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The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion ofFADto 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, whichmaymodulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria. [ABSTRACT FROM AUTHOR]

Prostaglandin E 2 (PGE 2 ) is the most abundant prostanoid synthesized in the kidney and plays an important role in renal function. Physiologically, PGE 2 regulates renal hemodynamics, water and sodium metabolism, blood pressure, and so on. As a well-known proinflammatory lipid mediator, PGE 2 also substantially mediates renal injury under many pathophysiological conditions. Multiple enzymes are involved in renal PGE 2 biosynthesis, including the three main PGE 2 terminal synthases, i.e. microsomal PGE 2 synthase-1 (mPGES-1), mPGES-2 and cytosolic PGE 2 synthase (cPGES). In the kidney, mPGES-1 is highly expressed in the collecting duct where it is the dominant contributor of PGE 2 biosynthesis and participates in blood pressure regulation and renal hemodynamic maintenance. mPGES-2 protein is mainly expressed in the renal cortex and the outer stripe of the outer medulla. cPGES is diffusely expressed in all nephron segments. Roles of mPGES-2 and cPGES in renal function have not been clearly characterized. Here we summarize the role of PGE 2 in the kidney, highlight the contribution of the three PGE 2 synthases, particularly mPGES-1, in blood pressure regulation and renal hemodynamics, and outline the contribution of mPGES-1 to kidney diseases. A clearer understanding of the role of PGE 2 in the kidney could pave the way for development of new therapeutic approaches. [ABSTRACT FROM AUTHOR]

The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5:UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5:UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL (Molecule Interacting with CasL) also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD axon guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance. [ABSTRACT FROM AUTHOR]

Aims Hypoxic response mediated by hypoxia-inducible factor ( HIF) seems to contribute to the benefit of endurance training. To verify the direct contribution of HIF activation to running training without exposure to atmospheric hypoxia, we used prolyl hydroxylase domain 2 ( PHD2) conditional knockout mice (c KO), which exhibit HIF activation independent of oxygen concentration, and we examined their maximal exercise capacity before and after 4 weeks of treadmill exercise training. Methods Phd2 f/f mice ( n = 26) and Phd2 c KO mice ( n = 24) were randomly divided into two groups, trained and untrained, and were subjected to maximal running test before and after a 4-week treadmill-training regimen. Results Prolyl hydroxylase domain 2 deficiency resulted in HIF-α protein accumulation. Phd2 c KO mice exhibited marked increases in haematocrit values and haemoglobin concentrations, as well as an increase in the capillary number in the skeletal muscle. The 4-week training elicited an increase in the capillary-to-fibre (C/F) ratio and succinyl dehydrogenase activity of the skeletal muscle. Importantly, trained Phd2 c KO mice showed a significantly greater improvement in running time than trained control mice ( P < 0.05). Collectively, these data suggest that the combination of training and the activation of the HIF pathway are important for maximizing the effect of running training. Conclusion We conclude that the activation of the HIF pathway induced by PHD2 deficiency enhances the effect of running training. [ABSTRACT FROM AUTHOR]

Microsomal cytochrome P450 enzymes (CYPs) are membrane-attached enzymes that play indispensable roles in biotransformations of numerous endogenous and exogenous compounds. Although recent progress in experiments and simulations has allowed many important features of CYP-membrane interactions to be deciphered, many other aspects remain underexplored. Using microsecond-long molecular dynamics simulations, we analyzed interaction of CYP3A4 with bilayers composed of lipids differing in their polar head groups, i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. In the negatively charged lipids, CYP3A4 was immersed more deeply and was more inclined toward the membrane because of favorable electrostatic and hydrogen bonding interactions between the CYP catalytic domain and lipid polar head groups. We showed that electrostatics significantly contributes to positioning and orientation of CYP on the membrane and might contribute to the experimentally observed preferences of individual CYP isoforms to distribute in (dis)ordered membrane microdomains. [ABSTRACT FROM AUTHOR]

