the evaporation angle, substrate rotation, and deposition rate of metals onto anodized alumina nanoporous membranes as large as 27 mm. In addition, we present experimental results showing how a hybrid structure comprising of gold nanospheres embedded in a silver nano-pillar structure can be used to obtain a 50× SERS enhancement over the raw nanoparticles themselves.Surface enhanced Raman scattering (SERS) is an analytical sensing method that provides label-free detection, molecularly specific information, and extremely high sensitivity. The Raman enhancement that makes this method attractive is mainly attributed to the local amplification of the incident electromagnetic field that occurs when a surface plasmon mode is excited at a metallic nanostructure. Here, we present a simple, cost effective method for creating flexible, large area SERS-active substrates using a new technique we call shadow mask assisted evaporation (SMAE). The advantage of large, flexible SERS substrates such as these is they have more area for multiplexing and can be incorporated into irregular surfaces such as

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

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    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Application of a physiologically based pharmacokinetic model for the evaluation of single-point plasma phenotyping method of CYP2D6.

    Science.gov (United States)

    Chen, Rui; Rostami-Hodjegan, Amin; Wang, Haotian; Berk, David; Shi, Jun; Hu, Pei

    2016-09-20

    Determining metabolic ratio from single-point plasma is potentially a good phenotyping method of CYP2D6 to reduce the required time interval and increase the reliability of data. It is difficult to conduct large sample size clinical trials to evaluate this phenotyping method for multiple plasma points. A physiologically based pharmacokinetic (PBPK) model can be developed to do simulations based on the large virtual Chinese population and evaluate single-point plasma phenotyping method of CYP2D6. Pharmacokinetic data of dextromethorphan (DM) and its metabolite dextrorphan (DX) after oral administration were used for model development. The SimCYP® model incorporating Chinese demographic, physiological, and enzyme data was used to simulate DM and DX pharmacokinetics in different phenotype groups. The ratios of the simulated to the observed mean AUC and Cmax of DM were 1.01 and 0.81 for extensive metabolizers (EMs), 0.90 and 0.81 for intermediate metabolizers (IMs), and 1.12 and 0.84 for poor metabolizers (PMs). The ratios of the simulated to the observed mean AUC and Cmax of DX were 1.12 and 0.89 for EMs, 0.66 and 0.62 for IMs. All ratios were within the predefined criterion of 0.5-2. The simulations of DM and DX pharmacokinetic profiles in 1000 virtual Chinese subjects with reported frequencies of different phenotypes indicated that statistically significant correlations were found between metabolic ratio of DM to DX (MRDM/DX) from AUC and from single-point plasma from 1 to 30h (all p-values phenotyping method of CYP2D6 for EMs, IMs, and PMs. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Effect of substrate mechanical properties on T cell activation

    Science.gov (United States)

    Hui, King; Upadhyaya, Arpita

    2013-03-01

    T cell activation is a key process in cell-mediated immunity, and engagement of T cell receptors by peptides on antigen presenting cells leads to activation of signaling cascades as well as cytoskeletal reorganization and large scale membrane deformations. While significant advances have been made in understanding the biochemical signaling pathways, the effects imposed by the physical environment and the role of mechanical forces on cell activation are not well understood. In this study, we have used anti-CD3 coated elastic polyacrylamide gels as stimulatory substrates to enable the spreading of Jurkat T cells and the measurement of cellular traction forces. We have investigated the effect of substrate stiffness on the dynamics of T cell spreading and cellular force generation. We found that T cells display more active and sustained edge dynamics on softer gels and that they exert increased traction stresses with increasing gel stiffness. A dynamic actin cytoskeleton was required to maintain the forces generated during activation, as inferred from small molecule inhibition experiments. Our results indicate an important role for physical properties of the antigen presenting cell as well as cytoskeleton-driven forces in signaling activation.

  17. Steady-state pharmacokinetics of the enantiomers of perhexiline in CYP2D6 poor and extensive metabolizers administered Rac-perhexiline.

    Science.gov (United States)

    Davies, Benjamin J; Herbert, Megan K; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

    2008-03-01

    What is already known about this subject. Perhexiline (PHX) is administered as a racemic mixture and exhibits enantioselective pharmacokinetics in both poor and extensive metabolizers of CYP2D6 (PM and EM, respectively). Extensive metabolism by CYP2D6 is primarily responsible for the observed enantioselectivity in EM, but the process responsible in PM is unknown. Analysis of the steady-state plasma concentration-time profiles of the enantiomers of PHX in PM and EM was undertaken in order to elucidate the observed enantioselectivity, particularly with respect to PM. What this study adds. This is the first study to examine the steady-state plasma concentration-time profiles of the enantiomers of PHX in EM and PM over the course of an interdosing interval. The apparent oral clearance of each enantiomer was calculated from their respective AUC rather than from trough concentrations and was enantioselective in both phenotypes, with higher apparent oral clearances of (-)-than (+)-PHX. Renal clearance, calculated for EM and subsequently assumed for PM, constitutes a greater proportion of the total apparent oral clearance of each enantiomer in PM than EM, but was not enantioselective and thus unable to explain the enantioselectivity observed in PM. To determine the steady-state pharmacokinetics of perhexiline (PHX) enantiomers over one interdosing interval in CYP2D6 extensive and poor metabolizer (EM and PM, respectively) patients administered rac-PHX. To elucidate the processes responsible for enantioselectivity, particularly in PM patients. Blood samples were taken over one interdosing interval from six EM and two PM patients at steady-state with respect to rac-PHX metabolism. Complete urine collections were taken from five EM patients. PHX concentrations in plasma and urine were determined with enantioselective high-performance liquid chromatography methods. EM patients had 16- and 10-fold greater median apparent oral clearances of (+)- and (-)-PHX, respectively, than

  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

    Institute of Scientific and Technical Information of China (English)

    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. A general method for determining secondary active transporter substrate stoichiometry

    Science.gov (United States)

    Fitzgerald, Gabriel A; Mulligan, Christopher; Mindell, Joseph A

    2017-01-01

    The number of ions required to drive substrate transport through a secondary active transporter determines the protein’s ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter’s mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na+-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures. DOI: http://dx.doi.org/10.7554/eLife.21016.001 PMID:28121290

  20. Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

    Science.gov (United States)

    Stawikowski, Maciej J; Stawikowska, Roma; Fields, Gregg B

    2015-05-19

    Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

  1. 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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

    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...... forsøgspersoner afsluttet denne randomiseret, dobbeltblindt, trefaset, crossover forsøg. Kombinationer af escitalopram 20 mg / dag eller placebo sammen med tramadol 150 mg eller placebo blev anvendt. Blodprøver til farmakokinetik blev trukket på 0-24 timer efter medicinering. Den analgetiske virkning af (+)-M...... 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....

  4. The effect of the Taq1A variant in the dopamine D2 receptor gene and common CYP2D6 alleles on prolactin levels in risperidone-treated boys

    NARCIS (Netherlands)

    Roke, Y.; Harten, P.N. van; Franke, B.; Galesloot, T.E.; Boot, A.M.; Buitelaar, J.K.

    2013-01-01

    OBJECTIVE: To investigate the effect of the Taq1A variant in the Dopamine D2 receptor gene (DRD2) and common functional genetic variants in the cytochrome P450 2D6 gene (CYP2D6) on prolactin levels in risperidone-treated boys with autism spectrum disorders and disruptive behavior disorders. METHODS:

  5. The effect of the Taq1A variant in the dopamine D-2 receptor gene and common CYP2D6 alleles on prolactin levels in risperidone-treated boys

    NARCIS (Netherlands)

    Roke, Yvette; van Harten, Peter N.; Franke, Barbara; Galesloot, Tessel E.; Boot, Annemieke M.; Buitelaar, Jan K.

    2013-01-01

    Objective To investigate the effect of the Taq1A variant in the Dopamine D2 receptor gene (DRD2) and common functional genetic variants in the cytochrome P450 2D6 gene (CYP2D6) on prolactin levels in risperidone-treated boys with autism spectrum disorders and disruptive behavior disorders.Methods Fo

  6. The effect of the Taq1A variant in the dopamine D-2 receptor gene and common CYP2D6 alleles on prolactin levels in risperidone-treated boys

    NARCIS (Netherlands)

    Roke, Yvette; van Harten, Peter N.; Franke, Barbara; Galesloot, Tessel E.; Boot, Annemieke M.; Buitelaar, Jan K.

    2013-01-01

    Objective To investigate the effect of the Taq1A variant in the Dopamine D2 receptor gene (DRD2) and common functional genetic variants in the cytochrome P450 2D6 gene (CYP2D6) on prolactin levels in risperidone-treated boys with autism spectrum disorders and disruptive behavior disorders.Methods Fo

  7. The effect of the Taq1A variant in the dopamine D2 receptor gene and common CYP2D6 alleles on prolactin levels in risperidone-treated boys

    NARCIS (Netherlands)

    Roke, Y.; Harten, P.N. van; Franke, B.; Galesloot, T.E.; Boot, A.M.; Buitelaar, J.K.

    2013-01-01

    OBJECTIVE: To investigate the effect of the Taq1A variant in the Dopamine D2 receptor gene (DRD2) and common functional genetic variants in the cytochrome P450 2D6 gene (CYP2D6) on prolactin levels in risperidone-treated boys with autism spectrum disorders and disruptive behavior disorders. METHODS:

  8. Effects of Different Substrates on Lignocellulosic Enzyme Expression, Enzyme Activity, Substrate Utilization and Biological Efficiency of Pleurotus Eryngii

    Directory of Open Access Journals (Sweden)

    Chunliang Xie

    2016-09-01

    Full Text Available Background/Aims: Pleurotus eryngii is one of the most valued and delicious mushrooms which are commercially cultivated on various agro-wastes. How different substrates affect lignocellulosic biomass degradation, lignocellulosic enzyme production and biological efficiency in Pleurotus eryngii was unclear. Methods and Results: In this report, Pleurotus eryngii was cultivated in substrates including ramie stalks, kenaf stalks, cottonseed hulls and bulrush stalks. The results showed that ramie stalks and kenaf stalks were found to best suitable to cultivate Pleurotus eryngii with the biological efficiency achieved at 55% and 57%, respectively. In order to establish correlations between different substrates and lignocellulosic enzymes expression, the extracellular proteins from four substrates were profiled with high throughput TMT-based quantitative proteomic approach. 241 non-redundant proteins were identified and 74 high confidence lignocellulosic enzymes were quantified. Most of the cellulases, hemicellulases and lignin depolymerization enzymes were highly up-regulated when ramie stalks and kenaf stalks were used as carbon sources. The enzyme activities results suggested cellulases, hemicellulases and lignin depolymerization enzymes were significantly induced by ramie stalks and kenaf stalks. Conclusion: The lignocelluloses degradation, most of the lignocellulosic enzymes expressions and activities of Pleurotus eryngii had positive correlation with the biological efficiency, which depend on the nature of lignocellulosic substrates. In addition, the lignocellulosic enzymes expression profiles during Pleurotus eryngii growth in different substrates were obtained. The present study suggested that most of the lignocellulosic enzymes expressions and activities can be used as tools for selecting better performing substrates for commercial mushroom cultivation.

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

    KAUST Repository

    Khaled, Fathi

    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.

  10. Polymorphisms in NAT2, CYP2D6, CYP2C19 and GSTP1 and their association with prostate cancer

    DEFF Research Database (Denmark)

    Wadelius, Mia; Autrup, Judith; Stubbins, Michael J.;

    1999-01-01

    The development of prostate cancer is dependent on heredity, androgenic influences, and exposure to environmental agents. A high intake of dietary fat is associated with an increased risk of prostate cancer, either through influence on steroid hormone profiles or through production of carcinogenic...... compounds that require biotransformation by enzymes. The polymorphic glutathione S-transferase (GST), N-acetyltransferase (NAT), and cytochrome P450 (CYP) enzymes are of particular interest in prostate cancer susceptibility because of their ability to metabolize both endogenous and exogenous compounds......, including dietary constituents. Association between different NAT2, CYP2D6, CYP2C19 and GSTP1 genotypes and prostate cancer was studied in a Swedish and Danish case-control study comprising 850 individuals. The combined Swedish and Danish study population was analysed by polymerase chain reaction...

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    process. That transient phenomenon exhibits itself immediately upon addition of a substrate source to an endogenously respiring activated sludge sample and it usually takes a few minutes until the activated sludge reaches its maximum possible rate under given environmental conditions. This discrepancy......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...... with fast-alternating process conditions, although such studies are beyond the scope of this report. (C) 2004 Wiley Periodicals, Inc....

  12. Effect of the potent CYP2D6 inhibitor sarpogrelate on the pharmacokinetics and pharmacodynamics of metoprolol in healthy male Korean volunteers.

    Science.gov (United States)

    Cho, Doo-Yeoun; Bae, Soo Hyeon; Lee, Joeng Kee; Park, Jung Bae; Kim, Yang-Weon; Lee, Sukhyang; Oh, Euichaul; Kim, Bom-Taeck; Bae, Soo Kyung

    2015-03-01

    1. Recently, we demonstrated that sarpogrelate is a potent and selective CYP2D6 inhibitor in vitro. Here, we evaluated the effect of sarpogrelate on the pharmacokinetics and pharmacodynamics of metoprolol in healthy subjects. 2. Nine healthy male subjects genotyped for CYP2D6*1/*1 or *1/*2 were included in an open-label, randomized, three treatment-period and crossover study. A single oral dose of metoprolol (100 mg) was administered with water (treatment A) and sarpogrelate (100 mg bid.; a total dose of 200 mg and treatment B), or after pretreatment of sarpogrelate for three days (100 mg tid.; treatment C). Plasma levels of metoprolol and α-hydroxymetoprolol were determined using a validated LC-MS/MS method. Changes in heart rate and blood pressure were monitored as pharmacodynamic responses to metoprolol. 3. Metoprolol was well tolerated in the three treatment groups. In treatment B and C groups, the AUCt of metoprolol increased by 53% (GMR, 1.53; 90% CI, 1.17-2.31) and by 51% (1.51; 1.17-2.31), respectively. Similar patterns were observed for the increase in Cmax of metoprolol by sarpogrelate. However, the pharmacodynamics of metoprolol did not differ significantly among the three treatment groups. 4. Greater systemic exposure to metoprolol after co-administration or pretreatment with sarpogrelate did not result in clinically relevant effects. Co-administration of both agents is well tolerated and can be employed without the need for dose adjustments.

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

    CERN Document Server

    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...

  14. 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

    Directory of Open Access Journals (Sweden)

    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

  15. Substrate regulation of ascorbate transport activity in astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. (Univ. of Western Ontario, London (Canada))

    1990-10-01

    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.

  16. HPLC法测定人尿中右美沙芬及去甲右美沙芬的含量及CYP2D6表型区分研究%Determination of Dextromethorphan and Dextrophan in Human Urine by HPLC and Study on the Discrimination of Phenotype of Cytochrome CYP2D6

    Institute of Scientific and Technical Information of China (English)

    林凌云; 劳万生; 孟珺; 何光明

    2008-01-01

    目的:建立以高效液相色谱法测定人尿中右美沙芬及去甲右美沙芬含量的方法,并行CYP2D6表型区分.方法:尿样经酶水解,酸、碱提取纯化,直接进样.其中色谱柱为Waters Nova-Pak Phenyl,流动相为乙腈-水(60:40),流速为1.0mL·min-1,激发波长为280 nm,发射波长为310 nm,柱温为25℃.结果:右美沙芬、去甲右美沙芬尿药浓度分别在0.065~4.148(r=0.998 5)、0.353~22.592(r=0.999 7)μg·mL-1范围内线性关系良好,最低检测限分别为0.016、0.018μg·mL-1,平均加样回收率各为90.5%、93.5%,日内RSD各≤3.26%、≤3.47%,日间RSD各≤3.65%、≤4.05%.8位受试者均为CYP2D6快代谢型.结论:本方法简便可靠,适用于人尿中右美沙芬及去甲右美沙芬含量测定和CYP2D6多态性的表型研究.

  17. New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases.

    Science.gov (United States)

    Meux, Edgar; Morel, Mélanie; Lamant, Tiphaine; Gérardin, Philippe; Jacquot, Jean-Pierre; Dumarçay, Stéphane; Gelhaye, Eric

    2013-02-01

    Omega glutathione transferases (GSTO) constitute a family of proteins with variable distribution throughout living organisms. It is notably expanded in several fungi and particularly in the wood-degrading fungus Phanerochaete chrysosporium, raising questions concerning the function(s) and potential redundancy of these enzymes. Within the fungal families, GSTOs have been poorly studied and their functions remain rather sketchy. In this study, we have used fluorescent compounds as activity reporters to identify putative ligands. Experiments using 5-chloromethylfluorescein diacetate as a tool combined with mass analyses showed that GSTOs are able to cleave ester bonds. Using this property, we developed a specific activity-based profiling method for identifying ligands of PcGSTO3 and PcGSTO4. The results suggest that GSTOs could be involved in the catabolism of toxic compounds like tetralone derivatives. Biochemical investigations demonstrated that these enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteine residue. To access the physiological function of these enzymes and notably during the wood interaction, recombinant proteins have been immobilized on CNBr Sepharose and challenged with beech wood extracts. Coupled with GC-MS experiments this ligand fishing method allowed to identify terpenes as potential substrates of Omega GST suggesting a physiological role during the wood-fungus interactions.

  18. Lignocellulolytic enzyme activity, substrate utilization, and mushroom yield by Pleurotus ostreatus cultivated on substrate containing anaerobic digester solids.

    Science.gov (United States)

    Isikhuemhen, Omoanghe S; Mikiashvilli, Nona A

    2009-11-01

    Solid waste from anaerobic digestion of litter from the commercial production of broiler chickens has limited use as fertilizer. Its disposal is a major problem for digester operators who are seeking alternative use for anaerobic digester solids, also referred to as solid waste (SW). The use of SW as substrates for the cultivation of Pleurotus ostreatus strain MBFBL400 was investigated. Lignocellulolytic enzymes activity, substrate utilization, and mushroom yield were evaluated in ten different substrate combinations (SCs) containing varying amounts of solid waste, wheat straw, and millet. Nutritional content of mushrooms produced on the different substrates was also determined. Substrates containing 70-80% wheat straw, 10-20% SW, and 10-20% millet were found to produce the highest mushroom yield (874.8-958.3 g/kg). Loss of organic matter in all SCs tested varied from 45.8% to 56.2%, which had positive correlation with the biological efficiency. Laccase, peroxidase, and carboxymethylcellulase (CMCase) activities were higher before fruiting, whereas xylanase showed higher activities after mushroom fruiting. SW increased the nutritional content in mushrooms harvested, and the combination of wheat straw and SW with millet significantly improved mushroom yield. Our findings demonstrated the possibility of utilizing anaerobic digester solids in mushroom cultivation. The application of SW as such could improve the financial gains in the overall economy of anaerobic digester plants.

  19. Cancer cachexia raises the plasma concentration of oxymorphone through the reduction of CYP3A but not CYP2D6 in oxycodone-treated patients.

    Science.gov (United States)

    Naito, Takafumi; Tashiro, Masaki; Ishida, Takuya; Ohnishi, Kazunori; Kawakami, Junichi

    2013-08-01

    This study evaluated the plasma concentrations of oxycodone and its demethylates and opioid-induced adverse effects based on cachexia stage in cancer patients receiving oxycodone. Seventy patients receiving oxycodone for cancer pain were enrolled. Cachexia was evaluated using the Glasgow Prognostic Score (GPS). Predose plasma concentrations of oxycodone, oxymorphone, and noroxycodone were determined at the titration dose. Opioid-induced adverse effects were monitored for 2 weeks after the titration. Plasma concentrations of oxycodone and oxymorphone but not noroxycodone in patients with a GPS of 2 were significantly higher than that with a GPS of 0. The metabolic ratios of noroxycodone but not oxymorphone to oxycodone in patients with a GPS of 1 and 2 were significantly lower than in those with a GPS of 0. A higher GPS was associated with a higher incidence of somnolence, while the GPS did not affect the incidence of vomiting. Plasma concentrations of oxycodone and oxymorphone were not associated with the incidence of adverse effects. In conclusion, cancer cachexia raised the plasma exposures of oxycodone and oxymorphone through the reduction of CYP3A but not CYP2D6. Although the cachexia elevated the incidence of somnolence, alterations in their pharmacokinetics were not associated with the incidence.

  20. Effect of cytochrome P450 2D6* 10 genetic polymorphism on tramadol pharmacokinetics%细胞色素P450 2D6*10基因多态性对曲马多药代动力学的影响

    Institute of Scientific and Technical Information of China (English)

    虞建刚; 王俊科; 李平; 孙新艳; 王秋石

    2008-01-01

    Objective To investigate the effect of eytochrome P450 2D6* 10 (CYP2D6* 10) genetic polymorphism on tramadol pharmacokinetics. Methods Nineteen healthy volunteers from Liaoning province aged 22-45 yr weighing 50-82 kg height 161-179 cm were enrolled in this study. Blood samples were taken from left ulnar vein for DNA extraction and CYP2D6* 10 genotyping using phenol chloroform extracting method and polymerase chain reaction-restriction fragment length polymorphism respectively. The volunteers were divided into 3 groups according to their genotypes: wild hemozygote group (w/w), heterozygote group (m/w) and mutation homozygote group (m/m). Tramadol 1.5 mg/kg was injected intravenously over 3 rain through a vein in right upper extremity. Blood samples were taken before tramadol injection and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 32 h after injection for determination of plasma tramadol and O-demethyl tramadol concentration by high-performance liquid chromatography. Pharmacokinetie analysis was performed using nonparametric methods. Area under concentration curve (AUC0→∞), plasma clearance (CL), elimination half-life (t1/2) and mean residence time (MRT) and metabolic rate (MR) of tramedol and peak concentration (Cmax) peak time (Tmax), AUC0→∞ and MRT of O-demethyl tramadol were calculated. Results MR of tramadol was significantly lower in m/w and m/m group, t1/2 and MRT of tramadol were significantly longer in m/m group and AUC0→∞ of O-demethyl tramadol was significantly lower in m/m group than in w/w group. MR of tramadol was significantly higher in m/w group than in m/m group. Conclusion CYP2D6* 10 genetic polymorphism is one of the genetic factors producing individual variation in tramadol pharmacokinetics.%目的 探讨细胞色素P450 2D6*10(CYP2D6*10)基因多态性对曲马多药代动力学的影响.方法 辽宁籍汉族健康志愿者,性别不限,年龄22~45岁,体重50~82 kg,身高161~179 cm,经左肘静脉采血2 ml,采用酚/

  1. CYP2D6, GST-M1 and GST-T1 enzymes: expression in parathyroid gland and association with the parathyroid hormone concentration during early renal replacement therapy

    Science.gov (United States)

    Yan, Feng-Xiang; Langub, M Chris; Ihnen, Mark A; Hornung, Carlton; Juronen, Erkki; Rayens, Mary K; Cai, Wei-Min; Wedlund, Peter J; Fanti, Paolo

    2003-01-01

    Aims The purpose of this research was to characterize CYP2D6, GST-M1 and GST-T1 enzyme expression in human parathyroid tissue, and to determine whether or not there is any association between deficiencies in these enzymes and serum parathyroid hormone concentrations in patients with end-stage renal disease. Methods Surgical human parathyroid tissue was obtained and evaluated by immunohistochemistry for cellular localization of CYP2D6, GST-M1 and GST-T1 and colocalization of CYP2D6 with parathyroid hormone. Blood samples were collected from 328 Caucasian patients with end-stage renal disease for genetic testing of CYP2D6*3, *4, *5, *6, *7 and GST-M1*0 and GST-T1*0 alleles. Clinical chemistry data and serum intact parathyroid hormone (iPTH) concentrations were obtained from patient medical records. In 277 of the patients, the same laboratory performed all clinical tests. Results CYP2D6, GST-M1 and GST-T1 were present in human parathyroid tissue. CYP2D6 was colocalized with parathyroid hormone in parathyroid chief cells. Within the end-stage renal disease population, a nonfunctional CYP2D6 genotype was present in 18.2%[95% confidence interval (CI) 8.0, 28.4] of patients in the 1st iPTH concentration quintile (iPTH 347 pg mL−1) (P = 0.001). Out of 12 CYP2D6-deficient females, seven were in the 1st iPTH concentration quintile and the remaining five were in the 5th quintile. Patients deficient in the GST-M1 and GST-T1 enzymes displayed a far more uniform frequency distribution relative to serum iPTH concentrations. Conclusions The presence of CYP2D6, GST-M1 and GST-T1 in parathyroid cells was observed. An association is reported between a lack of CYP2D6 and iPTH concentrations in newly diagnosed end-stage renal disease patients. Gender and concomitant deficiency in GST-M1 and/or GST-T1 appear to define this association further. It remains to be established whether these associations reflect a cause-effect relationship between deficient expression of metabolizing

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

    NARCIS (Netherlands)

    Aarnoutse, R.E.; Kleinnijenhuis, J.; Koopmans, P.P.; Touw, D.J.; Wieling, J.; Hekster, Y.A.; Burger, D.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. Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers

    NARCIS (Netherlands)

    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

  4. In silico prediction of cytochrome P450 2D6 and 3A4 inhibition using Gaussian kernel weighted k-nearest neighbor and extended connectivity fingerprints, including structural fragment analysis of inhibitors versus noninhibitors.