At present, there is controversy regarding the efficacy of tamoxifen in breast cancer patients who are carriers of cytochrome P450 2D6 (CYP2D6) gene polymorphisms, in terms of recurrence and overall survival. Thus, the aim of the present study was to investigate the association of the CYP2D6 *4, *10 and *17 gene polymorphisms with breast cancer recurrence in a Brazilian population. The cohort comprised 40 receptor-positive breast cancer patients without recurrence and 40 with distant recurrence. A 3-ml sample of peripheral blood was collected from each patient to determine the presence of the *4, *10 and *17 single nucleotide polymorphisms of the CYP2D6 gene by quantitative polymerase chain reaction analysis. There was no statistically significant difference between the two groups regarding the polymorphism frequency (P=0.246). The results revealed that intermediate metabolizers occurred in 5% of patients without recurrence and in 15% of those with distant recurrence. Poor metabolizers occurred in only 1 patient (2.5%) per group, and there was no significant difference between the groups (P=0.789). Thein women with hormone-sensitive breast cancer treated with tamoxifen was not associated with disease recurrence. present study concluded that the CYP2D6 gene polymorphism in women with hormone-sensitive breast cancer treated with tamoxifen was not associated with disease recurrence. [ABSTRACT FROM AUTHOR]

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2′-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 μM. Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed. [ABSTRACT FROM AUTHOR]

During cytokinesis, the antiparallel array of microtubules forming the central spindle organizes the midbody, a structure that anchors the ingressed cleavage furrow and guides the assembly of abscission machinery. Here, we identified a role for the flavoprotein monooxygenase MICAL3, an actin disassembly factor, in organizing midbody-associated protein complexes. By combining cell biological assays with cross-linking mass spectrometry, we show that MICAL3 is recruited to the central spindle and the midbody through a direct interaction with the centralspindlin component MKLP1. Knock-out of MICAL3 leads to an increased frequency of cytokinetic failure and a delayed abscission. In a mechanism independent of its enzymatic activity, MICAL3 targets the adaptor protein ELKS and Rab8A-positive vesicles to the midbody, and the depletion of ELKS and Rab8A also leads to cytokinesis defects. We propose that MICAL3 acts as a midbody-associated scaffold for vesicle targeting, which promotes maturation of the intercellular bridge and abscission. [ABSTRACT FROM AUTHOR]

Background: Molecules Interacting with CasL (MICAL1), a multidomain flavoprotein monoxygenase, is strongly involved in the mechanisms that promote cancer cell proliferation and survival. Activation of MICAL1 causes an up-regulation of reactive oxygen species (ROS) in HeLa cells. ROS can function as a signaling molecule that modulates protein phosphorylation, leading to malignant phenotypes of cancer cells such as invasion and metastasis. Herein, we tested whether MICAL1 could control cell migration and invasion through regulating ROS in breast cancer cell lines.Methods: The effects of depletion/overexperssion of MICAL1 on cell invasion rate were measured by matrigel-based transwell assays. The contents of ROS in breast cancer cells were evaluated by CM2-DCFHDA staining and enhanced lucigenin chemiluminescence method. RAB35 activity was assessed by pulldown assay. The relationship of RAB35 and MICAL1 was evaluated by immunofluorescence, coimmunoprecipitation, immunoblotting and co-transfection techniques. Immunoblotting assays were also used to analyze Akt phosphorylation level.Results: In this study, we found that depletion of MICAL1 reduced cell migration and invasion as well as ROS generation. Phosphorylation of Akt was also attenuated by MICAL1 depletion. Likewise, the over-expression of MICAL1 augmented the generation of ROS, increased Akt phosphorylation, and favored invasive phenotype of breast cancer cells. Moreover, we investigated the effect of EGF signaling on MICAL1 function. We demonstrated that EGF increased RAB35 activation and activated form of RAB35 could bind to MICAL1. Silencing of RAB35 repressed ROS generation, prevented Akt phosphorylation and inhibited cell invasion in response to EGF.Conclusions: Taken together, our results provide evidence that MICAL1 plays an essential role in the activation of ROS/Akt signaling and cell invasive phenotype and identify a novel link between RAB35 and MICAL1 in regulating breast cancer cell invasion. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Ewing sarcoma ( ES) is an aggressive pediatric tumor driven by the fusion protein EWS- FLI1. We report that EWS- FLI1 suppresses TDO2-mediated tryptophan (TRP) breakdown in ES cells. Gene expression and metabolite analyses reveal an EWS- FLI1-dependent regulation of TRP metabolism. TRP consumption increased in the absence of EWS- FLI1, resulting in kynurenine and kynurenic acid accumulation, both aryl hydrocarbon receptor ( AHR) ligands. Activated AHR binds to the promoter region of target genes. We demonstrate that EWS- FLI1 knockdown results in AHR nuclear translocation and activation. Our data suggest that EWS- FLI1 suppresses autocrine AHR signaling by inhibiting TDO2-catalyzed TRP breakdown. [ABSTRACT FROM AUTHOR]

Background: This study aimed to investigate genetic polymorphisms of CYP2D6 among healthy Uygur individuals. Genetic polymorphisms of CYP2D6 could greatly affect CYP2D6 activity and lead to differences among individuals in drug efficacy or side effects. To investigate genetic polymorphisms of CYP2D6 in the Uygur population, we directly sequenced the whole gene in 96 unrelated, healthy Uygur volunteers from the Xinjiang Uygur Autonomous Region and screened for genetic variants in the promoter, intron, exons, and 3'UTR. Results: We detected 62 genetic polymorphisms of CYP2D6, 16 of which were novel SNP with three novel non-synonymous mutations detected for the first time. The allelic frequencies of CYP2D6*1, *10, *39, and *48 were 0.542, 0.156, 0.068, 0.229, and 0.073, respectively. The frequency of CYP2D6*1/*10 which decreased CYP2D6 enzyme activity was 31.3 %. Conclusions: Our results provided basic information about CYP2D6 polymorphisms, suggested that the enzymatic activities of CYP2D6 might be different within the Uygur ethnic group, and provide a basis for safer drug administration and better therapeutic treatment of Uygur individuals. [ABSTRACT FROM AUTHOR]

3-Hydroxybenzoate 6-hydroxylase (3 HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that contains FAD as a redox-active cofactor. The enzyme catalyzes para-hydroxylation of 3-hydroxybenzoate (3 HB) to form 2,5-dihydroxybenzoate (2,5- DHB). Based on the enzyme crystal structure, residue His213 is located close to the hydroxyl moiety, whereas Tyr217 is close to the carboxylate group of 3 HB. Y217A and Y217S did not show any perturbation of flavin absorption upon addition of 3 HB, whereas Y217F has a Kd value for 3 HB binding of 7.5 m m, which is ~ 50-fold larger than that found for wild-type enzyme. The results clearly indicate that Tyr217 is necessary for substrate binding. All His213 variants can bind to 3 HB with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. H213S, H213D and H213E produce 2,5- DHB with yields of 28 ± 5%, 52 ± 7% and 92 ± 6%, respectively, whereas H213A cannot catalyze hydroxylation. The results indicate that the interaction between the hydroxyl group of 3 HB and residue 213 is important for substrate hydroxylation. Interestingly, the hydroxylation rate constant of H213E (35 s−1) is similar to that of wild-type enzyme (36 s−1) and this variant has an efficiency of hydroxylation (92 ± 6%) similar to the wild-type enzyme (86 ± 2%). Difference spectra of enzyme-bound substrate suggest that 3 HB binds to H213E in the phenolic form. The results indicate that His213 and Glu213 in H213E may act as a catalytic base to initiate the substrate deprotonation and facilitate the electrophilic aromatic substitution of 3 HB. [ABSTRACT FROM AUTHOR]

Purpose: Polymorphisms in the genes coding for the carcinogen metabolizing enzymes may affect enzyme activities and alter the activation and detoxification rates of the carcinogens. AKR1C3 is one of the very polymorphic xenobiotic metabolizing enzymes involved in the bioactivation process. Here we aimed to investigate the association of two single nucleotide polymorphisms in AKR1C3, rs12529 (c.15C > G) and rs1937920 (12259 bp 3' of STP A > G) with urinary bladder cancer (UBC). Materials and Methods: Two-hundred fifty UBC cases and 250 control subjects were genotyped using the Polymerase Chain Reaction and Restriction Fragment Length method. Associations of the genotypes with UBC risk and tumor characteristics were assessed using logistic regression and Fisher's exact test. The results are corrected for multiple testing. Results: We identified strong associations between the studied AKR1C3 variants and UBC risk. The homozygous variant genotype of rs12529 was found to be inversely associated with UBC, and rs1937920 was shown to be associated with increased risk of UBC. None of the genotypes were found to be significantly associated with tumor characteristics. Conclusion: We provided evidence that rs12529 and rs1937920 are significant in the molecular pathogenesis of UBC. However, the results presented here should be regarded as preliminary and might represent a first step of future larger studies aiming to better elucidate the role of AKR1C3 polymorphisms in the susceptibility to bladder cancer. [ABSTRACT FROM AUTHOR]