    Science.gov (United States)

    Jensen, Berith F; Vind, Christian; Padkjaer, Søren B; Brockhoff, Per B; Refsgaard, Hanne H F

    2007-02-08

    Inhibition of cytochrome P450 (CYP) enzymes is unwanted because of the risk of severe side effects due to drug-drug interactions. We present two in silico Gaussian kernel weighted k-nearest neighbor models based on extended connectivity fingerprints that classify CYP2D6 and CYP3A4 inhibition. Data used for modeling consisted of diverse sets of 1153 and 1382 drug candidates tested for CYP2D6 and CYP3A4 inhibition in human liver microsomes. For CYP2D6, 82% of the classified test set compounds were predicted to the correct class. For CYP3A4, 88% of the classified compounds were correctly classified. CYP2D6 and CYP3A4 inhibition were additionally classified for an external test set on 14 drugs, and multidimensional scaling plots showed that the drugs in the external test set were in the periphery of the training sets. Furthermore, fragment analyses were performed and structural fragments frequent in CYP2D6 and CYP3A4 inhibitors and noninhibitors are presented.

  5. Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates.

    Science.gov (United States)

    Stresser, D M; Blanchard, A P; Turner, S D; Erve, J C; Dandeneau, A A; Miller, V P; Crespi, C L

    2000-12-01

    Inhibition of cytochrome P450 catalytic activity is a principal mechanism for pharmacokinetic drug-drug interactions. Rapid, in vitro testing for cytochrome P450 inhibition potential is part of the current paradigm for identifying drug candidates likely to give such interactions. We have explored the extent that qualitative and quantitative inhibition parameters are dependent on the cytochrome P450 (CYP) 3A4 probe substrate. Inhibition potential (e.g., IC(50) values from 8-point inhibition curves) or activation potential for most compounds varied dramatically depending on the fluorometric probe substrates for CYP3A4 [benzyloxyresorufin (BzRes), 7-benzyloxy-4-trifluoromethylcoumarin (BFC), 7-benzyloxyquinoline (BQ), and dibenzylfluorescein (DBF)]. For 21 compounds that were primarily inhibitors, the range of IC(50) values for the four substrates varied from 2.1- to 195-fold with an average of 29-fold. While the rank order of sensitivity among the fluorometric substrates varied among the individual inhibitors, on average, BFC dealkylation was the most sensitive to inhibition, while BQ dealkylation was least sensitive. Partial inhibition was observed with BzRes and BQ but not for BFC and DBF. BzRes was more prone to activation, whereas dramatic changes in IC(50) values were observed when the BQ concentration was below the S(50). Three different correlation analyses indicated that IC(50) values with BFC, BQ, and DBF correlated well with each other, whereas the response with BzRes correlated more weakly with the other substrates. One of these correlation analyses was extended to the percent inhibition of 10 microM inhibitor with the standard CYP3A4 probe substrates testosterone, midazolam, and nifedipine. In this analysis the responses with BQ, BFC and DBF correlated well with testosterone and midazolam but more poorly with nifedipine. In the aggregate, BFC and DBF appear more suitable as an initial screen for CYP3A4 inhibition. However, the substrate-dependent effects

  6. 闽南地区抑郁症患者CYP1A2和CYP2D6基因多态性与度洛西汀疗效的关联研究%Association of CYP1A2 and CYP2D6 gene polymorphism with therapeutic efficacy of duloxetine on depression in southern region of Fujian

    Institute of Scientific and Technical Information of China (English)

    陈进东; 徐象珍; 陈彬彬

    2015-01-01

    Objective To explore the influence of CYP1A2 and CYP2D6 gene polymorphism on blood concentration,therapeutic efficacy and adverse effects of anti-depression drug duloxetine on depression patients in southern region of Fujian.Methods 82 patients with depression were selected from southern region of FuJian, China,and all participates received duloxetine for two weeks.Blood concentrations of duloxetine were detected by HPLC-MS,and CYP1A2 and CYP2D6 genotypes were determined by sequencing with the amplified PCR products from peripheral blood DNA.The therapeutic efficacy and adverse effects of duloxetine were evaluated by Hamilton depression scale (HAMD) and treatment emergent symptom scale (TESS) respectively.Results Subjects were divided into GG,GA and AA three groups based on CYP1A2 * 1C genotyping.There was no significant difference in blood concentrations of duloxetine, dose-corrected blood concentrations, the reduction rate of HAMD and the reduction rate of TESS among the three groups.Results were the same with CYP1A2 * 1F,which were divided into CC, CA and AA three groups.Subjects was divided into CC, CT,TT three groups based on CYP2D6 * 10 locus genotyping.Blood concentrations of duloxetine were (13.89±3.22) ng · ml-1 , (16.08±4.24) ng · ml-1 , (17.25±4.62) ng · ml-1 respectively and there was significant difference(F=3.21, P<0.05).CC group was significantly lower than TT group(P<0.05) , and CT group was lower than TT group but without significant difference (P>0.05).Dose-corrected blood concentrations were (304.84± 103.76), (368.13± 143.49), (444.50± 195.58) respectively and there was significant difference(F=4.19, P<0.05), and CC group was significantly lower than TT group (P<0.05).The reduction rate of HAMD were 0.42±0.11,0.46±0.11,0.52±0.09 respectively and there was significant difference (F =6.29, P<0.05), and CC and CT group was significantly lower than TT group(P<0.05).The reduction rate of TESS were 1.14±0.66,1.48±0.69, 1.69

  7. Glucuronoyl esterases are active on polymeric substrate, methyl esterified glucuronoxylan

    Science.gov (United States)

    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 d...

  8. Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

    Science.gov (United States)

    Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

    2016-11-01

    Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Modeling of organic substrate transformation in the high-rate activated sludge process.

    Science.gov (United States)

    Nogaj, Thomas; Randall, Andrew; Jimenez, Jose; Takacs, Imre; Bott, Charles; Miller, Mark; Murthy, Sudhir; Wett, Bernhard

    2015-01-01

    This study describes the development of a modified activated sludge model No.1 framework to describe the organic substrate transformation in the high-rate activated sludge (HRAS) process. New process mechanisms for dual soluble substrate utilization, production of extracellular polymeric substances (EPS), absorption of soluble substrate (storage), and adsorption of colloidal substrate were included in the modified model. Data from two HRAS pilot plants were investigated to calibrate and to validate the proposed model for HRAS systems. A subdivision of readily biodegradable soluble substrate into a slow and fast fraction were included to allow accurate description of effluent soluble chemical oxygen demand (COD) in HRAS versus longer solids retention time (SRT) systems. The modified model incorporates production of EPS and storage polymers as part of the aerobic growth transformation process on the soluble substrate and transformation processes for flocculation of colloidal COD to particulate COD. The adsorbed organics are then converted through hydrolysis to the slowly biodegradable soluble fraction. Two soluble substrate models were evaluated during this study, i.e., the dual substrate and the diauxic models. Both models used two state variables for biodegradable soluble substrate (SBf and SBs) and a single biomass population. The A-stage pilot typically removed 63% of the soluble substrate (SB) at an SRT <0.13 d and 79% at SRT of 0.23 d. In comparison, the dual substrate model predicted 58% removal at the lower SRT and 78% at the higher SRT, with the diauxic model predicting 32% and 70% removals, respectively. Overall, the dual substrate model provided better results than the diauxic model and therefore it was adopted during this study. The dual substrate model successfully described the higher effluent soluble COD observed in the HRAS systems due to the partial removal of SBs, which is almost completely removed in higher SRT systems.

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

    CERN Document Server

    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.

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

    NARCIS (Netherlands)

    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

  13. Effect of Spent Mushroom Substrate on Physical and Chemical Properties and Enzymic Activity of Rice

    Institute of Scientific and Technical Information of China (English)

    Hairu YU; Xue LI; Xin ZHANG; Changming GE; Renzhe PIAO; Meishan LI; Zongjun CUI; Hongyan ZHAO

    2016-01-01

    In order to explore the substitution substrate for rice seedling on upland fields,this paper uses spent mushroom substrate to study the physical and chemical properties of substrate,enzymic activity and number of tillers during the cultivation of rice seedling on upland fields.The results show that at the three stages of rice seedling cultivation( two-leaf stage,three-leaf stage,four-leaf stage),the content of organic matter and EC in spent mushroom substrate is higher than in the control soil,p H is within the range suitable for the growth of rice,and other nutrients( total nitrogen,total phosphorus,total potassium,available nitrogen,available phosphorus) are slightly different in different periods;except phosphatase,there are significant differences in urease,catalase and sucrase between spent mushroom substrate and the control soil; the number of tillers under spent mushroom substrate is larger than under the control.

  14. A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity

    Science.gov (United States)

    Takao, Haruna; Hirabayashi, Kei; Nishigaya, Yuki; Kouriki, Haruna; Nakaniwa, Tetsuko; Hagiwara, Yoshinori; Harada, Jiro; Sato, Hideaki; Yamazaki, Toshimasa; Sakakibara, Yoichi; Suiko, Masahito; Asada, Yujiro; Takahashi, Yasuhiro; Yamamoto, Ken; Fukuyama, Keiichi; Sugishima, Masakazu; Wada, Kei

    2017-01-01

    Biliverdin reductase catalyses the last step in haem degradation and produces the major lipophilic antioxidant bilirubin via reduction of biliverdin, using NAD(P)H as a cofactor. Despite the importance of biliverdin reductase in maintaining the redox balance, the molecular details of the reaction it catalyses remain unknown. Here we present the crystal structure of biliverdin reductase in complex with biliverdin and NADP+. Unexpectedly, two biliverdin molecules, which we designated the proximal and distal biliverdins, bind with stacked geometry in the active site. The nicotinamide ring of the NADP+ is located close to the reaction site on the proximal biliverdin, supporting that the hydride directly attacks this position of the proximal biliverdin. The results of mutagenesis studies suggest that a conserved Arg185 is essential for the catalysis. The distal biliverdin probably acts as a conduit to deliver the proton from Arg185 to the proximal biliverdin, thus yielding bilirubin. PMID:28169272

  15. Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase.

    Science.gov (United States)

    Versées, Wim; Spaepen, Stijn; Wood, Martin D H; Leeper, Finian J; Vanderleyden, Jos; Steyaert, Jan

    2007-11-30

    Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

  16. A novel fluorogenic substrate for the measurement of endothelial lipase activity.

    Science.gov (United States)

    Darrow, Andrew L; Olson, Matthew W; Xin, Hong; Burke, Sharon L; Smith, Charles; Schalk-Hihi, Celine; Williams, Robyn; Bayoumy, Shariff S; Deckman, Ingrid C; Todd, Matthew J; Damiano, Bruce P; Connelly, Margery A

    2011-02-01

    Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.

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

    Directory of Open Access Journals (Sweden)

    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

  18. Substrate elasticity affects bovine satellite cell activation kinetics in vitro.

    Science.gov (United States)

    Lapin, M R; Gonzalez, J M; Johnson, S E

    2013-05-01

    Satellite cells support efficient postnatal skeletal muscle hypertrophy through fusion into the adjacent muscle fiber. Nuclear contribution allows for maintenance of the fiber myonuclear domain and proficient transcription of myogenic genes. Niche growth factors affect satellite cell biology; however, the interplay between fiber elasticity and microenvironment proteins remains largely unknown. The objective of the experiment was to examine the effects of hepatocyte growth factor (HGF) and surface elasticity on bovine satellite cell (BSC) activation kinetics in vitro. Young's elastic modulus was calculated for the semimembranosus (SM) and LM muscles of young bulls (5 d; n = 8) and adult cows (27 mo; n = 4) cattle. Results indicate that LM elasticity decreased (P Young's modulus for the SM was noted. Bovine satellite cells were seeded atop polyacrylamide bioscaffolds with surface elasticities that mimic young bull and adult cow LM or traditional cultureware. Cells were maintained in low-serum media supplemented with 5 ng/mL HGF or vehicle only for 24 or 48 h. Activation was evaluated by proliferating cell nuclear antigen (PCNA) immunocytochemistry. Results indicate that BSC maintained on rigid surfaces were activated at 24 h and refractive to HGF supplementation. By contrast, fewer (P young bull (8.1 ± 1.7 kPa) or adult cow (14.6 ± 1.6 kPa) LM. Supplementation with HGF promoted activation of BSC cultured on bioscaffolds as measured by an increase (P muscle stem cells (P > 0.05). However, with increasing surface elasticity, an increase (P muscle progenitors was observed. These results confirm that biophysical and biochemical signals regulate BSC activation.

  19. A substrate-driven allosteric switch that enhances PDI catalytic activity.

    Science.gov (United States)

    Bekendam, Roelof H; Bendapudi, Pavan K; Lin, Lin; Nag, Partha P; Pu, Jun; Kennedy, Daniel R; Feldenzer, Alexandra; Chiu, Joyce; Cook, Kristina M; Furie, Bruce; Huang, Mingdong; Hogg, Philip J; Flaumenhaft, Robert

    2016-08-30

    Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum. The domain structure of PDI is a-b-b'-x-a', wherein the thioredoxin-like a and a' domains mediate disulfide bond shuffling and b and b' domains are substrate binding. The b' and a' domains are connected via the x-linker, a 19-amino-acid flexible peptide. Here we identify a class of compounds, termed bepristats, that target the substrate-binding pocket of b'. Bepristats reversibly block substrate binding and inhibit platelet aggregation and thrombus formation in vivo. Ligation of the substrate-binding pocket by bepristats paradoxically enhances catalytic activity of a and a' by displacing the x-linker, which acts as an allosteric switch to augment reductase activity in the catalytic domains. This substrate-driven allosteric switch is also activated by peptides and proteins and is present in other thiol isomerases. Our results demonstrate a mechanism whereby binding of a substrate to thiol isomerases enhances catalytic activity of remote domains.

  20. Nanoporous gold as a highly active substrate for surface-enhanced Raman scattering spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kucheyev, S O; Hayes, J R; Biener, J; Hamza, A V

    2006-03-28

    Colloidal solutions of metal nanoparticles are currently among most studied substrates for sensors based on surface-enhanced Raman scattering (SERS). However, such substrates often suffer from not being cost-effective, reusable, or stable. Here, we develop nanoporous Au as a highly active, tunable, a.ordable, stable, bio-compatible, and reusable SERS substrate. Nanoporous Au is prepared by a facile process of free corrosion of AgAu alloys followed by annealing. Results show that nanofoams with average pore sizes of {approx} 250 nm exhibit the largest SERS signal for 632.8 nm excitation. This is attributed to the electromagnetic SERS enhancement mechanism with additional field localization within pores.

  1. Exosome-mediated quality control : substrate recruitment and molecular activity

    OpenAIRE

    Lebreton, Alice; Séraphin, Bertrand

    2008-01-01

    International audience; The eukaryotic exosome is a multisubunit complex that is mainly responsible for 3'-5' exonucleolytic degradation of RNAs, both in the nucleus and the cytoplasm. In this review we summarize the recent experiments that have provided information on the organisation, structure and activity of this large assembly. Interestingly, eukaryotic exosomes have been implicated in a large number of RNA degradation pathways including recently discovered RNA quality control mechanisms...

  2. Electrochemically prepared surface-enhanced Raman scattering-active silver substrates with improved stabilities

    Science.gov (United States)

    Yang, Kuang-Hsuan; Liu, Yu-Chuan; Yu, Chung-Chin; Chen, Bo-Chuen

    2011-01-01

    In this work, SiO 2 nanoparticles-modified surface-enhanced Raman scattering (SERS)-active silver substrates were prepared by electrochemical oxidation-reduction cycles (ORC) methods in 0.1 N HCl aqueous solutions containing 1 mM SiO 2 nanoparticles to improve their thermal stabilities and anti-aging abilities in SERS performances. Then these SERS-active substrates were further modified with different contents of SiO 2 nanoparticles to improve their corresponding SERS performances. Experimental results indicate that the operation temperature can be significantly raised from 125 to 175 °C based on this modified SERS-active Ag substrate. Also, the aging in SERS intensity is also depressed on this modified Ag substrate due to the contribution of SiO 2 nanoparticles. Moreover, the SERS enhancement capability on this modified Ag substrate is gradually raised from 25 °C to a maximum at 55 °C and monotonically decreased from 55 to 60 °C. This is a 10 °C delay as compared with the similar phenomenon observed on the unmodified Ag substrate.

  3. Substrate Stiffness Regulates Proinflammatory Mediator Production through TLR4 Activity in Macrophages

    Science.gov (United States)

    Previtera, Michelle L.; Sengupta, Amitabha

    2015-01-01

    Clinical data show that disease adversely affects tissue elasticity or stiffness. While macrophage activity plays a critical role in driving disease pathology, there are limited data available on the effects of tissue stiffness on macrophage activity. In this study, the effects of substrate stiffness on inflammatory mediator production by macrophages were investigated. Bone marrow–derived macrophages were grown on polyacrylamide gels that mimicked the stiffness of a variety of soft biological tissues. Overall, macrophages grown on soft substrates produced less proinflammatory mediators than macrophages grown on stiff substrates when the endotoxin LPS was added to media. In addition, the pathways involved in stiffness–regulated proinflammation were investigated. The TLR4 signaling pathway was examined by evaluating TLR4, p–NF–κB p65, MyD88, and p–IκBα expression as well as p–NF–κB p65 translocation. Expression and translocation of the various signaling molecules were higher in macrophages grown on stiff substrates than on soft substrates. Furthermore, TLR4 knockout experiments showed that TLR4 activity enhanced proinflammation on stiff substrates. In conclusion, these results suggest that proinflammatory mediator production initiated by TLR4 is mechanically regulated in macrophages. PMID:26710072

  4. Substrate-induced activation of a trapped IMC-mediated protein folding intermediate.

    Science.gov (United States)

    Inouye, M; Fu, X; Shinde, U

    2001-04-01

    While several unfolded proteins acquire native structures through distinct folding intermediates, the physiological relevance and importance of such states in the folding kinetics remain controversial. The intramolecular chaperone (IMC) of subtilisin was used to trap a partially folded, stable crosslinked intermediate conformer (CLIC) through a disulfide bond between mutated IMC and subtilisin. The trapped CLIC contains non-native interactions. Here we show that CLIC can be induced into a catalytically active form by incubating it with small peptide substrates. The structure and catalytic properties of the activated crosslinked intermediate conformer (A-CLIC) differ from those of the fully folded enzyme in that A-CLIC lacks any endopeptidase activity toward a large protein substrate. Our results show that a disulfide-linked partially folded protein can be induced to acquire catalytic activity with a substrate specificity that is different from completely folded subtilisin. These results also suggest that protein folding intermediates may also participate in catalytic reactions.

  5. Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine.

    Science.gov (United States)

    Kim, Hye-Lim; Park, Mi-Bee; Kim, Yumin; Yang, Yun Gyeong; Lee, Soo-Woong; Zhuang, Ningning; Lee, Kon Ho; Park, Young Shik

    2012-10-19

    We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

  6. The clinical effectiveness and cost-effectiveness of genotyping for CYP2D6 for the management of women with breast cancer treated with tamoxifen: a systematic review

    OpenAIRE

    Fleeman, N.; Martín Saborido, Carlos; Payne, K; Boland, A; Dickson, R.; Dundar, Y.; Fernández Santander, Ana; Howell, S J; Newman, W G; Oyee, J.; Walley, T

    2011-01-01

    Background: Objectives: Data Sources: Review Methods: Results: Conclusion: Breast cancer is the most common cancer affecting women in the UK. Tamoxifen (TAM) is considered as the standard of care for many women with oestrogen receptor positive breast cancer. However, wide variability in the response of individuals to drugs at the same doses may occur, which may be a result of interindividual genetic differences (pharmacogenetics). TAM is known to be metabolised to its active metabolites N-des...

  7. Impact of Substrate Bias on Fixed-Pattern-Noise in Active Pixel Sensor Cells

    Science.gov (United States)

    Terauchi, Mamoru

    2007-11-01

    The effect of substrate (body) bias on fixed-pattern-noise (FPN) in active pixel sensor (APS) cells is studied. Through measuring test devices consisting of two metal-oxide-semiconductor field-effect transistors (MOSFETs) connected in series with each of the transistors located in the same well region, it has been revealed that substrate bias, which is inevitably applied in a normal circuit configuration in conventional APS cells, worsens the characteristics fluctuation in source-follower amplifiers in APS cells, leading to FPN that cannot be mitigated by conventional correction methods such as correlated double sampling. In addition it has been confirmed that the current-voltage characteristics of logarithmic converters, each of which is realized using a MOSFET with gate and drain terminals connected together, are also affected by substrate bias, resulting in increased characteristics fluctuation as compared with the case with no substrate bias.

  8. Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates.

    Science.gov (United States)

    Burda, Edyta; Rademann, Jörg

    2014-11-18

    Sensitive detection of small molecule fragments binding to defined sites of biomacromolecules is still a considerable challenge. Here we demonstrate that protein-binding fragments are able to induce enzymatic reactions on the protein surface via dynamic fragment ligation. Fragments binding to the S1 pocket of serine proteases containing a nitrogen, oxygen or sulphur nucleophile are found to activate electrophilic pre-substrates through a reversible, covalent ligation reaction. The dynamic ligation reaction positions the pre-substrate molecule at the active site of the protein thereby inducing its enzymatic cleavage. Catalytic activation of pre-substrates is confirmed by fluorescence spectroscopy and by high-performance liquid chromatography. The approach is investigated with 3 pre-substrates and 14 protein-binding fragments and the specific activation and the templating effect exerted by the enzyme is quantified for each protease-fragment-pre-substrate combination. The described approach enables the site-specific identification of protein-binding fragments, the functional characterization of enzymatic sites and the quantitative analysis of protein template-assisted ligation reactions.