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have previously been associated with a predisposition to pituitary adenomas. However, to the best of our knowledge, mutations in AIP that relate specifically to sporadic non-functioning pituitary adenomas (NFPAs) have yet to be reported. Therefore, the present study aimed to identify single nucleotide polymorphisms (SNPs) in the AIP gene that may be associated with NFPAs. Peripheral blood samples and the entire coding sequence of the AIP gene from 56 patients with NFPAs and 56 controls were analyzed in triplicate. Of the 56 patients with NFPAs, 9 patients (16.1%) were identified as harboring five different SNPs, although no germline mutations in the AIP gene were detected in any of the patients. Three different SNPs (7051C>T, 8012G>C and 8020G>C) were identified in exons 4 and 6 in 3 different patients (each in 1 patient). Two different SNPs (7318C>A and 7886A>G) were identified in exons 5 and 6, respectively, in 6 different patients (each in 3 patients). No SNPs or germline mutations in the AIP gene were identified in the controls. The results of the present study suggested that mutations in the AIP gene might not have an important role in the tumorigenesis of NFPAs. However, further studies are required in order to investigate potential molecular and genetic mechanisms that may underlie the involvement of AIP in NFPA. [ABSTRACT FROM AUTHOR]

Objective There are conflicting reports regarding the effects of cytochrome P450 (P450, CYP) genotypes on the plasma concentrations of risperidone and its pharmacologically active metabolite, 9-hydroxyrisperidone (paliperidone), in clinical patients. The aim of this study was to investigate individual differences in the metabolic clearance of risperidone in vitro and in vivo. Methods In vitro liver microsomal risperidone 9-hydroxylation activities and in vivo plasma concentrations of risperidone and paliperidone were investigated in 15 male and 12 female Japanese subjects (mean age 52 years, range: 24-75 years) genotyped for CYP2D6 and CYP3A5. Results CYP2D6 intermediate and poor metabolizers showed significantly lower liver microsomal risperidone 9-hydroxylation activities than extensive metabolizers did at 5 μM of risperidone; this difference was not evident at 50 μM of risperidone. The recombinant CYP3A5 Vmax/ Km value for risperidone 9-hydroxylation was 30% that of CYP3A4, and liver microsomes from CYP3A5 expressers had similar risperidone 9-hydroxylation activities to those of CYP3A5 poor expressers. The plasma concentration/dose ratios for risperidone and paliperidone in 27 Japanese patients were not significantly influenced by the CYP2D6 or CYP3A5 genotypes. Conclusions Individual differences in metabolic clearance of risperidone under the present conditions were not significantly influenced by the genotypes of CYP2D6 or CYP3A5. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Streptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of nonglycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in biological membranes and as a tool for the diagnosis of Niemann-Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have proven useful for the discovery of products with improved properties. Here, we describe the late biosynthetic steps for filipin III biosynthesis and strategies for the generation of bioactive filipin III derivatives at high yield. Results: A region of 13,778 base pairs of DNA from the S. filipinensis genome was isolated, sequenced, and characterized. Nine complete genes and two truncated ORFs were located. Disruption of genes proved that this genomic region is part of the biosynthetic cluster for the 28-membered ring of the polyene macrolide filipin. This set of genes includes two cytochrome P450 monooxygenase encoding genes, filC and filD, which are proposed to catalyse specific hydroxylations of the macrolide ring at C26 and C1' respectively. Gene deletion and complementation experiments provided evidence for their role during filipin III biosynthesis. Filipin III derivatives were accumulated by the recombinant mutants at high yield. These have been characterized by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification thus revealing the post-polyketide steps during polyene biosynthesis. Two alternative routes lead to the formation of filipin III from the initial product of polyketide synthase chain assembly and cyclization filipin I, one trough filipin II, and the other one trough 1'-hydroxyfilipin I, all filipin III intermediates being biologically active. Moreover, minimal inhibitory concentration values against Candida utilis and Saccharomyces cerevisiae were obtained for all filipin derivatives, finding that 1'-hydroxyfilipin and especially filipin II show remarkably enhanced antifungal bioactivity. Complete nuclear magnetic resonance assignments have been obtained for the first time for 1'-hydroxyfilipin I. Conclusions: This report reveals the existence of two alternative routes for filipin III formation and opens new possibilities for the generation of biologically active filipin derivatives at high yield and with improved properties. [ABSTRACT FROM AUTHOR]

A rapid method for the simultaneous determination of the in vitro activity of the 10 major human liver UDP-glucuronosyltransferase (UGT) enzymes was developed based on the cocktail approach. Specific substrates were first selected for each UGT: etoposide for UGT1A1, chenodeoxycholic acid for UGT1A3, trifluoperazine for UGT1A4, serotonin for UGT 1A6, isoferulic acid for UGT1A9, codeine for UGT2B4, azidothymidine for UGT2B7, levomedetomidine for UGT2B10, 4-hydroxy-3-methoxymethamphetamine for UGT2B15 and testosterone for UGT2B17. Optimal incubation conditions, including time-based experiments on cocktail metabolism in pooled HLMs that had been performed, were then investigated. A 45-min incubation period was found to be a favorable compromise for all the substrates in the cocktail. Ultra-high pressure liquid chromatography coupled to an electrospray ionization time-of-flight mass spectrometer was used to separate the 10 substrates and their UGT-specific glucuronides in less than 6 min. The ability of the cocktail to highlight the UGT inhibitory potential of xenobiotics was initially proven by using well-known UGT inhibitors (selective and nonselective) and then by relating some of the screening results obtained by using the cocktail approach with morphine glucuronidation (substrate highly glucuronidated by UGT 2B7). All the results were in agreement with both the literature and with the expected effect on morphine glucuronidation. [ABSTRACT FROM AUTHOR]

Flavin-containing mono-oxygenases (FMOs) are a family of microsomal chemical- and drug-metabolizing enzymes. FMO3 is a major FMO form in adult mouse and human liver. FMO3 mutations have been associated with the incidence and severity of trimethylaminuria (TMAU), a metabolic disorder characterized by the inability of the affected individual to metabolize the odorous trimethylamine to its non-odorous N-oxide. In addition to this primary genetic form, there are other forms of TMAU that support the hypothesis that FMO3 activity may be modulated by steroid hormones. To understand the molecular mechanism involved in the regulation of Fmo3 gene expression by steroid hormones, we performed this study in an in vitro cellular system, mouse liver cells, and on the human FMO3 gene. Dexamethasone, 5α-dihydrotestosterone, thyroid hormone, and progesterone had no effect on the accumulation of Fmo3 mRNA. The use of increased concentration of theophylline inhibited estrogen receptor α (ERα)-mediated transcription of Fmo3 mRNA. 17β-Estradiol inhibited Fmo3 mRNA accumulation. The use of ICI 164,384 abolished the inhibitory effect induced by estrogen. Gel-shift analyses showed a binding in the 5′ region of the Fmo3 gene. This binding was abrogated by an excess of a cDNA containing an estrogen-responsive element. An estrogen-binding site was also present in the first intron of the human gene, as demonstrated by the gel-shift assay. Supershift experiments confirmed the binding of ERα in both mouse and human samples. Furthermore, chromatin immunoprecipitation assay confirmed the binding of ERα in the promoter region of mouse Fmo3 and in the first intron of the human FMO3 gene. Thus, 17β-estradiol plays a fundamental role in the regulation of Fmo3 gene transcription. [ABSTRACT FROM AUTHOR]

The article focuses on the inhibition of cytochrome P-450 CYP2D6 with low dose paroxetine in patients with high 10-hydroxynortriptyline serum levels. Topics discussed include the metabolism of the tricyclic antidepressant (TCA) nortriptyline by CYP2D6 to active metabolites, the impact of 5 milligrams (mg) parotexine on nortriptyline serum levels in four patients, and an illustration on the increase of nortriptyline and hydroxynortriptyline serum levels after the additionof 5 mg of paroxetine.