  9. Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.

    Science.gov (United States)

    Lonhienne, T; Baise, E; Feller, G; Bouriotis, V; Gerday, C

    2001-02-09

    Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

  10. An evaluation of the CYP2D6 and CYP3A4 inhibition potential of metoprolol metabolites and their contribution to drug-drug and drug-herb interaction by LC-ESI/MS/MS.

    Science.gov (United States)

    Borkar, Roshan M; Bhandi, Murali Mohan; Dubey, Ajay P; Ganga Reddy, V; Komirishetty, Prashanth; Nandekar, Prajwal P; Sangamwar, Abhay T; Kamal, Ahmed; Banerjee, Sanjay K; Srinivas, R

    2016-10-01

    The aim of the present study was to evaluate the contribution of metabolites to drug-drug interaction and drug-herb interaction using the inhibition of CYP2D6 and CYP3A4 by metoprolol (MET) and its metabolites. The peak concentrations of unbound plasma concentration of MET, α-hydroxy metoprolol (HM), O-desmethyl metoprolol (ODM) and N-desisopropyl metoprolol (DIM) were 90.37 ± 2.69, 33.32 ± 1.92, 16.93 ± 1.70 and 7.96 ± 0.94 ng/mL, respectively. The metabolites identified, HM and ODM, had a ratio of metabolic area under the concentration-time curve (AUC) to parent AUC of ≥0.25 when either total or unbound concentration of metabolite was considered. In vitro CYP2D6 and CYP3A4 inhibition by MET, HM and ODM study revealed that MET, HM and ODM were not inhibitors of CYP3A4-catalyzed midazolam metabolism and CYP2D6-catalyzed dextromethorphan metabolism. However, DIM only met the criteria of >10% of the total drug related material and <25% of the parent using unbound concentrations. If CYP inhibition testing is solely based on metabolite exposure, DIM metabolite would probably not be considered. However, the present study has demonstrated that DIM contributes significantly to in vitro drug-drug interaction. Copyright © 2016 John Wiley & Sons, Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    Science.gov (United States)

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

    2014-04-01

    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

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

    NARCIS (Netherlands)

    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

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

    NARCIS (Netherlands)

    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

  15. Mammalian fatty acid activation : studies on substrate specificity and subcellular localization

    NARCIS (Netherlands)

    P.H.E. Groot

    1975-01-01

    textabstractThis thesis is dealing with two aspects of the ATPdependent fatty acid activation in mammalian tissues: a) the fatty acid substrate specificity of the different type of acyl-CoA synthetases, b) the tissue- and intracellular localization of acyl-CoA synthetases

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

    Directory of Open Access Journals (Sweden)

    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.

  17. An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

    Science.gov (United States)

    Szewczak, A A; Ortoleva-Donnelly, L; Zivarts, M V; Oyelere, A K; Kazantsev, A V; Strobel, S A

    1999-09-28

    Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.

  18. Enhanced transparency, mechanical durability, and antibacterial activity of zinc nanoparticles on glass substrate

    Science.gov (United States)

    Choi, Hyung-Jin; Choi, Jin-Seok; Park, Byeong-Ju; Eom, Ji-Ho; Heo, So-Young; Jung, Min-Wook; An, Ki-Seok; Yoon, Soon-Gil

    2014-09-01

    Homogeneously distributed zinc nanoparticles (NPs) on the glass substrate were investigated for the transmittance, mechanical durability, and antibacterial effect. The buffered Ti NPs between Zn NPs and glass substrate were studied for an enhancement of the transmittance and mechanical endurance. The Ti NPs buffered Zn NPs showed a high transmittance of approximately 91.5% (at a wavelength of 550 nm) and a strong antibacterial activity for Staphylococcus aureus and Escherichia coli bacteria. The buffered Ti NPs are attractive for an excellent mechanical endurance of the Zn NPs. The Zn NPs did not require the protection layer to prevent the degradation of the performance for both the antibacterial effect and the transmittance.

  19. Luminal starch substrate "brake" on maltase-glucoamylase activity is located within the glucoamylase subunit.

    Science.gov (United States)

    Quezada-Calvillo, Roberto; Sim, Lyann; Ao, Zihua; Hamaker, Bruce R; Quaroni, Andrea; Brayer, Gary D; Sterchi, Erwin E; Robayo-Torres, Claudia C; Rose, David R; Nichols, Buford L

    2008-04-01

    The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.

  20. Kinetics and activation thermodynamics of methane monooxygenase compound Q formation and reaction with substrates.

    Science.gov (United States)

    Brazeau, B J; Lipscomb, J D

    2000-11-07

    The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of

  1. Characterization of 107 genomic DNA reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: a GeT-RM and Association for Molecular Pathology collaborative project.

    Science.gov (United States)

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

    2010-11-01

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

  2. Simultaneous determination of metoprolol and its metabolites, α-hydroxymetoprolol and O-desmethylmetoprolol, in human plasma by liquid chromatography with tandem mass spectrometry: Application to the pharmacokinetics of metoprolol associated with CYP2D6 genotypes.

    Science.gov (United States)

    Bae, Soo Hyeon; Lee, Joeng Kee; Cho, Doo-Yeoun; Bae, Soo Kyung

    2014-06-01

    A rapid and simple LC with MS/MS method for the simultaneous determination of metoprolol and its two CYP2D6-derived metabolites, α-hydroxy- and O-desmethylmetoprolol, in human plasma was established. Metoprolol (MET), its two metabolites, and the internal standard chlorpropamide were extracted from plasma (50 μL) using ethyl acetate. Chromatographic separation was performed on a Luna CN column with an isocratic mobile phase consisting of distilled water and methanol containing 0.1% formic acid (60:40, v/v) at a flow rate of 0.3 mL/min. The total run time was 3.0 min per sample. Mass spectrometric detection was conducted by ESI in positive ion selected-reaction monitoring mode. The linear ranges of concentration for MET, α-hydroxymetoprolol, and O-desmethylmetoprolol were 2-1000, 2-500, and 2-500 ng/mL, respectively, with a lower limit of quantification of 2 ng/mL for all analytes. The coefficient of variation for the assay's precision was ≤ 13.2%, and the accuracy was 89.1-110%. All analytes were stable under various storage and handling conditions and no relevant cross-talk and matrix effect were observed. Finally, this method was successfully applied to assess the influence of CYP2D6 genotypes on the pharmacokinetics of MET after oral administration of 100 mg to healthy Korean volunteers.

  3. Nanoscale Patterning of Antigen on Silicon Substrate to Examine Mast Cell Activation

    Science.gov (United States)

    2002-04-01

    Examine Mast Cell Activation DISTRIBUTION: Approved for public release, distribution unlimited This paper is part of the following report: TITLE...Materials Research Society N4.3 Nanoscale Patterning of Antigen on Silicon Substrate to Examine Mast Cell Activation Reid N. Orth", Min Wu2 , Theodore G...nanometer scale to spatially control the stimulation of specific immunoreceptors on RBL mast cells . This work was motivated by previous research to

  4. Substrate activation for O2 reactions by oxidized metal centers in biology.

    Science.gov (United States)

    Pau, Monita Y M; Lipscomb, John D; Solomon, Edward I

    2007-11-20

    The uncatalyzed reactions of O(2) (S = 1) with organic substrates (S = 0) are thermodynamically favorable but kinetically slow because they are spin-forbidden and the one-electron reduction potential of O(2) is unfavorable. In nature, many of these important O(2) reactions are catalyzed by metalloenzymes. In the case of mononuclear non-heme iron enzymes, either Fe(II) or Fe(III) can play the catalytic role in these spin-forbidden reactions. Whereas the ferrous enzymes activate O(2) directly for reaction, the ferric enzymes activate the substrate for O(2) attack. The enzyme-substrate complex of the ferric intradiol dioxygenases exhibits a low-energy catecholate to Fe(III) charge transfer transition that provides a mechanism by which both the Fe center and the catecholic substrate are activated for the reaction with O(2). In this Perspective, we evaluate how the coupling between this experimentally observed charge transfer and the change in geometry and ligand field of the oxidized metal center along the reaction coordinate can overcome the spin-forbidden nature of the O(2) reaction.

  5. Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

    Science.gov (United States)

    Ranaweera, Ruchira S.; Yang, Xiaolu

    2013-01-01

    The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

  6. Correlations of CYP2C9*3/CYP2D6*10/CYP3A5*3 gene polymorphisms with efficacy of etanercept treatment for patients with ankylosing spondylitis: A case-control study.

    Science.gov (United States)

    Chen, Yuan-Yuan

    2017-03-01

    The tumor necrosis factor alpha (TNF-α) inhibitor etanercept has been proven to be effective in the treatment of ankylosing spondylitis (AS), while genetic polymorphism may affect drug metabolism or drug receptor, resulting in interindividual variability in drug disposition and efficacy. The purpose of this study is to investigate the correlations between CYP2C9*3/CYP2D6*10/CYP3A5*3 gene polymorphisms and the efficacy of etanercept treatment for patients with AS. From March 2012 to June 2015, 312 AS patients (174 males and 138 females, mean age: 35.2 ± 5.83 years) from 18 to 56 years old were enrolled in this study. Polymerase chain reaction-restriction fragment length polymorphism was applied to detect the allele and genotype frequencies of CYP2C93, CYP2D610, and CYP3A53 gene polymorphisms. The joint swelling score, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) level of AS patients were compared before and after 24-week etanercept treatment. Assessment in Ankylosing Spondylitis (ASAS) and bath ankylosing spondylitis disease activity index (BASDAI) scores were recorded to assess the efficacy of etanercept treatment. The AS patients with wild-type 1/1 and heterozygous 1/3 genotypes of CYP2C93 polymorphism accounted for 93.59% and 6.41%, respectively, without 3/3 genotype. The AS patients with wild-type CC, heterozygous CT, and mutation homozygous TT genotypes of CYP2D610 polymorphism accounted for 19.23%, 39.10%, and 41.67%, respectively. The AS patients with wild-type 1/1, heterozygous 1/3, and mutation homozygous 3/3 genotypes of CYP3A53 polymorphism accounted for 7.69%, 36.22%, and 56.09%, respectively. After 24-week treatment, AS patients with wild-type 1/1 genotype of CYP2C93, CC genotype of CYP2D610, and 3/3 genotype of CYP3A53 polymorphisms had lower joint swelling score, ESR, and CRP level. The joint swelling score, ESR, and CRP levels were significantly lower in the patients with CC genotype of CYP2D610 polymorphism than in CT and

  7. Investigation of the Photocatalytic Activity of ZnO Nanowires: Substrate Effect and Kinetics Analysis

    Directory of Open Access Journals (Sweden)

    Jan-Hau Chang

    2014-01-01

    Full Text Available The photocatalytic activity of substrate-supported ZnO nanowires is investigated with a focus on the substrate effect and the photodegradation kinetics. The employed NWs are grown on various substrates by thermal evaporation and a 50 μM rhodamine B solution is used as the target solution. It is found that the photocatalytic activity is facilitated by using a conductive substrate and such a result can be reasonably attributed to the improvement of charge separation. The photodegradation is found to follow a zeroth-order kinetics at high concentrations and a first-order kinetics at low concentrations with a transition at around 10 μM. The present observation is opposed to the commonly accepted first-order kinetics. Degradation constants of 0.58 μM·min−1 for the zeroth-order and 0.028 min−1 for the first-order have been achieved. The first-order constant also compares favorably with reported values.

  8. Characterization of a commercialized SERS-active substrate and its application to the identification of intact Bacillus endospores

    Science.gov (United States)

    Alexander, Troy A.; Le, Dianna M.

    2007-06-01

    Surface-enhanced-Raman-spectroscopy (SERS) can be made an attractive approach for the identification of Raman-active compounds and biological materials (i.e., toxins, viruses, or intact bacterial cells or spores) through development of reproducible, spatially uniform SERS-active substrates. Recently, reproducible (from substrate to substrate), spatially homogeneous (over large areas) SERS-active substrates have been commercialized and are now available in the marketplace. Scanning electron microscopy and high-resolution, tapping-mode atomic force microscopy have been used to analyze these novel plasmonic surfaces for topographical consistency. Additionally, we have assessed, by wavelength-tunable microreflectance spectrometry, the spatial distribution of the localized surface plasmon resonance (LSPR) across a single substrate surface as well as the LSPR λMAX variance from substrate to substrate. These analyses reveal that these surfaces are topologically uniform with small LSPR variance from substrate to substrate. Further, we have utilized these patterned surfaces to acquire SERS spectral signatures of four intact, genetically distinct Bacillus spore species cultivated under identical growth conditions. Salient spectral signature features make it possible to discriminate among these genetically distinct spores. Additionally, partial least squares, a multivariate calibration method, has been used to develop personal-computer-borne algorithms useful for classification of unknown spore samples based solely on SERS spectral signatures. To our knowledge, this is the first report detailing application of these commercially available SERS-active substrates to identification of intact Bacillus spores.

  9. Esterase catalysis of substrate vapour: enzyme activity occurs at very low hydration.

    Science.gov (United States)

    Lind, Penelope A; Daniel, Roy M; Monk, Colin; Dunn, Rachel V

    2004-10-01

    It has been generally accepted that enzyme activity requires a minimal hydration of about 0.2 g H2O g(-1) protein. This fits well with evidence that hydration above this level is associated with the onset of intramolecular motions. The influence of enzyme hydration on the hydrolysis of substrate by Candida rugosa Lipase B and pig liver esterase was investigated. Each enzyme was studied as a powder at various hydration levels, using vapour phase ethyl butyrate as substrate. This procedure allows the separation of those effects that are due to hydration from those arising from diffusional constraints. We found hydrolytic activity in both enzymes at all hydration levels above zero (between 0.054-0.47 and 0.029-0.60 g H2O g(-1) protein, respectively) that were investigated. The lowest hydration level investigated, hydrolysis of ethyl butyrate requires water as a second substrate, the absence of activity at zero hydration does not rule out the possibility of enzyme activity in the absence of water. These results suggest that the properties conferred on proteins by water, at least above 10% surface coverage (in this case corresponding to a hydration level of 0.03 g H2O g(-1) protein), are not a requirement for enzyme catalysis.

  10. Effect of Temperature, pH, Enzyme to Substrate Ratio, Substrate Concentration and Time on the Antioxidative Activity of Hydrolysates from Goat Milk Casein by Alcalase

    Directory of Open Access Journals (Sweden)

    Shu Guowei

    2016-12-01

    Full Text Available The effect of hydrolysis temperature (45, 50, 55, 60 and 65°C, pH (7.0, 7.5, 8.0, 8.5 and 9.0, enzyme to substrate (E/S ratio (1.0, 1.5, 2.0, 2.5 and 3.0%, substrate concentration (2, 3, 4, 5 and 6% and hydrolysis time (30-240min on antioxidant peptides hydrolysated from goat’s milk casein by Alcalase was investigated using single factor experiment. In order to obtain high DPPH radical-scavenging activity, metal-chelating activity and superoxide radical scavenging activity, the optimal conditions were hydrolysis time of 150 min, temperature of 50°C, pH 8.0, E/S ratio of 2.0% and substrate concentration of 4.0%. The hydrolysis time, hydrolysis temperature, pH, E/S ratio and substrate concentration had a significant influence on degree of hydrolysis, metal-chelating activity, DPPH and superoxide radical scavenging activity on casein hydrolysate of goat milk by Alcalase, the results were beneficial for further provide theoretical basis for production of antioxidant peptides.

  11. A novel fluorogenic substrate for the measurement of endothelial lipase activity

    OpenAIRE

    2011-01-01

    Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic ...

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

    Science.gov (United States)

    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.

  13. Niobium pentoxide: a promising surface-enhanced Raman scattering active semiconductor substrate

    Science.gov (United States)

    Shan, Yufeng; Zheng, Zhihui; Liu, Jianjun; Yang, Yong; Li, Zhiyuan; Huang, Zhengren; Jiang, Dongliang

    2017-03-01

    Surface-enhanced Raman scattering technique, as a powerful tool to identify the molecular species, has been severely restricted to the noble metals. The surface-enhanced Raman scattering substrates based on semiconductors would overcome the shortcomings of metal substrates and promote development of surface-enhanced Raman scattering technique in surface science, spectroscopy, and biomedicine studies. However, the detection sensitivity and enhancement effects of semiconductor substrates are suffering from their weak activities. In this work, a semiconductor based on Nb2O5 is reported as a new candidate for highly sensitive surface-enhanced Raman scattering detection of dye molecules. The largest enhancement factor value greater than 107 was observed with the laser excitation at 633 and 780 nm for methylene blue detection. As far as literature review shows, this is in the rank of the highest sensitivity among semiconductor materials; even comparable to the metal nanostructure substrates with "hot spots". The impressive surface-enhanced Raman scattering activities can be attributed to the chemical enhancement dominated by the photo-induced charge transfer, as well as the electromagnetic enhancement, which have been supported by the density-functional-theory and finite element method calculation results. The chemisorption of dye on Nb2O5 creates a new highest occupied molecular orbital and lowest unoccupied molecular orbital contributed by both fragments in the molecule-Nb2O5 system, which makes the charge transfer more feasible with longer excitation wavelength. In addition, the electromagnetic enhancement mechanism also accounts for two orders of magnitude enhancement in the overall enhancement factor value. This work has revealed Nb2O5 nanoparticles as a new semiconductor surface-enhanced Raman scattering substrate that is able to replace noble metals and shows great potentials applied in the fields of biology related.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Chemoenzymatic synthesis of branched oligo- and polysaccharides as potential substrates for starch active enzymes.

    Science.gov (United States)

    Greffe, Lionel; Jensen, Morten T; Bosso, Claude; Svensson, Birte; Driguez, Hugues

    2003-12-05

    Oligo- and polysaccharides embodying the alpha-maltotriosyl-6(II)-maltotetraosyl structure were readily synthesized by transglycosylation of maltosyl fluoride onto panose and pullulan catalysed by the bacterial transglycosylase cyclodextrin glycosyltransferase (CGTase). The two products obtained proved useful for increasing the knowledge of substrate binding and processing at the active site of barley limit dextrinase that is involved in the metabolism of amylopectin by acting upon its branch points.

  16. Growth and endoglucanase activity of Acetivibrio cellulolyticus grown in three different cellulosic substrates

    Directory of Open Access Journals (Sweden)

    Sanchez Cássia Regina

    1999-01-01

    Full Text Available The growth kinetics of Acetivibrio cellulolyticus grown in medium containing different carbon sources (cellobiose, amorphous or crystalline cellulose was investigated. The specific growth rate was higher in cellobiose fed cultures than in the presence of the other two substrates. Endoglucanase production was greater in cultures grown on amorphous cellulose; enzyme activity increased during the stationary phase in cultures grown on crystalline cellulose.

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

    Science.gov (United States)

    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.

  18. Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

    OpenAIRE

    Kazlauskaite, Agne; Kelly MR; Johnson, Clare; Baillie, Carla; Hastie, C James; Peggie, Mark; Macartney, Thomas; Woodroof, Helen I.; Alessi, Dario R; Pedrioli, Patrick G.A.; Muqit, Miratul M.K.

    2014-01-01

    Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Mir...

  19. [Preclinical investigation of pharmaceuticals impact against cytochrome P450 activity and prognosis of substrate affinity as means for providing substrate therapy safety].

    Science.gov (United States)

    Fomin, E V; Baĭchorov, I Kh; Shikh, E V; Sizova, Zh M

    2013-01-01

    Tricyclic antidepressants, not influencing the P450 3A4 activity, are safe in combination with drugs of other groups used in the treatment of comorbid patients. Azaphen is is one of the agents most widely used in the clinical practice. The in vitro electrochemical analysis showed that pipofezin (azaphen) was not a substrate, inductor, and/or inhibitor of cytochrome P450 CYP3A4 isoenzymes. The Guzar programme computer modelling and the literature data demonstrated the substrate affinity of pipifezin to CYP1A2.

  20. Substrate binding of gelatinase B induces its enzymatic activity in the presence of intact propeptide.

    Science.gov (United States)

    Bannikov, Gregory A; Karelina, Tatiana V; Collier, Ivan E; Marmer, Barry L; Goldberg, Gregory I

    2002-05-03

    Expression of gelatinase B (matrix metalloprotease 9) in human placenta is developmentally regulated, presumably to fulfill a proteolytic function. Here we demonstrate that gelatinolytic activity in situ, in tissue sections of term placenta, is co-localized with gelatinase B. Judging by molecular mass, however, all the enzyme extracted from this tissue was found in a proform. To address this apparent incongruity, we examined the activity of gelatinase B bound to either gelatin- or type IV collagen-coated surfaces. Surprisingly, we found that upon binding, the purified proenzyme acquired activity against both the fluorogenic peptide (7-methoxycoumarin-4-yl)-acetic acid (MCA)-Pro-Leu-Gly-Leu-3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl-Ala-Arg-NH(2) and gelatin substrates, whereas its propeptide remained intact. These results suggest that although activation of all known matrix metalloproteases in vitro is accomplished by proteolytic processing of the propeptide, other mechanisms, such as binding to a ligand or to a substrate, may lead to a disengagement of the propeptide from the active center of the enzyme, causing its activation.

  1. Beta-hexosaminidase activity of the oral pathogen Tannerella forsythia influences biofilm formation on glycoprotein substrates.

    Science.gov (United States)

    Roy, Sumita; Phansopa, Chatchawal; Stafford, Prachi; Honma, Kiyonobu; Douglas, C W Ian; Sharma, Ashu; Stafford, Graham P

    2012-06-01

    Tannerella forsythia is an important pathogen in periodontal disease. Previously, we showed that its sialidase activity is key to utilization of sialic acid from a range of human glycoproteins for biofilm growth and initial adhesion. Removal of terminal sialic acid residues often exposes β-linked glucosamine or galactosamine, which may also be important adhesive molecules. In turn, these residues are often removed by a group of enzymes known as β-hexosaminidases. We show here that T. forsythia has the ability to cleave glucosamine and galactosamine from model substrates and that this activity can be inhibited by the hexosaminidase inhibitor PugNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate). We now demonstrate for the first time that β-hexosaminidase activity plays a role in biofilm growth on glycoprotein-coated surfaces because biofilm growth and initial cell adhesion are inhibited by PugNAc. In contrast, adhesion to siallo-glycoprotein-coated surfaces is unaltered by PugNAc in the absence of sialidase activity (using a sialidase-deficient mutant) or surprisingly on the clinically relevant substrates saliva or serum. These data indicate that β-hexosaminidase activity has a significant role in biofilm formation in combination with sialidase activity in the biofilm lifestyle of T. forsythia.

  2. Multi-substrate-activity space and quasi-species in enzyme evolution: Ohno's dilemma, promiscuity and functional orthogonality.

    Science.gov (United States)

    Mannervik, Bengt; Runarsdottir, Arna; Kurtovic, Sanela

    2009-08-01

    A functional enzyme displays activity with at least one substrate and can be represented by a vector in substrate-activity space. Many enzymes, including GSTs (glutathione transferases), are promiscuous in the sense that they act on alternative substrates, and the corresponding vectors operate in multidimensional space. The direction of the vector is governed by the relative activities of the diverse substrates. Stochastic mutations of already existing enzymes generate populations of variants, and clusters of functionally similar mutants can serve as parents for subsequent generations of enzymes. The proper evolving unit is a functional quasi-species, which may not be identical with the 'best' variant in its generation. The manifestation of the quasi-species is dependent on the substrate matrix used to explore catalytic activities. Multivariate analysis is an approach to identifying quasi-species and to investigate evolutionary trajectories in the directed evolution of enzymes for novel functions.

  3. New photocathode using ZnSe substrates with GaAs active layer

    Science.gov (United States)

    Jin, Xiuguang; Takeda, Yoshikazu; Fuchi, Shingo

    2017-03-01

    GaAs active layers were successfully fabricated on ZnSe substrates using a metalorganic vapor phase epitaxy system. As a photocathode, a GaAs active layer shows a high quantum efficiency (QE) of 9% at 532 nm laser light illumination, which is comparable to a QE of 11% from GaAs bulk. In addition, a photoemission current of 10 µA was obtained from this photocathode. One more important point is that this photocathode could realize back-side illumination of 532 nm laser light, and thus its widespread applications are expected in microscopy and accelerator fields.

  4. Effects of combined growth of biogenic and xenobiotic substrates on degradation of xenobiotic by activated sludge

    Directory of Open Access Journals (Sweden)

    Thien Nguyen Phuc

    2017-01-01

    Full Text Available The purpose of this study was to research about supplementation of different concentrations of the substrate on the degradation rate of xenobiotic and to determine the optimal concentrations of the auxiliary substrates that are most beneficial of xenobiotic degradation rate. 2,4-dichlorophenol acid (2,4-D was used representative xenobiotic organic compounds, while peptone and sugar used for auxiliary substrates. The activated sludge was completely break down 100 mg/l of 2,4-D for three consecutive times. The different concentrations between biogenic substracts of sucrose and peptone were fed separately or combined into the medium containing 200 mg/l of 2,4-D and 140 mg SS/l of activated sludge. The results showed that sugar and peptone could affect 2,4-D degradation rate to several different degree at different concentrations. In separate supplementation, 2,4-D degradation completed within 25 hours, 40 mg/l sugar and 150 mg/l peptone concentrations were found to be the optimal concentrations. In combined case, 2,4-D was consumed totally within 20 hours and the optimal concentration of the combined sugar and peptone concentrations were 40 and 150 mg/l, respectively.

  5. Enhanced transparency, mechanical durability, and antibacterial activity of zinc nanoparticles on glass substrate

    Science.gov (United States)

    Choi, Hyung-Jin; Choi, Jin-Seok; Park, Byeong-Ju; Eom, Ji-Ho; Heo, So-Young; Jung, Min-Wook; An, Ki-Seok; Yoon, Soon-Gil

    2014-01-01

    Homogeneously distributed zinc nanoparticles (NPs) on the glass substrate were investigated for the transmittance, mechanical durability, and antibacterial effect. The buffered Ti NPs between Zn NPs and glass substrate were studied for an enhancement of the transmittance and mechanical endurance. The Ti NPs buffered Zn NPs showed a high transmittance of approximately 91.5% (at a wavelength of 550 nm) and a strong antibacterial activity for Staphylococcus aureus and Escherichia coli bacteria. The buffered Ti NPs are attractive for an excellent mechanical endurance of the Zn NPs. The Zn NPs did not require the protection layer to prevent the degradation of the performance for both the antibacterial effect and the transmittance. PMID:25183360

  6. Substrate activation of brewers' yeast pyruvate decarboxylase is abolished by mutation of cysteine 221 to serine.

    Science.gov (United States)

    Baburina, I; Gao, Y; Hu, Z; Jordan, F; Hohmann, S; Furey, W

    1994-05-10

    Brewers' yeast pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222. Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation [Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W. (1993) Biochemistry 32, 2704-2709], indicating that if Cys was responsible for this activation, it had to be C221. To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed. It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state. On the basis of the three-dimensional structure of the enzyme [Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F. (1993) Biochemistry 32, 6165-6170], it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains. In fact, residues in one or both domains must be dislocated by this adduct formation. It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    Science.gov (United States)

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

    2015-01-01

    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 solved 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 steric bulk and charge of the residue at position 200 appear capable of altering the rate-limiting step in catalysis, and perhaps, the nature of the reactive species. PMID:26267790

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

    Science.gov (United States)

    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.

  9. Generation and characterization of a highly effective protein substrate for analysis of FLT3 activity

    Directory of Open Access Journals (Sweden)

    Chen Yun

    2012-07-01

    Full Text Available Abstract Background Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in acute myeloid leukemia (AML. This has made FLT3 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to generate a sensitive substrate for assays of the FLT3 enzymatic activity. Methods We expressed in Escherichia coli cells a glutathione S-transferase (GST fusion protein designated GST-FLT3S, which contains a peptide sequence derived from an autophosphorylation site of FLT3. The protein was used to analyze tyrosine kinase activity of baculovirus-expressed FLT3 and crude cell extracts of bone marrow cells from AML patients. It was also employed to perform FLT3 kinase assays for FLT3 inhibitor screening. Results GST-FLT3S in solution or on beads was strongly phosphorylated by recombinant proteins carrying the catalytic domain of wild type FLT3 and FLT3D835 mutants, with the latter exhibiting much higher activity and efficiency. GST-FLT3S was also able to detect elevated tyrosine kinase activity in bone marrow cell extracts from AML patients. A small-scale inhibitor screening led to identification of several potent inhibitors of wild type and mutant forms of FLT3. Conclusions GST-FLT3S is a sensitive protein substrate for FLT3 assays. It may find applications in diagnosis of diseases related to abnormal FLT3 activity and in inhibitor screening for drug development.

  10. Glucose production and substrate cycle activity in a fasting adapted animal, the northern elephant seal.

    Science.gov (United States)

    Champagne, Cory D; Houser, Dorian S; Crocker, Daniel E

    2005-03-01

    During prolonged fasting physiological mechanisms defend lean tissue from catabolism. In the fasting state, glucose is derived solely from gluconeogenesis, requiring some catabolism of amino acids for gluconeogenic substrates. This creates a conflict in animals undergoing fasts concurrently with metabolically challenging activities. This study investigated glucose metabolism in fasting and developing neonatal elephant seals. Glucose production and glucose cycle activity were measured early (2 weeks) and late (6 weeks) in the postweaning fasting period. Additionally the role of regulatory hormones on glucose production and glucose cycle activity were investigated. Glucose cycle activity was highly variable throughout the study period, did not change over the fasting period, and was not correlated with insulin or glucagon level. Endogenous glucose production (EGP) was 2.80+/-0.65 mg kg(-1) min(-1) early and 2.21+/-0.12 during late fasting. Insulin to glucagon molar ratio decreased while cortisol levels increased over the fast (t=5.27, 2.84; P=0.003, 0.04; respectively). There was no relationship between EGP and hormone levels. The glucose production values measured in this study were high and exceeded the estimated gluconeogenic substrate available. These data suggest extensive glucose recycling via Cori cycle activity occurring in northern elephant seals, and we propose a possible justification for this recycling.

  11. Biofuel cell operating on activated THP-1 cells: A fuel and substrate study.

    Science.gov (United States)

    Javor, Kristina; Tisserant, Jean-Nicolas; Stemmer, Andreas

    2017-01-15

    It is known that electrochemical energy can be harvested from mammalian cells, more specifically from white blood cells (WBC). This study focuses on an improved biofuel cell operating on phorbol myristate acetate (PMA) activated THP-1 human monocytic cells. Electrochemical investigation showed strong evidence pointing towards hydrogen peroxide being the primary current source, confirming that the current originates from NADPH oxidase activity. Moreover, an adequate substrate for differentiation and activation of THP-1 cells was examined. ITO, gold, platinum and glass were tested and the amount of superoxide anion produced by NADPH oxidase was measured by spectrophotometry through WST-1 reduction at 450nm and used as an indicator of cellular activity and viability. These substrates were subsequently used in a conventional two-compartment biofuel cell where the power density output was recorded. The material showing the highest cell activity compared to the reference cell culture plate and the highest power output was ITO. Under our experimental conditions, a power density of 4.5μW/cm(2) was reached. To the best of our knowledge, this is a threefold higher power output than other leukocyte biofuel cells.

  12. Catalytic irreversible inhibition of bacterial and plant arginine decarboxylase activities by novel substrate and product analogues.

    Science.gov (United States)

    Bitonti, A J; Casara, P J; McCann, P P; Bey, P

    1987-02-15

    Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed.

  13. Blonanserin, a novel atypical antipsychotic agent not actively transported as substrate by P-glycoprotein.

    Science.gov (United States)

    Inoue, Tomoko; Osada, Kenichi; Tagawa, Masaaki; Ogawa, Yuriko; Haga, Toshiaki; Sogame, Yoshihisa; Hashizume, Takanori; Watanabe, Takashi; Taguchi, Atsushi; Katsumata, Takashi; Yabuki, Masashi; Yamaguchi, Noboru

    2012-10-01

    Although blonanserin, a novel atypical antipsychotic agent with dopamine D(2)/serotonin 5-HT(2A) antagonistic properties, displays good brain distribution, the mechanism of this distribution has not been clarified. P-glycoprotein [(P-gp) or multidrug resistance protein 1 (MDR1)] is an efflux transporter expressed in the brain and plays an important role in limiting drug entry into the central nervous system (CNS). In particular, P-gp can affect the pharmacokinetics and efficacy of antipsychotics, and exacerbate or soothe their adverse effects. In this study, we conducted in vitro and in vivo experiments to determine whether blonanserin is a P-gp substrate. Risperidone and its active metabolite 9-hydroxyrisperidone, both of which are P-gp substrates, were used as reference drugs. Affinity of blonanserin, risperidone, and 9-hydroxyrisperidone for P-gp was evaluated by in vitro transcellular transport across LLC-PK1, human MDR1 cDNA-transfected LLC-PK1 (LLC-MDR1), and mouse Mdr1a cDNA-transfected LLC-PK1 (LLC-Mdr1a). In addition, pharmacokinetic parameters in the brain and plasma (B/P ratio) of test compounds were measured in mdr1a/1b knockout (KO) and wild-type (WT) mice. The results of in vitro experiments revealed that P-gp does not actively transport blonanserin as a substrate in humans or mice. In addition, blonanserin displayed comparable B/P ratios in KO and WT mice, whereas B/P ratios of risperidone and 9-hydroxyrisperidone differed markedly in these animals. Our results indicate that blonanserin is not a P-gp substrate and therefore its brain distribution is unlikely to be affected by this transporter. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. LAT1 activity of carboxylic acid bioisosteres: Evaluation of hydroxamic acids as substrates.

    Science.gov (United States)

    Zur, Arik A; Chien, Huan-Chieh; Augustyn, Evan; Flint, Andrew; Heeren, Nathan; Finke, Karissa; Hernandez, Christopher; Hansen, Logan; Miller, Sydney; Lin, Lawrence; Giacomini, Kathleen M; Colas, Claire; Schlessinger, Avner; Thomas, Allen A

    2016-10-15

    Large neutral amino acid transporter 1 (LAT1) is a solute carrier protein located primarily in the blood-brain barrier (BBB) that offers the potential to deliver drugs to the brain. It is also up-regulated in cancer cells, as part of a tumor's increased metabolic demands. Previously, amino acid prodrugs have been shown to be transported by LAT1. Carboxylic acid bioisosteres may afford prodrugs with an altered physicochemical and pharmacokinetic profile than those derived from natural amino acids, allowing for higher brain or tumor levels of drug and/or lower toxicity. The effect of replacing phenylalanine's carboxylic acid with a tetrazole, acylsulfonamide and hydroxamic acid (HA) bioisostere was examined. Compounds were tested for their ability to be LAT1 substrates using both cis-inhibition and trans-stimulation cell assays. As HA-Phe demonstrated weak substrate activity, its structure-activity relationship (SAR) was further explored by synthesis and testing of HA derivatives of other LAT1 amino acid substrates (i.e., Tyr, Leu, Ile, and Met). The potential for a false positive in the trans-stimulation assay caused by parent amino acid was evaluated by conducting compound stability experiments for both HA-Leu and the corresponding methyl ester derivative. We concluded that HA's are transported by LAT1. In addition, our results lend support to a recent account that amino acid esters are LAT1 substrates, and that hydrogen bonding may be as important as charge for interaction with the transporter binding site. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Lipolytic activity of porcine pancreas lipase on fatty acid esters of dialkylglycerols: a structural basis for the design of new substrates for the assay of pancreatic lipases activity.

    Science.gov (United States)

    Ciuffreda, P; Loseto, A; Manzocchi, A; Santaniello, E

    2001-06-01

    For the design of new synthetic substrates for the assay of pancreatic lipases activity, acyl dialkylglycerols of variable chain length were prepared. Titrimetric assay of these substrates showed the highest lipolytic activity of porcine pancreas lipase (pPL) with butanoyl dibutylglycerol. The activity is lower but comparable to that shown by pPL towards the classical substrate tributyrin. The 4-nitrophenylcarbonate of 1,2-di-O-butylglycerol, has been prepared and proposed as synthetic substrate for a new spectrophotometric assay of pancreatic lipases.

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

    DEFF Research Database (Denmark)

    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. F...

  17. Changes in enzymes activity, substrate utilization pattern and diversity of soil microbial communities under cadmium pollution

    Institute of Scientific and Technical Information of China (English)

    Muhammad Akmal; WANG Hai-zhen; WU Jian-jun; XU Jian-ming; XU De-fu

    2005-01-01

    Heavy metal pollution has received increasing attention in recent years mainly because of the public awareness of environmental issues. In this study we have evaluated the effect of cadmium(Cd) on enzymes activity, substrate utilization pattern and diversity of microbial communities in soil spiked with 0, 20, 40, 60, 80, and 100 mg/kg Cd, during 60 d of incubation at 25℃. Enzyme activities determined at 0, 15, 30, 45, and 60 d after heavy metal application(DAA) showed marked declines for various Cd treatments, and up to 60 DAA, 100 mg/kg Cd resulted in 50.1%, 47.4%, and 39.8 % decreases in soil urease, acid phosphatase and dehydrogenase activities,respectively to control. At 60 DAA, substrate utilization pattern of soil microbial communities determined by inoculating Biolog ECO plates indicated that Cd addition had markedly inhibited the functional activity of soil microbial communities and multivariate analysis of sole carbon source utilization showed significantly different utilization patterns for 80 and 100 mg/kg Cd treatments. The structural diversity of soil microbial communities assessed by PCR-DGGE method at 60 DAA, illustrated that DGGE patterns in soil simplified with increasing Cd concentration, and clustering of DGGE profiles for various Cd treatments revealed that they had more than 50% difference with that of control.

  18. Characterization of phytase activity from cultivated edible mushrooms and their production substrates.

    Science.gov (United States)

    Collopy, Patrick D; Royse, Daniel J

    2004-12-15

    Phytase is used commercially to maximize phytic acid degradation and to decrease phosphorus levels in poultry and swine manure. To determine phytase content in edible mushrooms, basidiomata of Agaricus bisporus and three specialty mushrooms (Grifola frondosa, Lentinula edodes, and Pleurotus cornucopiae) and spent mushroom substrate (SMS) were surveyed. Enzyme activity ranged from 0.046 to 0.074 unit/g of tissue for four A. bisporus types (closed and open whites and closed and open browns) grown at The Pennsylvania State University's Mushroom Test Demonstration Facility (MTDF). The addition of various nutrient supplements to phase II mushroom production substrate did not alter phytase activity in A. bisporus. Portabella mushrooms (open brown) obtained from a commercial farm had significantly higher levels of phytase activity (0.211 unit/g of tissue) compared to A. bisporus grown at the MTDF. Of the specialty mushrooms surveyed, maitake (G. frondosa) had 20% higher phytase activity (0.287 unit/g of tissue) than commercial portabella mushrooms. The yellow oyster mushroom (P. cornucopiae) ranked second in level of phytase activity (0.213 unit/g of tissue). Shiitake (L. edodes) contained the least amount of phytase in basidiomata (0.107 unit/g of tissue). Post-crop steam treatment (60 degrees C, 24 h) of SMS reduced phytase activity from 0.074 to 0.018 unit/g. Phytase was partially purified from commercially grown portabella basidiomata 314-fold with an estimated molecular mass of 531 kDa by gel filtration chromatography. The optimum pH for activity was 5.5, but appreciable phytase activity was observed over the range of pH 5.0-8.0. Partially purified A. bisporus phytase was inactivated following a 10-min incubation at > or =60 degrees C.

  19. SfDredd, a Novel Initiator Caspase Possessing Activity on Effector Caspase Substrates in Spodoptera frugiperda.

    Directory of Open Access Journals (Sweden)

    Zhouning Yang

    Full Text Available Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells.

  20. SfDredd, a Novel Initiator Caspase Possessing Activity on Effector Caspase Substrates in Spodoptera frugiperda.

    Science.gov (United States)

    Yang, Zhouning; Zhou, Ke; Liu, Hao; Wu, Andong; Mei, Long; Liu, Qingzhen

    2016-01-01

    Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells.

  1. Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

    Science.gov (United States)

    Ichibangase, Tomoko; Ohba, Yoshihito; Kishikawa, Naoya; Nakashima, Kenichiro; Kuroda, Naotaka

    2004-01-01

    A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of 4.7% for within-day and 6.0% for between-day assays.

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

    Science.gov (United States)

    Longtin, Rémi; Sanchez-Valencia, Juan Ramon; Shorubalko, Ivan; Furrer, Roman; Hack, Erwin; Elsener, Hansrudolf; Gröning, Oliver; Greenwood, Paul; Rupesinghe, Nalin; Teo, Kenneth; Leinenbach, Christian; Gröning, Pierangelo

    2015-02-01

    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.

  3. MMP-9 and MMP-2 activities in stomach and breast tumours, as measured by a novel MMP activity assay using modified urokinase as a substrate

    NARCIS (Netherlands)

    Hanemaaijer, R.; Visser, H.; Duffy, J.; Verspaget, H.W.; Verheijen, J.H.; Maguire, T.

    1998-01-01

    Matrix metalloproteinases (MMPs) play an important role in many pathological processes. However, MMP activities are difficult to determine since no simple specific and/or chromogenic substrates exist. Therefore, we have developed a novel MMP activity assay using a modified urokinase as a substrate.

  4. Investigation of photocatalytic activity of titanium dioxide coating deposited on aluminium alloy substrate by plasma technique

    DEFF Research Database (Denmark)

    Daviðsdóttir, Svava; Soyama, Juliano; Dirscherl, Kai

    2011-01-01

    . 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...... of the coating strongly influences the photocatalytic properties. In general, the photocatalytic activity increased with thickness. Quantification of images scanned with Atomic Force Microscope (AFM) revealed that there is a linear relationship between the thickness of the coating and the average cell size...

  5. Silver nanoparticles deposited on porous silicon as a surface-enhanced Raman scattering (SERS) active substrate.

    Science.gov (United States)

    Zeiri, Leila; Rechav, Katya; Porat, Ze'ev; Zeiri, Yehuda

    2012-03-01

    Silver nanoparticles were deposited spontaneously from their aqueous solution on a porous silicon (PS) layer. The PS acts both as a reducing agent and as the substrate on which the nanoparticles nucleate. At higher silver ion concentrations, layers of nanoparticle aggregates were formed on the PS surface. The morphology of the metallic layers and their SERS activity were influenced by the concentrations of the silver ion solutions used for deposition. Raman measurements of rhodamine 6G (R6G) and crystal violet (CV) adsorbed on these surfaces showed remarkable enhancement of up to about 10 orders of magnitude.

  6. Collective transport for active matter run-and-tumble disk systems on a traveling-wave substrate

    Science.gov (United States)

    Sándor, Cs.; Libál, A.; Reichhardt, C.; Reichhardt, C. J. Olson

    2017-01-01

    We examine numerically the transport of an assembly of active run-and-tumble disks interacting with a traveling-wave substrate. We show that as a function of substrate strength, wave speed, disk activity, and disk density, a variety of dynamical phases arise that are correlated with the structure and net flux of disks. We find that there is a sharp transition into a state in which the disks are only partially coupled to the substrate and form a phase-separated cluster state. This transition is associated with a drop in the net disk flux, and it can occur as a function of the substrate speed, maximum substrate force, disk run time, and disk density. Since variation of the disk activity parameters produces different disk drift rates for a fixed traveling-wave speed on the substrate, the system we consider could be used as an efficient method for active matter species separation. Within the cluster phase, we find that in some regimes the motion of the cluster center of mass is in the opposite direction to that of the traveling wave, while when the maximum substrate force is increased, the cluster drifts in the direction of the traveling wave. This suggests that swarming or clustering motion can serve as a method by which an active system can collectively move against an external drift.

  7. Energy substrate utilization during nightly vocal activity in three species of Scinax (Anura/Hylidae).

    Science.gov (United States)

    Carvalho, José Eduardo; Gomes, Fernando Ribeiro; Navas, Carlos Arturo

    2008-05-01

    Anuran amphibians exhibit different patterns of energy substrate utilization that correlate with the intensity of vocal and locomotor activities. Given the remarkable differences among species in breeding and feeding strategies, and the different ways energy is used in the whole animal, the suggested correlations between calling and locomotor behavior and the level of energy substrates in the muscles responsible for such activities are more complex than previously reported. We explored the relationships between calling and locomotor behavior and energy supply to trunk and hindlimb muscles, respectively, within the ecologically diverse tree-frog genus Scinax. Specifically, we measured the relative amount of carbohydrates and lipids in these two groups of muscles, and in the liver of three species of Scinax that differ in vocal and locomotor performance, and compared our results with those of two other species for which comparable data are available. We also compared the contents of lipids and carbohydrates of conspecific males collected at the beginning and after 4 h of calling activity. The stomach content to potential feeding opportunities across species was also assessed in both groups of males. Scinax hiemalis and S. rizibilis exhibit comparatively low and episodic calling during long periods of activity whereas S. crospedospilus calls at higher rates over shorter periods. Male S. hiemalis had highest levels of trunk muscle glycogen followed by those of S. rizilbilis and S. crospedospilus, respectively. There was no correlation between total lipid content in trunk muscle and calling rate among different species, suggesting that other metabolic aspects may be responsible for the energetic support for vocal activity. The levels of lipids and carbohydrates in trunk and hindlimb muscles and liver of males collected at the beginning and 4 h into the calling period were similar across species, so the extent of energetic reserves does not appear to constrain vocal or

  8. Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity

    National Research Council Canada - National Science Library

    Mark A. Tarnopolsky; Courtney D. Rennie; Holly A. Robertshaw; Stephanie N. Fedak-Tarnopolsky; Michaela C. Devries; Mazen J. Hamadeh

    .... We examined whether endurance exercise training and sex influenced IMCL and mitochondrial morphology using electron microscopy, whole-body substrate use, and mitochondrial enzyme activity. Untrained men (n = 5) and women (n = 7...

  9. Determinación simultánea del fenotipo para las enzimas citocromo p450 1a2, 2d6, n-acetiltranferasa y xantina oxidasa en una población colombiana

    Directory of Open Access Journals (Sweden)

    Jesualdo Fuentes

    2001-04-01

    Full Text Available Las enzimas responsables del metabolismo de los fármacos presentan variabilidad en su actividad en diferentes poblaciones; ésta es una de las causas de la mayoría de las fallas terapéuticas. Las enzimas citocrómicas participan en el metabolismo del 90% de
    los fármacos en uso, razón por la cual se hace indispensable conocer su capacidad metabólica, puesto que han sido reportadas por ser altamente variables debido a la presencia de polimorfismos (1. De igual forma, enzimas como la NAT-2 han sido reportadas como polimórficas. El objetivo de este trabajo es determinar en una población de colombianos sanos, la actividad de las enzimas CYP1A2, NAT-2 y XO, de manera simultánea, empleando cafeína como fármaco prueba, así como a la enzima CYP2D6 en individuos de la misma población.