Background and Purpose: CYP2D6 metabolizes many centrally acting drugs, neurotoxins and endogenous neurochemicals, and differences in brain levels of CYP2D have been associated with brain function and drug response. Alcohol consumers and smokers have higher levels of CYP2D6 in brain, but not liver, suggesting ethanol and/or nicotine may induce human brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self‐administration and nicotine treatment on CYP2D expression in African green monkeys. Experimental Approach: Forty monkeys were randomized into control, ethanol‐only, nicotine‐only and ethanol + nicotine groups. Two groups voluntarily self‐administered 10% ethanol in sucrose solution for 4 h·day−1, whereas two groups consumed sucrose solution on the same schedule. Two groups received daily s.c. injections of 0.5 mg·kg−1 nicotine in saline bid, whereas two groups were injected with saline on the same schedule. Key Results: Both nicotine and ethanol dose‐dependently increased CYP2D in brain; brain mRNA was unaffected, and neither drug altered hepatic CYP2D protein or mRNA. The combination of ethanol and nicotine increased brain CYP2D protein levels to a greater extent than either drug alone (1.2–2.2‐fold, P < 0.05 among the eight brain regions assessed). Immunohistochemistry revealed the induction of brain CYP2D protein within specific cell types and regions in the treatment groups. Conclusions and Implications: Ethanol and nicotine increase brain CYP2D protein levels in monkeys, in a region and treatment‐specific manner, suggesting that CNS drug responses, neurodegeneration and personality may be affected among people who consume alcohol and/or nicotine. [ABSTRACT FROM AUTHOR]

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the [sup 32] P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 10[sup 8] nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min[sup -1] 10[sup -6] cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n= 42, r= 0.25, p= 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n= 13, r= 0.70, p< 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n= 17, r= 0.44, p= 0.07) and GSTP1-AA genotype (n= 29, r= 0.37, p= 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes. [ABSTRACT FROM AUTHOR]

Benzo(a)pyrene diol-epoxide (BPDE)-DNA adducts and aryl hydrocarbon hydroxylase (AHH) activity were determined in white blood cells (WBCs) obtained from non-smokers, ex-smokers and smokers. DNA adduct levels ranged from non-detectable (ND) to 8.8 per 108 nucleosides in all samples analysed. The mean level in WBC from smokers (1.93 +/- 0.55 mean +/- SE) was similar to that from non-smokers (1.18 +/- 0.40) and ex-smokers (1.83 +/- 0.50). The distribution frequency of AHH activity in non-smokers was different from that in smokers, while 50% of non-smokers had AHH activity ranging from ND to 20 fmol mg-1 min-1. This low AHH activity was not found in smokers; no correlation was found between AHH activity and tobacco consumption (r=0.097; P=0.76; n=12). On the contrary, AHH activity was correlated with BPDE-DNA adduct levels in WBCs from the same subjects (r=0.457; P=0.019; n=26). Our findings indicate that there are limitations to the use of WBCs as surrogate cells for determining internal PAH exposure in target organs. [ABSTRACT FROM AUTHOR]

The Ah receptor protein, important in the mechanism of induction of aryl hydrocarbon hydroxylase activity, has been identified and partially characterized in hepatic cytosolic preparations from rat, BALB/c mouse, gerbil, hamster, rabbit, ferret and guinea-pig by means of sucrose density centrifugation analysis and hydroxypatite binding assays. Using 2,3,7,8-tetrachloro [³H] dibenzo-p-dioxin (TCDD) as the ligand, total specific binding capacities ranged over 74-691 fmol [³H]TCDD/mg cytosolic protein and apparent dissociation constants ranged over 0.30-7.8 nM. There was no quantitative correlation between the concentration of cytosolic Ah receptors and the 3-methylcholanthrene-mediated induction of aryl hydrocarbon hydroxylase activity in the species studied. Competitive binding studies with a series of monohydroxylated activity in the species studied. Competitive binding studies with a series of monohydroxylated benzo[a] pyrene derivatives suggested the importance of electronic character in their ability to bind to the Ah receptor and to compete with TCDD for specific binding sites on the receptor. [ABSTRACT FROM AUTHOR]