  10. Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes [v1; ref status: indexed, http://f1000r.es/4zj

    Directory of Open Access Journals (Sweden)

    Ron Ammar

    2015-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract.

    Science.gov (United States)

    Baik, Joo Hyun; Suh, Hyung Joo; Cho, So Young; Park, Yooheon; Choi, Hyeon-Son

    2014-11-01

    Tannases are important enzymes in the antioxidant potential of tea leaves. In this study, we evaluated the effect of two tannases (T1 and T2) on biotransformation of tea polyphenols and antioxidative activities from catechins in green tea extract (GTE). The T1 tannase-catalyzed reaction was inhibited by the addition of >2.0% GTE substrate, whereas the T2-catalyzed reaction was not inhibited, even by addition of 5.0% GTE. Furthermore, the T1 tannase-catalyzed reaction was inhibited by addition of 10 mg mL(-1) EGCG, whereas the T2 tannase-catalyzed reaction did not display any inhibitory effect. These results indicate that T2 tannase was more tolerant than T1 tannase to substrate inhibition in degallation reactions. Specifically, the substrate EGCG (90,687.1 μg mL(-1)) was transformed into gallic acid (50,242.9 μg mL(-1)) and EGC (92,598.3 μg mL(-1)) after 1-h treatment with T2 tannase (500 U g(-1)). The tannase-mediated product displayed higher in vitro radical-scavenging activity than the control. IC50 value of GTE on ABTS and DPPH radicals (46.1 μg mL(-1) and 18.4 μg mL(-1), respectively) decreased markedly after T2 tannase treatment (to 35.8 μg mL(-1) and 15.1 μg mL(-1), respectively). These results indicate that T2 tannase treatment of GTE enhanced its radical-scavenging activity, an increase that was also observed in the reaction using EGCG substrate. Taken together, our results revealed that T2 tannase is more suitable for biotransformation of catechins in GTE than T1 tannase, and T2 treatment provides an enhanced radical-scavenging effect.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Ag-SiO2 Janus particles based highly active SERS macroscopic substrates

    Science.gov (United States)

    Panwar, Kamlesh; Jassal, Manjeet; Agrawal, Ashwini K.

    2017-07-01

    Macroscopic SERS (M-SERS) substrate with high activity was fabricated by attaching Ag-SiO2 Janus particles with ∼3 nm sized silver nanoparticles (AgNPs) on a cellulosic film. The synthesized Ag-SiO2 Janus particles had in-situ deposited AgNPs on one-half and amine functionalization on the other-half, which was utilized for their fixation as a monolayer on cellulosic substrate. Raman spectrum of Rhodamine B (RhB) showed an enhancement factor of ∼5 × 104 for dye tagged on both Janus particles and M-SERS. In comparison, AgNPs with almost the same diameter, even after aggregation, did not show enhancement of Raman signals. The enhancement is attributed to in-situ reduction of silver precursor on the SiO2 surface leading to deposition of AgNPs and generation of a large number of fixed hot-spots in a controlled manner. The Janus particles and M-SERS with ∼3 nm sized AgNPs can find application as both SERS active as well as catalytic material.

  15. Directed enzyme evolution guided by multidimensional analysis of substrate-activity space.

    Science.gov (United States)

    Larsson, Anna-Karin; Emrén, Lars O; Bardsley, William G; Mannervik, Bengt

    2004-01-01

    The directed evolution of protein function frequently involves identification of mutants with improved properties from a population of variants obtained by mutagenesis. The selection of clones to parent the subsequent generation is crucial to the continued creation of superior progeny. In the present study, multivariate analysis guided the evolution of human glutathione transferase (GST) T1-1 to 65-fold enhanced alkyltransferase activity. Six alternative substrates monitored the substrate-activity space that characterized a mutant library of enzymes, obtained by recombination of DNA and heterologous expression in Escherichia coli. A subset of mutants was identified by their proximity in the targeted region of six-dimensional factor space. DNA from these mutants was recombined to create a new generation of GST variants from which an improved enzyme was isolated. The multidimensional cluster analysis is applicable to quantitative properties in any population of molecules undergoing evolution and can guide the tailoring of proteins, nucleic acids and other chemical structures to novel and improved functions.

  16. Directed evolution of a histone acetyltransferase--enhancing thermostability, whilst maintaining catalytic activity and substrate specificity.

    Science.gov (United States)

    Leemhuis, Hans; Nightingale, Karl P; Hollfelder, Florian

    2008-11-01

    Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of opposing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P/CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from mutations predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein's surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers.

  17. Activation energy of thermal desorption of silicon oxide layers on silicon substrates

    Science.gov (United States)

    Enta, Yoshiharu; Osanai, Shodai; Ogasawara, Takahito

    2017-02-01

    Thermal desorption rates of silicon oxide layers, from 20 to 120 nm in thickness, on silicon substrates in vacuum have been accurately obtained from intervals between ring structures formed inside voids on the oxide layers. From the temperature dependence of the desorption rate, the activation energy and frequency factor of the desorption reaction have been derived as a function of the oxide thickness. The obtained values are compared with the previous studies, and as a result, the activation energy is found to be almost constant ( 4 eV) in a wide range of the oxide thickness. The frequency factor decreases as the inverse square of the oxide thickness. The decomposition kinetics of the oxide layer is also discussed from the obtained results.

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

    OpenAIRE

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

    2015-01-01

    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 (homo...

  19. Carbohydrate Bis-acetal-Based Substrates as Tunable Fluorescence-Quenched Probes for Monitoring exo-Glycosidase Activity.

    Science.gov (United States)

    Cecioni, Samy; Vocadlo, David J

    2017-06-28

    Tunable Förster resonance energy transfer (FRET)-quenched substrates are useful for monitoring the activity of various enzymes within their relevant physiological environments. Development of FRET-quenched substrates for exo-glycosidases, however, has been hindered by their constrained pocket-shaped active sites. Here we report the design of a new class of substrate that overcomes this problem. These Bis-Acetal-Based Substrates (BABS) bear a hemiacetal aglycon leaving group that tethers fluorochromes in close proximity, also positioning them distant from the active site pocket. Following cleavage of the glycosidic bond, the liberated hemiacetal spontaneously breaks down, leading to separation of the fluorophore and quencher. We detail the synthesis and characterization of GlcNAc-BABS, revealing a striking 99.9% quenching efficiency. These substrates are efficiently turned over by the human exo-glycosidase O-GlcNAcase (OGA). We find the hemiacetal leaving group rapidly breaks down, enabling quantitative monitoring of OGA activity. We expect this strategy to be broadly useful for the development of substrate probes for monitoring exo-glycosidases, as well as a range of other enzymes having constrained pocket-shaped active sites.

  20. Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression

    Directory of Open Access Journals (Sweden)

    Takahiro Ebata

    2017-01-01

    Full Text Available The physical properties of the extracellular matrix (ECM, such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin. Cell growth inhibition by doxorubicin was increased in response to ECM rigidity in a p53-dependent manner. The expression of Rho-associated coiled coil-containing protein kinase (ROCK 2, which induces the activation of myosin II, was significantly higher when cells were cultured on stiffer ECM substrates. Knockdown of ROCK2 expression or pharmacological inhibition of ROCK decreased doxorubicin-induced p53 activation. Our results suggest that a soft ECM causes downregulation of ROCK2 expression, which drives resistance to chemotherapy by repressing p53 activation.

  1. Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

    Science.gov (United States)

    Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

    2011-08-15

    Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.

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

    Science.gov (United States)

    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.

  3. Evidence that the RNAseH activity of the duck hepatitis B virus is unable to act on exogenous substrates

    Directory of Open Access Journals (Sweden)

    Tavis John E

    2001-07-01

    Full Text Available Abstract Background The hepadnaviral reverse transcriptase can synthesize DNA on its native RNA template within viral cores but it is usually unable to synthesize DNA employing exogenous nucleic acids as a template. The mechanism of this template commitment is unknown. Here we provide evidence that the RNAseH activity of duck hepatitis B virus reverse transcriptase may also be unable to act on exogenous substrates. Results RNAseH assays were performed under a wide variety of conditions employing substrate RNAs of Duck Hepatitis B Virus sequence annealed to complementary DNA oligonucleotides and permeabilized intracellular viral core particles. Temperature, pH, cation type, salt concentration, substrate concentration, and the sequences of the cleavage sites were varied, and the effects of ATP and dNTPs on RNAseH activity were examined. duck hepatitis B virus RNAseH activity was not detected under any of these conditions, although E. coli or Avian Myeloblastosis Virus RNAseH activity could be detected under all conditions. Access of the RNA substrate to the enzyme within the viral cores was confirmed. Conclusions These results imply that the RNAseH activity of the DHBV reverse transcriptase may not be able to degrade exogenous RNA:DNA heteroduplexes, although it can degrade heteroduplexes of the same sequence generated during reverse transcription of the endogenous RNA template. Therefore, the RNAseH activity appears to be "substrate committed" in a manner similar to the template commitment observed for the DNA polymerase activity.

  4. Substrate Utilization is Influenced by Acute Dietary Carbohydrate Intake in Active, Healthy Females.

    Science.gov (United States)

    Gregory, Sara; Wood, Richard; Matthews, Tracey; Vanlangen, Deborah; Sawyer, Jason; Headley, Samuel

    2011-01-01

    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. Key pointsThe relative carbohydrate content of a single meal has a significant impact on postprandial metabolism and substrate utilization in healthy, active females.A single bout of aerobic exercise performed within an hour of meal ingestion has the potential to modify the postprandial response.Interventions aimed at improving body composition and preventing chronic disease should focus on dietary

  5. Proteinase K activity determination with β-galactosidase as sensitive macromolecular substrate.

    Science.gov (United States)

    Ghéczy, Nicolas; Küchler, Andreas; Walde, Peter

    2016-11-15

    Proteinase K from Engyodontium album (proK) is a relatively unspecific serine endopeptidase which is known to attack proteins yet in their native states. If the attacked protein is an enzyme, even a partial hydrolysis by proK may lead to an inactivation of the enzyme, which can be monitored by measuring the loss of catalytic activity of the attacked enzyme. E. coli β-galactosidase (β-Gal) was used in this work as such enzyme. It was found to be a convenient and sensitive macromolecular model substrate for comparing the "native protein-attacking ability" of free and immobilized proK at pH = 7.0 and 23 °C. The β-Gal activity was measured spectrophotometrically with o-nitrophenyl-β-galactopyranoside. Reproducible proK determinations were possible for as little as 4.3 ng proK by using a proK analyte solution of 10 nM. Compared to free proK, immobilized proK was much less efficient in inactivating β-Gal, most likely due to a decreased mobility of immobilized proK and a restricted accessibility of β-Gal to the active site of proK. Worth noting is, that under conditions at which β-Gal was completely inactivated by proK, the activity of hen egg lysozyme, horseradish peroxidase, or Aspergillus sp. glucose oxidase remained unaltered.

  6. Graphdiyne oxides as excellent substrate for electroless deposition of Pd clusters with high catalytic activity.

    Science.gov (United States)

    Qi, Hetong; Yu, Ping; Wang, Yuexiang; Han, Guangchao; Liu, Huibiao; Yi, Yuanping; Li, Yuliang; Mao, Lanqun

    2015-04-29

    Graphdiyne (GDY), a novel kind of two-dimensional carbon allotrope consisting of sp- and sp(2)-hybridized carbon atoms, is found to be able to serve as the reducing agent and stabilizer for electroless deposition of highly dispersed Pd nanoparticles owing to its low reduction potential and highly conjugated electronic structure. Furthermore, we observe that graphdiyne oxide (GDYO), the oxidation form of GDY, can be used as an even excellent substrate for electroless deposition of ultrafine Pd clusters to form Pd/GDYO nanocomposite that exhibits a high catalytic performance toward the reduction of 4-nitrophenol. The high catalytic performance is considered to benefit from the rational design and electroless deposition of active metal catalysts with GDYO as the support.

  7. Influence of protonation on substrate and inhibitor interactions at the active site of human monoamine oxidase-A.

    Science.gov (United States)

    Zapata-Torres, Gerald; Fierro, Angelica; Miranda-Rojas, Sebastian; Guajardo, Carlos; Saez-Briones, Patricio; Salgado, J Cristian; Celis-Barros, Cristian

    2012-05-25

    Although substrate conversion mediated by human monoaminooxidase (hMAO) has been associated with the deprotonated state of their amine moiety, data regarding the influence of protonation on substrate binding at the active site are scarce. Thus, in order to assess protonation influence, steered molecular dynamics (SMD) runs were carried out. These simulations revealed that the protonated form of the substrate serotonin (5-HT) exhibited stronger interactions at the protein surface compared to the neutral form. The latter displayed stronger interactions in the active site cavity. These observations support the possible role of the deprotonated form in substrate conversion. Multigrid docking studies carried out to rationalize the role of 5-HT protonation in other sites besides the active site indicated two energetically favored docking sites for the protonated form of 5-HT on the enzyme surface. These sites seem to be interconnected with the substrate/inhibitor cavity, as revealed by the tunnels observed by means of CAVER program. pK(a) calculations in the surface loci pointed to Glu³²⁷, Asp³²⁸, His⁴⁸⁸, and Asp¹³² as candidates for a possible in situ deprotonation step. Docking analysis of a group of inhibitors (structurally related to substrates) showed further interactions with the same two docking access sites. Interestingly, the protonated/deprotonated amine moiety of almost all compounds attained different docking poses in the active site, none of them oriented to the flavin moiety, thus producing a more variable and less productive orientations to act as substrates. Our results highlight the role of deprotonation in facilitating substrate conversion and also might reflect the necessity of inhibitor molecules to adopt specific orientations to achieve enzyme inhibition.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Substrate-bound Structures of Benzylsuccinate Synthase Reveal How Toluene Is Activated in Anaerobic Hydrocarbon Degradation*

    Science.gov (United States)

    Funk, Michael A.; Marsh, E. Neil G.; Drennan, Catherine L.

    2015-01-01

    Various bacteria perform anaerobic degradation of small hydrocarbons as a source of energy and cellular carbon. To activate non-reactive hydrocarbons such as toluene, enzymes conjugate these molecules to fumarate in a radical-catalyzed, C—C bond-forming reaction. We have determined x-ray crystal structures of the glycyl radical enzyme that catalyzes the addition of toluene to fumarate, benzylsuccinate synthase (BSS), in two oligomeric states with fumarate alone or with both substrates. We find that fumarate is secured at the bottom of a long active site cavity with toluene bound directly above it. The two substrates adopt orientations that appear ideal for radical-mediated C—C bond formation; the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSSβ, a small protein subunit that forms a tight complex with BSSα, the catalytic subunit. Enzymes related to BSS are able to metabolize a wide range of hydrocarbons through attachment to fumarate. Using our structures as a guide, we have constructed homology models of several of these “X-succinate synthases” and determined conservation patterns that will be useful in understanding the basis for catalysis and specificity in this family of enzymes. PMID:26224635

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

    DEFF Research Database (Denmark)

    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......A was dramatically altered in Delta ptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets.......-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...

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

    Science.gov (United States)

    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.

  12. Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.

    Science.gov (United States)

    Leight, Jennifer L; Alge, Daniel L; Maier, Andrew J; Anseth, Kristi S

    2013-10-01

    Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated degradation in these materials has remained challenging. There are 20+ MMP family members whose activity is regulated in space and time by a number of biochemical and biophysical cues. Thus, the typical approach of characterizing cleavage of degradable moieties in solution with recombinant enzymes does not easily translate to three-dimensional cell-mediated matrix remodeling. To address this challenge, we report here the synthesis of a cell-laden hydrogel matrix functionalized with a fluorogenic peptide substrate to provide real-time, quantitative monitoring of global MMP activity. Using this system, stimulation of MMP activity was observed with growth factor treatment in mammary epithelial cells and compared to classical zymography results. Further, the effect of biophysical cues on MMP activity of human mesenchymal stem cells was also investigated where more rigid hydrogels were observed to increase MMP activity. The regulation of MMP activity by these biochemical and biophysical cues highlights the need for in situ, real-time measurement of hydrogel degradation, and use of these functionalized hydrogels will aid in future rational design of degradable synthetic hydrogels for in vitro cell studies and tissue engineering applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. A Study on Recycling of Spent Mushroom Substrate to Prepare Chars and Activated Carbon

    Directory of Open Access Journals (Sweden)

    Yuhui Ma

    2014-05-01

    Full Text Available Chars were obtained from spent mushroom substrate (SMS via pyrolysis. It was found that as the pyrolysis temperature increased from 400 to 700 °C, the char yield decreased from 45.10 to 33.79 wt.% and the higher heating value increased from 17.32 to 22.72 MJ/kg. The largest BET surface area (13 m2/g was created at 500 °C. Hydrogen atoms were continuously lost during pyrolysis, whereas oxygen atoms were difficult to eliminate. Whewellite, calcite, lime, and quartz were the minerals in the chars, and their forms and crystallinity changed with changing pyrolysis temperature. Activated carbon with a BET surface area of 1023 m2/g and a total pore volume of 0.595 cm3/g was obtained from the char prepared at 500 °C. Its characteristics were studied by N2-adsorption, Fourier transform infrared spectroscopy (FTIR, and X-ray diffraction (XRD. The pyrolysis and KOH-activation processes were investigated by thermogravimetric analysis (TGA. The results showed that the pyrolysis of SMS occurred primarily between 217 and 375 °C and that the energies needed for the pyrolysis reactions were relatively low due to the prior mushroom cultivation. Furthermore, lignin was incompletely decomposed in the char prepared at 500 °C, and KOH suppressed tar evolution and reduced the energy needed to decompose the residual lignin during activation.

  14. A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

    Science.gov (United States)

    Loch, Christian M; Strickler, James E

    2012-11-01

    Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

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

    Science.gov (United States)

    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}

    Energy Technology Data Exchange (ETDEWEB)

    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. Modulation of PC1/3 activity by self-interaction and substrate binding.

    Science.gov (United States)

    Hoshino, Akina; Kowalska, Dorota; Jean, François; Lazure, Claude; Lindberg, Iris

    2011-04-01

    Prohormone convertase (PC)1/3 is a eukaryotic serine protease in the subtilase family that participates in the proteolytic maturation of prohormone and neuropeptide precursors such as proinsulin and proopiomelanocortin. Despite the important role of this enzyme in peptide synthesis, how PC1/3 activity is regulated is still poorly understood. Using ion exchange chromatography and two-dimensional gel electrophoresis we found that natural PC1/3 present in AtT-20 cells and bovine chromaffin granules, as well as recombinant PC1/3 secreted from overexpressing Chinese hamster ovary cells, exists as multiple ionic forms. Gel filtration and cross-linking studies revealed that protein oligomerization and aggregation contribute greatly to variability in surface charge. The most acidic forms of PC1/3 contained both inactive aggregates as well as oligomerized 87-kDa PC1/3 that exhibited stable activity which was partially latent and could be revealed by dilution. No such latency was observed for the more basic, 66/74-kDa forms of PC1/3. Fractions containing these species were stabilized by preincubation with micromolar concentrations of either fluorogenic substrate or peptides containing pairs of basic residues. In addition, the most active form of 87-kDa PC1/3, a probable homodimer, was activated by preincubation with these same peptides. Cleavage by PC1/3 is often the initiating step in the biosynthetic pathway for peptide hormones, implying that this is a natural step for regulation. Our data suggest that enzyme oligomerization and peptide stabilization represent important contributing factors for the control of PC1/3 activity within secretory granules.

  18. A Rational Design Strategy for the Selective Activity Enhancement of a Molecular Chaperone toward a Target Substrate.

    Science.gov (United States)

    Aprile, Francesco A; Sormanni, Pietro; Vendruscolo, Michele

    2015-08-18

    Molecular chaperones facilitate the folding and assembly of proteins and inhibit their aberrant aggregation. They thus offer several opportunities for biomedical and biotechnological applications, as for example they can often prevent protein aggregation more effectively than other therapeutic molecules, including small molecules and antibodies. Here we present a method of designing molecular chaperones with enhanced activity against specific amyloidogenic substrates while leaving unaltered their functions toward other substrates. The method consists of grafting onto a molecular chaperone a peptide designed to bind specifically an epitope in the target substrate. We illustrate this strategy by describing Hsp70 variants with increased affinities for α-synuclein and Aβ42 but otherwise unaltered affinities for other substrates. These designed variants inhibit protein aggregation and disaggregate preformed fibrils significantly more effectively than wild-type Hsp70 indicating that the strategy presented here provides a possible route for tailoring rationally molecular chaperones for specific purposes.

  19. Rapid shifts in Atta cephalotes fungus-garden enzyme activity after a change in fungal substrate (Attini, Formicidae)

    DEFF Research Database (Denmark)

    Kooij, P W; Schiøtt, M; Boomsma, J J

    2011-01-01

    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......, 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...... from the rice diet, relative to the leaf diet controls. Enzyme activity in the older, bottom sections of fungus gardens decreased, indicating a faster processing of the rice substrate compared to the leaf diet. These results suggest that leaf-cutting ant fungus gardens can rapidly adjust enzyme...

  20. Impact of carrier material on fermentative activity of encapsulated yoghurt culture in whey based substrate

    Directory of Open Access Journals (Sweden)

    Krunić Tanja Ž.

    2017-01-01

    Full Text Available The main objectives of this paper were to study the influence of the carrier material used for encapsulation and of bead size to fermentative activity and viability of the dairy starter culture ‘Lactoferm ABY 6’. Encapsulation of yoghurt culture in beads with diameter of 1mm provides better results than encapsulation in beads with larger diameter. Alginate beads and chitosan coated beads have proved to be a strong barrier for nutrients from substrate, so samples with those beads have lower viable cell count, lower titratable acidity and higher pH value after 5h of fermentation at 42°C, than samples with WPC-alginate beads. Also those beads have significantly (P < 0.05 lower cell leaking, than WPC-alginate beads and lower antioxidant capacity. Encapsulation of yoghurt culture in WPC-alginate carrier with diameter of approximately 1mm provided the best characteristics for fermented product. Samples with these beads have significantly (P < 0.05 higher increase of viable cell number after fermentation, despite of major cell leaking (19.7 %. Moreover, sample with these beads have the highest titratable acidity, the lowest pH value after fermentation (the best fermentative activity and the best antioxidant characteristics. [Projekat Ministarstva nauke Republike Srbije, br. TR 31017 i br. III 46010

  1. Holocellulase activity from Schizophyllum commune grown on bamboo: a comparison with different substrates.

    Science.gov (United States)

    Arboleda Valencia, Jorge William; Valencia Jiménez, Arnubio; Gonçalves de Siqueira, Félix; Dussan Medina, Kelly; Restrepo Franco, Gloria M; Filho, Edivaldo Ximenes Ferreira; Siegfried, Blair D; Grossi-de-Sa, Maria Fatima

    2011-12-01

    The natural biodiversity that is found in tropical areas offers countless biotechnological opportunities; especially if we take in account that many biomolecules from several microorganisms have supported for many years, different industrial applications in areas such as pharmacology, agro-industry, bioprocess, environmental technology, and bioconversion. In order to find new lignocellulolytic enzymes and evaluate bamboo fibers as substrate, Schizophyllum commune a fungus with broad distribution was isolated and grown during 15 days in liquid culture medium containing 1% lignocellulosic fibers from bamboo, banana stem, and sugarcane bagasse. The enzymatic activity of xylanase, mannanase, polygalacturonase, CMCase, FPase, and avicelase were evaluated. Sugarcane bagasse and banana stem showed to induce higher hollocellulase activity when compared with bamboo as the main carbon source. The physical mechanism that the fungus uses to degrade bamboo was observed not only in fibers naturally infected but also in healthy fibers that were treated and untreated with enzyme solution. SEM analysis showed the structural disruption and invasion of the vascular bundles, parenchyma cells, and parenchymatous tissues as a consequence of the presence of this fungus and the catalytic action of its enzymes into the plant tissue.