The subcellular distribution of aryl-hydrocarbon hydroxylase, NADPH-cytochrome c reductase, NADH :cytochrome c reductase, and NADH :cytochrome b5 reductase activities in mouse liver was studied using the biochemical membrane markers microsomal glucose-6-phosphatase, mitochondrial cytochrome c oxidase, and plasma membrane 5′-nucleotidase. The rate of appearance of activity of 3-methylcholanthrene-induced aryl-hydrocarbon hydroxylase in the microsomes of C57BL/6N mice is more than twice as rapid as that in the nuclear envelope. The nuclear fraction contains less than 1% of the total cellular activities of the hydroxylase and all three reductases. All detectable basal activity of aryl-hydrocarbon hydroxylase in the nuclear fraction of control C57BL/6N and DBA′2N and 3-methylcholanthrene-treated DBA/2N mice and all detectable activities of NADPH :cytochrome c, NADH :cytochrome c, and NADH :catochrome b5 reductase in the nuclear fraction of control and 3-methylcholanthrene-treated C57BL/6N and DBA/2N mice can be completely accounted for by the degree of microsomal fragment contamination (as assessed by the microsomal marker glucose-6-phosphatase). These data raise doubts about certain previous reports of ‘nuclear’ enzyme activities in which microsomal contamination was not taken into account. However, there is more induced activity of aryl-hydrocarbon hydroxylase in the nuclear fraction of 3-methylcholanthrene-treated C57BL/6N mice than can be accounted for by the degree of microsomal membrane contribution. The routine Ah locus is known to regulate the induction by certain polycyclic aromatic chemicals of numerous drug-metabolizing enzyme activities such as aryl-hydrocarbon hydroxylase associated with cytochrome P1-450. The expression of 3-methylcholanthrene-inducible hydroxylase in nuclear membranes, like that in microsomal membranes, thus appears to be controlled by the Ah regulatory gene. [ABSTRACT FROM AUTHOR]

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed. [ABSTRACT FROM AUTHOR]

A review of the article "Activation of the hypoxia-inducible factor pathway induced by prolyl hydroxylase domain 2 deficiency enhances the effect of running training in mice" by Aki Nunomiya et al., which appeared in the periodical "Acta Physiologica" in 2016, is presented.

The article focuses on a research related to the development of pharmacogenetic and clinical algorithms for warfarin dosing by genotype as of March 2014. Topics discussed include a personalized approach to clinical management by pharmacogenetics due to the increasing ability to determine genotypes, the impact of polymorphisms of the cytochrome P-450 system on vitamin K antagonist (VAK) and treatment of venous thromboembolism (VTE).

The article focuses on a research related to the role of genotype in therapeutic range in patients with atrial fibrillation (AF) and venous thromboembolism (VTE) as of March 2014. Topics discussed include the impact of polymorphisms of the cytochrome P-450 system (CYP2C9) on the sensitivity to vitamin K antagonist (VAK), increase in risk for international normalized ratio (INR) due to genotype-based dosing and the effect of the indication for warfarin on genotype-guided dosing.

The article focuses on a research related to the effect of acenocoumarol or phenprocoumon dosing by genotype on therapeutic range in patients with atrial fibrillation (AF) and venous thromboembolism (VTE) as of March 2014. Topics discussed include the effect of CYP2C9 enzyme on phenprocoumon metabolism as compared to warfarin, parenteral heparin to occur in VTE patients than AF patients and the presence of a structured anticoagulation clinic in high-quality warfarin management.

The article discusses a review of peer-reviewed observational studies or clinical trials which examined the link between cytochrome P450(CYP)2C19 variant genotypes and clinical outcomes in patients with coronary artery disease under clopidogrel treatment regimen. Study authors found that reduced function CYP2C19 genotypes may be associated with increased risk for stent thrombosis.