  2. Active Mode Remote Infrared Spectroscopy Detection of TNT and PETN on Aluminum Substrates

    Directory of Open Access Journals (Sweden)

    John R. Castro-Suarez

    2017-01-01

    Full Text Available Two standoff detection systems were assembled using an infrared telescope coupled to a Fourier transform infrared spectrometer, a cryocooled mercury-cadmium telluride detector, and a telescope-coupled midinfrared excitation source. Samples of the highly energetic materials (HEMs 2,4,6-trinitrotoluene (TNT and pentaerythritol tetranitrate (PETN were deposited on aluminum plates and detected at several source-target distances by carrying out remote infrared spectroscopy (RIRS measurements on the aluminum substrates in active mode. The samples tested were placed at 1–30 m for the RIRS detection experiments. The effect of the angle of incidence/collection of the IR beams on the vibrational band intensities and the signal-to-noise ratios (S/N were investigated. Experiments were performed at ambient temperature. Surface concentrations from 50 to 400 μg/cm2 were studied. Partial least squares regression analysis was applied to the spectra obtained. Overall, RIRS detection in active mode was useful for quantifying the HEMs deposited on the aluminum plates with a high confidence level up to the target-collector distances of 1–25 m.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Evaluation of organic substrates to enhance the sulfate-reducing activity in phosphogypsum.

    Science.gov (United States)

    Castillo, Julio; Pérez-López, Rafael; Sarmiento, Aguasanta M; Nieto, José M

    2012-11-15

    Several experiments were conducted to evaluate the activity and growth of sulfate-reducing bacteria (SRB) in a metal-rich culture medium (approx. 250 mg/L Fe, 75 mg/L Zn and Cu, 10mg/L Cd) with phosphogypsum as bacterial inoculum. Phosphogypsum was collected from the stack covering the salt-marshes of the Tinto river (SW Spain). Three organic amendments were used as carbon sources, two low-cost wastes (horse manure and legume compost) and one sample of natural soil (vegetal cover). In the experiments, sulfate was reduced to sulfide during the growth of SRB populations, and concentrations were decreased in the solution. Metal concentrations also decreased to values below the detection limit. Metal removal took place by precipitation of newly-formed sulfides. Pyrite-S was the main sulfide component (approx. 200 μmol/g and 80% of pyritization) and occurred mainly as framboidal grains and rarely as isolated polyhedral crystals. Horse manure was the most successful organic substrate to promote SRB activity (sulfate removal of 61%), followed by vegetal cover (49%) and legume compost (31%). These findings propose the possibility of using naturally-occurring SRB in the phosphogypsum for bioremediation strategies based on natural soil covers with organic amendments.

  7. Comparative study of photocatalytic activities of hydrothermally grown ZnO nanorod on Si(001) wafer and FTO glass substrates

    Science.gov (United States)

    Jeon, Eun Hee; Yang, Sena; Kim, Yeonwoo; Kim, Namdong; Shin, Hyun-Joon; Baik, Jaeyoon; Kim, Hyun Sung; Lee, Hangil

    2015-09-01

    ZnO nanorods have been grown on Si(001) wafer and fluorine-doped tin oxide (FTO) glass substrates for 1 and 4 h with the hydrothermal methods. The morphologies and photocatalytic activities of the ZnO nanorods were found to depend on the substrates. We investigated their properties by using spectroscopic analysis and demonstrated that the shape of nanorod and the ratios of external defects can be controlled by varying the substrates. Our experiments revealed that the nanorods grown on Si(001) have a single-crystalline wurtzite structure with (002) facets and that the number of surface oxygen defects increases with their length as the growth time increases. The nanorods grown on Si(001) have different facets, in particular wider (002) facets, and a higher ratio of the oxygen defect than the nanorods on FTO glass substrate. Moreover, the photocatalytic activities with respect to 2-aminothiophenol (2-ATP) of these nanorods were investigated with high-resolution photoemission spectroscopy (HRPES). We demonstrated that their photocatalytic activity is influenced by the ratios of surface oxygen defects, which varies with the substrate surface.

  8. Deubiquitylase, deSUMOylase, and deISGylase activity microarrays for assay of substrate preference and functional modifiers.

    Science.gov (United States)

    Loch, Christian M; Cuccherini, Charles L; Leach, Craig A; Strickler, James E

    2011-01-01

    Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  11. Ultralong In2S3 Nanotubes on Graphene Substrate with Enhanced Electrocatalytic Activity.

    Science.gov (United States)

    Guo, Sheng-qi; Chen, Xue; Hu, Fang-zhong; Zhang, Qi-chun; Liu, Lu

    2015-09-16

    Ultralong one-dimensional (1D) nanostructures including nanowires or nanotubes have been extensively studied because of their widespread applications in many fields. Although a lot of methods have been reported to prepare In2S3 nanotubes, approaching these nanotubes through one-pot solution synthesis is still extremely difficult, probably because of the intrinsic isotropic crystal growth characteristic of In2S3. In this article, we demonstrated a self-assembly approach for hydrothermal synthesis of In2S3 nanotubes/graphene composites, which contain ultralong (up to 10 μm) In2S3 nanotubes on graphene substrate. The influence of several important synthetic parameters on the final products has been systematically investigated. Importantly, the as-prepared In2S3 nanotubes/graphene composites can be easily cast on FTO to form a film, which can be used as a counter electrode. Our research indicates that the as-fabricated counter electrode exhibits excellent electrocatalytic activity toward the iodide species (I-/I3-) reduction reaction and very high energy conversion efficiency (8.01%) in dye-sensitized solar cells.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  14. Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes.

    Science.gov (United States)

    Ivanova, Irina M; Nepogodiev, Sergey A; Saalbach, Gerhard; O'Neill, Ellis C; Urbaniak, Michael D; Ferguson, Michael A J; Gurcha, Sudagar S; Besra, Gurdyal S; Field, Robert A

    2017-01-13

    Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and β-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes.

  15. Fe-Impregnated Mineral Colloids for Peroxide Activation: Effects of Mineral Substrate and Fe Precursor.

    Science.gov (United States)

    Li, Yue; Machala, Libor; Yan, Weile

    2016-02-02

    Heterogeneous iron species at the mineral/water interface are important catalysts for the generation of reactive oxygen species at circumneutral pH. One significant pathway leading to the formation of such species arises from deposition of dissolved iron onto mineral colloids due to changes in redox conditions. This study investigates the catalytic properties of Fe impregnated on silica, alumina, and titania nanoparticles (as prototypical mineral colloids). Fe impregnation was carried out by immersing the mineral nanoparticles in dilute Fe(II) or Fe(III) solutions at pH 6 and 3, respectively, in an aerobic environment. The uptake of iron per unit surface area follows the order of nTiO2 > nAl2O3 > nSiO2 for both types of Fe precursors. Impregnation of mineral particles in Fe(II) solutions results in predominantly Fe(III) species due to efficient surface-mediated oxidation. The catalytic activity of the impregnated solids to produce hydroxyl radical (·OH) from H2O2 decomposition was evaluated using benzoic acid as a probe compound under dark conditions. Invariably, the rates of benzoic acid oxidation with different Fe-laden particles increase with the surface density of Fe until a critical density above which the catalytic activity approaches a plateau, suggesting active Fe species are formed predominantly at low surface loadings. The critical surface density of Fe varies with the mineral substrate as well as the aqueous Fe precursor. Fe impregnated on TiO2 exhibits markedly higher activity than its Al2O3 and SiO2 counterparts. The speciation of interfacial Fe is analyzed with diffuse reflectance UV-vis analysis and interpretation of the data in the context of benzoic oxidation rates suggests that the surface activity of the solids for ·OH generation correlates strongly with the isolated (i.e., mononuclear) Fe species. Therefore, iron dispersed on mineral colloids is a significant form of reactive iron surfaces in the aquatic environment.

  16. Human plasma platelet-activating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates.

    Science.gov (United States)

    Stremler, K E; Stafforini, D M; Prescott, S M; McIntyre, T M

    1991-06-15

    Human plasma platelet-activating factor (PAF) acetylhydrolase hydrolyzes the sn-2 acetyl residue of PAF, but not phospholipids with long chain sn-2 residues. It is associated with low density lipoprotein (LDL) particles, and is the LDL-associated phospholipase A2 activity that specifically degrades oxidatively damaged phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1989) J. Biol. Chem. 264, 5331-5334). To identify potential substrates, we synthesized phosphatidylcholines with sn-2 residues from two to nine carbon atoms long, and found the V/k ratio decreased as the sn-2 residue was lengthened: the C5 homolog was 50%, the C6 20%, while the C9 homolog was only 2% as efficient as PAF. However, the presence of an omega-oxo function radically affected hydrolysis: the half-life of the sn-2 9-aldehydic homolog was identical to that of PAF. We oxidized [2-arachidonoyl]phosphatidylcholine and isolated a number of more polar phosphatidylcholines. We treated these with phospholipase C, derivatized the resulting diglycerides for gas chromatographic/mass spectroscopic analysis, and found a number of diglycerides where the m/z ratio was consistent with a series of short to medium length sn-2 residues. We treated the polar phosphatidylcholines with acetylhydrolase and derivatized the products for analysis by gas chromatography/mass spectroscopy. The liberated residues were more polar than straight chain standards and had m/z ratios from 129 to 296, consistent with short to medium chain residues. Therefore, oxidation fragments the sn-2 residue of phospholipids, and the acetylhydrolase specifically degrades such oxidatively fragmented phospholipids.

  17. Ligninolytic enzymes activities of Oyster mushrooms cultivated on OMW (olive mill waste) supplemented media, spawn and substrates

    NARCIS (Netherlands)

    Ruiz-Rodrigues, A.; Polonia, I.; Soler-Rivas, C.; Wichers, H.J.

    2011-01-01

    Ligninolytic enzymes activities (laccases, peroxidases (total, MnP and MiP) and aryl-alcohol oxidase (AAO)) were measured during the cultivation of six commercial Pleurotus sp. strains on MMP media, on cereal grains (spawn) and on straw substrates (the three commonly utilized cultivation steps to ob

  18. Spectroscopic characterization of the catalytically competent ferrous site of the resting, activated, and substrate-bound forms of phenylalanine hydroxylase

    Energy Technology Data Exchange (ETDEWEB)

    Loeb, K.E.; Westre, T.E.; Hedman, B.; Hodgson, K.O.; Solomon, E.I. [Stanford Univ., CA (United States); Kappock, T.J.; Mitic, N.; Glasfeld, E.; Caradonna, J.P. [Yale Univ., New Haven, CT (United States)

    1997-02-26

    The geometric structure of the catalytically relevant ferrous active site of phenylalanine hydroxylase (PAH) has been investigated using magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies. From the excited state ligand field transitions in the MCD spectrum, the temperature and field dependence of these transitions, and the XAS pre-edge shapes and intensities, the resting ferrous site of the `tense` from PAH is six-coordinate distorted octahedral. The low ligand field strength observed in the MCD spectrum results from significant oxygen ligation and longer Fe-O/N bond distances relative to model complexes as determined from an EXAFS analysis. Substrate-induced allosteric activation ({approx}34 kcal/mol) does not alter the structure of the iron site in the `relaxed` form of PAH compared to the substrate-bound `tense` state. Thus, while activation is necessary for the enzyme to achieve complete catalytic competence, it does not appear to affect the geometry of the catalytically relevent six-coordinate ferrous active site and only directly influences the surrounding protein conformation. In contrast, substrate addition results in a geometric and electronic structural change at the iron center which may help orient the substrate for completely coupled hydroxylation. 106 refs., 10 figs., 6 tabs.

  19. Glial and Neuronal Glutamate Transporters Differ in the Na+ Requirements for Activation of the Substrate-Independent Anion Conductance

    Directory of Open Access Journals (Sweden)

    Christopher B. Divito

    2017-05-01

    Full Text Available Excitatory amino acid transporters (EAATs are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na+ and substrate binding. The activation of the anion channel by binding of Na+ alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3 and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na+-dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or “leak” channel activity. Choline or N-methyl-D-glucamine replacement of external Na+ ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na+ with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na+-independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system.

  20. Engineering the Activity and Lifetime of Heterogeneous Catalysts for Carbon Nanotube Growth via Substrate Ion Beam Bombardment (Postprint)

    Science.gov (United States)

    2014-07-31

    11,25 and chirality.19,20 CNTs are grown via heterogeneous catalysis using a thin film of catalyst on a wide variety of catalyst supports. Films of...another method in catalysis science to engineer supports to enhance both catalytic activity and lifetime with general implications for heterogeneous ...AFRL-RX-WP-JA-2014-0159 ENGINEERING THE ACTIVITY AND LIFETIME OF HETEROGENEOUS CATALYSTS FOR CARBON NANOTUBE GROWTH VIA SUBSTRATE ION BEAM

  1. QM/MM studies of xanthine oxidase: variations of cofactor, substrate, and active-site Glu802.

    Science.gov (United States)

    Metz, Sebastian; Thiel, Walter

    2010-01-28

    In continuation of our previous QM/MM study on the reductive half-reaction of wild-type xanthine oxidase, we consider the effects of variations in the cofactor, the substrate, and the active-site Glu802 residue on the reaction mechanism. Replacement of the sulfido ligand in the natural cofactor by an oxo ligand leads to a substantial increase in the computed barriers, consistent with the experimentally observed inactivity of this modified cofactor, whereas the selenido form is predicted to have lower barriers and hence higher activity. For the substrate 2-oxo-6-methylpurine, the calculated pathways for three different tautomers show great similarity to those found previously for xanthine, contrary to claims in the literature that the mechanisms for these two substrates are different. Compared with the wild-type enzyme, the conversion of xanthine to uric acid follows a somewhat different pathway in the Glu802 --> Gln mutant which exhibits a lower overall activity, in agreement with recently published kinetic data. The present results confirm the basic stepwise reaction mechanism and the orientation of the substrate that has been proposed in our previous QM/MM work on aldehyde oxidoreductase and xanthine oxidase.

  2. Effect of substrate temperature on structural properties and photocatalytic activity of TiO2 thin films

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Titanium dioxide (TiO2) films with anatase structure were prepared on quartz glass substrates by pulse laser ablating titanium (99.99%) target under oxygen pressure of 10 Pa at substrate temperature of 500-800 ℃. The structural properties of the films were characterized by X-ray diffractometry(XRD), X-ray photoelectron spectroscopy(XPS) and field emission scan electron microscopy(FESEM). The results show that, as the substrate temperature is increased from 600 ℃ to 800 ℃, the anatase structure of the films changes from random growth to (211)-oriented growth. The absorption edge tested by UV-Vis Spectrometer has a blue shift. The photocatalytic activity of the films was tested on the degradation of methyl orange. It is found that the film with random growth structure exhibits better photo-degradation efficiency than that with (211)-oriented growth structure.

  3. Fabrication of highly active and cost effective SERS plasmonic substrates by electrophoretic deposition of gold nanoparticles on a DVD template

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.

    Science.gov (United States)

    Dias, Marcio V B; Huang, Fanglu; Chirgadze, Dimitri Y; Tosin, Manuela; Spiteller, Dieter; Dry, Emily F V; Leadlay, Peter F; Spencer, Jonathan B; Blundell, Tom L

    2010-07-16

    The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr(42), His(76), and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg(120) located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.

  5. Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates.

    Science.gov (United States)

    Mouchlis, Varnavas D; Bucher, Denis; McCammon, J Andrew; Dennis, Edward A

    2015-02-10

    Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein-lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein-lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme-substrate-membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme's interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface.

  6. Synthesis and structure-activity relationship of liposomal substrates for phospholipase A(2)

    DEFF Research Database (Denmark)

    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...... 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. APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    Cheila Denise Ottonelli Stopiglia

    2013-09-01

    Full Text Available Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence and 6-hydroxylaminocoumarin (blue fluorescence in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

  8. Investigation of photocatalytic activity of titanium dioxide deposited on metallic substrates by DC magnetron sputtering

    DEFF Research Database (Denmark)

    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...

  9. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    DEFF Research Database (Denmark)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper

    2016-01-01

    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) as ATM...

  10. Active Control of Cellular Orientation through In-Situ Stress in the Substrate

    Institute of Scientific and Technical Information of China (English)

    WU Heng-An; WANG Xiu-Xi; YAN Shun-Ping

    2007-01-01

    We investigate the orientation of cells on substrates to find possible methods for controlling the cellular orientation.The force dipole model is employed in our modelling and simulation.The elastic interaction between cells as well as the elastic interaction between the cell and in-situ stress field in the substrate are found to be the two main physical mechanisms to control the cellular orientation,The former interaction dominates the cellular orientation when the in-situ stress is small.while the later dominates when the in-situ stress is large enough.Two cells tend to align perpendicularly on a free substrate,but the cellular orientation varies with the increasing in-situ stress.Two cells tend to align in parallel when the normal stress is large enough.Their direction is perpendicular to the extension stress direction or parallel to the compression stress direction.When the positive in-situ shear stress is large enough,the two cells tend to align at-45°.Based on this theoretical simulation,it is believed that the cellular orientation on substrates can be controlled by the in-situ stresses.

  11. Molecular Modeling of Peroxidase and Polyphenol Oxidase: Substrate Specificity and Active Site Comparison

    Directory of Open Access Journals (Sweden)

    Lalida Shank

    2010-09-01

    Full Text Available 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 pea (PDB ID:1APX, whereas the model of grape polyphenol oxidase was obtained directly from the available X-ray structure (PDB ID:2P3X. Molecular docking of common substrates of these two enzymes was subsequently studied. It was found that epicatechin and catechin exhibited high affinity with both enzymes, even though POD and PPO have different binding pockets regarding the size and the key amino acids involved in binding. Predicted binding modes of substrates with both enzymes were also compared. The calculated docking interaction energy of trihydroxybenzoic acid related compounds shows high affinity, suggesting specificity and potential use as common inhibitor to grape ascorbate peroxidase and polyphenol oxidase.

  12. Repositioning the substrate activity screening (SAS) approach as a fragment-based method for identification of weak binders.

    Science.gov (United States)

    Gladysz, Rafaela; Cleenewerck, Matthias; Joossens, Jurgen; Lambeir, Anne-Marie; Augustyns, Koen; Van der Veken, Pieter

    2014-10-13

    Fragment-based drug discovery (FBDD) has evolved into an established approach for "hit" identification. Typically, most applications of FBDD depend on specialised cost- and time-intensive biophysical techniques. The substrate activity screening (SAS) approach has been proposed as a relatively cheap and straightforward alternative for identification of fragments for enzyme inhibitors. We have investigated SAS for the discovery of inhibitors of oncology target urokinase (uPA). Although our results support the key hypotheses of SAS, we also encountered a number of unreported limitations. In response, we propose an efficient modified methodology: "MSAS" (modified substrate activity screening). MSAS circumvents the limitations of SAS and broadens its scope by providing additional fragments and more coherent SAR data. As well as presenting and validating MSAS, this study expands existing SAR knowledge for the S1 pocket of uPA and reports new reversible and irreversible uPA inhibitor scaffolds.

  13. Synthesis of cyclic imides from nitriles and diols using hydrogen transfer as a substrate-activating strategy.

    Science.gov (United States)

    Kim, Jaewoon; Hong, Soon Hyeok

    2014-09-05

    An atom-economical and versatile method for the synthesis of cyclic imides from nitriles and diols was developed. The method utilizes a Ru-catalyzed transfer-hydrogenation reaction in which the substrates, diols, and nitriles are simultaneously activated into lactones and amines in a redox-neutral manner to afford the corresponding cyclic imides with evolution of H2 gas as the sole byproduct. This operationally simple and catalytic synthetic method provides a sustainable and easily accessible route to cyclic imides.

  14. Modulation of γ-secretase activity by multiple enzyme-substrate interactions: implications in pathogenesis of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Zeljko M Svedružić

    Full Text Available BACKGROUND: We describe molecular processes that can facilitate pathogenesis of Alzheimer's disease (AD by analyzing the catalytic cycle of a membrane-imbedded protease γ-secretase, from the initial interaction with its C99 substrate to the final release of toxic Aβ peptides. RESULTS: The C-terminal AICD fragment is cleaved first in a pre-steady-state burst. The lowest Aβ42/Aβ40 ratio is observed in pre-steady-state when Aβ40 is the dominant product. Aβ42 is produced after Aβ40, and therefore Aβ42 is not a precursor for Aβ40. The longer more hydrophobic Aβ products gradually accumulate with multiple catalytic turnovers as a result of interrupted catalytic cycles. Saturation of γ-secretase with its C99 substrate leads to 30% decrease in Aβ40 with concomitant increase in the longer Aβ products and Aβ42/Aβ40 ratio. To different degree the same changes in Aβ products can be observed with two mutations that lead to an early onset of AD, ΔE9 and G384A. Four different lines of evidence show that γ-secretase can bind and cleave multiple substrate molecules in one catalytic turnover. Consequently depending on its concentration, NotchΔE substrate can activate or inhibit γ-secretase activity on C99 substrate. Multiple C99 molecules bound to γ-secretase can affect processive cleavages of the nascent Aβ catalytic intermediates and facilitate their premature release as the toxic membrane-imbedded Aβ-bundles. CONCLUSIONS: Gradual saturation of γ-secretase with its substrate can be the pathogenic process in different alleged causes of AD. Thus, competitive inhibitors of γ-secretase offer the best chance for a successful therapy, while the noncompetitive inhibitors could even facilitate development of the disease by inducing enzyme saturation at otherwise sub-saturating substrate. Membrane-imbedded Aβ-bundles generated by γ-secretase could be neurotoxic and thus crucial for our understanding of the amyloid hypothesis and AD

  15. Effects of Hard Surface Grinding and Activation on Electroless-Nickel Plating on Cast Aluminium Alloy Substrates

    Directory of Open Access Journals (Sweden)

    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.

  16. Effects of Macromolecular Crowding on Alcohol Dehydrogenase Activity Are Substrate-Dependent.

    Science.gov (United States)

    Wilcox, A E; LoConte, Micaela A; Slade, Kristin M

    2016-06-28

    Enzymes operate in a densely packed cellular environment that rarely matches the dilute conditions under which they are studied. To better understand the ramifications of this crowding, the Michaelis-Menten kinetics of yeast alcohol dehydrogenase (YADH) were monitored spectrophotometrically in the presence of high concentrations of dextran. Crowding decreased the maximal rate of the reaction by 40% for assays with ethanol, the primary substrate of YADH. This observation was attributed to slowed release of the reduced β-nicotinamide adenine dinucleotide product, which is rate-limiting. In contrast, when larger alcohols were used as the YADH substrate, the rate-limiting step becomes hydride transfer and crowding instead increased the maximal rate of the reaction by 20-40%. This work reveals the importance of considering enzyme mechanism when evaluating the ways in which crowding can alter kinetics.

  17. Structure of active IspH enzyme from escherichia coli provides mechanistic insights into substrate reduction

    KAUST Repository

    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.

  18. Gold nano-island arrays on silicon as SERS active substrate for organic molecule detection

    Energy Technology Data Exchange (ETDEWEB)

    Ignat, Teodora, E-mail: teodora.ignat@imt.ro [National Institute for Research and Development in Microtechnologies, Laboratory of Nanobiotechnology, 126A, Erou Iancu Nicolae Street, 077190 (Romania); Husanu, Marius-Adrian, E-mail: adrianhusanu@gmail.com [National Institute of Materials Physics, Atomistilor Str. 105bis, PO Box MG 7, Magurele, Bucharest 077125 (Romania); Munoz, Roberto, E-mail: rmunoz@icmm.csic.es [Inasmet Fdn, Dept. Biomat and Nanotechnol, San Sebastian (Spain); Kusko, Mihaela, E-mail: mihaela.kusko@imt.ro [National Institute for Research and Development in Microtechnologies, Laboratory of Nanobiotechnology, 126A, Erou Iancu Nicolae Street, 077190 (Romania); Danila, Mihai, E-mail: mihai.danila@imt.ro [National Institute for Research and Development in Microtechnologies, Laboratory of Nanobiotechnology, 126A, Erou Iancu Nicolae Street, 077190 (Romania); Teodorescu, Cristian Mihail, E-mail: teodorescu@infim.ro [National Institute of Materials Physics, Atomistilor Str. 105bis, PO Box MG 7, Magurele, Bucharest 077125 (Romania)

    2014-01-01

    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.

  19. Substrate-selective COX-2 inhibition decreases anxiety via endocannabinoid activation.

    Science.gov (United States)

    Hermanson, Daniel J; Hartley, Nolan D; Gamble-George, Joyonna; Brown, Naoko; Shonesy, Brian C; Kingsley, Phillip J; Colbran, Roger J; Reese, Jeffrey; Marnett, Lawrence J; Patel, Sachin

    2013-09-01

    Augmentation of endogenous cannabinoid (eCB) signaling represents an emerging approach to the treatment of affective disorders. Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid to form prostaglandins, but also inactivates eCBs in vitro. However, the viability of COX-2 as a therapeutic target for in vivo eCB augmentation has not been explored. Using medicinal chemistry and in vivo analytical and behavioral pharmacological approaches, we found that COX-2 is important for the regulation of eCB levels in vivo. We used a pharmacological strategy involving substrate-selective inhibition of COX-2 to augment eCB signaling without affecting related non-eCB lipids or prostaglandin synthesis. Behaviorally, substrate-selective inhibition of COX-2 reduced anxiety-like behaviors in mice via increased eCB signaling. Our data suggest a key role for COX-2 in the regulation of eCB signaling and indicate that substrate-selective pharmacology represents a viable approach for eCB augmentation with broad therapeutic potential.

  20. Modulation of Pantothenate Kinase 3 Activity by Small Molecules that Interact with the Substrate/Allosteric Regulatory Domain

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing

    Science.gov (United States)

    Liu, Aizhuo; Sui, Dexin; Wu, Dianqing; Hu, Jian

    2016-01-01

    Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K’s activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with the 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane-sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane-sensing mechanism on PIP5K. The activation loop, surprisingly functioning as a membrane sensor, represents a new paradigm of kinase regulation by the activation loop through protein-membrane interaction, which also lays a foundation on the regulation of PIP5K (and other PIPKs) by membrane lipids for future studies. PMID:28138522

  2. Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase.

    Science.gov (United States)

    Srivastava, Sandeep Kumar; Rajasree, Kalagiri; Gopal, B

    2011-10-01

    Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug-an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme.

  3. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators.

    Science.gov (United States)

    Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C Y; Chang, Ta-Yuan

    2015-07-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  4. Neural substrates associated with evaluative processing during co-activation of positivity and negativity: a PET investigation.

    Science.gov (United States)

    Jung, Young Chul; An, Suk Kyoon; Seok, Jeong Ho; Kim, Jae Seung; Oh, Seung Jun; Moon, Dae Hyuk; Kim, Jae-Jin

    2006-10-01

    Affective symmetries, such as the positivity offset and negativity bias, have been postulated to be attributable to distinct activation functions of the positive and negative affect systems. We investigated the neural substrates that are engaged when the positive and negative affect systems undergo parallel and integrative processing. Eleven subjects were scanned using H(2)(15)O PET during choosing the subjective feeling produced by a stimulation pair of pictures or words. Four different conditions were designed for contrast: pure positivity, pure negativity, positivity offset, and negativity bias. The dorsolateral prefrontal activation was associated with positivity offset and negativity bias condition, whereas the ventromedial prefrontal activation, together with limbic and subcortical activations, was associated with pure positivity and pure negativity condition. The results indicated that positivity offset and negativity bias are not merely due to asymmetric activations of the positive and negative systems, but integrative processing of higher neocortical levels is involved.

  5. Fuel-Stimulated Insulin Secretion Depends upon Mitochondria Activation and the Integration of Mitochondrial and Cytosolic Substrate Cycles

    Directory of Open Access Journals (Sweden)

    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.

  6. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    Energy Technology Data Exchange (ETDEWEB)

    Marcondes, Marcelo F. [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Torquato, Ricardo J.S. [Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Assis, Diego M.; Juliano, Maria A. [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Hayashi, Mirian A.F. [Department of Pharmacology, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Oliveira, Vitor, E-mail: vitor@biofis.epm.br [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil)

    2010-01-01

    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.

  7. Inhibition of spontaneous network activity in neonatal hippocampal slices by energy substrates is not correlated with intracellular acidification.

    Science.gov (United States)

    Mukhtarov, Marat; Ivanov, Anton; Zilberter, Yuri; Bregestovski, Piotr

    2011-01-01

    Several energy substrates complementary to glucose, including lactate, pyruvate and β-hydroxybutyrate, serve as a fuel for neurons. It was reported recently that these substrates can substantially modulate cortical excitability in neonatal slices. However, complementary energy substrates (CES) can also induce an intracellular acidification when added exogenously. Therefore, action of CES on the neuronal properties governing excitability in neonatal brain slices may be underlain by a change in the cell energy status or by intracellular acidification, or both. Here, we attempt to elucidate these possibilities in neonatal hippocampus by recording neuronal population activity and monitoring intracellular pH. We show that a spontaneous network activity pattern, giant depolarizing potentials (GDPs), characteristic for the neonatal hippocampal slices exposed to artificial cerebrospinal fluid, is strongly inhibited by CES and this effect is unlikely to be caused by a subtle intracellular acidification induced by these compounds. Indeed, a much stronger intracellular acidification in the HCO(3) -free solution inhibited neither the GDP frequency nor the GDP amplitude. Therefore, modulation of neuronal energy homeostasis is the most likely factor underlying the effect of lactate, pyruvate and β-hydroxybutyrate on network excitability in neonatal brain slices.

  8. Fabrication of SERS-active substrates using silver nanofilm-coated porous anodic aluminum oxide for detection of antibiotics.

    Science.gov (United States)

    Chen, Jing; Feng, Shaolong; Gao, Fang; Grant, Edward; Xu, Jie; Wang, Shuo; Huang, Qian; Lu, Xiaonan

    2015-04-01

    We have developed a silver nanofilm-coated porous anodic aluminum oxide (AAO) as a surface-enhanced Raman scattering (SERS)-active substrate for the detection of trace level of chloramphenicol, a representative antibiotic in food systems. The ordered aluminum template generated during the synthesis of AAO serves as a patterned matrix on which a coated silver film replicates the patterned AAO matrix to form a 2-dimensional ordered nanostructure. We used atomic force microscopy and scanning electron microscopy images to determine the morphology of this nanosubstrate, and characterized its localized surface plasmon resonance by ultraviolet-visible reflection. We gauged the SERS effect of this nanosubstrate by confocal micro-Raman spectroscopy (782-nm laser), finding a satisfactory and consistent performance with enhancement factors of approximately 2 × 10(4) and a limit of detection for chloramphenicol of 7.5 ppb. We applied principal component analysis to determine the limit of quantification for chloramphenicol of 10 ppb. Using electromagnetic field theory, we developed a detailed mathematical model to explain the mechanism of Raman signal enhancement of this nanosubstrate. With simple sample pretreatment and separation steps, this silver nanofilm-coated AAO substrate could detect 50 ppb chloramphenicol in milk, indicating good potential as a reliable SERS-active substrate for rapid detection of chemical contaminants in agricultural and food products.

  9. Astronomical liquid mirrors as highly ultrasensitive, broadband-operational surface-enhanced Raman scattering-active substrates.

    Science.gov (United States)

    Lu, Tai-Yen; Lee, Yang-Chun; Yen, Yu-Ting; Yu, Chen-Chieh; Chen, Hsuen-Li

    2016-03-15

    In this study, we found that an astronomical liquid mirror can be prepared as a highly ultrasensitive, low-cost, highly reproducible, broadband-operational surface-enhanced Raman scattering (SERS)-active substrate. Astronomical liquid mirrors are highly specularly reflective because of their perfectly dense-packed silver nanoparticles; they possess a large number and high density of hot spots that experience a very high intensity electric field, resulting in excellent SERS performance. When using the liquid mirror-based SERS-active substrate to detect 4-aminothiophenol (4-ATP), we obtained measured analytical enhancement factors (AEFs) of up to 2.7×10(12) and detection limits as low as 10(-15) M. We also found that the same liquid mirror could exhibit superior SERS capability at several distinct wavelengths (532, 632.8, and 785 nm). The presence of hot spots everywhere in the liquid mirror provided highly repeatable Raman signals from low concentrations of analytes. In addition, the astronomical liquid mirrors could be transferred readily onto cheap, flexible, and biodegradable substrates and still retain their excellent SERS performance, suggesting that they might find widespread applicability in various (bio)chemical detection fields.

  10. Eelgrass slabs, a soilless culture substrate that inhibits adhesion of fungi and oomycetes and enhances antioxidant activity in tomato.

    Science.gov (United States)

    Meot-Duros, Laetitia; Le Floch, Gaëtan; Meot, Benoit; Letousey, Patricia; Jacob, Bruno; Barbier, Georges

    2011-10-26

    Composed of a marine plant, Zostera sp., eelgrass slabs are a novel organic substrate for soilless cultures used in tomato production. The benefit of using eelgrass slabs for growing tomatoes was assessed by comparing it with coconut fiber slabs in regard to contamination by Pythium spp. and to the antioxidant properties of tomato fruits. First, tomato root contamination by Pythium spp. was studied by direct plate counting, and a molecular comparison of fungal and oomycete communities was conducted using PCR-DHPLC. Second, the antioxidant properties of tomato fruits were analyzed by measuring total phenol and carotenoid contents and by evaluating radical scavenging activity. Compared to plants grown on coconut fiber slabs, those on eelgrass slabs presented a lower rate of Pythium spp. root contamination. Moreover, culture on eelgrass slabs produced fruits with better radical scavenging activity and higher total phenol content compared to controls. Carotenoid content was not affected by the type of substrate. This study highlights the value of detrital leaves of Zostera sp. as a substrate for soilless culture that reduces root contamination and also promotes the production of tomato fruits with better nutritional value.

  11. Salinosporamides A and B Inhibit Proteasome Activity and Delay the Degradation of N-end Rule Model Substrates

    Energy Technology Data Exchange (ETDEWEB)

    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. Integrated active mixing and biosensing using surface acoustic waves (SAW) and surface plasmon resonance (SPR) on a common substrate.

    Science.gov (United States)

    Renaudin, Alan; Chabot, Vincent; Grondin, Etienne; Aimez, Vincent; Charette, Paul G

    2010-01-07

    This article presents a device incorporating surface plasmon resonance (SPR) sensing and surface acoustic wave (SAW) actuation integrated onto a common LiNbO(3) piezoelectric substrate. The device uses Rayleigh-type SAW to provide active microfluidic mixing in the fluid above the SPR sensor. Validation experiments show that SAW-induced microfluidic mixing results in accelerated binding kinetics of an avidin-biotin assay. Results also show that, though SAW action causes a parasitic SPR response due to heat injection into the fluid, a relatively brief relaxation time following the SAW pulses allows the effect to dissipate, without affecting the overall assay response. Since both SPR sensors and SAW transducers can be fabricated simultaneously using low-cost microfabrication methods on a single substrate, the proposed design is well-suited to lab-on-chip applications.

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

    DEFF Research Database (Denmark)

    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 addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has about 10 glucopyranose sub...

  14. 6-Methylpurine derived sugar modified nucleosides: Synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases.

    Science.gov (United States)

    Hassan, Abdalla E A; Abou-Elkhair, Reham A I; Parker, William B; Allan, Paula W; Secrist, John A

    2016-04-01

    6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

  15. Plasmodium falciparum Plasmepsin V (PfPMV): Insights into recombinant expression, substrate specificity and active site structure.

    Science.gov (United States)

    Boonyalai, Nonlawat; Sittikul, Pichamon; Yuvaniyama, Jirundon

    2015-05-01

    Plasmepsin V from Plasmodium falciparum (PfPMV) is responsible for the cleavage of the Plasmodium export element (PEXEL) motif at the N-terminus of several hundreds of the exported proteins. PfPMV is necessary for parasite viability and has become a novel promising target for antimalarial therapy. The first recombinant expression of soluble, active PfPMV as thioredoxin fusion proteins is reported herein. Two truncated forms of PfPMV were fused to thioredoxin (Trx) to generate Trx-PfPMVp37 and Trx-PfPMVm84. The fusion proteins were successfully purified using Ni(2+) affinity chromatography in combination with ATP treatment to eliminate Escherichia coli HSP60 contaminant. Trx-PfPMVm84 could hydrolyze the PEXEL-containing peptides more efficiently than Trx-PfPMVp37. Interestingly, both Trx-PfPMVs preferred to cleave PfEMP2 peptide over HRPII peptide. The replacement of Ser with Val or Glu at P1' position created a substrate with 75% reduction in the enzyme activity, whereas the substitution of Ile with Lys or Glu at P2 position reduced the cleavage efficiency by 30%. The activity of Trx-PfPMVm84 was inhibited by PMSF and nelfinavir but not by pepstatin A. After the removal of Trx domain, activities of both enzymes toward PfEMP2 and HRPII peptides were fitted to the Michaelis-Menten model to determine kinetic parameters. The Km values toward both peptides were apparently much lower than the previously reported data although with similar kcat values. Along with an improved PfPMV preparation protocol, these findings have provided insights into its substrate specificity at P2 and P1' positions as well as interactions among the enzyme, substrates, and inhibitors.

  16. A Sensitive Method Using 4-Methylumbelliferyl-(beta)-Cellobiose as a Substrate To Measure (1,4)-(beta)-Glucanase Activity in Sediments

    NARCIS (Netherlands)

    Boschker, H.T.S.; Cappenberg, T.E.

    1994-01-01

    A sensitive method to measure (1,4)-beta-glucanase activity in organic matter-rich sediments, using 4-methylumbelliferyl-beta- cellobiose as a substrate, is described. beta-Glucosidases, which were also able to hydrolyze this substrate, were inhibited with D-glucono-delta-lactone. The produced 4- me

  17. A Sensitive Method Using 4-Methylumbelliferyl-(beta)-Cellobiose as a Substrate To Measure (1,4)-(beta)-Glucanase Activity in Sediments

    NARCIS (Netherlands)

    Boschker, H.T.S.; Cappenberg, T.E.

    1994-01-01

    A sensitive method to measure (1,4)-beta-glucanase activity in organic matter-rich sediments, using 4-methylumbelliferyl-beta- cellobiose as a substrate, is described. beta-Glucosidases, which were also able to hydrolyze this substrate, were inhibited with D-glucono-delta-lactone. The produced 4- me

  18. Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples

    Science.gov (United States)

    Mazzola, Priscila Gava

    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

  19. Substrate-mediated enhanced activity of Ru nanoparticles in catalytic hydrogenation of benzene

    KAUST Repository

    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.

  20. Microbial community dynamics linked to enhanced substrate availability and biogas production of electrokinetically pre-treated waste activated sludge.

    Science.gov (United States)

    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

  1. Photo- and Electrochromic Properties of Activated Reactive Evaporated MoO3 Thin Films Grown on Flexible Substrates

    Directory of Open Access Journals (Sweden)

    K. Hari Krishna

    2008-01-01

    Full Text Available The molybdenum trioxide (MoO3 thin films were grown onto ITO-coated flexible Kapton substrates using plasma assisted activated reactive evaporation technique. The film depositions were carried out at constant glow power and oxygen partial pressures of 8 W and 1×10−3 Torr, respectively. The influence of substrate temperature on the microstructural and optical properties was investigated. The MoO3 thin films prepared at a substrate temperature of 523 K were found to be composed of uniformly distributed nanosized grains with an orthorhombic structure of α-MoO3. These nanocrystalline MoO3 thin films exhibited higher optical transmittance of about 80% in the visible region with an evaluated optical band gap of 3.29 eV. With the insertion of 12.5 mC/cm2, the films exhibited an optical modulation of 40% in the visible region with coloration efficiency of 22 cm2/C at the wavelength of 550 nm. The MoO3 films deposited at 523 K demonstrated better photochromic properties and showed highest color center concentration for the irradiation time of 30 minutes at 100 mW/cm2.

  2. Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.

    Science.gov (United States)

    Montano, Sergio J; Lu, Jun; Gustafsson, Tomas N; Holmgren, Arne

    2014-03-15

    Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), which displayed higher fluorescence on disulfide reduction. Di-E-GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC-insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.

  3. A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser-His-Asp Containing C-C Bond Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Immunoassay for tumor markers in human serum based on Si nanoparticles and SiC@Ag SERS-active substrate.

    Science.gov (United States)

    Zhou, Lu; Zhou, Jun; Feng, Zhao; Wang, Fuyan; Xie, Shushen; Bu, Shizhong

    2016-04-21

    Based on a sandwich structure consisting of nano-Si immune probes and a SiC@Ag SERS-active immune substrate, a kind of ultra-sensitive immunoassay protocol is presented to detect tumor markers in human serum. The nano-Si immune probes were prepared by immobilizing the detecting antibodies onto the surfaces of SiO2-coated Si nanoparticles (NPs) which were modified with 3-(aminopropyl)trimethoxysilane, and the SiC@Ag SERS-active immune substrates were prepared by immobilizing the captured antibodies on Ag film sputtered on SiC sandpaper. To the best of our knowledge, it is the first time that Si NPs are directly used as Raman tags in an immunoassay strategy. And, the SiC@Ag SERS-active substrates exhibit excellent surface enhanced Raman scattering (SERS) performances with an enhancement factor of ∼10(5), owing to the plasmonic effect of the Ag film on the rough surface of the SiC sandpaper. In our experiments, the sandwich immunoassay structure has been successfully applied to detect prostate specific antigen (PSA), α-fetoprotein (AFP) and carbohydrate antigen 19-9 (CA19-9) in a human serum sample and the limit of detections are as low as 1.79 fg mL(-1), 0.46 fg mL(-1) and 1.3 × 10(-3) U mL(-1), respectively. It reveals that the proposed immunoassay protocol has demonstrated a high sensitivity for tumor markers in human serum and a potential practicability in biosensing and clinical diagnostics.

  5. Investigation of photocatalytic activity of titanium dioxide coating deposited on aluminium alloy substrate by plasma technique

    DEFF Research Database (Denmark)

    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 photoca......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...... have wide spread technological applications, where a combination of self-cleaning properties has a huge business potential. The results presented in this paper demonstrate superior photocatalytic properties of TiO2 coated aluminium compared to nano-scale TiO2 coating on glass substrate. The thickness...

  6. Oxidative enzymes activities from Lentinula edodes on agribusiness substrate / Atividade de enzimas oxidativas do Lentinula edodes em substratos agroindustriais

    Directory of Open Access Journals (Sweden)

    Claudio Perani

    2009-12-01

    Full Text Available Basidyomycete Lentinula edodes has its related enzymatic activity mainly on the agribusiness waste kind used as a substrate. The objective of this work was to verify the activity of oxidative enzymes lacase (Lac, lignina peroxidase (LiP and manganese peroxidase (MnP of three L. edodes strains, in stationary system, cultivated the 25 ºC, in the absence of light, in substrates wtih 20% of bran of rice, 1% of CaCO3 and 79% of rice husk (CA, eucalyptus sawdust (SE, cassava bagasse (BM and sugarcane bagasse (BC, adjusted to 60% of humidity. The Lac and MnP activities were bigger in eucalyptus sawdust (SE and sugarcane bagasse (BC. The LiP activity was not induced for tested substrates. The rice husk (CA and cassava bagasse (BM substrates, although are not adequate to produce Lac or MnP, can be used as additives to increase the porosity, air availability and easy metabolism polysaccharides.O basidiomiceto Lentinula edodes tem sua atividade enzimática relacionada, principalmente, ao tipo de resíduo agroindustrial utilizado como substrato. O objetivo deste trabalho foi verificar a atividade das enzimas oxidativas lacase (Lac, lignina peroxidase (LiP e manganês peroxidase (MnP de três linhagens de L. edodes, em sistema estacionário, cultivadas a 25 ºC, na ausência de luz, em substratos compostos de 20% de farelo de arroz, 1% de CaCO3 e 79% de casca de arroz (CA, serragem de eucalipto (SE, bagaço de mandioca (BM e bagaço de cana-de-açúcar (BC, ajustados à 60% de umidade. As atividades de Lac e MnP foram maiores nos substratos a base de serragem de eucalipto (SE e bagaço de cana-de-açúcar (BC. A atividade de LiP não foi induzida pelos substratos testados. Os substratos à base de casca de arroz (CA e bagaço de mandioca (BM, não se mostraram adequados na indução da produção de Lac ou MnP, mas podem ser utilizados como aditivos para aumentar a porosidade, disponibilidade de ar e fornecer polissacarídeos de fácil metabolização.

  7. Assessment of SERS activity and enhancement factors for highly sensitive gold coated substrates probed with explosive molecules

    Science.gov (United States)

    Botti, Sabina; Cantarini, Luciano; Almaviva, Salvatore; Puiu, Adriana; Rufoloni, Alessandro

    2014-01-01

    The surface enhanced Raman spectroscopy (SERS) is one of the most sensitive tools for trace analysis and the fabrication of effective and robust SERS substrates is the subject of intensive research in view of their possible applications. In this Letter, the SERS activity of an ordered structure of inverted pyramids (Klarite, Ltd) was examined by using nitro-based explosives, as Raman probe molecules. Enhancement factors up to 106 and detection limits of tens of pg were measured with an excellent run to run reproducibility. These findings provide new opportunities for the utilization of SERS for analytical detection at trace level.

  8. Microfluidic setup for on-line SERS monitoring using laser induced nanoparticle spots as SERS active substrate

    Directory of Open Access Journals (Sweden)

    Oana-M. Buja

    2017-01-01

    Full Text Available A microfluidic setup which enables on-line monitoring of residues of malachite green (MG using surface-enhanced Raman scattering (SERS is reported. The SERS active substrate was prepared via laser induced synthesis of silver or gold nanoparticles spot on the bottom of a 200 μm inner dimension glass capillary, by focusing the laser beam during a continuous flow of a mixture of silver nitrate or gold chloride and sodium citrate. The described microfluidic setup enables within a few minutes the monitoring of several processes: the synthesis of the SERS active spot, MG adsorption to the metal surface, detection of the analyte when saturation of the SERS signal is reached, and finally, the desorption of MG from the spot. Moreover, after MG complete desorption, the regeneration of the SERS active spot was achieved. The detection of MG was possible down to 10−7 M concentration with a good reproducibility when using silver or gold spots as SERS substrate.

  9. ACTIVITIES OF AMMONIA ASSIMILATION ENZYMES AS INDICATORS OF THE RELATIVE SUPPLY OF NITROGEN SUBSTRATES FOR MARINE BACTERIOPLANKTON IN SUB-TROPICAL COASTAL WATER

    Science.gov (United States)

    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. A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method.

    Science.gov (United States)

    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.

  11. Evaluating the addition of activated carbon to heat-treated mushroom casing for grain-based and compost-based substrates.

    Science.gov (United States)

    Bechara, Mark A; Heinemann, P H; Walker, P N; Demirci, A; Romaine, C P

    2009-10-01

    Two substrates, a non-composted grain spawn substrate and a traditional composted substrate, each covered with peat-based casing that contained varying amounts of activated carbon (AC) and each receiving different heat-treatment durations, were tested for Agaricus bisporus mushroom production. The amounts of AC were 0, 5, 10, 15, and 20% v/v, and the heat treatments were 0, 60, and 180 min at 121 degrees C and 103.4 kPa. Overall, the addition of AC up to 10-15% of casing for a grain spawn substrate increased mushroom yield. However, the addition of AC to the casing for compost substrates had no significant effect on yield, whereas heat-treating the casing increased yield. The onset of fruiting was retarded in grain spawn treatments not receiving AC with heat-treatment durations of 60 and 180 min, whereas this effect was not as apparent for the compost substrates. On average, mushroom yield was greater for the grain spawn substrate (366 g) than for compost substrate (287 g). For grain spawn substrate, the results show that the addition of AC ranging from 5% to 10% was adequate for maximum mushroom production.

  12. Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V. (Duke)

    2012-04-18

    Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.

  13. An oxidized derivative of phosphatidylcholine is a substrate for the platelet-activating factor acetylhydrolase from human plasma.

    Science.gov (United States)

    Stremler, K E; Stafforini, D M; Prescott, S M; Zimmerman, G A; McIntyre, T M

    1989-04-05

    Platelet-activating factor (PAF) is a glycerophospholipid that has diverse potent biological actions. A plasma enzyme catalyzes the hydrolysis of the sn-2 acetoyl group of PAF and thereby abolishes its bioactivity. This PAF acetylhydrolase is specific for phospholipids, such as PAF, with a short acyl group at the sn-2 position. The majority of it (60-70%) is associated with low density lipoprotein (LDL), and the remainder is with high density lipoprotein (HDL). LDL also has a phospholipase A2 activity that is specific for oxidized polyunsaturated fatty acids, which may be important in determining how LDL is recognized by cellular receptors. We previously have purified and characterized the PAF acetylhydrolase from human plasma. We now have found that the purified PAF acetylhydrolase catalyzes the hydrolysis of the oxidized fragments of arachidonic acid from the sn-2 position of phosphatidylcholine. One of the preferred substrates appeared by mass spectrometry to have 5-oxovalerate at the sn-2 position. We synthesized 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine and found that the PAF acetylhydrolase had the same apparent Km for it (11.3 microM) as for PAF (12.5 microM), with Vmax values of 100 and 167 mumol/h/mg of protein, respectively. We also conclude that the PAF acetylhydrolase is the sole activity in LDL that degrades oxidized phospholipids since we found co-localization of the activity against both substrates to LDL and HDL, and precipitation of enzyme activity with an antibody to the PAF acetylhydrolase. Thus, the PAF acetylhydrolase in human plasma degrades oxidized phospholipids, which may be involved in the modification of apolipoprotein B100 and other pathological processes.

  14. Arrayed SU-8 polymer thermal actuators with inherent real-time feedback for actively modifying MEMS’ substrate warpage

    Science.gov (United States)

    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. Effective SERS-active substrates composed of hierarchical micro/nanostructured arrays based on reactive ion etching and colloidal masks

    Science.gov (United States)

    Zhang, Honghua; Liu, Dilong; Hang, Lifeng; Li, Xinyang; Liu, Guangqiang; Cai, Weiping; Li, Yue

    2016-09-01

    A facile route has been proposed for the fabrication of morphology-controlled periodic SiO2 hierarchical micro/nanostructured arrays by reactive ion etching (RIE) using monolayer colloidal crystals as masks. By effectively controlling the experimental conditions of RIE, the morphology of a periodic SiO2 hierarchical micro/nanostructured array could be tuned from a dome-shaped one to a circular truncated cone, and finally to a circular cone. After coating a silver thin layer, these periodic micro/nanostructured arrays were used as surface-enhanced Raman scattering (SERS)-active substrates and demonstrated obvious SERS signals of 4-Aminothiophenol (4-ATP). In addition, the circular cone arrays displayed better SERS enhancement than those of the dome-shaped and circular truncated cone arrays due to the rougher surface caused by physical bombardment. After optimization of the circular cone arrays with different periodicities, an array with the periodicity of 350 nm exhibits much stronger SERS enhancement and possesses a low detection limit of 10-10 M 4-ATP. This offers a practical platform to conveniently prepare SERS-active substrates.

  16. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. Simple synthetic route for SERS-active gold nanoparticles substrate with controlled shape and organization.

    Science.gov (United States)

    Bechelany, Mikhael; Brodard, Pierre; Elias, Jamil; Brioude, Arnaud; Michler, Johann; Philippe, Laetitia

    2010-09-07

    We report a simple synthetic route based on electroless deposition (galvanic displacement) and natural lithography to simultaneously control the shape and organization of Au nanoparticles (NPs). We show for the first time the formation of organized extended domains of Au nanoflowers and nanocrowns with single crystalline tips. The dimension and morphology of the desired nanostructures (NSs) can be tuned easily by controlling the deposition conditions at room temperature using saccharin as an organic additive. The exact role of saccharin on the crystal growth process of Au NPs is also discussed. A systematic surface enhancement Raman spectroscopy (SERS) study of large, ordered areas of organized gold nanoflowers using p-mercaptoaniline (pMA) as the probe molecule shows massive and reproducible enhancements of the Raman signal. By comparing the relative enhancement of the different vibrational modes as a function of the morphology, the specific charge-transfer (chemical effect) SERS mechanism can be distinguished from the general electromagnetic field enhancement (physical effect). A wide range of applications can be envisaged for these SERS substrates.

  19. Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

    DEFF Research Database (Denmark)

    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...

  20. Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

    Institute of Scientific and Technical Information of China (English)

    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.

  1. Carnitine palmitoyl transferase activity and whole muscle oxidation rates vary with fatty acid substrate in avian flight muscles.

    Science.gov (United States)

    Price, Edwin R; Staples, James F; Milligan, C Louise; Guglielmo, Christopher G

    2011-05-01

    Birds primarily fuel migratory flights with fat, and the composition of that fat has the potential to affect overall lipid oxidation rates. We measured the whole muscle lipid oxidation rates in extensor digitorum communis muscles from white-throated sparrows (Zonotrichia albicollis Gmelin) incubated for 20 min at 20°C with radiolabeled stearate (18:0), oleate (18:1ω9), or linoleate (18:2ω6). Lipid oxidation rates were ~40% higher with linoleate than oleate (oleate: 36 ± 8.54 μmol CO(2) g(-1) h(-1)), and ~75% lower with stearate compared with oleate, indicating that maximal lipid oxidation rates can indeed be affected by the type of fatty acid supplied to the muscle. Additionally, we investigated the activity of the mitochondrial fatty acid transport-associated enzyme carnitine palmitoyl transferase (CPT) in pectoralis muscles of 5 bird species (Zonotrichia albicollis, Philomachus pugnax, Sturnus vulgaris, Taeniopygia guttata, Passer domesticus). Activity was measured in homogenized samples using various fatty acyl-CoA substrates (16:0, 16:1, 18:0, 18:1ω9, 18:2ω6, 18:3ω3, 18:3ω6, 20:0, 20:4ω6, 22:6ω3) in a spectrophotometric assay. CPT activity increased with the degree of unsaturation and decreased with chain length. CPT activity did not differ between ω3 and ω6 isomers of 18:3, nor was the pattern of CPT substrate preference different between captive white-throated sparrows in a migratory (i.e., displaying Zugunruhe) or non-migratory state. These findings can explain previously observed differences in peak performance induced by dietary fat composition and suggest that lipid supply is limiting to maximal exercise performance in birds.

  2. Interaction between substrates suggests a relationship between organophosphorus-sensitive phenylvalerate- and acetylcholine-hydrolyzing activities in chicken brain.

    Science.gov (United States)

    Benabent, Mónica; Vilanova, Eugenio; Mangas, Iris; Sogorb, Miguel Ángel; Estévez, Jorge

    2014-10-15

    Organophosphorus compounds (OPs) induce neurotoxic disorders through interactions with well-known target esterases, such as acetylcholinesterase and neuropathy target esterase (NTE). However, OPs interact with other esterases of unknown biological function. In soluble chicken brain fractions, three components of enzymatic phenylvalerate esterase activity (PVase) called Eα, Eβ and Eγ, have been kinetically discriminated. These components are studied in this work for the relationship with acetylcholine-hydrolyzing activity. When Eα PVase activity (resistant PVase activity to 1500 μM PMSF for 30 min) was tested with different acetylthiocholine concentrations, inhibition was observed. The best-fitting model to the data was the non-competitive inhibition model (Km=0.12, 0.22 mM, Ki=6.6, 7.6 mM). Resistant acetylthiocholine-hydrolyzing activity to 1500 μM PMSF was inhibited by phenylvalerate showing competitive inhibition (Km=0.09, 0.11 mM; Ki=1.7, 2.2 mM). Eβ PVase activity (resistant PVase activity to 25 μM mipafox for 30 min) was not affected by the presence of acetylthiocholine, while resistant acetylthiocholine-hydrolyzing activity to 25 μM mipafox showed competitive inhibition in the presence of phenylvalerate (Km=0.05, 0.06 mM; Ki=0.44, 0.58 mM). The interactions observed between the substrates of AChE and PVase suggest that part of PVase activity might be a protein with acetylthiocholine-hydrolyzing activity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Wetland restoration and methanogenesis: the activity of microbial populations and competition for substrates at different temperatures

    Directory of Open Access Journals (Sweden)

    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.

  4. Wetland restoration and methanogenesis: the activity of microbial populations and competition for substrates at different temperatures

    Directory of Open Access Journals (Sweden)

    V. Jerman

    2009-06-01

    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 of 84 d at 15°C and a minimum of 7 d at 37°C, the optimum temperature for methanogenesis. This lag 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.

  5. Thermodynamics of the localized D2-D6 system

    Energy Technology Data Exchange (ETDEWEB)

    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}.

  6. #608902 DRUG METABOLISM, POOR, CYP2D6-RELATED [OMIM

    Lifescience Database Archive (English)

    Full Text Available henotype are tests using oral ingestion of debrisoquine (approximately 5 mg) and dextromethorphan (approxima...-hydroxydebrisoquine and dextromethorphan-to-dextrorphan. Poor metabolizers are defined by an MR of greater ...than 12.6 for debrisoquine and an MR of greater than 0.3 for dextromethorphan (Sachse et al., 1997). Ultrara

  7. CYP2D6 genotype determination in the Danish population

    DEFF Research Database (Denmark)

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

    1994-01-01

    .741 (0.026) in the Danish population and the second most common allele was the D6-B with an apparent frequency of 0.194 (0.024). The median (range) of the sparteine metabolic ratio (MR) in 47 homozygous D6-wt EM was 0.28 (0.11-4.10) and the corresponding value in heterozygous EM was 0.36 (0...

  8. Thermodynamics of the localized D2-D6 system

    CERN Document Server

    Gómez-Reino, Marta; Schnitzer, H; 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 entropy, which is proportional to N_2^{3/2} N_6^{1/2} T_H^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_2 ~ N_6.

  9. Influence of soil management practices and substrate availability on microbial biomass and its activities in some haplic luvisols

    Energy Technology Data Exchange (ETDEWEB)

    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

  10. Rapid functional definition of extended spectrum β-lactamase activity in bacterial cultures via competitive inhibition of fluorescent substrate cleavage.

    Science.gov (United States)

    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 40× 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 (C(i)) in 30 min. Significant differences in the relative C(i) values of the panel of β-lactams were determined in vitro for Bacillus cereus penicillinase. Additionally, the relative C(i) values for whole bacterial cell suspensions of B. cereus 5/β were compared with the relative minimal inhibitory concentration (MIC) values and a correlation coefficient of 0.899 was determined. We further demonstrated the ability of β-LEAP to probe the capacity of ceftazidime to inhibit the enzyme activity of a panel of ESBL-producing Escherichia coli. The bacteria were assayed for susceptibility to ceftazidime and the relative MIC values were compared with the relative C(i) values for ceftazidime yielding a correlation coefficient of 0.984. This work demonstrates for the first time the whole cell assay of the competitive inhibition of β-lactamase enzyme activity and derivation of associated constants.

  11. A Study of the Antibacterial Activity of Polyhexamethylene Bigaunide on Cotton Substrate

    NARCIS (Netherlands)

    Amrit, Usha; Hendrix, R.; Dutschk, Victoria; Warmoeskerken, Marinus; Mijovic, Budimir; Ujevic, Darko; Petrak, Slavenka; Marija Grancaric, Ana; Ira Glogar, Martinia; Salopek Cubric, Ivana

    2012-01-01

    Interest in antibacterial textiles has been increasing recently. Different kinds of antibacterial textiles with different effectivity are being sold in the market. The influence of time on the antibacterial activity of such textiles is not known generally. The goal of this research was to study the

  12. Human intestinal P-glycoprotein activity estimated by the model substrate digoxin

    DEFF Research Database (Denmark)

    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...

  13. A Study of the Antibacterial Activity of Polyhexamethylene Bigaunide on Cotton Substrate

    NARCIS (Netherlands)

    Amrit, U.R.; Hendrix, R.; Dutschk, V.; Warmoeskerken, M.M.C.G.; Mijovic, Budimir; Ujevic, Darko; Petrak, Slavenka; Grancaric, Marija Ana; Glogar, Ira Martinia; Salopek Cubric, Ivana

    2012-01-01

    Interest in antibacterial textiles has been increasing recently. Different kinds of antibacterial textiles with different effectivity are being sold in the market. The influence of time on the antibacterial activity of such textiles is not known generally. The goal of this research was to study the

  14. Correlation between enzyme activity and substrate storage in a cell culture model system for Gaucher disease.

    Science.gov (United States)

    Schueler, U H; Kolter, T; Kaneski, C R; Zirzow, G C; Sandhoff, K; Brady, R O

    2004-01-01

    Gaucher disease, the most common sphingolipidosis, is caused by a decreased activity of glucosylceramide beta-glucosidase, resulting in the accumulation of glucosylceramide in macrophage-derived cells known as Gaucher cells. Much of the storage material is thought to originate from the turnover of cell membranes, such as phagocytosed red and white blood cells. In this study, an in vitro model of Gaucher disease was developed by treating the murine macrophage cell line J774 with a specific inhibitor of glucosylceramide beta-glucosidase, conduritol B-epoxide, and feeding red blood cell ghosts, in order to mimic the disease state. It was found in this model system that glucosylceramide beta-glucosidase activity could be reduced to about 11-15% of the normal control level before increased storage of glucosylceramide occurred. This in vitro system allows insight into the correlation between enzyme activity and lipid storage as predicted by the theory of residual enzyme activity that was proposed by Conzelmann and Sandhoff.

  15. Silver overlayer-modified surface-enhanced Raman scattering-active gold substrates for potential applications in trace detection of biochemical species.

    Science.gov (United States)

    Ou, Keng-Liang; Hsu, Ting-Chu; Liu, Yu-Chuan; Yang, Kuang-Hsuan; Tsai, Hui-Yen

    2014-01-02

    Because Ag and Au nanoparticles (NPs) possess well-defined localized surface plasmon resonance (LSPR) they are popularly employed in the studies of surface-enhanced Raman scattering (SERS). As shown in the literature and in our previous studies, the advantage of SERS-active Ag NPs is their higher SERS enhancement over Au NPs. On the other hand, the disadvantage of SERS-active Ag NPs compared to Au NPs is their serious decay of SERS enhancement in ambient laboratory air. In this work, we develop a new strategy for preparing highly SERS-active Ag NPs deposited on a roughened Au substrate. This strategy is derived from the modification of electrochemical underpotential deposition (UPD) of metals. The coverage of Ag NPs on the roughened Au substrate can be as high as 0.95. Experimental results indicate that the SERS of Rhodamine 6G (R6G) observed on this developed substrate exhibits a higher intensity by ca. 50-fold of magnitude, as compared with that of R6G observed on the substrate without the deposition of Ag NPs. The limit of detection (LOD) for R6G measured on this substrate is markedly reduced to 2×10(-15)M. Moreover, aging of SERS effect observed on this developed substrate is significantly depressed, as compared with that observed on a generally prepared SERS-active Ag substrate. These aging tests were performed in an atmosphere of 50% relative humidity (RH) and 20% (v/v) O2 at 30°C for 60 day. Also, the developed SERS-active substrate enables it practically applicable in the trace detection of monosodium urate (MSU)-containing solution in gouty arthritis without a further purification process.

  16. Structure of Human Acid Sphingomyelinase Reveals the Role of the Saposin Domain in Activating Substrate Hydrolysis.

    Science.gov (United States)

    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.

  17. The production and activity test of cellulases using bagasse substrate on Aspergillus niger isolated from Clove field, Kare, Madiun

    Science.gov (United States)

    Ardhi, Muh. Waskito; Sulistyarsi, Ani; Pujiati

    2017-06-01

    Aspergillus sp is a microorganism which has a high ability to produce cellulase enzymes. In producing Cellulase enzymes requires appropriate concentration and incubation time to obtain optimum enzyme activity. This study aimed to determine the effect of inoculum concentration and incubation time towards production and activity of cellulases from Aspergillus sp substrate bagasse. This research used experiments method; completely randomized design with 2 factorial repeated 2 times. The treatment study include differences inoculum (K) 5% (K1), 15% (K2) 25%, (K3) and incubation time (F) that is 3 days (F1), 6 days (F2), 9 days (F3), 12 days (F4). The data taken from the treatment are glucose reduction and protein levels of crude cellulase enzyme activity that use Nelson Somogyi and Biuret methods. Analysis of variance ANOVA data used two paths with significance level of 5% then continued with LSD test. The results showed that: Fhit>Ftab. Thus, there is effect of inoculum concentrations and incubation time toward activity of crude cellulases of Aspergillus sp. The highest glucose reduction of treatment is K3F4 (concentration of inoculum is 25% with 12 days incubation time) amount 12.834 g / ml and the highest protein content is K3F4 (concentration of inoculum is 25% with with 12 days incubation time) amount 0.740 g / ml.

  18. Retraction: Characterization of cellulolytic activities of newly isolated Thelephora sowerbyi from North-Western Himalayas on different lignocellulosic substrates.

    Science.gov (United States)

    Sharma, Deepika; Goel, Gunjan; Bansal, Saurabh; Mahajan, Rishi; Sharma, B M; Chauhan, Rajinder Singh

    2016-12-01

    Characterization of cellulolytic activities of newly isolated Thelephora sowerbyi from North-Western Himalayas on different lignocellulosic substrate J. Basic Microbiol. 2015, 55, 1-11 - DOI: 10.1002/jobm.201500107 The above article from the Journal of Basic Microbiology, published online on 08 June 2015 in Wiley Online Library as Early View (http://onlinelibrary.wiley.com/doi/10.1002/jobm.201500107/pdf), has been retracted by agreement between the authors, the Editor-in-Chief and Wiley-VCH GmbH & Co. KGaA. The retraction has been agreed because the microorganism studied in the described experiments has been identified as the fungus Cotylidia pannosa (Gene Accession No. KT008117) instead of Thelephora sowerbyi. The culture has been identified on the basis of the sequence of the amplified ITS region of the microorganism which was submitted by the authors to the NCBI database.

  19. C60 as an active smart spacer material on silver thin film substrates for enhanced surface plasmon coupled emission.

    Science.gov (United States)

    Mulpur, Pradyumna; Podila, Ramakrishna; Ramamurthy, Sai Sathish; Kamisetti, Venkataramaniah; Rao, Apparao M

    2015-04-21

    In this study, we present the use of C60 as an active spacer material on a silver (Ag) based surface plasmon coupled emission (SPCE) platform. In addition to its primary role of protecting the Ag thin film from oxidation, the incorporation of C60 facilitated the achievement of a 30-fold enhancement in the emission intensity of rhodamine B (RhB) fluorophore. The high signal yield was attributed to the unique π-π interactions between C60 thin films and RhB, which enabled efficient transfer of energy of RhB emission to Ag plasmon modes. Furthermore, minor variations in the C60 film thickness yielded large changes in the enhancement and angularity properties of the SPCE signal, which can be exploited for sensing applications. Finally, the low-cost fabrication process of the Ag-C60 thin film stacks render C60 based SPCE substrates ideal, for the economic and simplistic detection of analytes.

  20. Room-temperature bonding method for polymer substrate of flexible electronics by surface activation using nano-adhesion layers

    Science.gov (United States)

    Matsumae, Takashi; Fujino, Masahisa; Suga, Tadatomo

    2015-10-01

    A sealing method for polymer substrates to be used in flexible electronics is studied. For this application, a low-temperature sealing method that achieves flexible bonding of inorganic bonding material is required, but no conventional technique satisfies these requirements simultaneously. In this study, a new polymer bonding method using thin Si and Fe layers and the surface activated bonding (SAB) method are applied to bond poly(ethylene naphthalate) (PEN) films to each other. PEN films can be bonded via the proposed method without voids at room temperature, and the bonded samples are bendable. The adhesion strength of the bonded samples is so strong that fracture occurs in the polymer bulk rather than at the bond interface. Investigations of the bonded samples by transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR) reveal that bonding is achieved by chemical interactions between the polymer surface and deposited atoms.

  1. Testing anti-fungal activity of a soil-like substrate for growing plants in bioregenerative life support systems

    Science.gov (United States)

    Nesterenko, E. V.; Kozlov, V. A.; Khizhnyak, S. V.; Manukovsky, N. S.; Kovalev, V. S.; Gurevich, Yu. L.; Liu, Hong; Xing, Yidong; Hu, Enzhu

    2009-10-01

    The object of this research is to study a soil-like substrate (SLS) to grow plants in a Bioregenerative Life Support System (BLSS). Wheat and rice straw were used as raw materials to prepare SLS. Anti-fungal activity of SLS using test cultures of Bipolaris sorokiniana, a plant-pathogenic fungus which causes wheat root rot was studied. Experiments were conducted with SLS samples, using natural soil and sand as controls. Infecting the substrates, was performed at two levels: the first level was done with wheat seeds carrying B. sorokiniana and the second level with seeds and additional conidia of B. sorokiniana from an outside source. We measured wheat disease incidence and severity in two crop plantings. Lowest disease incidence values were obtained from the second planting, SLS: 26% and 41% at the first and the second infection levels, respectively. For soil the values were 60% and 82%, respectively, and for sand they were 67% and 74%, respectively. Wheat root rot in the second crop planting on SLS, at both infection levels was considerably less severe (9% and 13%, respectively) than on natural soil (20% and